Priming with HDAC Inhibitors Sensitizes Ovarian Cancer Cells to Treatment with Cisplatin and HSP90 Inhibitors

Abstract

Ovarian cancer is the fifth leading cause of cancer deaths. Chemoresistance, particularly against platinum compounds, contributes to a poor prognosis. Histone deacetylase inhibitors (HDACi) and heat shock protein 90 inhibitors (HSP90i) are known to modulate pathways involved in chemoresistance. This study investigated the effects of HDACi (panobinostat, LMK235) and HSP90i (luminespib, HSP990) on the potency of cisplatin in ovarian cancer cell lines (A2780, CaOV3, OVCAR3 and cisplatin-resistant sub-clones). Preincubation with HDACi increased the cytotoxic potency of HSP90i, whereas preincubation with HSP90i had no effect. Preincubation with HSP90i or HDACi 48h prior to cisplatin enhanced the cisplatin potency significantly in all cell lines via apoptosis induction and affected the expression of apoptosis-relevant genes and proteins. For CaOV3CisR and A2780CisR, a preincubation with HDACi for 48-72 h led to complete reversal of cisplatin resistance. Furthermore, permanent presence of HDACi in sub-cytotoxic concentrations prevented the development of cisplatin resistance in A2780. However, triple combinations of HDACi, HSP90i and cisplatin were not superior to dual combinations. Overall, priming with HDACi sensitizes ovarian cancer cells to treatment with HSP90i or cisplatin and has an influence on the development of cisplatin resistance, both of which may contribute to an improved ovarian cancer treatment.

Keywords: HDAC inhibitors; HSP90 inhibitors; HSP990; LMK235; chemoresistance; cisplatin; epigenetics; luminespib; ovarian cancer; panobinostat.

Figure 1

Effects of HSP90i and HDACi on cell growth and protein expression in A2780 and A2780CisR cells. (A) A 72 h incubation with the HSP90i luminespib and HSP990 showed a slight decrease in cell proliferation for A2780CisR cells. In A2780, this effect was only achieved with HSP990. (B) A 72 h incubation with the HDACi panobinostat or LMK235 slightly reduced the cellular growth of A2780 and A2780CisR cells. (C) A 48 h incubation with luminespib with IC₅₀, three-fold IC₅₀ and five-fold IC₅₀ or time-dependent incubation with three-fold IC₅₀ of luminespib led to a decrease in the expression of the HSP90 client protein AKT and its phosphorylation (pAKT). The uncropped and labeled immunoblots are shown in Figure S1. (D) A 24 h incubation with panobinostat or LMK235 increased the acetylation of α-tubulin in both cell lines. Data shown are mean ± SEM of three independent experiments or one representative immunoblot out of three. Statistical analysis was performed using t-test. Levels of significance: * p ≤ 0.05, *** p ≤ 0.001.

Figure 2

Preincubation with HDACi increase cytotoxic potency of HSP90i. A 48 h preincubation (preinc) with panobinostat or LMK235 with the indicated concentrations decreased IC₅₀ values of HSP990 (A,B) and luminespib (C,D) in A2780 (A,C) and A2780CisR cells (B,D). IC₅₀ values, pIC₅₀ and SEM are shown in Table S1. Data shown are mean ± SEM of three independent experiments each carried out in triplicate. Statistical analysis was performed using t-test. Levels of significance: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Effects of HDACi or HSP90i incubation on apoptosis-related genes. Gene expression data were obtained by PCR. Cells were treated with 10 nM (A2780) or 20 nM (A2780CisR) panobinostat or 5 nM (A2780) or 10 nM (A2780CisR) luminespib for 24 or 48 h.

Figure 4

Caspase 3/7 activation by single treatment and combinations of panobinostat, luminespib, and cisplatin in A2780CisR cells. A2780CisR cells were preincubated with panobinostat and/or luminespib (lumi) for 48h. Cisplatin (23.4 µM, corresponding to the IC₅₀) or buffer control were added for a further incubation period of 24 h. Caspase 3/7 activation was analyzed by ArrayScan XTI. Cisplatin 50 µM (24 h) served as positive control for caspase 3/7 activation, 0.2% DMSO as vehicle control. Data are the mean ± SD. Statistical analysis to compare the caspase 3/7 activation of the two indicated treatments was performed using t-test. Levels of significance: ns p > 0.05, ** p ≤ 0.01.

Figure 5

Effects of HDACi or HSP90i preincubation prior to cisplatin on apoptosis-related genes. Gene expression data were obtained by PCR. (A,B) A2780 and A2780CisR cells were treated with the indicated inhibitors or their combinations. For combination treatments panobinostat and/or luminespib were administrated 48 h prior to 24 h cisplatin treatment in an IC₅₀. Cell line specific untreated controls were obtained by 72 h incubation with cell culture medium. The concentrations used (A,B) were the same as for cytotoxicity combination studies (Figure S6 and Table 2). One representative agarose gel is shown. Densitometric evaluation can be found in Figure S10B,C.

Figure 6

Effects of HDACi and HSP90i incubation or preincubation prior to cisplatin on protein expression levels of pro-/antiapoptotic proteins. Representative immunoblot analysis of APAF-1, p21, survivin and α-tubulin (as loading control) in A2780 and A2780CisR cells is shown. For combination treatments, panobinostat (pano) and/or luminespib (lumi) were administrated 48 h prior to 24 h cisplatin treatment with respective IC₅₀ concentrations. Cell line specific control was treated with vehicle for 72 h. Densitometric analysis of the Western blots are shown in Figure S11.

Figure 7

48 h preincubation with luminespib, HSP990, panobinostat or LMK235 prior to 72 h cisplatin treatment did not affect cisplatin sensitivity of the non-cancerous cell line HEK293. Data shown are mean ± SEM of three independent experiments. Results (IC₅₀, pIC₅₀, SEM, and shift factors) are summarized in Table S6.

Figure 8

Effects of prolonged (72 h instead of 48 h) preincubation with HDACi / HSP90i on cisplatin sensitivity in A2780CisR ovarian cancer cells. 72 h preincubation with panobinostat or combination of 72 h of panobinostat with 24 h of luminespib or HSP990 prior to 48 h cisplatin treatment increased cisplatin sensitivity in A2780CisR (A). pIC₅₀ ± SEM values of cisplatin in A2780CisR using 72 h preincubation with panobinostat compared to A2780 (B). Results (IC₅₀, pIC₅₀, SEM, and shift factor) are summarized in Table S5. Statistical analysis was performed using t-test. Levels of significance: ns p > 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 9

Effects of HDACi or HSP90i on short-term (A) or long-term (B) cisplatin stress-induced changes of cisplatin sensitivity in A2780 ovarian cancer cells. Average resistance factors of cisplatin after single cisplatin treatment cycle (“cisplatin stress”) (A) or after 16–21 cisplatin treatment cycles (averaged data over Treatment Cycles 16–21) in A2780 cells. Cisplatin treatment cycle means treatment for 6 h with an IC₅₀ of cisplatin alone (“control”) or in permanent presence of 3 nM luminespib, 5 nM panobinostat or 200 nM LMK235. Resistance factors were calculated by dividing the IC₅₀ of cisplatin obtained after a treatment cycle (“stressed” cells) by the IC₅₀ of cisplatin from unstressed A2780 cells. Statistical analysis was performed using t-test. Levels of significance: * p ≤ 0.05, *** p ≤ 0.001.