Prohibitin silencing reverses stabilization of mitochondrial integrity and chemoresistance in ovarian cancer cells by increasing their sensitivity to apoptosis

Abstract

Current approaches to the treatment of ovarian cancer are limited because of the development of resistance to chemotherapy. Prohibitin (Phb1) is a possible candidate protein that contributes to development of drug resistance, which could be targeted in neoplastic cells. Phb1 is a highly conserved protein that is associated with a block in the G0/G1 phase of the cell cycle and also with cell survival. Our study was designed to determine the role of Phb1 in regulating cellular growth and apoptosis in ovarian cancer cells. Our results showed that Phb1 content is differentially overexpressed in papillary serous ovarian carcinoma and endometrioid ovarian adenocarcinoma when compared to normal ovarian epithelium and was inversely related to Ki67 expression. Immunofluorescence microscopy and Western analyses revealed that Phb1 is primarily associated with the mitochondria in ovarian cancer cells. Over-expression of Phb1 by adenoviral Phb1 infection resulted in an increase in the percentage of ovarian cancer cells accumulating at G0/G1 phase of the cell cycle. Treatment of ovarian cancer cells with staurosporine (STS) induced apoptosis in a time-dependent manner. Phb1 over-expression induced cellular resistance to STS via the intrinsic apoptotic pathway. In contrast, silencing of Phb1 expression by adenoviral small interfering RNA (siRNA) sensitized ovarian cancer cells to STS-induce apoptosis. Taken together, these results suggest that Phb1 induces block at G0/G1 phase of the cell cycle and promotes survival of cancer cells. Furthermore, silencing of the Phb1 gene expression may prove to be a valuable therapeutic approach for chemoresistant ovarian cancer by increasing sensitivity of cancer cells to apoptosis.

Figure 1

Immunolocalization and Western blot analyses of prohibitin in normal and ovarian cancer tissues. (a) Immunolocalization of prohibitin (red), DAPI (blue) and Ki67 (green) in normal ovarian epithelium, endometroid, and serous ovarian tumors. Ovarian tissues were fixed in 10% formalin, embedded in paraffin and sectioned for immunohistochemical analyses as described in Material and methods section. Immunocolocalization of endogenous prohibitin was detected with Alexa fluor 594 (red) labeled antibody and the proliferative marker Ki67 was detected with Alexa fluor 488 (green) labeled secondary antibody. Nuclear DNA was counterstained with 4′,6′-dia-midino 2-phenylindole, DAPI (blue). Images d′, e′ and f ′ were higher magnification, demonstrating staining specificity. Bar = 100 μm (a, b and c) and 50 μm (d, e and f). (b) Western blot analyses of protein levels for prohibitin and XIAP in normal ovarian epithelium (lane 1), endometroid (lane 2) and serous (lane 3) ovarian tumors. Fifty micrograms of protein from normal and cancerous tissue samples were applied to each lane and analyzed for prohibitin and XIAP protein expression levels by Western blot analyses. Cyclophilin A served as an internal control for sample loading.

Figure 2

Localization of endogenous prohibitin in OVCAR3 and OVCAR8 cells. (a) Immunolocalization of endogenous prohibitin detected with Alexa fluor 594 (red). The DNA was counterstained with 4′,6′-diamidino 2-phenylindole, DAPI (blue). Note that the majority of prohibitin immunostaining is isomorphic with the mitochondrial reticular network. (b and c) Location of prohibitin in subcellular fractions of OVCAR3 and OVCAR 8 cells. Fifty micograms of protein from the cytosol (cyto), mitochondria (mito) and nuclei (nuc) of OVCAR3 and OVCAR8 cells were applied to each lane and subjected to Western blot analysis as described in Material and methods section. All blots were probed with porin, tubulin and lamin A/c antibodies.

Figure 3

Induction of apoptosis in OVCAR8 cells by STS. (a) OVCAR8 cells that were untreated (control) or treated with 1 μm STS for 3 hr were monitored using phase-contrast microscopy to identify morphological changes associated with apoptosis (cell rounding and shrinkage). (b) OVCAR8 cells were treated with 1 μm of STS for 0.5, 1.0, 2.0 and 3.0 hr and protein extracts (50 μg) were subjected to Western blot analysis as described in Material and methods section. Blots were probed with procaspase 3 antibody. (c) Caspase-3 activity in cytosolic protein extracts from OVCAR8 cells untreated or treated with STS was measured using the spectrophotometric substrate DEVD-pNA. Graphically, activities are represented as a percentage of the control group, and all numerical values are represented as mean ± SEM of three individual experiments (n = 3). An asterisk indicates a significant difference between the control and treated groups (p < 0.05).

Figure 4

Effects of recombinant adenovirus directed overexpression of prohibitin on procaspase-3 cleavage in OVCAR8 cells. (a) OVCAR8 cell lysates were obtained after 24 hr infection with GFP-prohibitin adenovirus at 10, 20, 40 MOI to assess for adenoviral efficacy. Equal amounts of protein (15 μg) from OVCAR8 cells infected with Ad-PHB1-eGFP were applied to each lane and analyzed for over-expressed prohibitin. Tubulin alpha was used as an internal control. (b) OVCAR8 cells were infected with sense Ad-PHB1-eGFP (MOI = 40) or Ad-eGFP vector control (MOI = 40) for 2 hr and maintained in culture for 24 hr. Thereafter, OVCAR8 cells were treated with STS (1 μm) for indicated time periods (0.5, 1, 2 and 3 hr) followed by Western blot analysis. Equal amounts of protein (15 μg) from STS treated OVCAR8 were applied to each lane, and the blots were analyzed for prohibitin (30-kDa) and cleaved caspase-3 protein levels. Tubulin was measured as the internal control. A representative of three individual experiments (n = 3) were performed for a and b. + = infection with Ad-PHB1-eGFP; − = infection with Ad-eGFP vector only.

Figure 5

Effects of recombinant adenovirus directed overexpression of prohibitin on OVCAR8 cell growth. OVCAR8 cells were infected with sense Ad-PHB1-eGFP (MOI = 40), Ad-eGFP vector control (MOI = 40) or uninfected for 2 hr and maintained in culture for 24, 48 and 72 hr. At each time point a MTT colorimetric assay was performed.

Figure 6

Effects of silencing the prohibitin gene in OVCAR8 cells. (a) OVCAR8 cells were infected with Ad-siRNA prohibitin (MOI = 80) or Ad-eGFP (MOI = 80) for 2 hr and maintained in culture for 48 hr. Expression of prohibitin protein level was assayed using Western-blotting techniques. (b) Effect of Ad-siRNA-PHB1 on the morphology of the mitochondria. Two days after infection with vector only (a′) or PHB1 siRNA (b′), OVCAR8 cells were fix and immunostained with prohibitin antibody. Note arrow depicting long tubular mitochondrial network (a′) and arrowhead showing punctiform mitochondria (b′). (c) Western blot analysis was conducted to detect levels of prohibitin and cleaved caspase-3, 0.5, 1, 2 and 3 hr posttreatment with 1 μM STS. Tubulin was used as an internal control. Prohibitin knockdown enhances apoptosis in OVCAR8 cells. Data are representative of three individual experiments. Bar = 20 mm.