Suberoylanilide hydroxamic acid (SAHA) potentiates paclitaxel-induced apoptosis in ovarian cancer cell lines.

OBJECTIVES: To determine if SAHA, a histone deacetylase inhibitor, decreases ovarian cancer cell viability when combined with paclitaxel in vitro, and to explore molecular alterations of combined paclitaxel+SAHA treatment. METHODS: SKOV3 and Hey ovarian cancer cell lines were treated for 24 h with paclitaxel, then re-treated with SAHA or paclitaxel for an additional 48 h. Protein extracts were prepared at 48 h for western blot analysis. Cell viability was assessed at 72 h using the ApoAlert Annexin V Apoptosis Kit. RESULTS: SAHA causes G1 and G2 cell cycle arrest in ovarian cancer cell lines. Cell viability was significantly reduced by combined paclitaxel+SAHA treatment. In Hey cells, viability was reduced to 67% with paclitaxel, and to 48% with paclitaxel+SAHA (p<0.001). In the SKOV3 cell line, viability was reduced to 70% with continuous paclitaxel treatment, and was further reduced to 57% in the combined treatment group (p<0.05). Increased PARP cleavage was noted in the paclitaxel+SAHA groups. SAHA increased expression of p21cip1/waf1 and p27Kip1, down regulated cyclins A and B, and suppressed CDK1. Paclitaxel induced expression of survivin, an inhibitor of apoptosis protein, was reduced to baseline control levels with the addition of SAHA. The pro-apoptotic protein, Bad, was also increased with SAHA. CONCLUSIONS: Paclitaxel+SAHA reduces cell viability in excess of either agent alone in ovarian cancer cell lines. Cell death is mediated via several mechanisms including G1/G2 arrest from CDK1 downregulation, inhibition of paclitaxel-induced survivin accumulation, and from increased Bad expression.

Paclitaxel induces the phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 through a Cdk1-dependent mechanism.

Initial chemotherapeutic treatment triggers a stress-related response, which can lead to an increase in the expression of survival proteins. In this study we examine whether paclitaxel (PTX) alters the expression and/or phosphorylation of the translation initiation proteins, eukaryotic initiation factor 4E (eIF-4E) and 4E-binding protein (4E-BP1), a suppressor of eIF-4E in the dephosphorylated state. We found that PTX induced the hyperphosphorylation of 4E-BP1 in the breast cancer cell line, MDA MB 231, which reduced its association with eIF-4E, but did not alter the expression and phosphorylation of eIF-4E. The hyperphosphorylation of 4E-BP1 correlated with G2/M accumulation and with an increase in the phosphorylation of cdk1 substrates. Cotreatment with a histone deacetylase inhibitor (an indirect inhibitor of cdk activity), purvalanol A and roscovitine (direct cdk inhibitors), and the reduction of cyclin B expression using RNA interference decreased the hyperphosphorylation of 4E-BP1 in PTX treated cells. The hyperphosphorylation of 4E-BP1 by PTX increased the association of eIF-4E with eIF-4G, whereas cotreatment with purvalanol A inhibited the association of eIF-4E with eIF-4G in PTX treated cells. Taken together, our data suggest that PTX-increases the functional level of eIF-4E by promoting the hyperphosphorylation and release of 4E-BP1 through a cdk1-dependent mechanism.

Histone deacetylase inhibitors enhance paclitaxel-induced cell death in ovarian cancer cell lines independent of p53 status.

BACKGROUND: Recurrence of drug-resistant disease contributes to the high mortality of ovarian cancer patients, which necessitates the identification of additional chemotherapeutic drugs. Histone deacetylase inhibitors (HDAIs) induce apoptosis in a number of malignant cell types and may represent a new class of drugs clinically relevant in the treatment of ovarian cancer. MATERIALS AND METHODS: Ovarian cancer cells were treated with various combinations of a HDAI and paclitaxel (PTX). Cell death was measured using annexin V/propidium iodide exclusion. RESULTS: The PTX/HDAI drug combination was as efficient in inducing cell death as continuous PTX treatment and superior to continuous HDAI treatment. Reversing the sequence of drug exposure reduced the cytotoxic efficacy of the drug combination. The p53 status of the cell lines did not alter the cytotoxic efficacy of the treatment protocols. CONCLUSION: These results suggest that HDAIs possess possible clinical applications as an adjuvant therapy in the treatment of ovarian cancer.

In vitro and in vivo histone deacetylase inhibitor therapy with suberoylanilide hydroxamic acid (SAHA) and paclitaxel in ovarian cancer.

OBJECTIVE: To determine effects of suberoylanilide hydroxamic acid (SAHA) with and without paclitaxel in ovarian cancer cells and a nude mouse model. METHODS: Cell viability and apoptosis of ovarian cancer cells (2774) were measured following exposure to control, SAHA, paclitaxel, or SAHA in combination with paclitaxel. Nude mice were injected intraperitoneally (IP) with cancer cells and then groups received variable SAHA doses (25-100 mg/kg/day). In a second experiment, mice were inoculated with cancer and treated IP with vehicle injection, SAHA, paclitaxel, paclitaxel followed by SAHA, or SAHA followed by paclitaxel. Survival, tumor weight, and ascites were evaluated. RESULTS: SAHA decreased viability and increased apoptosis similarly to paclitaxel, but the combination was not statistically significantly different from the single agents. The only significant difference in the SAHA alone mouse study was decreased survival in the 50 mg/kg/daily group. In the combination groups, SAHA followed by paclitaxel, paclitaxel alone, and paclitaxel followed by SAHA improved survival compared with control (p=0.0358, 0.0006, and 0.0001), but SAHA alone did not (p=0.524). The paclitaxel followed by SAHA group had improved survival compared to SAHA followed by paclitaxel (p=0.0002) but not compared to paclitaxel alone (p=0.166). CONCLUSIONS: In vitro, SAHA alone decreased viability and increased apoptosis similarly to paclitaxel. In vivo, paclitaxel followed by SAHA and paclitaxel alone increased survival compared with SAHA alone or SAHA followed by paclitaxel. This suggests adding SAHA to ovarian cancer chemotherapy could increase efficacy and that sequencing of agents is important.