Journal: Carcinogenesis

Article Title: Sildenafil triggers tumor lethality through altered expression of HSP90 and degradation of PKD2

doi: 10.1093/carcin/bgaa001

Figure Lengend Snippet: Sildenafil treatment is associated with altered HSP90 expression and PKD2 degradation. (A) Lysates of cancer cell lines incubated with 25 µM Sildenafil for 3 days were subjected to western blot analysis with HSP90β antibody. β-Actin was used as loading control. Densitometry analysis of three to four blots is presented. (B) Cleared lysates of tumor cell lines subjected to 25 µM Sildenafil for 3 days were used for western blot analysis with PKD2 antibody. β-Actin was used as loading control. (C) PKD2 overexpression was achieved via lentiviral-mediated transduction. Western blot analysis with PKD2-specific antibody after ending the selection is presented. (D–G) Cancer cells stably transduced with empty vector (Con) or PKD2 (PKD2 o.e.) were seeded in 12 well dishes. Cell proliferation was monitored for the next 3 days in the presence or absence of Sildenafil (Sil) when cell number was quantified. One representative of three experiments conducted in triplicate is shown.

Article Snippet: The following antibodies were used: PKD2 (Bethyl Laboratories, #A300-073A), cleaved poly (ADP ribose) polymerase (PARP; Cell Signaling, #9542S), HSP90β (D-19; Santa Cruz Biotechnology, #sc-1057) and β-actin (Sigma, #A1978).

Techniques: Expressing, Incubation, Western Blot, Control, Over Expression, Transduction, Selection, Stable Transfection, Plasmid Preparation

Journal: Cells

Article Title: Octyl Gallate Induces Pancreatic Ductal Adenocarcinoma Cell Apoptosis and Suppresses Endothelial-Mesenchymal Transition-Promoted M2-Macrophages, HSP90α Secretion, and Tumor Growth

doi: 10.3390/cells9010091

Figure Lengend Snippet: OG inhibits tumor growth, M2-type macrophages, and serum HSP90α levels in Panc 02 + EndoMT tumor model. ( A ) Measurement of the superficial tumor volumes from the C57BL/6 mice subcutaneously inoculated with Panc 02 + EndoMT cell grafts and treated with OG or control 40% PEG400 (CTRL). The measurement was started on Day 13 post-inoculation and continued every 3 days using Vernier caliper with the formula 1/2 × length × width 2 . ( B ) Tumors removed from the mice described in (A) on Day 32 post-inoculation. OG treatment significantly inhibited the tumor growth of Panc 02 + EndoMT cell grafts. ( C ) Representative H&E-stained tumor tissue sections of control and OG-treated mice. OG treatment did not obviously cause loosened cell arrangement and decreased chromatin basophilia which were observed in the Panc 02 tumor model. ( D ) Immunohistochemical staining of CK18 and α-SMA from the tumor tissues of control or OG-treated mice. OG treatment did not significantly change the CK18 + and α-SMA + cell levels of tumor tissues. ( E ) Immunohistochemical staining of CD11b + , F4/80 + , and CD163 + cells from the tumor tissues of control or OG-treated mice. ( F ) Quantitation of the levels of CD11b + , F4/80 + , and CD163 + cells from the tumor tissues of control or OG-treated mice. OG treatment caused a significant increase of F4/80 + macrophages but decreases of CD11b + myeloid cells and CD163 + macrophages in the tumor tissues. ( G ) Serum HSP90α levels of the mice described in ( A ). Mouse sera were collected every week and serum HSP90α levels were measured using ELISA assay. OG treatment resulted in a significant reduction of the serum HSP90α level. Data are the mean ± SD of 6 mice. # p < 0.001 when compared with “CTRL” group.

Article Snippet: Primary antibodies used for immunoblot analyses were listed as follows: Bcl-2 (BD Biosciences, #610539, 1:1000), Bcl-X L (Santa Cruz Biotechnology, sc-8392; 1:1000), Bax (Santa Cruz Biotechnology, sc-7480; 1:1000), prohibitin (NeoMarker, Fremont, CA, USA, MS-261-P; 1:500), BNIP3 (Cell Signaling, Danvers, MA, USA, #44060S; 1:1000), BNIP-3L (Cell Signaling, #12396S; 1:1000), cytochrome c (Santa Cruz Biotechnology, sc-13156; 1:2000), HIF-1α (GeneTex Inc., Hsinchu City, Taiwan, GTX127309; 1:1000), Nrf2 (Santa Cruz Biotechnology, sc-722; 1:1000), p62 (Abcam, Cambridge, UK, ab-56416; 1:4000), p-STAT-3 (Epitomics, Burlingame, CA, USA, #2236; 1:500), STAT-3 (EMD Millipore, #04-1014; 1:1000), and HSP90α (GeneTex Inc., GTX109753; 1:1000).

Techniques: Control, Staining, Immunohistochemical staining, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Octyl Gallate Induces Pancreatic Ductal Adenocarcinoma Cell Apoptosis and Suppresses Endothelial-Mesenchymal Transition-Promoted M2-Macrophages, HSP90α Secretion, and Tumor Growth

doi: 10.3390/cells9010091

Figure Lengend Snippet: OG inhibits EndoMT-induced macrophage M2-polarization and a feedforward loop of HSP90α secretion. ( A ) Immunoblot analysis of the eHSP90α levels from Endo-CM and EndoMT-CM. EndoMT-derived cells secreted more HSP90α when compared with Endo cells. Relative eHSP90α levels were presented after quantification of protein band intensities using ImageJ software. ( B ) mRNA levels of TNF-α, iNOS, CD163, TGF-β, Arg1, and HSP90α in the RAW264.7 cells treated 24 h with control medium (CTRL), EndoMT-CM, or EndoMT-CM plus 10 μM of OG. EndoMT-CM repressed mRNA levels of M1-associated TNF-α and iNOS, whereas those of M2-associated CD163, TGF-β, Arg1, and HSP90α were significantly up-regulated. The effects were abrogated by the presence of OG. @ p < 0.01 when compared with “CTRL” group. ^ p < 0.05 and # p < 0.01 when compared with “EndoMT-CM” group. ( C ) mRNA levels of TNF-α, iNOS, CD163, TGF-β, Arg1, and HSP90α in the RAW264.7 cells treated 24 h with PBS (CTRL) or 15 μg/mL of rHSP90α in the absence or presence of 10 μM of OG. rHSP90α treatment inhibited TNF-α and iNOS mRNA expressions but up-regulated CD163, TGF-β, Arg1, and HSP90α mRNA expression levels. The effects were antagonized by OG. @ p < 0.01 when compared with “CTRL” group. ^ p < 0.05 and # p < 0.01 when compared with “rHSP90α” group. ( D ) PLA showing that the physical association of eHSP90α with TLR4 but not with CD91 was prevented by OG on macrophages. Nuclei were stained with DAPI. @ p < 0.05 when compared with “CTRL” group. # p < 0.01 when compared with “rHSP90α + DMSO” group. ( E ) Immunoblot analysis of the eHSP90α levels from the conditioned media of the Panc 02 cells pretreated 24 h with control medium (CTRL), Endo-CM, or EndoMT-CM in the absence or presence of 5 μM of OG. The conditioned media were collected as described in Materials and Methods. The result revealed that EndoMT-CM was more than Endo-CM to enhance HSP90α secretion from Panc 02 cells, and this enhancement was inhibited by the presence of OG. ( F ) Immunoblot analysis of the eHSP90α levels from the conditioned media of the Panc 02 cells pretreated 24 h with 15 μg/mL of rHSP90α in the absence or presence of 5 μM of OG. rHSP90α treatment was able to stimulate HSP90α secretion from Panc 02 cells, which was inhibited by the presence of OG. ( G ) Immunoblot analysis of the phosphorylated and total STAT-3 levels from the Panc 02 cells treated 4 or 6 h with rHSP90α in the absence or presence of OG. Quantification of protein band intensities was performed using ImageJ software, and the relative p-STAT-3 levels were presented after normalization to total STAT-3. rHSP90α-induced STAT-3 phosphorylation (activation) was significantly suppressed by OG.

Article Snippet: Primary antibodies used for immunoblot analyses were listed as follows: Bcl-2 (BD Biosciences, #610539, 1:1000), Bcl-X L (Santa Cruz Biotechnology, sc-8392; 1:1000), Bax (Santa Cruz Biotechnology, sc-7480; 1:1000), prohibitin (NeoMarker, Fremont, CA, USA, MS-261-P; 1:500), BNIP3 (Cell Signaling, Danvers, MA, USA, #44060S; 1:1000), BNIP-3L (Cell Signaling, #12396S; 1:1000), cytochrome c (Santa Cruz Biotechnology, sc-13156; 1:2000), HIF-1α (GeneTex Inc., Hsinchu City, Taiwan, GTX127309; 1:1000), Nrf2 (Santa Cruz Biotechnology, sc-722; 1:1000), p62 (Abcam, Cambridge, UK, ab-56416; 1:4000), p-STAT-3 (Epitomics, Burlingame, CA, USA, #2236; 1:500), STAT-3 (EMD Millipore, #04-1014; 1:1000), and HSP90α (GeneTex Inc., GTX109753; 1:1000).

Techniques: Western Blot, Derivative Assay, Software, Control, Expressing, Staining, Phospho-proteomics, Activation Assay

Journal: Cells

Article Title: Octyl Gallate Induces Pancreatic Ductal Adenocarcinoma Cell Apoptosis and Suppresses Endothelial-Mesenchymal Transition-Promoted M2-Macrophages, HSP90α Secretion, and Tumor Growth

doi: 10.3390/cells9010091

Figure Lengend Snippet: A schematic illustration summarizing our studies on the anti-PDAC mechanisms of OG. In the Panc 02 tumor model, daily oral administration of OG was efficacious to prevent the tumor growth of PDAC cell grafts. The underlying mechanism could be partly at least attributed to the ability of OG to induce a mitochondrial-mediated apoptosis in PDAC cells. OG induced p62 in PDAC cells. p62 could cause HIF-1α stabilization through binding and inhibiting the VHL E3 ligase complex, and the increased HIF-1α could further transcriptionally activate BNIP3L expression. By inducing BNIP3L expression and binding to Bcl-2 and Bcl-X L , OG set the mitochondrial Bax/Bak channels open for cytochrome c release to induce PDAC cell apoptosis. In the Panc 02 + EndoMT tumor model, OG still exhibited its potent anti-cancer efficacy even though OG did not induce tumor apoptosis as well as observed in the Panc 02 tumor model. The involvement of EndoMT-derived CAFs facilitated the recruitment of myeloid-derived macrophages into Panc 02 cell grafts. HSP90α secreted by EndoMT-derived CAFs could further induce macrophage M2-polarization and more HSP90α secretion, which resulted not only in an immunosuppressive and proangiogenic microenvironment but also creates an eHSP90α-rich condition to enhance PDAC tumor growth and malignant progression. In our study, OG blocked the binding of eHSP90α to cell-surface receptor TLR4 and thus prevented eHSP90α-induced M2-macrophages, a feedforward loop of HSP90α secretion in macrophages and PDAC cells, and PDAC tumor growth. Altogether, the anti-PDAC effects exerted by OG should be valued since OG targeted not only PDAC cells but the tumor microenvironment as well.

Article Snippet: Primary antibodies used for immunoblot analyses were listed as follows: Bcl-2 (BD Biosciences, #610539, 1:1000), Bcl-X L (Santa Cruz Biotechnology, sc-8392; 1:1000), Bax (Santa Cruz Biotechnology, sc-7480; 1:1000), prohibitin (NeoMarker, Fremont, CA, USA, MS-261-P; 1:500), BNIP3 (Cell Signaling, Danvers, MA, USA, #44060S; 1:1000), BNIP-3L (Cell Signaling, #12396S; 1:1000), cytochrome c (Santa Cruz Biotechnology, sc-13156; 1:2000), HIF-1α (GeneTex Inc., Hsinchu City, Taiwan, GTX127309; 1:1000), Nrf2 (Santa Cruz Biotechnology, sc-722; 1:1000), p62 (Abcam, Cambridge, UK, ab-56416; 1:4000), p-STAT-3 (Epitomics, Burlingame, CA, USA, #2236; 1:500), STAT-3 (EMD Millipore, #04-1014; 1:1000), and HSP90α (GeneTex Inc., GTX109753; 1:1000).

Techniques: Binding Assay, Expressing, Derivative Assay, Cell Surface Receptor Assay

Journal: Molecular Endocrinology

Article Title: Luteinizing Hormone Receptor mRNA Down-Regulation Is Mediated through ERK-Dependent Induction of RNA Binding Protein

doi: 10.1210/me.2010-0366

Figure Lengend Snippet: Nuclear translocation of ERK½ in response to hCG treatment in human granulosa cells. A, Day 3 granulosa cells were serum-starved, then treated with hCG (10 IU/ml) for 15 min and 30 min. The nuclear and cytoplasmic fractions were separated and subjected to Western blot analysis using ERK 2 antibody (upper panel). The membranes were stripped and reprobed for CREB (middle panel) and HSP90β (lower panel). The blots shown are representative of three independent experiments. B, Immunofluorescence analysis of control (CTL) and hCG-treated (30 min) human granulosa cells incubated with antibody against p-ERK½, then incubated with secondary antibody (Alexa fluor 594-labeled goat anti-mouse IgG) and mounted with a DAPI containing anti-FADE reagent. Using confocal microscopy, the top panels show DAPI (A; blue fluorescence) and p-ERK½ (B; red fluorescence) labeling. Bottom panel (C) shows the merged images of the above two.

Article Snippet: Purified antibodies against ERK2, p-ERK½ (Santa Cruz, CA), CREB (Upstate, Chicago, IL), β tubulin (Sigma, St. Louis, MO), and HSP90β (Assay Designs, Ann Arbor, MI) were commercial products.

Techniques: Translocation Assay, Western Blot, Immunofluorescence, Incubation, Labeling, Confocal Microscopy, Fluorescence

Journal: British Journal of Cancer

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

doi: 10.1038/bjc.2013.710

Figure Lengend Snippet: STCA destabilises the HSP90 client proteins HER2, RAF1, and mutant p53, and stabilises WT p53. ( A ) Domain structure of human HSP90 β . The N-terminal domain (NTD) is the site of binding of ATP and some co-chaperones. The middle domain (MD) is where many of the HSP90 client proteins and co-chaperones interact, and the C-terminal domain (CTD) contains a dimerisation motif. The white bars indicate the distribution and positions of the cysteine residues. ( B – D ) MCF7 cells (2.5 × 10 5 per well) in six-well plates were exposed to vehicle (0.1% acetonitrile) or STCA for 16 h. Cells were then lysed in RIPA buffer, proteins in aliquots from cell lysates were resolved by SDS–PAGE, transferred to immobilon-P, and probed with antibodies against HER2, HSP70 ( B ), RAF1( C ), and glycogen synthase kinase 3 (GSK3) α /GSK3 β ( D ). ( E ) MDA-MB-231 cells (5 × 10 5 per well) in six-well plates were treated with vehicle (0.1% acetonitrile) or STCA for 24 h. The levels of mutant p53 and HSP70 were detected by western blot analyses. ( F ) MCF7 cells (2.5 × 10 5 per well) in six-well plates were treated with vehicle (0.1% acetonitrile) or STCA for 24 h. The levels of WT p53 were detected by western blot analyses. ( G ) MCF7 cells were treated with vehicle (0.1% acetonitrile) or 75 μ M STCA for 2, 4, 8 or 24 h. For all panels, the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were also determined as a loading control. The data represent results from three independent experiments.

Article Snippet: Human HSP90β clone was kindly provided by Dr Anutosh Chakraborty and Dr Solomon H Snyder (Johns Hopkins University).

Techniques: Mutagenesis, Binding Assay, SDS Page, Western Blot, Control

Journal: British Journal of Cancer

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

doi: 10.1038/bjc.2013.710

Figure Lengend Snippet: STCA does not affect the ability of HSP90 to bind ATP. MCF7 cells (5 × 10 5 per dish) in 6-cm dishes were exposed to vehicle (0.1% acetonitrile) or STCA for 24 h. Control treatments were also performed with vehicle (0.1% DMSO), 1 μ M GA and 0.8 μ M celastrol (CL). Cells were lysed and subjected to ATP pulldown using ATP-agarose beads. For the ATP pulldown and input samples, the levels HSP90 and GAPDH were determined by western blot analyses. The data represent results from three independent experiments.

Article Snippet: Human HSP90β clone was kindly provided by Dr Anutosh Chakraborty and Dr Solomon H Snyder (Johns Hopkins University).

Techniques: Control, Western Blot

Journal: British Journal of Cancer

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

doi: 10.1038/bjc.2013.710

Figure Lengend Snippet: STCA modifies cysteine residues of HSP90. Purified recombinant HSP90 β was incubated with STCA for 2 h, dialysed to remove STCA, subject to click reaction with biotin azide, and digested by trypsin. The tryptic digest was incubated with avidin-coated beads. The captured peptides were eluted by 0.5 M acetic acid and analysed by MALDI-mass spectroscopy.

Article Snippet: Human HSP90β clone was kindly provided by Dr Anutosh Chakraborty and Dr Solomon H Snyder (Johns Hopkins University).

Techniques: Purification, Recombinant, Incubation, Avidin-Biotin Assay, Mass Spectrometry

Journal: British Journal of Cancer

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation

doi: 10.1038/bjc.2013.710

Figure Lengend Snippet: S-4 induces transcription of hsp70a1a and hsp70a1b, causes destabilisation of HSP90 client proteins, and inhibits cell proliferation. ( A ) WT MEFs (2.1 × 10 5 per well) in six-well plates were exposed to vehicle (0.1% acetonitrile) or increasing concentrations of each sulphoxythiocarbamate for 8 h. Cells were then lysed and total RNA were extracted. The amount of mRNA for hsp70a1a and hsp70a1b was analysed by quantitative RT–PCR, using β -actin mRNA as an internal control. The mRNA from each sample was measured separately, in triplicate. Data represent means±s.d. and are expressed as ratio of the relative transcription units in treated over control samples. ( B – F ) Cells seeded in six-well plates at a density of 2.5 × 10 5 per well ( B , D and E ) or in 6-cm dishes at a density of 10 6 per dish ( C ) were exposed to vehicle (0.1% acetonitrile) or sulphoxythiocarbamates for either 24 h ( B and F ) or 16 h ( C – E ). Cells were lysed in RIPA buffer, proteins in aliquots from cell lysates were resolved by electrophoresis, transferred to immobilon-P, and probed with the specified antibodies. The data represent results from two independent experiments. ( G ) MCF7 cells (5 × 10 3 per well) in 96-well plates were treated with vehicle (0.1% acetonitrile, ○), 2.5 μ M (♦), 5 μ M (▴), or 10 μ M S-4 (◊) for 24 or 48 h. Cell proliferation was assessed using the Alamar Blue fluorometric assay. * P <0.001 relative to vehicle-treated cells.

Article Snippet: Human HSP90β clone was kindly provided by Dr Anutosh Chakraborty and Dr Solomon H Snyder (Johns Hopkins University).

Techniques: Quantitative RT-PCR, Control, Electrophoresis