prostate cancer-derived human epithelial cells Search Results


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  • 99
    ATCC prostate cancer derived human epithelial cells
    Expression of trypsinogen IV in <t>human</t> intact whole kidney and in HPTCs. PCR products for reverse-transcribed mRNA isolated from intact human kidney ( left ), from cultured HPTCs ( middle ), or from <t>prostate</t> <t>cancer-derived</t> human <t>epithelial</t> <t>cells</t> (PC3) ( right ) were obtained using the PCR primers and procedures outlined under “Experimental Procedures.” The sizes of the PCR products can be estimated from the base pair ( BP ) ladder standards shown.
    Prostate Cancer Derived Human Epithelial Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore human fgf 2
    Expression of trypsinogen IV in <t>human</t> intact whole kidney and in HPTCs. PCR products for reverse-transcribed mRNA isolated from intact human kidney ( left ), from cultured HPTCs ( middle ), or from <t>prostate</t> <t>cancer-derived</t> human <t>epithelial</t> <t>cells</t> (PC3) ( right ) were obtained using the PCR primers and procedures outlined under “Experimental Procedures.” The sizes of the PCR products can be estimated from the base pair ( BP ) ladder standards shown.
    Human Fgf 2, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    ATCC cell culture human prostate cancer epithelial cell lines
    Interactions of stromal fibroblasts and <t>prostate</t> <t>cancer.</t> Coculture of HS27A bone stromal fibroblasts with prostate cancer LNCaP (HS27A-LN) and C4-2 (HS27A-C4-2) cells as tumoroids or alone (HS27A) under 3D-RWV <t>culture</t> conditions ( a , b ). Microscopic images of cultures were taken on day 14, and the size ( a ) and number of tumoroids were calculated. Each condition was performed in duplicate vessels for three independent experiments. A representative image of each group is shown at the top. Quantitative data are represented as a box–whisker plot of triplicate experiments. Tumor-promoting effect of cancer-associated fibroblasts (CAFs) in a xenograph mouse model ( c , d ). Nude mice were subcutaneously implanted with C4-2 prostate cancer cells, either alone (C4-2) or mixed with HS27A pre-cultured with LNCaP (HS27A LN ) or C4-2 (HS27A C4-2 ) ( c ), or primary cultured prostate fibroblasts derived from benign/normal prostate (BAF) or prostate cancer (CAF) tissues of the same patients ( d ). Eight mice were used in each group and independently conducted twice. Tumor growth was monitored weekly, and the results are presented as representative images of two independent experiments (top) and a box–whisker plot of median tumor volumes (bottom) of each group ( n = 16 per group; two independent experiments pooled) at the end of the experiment (8 weeks after <t>cell</t> inoculation). Boxes ( a – d ) encompass the 25th to 75th percentiles, and the whiskers represent the 10%–90% range of observations. <t>Lines</t> within boxes represent median values. * p
    Cell Culture Human Prostate Cancer Epithelial Cell Lines, supplied by ATCC, used in various techniques. Bioz Stars score: 96/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    ATCC human prostate carcinoma epithelial cell line
    Interactions of stromal fibroblasts and <t>prostate</t> <t>cancer.</t> Coculture of HS27A bone stromal fibroblasts with prostate cancer LNCaP (HS27A-LN) and C4-2 (HS27A-C4-2) cells as tumoroids or alone (HS27A) under 3D-RWV <t>culture</t> conditions ( a , b ). Microscopic images of cultures were taken on day 14, and the size ( a ) and number of tumoroids were calculated. Each condition was performed in duplicate vessels for three independent experiments. A representative image of each group is shown at the top. Quantitative data are represented as a box–whisker plot of triplicate experiments. Tumor-promoting effect of cancer-associated fibroblasts (CAFs) in a xenograph mouse model ( c , d ). Nude mice were subcutaneously implanted with C4-2 prostate cancer cells, either alone (C4-2) or mixed with HS27A pre-cultured with LNCaP (HS27A LN ) or C4-2 (HS27A C4-2 ) ( c ), or primary cultured prostate fibroblasts derived from benign/normal prostate (BAF) or prostate cancer (CAF) tissues of the same patients ( d ). Eight mice were used in each group and independently conducted twice. Tumor growth was monitored weekly, and the results are presented as representative images of two independent experiments (top) and a box–whisker plot of median tumor volumes (bottom) of each group ( n = 16 per group; two independent experiments pooled) at the end of the experiment (8 weeks after <t>cell</t> inoculation). Boxes ( a – d ) encompass the 25th to 75th percentiles, and the whiskers represent the 10%–90% range of observations. <t>Lines</t> within boxes represent median values. * p
    Human Prostate Carcinoma Epithelial Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC human squamous cell carcinoma line siha
    Co-infection of Onc.Ad with HDPD-L1 ( CAd-VEC PD-L1) amplified PD-L1 mini-body while maintaining oncolysis in vitro and in vivo (A) <t>A549,</t> PC-3, <t>SiHa</t> and HepG2 were infected with a total 10 Vp/cell of HDPD-L1, Onc.Ad or with an CAd-VECPD-L1 (Onc.Ad:HDAd; 1:10). Medium samples were collected at 48 hours post-infection. Levels of PD-L1 mini-body in medium samples were quantified by ELISA-based assay for HA-tagged protein. Data are presented as means ± SD (n=4). *P =0.003, **P=0.002, ***P
    Human Squamous Cell Carcinoma Line Siha, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Lonza normal human prostate epithelial cells prec
    Interactions of <t>human</t> bone marrow-derived MSCs and <t>prostate</t> cancer <t>cells.</t> (A) Migratory response of human MSCs to the medium, <t>normal</t> prostatic <t>epithelial</t> cells (PrEC) and prostate cancer cell lines (DU145, PC3 and LNCaP). Migrated cells through the membrane of transwells were stained with crystal violet 8 h after cell plating. The lower surface of the membrane was photographed (100x), and a representative image from each condition is shown at top. Quantitative data are represented as the means ± SD of the number of cells per high-power field (100x) in triplicate experiments. (B) Macroscopic (upper) and microscopic (bottom) view of cellular aggregates under 3-D RWV culture conditions of 3A6 cells alone (3A6/3A6) or mixed with LNCaP (3A6/LNCaP), C4-2 (3A6/C4-2) or PC3 (3A6/PC3) cells. (C) Detection of mixed cellular components in the 3-D cultured prostate tumoroids by H E and immunohistochemical staining of E-cadherin (E-Cad) in the cell aggregate sections.
    Normal Human Prostate Epithelial Cells Prec, supplied by Lonza, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC culture conditions 22rv1 human prostate carcinoma epithelial cells
    RelB effects on cell death of <t>22Rv1</t> cells. A) Illustration of 22Rv1-derived cells cultured in suspension on poly-HEMA pre-coated plates. Representative fields at 200X magnificence. B) Cell death evaluation in suspension cell culture of 22Rv1-derived cells. Cells were cultured 7 days in 96-well plates pre-coated or not with poly-HEMA. Cell death was evaluated by bioluminescence using the CytoTOX-Glo cytotoxicity assay. Data represent the ratio of basal cell death/total cell death after complete lysis using digitonin reagent. Cell death in suspension culture conditions are illustrated in black bars and adherence control cell culture conditions in empty bars. The variation was significant at p
    Culture Conditions 22rv1 Human Prostate Carcinoma Epithelial Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC non tumorigenic epithelial cell line mcf10a
    RelB effects on cell death of <t>22Rv1</t> cells. A) Illustration of 22Rv1-derived cells cultured in suspension on poly-HEMA pre-coated plates. Representative fields at 200X magnificence. B) Cell death evaluation in suspension cell culture of 22Rv1-derived cells. Cells were cultured 7 days in 96-well plates pre-coated or not with poly-HEMA. Cell death was evaluated by bioluminescence using the CytoTOX-Glo cytotoxicity assay. Data represent the ratio of basal cell death/total cell death after complete lysis using digitonin reagent. Cell death in suspension culture conditions are illustrated in black bars and adherence control cell culture conditions in empty bars. The variation was significant at p
    Non Tumorigenic Epithelial Cell Line Mcf10a, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SAS institute oscc derived cell lines
    RelB effects on cell death of <t>22Rv1</t> cells. A) Illustration of 22Rv1-derived cells cultured in suspension on poly-HEMA pre-coated plates. Representative fields at 200X magnificence. B) Cell death evaluation in suspension cell culture of 22Rv1-derived cells. Cells were cultured 7 days in 96-well plates pre-coated or not with poly-HEMA. Cell death was evaluated by bioluminescence using the CytoTOX-Glo cytotoxicity assay. Data represent the ratio of basal cell death/total cell death after complete lysis using digitonin reagent. Cell death in suspension culture conditions are illustrated in black bars and adherence control cell culture conditions in empty bars. The variation was significant at p
    Oscc Derived Cell Lines, supplied by SAS institute, used in various techniques. Bioz Stars score: 92/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    ATCC prostate epithelial cell line rwpe 1
    Tipifarnib inhibits exosome biogenesis and secretion via both ESCRT dependent and independent pathways and disrupts RAS signaling. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with tipifarnib or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the qNano IZON. Tipifarnib shows a significant decrease in the concentration of exosomes in C4-2B cells both at 0.25 and 1 μM as well as in the PC-3 cells at 0.25 μM. ( B ) Tipifarnib at a 1 μM concentration significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in C4-2B cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Tipifarnib significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal <t>RWPE-1</t> cells. Full blots are presented in Supplementary Fig. S6 . ( D ) Tipifarnib significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p
    Prostate Epithelial Cell Line Rwpe 1, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    ATCC normal prostate epithelial cell line wpmy 1
    Tipifarnib inhibits exosome biogenesis and secretion via both ESCRT dependent and independent pathways and disrupts RAS signaling. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with tipifarnib or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the qNano IZON. Tipifarnib shows a significant decrease in the concentration of exosomes in C4-2B cells both at 0.25 and 1 μM as well as in the PC-3 cells at 0.25 μM. ( B ) Tipifarnib at a 1 μM concentration significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in C4-2B cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Tipifarnib significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal <t>RWPE-1</t> cells. Full blots are presented in Supplementary Fig. S6 . ( D ) Tipifarnib significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p
    Normal Prostate Epithelial Cell Line Wpmy 1, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore egf
    Tipifarnib inhibits exosome biogenesis and secretion via both ESCRT dependent and independent pathways and disrupts RAS signaling. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with tipifarnib or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the qNano IZON. Tipifarnib shows a significant decrease in the concentration of exosomes in C4-2B cells both at 0.25 and 1 μM as well as in the PC-3 cells at 0.25 μM. ( B ) Tipifarnib at a 1 μM concentration significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in C4-2B cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Tipifarnib significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal <t>RWPE-1</t> cells. Full blots are presented in Supplementary Fig. S6 . ( D ) Tipifarnib significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p
    Egf, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher human recombinant basic fibroblast growth factor bfgf
    Mel-18 negatively regulated the self-renew of gastric cancer stem like cells A. Patients with Mel-18 positive expression survived longer than those with Mel-18 negative expression. Kaplan-Meyer survival curves were plotted as cumulative survival vs months according to Mel-18 expression (negative or positive) in cancer samples in patients with gastric cancer. The expression of Mel-18 was detected by Immunohistochemical (IHC) Assay. B. The expression of Mel-18 mRNA was downregulated in spheroid cells (SC) compared with that in parental adherent cells (PC). Tumorigenic spheres were derived from SGC7901 gastric cancer cell line in serum-free media containing <t>EGF</t> and <t>bFGF</t> and then photographed (upper pane). Fold change of Mel-18 in PC and SC was analyzed by QRT-PCR (lower panel). Total RNA of parental adherent cells and spheroid cells were extracted using TRIzol reagent (Invitrogen) and cDNA synthesis were performed as manufacturer's protocol. For Mel-18 mRNA, GAPDH acted as an internal control. C. Overexpression of Mel-18 reduced self-renew of gastric cancer stem like cells. The self-renew ability was analyzed by serum-free culture spheres formation and photographed (upper pane). The percentage of spheres formed was calculated and plotted (lower left panel), and overexpression of Mel-18 in SGC-7901 cells was determined by Western blot ( lower right panel ). Stable cell lines expressing Mel-18 was generated by transfection of Mel-18 overexpressing plasmid and selected by puromycine. Con: cells transfected with control vector; Mel-18: cells transfected with Mel-18 overexpressing plasmids. D. Mel-18 overexpression inhibited in vivo tumorigenecity of SGC7901 cells. In vivo tumorigenecity was detected by subcutaneously cancer cells injected SCID mice model. Suppressed tumor size after SGC7901 injected subcutaneously in one rear flank of severe combined SCID mice. Mel-18-overexpressing SGC-7901 cells or control cells were injected subcutaneously into the flanks of severe combined SCID mice. Tumor sizes were detected terminally by vernier caliper. After 4 weeks, mice were sacrificed by cervical dislocation, and tumors were removed and imaged. All experiments involving animal abided by protocols approved by the Shanghai Medical Experimental Animal Care Commission.
    Human Recombinant Basic Fibroblast Growth Factor Bfgf, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression of trypsinogen IV in human intact whole kidney and in HPTCs. PCR products for reverse-transcribed mRNA isolated from intact human kidney ( left ), from cultured HPTCs ( middle ), or from prostate cancer-derived human epithelial cells (PC3) ( right ) were obtained using the PCR primers and procedures outlined under “Experimental Procedures.” The sizes of the PCR products can be estimated from the base pair ( BP ) ladder standards shown.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites *

    doi: 10.1074/jbc.M113.538470

    Figure Lengend Snippet: Expression of trypsinogen IV in human intact whole kidney and in HPTCs. PCR products for reverse-transcribed mRNA isolated from intact human kidney ( left ), from cultured HPTCs ( middle ), or from prostate cancer-derived human epithelial cells (PC3) ( right ) were obtained using the PCR primers and procedures outlined under “Experimental Procedures.” The sizes of the PCR products can be estimated from the base pair ( BP ) ladder standards shown.

    Article Snippet: Total RNA from human kidney tissue or cultured HPTCs or prostate cancer-derived human epithelial cells (PC3, CRL-1435TM ; ATCC (Manassas, VA)) was extracted using an RNAeasy kit (Qiagen) and transcribed to cDNA using Moloney murine leukemia virus transcriptase (Invitrogen) according to the manufacturer's protocol.

    Techniques: Expressing, Polymerase Chain Reaction, Isolation, Cell Culture, Derivative Assay

    Tipifarnib inhibits exosome biogenesis and secretion via both ESCRT dependent and independent pathways and disrupts RAS signaling. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with tipifarnib or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the qNano IZON. Tipifarnib shows a significant decrease in the concentration of exosomes in C4-2B cells both at 0.25 and 1 μM as well as in the PC-3 cells at 0.25 μM. ( B ) Tipifarnib at a 1 μM concentration significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in C4-2B cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Tipifarnib significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . ( D ) Tipifarnib significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p

    Journal: Scientific Reports

    Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

    doi: 10.1038/s41598-018-26411-7

    Figure Lengend Snippet: Tipifarnib inhibits exosome biogenesis and secretion via both ESCRT dependent and independent pathways and disrupts RAS signaling. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with tipifarnib or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the qNano IZON. Tipifarnib shows a significant decrease in the concentration of exosomes in C4-2B cells both at 0.25 and 1 μM as well as in the PC-3 cells at 0.25 μM. ( B ) Tipifarnib at a 1 μM concentration significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in C4-2B cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Tipifarnib significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . ( D ) Tipifarnib significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p

    Article Snippet: The human prostate cancer epithelial metastatic cell line PC-3 (ATCC CRL-1435) and the normal prostate epithelial cell line RWPE-1 (ATCC CRL-11609) were purchased from ATCC (Manassas, VA, USA).

    Techniques: Concentration Assay, Protein Concentration, Activation Assay, Derivative Assay

    Ketoconazole (prototype imidazole) inhibits exosome biogenesis through ESCRT dependent and independent pathway. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with ketoconazole or DMSO (vehicle control) for 48 hours at 5 μM. Concentration of exosomes was measured by qNano IZON. Ketoconazole shows a significant decrease in the concentration of exosomes in C4-2B cells at 5 μM as well as in the PC-3 cells ( B ) Ketoconazole significantly inhibited the protein concentration of Alix, nSMase2 and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Ketoconazole significantly inhibited activation p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B, and PC-3 cells, but not in the RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p

    Journal: Scientific Reports

    Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

    doi: 10.1038/s41598-018-26411-7

    Figure Lengend Snippet: Ketoconazole (prototype imidazole) inhibits exosome biogenesis through ESCRT dependent and independent pathway. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with ketoconazole or DMSO (vehicle control) for 48 hours at 5 μM. Concentration of exosomes was measured by qNano IZON. Ketoconazole shows a significant decrease in the concentration of exosomes in C4-2B cells at 5 μM as well as in the PC-3 cells ( B ) Ketoconazole significantly inhibited the protein concentration of Alix, nSMase2 and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Ketoconazole significantly inhibited activation p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B, and PC-3 cells, but not in the RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p

    Article Snippet: The human prostate cancer epithelial metastatic cell line PC-3 (ATCC CRL-1435) and the normal prostate epithelial cell line RWPE-1 (ATCC CRL-11609) were purchased from ATCC (Manassas, VA, USA).

    Techniques: Concentration Assay, Protein Concentration, Activation Assay, Derivative Assay

    Analysis of Proteins Contained in PC-3 Cell-released Microvesicles

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.012914

    Figure Lengend Snippet: Analysis of Proteins Contained in PC-3 Cell-released Microvesicles

    Article Snippet: The epithelial human prostate cancer cell line PC-3 ( ) obtained from the American Type Culture Collection was maintained in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques:

    Analysis of Proteins Contained in PC-3 Cell-released Microvesicles

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.012914

    Figure Lengend Snippet: Analysis of Proteins Contained in PC-3 Cell-released Microvesicles

    Article Snippet: The epithelial human prostate cancer cell line PC-3 ( ) obtained from the American Type Culture Collection was maintained in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques:

    Classification of proteins in PC-3 cell-released microvesicles by GOslim annotations. Proteins identified in PC-3 cell-derived microvesicles were classified by their cellular location ( A ) (er: endoplasmic reticulum) and biological processes ( B ) using

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.012914

    Figure Lengend Snippet: Classification of proteins in PC-3 cell-released microvesicles by GOslim annotations. Proteins identified in PC-3 cell-derived microvesicles were classified by their cellular location ( A ) (er: endoplasmic reticulum) and biological processes ( B ) using

    Article Snippet: The epithelial human prostate cancer cell line PC-3 ( ) obtained from the American Type Culture Collection was maintained in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques: Derivative Assay

    Analysis of Proteins Contained in PC-3 Cell-released Microvesicles

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.012914

    Figure Lengend Snippet: Analysis of Proteins Contained in PC-3 Cell-released Microvesicles

    Article Snippet: The epithelial human prostate cancer cell line PC-3 ( ) obtained from the American Type Culture Collection was maintained in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques:

    Microvesicles released by PC-3 cells. A , Microvesicles were isolated from PC-3 cells after 1, 2, and 3 days as indicated under “Experimental Procedures.” Caveolin-1 was then detected in lysates and microvesicles by Western blot. B , Microvesicles

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.012914

    Figure Lengend Snippet: Microvesicles released by PC-3 cells. A , Microvesicles were isolated from PC-3 cells after 1, 2, and 3 days as indicated under “Experimental Procedures.” Caveolin-1 was then detected in lysates and microvesicles by Western blot. B , Microvesicles

    Article Snippet: The epithelial human prostate cancer cell line PC-3 ( ) obtained from the American Type Culture Collection was maintained in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques: Isolation, Western Blot

    Analysis of Proteins Contained in PC-3 Cell-released Microvesicles

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.012914

    Figure Lengend Snippet: Analysis of Proteins Contained in PC-3 Cell-released Microvesicles

    Article Snippet: The epithelial human prostate cancer cell line PC-3 ( ) obtained from the American Type Culture Collection was maintained in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques:

    Analysis of CDCP1 as a potential prostate cancer biomarker. Microvesicles were isolated from PC-3 and RWPE-1 cells as indicated under “Experimental Procedures.” The amounts of protein loaded per lane are indicated in the figure. CDCP1

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.012914

    Figure Lengend Snippet: Analysis of CDCP1 as a potential prostate cancer biomarker. Microvesicles were isolated from PC-3 and RWPE-1 cells as indicated under “Experimental Procedures.” The amounts of protein loaded per lane are indicated in the figure. CDCP1

    Article Snippet: The epithelial human prostate cancer cell line PC-3 ( ) obtained from the American Type Culture Collection was maintained in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques: Biomarker Assay, Isolation

    Analysis of CD151 and CD147 as potential prostate cancer biomarkers. Microvesicles were isolated from PC-3 and RWPE-1 cells as indicated under “Experimental Procedures.” CD151 ( A ) and CD147 ( B ) were then detected in lysates (Lys) and microvesicles

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title:

    doi: 10.1074/mcp.M111.012914

    Figure Lengend Snippet: Analysis of CD151 and CD147 as potential prostate cancer biomarkers. Microvesicles were isolated from PC-3 and RWPE-1 cells as indicated under “Experimental Procedures.” CD151 ( A ) and CD147 ( B ) were then detected in lysates (Lys) and microvesicles

    Article Snippet: The epithelial human prostate cancer cell line PC-3 ( ) obtained from the American Type Culture Collection was maintained in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium supplemented with 7% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin.

    Techniques: Isolation

    Prostate tumors grown from Cav1-silenced PC3 cells were accompanied by a more reactive tumor stroma. ( A ) Tumors derived from shCav1 PC3 cells [PC3(−)] as well as from PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression were removed when tumor volumes reached a critical size (8–14 days after implantation) and were then subjected to immunohistochemistry with the indicated antibodies. Masson´s Goldner Trichrome (TC) was performed in order to visualize the collagenous stroma. Representative images are shown. Sections were counterstained using hematoxylin. Magnification Cav1, TC, FAP, Tagln 20x; Pcna 10x. ( B ) Subcutaneously grown tumors were further analyzed by immunofluorescence and confocal microscopy. Tumor stroma was stained for smooth muscle actin (ACTA2; red) and Cav1 (green). Arrows point towards Cav1-positive PC3(−) epithelial cells which were supposed to become immunoreactive for Cav1 upon tumor progression. Representative images from at least three independent experiments are shown. Magnification 63x (scale bar 50 μm).

    Journal: Scientific Reports

    Article Title: Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    doi: 10.1038/srep41138

    Figure Lengend Snippet: Prostate tumors grown from Cav1-silenced PC3 cells were accompanied by a more reactive tumor stroma. ( A ) Tumors derived from shCav1 PC3 cells [PC3(−)] as well as from PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression were removed when tumor volumes reached a critical size (8–14 days after implantation) and were then subjected to immunohistochemistry with the indicated antibodies. Masson´s Goldner Trichrome (TC) was performed in order to visualize the collagenous stroma. Representative images are shown. Sections were counterstained using hematoxylin. Magnification Cav1, TC, FAP, Tagln 20x; Pcna 10x. ( B ) Subcutaneously grown tumors were further analyzed by immunofluorescence and confocal microscopy. Tumor stroma was stained for smooth muscle actin (ACTA2; red) and Cav1 (green). Arrows point towards Cav1-positive PC3(−) epithelial cells which were supposed to become immunoreactive for Cav1 upon tumor progression. Representative images from at least three independent experiments are shown. Magnification 63x (scale bar 50 μm).

    Article Snippet: Cav1 silencing The human prostate epithelial cell lines PC3, LNCaP and the fibroblast cell line HS5 were from ATCC (Manassas, VA).

    Techniques: Derivative Assay, Expressing, Immunohistochemistry, Immunofluorescence, Confocal Microscopy, Staining

    Single dose irradiation (10 Gy) decreased growth of PC3 xenograft tumors more efficiently in Cav1-expressing PC3 tumors which was accompanied by a less reactive tumor stroma. ( A ) PC3 shCav1 tumor cells [PC3(−)] as well as PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression (0.5*10 6 cells each) were subcutaneously transplanted onto the hind limb of NMRI nude mice. One set of animals from each group received a single radiation dose of 10 Gy to the tumor after manifestation of the tumor at day 3. Tumor volume was determined at indicated time points using a sliding caliper (left diagram). Data are presented as mean ± SEM from 3 independent experiments (25 mice in total: PC3(+) 0 Gy n = 5; PC3(+) 10 Gy n = 5; PC3(−) 0 Gy n = 8; PC3(−) 10 Gy n = 7). Tumor growth and respective computed median growth delay was determined as time (days) until a four-fold tumor volume was reached (right diagram). *p

    Journal: Scientific Reports

    Article Title: Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    doi: 10.1038/srep41138

    Figure Lengend Snippet: Single dose irradiation (10 Gy) decreased growth of PC3 xenograft tumors more efficiently in Cav1-expressing PC3 tumors which was accompanied by a less reactive tumor stroma. ( A ) PC3 shCav1 tumor cells [PC3(−)] as well as PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression (0.5*10 6 cells each) were subcutaneously transplanted onto the hind limb of NMRI nude mice. One set of animals from each group received a single radiation dose of 10 Gy to the tumor after manifestation of the tumor at day 3. Tumor volume was determined at indicated time points using a sliding caliper (left diagram). Data are presented as mean ± SEM from 3 independent experiments (25 mice in total: PC3(+) 0 Gy n = 5; PC3(+) 10 Gy n = 5; PC3(−) 0 Gy n = 8; PC3(−) 10 Gy n = 7). Tumor growth and respective computed median growth delay was determined as time (days) until a four-fold tumor volume was reached (right diagram). *p

    Article Snippet: Cav1 silencing The human prostate epithelial cell lines PC3, LNCaP and the fibroblast cell line HS5 were from ATCC (Manassas, VA).

    Techniques: Irradiation, Expressing, Mouse Assay

    Reduction of Cav1 levels increased survival of clonogenic epithelial PC3 and stromal HS5 cells while proliferation was decreased in vitro. ( A ) Lentiviral expression of a Cav1-specific siRNA (shCav1) in stromal HS5 fibroblasts resulted in an efficient and sustained down-regulation of Cav1 expression compared to control-transduced (shCtrl) cells as shown by Western blot analysis. β-actin (bActin) was included as loading control. Representative blots of at least three different experiments are shown. ( A ) HS5 (shCav1-transfected fibroblasts [HS5(−)] as well as shCtrl control cells [HS5(+)] with normal Cav1 expression) cells were plated for colony formation assay, irradiated with indicated doses (0–8 Gy) and subsequently further incubated for additional 10 days. Data show the surviving fractions from three independent experiments measured in triplicates each (means ± SD). **** P ≤ 0.001 by two-tailed students T-test. ( B ) Cell proliferation was analyzed by cell counting in cultured shCav1-transfected HS5(−) and control-transfected HS5(+) fibroblasts cells at the indicated time points after irradiation with 10 Gy. Data are shown as means ± SEM of three independent experiments. ( C ) Expression levels of the indicated proteins were analyzed in whole protein lysates of cultured HS5 cells (+/−Cav1) with or without radiation (48 hours after XRT with 10 Gy) using Western blot analysis. Representative blots are shown. For quantification blots were analyzed by densitometry and the respective signal was related to beta-actin (n = 4–5 for each group). For determination of the Akt phosphorylation status the obtained phospho-specific signal was related to the signal of the total protein (phAkt/Akt). P-values were indicated: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005 **** P ≤ 0.001; by one-way ANOVA followed by post-hoc Tukey test.

    Journal: Scientific Reports

    Article Title: Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    doi: 10.1038/srep41138

    Figure Lengend Snippet: Reduction of Cav1 levels increased survival of clonogenic epithelial PC3 and stromal HS5 cells while proliferation was decreased in vitro. ( A ) Lentiviral expression of a Cav1-specific siRNA (shCav1) in stromal HS5 fibroblasts resulted in an efficient and sustained down-regulation of Cav1 expression compared to control-transduced (shCtrl) cells as shown by Western blot analysis. β-actin (bActin) was included as loading control. Representative blots of at least three different experiments are shown. ( A ) HS5 (shCav1-transfected fibroblasts [HS5(−)] as well as shCtrl control cells [HS5(+)] with normal Cav1 expression) cells were plated for colony formation assay, irradiated with indicated doses (0–8 Gy) and subsequently further incubated for additional 10 days. Data show the surviving fractions from three independent experiments measured in triplicates each (means ± SD). **** P ≤ 0.001 by two-tailed students T-test. ( B ) Cell proliferation was analyzed by cell counting in cultured shCav1-transfected HS5(−) and control-transfected HS5(+) fibroblasts cells at the indicated time points after irradiation with 10 Gy. Data are shown as means ± SEM of three independent experiments. ( C ) Expression levels of the indicated proteins were analyzed in whole protein lysates of cultured HS5 cells (+/−Cav1) with or without radiation (48 hours after XRT with 10 Gy) using Western blot analysis. Representative blots are shown. For quantification blots were analyzed by densitometry and the respective signal was related to beta-actin (n = 4–5 for each group). For determination of the Akt phosphorylation status the obtained phospho-specific signal was related to the signal of the total protein (phAkt/Akt). P-values were indicated: * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.005 **** P ≤ 0.001; by one-way ANOVA followed by post-hoc Tukey test.

    Article Snippet: Cav1 silencing The human prostate epithelial cell lines PC3, LNCaP and the fibroblast cell line HS5 were from ATCC (Manassas, VA).

    Techniques: In Vitro, Expressing, Western Blot, Transfection, Colony Assay, Irradiation, Incubation, Two Tailed Test, Cell Counting, Cell Culture

    Orthotopic tumors derived from Cav1-silenced PC3(−) cells showed a significantly increased tumor growth and epithelial Cav1 (re-) expressions. ( A ) PC3 (+/−Cav1) cells were injected into the right and left dorsolateral lobe (0.5 × 10 5 cells per lobe) of the prostate of NMRI nude mice. Tumor weight and volume was determined 14 days after tumor cell implantation. Data are presented as mean ± SEM from 3 independent experiments (16 mice in total: PC3(+) n = 7; PC3(−) n = 9); *P ≤ 0.05, **P ≤ 0.01 by two-tailed t-tests with Welch’s correction. ( B ) Expression levels of the indicated proteins were analyzed in whole protein lysates using Western blot analysis. Representative blots are shown. ( C ) Tumors derived from shCav1 PC3 cells as well as from shCtrl control cells with normal Cav1 expression were subjected to immunohistochemistry with Cav1 antibody. Representative images are shown. Sections were counterstained using hematoxylin. Arrows point towards Cav1-positive epithelial cells within tumors derived from implanted PC3(−) cells. Magnification 20x. ( D ) Orthotopic grown tumors were further analysed by immunofluorescence and confocal microscopy. Tumor stroma was stained for Tagln (red) and Cav1 (green). Representative images from at least three independent experiments are shown. Magnification 63x (scale bar 50 μm).

    Journal: Scientific Reports

    Article Title: Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    doi: 10.1038/srep41138

    Figure Lengend Snippet: Orthotopic tumors derived from Cav1-silenced PC3(−) cells showed a significantly increased tumor growth and epithelial Cav1 (re-) expressions. ( A ) PC3 (+/−Cav1) cells were injected into the right and left dorsolateral lobe (0.5 × 10 5 cells per lobe) of the prostate of NMRI nude mice. Tumor weight and volume was determined 14 days after tumor cell implantation. Data are presented as mean ± SEM from 3 independent experiments (16 mice in total: PC3(+) n = 7; PC3(−) n = 9); *P ≤ 0.05, **P ≤ 0.01 by two-tailed t-tests with Welch’s correction. ( B ) Expression levels of the indicated proteins were analyzed in whole protein lysates using Western blot analysis. Representative blots are shown. ( C ) Tumors derived from shCav1 PC3 cells as well as from shCtrl control cells with normal Cav1 expression were subjected to immunohistochemistry with Cav1 antibody. Representative images are shown. Sections were counterstained using hematoxylin. Arrows point towards Cav1-positive epithelial cells within tumors derived from implanted PC3(−) cells. Magnification 20x. ( D ) Orthotopic grown tumors were further analysed by immunofluorescence and confocal microscopy. Tumor stroma was stained for Tagln (red) and Cav1 (green). Representative images from at least three independent experiments are shown. Magnification 63x (scale bar 50 μm).

    Article Snippet: Cav1 silencing The human prostate epithelial cell lines PC3, LNCaP and the fibroblast cell line HS5 were from ATCC (Manassas, VA).

    Techniques: Derivative Assay, Injection, Mouse Assay, Two Tailed Test, Expressing, Western Blot, Immunohistochemistry, Immunofluorescence, Confocal Microscopy, Staining

    Cav1-deficient stromal fibroblasts mediated radiation resistance. ( A ) Co-implantations of PC3 tumor cells (0.5*10 6 cells each) after Cav1 silencing [shCav1, PC3(−)] in combination HS5 Cav1-silenced fibroblasts [shCav1, HS5(−)] (0.5*10 6 cells each) or control fibroblasts [shCtrl, HS5(+)] were performed by subcutaneously transplanted onto the hind limb of NMRI nude mice. One set of animals from each group received a single radiation dose of 10 Gy to the tumor after manifestation of the tumor at day 3. Tumor volume was determined at indicated time points using a sliding caliper (left diagram). Data are presented as mean ± SEM from 3 independent experiments (37 mice in total: PC3(−)HS5(+) 0 Gy n = 10; PC3(−)HS5(+) 10 Gy n = 8; PC3(−)HS5(−) 0 Gy n = 9; PC3(−)HS5(−) 10 Gy n = 10). Tumor growth and respective computed median growth delay was determined as time (days) until a four-fold tumor volume was reached (right diagram). *p

    Journal: Scientific Reports

    Article Title: Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    doi: 10.1038/srep41138

    Figure Lengend Snippet: Cav1-deficient stromal fibroblasts mediated radiation resistance. ( A ) Co-implantations of PC3 tumor cells (0.5*10 6 cells each) after Cav1 silencing [shCav1, PC3(−)] in combination HS5 Cav1-silenced fibroblasts [shCav1, HS5(−)] (0.5*10 6 cells each) or control fibroblasts [shCtrl, HS5(+)] were performed by subcutaneously transplanted onto the hind limb of NMRI nude mice. One set of animals from each group received a single radiation dose of 10 Gy to the tumor after manifestation of the tumor at day 3. Tumor volume was determined at indicated time points using a sliding caliper (left diagram). Data are presented as mean ± SEM from 3 independent experiments (37 mice in total: PC3(−)HS5(+) 0 Gy n = 10; PC3(−)HS5(+) 10 Gy n = 8; PC3(−)HS5(−) 0 Gy n = 9; PC3(−)HS5(−) 10 Gy n = 10). Tumor growth and respective computed median growth delay was determined as time (days) until a four-fold tumor volume was reached (right diagram). *p

    Article Snippet: Cav1 silencing The human prostate epithelial cell lines PC3, LNCaP and the fibroblast cell line HS5 were from ATCC (Manassas, VA).

    Techniques: Mouse Assay

    Reduction of Cav1 levels decreased survival of clonogenic epithelial PC3 while proliferation was increased in vitro . ( A ) PC3 (shCav1-transfected tumor cells [PC3(−)] as well as PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression) cells were plated for colony formation assay, irradiated with indicated doses (0–8 Gy) and subsequently further incubated for additional 10 days. Data show the surviving fractions from three independent experiments measured in triplicates each (means ± SD). *** P ≤ 0.005 by two-tailed students T-test. ( B ) Cell proliferation was analyzed by cell counting in cultured shCav1-transfected PC3(−) and control-transfected PC3(+) epithelial cells at the indicated time points after irradiation with 10 Gy. Data are shown as means ± SEM of three independent experiments. ( C ) Expression levels of the indicated proteins were analyzed in whole protein lysates of cultured PC3 cells (+/−Cav1) with or without radiation (48 hours after XRT with 10 Gy) using Western blot analysis. Representative blots are shown. For quantification blots were analyzed by densitometry and the respective signal was related to beta-actin (n = 4–5 for each group). For determination of the Akt phosphorylation status the obtained phospho-specific signal was related to the signal of the total protein (phAkt/Akt). P-values were indicated: * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.001, by one-way ANOVA followed by post-hoc Tukey test.

    Journal: Scientific Reports

    Article Title: Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    doi: 10.1038/srep41138

    Figure Lengend Snippet: Reduction of Cav1 levels decreased survival of clonogenic epithelial PC3 while proliferation was increased in vitro . ( A ) PC3 (shCav1-transfected tumor cells [PC3(−)] as well as PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression) cells were plated for colony formation assay, irradiated with indicated doses (0–8 Gy) and subsequently further incubated for additional 10 days. Data show the surviving fractions from three independent experiments measured in triplicates each (means ± SD). *** P ≤ 0.005 by two-tailed students T-test. ( B ) Cell proliferation was analyzed by cell counting in cultured shCav1-transfected PC3(−) and control-transfected PC3(+) epithelial cells at the indicated time points after irradiation with 10 Gy. Data are shown as means ± SEM of three independent experiments. ( C ) Expression levels of the indicated proteins were analyzed in whole protein lysates of cultured PC3 cells (+/−Cav1) with or without radiation (48 hours after XRT with 10 Gy) using Western blot analysis. Representative blots are shown. For quantification blots were analyzed by densitometry and the respective signal was related to beta-actin (n = 4–5 for each group). For determination of the Akt phosphorylation status the obtained phospho-specific signal was related to the signal of the total protein (phAkt/Akt). P-values were indicated: * P ≤ 0.05, ** P ≤ 0.01, **** P ≤ 0.001, by one-way ANOVA followed by post-hoc Tukey test.

    Article Snippet: Cav1 silencing The human prostate epithelial cell lines PC3, LNCaP and the fibroblast cell line HS5 were from ATCC (Manassas, VA).

    Techniques: In Vitro, Transfection, Expressing, Colony Assay, Irradiation, Incubation, Two Tailed Test, Cell Counting, Cell Culture, Western Blot

    Treatment of cultured PC3 (+/−Cav1) or Cav1-deficient LNCaP malignant epithelial cells with supernatants derived from Cav1-silenced HS5 fibroblasts fostered radiation resistance. ( A , B ) PC3 (shCav1-transfected tumor cells [PC3(−)] as well as PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression) and Cav1-deficient LNCaP cells were irradiated with 10 Gy and subsequently treated with supernatants (SN) derived from cultured Cav1-silenced HS5(−) or control transfected Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy). Western blot analysis of whole protein lysates was performed after 48 hours of treatment using the indicated antibodies. ( C , D ) The degree of apoptosis was quantified by measuring the SubG1 fraction 48 hours after radiation by flow cytometry analysis. Therefore, PC3cells (+/−Cav1) and Cav1-deficient LNCaP cells were left non-irradiated (white bars) or irradiated with 10 Gy (black bars) and subsequently treated with SN derived from cultured Cav1-silenced HS5(−) or Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy). ( E ) Quantitative Real Time RT-PCR (qRT-PCR) analysis of the reactive fibroblasts markers Acta2, Tagln as well as the tumor-promoting factors Vegf (vascular endothelial growth factor), Tgfb and Mmp2 (matrix metalloproteinase 2) were performed in total RNA isolates of Cav1-silenced HS5(−) or control transfected Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy) and were shown as relative expression to actin (set as 1) at 96 hours post irradiation. Shown are mean values ± SEM from 4 independent samples per group measured in duplicates each. * P ≤ 0.05, *** P ≤ 0.005, by one-way ANOVA followed by post-hoc Tukey’s test. ( F ) Expression levels of the Tgfb receptors TGFBR1 and TGFBR2 as well as the E-cadherin repressing EMT gene Snai2 were analyzed in whole RNA isolates of cultured PC3 cells (+/−Cav1) with or without radiation (48 hours after XRT with 10 Gy) using qRT-PCR analysis. Shown are mean values ± SEM from 4 independent samples per group measured in duplicates each. * P ≤ 0.05 by one-way ANOVA followed by post-hoc Tukey’s test.

    Journal: Scientific Reports

    Article Title: Progression-related loss of stromal Caveolin 1 levels fosters the growth of human PC3 xenografts and mediates radiation resistance

    doi: 10.1038/srep41138

    Figure Lengend Snippet: Treatment of cultured PC3 (+/−Cav1) or Cav1-deficient LNCaP malignant epithelial cells with supernatants derived from Cav1-silenced HS5 fibroblasts fostered radiation resistance. ( A , B ) PC3 (shCav1-transfected tumor cells [PC3(−)] as well as PC3 shCtrl control cells [PC3(+)] with normal Cav1 expression) and Cav1-deficient LNCaP cells were irradiated with 10 Gy and subsequently treated with supernatants (SN) derived from cultured Cav1-silenced HS5(−) or control transfected Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy). Western blot analysis of whole protein lysates was performed after 48 hours of treatment using the indicated antibodies. ( C , D ) The degree of apoptosis was quantified by measuring the SubG1 fraction 48 hours after radiation by flow cytometry analysis. Therefore, PC3cells (+/−Cav1) and Cav1-deficient LNCaP cells were left non-irradiated (white bars) or irradiated with 10 Gy (black bars) and subsequently treated with SN derived from cultured Cav1-silenced HS5(−) or Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy). ( E ) Quantitative Real Time RT-PCR (qRT-PCR) analysis of the reactive fibroblasts markers Acta2, Tagln as well as the tumor-promoting factors Vegf (vascular endothelial growth factor), Tgfb and Mmp2 (matrix metalloproteinase 2) were performed in total RNA isolates of Cav1-silenced HS5(−) or control transfected Cav1-expressing HS5(+) fibroblasts with or without radiation treatment (+/−XRT with 10 Gy) and were shown as relative expression to actin (set as 1) at 96 hours post irradiation. Shown are mean values ± SEM from 4 independent samples per group measured in duplicates each. * P ≤ 0.05, *** P ≤ 0.005, by one-way ANOVA followed by post-hoc Tukey’s test. ( F ) Expression levels of the Tgfb receptors TGFBR1 and TGFBR2 as well as the E-cadherin repressing EMT gene Snai2 were analyzed in whole RNA isolates of cultured PC3 cells (+/−Cav1) with or without radiation (48 hours after XRT with 10 Gy) using qRT-PCR analysis. Shown are mean values ± SEM from 4 independent samples per group measured in duplicates each. * P ≤ 0.05 by one-way ANOVA followed by post-hoc Tukey’s test.

    Article Snippet: Cav1 silencing The human prostate epithelial cell lines PC3, LNCaP and the fibroblast cell line HS5 were from ATCC (Manassas, VA).

    Techniques: Cell Culture, Derivative Assay, Transfection, Expressing, Irradiation, Western Blot, Flow Cytometry, Cytometry, Quantitative RT-PCR

    Pim-1 and Pim-3 enhance CXCR4 phosphorylation and cell surface expression in prostate cancer cells. Phosphorylation of CXCR4 at S339 as well as Pim levels were analysed by western blotting in the stable Pim-1 (P1), Pim-3 (P3) or control vector (C) overexpressing PC-3 cells or the parental PC-3 cell line treated with 0.1% DMSO or 10 μM DHPCC-9. Shown are results from one representative experiment with loading controls and molecular weight (kDa) markers (A-B). The ability of Pim family members to phosphorylate CXCR4 in vitro was analysed by incubating GST-tagged Pim-1 (P1), Pim-2 (P2) or Pim-3 (P3) proteins with GST-tagged fragments of wild-type (WT) or Ser339 > Ala (SA) mutant human CXCR4. Phosphorylated CXCR4 was detected by phospho(Ser339)-CXCR4 antibody and protein loading by Ponceau S staining. Shown are results from one representative experiment (C). Localization and signal intensity of phosphorylated versus overall CXCR4 expression was analysed by immunofluorescent (IF) staining of stably transfected cells treated with either 0.1% DMSO or 10 μM DHPCC-9. The experiment was controlled by parallel samples stained only with the secondary antibody (-ab). Stainings were repeated twice and stacks of images were taken by confocal microscopy from at least 30 cells per sample per experiment. Shown are the signal intensities of phospho-CXCR4 stainings compared to overall CXCR4 levels along with representative images from phospho-CXCR4 and CXCR4 stainings (D-E). Phosphorylation and localization of CXCR4 was also analysed by immunohistochemical staining of the paraffin-embedded tissue sections from orthopic prostate tumors. Shown is the relative increase in the amount of phospho-CXCR4-positive cells versus overall CXCR4 expression measured by whole tumor scanning. PBS instead of the primary antibody was used as a negative control. Representative images were taken to visualize the differences in phospho-CXCR4 (dark brown) stainings (F).

    Journal: PLoS ONE

    Article Title: Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts

    doi: 10.1371/journal.pone.0130340

    Figure Lengend Snippet: Pim-1 and Pim-3 enhance CXCR4 phosphorylation and cell surface expression in prostate cancer cells. Phosphorylation of CXCR4 at S339 as well as Pim levels were analysed by western blotting in the stable Pim-1 (P1), Pim-3 (P3) or control vector (C) overexpressing PC-3 cells or the parental PC-3 cell line treated with 0.1% DMSO or 10 μM DHPCC-9. Shown are results from one representative experiment with loading controls and molecular weight (kDa) markers (A-B). The ability of Pim family members to phosphorylate CXCR4 in vitro was analysed by incubating GST-tagged Pim-1 (P1), Pim-2 (P2) or Pim-3 (P3) proteins with GST-tagged fragments of wild-type (WT) or Ser339 > Ala (SA) mutant human CXCR4. Phosphorylated CXCR4 was detected by phospho(Ser339)-CXCR4 antibody and protein loading by Ponceau S staining. Shown are results from one representative experiment (C). Localization and signal intensity of phosphorylated versus overall CXCR4 expression was analysed by immunofluorescent (IF) staining of stably transfected cells treated with either 0.1% DMSO or 10 μM DHPCC-9. The experiment was controlled by parallel samples stained only with the secondary antibody (-ab). Stainings were repeated twice and stacks of images were taken by confocal microscopy from at least 30 cells per sample per experiment. Shown are the signal intensities of phospho-CXCR4 stainings compared to overall CXCR4 levels along with representative images from phospho-CXCR4 and CXCR4 stainings (D-E). Phosphorylation and localization of CXCR4 was also analysed by immunohistochemical staining of the paraffin-embedded tissue sections from orthopic prostate tumors. Shown is the relative increase in the amount of phospho-CXCR4-positive cells versus overall CXCR4 expression measured by whole tumor scanning. PBS instead of the primary antibody was used as a negative control. Representative images were taken to visualize the differences in phospho-CXCR4 (dark brown) stainings (F).

    Article Snippet: Cell culture and transfections The human androgen-independent prostate epithelial adenocarcinoma cell line PC-3 (American Type Culture Collection) was maintained in Roswell Park Memorial Institute (RPMI) -1640 medium, supplemented with 10% fetal bovine serum, L-glutamine and antibiotics.

    Techniques: Expressing, Western Blot, Plasmid Preparation, Molecular Weight, In Vitro, Mutagenesis, Staining, Stable Transfection, Transfection, Confocal Microscopy, Immunohistochemistry, Negative Control

    Pim inhibition by DHPCC-9 reduces the number of metastases in orthotopic prostate tumors overexpressing Pim-3. Different organs were collected from mice with orthotopic prostate tumor xenografts formed by PC-3 cells stably overexpressing an empty vector (C), Pim-1 (P1) or Pim-3 (P3). Paraffin-embedded tissue sections were stained with hematoxylin and eosin and analysed for the presence of metastases. Shown are representative images (A) from lymph node and lung sections (tumor cells indicated by arrows). The metastatic properties of xenografts from mice treated with DHPCC-9, BA-1a or their solvents were also analysed. Shown are percentages of mice positive for either lymph node metastases (B) or lung metastases (C) in each group.

    Journal: PLoS ONE

    Article Title: Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts

    doi: 10.1371/journal.pone.0130340

    Figure Lengend Snippet: Pim inhibition by DHPCC-9 reduces the number of metastases in orthotopic prostate tumors overexpressing Pim-3. Different organs were collected from mice with orthotopic prostate tumor xenografts formed by PC-3 cells stably overexpressing an empty vector (C), Pim-1 (P1) or Pim-3 (P3). Paraffin-embedded tissue sections were stained with hematoxylin and eosin and analysed for the presence of metastases. Shown are representative images (A) from lymph node and lung sections (tumor cells indicated by arrows). The metastatic properties of xenografts from mice treated with DHPCC-9, BA-1a or their solvents were also analysed. Shown are percentages of mice positive for either lymph node metastases (B) or lung metastases (C) in each group.

    Article Snippet: Cell culture and transfections The human androgen-independent prostate epithelial adenocarcinoma cell line PC-3 (American Type Culture Collection) was maintained in Roswell Park Memorial Institute (RPMI) -1640 medium, supplemented with 10% fetal bovine serum, L-glutamine and antibiotics.

    Techniques: Inhibition, Mouse Assay, Stable Transfection, Plasmid Preparation, Staining

    Pim-3 overexpression promotes metastatic growth of prostate tumor xenografts. PC-3-derived cell lines that had been stably transfected with an empty vector (C) or a vector expressing Pim-3 (P3) were subcutaneously or orthotopically injected into athymic nude mice. Tumors and isolated tissues were stained with Hematoxylin and Eosin for visualization of their structure. Additional stainings were carried out with anti-phospho-histone H3 antibody to visualize the number of mitotic cells. In the subcutaneous experiments, tumor formation was followed by fluorescence imaging of Tomato expression (A) and approximate tumor sizes were measured by palpation at different time-points (B). After 24 days, mice were sacrificed and their tumors and tissues were collected. Shown are average values from all fully imaged tumor sections from indicated numbers ( n ) of mice after staining of mitotic cells (C). In the orthotopic experiments, the stable PC-3 cells were allowed to grow in the prostates for three weeks. Thereafter mitotic cells (brown) were analysed from sample images (D), while metastases (indicated by arrows) were counted from prostate-draining lymph nodes and lungs (E).

    Journal: PLoS ONE

    Article Title: Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts

    doi: 10.1371/journal.pone.0130340

    Figure Lengend Snippet: Pim-3 overexpression promotes metastatic growth of prostate tumor xenografts. PC-3-derived cell lines that had been stably transfected with an empty vector (C) or a vector expressing Pim-3 (P3) were subcutaneously or orthotopically injected into athymic nude mice. Tumors and isolated tissues were stained with Hematoxylin and Eosin for visualization of their structure. Additional stainings were carried out with anti-phospho-histone H3 antibody to visualize the number of mitotic cells. In the subcutaneous experiments, tumor formation was followed by fluorescence imaging of Tomato expression (A) and approximate tumor sizes were measured by palpation at different time-points (B). After 24 days, mice were sacrificed and their tumors and tissues were collected. Shown are average values from all fully imaged tumor sections from indicated numbers ( n ) of mice after staining of mitotic cells (C). In the orthotopic experiments, the stable PC-3 cells were allowed to grow in the prostates for three weeks. Thereafter mitotic cells (brown) were analysed from sample images (D), while metastases (indicated by arrows) were counted from prostate-draining lymph nodes and lungs (E).

    Article Snippet: Cell culture and transfections The human androgen-independent prostate epithelial adenocarcinoma cell line PC-3 (American Type Culture Collection) was maintained in Roswell Park Memorial Institute (RPMI) -1640 medium, supplemented with 10% fetal bovine serum, L-glutamine and antibiotics.

    Techniques: Over Expression, Derivative Assay, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Injection, Mouse Assay, Isolation, Staining, Fluorescence, Imaging

    Pim overexpression increases and Pim inhibition by DHPCC-9 decreases growth of orthotopic prostate xenografts. PC-3-derived stably transfected cells (Mock = C, Pim-1 = P1, Pim-3 = P3) were orthotopically inoculated into the prostates of athymic nude mice. Mice were treated daily with either DMSO or DMA (control treatments) or with Pim inhibitors (50 mg/kg of DHPCC9 in DMSO or 20 mg/kg of BA-1a in DMA). After three weeks of treatments, mice were sacrificed and their tumor volumes were measured. First the volumes were compared between indicated numbers ( n ) of mice without inhibitor treatments (A). Then the volumes of tumors derived from inhibitor-treated animals were compared to tumors from animals with appropriate control treatments DMSO (B) or DMA (C). Before tumor fixation, representative images were taken (D). Later on paraffin-embedded tumor sections were stained with anti-phospho-histone H3 antibody to visualize mitotic cells (brown). Shown are average values combined from all fully imaged tumor tissue sections from DHPCC-9-treated and DMSO- or DMA-treated control groups as well as representative images from Pim-3-overexpressing tumors (E).

    Journal: PLoS ONE

    Article Title: Pim Kinases Promote Migration and Metastatic Growth of Prostate Cancer Xenografts

    doi: 10.1371/journal.pone.0130340

    Figure Lengend Snippet: Pim overexpression increases and Pim inhibition by DHPCC-9 decreases growth of orthotopic prostate xenografts. PC-3-derived stably transfected cells (Mock = C, Pim-1 = P1, Pim-3 = P3) were orthotopically inoculated into the prostates of athymic nude mice. Mice were treated daily with either DMSO or DMA (control treatments) or with Pim inhibitors (50 mg/kg of DHPCC9 in DMSO or 20 mg/kg of BA-1a in DMA). After three weeks of treatments, mice were sacrificed and their tumor volumes were measured. First the volumes were compared between indicated numbers ( n ) of mice without inhibitor treatments (A). Then the volumes of tumors derived from inhibitor-treated animals were compared to tumors from animals with appropriate control treatments DMSO (B) or DMA (C). Before tumor fixation, representative images were taken (D). Later on paraffin-embedded tumor sections were stained with anti-phospho-histone H3 antibody to visualize mitotic cells (brown). Shown are average values combined from all fully imaged tumor tissue sections from DHPCC-9-treated and DMSO- or DMA-treated control groups as well as representative images from Pim-3-overexpressing tumors (E).

    Article Snippet: Cell culture and transfections The human androgen-independent prostate epithelial adenocarcinoma cell line PC-3 (American Type Culture Collection) was maintained in Roswell Park Memorial Institute (RPMI) -1640 medium, supplemented with 10% fetal bovine serum, L-glutamine and antibiotics.

    Techniques: Over Expression, Inhibition, Derivative Assay, Stable Transfection, Transfection, Mouse Assay, Staining

    Interactions of stromal fibroblasts and prostate cancer. Coculture of HS27A bone stromal fibroblasts with prostate cancer LNCaP (HS27A-LN) and C4-2 (HS27A-C4-2) cells as tumoroids or alone (HS27A) under 3D-RWV culture conditions ( a , b ). Microscopic images of cultures were taken on day 14, and the size ( a ) and number of tumoroids were calculated. Each condition was performed in duplicate vessels for three independent experiments. A representative image of each group is shown at the top. Quantitative data are represented as a box–whisker plot of triplicate experiments. Tumor-promoting effect of cancer-associated fibroblasts (CAFs) in a xenograph mouse model ( c , d ). Nude mice were subcutaneously implanted with C4-2 prostate cancer cells, either alone (C4-2) or mixed with HS27A pre-cultured with LNCaP (HS27A LN ) or C4-2 (HS27A C4-2 ) ( c ), or primary cultured prostate fibroblasts derived from benign/normal prostate (BAF) or prostate cancer (CAF) tissues of the same patients ( d ). Eight mice were used in each group and independently conducted twice. Tumor growth was monitored weekly, and the results are presented as representative images of two independent experiments (top) and a box–whisker plot of median tumor volumes (bottom) of each group ( n = 16 per group; two independent experiments pooled) at the end of the experiment (8 weeks after cell inoculation). Boxes ( a – d ) encompass the 25th to 75th percentiles, and the whiskers represent the 10%–90% range of observations. Lines within boxes represent median values. * p

    Journal: Scientific Reports

    Article Title: Reactive oxygen species–mediated switching expression of MMP-3 in stromal fibroblasts and cancer cells during prostate cancer progression

    doi: 10.1038/s41598-017-08835-9

    Figure Lengend Snippet: Interactions of stromal fibroblasts and prostate cancer. Coculture of HS27A bone stromal fibroblasts with prostate cancer LNCaP (HS27A-LN) and C4-2 (HS27A-C4-2) cells as tumoroids or alone (HS27A) under 3D-RWV culture conditions ( a , b ). Microscopic images of cultures were taken on day 14, and the size ( a ) and number of tumoroids were calculated. Each condition was performed in duplicate vessels for three independent experiments. A representative image of each group is shown at the top. Quantitative data are represented as a box–whisker plot of triplicate experiments. Tumor-promoting effect of cancer-associated fibroblasts (CAFs) in a xenograph mouse model ( c , d ). Nude mice were subcutaneously implanted with C4-2 prostate cancer cells, either alone (C4-2) or mixed with HS27A pre-cultured with LNCaP (HS27A LN ) or C4-2 (HS27A C4-2 ) ( c ), or primary cultured prostate fibroblasts derived from benign/normal prostate (BAF) or prostate cancer (CAF) tissues of the same patients ( d ). Eight mice were used in each group and independently conducted twice. Tumor growth was monitored weekly, and the results are presented as representative images of two independent experiments (top) and a box–whisker plot of median tumor volumes (bottom) of each group ( n = 16 per group; two independent experiments pooled) at the end of the experiment (8 weeks after cell inoculation). Boxes ( a – d ) encompass the 25th to 75th percentiles, and the whiskers represent the 10%–90% range of observations. Lines within boxes represent median values. * p

    Article Snippet: Cell lines and cell culture Human prostate cancer epithelial cell lines, LNCaP, C4-2, PC3, PC3M, and DU145, and a human bone stromal cell line HS27A (ATCC, Manassas, VA) that were used in our previous studies – were maintained in T-medium (Invitrogen, Carlsbad, CA) with 5% fetal bovine serum (FBS; Invitrogen).

    Techniques: Whisker Assay, Mouse Assay, Cell Culture, Derivative Assay

    Co-infection of Onc.Ad with HDPD-L1 ( CAd-VEC PD-L1) amplified PD-L1 mini-body while maintaining oncolysis in vitro and in vivo (A) A549, PC-3, SiHa and HepG2 were infected with a total 10 Vp/cell of HDPD-L1, Onc.Ad or with an CAd-VECPD-L1 (Onc.Ad:HDAd; 1:10). Medium samples were collected at 48 hours post-infection. Levels of PD-L1 mini-body in medium samples were quantified by ELISA-based assay for HA-tagged protein. Data are presented as means ± SD (n=4). *P =0.003, **P=0.002, ***P

    Journal: Cancer research

    Article Title: Armed oncolytic adenovirus expressing PD-L1 mini-body enhances anti-tumor effects of chimeric antigen receptor T-cells in solid tumors

    doi: 10.1158/0008-5472.CAN-16-1577

    Figure Lengend Snippet: Co-infection of Onc.Ad with HDPD-L1 ( CAd-VEC PD-L1) amplified PD-L1 mini-body while maintaining oncolysis in vitro and in vivo (A) A549, PC-3, SiHa and HepG2 were infected with a total 10 Vp/cell of HDPD-L1, Onc.Ad or with an CAd-VECPD-L1 (Onc.Ad:HDAd; 1:10). Medium samples were collected at 48 hours post-infection. Levels of PD-L1 mini-body in medium samples were quantified by ELISA-based assay for HA-tagged protein. Data are presented as means ± SD (n=4). *P =0.003, **P=0.002, ***P

    Article Snippet: Human prostate cancer cell line PC-3, human non-small cell lung carcinoma cell line A549, human hepatocellular carcinoma cell line HepG2 and human squamous cell carcinoma line SiHa were obtained from ATCC (Manassas, VA) in 2014.

    Techniques: Infection, Amplification, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay

    HDAd-derived PD-L1 mini-body functions similarly to commercial anti-PD-L1 IgG (A) PC-3 and SiHa were co-cultured with HER2.CAR T-cells generated from 3 different donors (effector:target ratio of 1:20). Cells were harvested at 8, 24, 72 and 120 hours post-co-culture, and PD-1 on HER2.CAR T-cells and PD-L1 on cancer cells were analyzed by flow cytometry. Data are presented as means ± SD (n=3). (B) Schematic structure of HDAd encodes an anti-PD-L1 mini-body expression cassette (HD PD-L1 ). A549 cells were infected with different dosages of HD PD-L1 . Media and cells were collected at 48 hours post-infection. Samples were subjected to western blotting, and PD-L1 mini-body in media was detected by anti-HA antibody. Human GAPDH in cells was detected by anti-human GAPDH antibody, HD eGFP encoding an EGFP expression cassette was used as a control. (C) A549 media infected with 1,000 vp/cell of HD PD-L1 or HD eGFP were collected at 48 hours post-infection, and binding of PD-L1 mini-body to recombinant human PD-L1 was assessed by enzyme-linked immunosorbent assay (ELISA). Anti-PD-L1 IgG or Isotype IgG were controls (10 μg/mL; highest concentration). Data are presented as means ± SD (n=3). (D) Purified CD4 + T-cells were co-cultured with irradiated allogeneic mature DCs in the presence of PD-L1 mini-body-containing medium, 10 μg/mL anti-PD-L1 IgG or isotype IgG for 5 days (T-cell:DC ratio of 10:1). IFNγ levels in the medium were measured by ELISA. Data are presented as means ± SD (n=4). *P= 0.005. The experiment was triplicated with similar results.

    Journal: Cancer research

    Article Title: Armed oncolytic adenovirus expressing PD-L1 mini-body enhances anti-tumor effects of chimeric antigen receptor T-cells in solid tumors

    doi: 10.1158/0008-5472.CAN-16-1577

    Figure Lengend Snippet: HDAd-derived PD-L1 mini-body functions similarly to commercial anti-PD-L1 IgG (A) PC-3 and SiHa were co-cultured with HER2.CAR T-cells generated from 3 different donors (effector:target ratio of 1:20). Cells were harvested at 8, 24, 72 and 120 hours post-co-culture, and PD-1 on HER2.CAR T-cells and PD-L1 on cancer cells were analyzed by flow cytometry. Data are presented as means ± SD (n=3). (B) Schematic structure of HDAd encodes an anti-PD-L1 mini-body expression cassette (HD PD-L1 ). A549 cells were infected with different dosages of HD PD-L1 . Media and cells were collected at 48 hours post-infection. Samples were subjected to western blotting, and PD-L1 mini-body in media was detected by anti-HA antibody. Human GAPDH in cells was detected by anti-human GAPDH antibody, HD eGFP encoding an EGFP expression cassette was used as a control. (C) A549 media infected with 1,000 vp/cell of HD PD-L1 or HD eGFP were collected at 48 hours post-infection, and binding of PD-L1 mini-body to recombinant human PD-L1 was assessed by enzyme-linked immunosorbent assay (ELISA). Anti-PD-L1 IgG or Isotype IgG were controls (10 μg/mL; highest concentration). Data are presented as means ± SD (n=3). (D) Purified CD4 + T-cells were co-cultured with irradiated allogeneic mature DCs in the presence of PD-L1 mini-body-containing medium, 10 μg/mL anti-PD-L1 IgG or isotype IgG for 5 days (T-cell:DC ratio of 10:1). IFNγ levels in the medium were measured by ELISA. Data are presented as means ± SD (n=4). *P= 0.005. The experiment was triplicated with similar results.

    Article Snippet: Human prostate cancer cell line PC-3, human non-small cell lung carcinoma cell line A549, human hepatocellular carcinoma cell line HepG2 and human squamous cell carcinoma line SiHa were obtained from ATCC (Manassas, VA) in 2014.

    Techniques: Derivative Assay, Cell Culture, Generated, Co-Culture Assay, Flow Cytometry, Cytometry, Expressing, Infection, Western Blot, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay, Purification, Irradiation

    Interactions of human bone marrow-derived MSCs and prostate cancer cells. (A) Migratory response of human MSCs to the medium, normal prostatic epithelial cells (PrEC) and prostate cancer cell lines (DU145, PC3 and LNCaP). Migrated cells through the membrane of transwells were stained with crystal violet 8 h after cell plating. The lower surface of the membrane was photographed (100x), and a representative image from each condition is shown at top. Quantitative data are represented as the means ± SD of the number of cells per high-power field (100x) in triplicate experiments. (B) Macroscopic (upper) and microscopic (bottom) view of cellular aggregates under 3-D RWV culture conditions of 3A6 cells alone (3A6/3A6) or mixed with LNCaP (3A6/LNCaP), C4-2 (3A6/C4-2) or PC3 (3A6/PC3) cells. (C) Detection of mixed cellular components in the 3-D cultured prostate tumoroids by H E and immunohistochemical staining of E-cadherin (E-Cad) in the cell aggregate sections.

    Journal: PLoS ONE

    Article Title: Loss of Let-7 MicroRNA Upregulates IL-6 in Bone Marrow-Derived Mesenchymal Stem Cells Triggering a Reactive Stromal Response to Prostate Cancer

    doi: 10.1371/journal.pone.0071637

    Figure Lengend Snippet: Interactions of human bone marrow-derived MSCs and prostate cancer cells. (A) Migratory response of human MSCs to the medium, normal prostatic epithelial cells (PrEC) and prostate cancer cell lines (DU145, PC3 and LNCaP). Migrated cells through the membrane of transwells were stained with crystal violet 8 h after cell plating. The lower surface of the membrane was photographed (100x), and a representative image from each condition is shown at top. Quantitative data are represented as the means ± SD of the number of cells per high-power field (100x) in triplicate experiments. (B) Macroscopic (upper) and microscopic (bottom) view of cellular aggregates under 3-D RWV culture conditions of 3A6 cells alone (3A6/3A6) or mixed with LNCaP (3A6/LNCaP), C4-2 (3A6/C4-2) or PC3 (3A6/PC3) cells. (C) Detection of mixed cellular components in the 3-D cultured prostate tumoroids by H E and immunohistochemical staining of E-cadherin (E-Cad) in the cell aggregate sections.

    Article Snippet: Human bone marrow-derived MSCs (hMSCs) and normal human prostate epithelial cells (PrEC) purchased from Lonza (Rockland, ME) were maintained in MSCGM™ and PrEGM™, respectively, according to the manufacturer’s instructions (Lonza).

    Techniques: Derivative Assay, Staining, Cell Culture, Immunohistochemistry

    Effects of IL-6 on the reactive stromal phenotypes of MSCs. (A) Cytokine expression profile of the conditioned medium obtained from the normal 3A6 RWV and the prostate cancer-associated 3A6 LNCaP , 3A6 C4-2 , and 3A6 PC3 was analyzed by using a Human Cytokine Antibody Array C Series 2000. The autoradiograph of one set of Array VI containing IL-6 spots (rectangular boxes) is presented. (B) Quantitative detection of the expression level of IL-6 by ELISA assay. Data represent the means ± SD of triplicate determination. (C) Chemotactic response of PC3 cells induced by human recombinant IL-6 (rIL-6). Data are represented as the means ± SD of the number of cells per high-power field (100x) in triplicate experiments. * P

    Journal: PLoS ONE

    Article Title: Loss of Let-7 MicroRNA Upregulates IL-6 in Bone Marrow-Derived Mesenchymal Stem Cells Triggering a Reactive Stromal Response to Prostate Cancer

    doi: 10.1371/journal.pone.0071637

    Figure Lengend Snippet: Effects of IL-6 on the reactive stromal phenotypes of MSCs. (A) Cytokine expression profile of the conditioned medium obtained from the normal 3A6 RWV and the prostate cancer-associated 3A6 LNCaP , 3A6 C4-2 , and 3A6 PC3 was analyzed by using a Human Cytokine Antibody Array C Series 2000. The autoradiograph of one set of Array VI containing IL-6 spots (rectangular boxes) is presented. (B) Quantitative detection of the expression level of IL-6 by ELISA assay. Data represent the means ± SD of triplicate determination. (C) Chemotactic response of PC3 cells induced by human recombinant IL-6 (rIL-6). Data are represented as the means ± SD of the number of cells per high-power field (100x) in triplicate experiments. * P

    Article Snippet: Human bone marrow-derived MSCs (hMSCs) and normal human prostate epithelial cells (PrEC) purchased from Lonza (Rockland, ME) were maintained in MSCGM™ and PrEGM™, respectively, according to the manufacturer’s instructions (Lonza).

    Techniques: Expressing, Ab Array, Autoradiography, Enzyme-linked Immunosorbent Assay, Recombinant

    RelB effects on cell death of 22Rv1 cells. A) Illustration of 22Rv1-derived cells cultured in suspension on poly-HEMA pre-coated plates. Representative fields at 200X magnificence. B) Cell death evaluation in suspension cell culture of 22Rv1-derived cells. Cells were cultured 7 days in 96-well plates pre-coated or not with poly-HEMA. Cell death was evaluated by bioluminescence using the CytoTOX-Glo cytotoxicity assay. Data represent the ratio of basal cell death/total cell death after complete lysis using digitonin reagent. Cell death in suspension culture conditions are illustrated in black bars and adherence control cell culture conditions in empty bars. The variation was significant at p

    Journal: Cancer Cell International

    Article Title: The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo

    doi: 10.1186/1475-2867-14-67

    Figure Lengend Snippet: RelB effects on cell death of 22Rv1 cells. A) Illustration of 22Rv1-derived cells cultured in suspension on poly-HEMA pre-coated plates. Representative fields at 200X magnificence. B) Cell death evaluation in suspension cell culture of 22Rv1-derived cells. Cells were cultured 7 days in 96-well plates pre-coated or not with poly-HEMA. Cell death was evaluated by bioluminescence using the CytoTOX-Glo cytotoxicity assay. Data represent the ratio of basal cell death/total cell death after complete lysis using digitonin reagent. Cell death in suspension culture conditions are illustrated in black bars and adherence control cell culture conditions in empty bars. The variation was significant at p

    Article Snippet: Cell line and culture conditions 22Rv1 human prostate carcinoma epithelial cells were obtained from ATCC and cultured in RPMI-1640 complete media (Wisent, Montreal, Qc) containing 10% FBS (Fetal Bovine Serum) (Wisent, Montreal, Qc), 2.5 μg/mL amphotericin B and 50 μg/mL gentamicin (Gibco, Grand Island, NY), at 37°C with 5% CO2 .

    Techniques: Derivative Assay, Cell Culture, Cytotoxicity Assay, Lysis

    RelB sub-cellular localization and alternative NF-κB activity in 22Rv1-derived cells. A) Cellular distribution of the main NF-κB subunits analyzed by immunoblotting on protein extracts of cytoplasmic and nuclear compartments from RelB clones and GFP control cells. Actin was used as loading control for both cytoplasmic and nuclear protein extracts whereas GAPDH was used as a purity indicator for nuclear protein extracts. B) Immunoprecipitation of RelB from cytoplasmic and nuclear compartments protein extracts from RelB clones and GFP control cells. Immunoblot analyses of p100, p52 and p65 were performed from immunoprecipitated RelB fraction. C) NF-κB transcriptional activity by luciferase gene-reporter assay. RelB clones and GFP control cells were co-transfected with p3enh-κB-CONAluc (Firefly luciferase) and phRL-CMV (Renilla luciferase). Normalized data for each cell population presented are the luminescence ratio Firefly luc/Renilla luc . Experiments were done three times in triplicate. Error bars represent the standard error of the mean and p

    Journal: Cancer Cell International

    Article Title: The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo

    doi: 10.1186/1475-2867-14-67

    Figure Lengend Snippet: RelB sub-cellular localization and alternative NF-κB activity in 22Rv1-derived cells. A) Cellular distribution of the main NF-κB subunits analyzed by immunoblotting on protein extracts of cytoplasmic and nuclear compartments from RelB clones and GFP control cells. Actin was used as loading control for both cytoplasmic and nuclear protein extracts whereas GAPDH was used as a purity indicator for nuclear protein extracts. B) Immunoprecipitation of RelB from cytoplasmic and nuclear compartments protein extracts from RelB clones and GFP control cells. Immunoblot analyses of p100, p52 and p65 were performed from immunoprecipitated RelB fraction. C) NF-κB transcriptional activity by luciferase gene-reporter assay. RelB clones and GFP control cells were co-transfected with p3enh-κB-CONAluc (Firefly luciferase) and phRL-CMV (Renilla luciferase). Normalized data for each cell population presented are the luminescence ratio Firefly luc/Renilla luc . Experiments were done three times in triplicate. Error bars represent the standard error of the mean and p

    Article Snippet: Cell line and culture conditions 22Rv1 human prostate carcinoma epithelial cells were obtained from ATCC and cultured in RPMI-1640 complete media (Wisent, Montreal, Qc) containing 10% FBS (Fetal Bovine Serum) (Wisent, Montreal, Qc), 2.5 μg/mL amphotericin B and 50 μg/mL gentamicin (Gibco, Grand Island, NY), at 37°C with 5% CO2 .

    Techniques: Activity Assay, Derivative Assay, Clone Assay, Immunoprecipitation, Luciferase, Reporter Assay, Transfection

    RelB expression effect on 22Rv1 induced-tumor formation in a SCID mouse model. Analysis of 22Rv1-induced tumor growth. 250,000 22Rv1 RelB or GFP cells suspended in matrigel solution (5 mg/mL) were injected subcutaneously in the flank of 6-weeks old SCID mice. For each 22Rv1 cell population, 6 mice were injected. A) Tumor size was graphically represented across time. For each group of mice, the last point of the curve corresponds to the time of the first mouse sacrifice. Error bars represent the standard error of the mean. Significant variation between C2- and C3-RelB mouse groups individually compared to each GFP control (MP and clone) and C1-RelB mouse groups is illustrated by (*) on graph whereas (!) represents a significant variation between C2 and C3 RelB mice individually compared to MP GFP control group (Kruskal-Wallis test). B) Immunohistochemical staining of RelB illustrating its expression status on harvested 22Rv1-induced tumors. C) Time for 22Rv1-induced tumors to reach 500 mm 3 . On graph, (*) illustrated a p

    Journal: Cancer Cell International

    Article Title: The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo

    doi: 10.1186/1475-2867-14-67

    Figure Lengend Snippet: RelB expression effect on 22Rv1 induced-tumor formation in a SCID mouse model. Analysis of 22Rv1-induced tumor growth. 250,000 22Rv1 RelB or GFP cells suspended in matrigel solution (5 mg/mL) were injected subcutaneously in the flank of 6-weeks old SCID mice. For each 22Rv1 cell population, 6 mice were injected. A) Tumor size was graphically represented across time. For each group of mice, the last point of the curve corresponds to the time of the first mouse sacrifice. Error bars represent the standard error of the mean. Significant variation between C2- and C3-RelB mouse groups individually compared to each GFP control (MP and clone) and C1-RelB mouse groups is illustrated by (*) on graph whereas (!) represents a significant variation between C2 and C3 RelB mice individually compared to MP GFP control group (Kruskal-Wallis test). B) Immunohistochemical staining of RelB illustrating its expression status on harvested 22Rv1-induced tumors. C) Time for 22Rv1-induced tumors to reach 500 mm 3 . On graph, (*) illustrated a p

    Article Snippet: Cell line and culture conditions 22Rv1 human prostate carcinoma epithelial cells were obtained from ATCC and cultured in RPMI-1640 complete media (Wisent, Montreal, Qc) containing 10% FBS (Fetal Bovine Serum) (Wisent, Montreal, Qc), 2.5 μg/mL amphotericin B and 50 μg/mL gentamicin (Gibco, Grand Island, NY), at 37°C with 5% CO2 .

    Techniques: Expressing, Injection, Mouse Assay, Immunohistochemistry, Staining

    Effect of RelB expression on cell proliferation and anchorage-independent cell growth of 22Rv1 cells. A) Representative field of 22Rv1 colonies (macroscopic view) illustrating 22Rv1 RelB and GFP cells grown in soft agar. B) Soft agar growth assay. 22Rv1 cells were seeded in soft agar and incubated for 2 weeks. Colonies were stained with crystal violet. Graph represented the number of colonies in soft agar. Experiments were done three times in duplicate. Error bars represent the standard error of the mean and p

    Journal: Cancer Cell International

    Article Title: The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo

    doi: 10.1186/1475-2867-14-67

    Figure Lengend Snippet: Effect of RelB expression on cell proliferation and anchorage-independent cell growth of 22Rv1 cells. A) Representative field of 22Rv1 colonies (macroscopic view) illustrating 22Rv1 RelB and GFP cells grown in soft agar. B) Soft agar growth assay. 22Rv1 cells were seeded in soft agar and incubated for 2 weeks. Colonies were stained with crystal violet. Graph represented the number of colonies in soft agar. Experiments were done three times in duplicate. Error bars represent the standard error of the mean and p

    Article Snippet: Cell line and culture conditions 22Rv1 human prostate carcinoma epithelial cells were obtained from ATCC and cultured in RPMI-1640 complete media (Wisent, Montreal, Qc) containing 10% FBS (Fetal Bovine Serum) (Wisent, Montreal, Qc), 2.5 μg/mL amphotericin B and 50 μg/mL gentamicin (Gibco, Grand Island, NY), at 37°C with 5% CO2 .

    Techniques: Expressing, Growth Assay, Incubation, Staining

    Model of RelB influence in 22Rv1 cells in xenograft assays. In 22Rv1 cells, exogenous RelB induces the activation of alternative NF-κB pathway, resulting in undetermined target-genes and specific cell responses. The tumor growth initiation in vivo , cells are suspended in semi-solid media, mimicking anchorage free conditions that trigger autophagy in the RelB expressing cells (green lined pink colonies). At this time there is a low proportion of proliferating cells (red colonies) and the balance autophagy/proliferation weighs in favor of autophagy. Triggered autophagy then protects 22Rv1-RelB cells from stress induced by suspension conditions until they recover optimal growth conditions once they are anchored within mouse ECM. The colonies continue to grow and form the tumor mass (until 500 mm 3 of tumor size) where adherence conditions predominate. At this time the balance autophagy/proliferation weighs in favor of proliferation resulting in increase of tumor growth rate.

    Journal: Cancer Cell International

    Article Title: The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo

    doi: 10.1186/1475-2867-14-67

    Figure Lengend Snippet: Model of RelB influence in 22Rv1 cells in xenograft assays. In 22Rv1 cells, exogenous RelB induces the activation of alternative NF-κB pathway, resulting in undetermined target-genes and specific cell responses. The tumor growth initiation in vivo , cells are suspended in semi-solid media, mimicking anchorage free conditions that trigger autophagy in the RelB expressing cells (green lined pink colonies). At this time there is a low proportion of proliferating cells (red colonies) and the balance autophagy/proliferation weighs in favor of autophagy. Triggered autophagy then protects 22Rv1-RelB cells from stress induced by suspension conditions until they recover optimal growth conditions once they are anchored within mouse ECM. The colonies continue to grow and form the tumor mass (until 500 mm 3 of tumor size) where adherence conditions predominate. At this time the balance autophagy/proliferation weighs in favor of proliferation resulting in increase of tumor growth rate.

    Article Snippet: Cell line and culture conditions 22Rv1 human prostate carcinoma epithelial cells were obtained from ATCC and cultured in RPMI-1640 complete media (Wisent, Montreal, Qc) containing 10% FBS (Fetal Bovine Serum) (Wisent, Montreal, Qc), 2.5 μg/mL amphotericin B and 50 μg/mL gentamicin (Gibco, Grand Island, NY), at 37°C with 5% CO2 .

    Techniques: Activation Assay, In Vivo, Expressing

    RelB expression profile in 22Rv1-derived cells. A) RelB immunoblotting from whole protein extracts of wild type (WT) and transduced (RelB, GFP) 22Rv1 cells. B) Immunocytochemical staining of RelB on 22Rv1-derived cells: RelB clones (C1, C2, and C3) and GFP control cells (mixed population and clone). In the negative control, 1X PBS replaced RelB antibody.

    Journal: Cancer Cell International

    Article Title: The RelB alternative NF-kappaB subunit promotes autophagy in 22Rv1 prostate cancer cells in vitro and affects mouse xenograft tumor growth in vivo

    doi: 10.1186/1475-2867-14-67

    Figure Lengend Snippet: RelB expression profile in 22Rv1-derived cells. A) RelB immunoblotting from whole protein extracts of wild type (WT) and transduced (RelB, GFP) 22Rv1 cells. B) Immunocytochemical staining of RelB on 22Rv1-derived cells: RelB clones (C1, C2, and C3) and GFP control cells (mixed population and clone). In the negative control, 1X PBS replaced RelB antibody.

    Article Snippet: Cell line and culture conditions 22Rv1 human prostate carcinoma epithelial cells were obtained from ATCC and cultured in RPMI-1640 complete media (Wisent, Montreal, Qc) containing 10% FBS (Fetal Bovine Serum) (Wisent, Montreal, Qc), 2.5 μg/mL amphotericin B and 50 μg/mL gentamicin (Gibco, Grand Island, NY), at 37°C with 5% CO2 .

    Techniques: Expressing, Derivative Assay, Staining, Clone Assay, Negative Control

    Tipifarnib inhibits exosome biogenesis and secretion via both ESCRT dependent and independent pathways and disrupts RAS signaling. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with tipifarnib or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the qNano IZON. Tipifarnib shows a significant decrease in the concentration of exosomes in C4-2B cells both at 0.25 and 1 μM as well as in the PC-3 cells at 0.25 μM. ( B ) Tipifarnib at a 1 μM concentration significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in C4-2B cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Tipifarnib significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . ( D ) Tipifarnib significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p

    Journal: Scientific Reports

    Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

    doi: 10.1038/s41598-018-26411-7

    Figure Lengend Snippet: Tipifarnib inhibits exosome biogenesis and secretion via both ESCRT dependent and independent pathways and disrupts RAS signaling. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with tipifarnib or DMSO (vehicle control) for 48 hours. The concentration of exosomes was measured by the qNano IZON. Tipifarnib shows a significant decrease in the concentration of exosomes in C4-2B cells both at 0.25 and 1 μM as well as in the PC-3 cells at 0.25 μM. ( B ) Tipifarnib at a 1 μM concentration significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in C4-2B cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Tipifarnib significantly inhibited the protein concentration of Alix, nSMase2, and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . ( D ) Tipifarnib significantly inhibited the activation of p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p

    Article Snippet: The human prostate cancer epithelial metastatic cell line PC-3 (ATCC CRL-1435) and the normal prostate epithelial cell line RWPE-1 (ATCC CRL-11609) were purchased from ATCC (Manassas, VA, USA).

    Techniques: Concentration Assay, Protein Concentration, Activation Assay, Derivative Assay

    Ketoconazole (prototype imidazole) inhibits exosome biogenesis through ESCRT dependent and independent pathway. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with ketoconazole or DMSO (vehicle control) for 48 hours at 5 μM. Concentration of exosomes was measured by qNano IZON. Ketoconazole shows a significant decrease in the concentration of exosomes in C4-2B cells at 5 μM as well as in the PC-3 cells ( B ) Ketoconazole significantly inhibited the protein concentration of Alix, nSMase2 and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Ketoconazole significantly inhibited activation p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B, and PC-3 cells, but not in the RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p

    Journal: Scientific Reports

    Article Title: High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer

    doi: 10.1038/s41598-018-26411-7

    Figure Lengend Snippet: Ketoconazole (prototype imidazole) inhibits exosome biogenesis through ESCRT dependent and independent pathway. ( A ) C4-2B and PC-3 cells were maintained overnight in exosome free DMEM media and then treated with ketoconazole or DMSO (vehicle control) for 48 hours at 5 μM. Concentration of exosomes was measured by qNano IZON. Ketoconazole shows a significant decrease in the concentration of exosomes in C4-2B cells at 5 μM as well as in the PC-3 cells ( B ) Ketoconazole significantly inhibited the protein concentration of Alix, nSMase2 and Rab27a in a dose-dependent manner in C4-2B and PC-3 cells but not in the normal RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . ( C ) Ketoconazole significantly inhibited activation p-ERK (downstream effector molecule of the Ras/Raf/ERK signaling pathway) but not total ERK in C4-2B, and PC-3 cells, but not in the RWPE-1 cells. Full blots are presented in Supplementary Fig. S6 . Mean values and standard errors were derived from four independent experiments. *Denotes significance at p

    Article Snippet: The human prostate cancer epithelial metastatic cell line PC-3 (ATCC CRL-1435) and the normal prostate epithelial cell line RWPE-1 (ATCC CRL-11609) were purchased from ATCC (Manassas, VA, USA).

    Techniques: Concentration Assay, Protein Concentration, Activation Assay, Derivative Assay

    Mel-18 negatively regulated the self-renew of gastric cancer stem like cells A. Patients with Mel-18 positive expression survived longer than those with Mel-18 negative expression. Kaplan-Meyer survival curves were plotted as cumulative survival vs months according to Mel-18 expression (negative or positive) in cancer samples in patients with gastric cancer. The expression of Mel-18 was detected by Immunohistochemical (IHC) Assay. B. The expression of Mel-18 mRNA was downregulated in spheroid cells (SC) compared with that in parental adherent cells (PC). Tumorigenic spheres were derived from SGC7901 gastric cancer cell line in serum-free media containing EGF and bFGF and then photographed (upper pane). Fold change of Mel-18 in PC and SC was analyzed by QRT-PCR (lower panel). Total RNA of parental adherent cells and spheroid cells were extracted using TRIzol reagent (Invitrogen) and cDNA synthesis were performed as manufacturer's protocol. For Mel-18 mRNA, GAPDH acted as an internal control. C. Overexpression of Mel-18 reduced self-renew of gastric cancer stem like cells. The self-renew ability was analyzed by serum-free culture spheres formation and photographed (upper pane). The percentage of spheres formed was calculated and plotted (lower left panel), and overexpression of Mel-18 in SGC-7901 cells was determined by Western blot ( lower right panel ). Stable cell lines expressing Mel-18 was generated by transfection of Mel-18 overexpressing plasmid and selected by puromycine. Con: cells transfected with control vector; Mel-18: cells transfected with Mel-18 overexpressing plasmids. D. Mel-18 overexpression inhibited in vivo tumorigenecity of SGC7901 cells. In vivo tumorigenecity was detected by subcutaneously cancer cells injected SCID mice model. Suppressed tumor size after SGC7901 injected subcutaneously in one rear flank of severe combined SCID mice. Mel-18-overexpressing SGC-7901 cells or control cells were injected subcutaneously into the flanks of severe combined SCID mice. Tumor sizes were detected terminally by vernier caliper. After 4 weeks, mice were sacrificed by cervical dislocation, and tumors were removed and imaged. All experiments involving animal abided by protocols approved by the Shanghai Medical Experimental Animal Care Commission.

    Journal: Oncotarget

    Article Title: Mel-18 negatively regulates stem cell-like properties through downregulation of miR-21 in gastric cancer

    doi: 10.18632/oncotarget.11221

    Figure Lengend Snippet: Mel-18 negatively regulated the self-renew of gastric cancer stem like cells A. Patients with Mel-18 positive expression survived longer than those with Mel-18 negative expression. Kaplan-Meyer survival curves were plotted as cumulative survival vs months according to Mel-18 expression (negative or positive) in cancer samples in patients with gastric cancer. The expression of Mel-18 was detected by Immunohistochemical (IHC) Assay. B. The expression of Mel-18 mRNA was downregulated in spheroid cells (SC) compared with that in parental adherent cells (PC). Tumorigenic spheres were derived from SGC7901 gastric cancer cell line in serum-free media containing EGF and bFGF and then photographed (upper pane). Fold change of Mel-18 in PC and SC was analyzed by QRT-PCR (lower panel). Total RNA of parental adherent cells and spheroid cells were extracted using TRIzol reagent (Invitrogen) and cDNA synthesis were performed as manufacturer's protocol. For Mel-18 mRNA, GAPDH acted as an internal control. C. Overexpression of Mel-18 reduced self-renew of gastric cancer stem like cells. The self-renew ability was analyzed by serum-free culture spheres formation and photographed (upper pane). The percentage of spheres formed was calculated and plotted (lower left panel), and overexpression of Mel-18 in SGC-7901 cells was determined by Western blot ( lower right panel ). Stable cell lines expressing Mel-18 was generated by transfection of Mel-18 overexpressing plasmid and selected by puromycine. Con: cells transfected with control vector; Mel-18: cells transfected with Mel-18 overexpressing plasmids. D. Mel-18 overexpression inhibited in vivo tumorigenecity of SGC7901 cells. In vivo tumorigenecity was detected by subcutaneously cancer cells injected SCID mice model. Suppressed tumor size after SGC7901 injected subcutaneously in one rear flank of severe combined SCID mice. Mel-18-overexpressing SGC-7901 cells or control cells were injected subcutaneously into the flanks of severe combined SCID mice. Tumor sizes were detected terminally by vernier caliper. After 4 weeks, mice were sacrificed by cervical dislocation, and tumors were removed and imaged. All experiments involving animal abided by protocols approved by the Shanghai Medical Experimental Animal Care Commission.

    Article Snippet: Cells were seeded in wells (1000 cells per well or otherwise indicated) of ultra-low-attachment 6-well plates (Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences ) supplemented plus2ml of DMEM/F12 medium (Invitrogen) with 10mM HEPES, human recombinant epidermal growth factor (EGF) (Invitrogen) at the concentration of 20 ng/ml, and human recombinant basic fibroblast growth factor (bFGF) (Invitrogen) at the concentration of 10 ng/ml.

    Techniques: Expressing, Immunohistochemistry, Derivative Assay, Quantitative RT-PCR, Over Expression, Western Blot, Stable Transfection, Generated, Transfection, Plasmid Preparation, In Vivo, Injection, Mouse Assay