Journal: Pharmaceuticals

Article Title: Antitumor Activity of the ACC Inhibitor Firsocostat in Breast Cancer Cell Lines: A Proof-of-Concept In Vitro Study

doi: 10.3390/ph19020201

Figure Lengend Snippet: Morphological analysis of BC and non-tumorigenic cell lines after exposure to firsocostat. Panels ( a – d ) show representative phase-contrast micrographs (10X magnification) of MCF7 (1), SK-BR-3 (2), MDA-MB-231 (3), HCC1937 (4), and MCF-10A (5) cells after 72 h of treatment with firsocostat at ( b ) 25, ( c ) 100, and ( d ) 200 µM compared with ( a ) DMSO controls cells.

Article Snippet: Human breast cancer cell lines MCF7 (#HTB-22), SK-BR-3 (#HTB-30), MDA-MB-231 (#HTB-26), HCC1937 (#CRL-2336), and the normal-like breast epithelial MCF-10A (#CRL-10317) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) ( ).

Techniques:

Journal: Pharmaceuticals

Article Title: Antitumor Activity of the ACC Inhibitor Firsocostat in Breast Cancer Cell Lines: A Proof-of-Concept In Vitro Study

doi: 10.3390/ph19020201

Figure Lengend Snippet: Effect of firsocostat on BC cell viability assessed by Trypan Blue exclusion assay. Panels ( a – d ) show the dose- and time-dependent effects of firsocostat on BC cell viability: ( a ) MCF7, ( b ) SK-BR-3, ( c ) MDA-MB-231, and ( d ) HCC1937. Data represent mean values from three independent experiments, illustrating overall growth trends under control and treated conditions after 72 h with firsocostat at 25, 50, and 100 µM compared with DMSO control cells.

Article Snippet: Human breast cancer cell lines MCF7 (#HTB-22), SK-BR-3 (#HTB-30), MDA-MB-231 (#HTB-26), HCC1937 (#CRL-2336), and the normal-like breast epithelial MCF-10A (#CRL-10317) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) ( ).

Techniques: Trypan Blue Exclusion Assay, Control

Journal: Pharmaceuticals

Article Title: Antitumor Activity of the ACC Inhibitor Firsocostat in Breast Cancer Cell Lines: A Proof-of-Concept In Vitro Study

doi: 10.3390/ph19020201

Figure Lengend Snippet: Cell viability of BC and non-tumorigenic cell lines after firsocostat treatment assessed by MTS assay. Panels ( a – e ) show viability of MCF7 ( a ), SK-BR-3 ( b ), MDA-MB-231 ( c ), HCC1937 ( d ), and MCF-10A ( e ) cells following 72 h of exposure to increasing concentrations of firsocostat (25–200 µM). Data are expressed as mean ± SD of three independent experiments, normalized to control (CTRL, 100%). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. Statistical significance vs. CTRL is indicated as ns, not significant; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***); p < 0.0001 (****).

Article Snippet: Human breast cancer cell lines MCF7 (#HTB-22), SK-BR-3 (#HTB-30), MDA-MB-231 (#HTB-26), HCC1937 (#CRL-2336), and the normal-like breast epithelial MCF-10A (#CRL-10317) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) ( ).

Techniques: MTS Assay, Control

Journal: Pharmaceuticals

Article Title: Antitumor Activity of the ACC Inhibitor Firsocostat in Breast Cancer Cell Lines: A Proof-of-Concept In Vitro Study

doi: 10.3390/ph19020201

Figure Lengend Snippet: Dose–response curves of firsocostat in BC and non-tumorigenic cell lines. Panels ( a – e ) depicts nonlinear regression curves based on cell viability assessed by MTS assay after 72 h of treatment with increasing concentrations of firsocostat: MCF7 ( a ), SK-BR-3 ( b ), MDA-MB-231 ( c ), HCC1937 ( d ), and MCF-10A ( e ). IC 50 values (µM) were calculated using nonlinear regression (4-parameter logistic model) in GraphPad Prism. Data are presented as mean ± SD, normalized to untreated controls.

Article Snippet: Human breast cancer cell lines MCF7 (#HTB-22), SK-BR-3 (#HTB-30), MDA-MB-231 (#HTB-26), HCC1937 (#CRL-2336), and the normal-like breast epithelial MCF-10A (#CRL-10317) cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) ( ).

Techniques: MTS Assay

Journal: Journal of cellular physiology

Article Title: Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity.

doi: 10.1002/jcp.30329

Figure Lengend Snippet: FIGURE 1 Low glucose promotes TRAIL‐induced apoptosis through activation of caspases. (a) SW48 cells were treated with various doses of TRAIL (0–100 ng/ml) for 4 h. (b) SW48 cells were treated with various concentrations of glucose (0–25 mM) for 24 h. (c) SW48 cells were pretreated with various concentrations of glucose for 20 h and then treated with 10 ng/ml TRAIL for an additional 4 h. (d) SW48 cells were pretreated with a glucose‐free medium for 20 h and then treated with various doses (10 or 50 ng/ml) of TRAIL for an additional 4 h. Cell survival was determined using trypan blue exclusion assay (upper panels). Error bars represent the mean ± SD from triplicate experiments. p values: *, .05; **, .01; ***, .001. Cell lysates were analyzed with an immunoblotting assay using indicated antibodies (lower panels). Immunoblots from two gels were combined in each figure. Actin was used as a protein loading control in each lane. C‐Cas3, cleaved caspase‐3; C‐Cas8, cleaved caspase‐8; C‐Cas9, cleaved caspase‐9; CM, complete medium; GFM, glucose‐free medium; PARP‐1, poly(ADP‐ribose) polymerase‐1; TRAIL, tumor necrosis factor‐related apoptosis‐inducing ligand

Article Snippet: Human colorectal adenocarcinoma SW48, human colorectal adeno- carcinoma SW480, and human colorectal carcinoma HCT116 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Activation Assay, Trypan Blue Exclusion Assay, Western Blot, Control

Journal: Journal of cellular physiology

Article Title: Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity.

doi: 10.1002/jcp.30329

Figure Lengend Snippet: FIGURE 2 Low glucose induces ER stress in SW48 cells. (a) Cells were treated with various concentrations of glucose (0–25 mM) for 24 h. C, untreated control cells. (b) cells were treated with a glucose‐free medium for various times (0–24 h). (c) Cells were pretreated with/without glucose‐free medium for 20 h and treated with various doses (10 or 50 ng/ml) of TRAIL for an additional 4 h. Whole‐cell lysates were analyzed using an immunoblotting assay with the indicated antibodies. CHOP, CCAAT‐enhancer‐binding protein homologous protein; CM, complete medium; GFM, glucose‐free medium; HSPA5, heat shock protein family A (Hsp70) member 5; PUMA, p53 upregulated modulator of apoptosis; TRAIL, tumor necrosis factor‐related apoptosis‐inducing ligand

Article Snippet: Human colorectal adenocarcinoma SW48, human colorectal adeno- carcinoma SW480, and human colorectal carcinoma HCT116 cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Control, Western Blot, Binding Assay

Journal: Free radical biology & medicine

Article Title: p66Shc Protein through a Redox Mechanism Enhances the Progression of Prostate Cancer Cells towards Castration-Resistance

doi: 10.1016/j.freeradbiomed.2019.05.015

Figure Lengend Snippet: AS and AI LNCaP, MDA PCa2b, and VCaP cells were subjected to a kinetic growth assay in (A-C) regular and (D-F) SR conditions. (A and D) LNCaP, (B and E) MDA PCa2b, and (C and F) VCaP cells were plated at 2 × 104, 1 × 105, and 2 × 105 cells per well in 6-well plates, respectively. For SR conditions, cells were adjusted to SR medium for 48 hours before growth determination. Cells were harvested every 72 hours on days 0, 3, 6, and 9. Cell viability was measured using Trypan blue exclusion dye. Results presented are mean ± SD. n=3×3. *p<0.05, **p<0.005, ***p<0.0005. Cells were lysed to determine PCNA levels via western blot analysis to confirm cell growth results. β-actin protein level was used as a loading control.

Article Snippet: Human prostate cancer cell lines LNCaP, VCaP and MDA PCa2b were originally purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured according to the accompanied protocols and our publications [ 28 - 32 ].

Techniques: Kinetic Growth Assay, Western Blot, Control

Journal: Free radical biology & medicine

Article Title: p66Shc Protein through a Redox Mechanism Enhances the Progression of Prostate Cancer Cells towards Castration-Resistance

doi: 10.1016/j.freeradbiomed.2019.05.015

Figure Lengend Snippet: (A-C) AS and AI LNCaP, MDA PCa2b and VCaP cells were plated in 6-well plates at 3 × 103, 1 × 104 and 1 × 104 cells per well, respectively. LNCaP cells were maintained in regular RPMI 1640 medium for 9 days with a fresh change of medium every 3 days. MDA PCa2b and VCaP cell lines were maintained in HPC1 and DMEM, respectively, for 14 days, with a fresh change of medium every 3 days. Cells were stained with 0.02% crystal violet containing 50% methanol. The stained cells were then photographed at 40x magnification for counting. Results presented are mean ± SD. n=2×3. *p<0.05, **p<0.005, ***p<0.0005.

Article Snippet: Human prostate cancer cell lines LNCaP, VCaP and MDA PCa2b were originally purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured according to the accompanied protocols and our publications [ 28 - 32 ].

Techniques: Staining

Journal: Free radical biology & medicine

Article Title: p66Shc Protein through a Redox Mechanism Enhances the Progression of Prostate Cancer Cells towards Castration-Resistance

doi: 10.1016/j.freeradbiomed.2019.05.015

Figure Lengend Snippet: (A-C) DCF-DA analysis for general oxidant species levels. AS and AI (A) LNCaP, (B) MDA PCa2b and (C) VCaP cells were plated at 1 × 105, 5 × 105, and 3 × 105 cells per T25 flask and grown for 72 hours. Cells were stained with DCF-DA (20 μM) for 30 minutes in the dark and subjected to flow cytometry analysis. Results presented are mean ± SD. n=3, *p<0.05.

Article Snippet: Human prostate cancer cell lines LNCaP, VCaP and MDA PCa2b were originally purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured according to the accompanied protocols and our publications [ 28 - 32 ].

Techniques: Staining, Flow Cytometry

Journal: Free radical biology & medicine

Article Title: p66Shc Protein through a Redox Mechanism Enhances the Progression of Prostate Cancer Cells towards Castration-Resistance

doi: 10.1016/j.freeradbiomed.2019.05.015

Figure Lengend Snippet: (A-C) Trypan Blue Exclusion Assay. (A) LNCaP-AS, (B) MDA PCa2b-AS and (C) VCaP-AS cells were plated at 2 × 104, 2 × 104 and 2 × 104 cells per well, respectively, for 72 hours. Cells were treated with H2O2 (0-20 μM) or NAC (10 mM) for 72 hours before cells were harvested and counted via Trypan blue exclusion dye. Results presented are mean ± SD. n=3×3. *p<0.05, **p<0.005, ***p<0.0005.

Article Snippet: Human prostate cancer cell lines LNCaP, VCaP and MDA PCa2b were originally purchased from the American Type Culture Collection (Rockville, MD, USA) and cultured according to the accompanied protocols and our publications [ 28 - 32 ].

Techniques: Trypan Blue Exclusion Assay

Journal: Oncotarget

Article Title: Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression.

doi: 10.18632/oncotarget.23046

Figure Lengend Snippet: Figure 1: Artesunate (ART) promotes TRAIL-induced apoptosis. (A-F) PANC-1 (A), BxPC-3 (B), and HCT116 (C) cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (PANC-1, 100 ng/ml; BxPC-3, 2 ng/ml; HCT116, 1 ng/ml) for an additional 4 h. The cells were stained with propidium iodide (PI). Phase-contrast images or fluorescence images were visualized under a light or fluorescence microscope, respectively (upper panels). Representative images are shown (magnification, X200). Cell death was determined by counting PI-stained cells and plotted (lower panels). Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, Student’s t-test (two-sided, paired) was used. p-values: *, 0.05; **, 0.01; ***, 0.001. Cell lysates of PANC-1 (D), BxPC-3 (E), and HCT116 (F) cells were analyzed with immunoblotting assay using indicated antibodies.

Article Snippet: Cell lines and cell culture conditions Human pancreatic cancer PANC-1 and BxPC-3 cells and human colon cancer HCT116 cells were previously obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Staining, Fluorescence, Microscopy, Western Blot

Journal: Oncotarget

Article Title: Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression.

doi: 10.18632/oncotarget.23046

Figure Lengend Snippet: Figure 2: Erastin promotes TRAIL-induced apoptosis. (A and B) HCT116 cells were treated with various doses of erastin for 24 h (A) or TRAIL for 4 h (B) and then the cell death rate was determined, respectively (upper panels). Cell lysates were analyzed with immunoblotting assay using indicated antibodies (lower panels). (C and D) HCT116 cells were pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Cell death was determined by counting and plotted. Whole-cell extracts were then analyzed with immunoblotting assay using indicated antibodies (C). The cells were stained with annexin V and PI and then analyzed using flow cytometry (D). (E) BxPC-3 cells were pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (6 ng/ ml) for an additional 4 h. Whole-cell extracts were then analyzed with immunoblotting assay using indicated antibodies.

Article Snippet: Cell lines and cell culture conditions Human pancreatic cancer PANC-1 and BxPC-3 cells and human colon cancer HCT116 cells were previously obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression.

doi: 10.18632/oncotarget.23046

Figure Lengend Snippet: Figure 3: ART and erastin, but not TRAIL, induce lipid peroxidation. (A-C) HCT116 cells were treated with various doses of erastin for 24 h (A) or TRAIL for 4 h (B). Cells were pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h (C). Lipid peroxidation (upper panels) and heme oxygenase (HO-1, lower panels) levels were analyzed by malondialdehyde (MDA) assay and immunoblotting assay, respectively. (D and E) HCT116 cells were treated with various doses of ART (D) or pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for additional 4 h (E). (F) BxPC-3 cells were pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for additional 4 h. MDA levels were determined and plotted. Error bars represent the mean ± SD from triplicate experiments. For statistical analysis, Student’s t-test (two-sided, paired) was used. p-values: *, 0.05; **, 0.01.

Article Snippet: Cell lines and cell culture conditions Human pancreatic cancer PANC-1 and BxPC-3 cells and human colon cancer HCT116 cells were previously obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Malondialdehyde MDA Assay, Western Blot

Journal: Oncotarget

Article Title: Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression.

doi: 10.18632/oncotarget.23046

Figure Lengend Snippet: Figure 4: Ferroptotic agents induce ER stress in HCT116 cells. (A) Microarray assay for detection of ART-induced gene expression. Cells were treated with 50 μM ART for 24 h and triplicate Illumina gene expression microarrays were performed with BeadArray microarray technology. (B) Ferroptotic agents induce the unfolded protein response (UPR). Cells were treated with 50 μM ART or 50 μM erastin for 24 h in the presence/absence of 250 nM MG132. Cell lysates were subjected to immunoblotting analysis using FK2 antibody specific to ubiquitin-conjugated proteins. Actin was used to confirm equal amounts of proteins loaded in each lane. (C and D) Cells were treated with ART (50 μM, B) or erastin (50 μM, C) for various times (1-24 h). Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies. (E) Cells were treated with various doses of erastin for 24 h. Whole-cell extracts were analyzed using immunoblotting assay with indicated antibodies. (F) Cells were treated with erastin alone (10 or 50 μM) for 24 h, TRAIL (1 ng/ml) alone for 4 h, or pretreated with erastin (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Whole-cell lysates were analyzed using immunoblotting assay with indicated antibodies.

Article Snippet: Cell lines and cell culture conditions Human pancreatic cancer PANC-1 and BxPC-3 cells and human colon cancer HCT116 cells were previously obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Microarray, Gene Expression, Western Blot, Ubiquitin Proteomics

Journal: Oncotarget

Article Title: Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression.

doi: 10.18632/oncotarget.23046

Figure Lengend Snippet: Figure 5: The combinatorial treatment of ferroptotic agent and TRAIL promotes apoptosis via ER stress, but not lipid peroxidation in HCT116 cells. (A and B) Cells were treated with 50 μM erastin (A) or ART (B) for 24 h in the absence/presence of DFO (100 μM), ferrostatin-1 (Fer-1, 10 μM), or liproxstatin-1 (Lip-1, 2 μM). Lipid peroxidation level was detected by MDA assay (left panels) and whole-cell lysates were analyzed with immunoblotting assay using indicated antibodies (right panels). (C and D) Cells were pretreated with erastin (C) and ART (D) for 20 h in the absence/presence of Z-VAD (1 μM), DFO (100 μM), Fer-1 (10 μM), or Lip-1 (2 μM) and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Cell death was determined using trypan blue exclusion assay and plotted.

Article Snippet: Cell lines and cell culture conditions Human pancreatic cancer PANC-1 and BxPC-3 cells and human colon cancer HCT116 cells were previously obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Multiple Displacement Amplification, Western Blot, Trypan Blue Exclusion Assay

Journal: Oncotarget

Article Title: Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression.

doi: 10.18632/oncotarget.23046

Figure Lengend Snippet: Figure 6: ART increases PUMA protein level. PANC-1 (A), BxPC-3 (B), and HCT116 (C) cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (PANC-1, 100 ng/ml; BxPC-3, 2 ng/ml; HCT116, 1 ng/ml) for additional 4 h. Whole-cell lysates were analyzed with immunoblotting assay using indicated antibodies. (D) HCT116 cells were treated with erastin (1-50 μM) or mitomycin C (1-20 μM, MMC) for 24 h. Whole-cell lysates were analyzed with immunoblotting assay using indicated antibodies.

Article Snippet: Cell lines and cell culture conditions Human pancreatic cancer PANC-1 and BxPC-3 cells and human colon cancer HCT116 cells were previously obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot

Journal: Oncotarget

Article Title: Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression.

doi: 10.18632/oncotarget.23046

Figure Lengend Snippet: Figure 7: ART promotes TRAIL-induced apoptosis via the p53-independent CHOP/PUMA pathway. (A) Mouse embryonic fibroblasts (MEFs) wild-type (WT) or MEF CHOP knockout (KO) cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to recombinant murine TRAIL (mTRAIL; 100 ng/ml) for an additional 4 h. Cell death was determined using trypan blue exclusion assay and plotted (left panel). Whole-cell extracts were analyzed with immunoblotting assay using indicated antibodies (right panel). (B) HCT116 WT or HCT116 PUMA KO cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for additional 4 h. Cell death was determined by counting and plotted (left panel). Whole-cell extracts were then analyzed with immunoblotting using indicated antibodies (right panel). (C) HCT116 WT or HCT116 p53 KO cells were pretreated with ART (10 or 50 μM) for 20 h and then exposed to TRAIL (1 ng/ml) for an additional 4 h. Cell death was determined using trypan blue exclusion assay and plotted (left panel). Whole-cell extracts were then analyzed with immunoblotting assay using indicated antibodies (right panel).

Article Snippet: Cell lines and cell culture conditions Human pancreatic cancer PANC-1 and BxPC-3 cells and human colon cancer HCT116 cells were previously obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Knock-Out, Recombinant, Trypan Blue Exclusion Assay, Western Blot

Journal: Oncotarget

Article Title: Molecular crosstalk between ferroptosis and apoptosis: emerging role of ER stress-induced p53-independent PUMA expression.

doi: 10.18632/oncotarget.23046

Figure Lengend Snippet: Figure 8: The combinatorial treatment of ART and TRAIL synergistically inhibits tumor growth. Nude mice were subcutaneously inoculated with 1×106 HCT116-luc cells. When the tumor volume reached approximately 200 mm3, tumor bearing mice were treated with ART (A, 200 mg/kg, twice per week, oral gavage) alone, TRAIL (T, 100 μg/kg, twice per week, intra-tumoral) alone, or the combination of ART and TRAIL (A + T). (A) Mice were imaged using the NightOWL LB983 bioluminescence imaging (BLI) system. Representative images are shown on day 19. (B) Tumor tissues were harvested on day 19 and displayed. (C) Line graph illustrating the tumor volume (mm3) in HCT116-luc tumor-bearing mice treated with PBS alone, ART alone, TRAIL alone, or the combination from day 0 to day 19. Error bars represent the mean ± SD from five mice. For statistical analysis, Student’s t-test (two-sided, paired) was used. p-values: *, 0.05; **, 0.01; ***, 0.001. (D and E) Tumor tissues were harvested on day 19 and subjected to TUNEL assay and DAPI (4’,6-diamidino- 2-phenylindole) staining. Cell nuclei were stained with DAPI. Apoptosis was detected using TUNEL assay (D) and percents of TUNEL- positive cells were plotted (E). (F) Line graph illustrating the body weight (gram) in HCT116-luc tumor-bearing mice treated with PBS alone, ART alone, TRAIL alone, or the combination from day 0 to day 19.

Article Snippet: Cell lines and cell culture conditions Human pancreatic cancer PANC-1 and BxPC-3 cells and human colon cancer HCT116 cells were previously obtained from American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Imaging, TUNEL Assay, Staining