Anticancer potential of diarylidenyl piperidone derivatives, HO-4200 and H-4318, in cisplatin resistant primary ovarian cancer

ABSTRACT

We have previously developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group capable of selectively inhibiting STAT3 activation, translocation, and DNA binding activity. HO-4200 and H-4318 are 2 such derivatives capable of inducing apoptosis in ovarian cancer cells through this mechanism and demonstrated efficacy in platinum resistant primary ovarian cancer cell populations and tumor tissues. The improved absorption and cellular uptake of HO-4200 by cancer cells was determined using optical and electron paramagnetic resonance spectrometry. Treatment of ovarian cancer cells with HO-4200 and H-4318 resulted in cleavage of caspase proteins 3, 7, and 9, as well as PARP and inhibition of the pro-survival protein, Bcl-xL, resulting in significantly decreased cell survival and increased apoptosis. HO-4200 and H-4318 significantly inhibit fatty acid synthase (FAS) and pSTAT3 and decreased the expression of STAT3 target proteins: Survivin, c-myc, Bcl-xl, Bcl-2, cyclin D1/D2, and VEGF were suppressed as analyzed using quantitative real time PCR. In addition, HO-4200 and H-4318 significantly inhibited migration/invasion, in primary ovarian cancer cell populations isolated from primary and recurrent ovarian cancer patients. Treatment of freshly collected human ovarian tumor sections with HO-4200 demonstrated significant suppression of pSTAT3 Tyr 705, angiogenesis (VEFG), and markers of proliferation (Ki-67) in ex vivo models. We have shown, for the first time, that the DAP compounds, HO-4200 and H-4318, inhibit cell migration/invasion and induce apoptosis by targeting FAS/STAT3 in human ovarian cancer cells, including primary ovarian cancer cell populations and tumor tissues. Therefore, our results highlight the clinical anti-cancer potential of HO-4200 and H-4318.

Introduction

There are nearly 22,000 new cases of ovarian cancer annually.^1,2 Patients are managed with a combination of surgery and chemotherapy and approximately 70% of patients will have an initial response to treatment. Unfortunately, an equal percentage of patients will have recurrent, and ultimately platinum resistant disease.³ In patients with platinum-resistance, response rates range between 10 and 30 percent and carry a median survival of 6 to 9 months.^4,5 This response decreases with each successive treatment.⁶ Given the poor prognosis of recurrent, platinum-resistant disease, it becomes important to balance quality of life with treatment. For this reason, new therapies that are well tolerated and effective are needed.

We have previously developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to -NOH group capable of selectively inhibiting STAT3 activation, translocation, and DNA binding activity.^7-10 These derivatives are designed to take advantage of molecular pathways involved in the pathophysiology of ovarian cancer and more selectively target cancerous cells over healthy tissues; thus preventing toxic side effects that lead to dose reductions, treatment delays, and even the discontinuation of therapy. Previous work found that DAP compounds exhibit more toxicity toward cancer cells when compared with noncancerous cells.^8,11 This selectivity occurs through the addition of the –NOH moiety, which functions as a modulator of cytotoxicity, imparting antioxidant protection to noncancerous tissues, while allowing the compound to maintain toxicity toward cancer cells. Previously, 2 compounds, HO-3867 (possessing the –NOH moiety) and H-4073 (lacking the –NOH moiety) with the same DAP backbone, have been compared.^8,12

DAP compounds HO-4200 and H-4318 are 2 new derivatives capable of inhibiting ovarian cancer cell proliferation and inducing apoptosis through the targeting of FAS/STAT3 signaling and have demonstrated efficacy in primary ovarian cancer cell populations and tumor tissues. Results of this study will expand the derivatives available as this novel therapeutic moves into the preclinical phase of drug development for the safe, targeted treatment of ovarian cancer.

Results

STAT3 inhibitors HO-4200 and H-4318 induce apoptosis in TR-127 and TR-182

Two new DAP compounds were synthesized by linking 2 diarylidene groups with a piperidone group and substituting the fluorine of base compounds H-4073 and HO-3867 with a triflouromethyl group to create H-4318 and HO-4200, respectively.⁸ Cisplatin resistant ovarian cancer cell lines, TR-127 and TR-182, were seeded in 96-well plates. Twenty four hours after seeding, cells were treated with H-4318 and HO-4200. After 24 and 48 hours of drug exposure, the sulforhodamine B (SRB) chemosensitivity assay was performed and OD at 492 nm was determined. Growth inhibition curves were generated, where each point represents mean ± SEM of at least 3 replicates (Fig. 1A). TR-127 cells were 40% inhibited in the first 24 hours by 1µM HO-4200 and up to 17% when the concentration increased to 10 µM. Growth in TR-182 cells was reduced to 20% in the first 24 hours, even with the lowest concentration of 1 µM. When treated with H-4318, cell growth of TR-127 reduced to 24%, even with the lowest concentration of 1 µM. TR-182 cells were reduced 33 percent. Based on these SRB and apoptotic assays, we decided to move forward with 10 and 20 µM of the doses for both the drugs.

Figure 1.

STAT3 inhibitors - HO-4200 and H-4318 induce apoptosis in TR-127 and TR-182. (A) Cytotoxic activity of potent STAT3 inhibitors, H-4318 and HO-4200, in TR-127 and TR-182 cells was determined by the SRB assay. Cells were exposed to 0 to 20 μmol H-4318 or HO-4200 for 24 or 48 h. Results are normalized against vehicle control and initial plating density. Experiments were done in quadruplicate wells per condition, and each graph is representative of 3 independent experiments. (B) Cytograms obtained from Annexin V assays of H-4318 and HO-4200 (20 µM) -treated TR-127 and TR-182 cells. Each cytogram consists of data showing live cells (PI and FITC negative) bottom left; early apoptotic population (FITC positive) bottom right; mid-late stage apoptosis (PtdIns and FITC positive) top left; necrotic/end stage apoptotic cells (PI positive, FITC negative) top right. 1^st column: control untreated cells; Middle column: H-4318-treated cells; Right column: HO-4200-treated cells. The live cell population is indicated for each sample and is representative of typical data obtained, (p < 0.005; n = 3). (C) Bar graphs (derived from A) showing percent of Annexin V positive TR-127 and TR-182 cells post treatment with 20 µM H-4318 or HO-4200 (D) Bar graphs displaying a comparison of the percent of Annexin V positive cells after treatment with HO-4200 or H-4318 compared to CDDP and the commercially available STAT3 inhibitor, STATIC. Data shown are representative of triplicate experiments and the numbers refer to the mean (and standard deviation) live (PtdIns and FITC negative) populations (expressed as a percentage of the untreated controls). (E) ICC/IF image of 8OHdG stained TR-127 cells. The secondary antibody (green) was anti-rabbit Alexa Fluor® 488. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. The pictures were captured using a fluorescence microscope using 20X magnification. (F) Protein lysates were prepared from TR-127 cells after treatment with 20 µM H-4318 or HO-4200 and protein expression levels were analyzed by Western blotting. Results represent a number of attempts, which showed similar trends. Increased cleaved PARP and cleaved Caspases 3, 7 and 9 were observed post treatment.

The same cell lines were then grown in 10 cm petri dishes and treated with 20 µM of H-4318 and HO-4200 for 24 hours. Apoptosis detection was carried out by Annexin V apoptosis assay which relies upon the fact that upon the initiation of apoptosis, PS loses its asymmetric distribution in the phospholipid bilayer and translocated to the extracellular membrane, which is detectable with fluorescently labeled Annexin V. The harvested cells were labelled with Annexin V and Propidium Iodide as per the manufacturer's manual and analyzed using Aria flow cytometer. Fig. 1B shows the ANNEXIN V results using FACS for the untreated TR-127 (top row) and TR 182 (bottom row). We observed that both H-4318 and HO-4200 caused significant apoptosis (Fig. 1B) and the apoptosis was dose dependent with 40–70% annexin V positive cells in the higher dose of 20 µM and 40–50% apoptosis in the lower dose of 10µM (Fig. 1C). When compared to other commercially available STAT3 inhibitors like STATIC and HO-4200 was at par with STATIC and both HO-4200 and H-4318 were significantly better than CDDP (Fig. 1D). The efficiency of HO-4200 and H-4318 is further evident by the immunocytochemistry experiments (Fig. 1E). 8-hydroxyguanosine (8OHdG) is a sensitive, stable, and integral marker of oxidative damage in cellular DNA, produced by reactive oxygen and nitrogen species, including hydroxyl radical and peroxynitrite. Staining with 8OHdG (green fluorescence) revealed heavy DNA damage post treatment with HO-4200, as well as H-4318, which is absent from the control untreated cells. We used Western Blot to determine the drug mediated expression of caspases–which are the hallmark of apoptosis. Caspase 9 is an initiator caspase which activates the downstream executioner caspases like caspase 3 and 7, which in turn are responsible for the proteolytic cleavage of many key proteins; such as the nuclear enzyme poly(ADP-ribose) polymerase (PARP). Treatment with HO-4200 and H-4318 clearly increased the expression of cleaved PARP, cleaved caspase 9, cleaved caspase 7 and cleaved caspase 3 (Fig. 1F).

Cellular absorption of HO-4200 in cisplatin resistant primary ovarian cancer cells

Chemoresistance is often mediated by the limited bio-absorption or extravascular penetration of synthetic small molecule or natural compound anti-cancer agents.^13,14 Therefore, in vitro and and in vivo bioabsorption and bioavailability are important factors that must be considered when evaluating efficacy. Electron paramagnetic resonance (EPR) was used to detect bio-absorption in cisplatin resistant, primary ovarian cancer cells isolated from patient ascites. We have previously demonstrated the improved uptake of the –NOH moiety containing DAP compound HO-3867, relative to the –NOH moiety lacking H-4073.¹¹ For this reason, we focused our absorption experiments on HO-4200. The N-hydroxypyrroline moiety is capable of undergoing a reversible, one-electron oxidation to its nitroxide form (−NO), which is paramagnetic and detectable by EPR spectroscopy. Our results showed a substantial, time dependent, cellular uptake of the nitroxide form of HO-4200 in cisplatin resistant, primary ovarian cancer cells (Fig. 2), demonstrating that HO-4200 is greatly absorbed within 1 hour treatment, even in drug resistant cells.

Figure 2.

Cellular absorption of HO-4200 on a time dependent manner: We have used electron paramagnetic resonance (EPR) spectrometry to quantify the intracellular content of HO-4200 in various time points in platinum resistant ovarian cancer cell lines. Results showed that HO-4200 rapidly entered the cells during the 1 hour treatment period.

H-4318 and HO-4200 target the migratory and invasive capacity of primary ovarian cancer cell populations

In depth analysis of the migration and invasion of the tumor into the stromal cell layer, along with the underlying mechanisms, is imperative for novel strategies in cancer management. In line with this, we analyzed the migration-invasion capabilities of these cells pre- and post- treatment. Wound healing assays were performed to create a defined scratch across which cells migrate. The cells were allowed to grow for 24 hours in the absence (control) or presence of 20 µM of H-4318 and HO-4200. The gap was pictured and monitored after 24 hours (Fig. 3A and B). We found that for the untreated control well, the gap size was reduced to 4% within the first 24 hours, indicating the very high migration efficiency of these cells. However the presence of HO-4200 or H-4318 prevented the migration of cells and the gap width was still 87% and 83%, respectively, after 24 hours, demonstrating the anti-migratory efficacy of these drugs.

Figure 3.

H-4318 and HO-4200 target the migratory and invasive capacity of the primary ovarian cancer cell populations: (A) TR 127 cells were grown into monolayers in triplicate in 6-well plates up to 70% confluence and treated with 20 µM H-4318 and HO-4200. They were then subjected to in vitro scratch assay with images captured at 0 and 24 h after incubation using phase-contrast microscope. The rate of migration was measured by quantifying the total distance that the cells (as indicated by arrows) moved from the edge of the scratch toward the center of the scratch (marked by imaginary solid lines). (B) The percent of gap size was then plotted to generate the bar graph. (C) Cell invasion was assayed in transwells coated with matrigel. After incubation, cells which had migrated to the bottom side of the chamber inserts were fixed and stained with crystal violet. The number of cells in 5 random microscopic fields (x200) were counted for each group. The results presented are an average of 5 random microscopic fields from 3 independent experiments. Data shown represent the means ± standard errors of the means (SEM) of data from at least 3 independent experiments.

Cell invasion, which is related to, and encompasses, cell migration; is a process where the invasive cells move through the extracellular matrix into neighboring tissues in a process that involves ECM degradation and proteolysis. The high migration rate of these cells prompted us to look for the invasive capabilities of these cells and if HO-4200 and H-4318 would have an inhibitory effect. Boyden Chamber Assays were used to study the invasive capacity of these cells. Cells were seeded on specialized inserts that were then nested in the well of cell culture plates. Cells were allowed to migrate through the pores of the membrane at the bottom of the insert. Migratory cells that moved through the pores toward the chemoattractant below were stained and counted. For the control, untreated cells, a 90 to 100% invasion was observed. Conversely, invasion was 20% and 25% when treated with HO-4200 and H-4318, respectively (Fig. 3C). This demonstrates that both novel compounds are capable of inhibiting cell growth and proliferation, as well as the migratory and invasive abilities of the ovarian cancer cells.

Effect of HO-4200 and H-4318 on expression of STAT3 and associated genes

To evaluate the biochemical pathways governing drug efficacy, western blot analysis of control and treated cells was used. This revealed a decreased FAS and TYK2 for both H-4318 and HO-4200. H-4318 reduced the STAT3, pSTAT3 and c-myc expression (Fig 4A). Collectively, we can infer that our novel drugs target STAT3 and associated genes and cause a remarkable decrease in the same, which in turn is directly linked to blocking the proliferation and spread of the disease.

Figure 4.

Effect of HO-4200 and H-4318 on expression of STAT3 and associated genes: (A) Expression of STAT3 and regulatory proteins in TR 127 cells with/without treatments with 20 µM HO-4200 or H-4318. Cells were collected 24 hours post treatments and 60µg used for protein gel blot analysis as described. (B) Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed to determine gene expression of BCl2, Cyc-D1, ATF-2, c-myc, Cyc-D2, Akt, Survivin, VEGF, STAT3 TR-127 cells that underwent treatment with 20 µM HO-4200 or H-4318. For all 9 genes, relative expression was decreased manifold post-treatment, irrespective of the drug; as compared to the untreated control. The data presented in Y-axis indicates the relative mRNA expression and calculated as means of 3 experiments. In the real time quantitative PCR experiments described here, expression of all the genes of interest was normalized to GAPDH expression levels within the same sample, and separately for each sample, to determine δCq. δδCq method to determine relative gene expression from q PCR data with GAPDH as an endogenous REF gene. The relative mRNA levels for each gene were normalized to GAPDH. ** over the bars in the graph indicates a p value< 0.005, * < 0.05 and NS – non significant.

Further experiments were conducted to confirm the Western Blot results at the level of mRNA using real time quantitative PCR. RNA extracted from the untreated control and treated cells was reverse transcribed and subjected to gene specific real time-quantitative PCR. Overall, both the treatments resulted in a drastic decrease in the expression of STAT3 and other associated genes like BCl2, VEGF, c-myc, Akt, ATF2, Survivin, Cyc-D1, Cyc-D2 (Fig 4B). This further demonstrates the inhibitory properties of our novel agent on STAT3 and the associated genes.

HO-4200 reduces expression of pSTAT3 Tyr705 in ex-vivo human tumor specimens

Fresh human ovarian cancer tissue was collected from consented patients undergoing surgery and the uniform tissue sections were cut using a microtome. The sections were treated with HO-4200 for 72 hours. 10 micron sections were obtained and stained with pSTAT3 705, Ki67 (proliferation marker), and VEGF (angiogenesis marker). Fig. 5A shows the haematoxylin and eosin (H&E) stain for the untreated and treated tissues at 0 and 72 hours. Total disorganization of the tissue architecture and increased necrosis was observed within 72 hours post treatment (Fig. 5A, extreme right and inset). Analysis of expression of pSTAT3 Tyr705, Ki67, and VEGF in human ovarian cancer specimens was examined via immunohistochemical or immunoflourescent analysis. Treatment of human tumor samples with HO-4200 for 72 hours resulted in decreased expression of pSTAT3 Tyr705, Ki67, and VEGF (Fig. 5B & C. Collectively, the data presented in this section demonstrates the efficiency of HO-4200 in inhibiting STAT3, which in turn significantly inhibits the growth of primary tumor.

Figure 5.

Effect of HO-4200 in ex vivo human ovarian tumor tissues. (A) H&E staining of human ovarian cancer tissue showing the tissue morphology at 72 hours in untreated control or after treatment with HO-4200. Note the intact tissue architecture in sections with no treatment and dissociation of tissue after treatment. (B) Decreased pSTAT3, Ki67, and VEGF staining in representative human tissue. (C) Quantification of pSTAT3, ki67 and VEGF (p < 0.05; n = 3).

Discussion

Our current study demonstrated the cytotoxicity of this novel class of antioxidant-conjugated difluorodiarylidenyl piperidones in ovarian cancer cells obtained from recurrent, platinum resistant tissue (TR-127) and primary ovarian cancer ascites (TR-182). Furthermore, we have demonstrated that HO-4200 is well absorbed in platinum resistant primary cell lines. In addition, we have also observed that our compounds induced apoptosis and inhibited migration/invasion, possibly by down-regulating FAS and STAT3 signaling pathways.

The bioabsorption and bioavailability of chemotherapeutic agents are important factors to study in -vitro and -vivo,^15-17 as they carry significant ramifications as anticancer agent move forward in pre-clinical studies. Current research has demonstrated that many small molecule, anti-cancer agents are limited in their efficacy due to poor bioavailability.^18,19 For example, the base compound of curcumin, has effective anticancer potential to various cancer cell lines; however, low absorption into the vascular system and very low serum concentration of curcumin limits its efficacy.^13,20 Thus we have begun creating a novel class of curcumin analogs, which have showed greater bio-absorption in both in vitro and in vivo studies in ovarian and breast cancer.^11,12,21 Our results demonstrate excellent absorption of HO-4200 via EPR and the increased apoptosis when compared to STATIC and CDDP via Annexin V assay.

Drug resistance is a major obstacle in the treatment of recurrent ovarian tumor.^22,23 Specific to the management of ovarian cancer, platinum resistance is the defining prognostic feature at the time of recurrence. In this subset, single agent therapy is often the preferred approach due to the additive toxicity risks and lack of high-quality evidence that combination therapy improves survival. Anthracyclines (particularly when formulated as PLD),²⁴ taxanes,^25,26 topotecan,²⁷ and gemcitabine ²⁸ have all been demonstrated to have response rates between 10–20%. This is in stark contrast to the 70% expected overall response rate in patients with platinum sensitive disease.²⁹ Non- platinum combination therapy with bevacizumab appears to improve response rates, but without significant improvement in overall survival.³⁰ Given the poor response rates to chemotherapy, whether single agent or in combination, new compounds are needed. The present study provides experimental evidence that our novel compounds, HO-4200 and H-4318, are able to inhibit cell survival and induce apoptosis in primary, platinum resistant ovarian cancer cell lines. Another important finding was the ability of both DAP compounds to significantly suppress cell migration and invasion, which are important factors to ovarian tumor progression and metastasis in this highly invasive (metastatic) disease.^31,32

The expression of FAS and constitute activation of STAT3 has been reported in several cancers, most notably ovarian cancer where the higher activation is associated with tumor aggressiveness and poor survival.^9,33,34 FAS and STAT3 have been demonstrated to promote tumor progression, metastasis, and drug resistance through MMP-2, -9, VEGF; regulating anti-apoptotic proteins such as Bcl-2; protecting against drug induced apoptosis in ovarian cancer cells.^35,36 Inhibition of migration/invasion and induction of apoptosis by blocking the activation of FAS and STAT3 signaling pathways has also been demonstrated.^37,38 In the current study, HO-4200 and H-4318 effectively suppress ovarian cancer cell migration and invasion through inhibition of FAS, STAT3, and their target genes. Recent evidence has demonstrated crosstalk of FAS and HER2 expression through PI3k-Akt pathway in ovarian cancer tumors.³³ While HER2 expression was not specifically evaluated in this study, the ability of HO-4200 and H-4318 to suppress Akt and FAS would be expected to also decrease HER2 mediated tumorigenesis.³⁹

In summary, the present study demonstrated the ability of novel DAP compounds, HO-4200 and H-4318, to be highly absorbed in primary, platinum resistant ovarian cancer cell lines, significantly inhibit cell survival and migration/invasion, and induce apoptosis. This is mediated largely by targeting FAS and STAT3 signaling pathways. Overall, our results highlight the therapeutic potential of HO-4200 and H-4318 for the treatment of platinum-resistant ovarian cancer. Further research studies are required in animal models to assess whether DAP mediated FAS/STAT3 inhibition allows for meaningful response rates, is tolerable, and improves survival in ovarian cancer.

Materials and methods

Cell lines and cultures

The TR-127 and TR-182 human epithelial primary ovarian cancer cell lines were used for this study and were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 2% sodium pyruvate, 1% penicillin, and 1% streptomycin. Cells were grown in a 75-mm flask or culture plate to 70% confluence at 37°C in an atmosphere of 5% CO2 and 95% air. Cells were routinely trypsinized (0.05% trypsin/EDTA) and counted using an automated counter (NucleoCounter, New Brunswick Scientific).

Immunocytochemistry

Cells in RPMI medium were seeded onto sterile glass coverslips in 6-well plates with an average population of 50,000 cells/well. After 24 hours of culture, cells were treated with 10 μM HO-4200 for 24 hours. The cells were then washed, fixed, and incubated with primary antibody according to a previously described protocol.⁴⁰

Immunohistochemistry

Human ovarian tumor tissues were embedded in OCT medium (Tissue Tek 4583) and stored at −70° C until sectioning. Consecutive, 5 μm tissue sections were obtained for H&E and immunohistochemical (IHC) staining, following previously-described methods.⁴¹

Flowcytometry

TR-127 and 172 cells were treated with 10 μM HO-4200 and H-4318 for 24 hours. Apoptotic cells were measured by flow cytometry using Annexin V. All sampleswere analyzed on an LSRII flowcytometer (BD Biosciences) and data was evaluated using FlowJo software (Tree Star, Inc., Ashland, OR).

Immunoblot

Cells in RPMI 1640 medium were treated with DMSO (control) or HO-4200 and H-4318 (10 μM or 20 μM) for 24 hours. Equal volumes of DMSO (0.1% v/v) were present as controls in each treatment. Following treatment, the cell lysates were prepared in non-denaturing lysis buffer as previously described.⁷

Migration and invasion assay

Cell migration assays were performed on both treated and non-treated cells using a wound-healing method.³⁸ The cell invasion assay was performed using the Boyden chamber method.³⁸ Experimental wells were treated using 10 µM of HO-4200 and H-4318 added to the culture medium.

Evaluate the bio-absorption of HO-4200 in primary ovarian cancer cells using EPR

Our previous study showed that cellular uptake of HO-3867 was significantly greater than curcumin.²¹ We evaluated the bioabsorption of HO-4200 compounds in drug resistant ovarian cancer cells using EPR, as previously described.^8,21

Ex vivo slice culture

Fresh specimens of human ovarian carcinoma were obtained under an institutional review board approved protocol with the written informed consent of the patients undergoing surgery. Patients were informed about the nature of the experiments related to ex vivo slice cultures and offered the opportunity to participate by consenting to provide residual tissue obtained from surgery for this study including non-tumor tissue. The tissue specimen was rapidly transported to the laboratory on ice, transferred into ice cold RPMI medium. Living ovarian carcinoma slices (300 µM thick) were generated using a vibrating microtome (Vibratome 1500 series, Vibratome, St. Louis, MO) and transferred to tissue culture dishes containing RPMI/ 5x more antibiotic and allowed to equilibrate at 37°C for 2 hrs and allowed to equilibrate 2 hrs.^42-44 Subsequently, slices were transferred to cell culture dishes containing RPMI medium with or without 10µM dosage for HO-4200 for a 72 hr. In our experiments, we utilized 3–5 slices per condition (0.5 cm × 0.5 cm × 300 µm), and over 72 h period which was within the viability period for the slices. At end of treatment period, tumor slices were formalin fixed and paraffin embedded for histological processing and immunohistochemial analysis.

Statistical analysis

All data were expressed as mean ± SE. Comparisons among groups were performed using a Student's t-test. The significance level was set at **< 0.005, * < 0.05 and NS – non significant.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Dr. G. Mor of Yale University for providing the primary ovarian cancer cell populations. The authors also thank graduate students Roman Zingarelli and Maria Riley for the cell culture and basic assay help.

Funding

This work was funded by Ovarian Cancer Research Fund (OCRF), and NCI RO1-CA176078 grant (K.S and D.E.C).