Gynecologic Oncology 116 (2010) 126–130
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Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
Suberoylanilide hydroxamic acid (SAHA) potentiates paclitaxel-induced apoptosis in ovarian cancer cell lines☆,☆☆ Charles S. Dietrich III a,⁎, Victoria L. Greenberg b, Christopher P. DeSimone b, Susan C. Modesitt c, John R. van Nagell b, Rolf Craven b, Stephen G. Zimmer b a
Gynecologic Oncology Service, Department of Obstetrics and Gynecology, Tripler Army Medical Center, 1 Jarrett White Road, Honolulu, HI 96859-5000, USA Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Kentucky Markey Cancer Center, 800 Rose Street, 333 Whitney-Hendrickson Building, Lexington, KY 40536-0298, USA c Division of Gynecologic Oncology, University of Virginia Health System, P.O. Box 800712, Charlottesville, VA 22908, USA b
a r t i c l e
i n f o
Article history: Received 17 July 2009 Available online 28 October 2009 Keywords: Ovarian cancer Histone deacetylase inhibitor SAHA Paclitaxel Survivin Bad
a b s t r a c t 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 b 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 b 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. Published by Elsevier Inc.
Introduction Ovarian cancer is the second most common gynecologic malignancy in the United States with 21,650 new cases estimated in 2008 [1]. Due to the lack of an effective screening algorithm and a paucity of definitive presenting symptoms, most patients are diagnosed in advanced stages. Standard therapy involves extensive surgical debulking followed by combination chemotherapy, usually with paclitaxel and a platinum-based agent [2,3]. While initial response to therapy approaches 80%, recurrences are common. Unfortunately, the 5-year survival rate for advanced stage disease approaches 30% [1]. Recurrent disease quickly develops chemo-resistance and
☆ Presented as a poster at the Society of Gynecologic Oncologists Annual Meeting, March 2006, Palm Springs, CA. ☆☆ The views expressed in this manuscript are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the United States Government. ⁎ Corresponding author. Fax: +1 808 433 1552. E-mail address:
[email protected] (C.S. Dietrich). 0090-8258/$ – see front matter. Published by Elsevier Inc. doi:10.1016/j.ygyno.2009.09.039
responds poorly to salvage regimens. Novel modalities are needed to improve treatment responses. Histones, the core proteins of the nucleosome packaging DNA, play a key role in the regulation of gene transcription and expression [4]. Competing acetyltransferases and deacetylases tightly control modification of histones. Deacetylation maintains chromatin in a compacted state, limiting accessibility of DNA targets to transcription factors critical for cell regulation [5]. Histone deacetylase inhibitors (HDACI) are gaining attention as anti-neoplastic agents as they induce cell differentiation, cell cycle arrest, and apoptosis [6–12]. Suberoylanilide hydroxamic acid (SAHA, vorinostat) is a potent inhibitor of histone deacetylase (HDAC) 1, 2, 3, and 6 [13]. In phase I/II trials, SAHA has shown promising responses with limited toxicities and has recently been FDA approved for treatment of recurrent cutaneous T-cell lymphoma [14–16]. Pilot studies with ovarian cancer cell lines demonstrate an encouraging decrease in cell viability with the combination of paclitaxel and an HDACI (sodium butyrate and trichostatin-A) [17–19]. The purpose of this study was to determine if SAHA decreases cell viability and increases apoptosis when combined with
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paclitaxel in vitro and to further explore molecular alterations of combined paclitaxel and SAHA treatment. Materials and methods
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FITC and propidium iodide for 15 min in the dark at room temperature. Cell samples were analyzed by two-color flow cytometry using a FACScan flow cytometer (Becton Dickinson). Viable cell percentages were calculated from the proportion of cells that were annexin V and propidium iodide negative.
Cell culture SKOV3 and Hey ovarian cancer cells were cultured in complete RPMI medium 1640 (Gibco, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 100 Units/mL of Penicillin G and 100 mcg/mL of Streptomycin sulfate (Gibco). Incubation occurred at 37°C in 5% CO2 and 100% humidity. Confluent flasks were harvested with trypsin, and cells were then plated at a density of 5.0x105 cells per 12 mL of RPMI or MEM in 100 mm tissue culture dishes.
Statistical analysis One way analysis of variance (ANOVA) was used to compare means between treatment groups. Paired Student's t-test was used to evaluate statistically significant differences between groups. A p value b 0.05 was considered significant. Results
Treatment protocol
SAHA elevates paclitaxel toxicity in ovarian cancer cell lines
At the time of initial cell plating, one half of the tissue culture dishes were treated with paclitaxel (Sigma) [SKOV3: 25 nM, Hey: 50 nM], and the control half with vehicle, dimethyl sulfoxide (DMSO) (FisherScientific, Fair Lawn, NJ). Twenty-four hours after plating, the medium was removed and replaced. Each plate was then retreated with DMSO, paclitaxel or SAHA (Biovision, Mountain View, CA). SKOV3 cells were also treated with Purvalanol A [10 μM] (Calbiochem, San Diego, CA). Analysis was conducted on duplicate samples, and each experiment was performed three times.
Single agent SAHA was evaluated in SKOV3 and Hey cell lines and a dose response curve was demonstrated. The lowest dose of SAHA reducing the S-phase fraction to less than 10% was selected for subsequent experiments (SKOV3: 10 μM, Hey: 2.5 μM). In the Hey cell line (Fig. 1A), cells treated with paclitaxel or SAHA alone demonstrated 73% and 67% viability, respectively. Paclitaxel followed by SAHA, however, showed further reduction in viability to 48% (p b 0.001). In the SKOV3 cell line (Fig. 1B) single agent paclitaxel and SAHA caused similar reductions in cell viability, 78% and 77% respectively. Combined treatment showed only 57% viability (p b 0.001) which even exceeded the reduced viability seen with continuous 72-h paclitaxel treatment (70%, p b 0.05).
Cell cycle analysis Representative treatment groups were harvested at 24 and 48 h. The culture medium and trypsinized monolayer were combined and centrifuged at 5000 rpm for 10 min. Cell pellets were washed in 5 mL of cold phosphate-buffered saline (PBS), centrifuged again for 10 min, and then re-suspended in 1 mL of PBS. Following re-suspension, cells were fixed in a 70% ethanol solution for N15 min. A PBS solution was made with 1/50 volume of 0.5 mg/mL propidium iodide (Sigma) in 3.8 × 10− 2 M sodium citrate and 1/40 volume of 10 mg/mL RNase A (Sigma) in 10 mM Tris–HCl pH 7.5/15 mM sodium chloride. Ethanol fixed samples were centrifuged, washed in PBS, and then re-suspended in 250–500 μL of the propidium iodide solution. The samples were incubated for 30 min at 37°C and DNA content was analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Western blot analysis Protein extracts were prepared at 48 h. Two plates from each treatment group were collected as described above, pooled and lysed in SDS-PAGE sample buffer. Extracts were then analyzed by western blot. Antibodies specific to the following proteins were obtained from Santa Cruz Biotechnology, Santa Cruz, CA: Bad (SC-8044), PARP (SC-7150), and Survivin (SC-10811). The following protein-specific antibodies were obtained from BD Transduction Laboratories, Los Angeles, CA: Cdk1/cdc2 (c12720), Cyclin B (610220), p21cip1/waf1 (c24420), p27kip1 (k25020). Protein extracts from induced HL60 cells (BioMol, Plymouth Meeting, PA) were utilized as a PARP control. Analysis of apoptosis Seventy-two hours after initial treatment, representative samples were harvested. Both floating and adherent cells were collected and centrifuged. Apoptosis was measured using the ApoAlert Annexin V Apoptosis Kit (CLONTECH Laboratories, Palo Alto, CA) according to manufacturer instructions. After washing the cell pellets in binding buffer and re-suspending them, they were incubated with annexin V-
Fig. 1. Cell viability is decreased with combined paclitaxel + SAHA. (A) Hey ovarian cancer cell line. (B) SKOV3 ovarian cancer cell line. V/V - control; V/S - SAHA alone; V/P - paclitaxel (24 h) alone; S/P - Paclitaxel + SAHA; P/P - continuous paclitaxel.
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Cell cycle analysis in Hey cells revealed that paclitaxel caused a sharp G2 cell cycle arrest after 24 h (Fig. 2B) when compared to untreated cells (Fig. 2A). After 48 h the cells progressed through the cell cycle and showed little evidence of arrest (Fig. 2D). In contrast, SAHA treated cells arrested sharply in G1 and G2 (Fig. 2E). Cells treated with combined paclitaxel and SAHA displayed a broad increase in cells with sub-G1 DNA content, increased G2 arrested cells, and a third peak of cells with N2N DNA content (Fig. 2F). Apoptosis was verified by western blotting for PARP cleavage in the Hey cell line. As expected, increased levels of cleaved PARP (85kDa) following sequential treatment with paclitaxel and SAHA were noted (Fig. 3A, lane 4) when compared with either SAHA or paclitaxel alone (Fig. 3A, lanes 2 and 3, respectively). In addition, increased expression of survivin, a caspase inhibitor, was noted in paclitaxel
treated cells (Fig. 3B, lane 3) but was not induced by SAHA (Fig. 3B, lane 2). When cells were treated with combined paclitaxel and SAHA, survivin levels were reduced to baseline control levels (Fig. 3B, lane 4). In contrast to survivin, the pro-apoptotic protein Bad was induced by both SAHA and paclitaxel (Fig. 3C, lanes 2 and 3). Additive levels were induced when the two drugs were combined (Fig. 3C, lane 4).
SAHA alters the expression of CDK proteins and their inhibitors SAHA reduced CDK1 expression when used as a single agent and largely reversed CDK1 expression after paclitaxel treatment (Fig. 4A). Similarly, SAHA reduced the expression of cyclin B when used alone and reversed cyclin B induction by paclitaxel (Fig. 4B). Furthermore,
Fig. 2. Cell cycle analysis (Hey ovarian cancer cell line). (A) Control at 24 h. (B) Paclitaxel at 24 h. (C) Control at 48 h. (D) Paclitaxel at 48 h. (E) SAHA at 24 h. (F) Paclitaxel + SAHA at 48 hrs.
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Fig. 3. Apoptosis is increased with paclitaxel and SAHA. (A) Increased PARP cleavage. (B) Suppression of paclitaxel-induced survivin expression. (C) Increased expression of Bad.
SAHA inhibited cyclin A levels when used alone or in combination with paclitaxel (Fig. 4C). In contrast, SAHA induced CDK-cyclin inhibitor proteins. SAHA increased the expression of p21cip1/waf1 as a single agent (Fig. 4D, lane 2) as did paclitaxel (Fig. 4D, lane 3), and the effect of both drugs was approximately additive when used in combination (Fig. 4D, lane 4). Similarly, p27Kip1 was induced by both SAHA (Fig. 4E, lane 2) and paclitaxel (Fig. 4E, lane 3), but the levels of p27Kip1 were not additive when the drugs were combined (Fig. 4E, lane 4). This suggests the pathways that induce p21cip1/waf1 and p27Kip1 are distinct, and they differ markedly from the regulation of CDK1, cyclin A and cyclin B. Direct inhibition of cyclin B in the SKOV-3 cell line was assessed by treating them with purvalanol A (PA) in combination with paclitaxel. Viability was reduced from 90% in control groups to 57% in the paclitaxel/PA groups (p b 0.0001). Discussion Ovarian cancer is the most lethal gynecologic malignancy in industrialized nations. While excellent responses to initial surgical
Fig. 4. SAHA alters expression of CDK proteins and their inhibitors. (A) CDK1. (B) Cyclin B. (C) Cyclin A. (D) p21. (E) p27.
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debulking and primary cytotoxic chemotherapy are often obtained, recurrent disease usually develops and becomes refractory to multiple chemotherapeutic options. Future successes will depend on novel treatment modalities that target various intracellular processes. Histone deacetylase inhibitors offer an exciting approach in the treatment of ovarian cancer because of their unique mechanisms of action and lower toxicities [6,9]. SAHA is an organic hydroxamic acid related in structure to trichostatin A [20]. While it is a known inhibitor of HDAC 1, 2, 3 and 6, the underlying mechanisms for its anti-neoplastic effects are poorly understood [13]. In vitro antitumor activity has been reported in a number of hematogenous and solid malignancies including leukemia, cutaneous T-cell lymphoma, transitional cell carcinoma, breast cancer, prostate cancer, colon cancer and ovarian cancer [18]. In a clinical phase I study by Kelly et al., daily intravenous SAHA was well tolerated. Dose limiting toxicities were mainly hematologic with only 4% of solid tumor patients experiencing Grade 3 thrombocytopenia and 8% showing Grade 3 anemia [14]. SAHA also has excellent oral bioavailability and an acceptable safety profile when administered chronically [15]. A Phase II clinical trial involving patients with advanced or recurrent cutaneous T-cell lymphoma (CTCL) showed an overall response rate of 29.7% with a median time to tumor progression of 202 days [16]. This pivotal study led the FDA to approve vorinostat (SAHA) for the treatment of advanced CTCL in October 2006 [16]. Unfortunately, a Gynecologic Oncology Group sponsored phase II trial did not show significant activity when SAHA was used as a single agent in patients with recurrent ovarian cancer [21]. It is unclear at this time why hematologic malignancies seem more susceptible to SAHA when compared to solid tumors. In the current study, we have demonstrated that the combination of paclitaxel followed by SAHA reduces cell viability in excess of either agent alone in ovarian cancer cell lines, and even exceeds the cell kill obtained by continuous paclitaxel treatment. Our findings confirm the synergistic effects of dual therapy noted in several other preclinical trials studying gynecologic malignancies. Chobanian et al. first demonstrated reduced survival in ovarian cancer cell lines when paclitaxel was followed by a HDACI (sodium butyrate and trichostatin A). Reversing the sequence of drug exposure lessened the cytotoxic efficacy of the drug combination [17]. Similar findings have also been reported for papillary serous endometrial cancer cells [22]. Cooper et al., employing an ovarian cancer mouse model, showed increased survival with sequential paclitaxel + SAHA [18]. Finally, Sonnemann et al. found SAHA activity in paclitaxel-resistant ovarian cancer cell lines [23]. Understanding the biochemical pathways responsible for HDACI induced apoptosis is an area of intense investigation [20,24,25]. Our results suggest that SAHA functions to sustain the cell cycle arrest that occurs in the first 24 h of paclitaxel treatment, as evidenced by the elevated proportion of G2 phase cells in the paclitaxel + SAHA group. Furthermore, the cells that escape G2 arrest and continue into the cell cycle appear to undergo apoptosis or an abnormal second round of DNA synthesis without cell division. Cell cycle arrest may result from increased expression of p21cip1/waf1 and p27Kip1 that was noted in our study as well as other reports [20]. These proteins block cdkactivating kinase access to cdk, preventing phosphorylation. We found that cyclins A and B were also down regulated with SAHA, further limiting cdk phosphorylation. To confirm cyclin importance in SAHA-mediated apoptosis, we repeated our experiments, substituting purvalanol A, a direct inhibitor of cyclin B, for SAHA. Similar reductions in cell viability in purvalanol A-treated lines were found. Our results also suggest a role for survivin in paclitaxel/SAHAinduced apoptosis. Survivin, an inhibitor of apoptosis (IAP) protein, acts as an endogenous inhibitor of caspases. Its expression is cell-cycle dependent, peaking during mitosis. Over-expression of survivin has been shown to interfere with cell death initiated by both intrinsic and extrinsic apoptotic pathways [26]. Not surprisingly, survivin is
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differentially expressed in cancer versus normal tissues, and overexpression has been documented in ovarian malignant tumors [27]. Furthermore, paclitaxel induces persistent activation of cyclin B-CDK1 and can lead to up regulation of survivin [28,29]. Survivin, therefore, acts as an antagonist of paclitaxel-induced apoptosis [30], and increased expression of survivin has been correlated with taxane resistance in ovarian cancer [31]. Our study, as in other research [26], demonstrates that survivin levels are increased in paclitaxel-treated cells. When SAHA is added however, survivin expression is reduced to baseline control levels. Other investigations have looked at using CDK1/cdc2 antagonists to block survivin phosphorylation as an anticancer modality [26,32–34]. Vorinostat resistance has been studied in colon cancer cell lines. One study revealed expression of a multidrug resistance protein that inhibited acetylation of histones, caused loss of the G2 checkpoint, and blocked caspase dependent apoptosis [35]. This potentially could explain the disappointing results when SAHA was used alone in the GOG trial, as most patients enrolled had paclitaxel resistance. Further studies will need to be explored in the ovarian cancer model to help identify patients most likely to benefit from HDAC inhibitors and to determine if SAHA effects are transcriptional, translational or posttranslational. In conclusion, we have found that SAHA potentiates paclitaxelinduced cytotoxicity in ovarian cancer cell lines and have contributed to the growing body of evidence establishing HDACIs as viable antineoplastic agents in the treatment of gynecologic malignancies. SAHA sustains the cell cycle arrest induced by paclitaxel, increasing the apoptosis potential. Possible mechanisms of action include alteration of CDK proteins and their inhibitors as well as down regulation of survivin. While SAHA alone has not shown significant responses in phase II trials in ovarian cancer patients, our study suggests that combined therapy may be more efficacious. Conflict of interest statement Dr. Modesitt received an investigational grant from Merck after this project was completed to further study SAHA. The remaining authors declare no conflicts of interest.
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