Figure 1.
Genetic variants among the cisplatin-sensitive and -resistant ovarian cancer cells.
(A) Western blotting was performed for the expression variations of prosurvival Bcl-2, Bcl-xL and Mcl-1 and proapoptotic Bak and Bax proteins among the cisplatin-sensitive (A2780s, IGROV1 and OAW42) and -resistant (A2780cp, OVCAR-3 and SKOV3) ovarian cancer cells. β-actin was used as a loading control. (B) A2780s (p53 WT) and SKOV3 (p53 -/-) cells were treated with cisplatin (5 µg/mL) for 24 hours and analyzed for the expression of p53, p73, p21waf1/cip1, NOXA and Bax by Western blotting. β-actin was used as a loading control. (C) OVCAR3 (harboring mutant p53 R248Q) and A2780cp (containing p53 wild-type gene sequence but showing loss of p53 function) cells were treated with cisplatin (5 µg/mL) for 24 hours and analyzed for the expression of p53, p73, p21waf1/cip1, NOXA and Bax by Western blotting. β-actin was used as a loading control.
Figure 2.
Reduced viability of ovarian cancer cells In Vitro by hNOXA and cisplatin.
(A) RT-PCR analysis of hNOXA expression in vitro after transfection of A2780s cells. GAPDH was used as a loading control. (B) Western blotting analysis of hNOXA expression in vitro after transfection of A2780s cells. β-actin was used as a loading control. (C) The treatment of cisplatin at indicated concentrations and periods reduced A2780s cell viability, showing that the dose of IC50 ranged from 5 µg/ml to 10 µg/ml. (D) The treatment of hNOXA plus cisplatin reduced A2780s cell viability more significantly than the treatment of hNOXA alone or cisplatin alone did. Significant differences compared with the control group (24 h, **P<0.001; 48 h, ##P<0.001). (E) The treatment of cisplatin alone had little effect on survival of SKOV3 cells, and the combination of hNOXA plus cisplatin reduced SKOV3 cell viability more significantly than the treatment of hNOXA alone or cisplatin alone did. Significant differences compared with the control group (24 h, **P<0.001; 48 h, ##P<0.001). Percentage of survival was calculated. Results are shown as means ± SD of three wells and triplicate experiments. In each experiment, the medium-only treatment (untreated) indicates 100% cell viability.
Figure 3.
Induction of apoptosis of tumor cells in vitro by hNOXA and cisplatin.
(A) Representative DNA fluorescence histograms of PI-stained cells. A2780s cells were treated with hNOXA for 24 h, then with 5 µg/ml cisplatin for an additional 24 h. A2780s cells were treated with pcDNA3.1 or hNOXA, cisplatin alone or hNOXA plus cisplatin and groups Ctrl, pc3.1, hNOXA, Cis and hNOXA+Cis correspond to these five treatments (the same as shown in the subsequent panels), with 8.7% (Ctrl), 15.6% (pc3.1), 34.6% (hNOXA), 48.3% (Cis) and 63.6% (hNOXA+Cis) sub-G1 cells (apoptotic cells), respectively, as assessed by flow cytometry. (B) SKOV3 cells were treated with hNOXA for 24 h, then with 5 µg/ml cisplatin for an additional 24 h. SKOV3 cells were untreated, treated with empty vector or hNOXA, cisplatin alone or hNOXA plus cisplatin, with 4.9% (Ctrl), 6.4% (pc3.1), 38.4% (hNOXA), 9.1% (Cis) and 52.3% (hNOXA+Cis) sub-G1 cells (apoptotic cells), respectively, as assessed by flow cytometry. (C) Normal and apoptotic nuclear morphology of A2780s cells was analyzed by Hoechst 33258 staining. A2780s cells were treated with the same conditions as described above. (D) Normal and apoptotic nuclear morphology of SKOV3 cells was analyzed by Hoechst 33258 staining. SKOV3 cells were treated with the same conditions as mentioned above.
Figure 4.
The sensitizing effects of hNOXA are mediated by enhanced caspase activation and release of Cyt C and Smac into the cytosol.
(A) A2780s and SKOV3 cells were subjected to the indicated treatments as described in Materials and Methods. Caspase activation were analyzed by Western blotting. Arrows indicate active forms of caspases. β-actin was used as a loading control. (B) A2780s and SKOV3 cells were subjected to the indicated treatments as described in Materials and Methods. Release of Cyt C and Smac into the cytosol were analyzed by Western blotting. Cox-IV blots indicate mitochondrial loading controls while β-actin was used as a loading control for the cytosolic fraction.
Figure 5.
Alterations in the Bax/Smac Axis affect sensitivity of Ovarian Cancer Cells to Cisplatin.
(A) siRNAs targeting Bax significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells (*P<0.01; **P<0.001). (B) siRNAs targeting Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells (#P<0.05; *P<0.01; **P<0.001). (C) Down-regulation of Bax or Smac by Bax siRNA or Smac siRNA were confirmed by Western blot. (D) Overexpression of Bax was confirmed by Western blot. (E) SKOV3 cells were co-transfected with NOXA and pc3.1-Bax plasmids for 12 hours, followed by 5 µg/mL cisplatin treatment for additional 12 hours. Overexpression of Bax significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells (*P<0.01). (F) SKOV3 cells were treated with NOXA and/or cisplatin as described above, then with 20 µmol/L Smac-N7 peptide for an additional 3 hours. Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells (#P<0.05; *P<0.01).
Figure 6.
Enhanced antitumor efficacy of the combination of hNOXA and Cisplatin In Vivo (A–D)
Tumor suppression and survival advantage in mice. A2780s cells (A, C) or SKOV3 cells (B, D) of 2×106 were inoculated subcutaneously into female nude mice at 6–8 weeks of age. Mice (five per group) were treated with PBS, pcDNA3.1, pcDNA3.1-hNOXA, Cisplatin and pcDNA3.1-hNOXA+cisplatin. In A2780s tumor model, significant differences in tumor suppression (**P<0.001) and survival time (ΔP<0.05) in mice treated with hNOXA or cisplatin versus PBS and pcDNA3.1 controls; significant difference for tumors treated with hNOXA+cisplatin versus PBS and pcDNA3.1 controls (**P<0.001; ΔΔP<0.01), and significant difference for the combination therapy versus hNOXA or cisplatin monotherapy (#P<0.05; †P<0.05). Similar results were also found in SKOV3 model, except that no significant differences in tumor suppression (P = 0.222) and survival time (P = 0.433) between cisplatin- and pcDNA3.1-treated tumors were found. (E–F) TUNEL staining of tumor tissues. Representative sections were taken from A2780s (E) and SKOV3 (F) tumor tissues of mice receiving PBS, pcDNA3.1, hNOXA, cisplatin and hNOXA+cisplatin. (G) Apoptotic index within A2780s and SKOV3 tumor tissues were counted. In A2780s model, statistically significant difference in the apoptotic index for tumors treated with hNOXA or cisplatin versus PBS and pcDNA3.1 controls (**P<0.001); significant difference for tumors treated with hNOXA+cisplatin versus the two controls (**P<0.001); and significant difference for the combination therapy versus hNOXA or cisplatin monotherapy (##P<0.001). Similar results were also found in SKOV3 model, except that no significant differences in the apoptotic index between cisplatin-treated tumor and pcDNA3.1-treated tumor (P = 0.981) or PBS-treated tumor (P = 0.705) were found. The apoptotic index was calculated as a ratio of the apoptotic cell number to the total cell number in each field. (H) RT-PCR analysis of expression of exogenous hNOXA in vivo.