Journal: Molecular Oncology

Article Title: HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer

doi: 10.1002/1878-0261.12883

Figure Lengend Snippet: HSP90 pharmacological inhibition or HSP90α knockout increases the pro‐apoptotic and DNA damage effect of CDDP in Pt‐res TOV‐112D cells. (A) Apoptosis and necrosis evaluated by flow cytometry after Annexin V‐FITC and propidium iodide staining in TOV‐112D Pt‐res cl. 7 and (B) cl. 2 cells, untreated or treated for 48 h (upper panels) or 72 h (lower panels), with CDDP and/or ganetespib at IC 50 72 h doses of parental cells. (C) Western blot analysis of γH2AX, PARP1 and caspase 3 cleavage, BAX and BCL2 expression, in TOV‐112D Pt‐res pool 2 cells untreated or treated with CDDP and/or ganetespib at the doses indicated above. β‐Actin expression serves as loading control. Western blot quantification was performed by imagej software. (D) Apoptosis and necrosis evaluated by flow cytometry after Annexin V‐FITC and propidium iodide staining in TOV‐112D, TOV‐112D Pt‐res cl. 7 and in HSP90α knockout TOV‐112D Pt‐res KO#2 cells, untreated or treated for 48 or 72 h with CDDP at IC 50 72 h doses of parental cells.

Article Snippet: Primary antibodies were purchased as follows: HSP90 alpha 2G5.G3 (#SMC‐147) and HSP90 (total) 4F3.E8 (#SMC‐149) from StressMarq Biosciences (Victoria, BC, Canada); GRP75 (#3593), LMN A/C (#2032), ANXA1 (#3299), Bax (#2774), CASPASE 3 (#9662), cleaved PARP1 (Asp214; #5625), PARP1 (#9542), Bcl‐2 (#4223S) from Cell Signaling Technology (Leiden, Netherlands); VIM (ab 16700), prohibitin (PHB; ab55618), CALR (ab2907), heat‐shock cognate 71‐kDa protein (HSP7C; ab19136), PGK1 (ab67335), PRDX4 (ab15574) from Abcam (Cambridge, UK); GRP78 C‐20 (sc‐1051), EZR H‐276 (sc‐20773), β‐actin C4 (sc‐47778), and GAPDH (FL‐335) from Santa Cruz Biotechnology Inc., (Dallas, TX, USA); VINC (10C‐CR1199M1) from Fitzgerald (Acton, MA, USA); ɣH2AX (Ser139) clone JBW301 (#05‐636) from Millipore (Burlington, MA, USA); and heterogeneous nuclear ribonucleoprotein L (HNRPL; A303‐895A) from Bethyl (Montgomery, TX, USA).

Techniques: Inhibition, Knock-Out, Flow Cytometry, Staining, Western Blot, Expressing, Software

Journal: Molecular Oncology

Article Title: HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer

doi: 10.1002/1878-0261.12883

Figure Lengend Snippet: Potentiation of CDDP antitumor effect induced by ganetespib in vivo CDDP‐resistant TOV‐112D xenograft model. TOV‐112D Pt‐res pool 2 cells (5 × 10 6 ) were s.c. injected into NSG mice as described in . When tumors were established, mice (five/group) were treated once a week for two weeks, with CDDP (2.5 mg·kg −1 i.p.), ganetespib (GANE; 75 mg·kg −1 i.p.), both drugs in combination, or their respective vehicles (UNT). (A) Relative TV measured at prespecified time points (Means ± SEM). (B) TVs at cutoff when mice were sacrificed. Data are shown as means ± SEM. (C) Mice body weight as surrogate indicator of toxicity for in vivo experiment reported in A. Body weight was measured three times/week. (D) TGD, indicating the mean rate of tumor growth in the treatment groups relative to control untreated mice (see ). Statistically significant results calculated with one‐way ANOVA test are reported (* P < 0.05, ** P < 0.01, and *** P < 0.001). (E) Western blot analysis of γH2AX, cleaved PARP1, cleaved caspase 3, HSP90α, and total HSP90, in lysates from three representative xenograft tumor samples from each treatment group (see ). β‐Actin expression serves as loading control. Western blot quantification was performed by imagej software using the mean value of the three samples for each experimental group.

Article Snippet: Primary antibodies were purchased as follows: HSP90 alpha 2G5.G3 (#SMC‐147) and HSP90 (total) 4F3.E8 (#SMC‐149) from StressMarq Biosciences (Victoria, BC, Canada); GRP75 (#3593), LMN A/C (#2032), ANXA1 (#3299), Bax (#2774), CASPASE 3 (#9662), cleaved PARP1 (Asp214; #5625), PARP1 (#9542), Bcl‐2 (#4223S) from Cell Signaling Technology (Leiden, Netherlands); VIM (ab 16700), prohibitin (PHB; ab55618), CALR (ab2907), heat‐shock cognate 71‐kDa protein (HSP7C; ab19136), PGK1 (ab67335), PRDX4 (ab15574) from Abcam (Cambridge, UK); GRP78 C‐20 (sc‐1051), EZR H‐276 (sc‐20773), β‐actin C4 (sc‐47778), and GAPDH (FL‐335) from Santa Cruz Biotechnology Inc., (Dallas, TX, USA); VINC (10C‐CR1199M1) from Fitzgerald (Acton, MA, USA); ɣH2AX (Ser139) clone JBW301 (#05‐636) from Millipore (Burlington, MA, USA); and heterogeneous nuclear ribonucleoprotein L (HNRPL; A303‐895A) from Bethyl (Montgomery, TX, USA).

Techniques: In Vivo, Injection, Western Blot, Expressing, Software

Journal:

Article Title: Direct activation of the apoptosis machinery as a mechanism to target cancer cells

doi: 10.1073/pnas.1031631100

Figure Lengend Snippet: Compounds activate caspases in cell lysates. (A) Chemical structures of the apoptosis activators. (B) Compound activation is cyto c-dependent. cyto c was titrated into S-100 cytoplasmic extracts with vehicle alone or 20 μM compound, and procaspase-3 processing was assayed by capture ELISA. (C) Compounds or vehicle were titrated into S-100 cytoplasmic extracts at a cyto c concentration of 1.25 μM, and procaspase-3 processing was assayed by capture ELISA. (D) Immnunoblot of compound-induced caspase activation. Samples at the 20 μM compound concentration in B were probed with antibodies to caspase-9, the large subunit of active caspase-3, and phosphatidylinositol 3-kinase as a loading control.

Article Snippet: Bovine heart cyto c was purchased from Sigma, and procaspase-3 was purchased from Biomol (Plymouth Meeting, PA).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal:

Article Title: Direct activation of the apoptosis machinery as a mechanism to target cancer cells

doi: 10.1073/pnas.1031631100

Figure Lengend Snippet: Compounds promote the oligomerization of Apaf-1. (A) cyto c was titrated into a reconstituted system containing 31 nM procaspase-3, 4 μM procaspase-9, 160 nM Apaf-1, and 300 μM dATP, with or without 20 μM compounds. Procaspase-3 processing was assayed by capture ELISA. (B) Compounds were titrated into reactions containing procaspase-3, procaspase-9, Apaf-1, dATP, and 0.15 μM cyto c, and procaspase-3 processing was assayed by capture ELISA. (C) Apaf-1 was incubated with dATP, with or without cyto c or 20 μM compound as indicated, and then separated by gel filtration. Individual fractions were assayed for relative Apaf-1 concentration by capture ELISA (bar graph) or the ability to activate procaspase-3 processing in lysates (line graph). The percent of Apaf-1 in the apoptosome was determined by dividing the amount of Apaf-1 in fractions 8–12 by the total amount of Apaf-1. The extent of caspase-3 activation (given in arbitrary units) corresponds to the area under the curve for fractions 8–12 in each panel. Black bars represent fractions used for calculations.

Article Snippet: Bovine heart cyto c was purchased from Sigma, and procaspase-3 was purchased from Biomol (Plymouth Meeting, PA).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Filtration, Concentration Assay, Activation Assay

Journal:

Article Title: Direct activation of the apoptosis machinery as a mechanism to target cancer cells

doi: 10.1073/pnas.1031631100

Figure Lengend Snippet: Compounds induce apoptosis in whole cells. Jurkat cells were incubated with vehicle, 1 μM staurosporin, or 50 μM compound for 6 h and then lysed. Samples were then probed by immunoblot for cleavage of procaspase-3 (A) or PARP (B). (C) Jurkat cells were incubated with vehicle, 1 μM staurosporin, or 50 μM compound for 8 h and then lysed. The DNA was isolated and visualized by ethidium bromide staining. The last lane (M) is a 250-bp DNA molecular weight marker. (D) Jurkat cells were incubated with different concentrations of compound or vehicle for 22 h and assayed for viability by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test (Sigma) according to the manufacturer's protocol.

Article Snippet: Bovine heart cyto c was purchased from Sigma, and procaspase-3 was purchased from Biomol (Plymouth Meeting, PA).

Techniques: Incubation, Western Blot, Isolation, Staining, Molecular Weight, Marker