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. Author manuscript; available in PMC: 2014 Dec 17.
Published in final edited form as: J Exp Ther Oncol. 2014;10(4):275–283.

Anticancer activity of pristimerin in ovarian carcinoma cells is mediated through the inhibition of prosurvival Akt/NF-κB/mTOR Signaling

Xiaohua Gao 1, Yongbo Liu 1, Dorrah Deeb 1, Ali S Arbab 2, Subhash C Gautam 1
PMCID: PMC4268656  NIHMSID: NIHMS646887  PMID: 25509983

Abstract

Pristimerin is a quinonemethide triterpenoid that has shown anticancer activity against some cancer types. However, the antitumor effects of pristimerin (PM) in ovarian cancer cells have not been adequately studied. The objective of the present study was to determine the anticancer activity and its mechanism of action in human ovarian carcinoma cell lines. PM strongly inhibited the proliferation of ovarian cancer cells by inducing apoptosis characterized by increased annexin V-binding, cleavage of poly (ADP-ribose) polymerase (PARP-1) and procaspases-3, -8 and -9. Furthermore, PM caused mitochondrial depolarization. Western blot analysis showed inhibition of prosurvival phospho-AKT (p-AKT), nuclear factor kappa B (NF-κB) (p65) and phospho-mammalian target of rapamycin (p-mTOR) signaling proteins in cells treated with PM. Treatment with PM also inhibited the expression of NF-κB-regulated antiapoptotic Bcl-2, Bcl-xL, c-IAP1 and survivin. Thus, our data showing potent antiproliferative and apoptosis-inducing activity of PM in ovarian carcinoma cells through the inhibition of AKT/NF-κB/mTOR signaling pathway warrant further investigation of PM for the management of ovarian cancer.

Keywords: Ovarian cancer, pristimerin, apoptosis, Akt, NF-κB, mTOR

INTRODUCTION

Carcinoma of the ovary is the fifth most common cause of cancer related death in women in the United States. It is also the most common gynecologic malignancy. It is estimated that 22,240 new cases of ovarian cancer will be diagnosed and 14,030 women will die from the disease in the United States in 2013 (1). While surgical resection of tumors confined to the ovary can result in 5-year survival rate of approximately 90%, unfortunately most patients have widespread disease at the time of diagnosis (2). Carboplatin/paclitaxel is the current standard of care for first line treatment of ovarian cancer; however development of drug resistance limits the effectiveness of these chemotherapeutic agents, underscoring the urgent need for developing new strategies and novel agents for prevention and treatment of ovarian cancer (3,4)

Novel agents that promote apoptosis in ovarian cancer cells could compliment chemotherapy and lead to tumor regression and improved prognosis. Medicinal plants and herbal products derived from them are used in traditional medicine to treat a variety of human diseases including cancer. Isolation and identification of bioactive components in medicinal plants have led to the synthesis and development of several potent anticancer compounds, such as Vinca alkaloids, taxol, camtothecan, etoposide and retinoids. Triterpenoids are members of a larger family of structurally related compounds known as cyclosqualenoids that are widely distributed in nature. Pristimerin is a quinonemethide triterpenoid present in various plant species in the Celastraceae and Hippocrateaceae families (5,6). Pristimerin has been used for long time in traditional medicine as an anti-inflammatory, antioxidant and antimalarial agent (7–9). Recent studies have shown potent antiproliferative and apoptosis-inducing activity of pristimerin in diverse types of tumor cell lines, including glioma, leukemia, breast, lung, prostate and pancreatic cancer cell lines (10–13). Apoptosis induction by pristimerin involves activation of caspases, mitochondrial dysfunction, inhibition of antiapoptotic nuclear factor kappa B (NF-κB) and Akt signaling pathways (14–16). Pristimerin activates c-Jun N-terminal kinase (JNK) and poly (ADP-ribose) polymerase-1 (PARP-1) through generation of reactive oxygen species (17). Pristimerin is also capable of inhibiting cell cycle progression, proteosome, tumor cell migration and angiogenesis (11,18–20). We found only one study in which a fraction of Reissantia buchananii extract containing methyl ester derivative of celastrol (pristimerin) showed significant cytotoxicity towards an ovarian cancer cell line (6). Since this report, there has been no other published report on the anticancer activity or the mechanism of action of pristimerin in ovarian cancer cells. In the present study, we investigated the anticancer activity of pristimerin in vitro using four human ovarian cancer cell lines. The results demonstrate that pristimerin inhibits the growth and induces apoptosis in ovarian cancer cells through the inhibition of prosurvival Akt/NF-κB/mTOR signaling, indicating the potential of pristimerin in the treatment and/or prevention of ovarian cancer.

MATERIALS AND METHODS

Reagents and antibodies

Pristimerin (PM) was purchased from Sigma Chemicals (Saint Louis, MO). Anti-caspase-3, caspase-8, and caspase-9 antibodies were purchased from BD Pharmingen (San Diego, CA). Anti-p-Akt (ser473) and anti-p-mTOR (ser2448) antibodies were from Cell Signaling Technology (Danvers, MA). Anti-NF-κB (p65), anti-PARP-1, anti-Bcl-2, anti-Bcl-xL, c-IAP1 and anti-survivin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). 96 AQueous One Solution Proliferation Assay System was from Promega (Madison, WI). Annexin V-FITC apoptosis detection kit was purchased from BD Pharmingen (San Diego, CA) and mitochondrial potential sensor JC-1 was obtained from Molecular Probes, Invitrogen (San Diego, CA).

Cell lines

Human ovarian cancer cell lines OVCAR-5, MDAH-2774, OVCAR-3 and SK-OV-3 were obtained from the American Type Tissue Collection (Rockville, MD). Cells were maintained in tissue culture using fully supplemented cell line specific tissue culture medium.

Measurement of cell viability (MTS assay)

Tumor cells (1×104) were seeded into each well of a 96-well plate in 100 μl of tissue culture medium. After 24 h incubation, cells were treated with PM at concentrations of 0.625 to 10 μM for 48–72 h. Cell viability was then determined by the colorimetric MTS assay using CellTiter 96 AQueous One Solution Proliferation Assay System.

Annexin V-FITC binding

Tumor cells treated with PM for 20 h were suspended in the binding buffer provided in the annexin V-FITC apoptosis detection kit and reacted with 5 μl of annexin V-FITC reagent plus 5 μl of propidium iodide (PI) for 30 min at room temperature in the dark. Stained cells were analyzed by flow cytometry.

Mitochondrial depolarization assay

Change in mitochondrial potential by PM was determined by flow cytometry. Briefly, after treating with PM for 20 h, cells were loaded with mitochondrial potential sensor JC-1 (10 μg/ml) for 10 minutes at 22°C cells and analyzed by flow cytometry. In normal cells, dye is aggregated in mitochondria, fluoresces red, and detected in the FL2 channel. In cells with altered mitochondrial potential, the dye fails to accumulate in the mitochondria, remains as monomers in the cytoplasm, fluoresces green, and is detected in the FL1 channel.

Western blotting

Total cellular proteins were obtained by detergent lysis. Samples (50 μg) were boiled in an equal volume of sample buffer (20% glycerol, 4% SDS, 0.2% Bromophenol Blue, 125 mM Tris-HCl (pH 7.5), and 640 mM 2-mercaptoethanol) and separated on pre-casted Tris-glycine polyacrylamide gels (6–10%) using the XCell Surelock™ Mini-Cell, in Tris-Glycine SDS running buffer, all from Novex (Invitrogen, Carlsbad, CA). Proteins resolved on the gels were transferred to nitrocellulose membranes, which were then probed with protein specific antibody or anti-β-actin antibody as loading control. Immune complexes were visualized by chemiluminescence. Protein band densities were analyzed using the NIH/Scion image analysis software and normalized to the corresponding β-actin band densities.

Statistical analysis

Most data are presented as means ± S.D. and outcomes for treated and untreated cells were compared by Student’s t-test. Differences were considered significant at P<0.05.

RESULTS

Pristimerin inhibits proliferation of ovarian cancer cells

Effect of PM on the proliferation of OVCAR-5, MDAH-2774, SK-OV-3 and OVCAR-3 ovarian cancer cells was measured by MTS assay. As shown in Figure 1, PM at concentrations of 1.25 to 10 μM inhibited the growth of all ovarian cancer cell lines in a dose dependent manner. OVCAR-5 and MDAH-2774 cells were more sensitive to PM than SK-OV-3 and OVCAR-3 cells since significant inhibition was observed in both cell lines at 1.25 μM PM (44% and 28%, respectively) compared to untreated cells (P<0.05). In SK-OV-3 and OVCAR-3 cells significant reduction in proliferation was observed at 2.5 μM PM (36% and 27%, respectively). However, inhibition of proliferation in all cell lines ranged from 80% to 90% at 5 to 10 μM PM (P<0.01). These data indicated strong antiprolifertive effect of PM on ovarian cancer cells at concentrations of 2.5 to 10 μM.

Figure 1. PM inhibits proliferation of ovarian cancer cells.

Figure 1

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Ovarian cancer cells (OVCAR-5, MDAH 2774, SK-OV and OVCAR-3) were treated with PM at concentrations ranging from 0 to 10 μM for 72 h in triplicate in 96-well plates. Cell viability was measured by MTS assay using CellTiter AQueous Assay System. Similar results were obtained in three independent experiments. *P<0.01 compared to control cells at 5 and 10 μM PM.

Pristimerin induces apoptosis in ovarian cancer cells

Whether PM induces apoptosis in ovarian cancer cells was measured by the binding of annexin V-FITC to cancer cells by flow cytometry. For this OVCAR-5 and MDAH 2774 cells were treated with PM at 0.625 to 5 μM for 20 h. As shown in Figure 2A, no binding of annexin V-FITC was detected in untreated OVCAR-5 and MDAH 2774 cells. On the other hand, treatment with PM increased the percentage of annexin V-FITC binding OVCAR-5 cells from 4 to 77% and MADH 2774 cells from 9 to 73% at concentrations of 0.625 to 5 μM.

Figure 2. Treatment with PM induces apoptosis in ovarian cancer cells.

Figure 2

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OVCAR-5 and MDAH 2774 cells were treated with PM at 0 to 5 μM for 20 h. Cells were then reacted with 5 μl of annexin V-FITC and 5 μl PI for 30 min and percentage of annexin V-FITC binding cells was determined by flow cytometry (A). Cleavage of PARP-1 in cells treated with PM (0 to 5 μM) for 20 h was analyzed by western blotting (B). Each experiment was repeated at least three times.

To confirm induction of apoptosis by PM we also measured the cleavage of PARP-1 in cells treated with PM. Thus OVCAR-5 and MDAH-2774 cells were treated with PM at 0 to 5 μM for 20 h and PARP-1 was analyzed by western blotting. As shown in Figure 2B, PM induced the cleavage of native PARP-1 as identified by the appearance of the cleavage product (89 kDa) in both cell lines at 1.25 to 5 μM. Taken together, increase in binding of annexin V-FITC and cleavage of PARP-1 in cells treated with PM showed induction of apoptosis in ovarian cancer cells by PM.

PM activates procaspases in ovarian cancer cells

Since apoptotic cell death is mediated through the activation of cysteine caspases that digest and destroy numerous cellular substrates, we measured the processing/activation of procaspases 3,-8, and -9 by PM. OVCAR-5 and MDAH 2774 cells were treated with PM at 0.625 to 5 μM for 20 h and processing of these pro-caspases was analyzed by western blotting. As shown in Figure 3, PM caused significant to complete processing of procaspases 3, -8, and -9 at 2.5 to 5 μM in both cell lines. These data demonstrated that PM induces activation of both initiator (caspases-8 and -9) and effector (caspase-3) caspases.

Figure 3. Treatment with PM cleaves procaspases-3, -8 and -9.

Figure 3

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OVCAR-5 and MDAH 2774 cells were treated with PM at concentrations of 0 to 5 μM for 20 h. Cellular lysates prepared from untreated (control) and PM treated cells were analyzed for processing of procaspase-3, -8 and -9 by western blotting. The processing of procaspases was identified by decrease in levels of native proteins. Each experiment was repeated at least two times.

PM induces mitochondrial depolarization

Whether mitochondria are involved in induction of apoptosis by PM was investigated next. For this, we measured the change in mitochondrial membrane potential in OVCAR-5 and MDAH 2774 cells treated with PM at 0 to 5 μM for 20 h. After treatment with PM, cells were loaded with JC-1 probe and fluorescent shift was measured by flow cytometry. As shown in Figure 4, the percentage of cells emitting green fluorescence significantly increased after treatment with PM at 2.5 and 5 μM compared to control cells (OVCAR-5: 14%, 41%, and 87%; MDAH-2774: 9%, 53%, and 78% at 0, 2.5 and 5 μM PM, respectively, p<0.01). These data showed that depolarization of mitochondria plays a role in induction of apoptosis in ovarian cancer cells by PM.

Figure 4. PM induces mitochondrial depolarization.

Figure 4

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OVCAR-5 and MDAH 2774 cells were treated with PM at 0 to 5 μM for 20 h. Cells were loaded with mitochondrial potential sensor JC-1 (10 μg/ml) for 10 minutes at 22°C and analyzed by flow cytometry for cells fluorescing red (FL2 channel) or green (FL1 channel) (A). Bar graphs show the percentage of cells with loss of mitochondrial potential difference (B). Similar results were obtained in two separate experiments. *P<0.05 compared to no PM controls.

PM inhibits antiapoptotic p-Akt, p-mTOR and NF-κB signaling proteins in ovarian cancer cells

Akt/NF-κB/mTOR signaling constitutes a major antiapoptotic mechanism that confers survival advantage in cancer cells. We analyzed the effect of PM on these signaling proteins in OVCAR-5 and MDAH 2774 cells treated with PM by western blotting. As shown in Figure 5A, PM markedly to completely reduced the levels of p-Akt, p-mTOR and NF-κB in both cell lines at concentrations of 1.25 to 5 μM without significantly altering the levels of β-actin. In addition, PKC-ε, which has been invoked in induction of apoptosis was also reduced in both cell lines treated with PM at 2.5 and 5 μM (Fig. 5B).

Figure 5. PM inhibits prosurvival p-Akt, NF-κB and p-mTOR.

Figure 5

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A. OVCAR-5 and MDAH 2774 cells were treated with PM at 0 to 5 μM for 20 h. After treatment, cell lysates and nuclear lysates were prepared and analyzed by western blotting for p-Akt and p-mTOR (cell lysates), NF-κB (nuclear lysates) or β-actin (loading control). B. Western blot analysis of PM effect on PKC-ε expression. The change in signal strength relative to control cells (0 PM) is indicated by numbers at the top of each signal. Each experiment was repeated twice.

Effect of PM on antiapoptotic proteins

Transcription factor NF-κB regulates the expression of several members of the Bcl-2 and inhibitors of apoptosis families of proteins that negatively regulate apoptosis. We measured the effect of PM on expression of NF-κB-regulated antiapoptotic Bcl-2, Bcl-xL, c-IAP1 and survivin in OVCAR-5 and MDAH 2774 cells. As shown in Figure 6, levels of most of these antiapoptotic proteins were significantly to completely reduc ed by PM in both cell lines at concentrations of 1.25 to 5 μM. These data indicated that inhibition of antiapoptotic proteins plays a role the apoptotic death of ovarian cancer cells by PM.

Figure 6. Effect of PM on expression of antiapoptotic proteins.

Figure 6

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OVCAR-5 and MDAH 2774 cells were treated with PM at 0 to 5 μM for 20 h. Cellular lysates were analyzed for Bcl-2, Bcl-xL, c-IAP1, survivin or β-actin (loading control) by western blotting. The change in signal strength relative to control cells (0 PM) is indicated by numbers above each signal. Similar results were obtained in two independent experiments.

DICUSSION

Only a small number of studies have been reported on the anticancer activity of pristimerin. One of these studies showed sensitivity of an ovarian cancer cell line to PM isolated from R. buchananii (6). The mechanism of the antitumor activity of PM in ovarian cancer cells was not evaluated in this study. In the present study, we investigated the antitumor activity of PM in four human ovarian carcinoma cell lines. Data showed that PM has potent antiproliferative and apoptosis-inducing effects on all of the ovarian cancer cell lines. PM significantly inhibited the proliferation of OVCAR-5 and MADH 2774 at 1.25 to 10 μM, whereas in SK-OV-3 and OVCAR-3 significant antiproliferative effect was observed at 2.5 and 5 μM PM. The antiproliferative effect reached plateau at 5 to 10 μM PM. Although the mechanisms of the antiproliferative activity of PM are not fully understood, in pancreatic cancer cells it caused cell cycle arrest in G1 phase (18). Therefore, the possibility exists that PM inhibits the proliferation of ovarian cancer cells through cell cycle arrest.

PM has been shown to induce apoptosis in cancer cells (11–15). We also observed that inhibition of cell proliferation by PM was associated with induction of apoptosis in ovarian cancer cells. The induction of apoptosis by PM in ovarian cancer cells was demonstrated by the increased binding of annexin V due to the externalization of phosphatidylserine and cleavage of PARP-1. This result indicated that induction of apoptosis is part of the mechanism by which PM destroys ovarian cancer cells. Two major pathways of apoptotic cell death program have been identified, namely receptor-mediated (extrinsic) and chemical-induced mitochondrial (intrinsic) pathway (21). In both cases, caspases, a family of cysteine proteases, play an important role in the apoptotic cell death. In the extrinsic pathway, binding of the death ligands (e.g., TNF-α, FasL, TRAIL) with their cognate receptors activates initiator caspase-8 which then cleaves and activates effector caspases -3, -6, and -7 leading to apoptosis (22). In chemical-induced (chemotherapeutic agents) apoptosis (intrinsic pathway), undefined signals induce release of cytochrome c from mitochondria, which in conjunction with Apaf-1 causes activation of initiator caspase-9. Activated caspase-9, in turn, activates effector caspases -3, -6, and -7 (21). PM caused the cleavage of the initiator procaspase-8 and effector procaspase-3 in ovarian cancer cells. The cleavage of these procaspases suggested that death receptor-signaling pathway (extrinsic) of apoptosis may play a role in apoptotic death of ovarian cancer cells by PM. Whether PM increases the expression of death receptors DR4 and DR5 remains to be determined. PM also induced the cleavage of pro-caspase-9 and mitochondrial depolarization, indicating that the intrinsic pathway of apoptosis is also induced by PM in ovarian cancer cells. The induction of both pathways of apoptosis by PM in ovarian cancer cells is consistent with previous reports showing similar effects of PM in other tumor types (14,15). In addition, generation of reactive oxygen species (ROS) by PM also plays a role in induction of apoptosis in cancer cells (12,16). Whether ROS are generated in response to PM and mediate induction of apoptosis in ovarian cancer cells remains to be investigated

PI3K/Akt is a major signal transduction pathway that controls cell proliferation, survival, apoptosis, and malignant transformation and is frequently hyperactivated in most cancers (23). Activated Akt promotes cell growth and survival by inactivating downstream substrates such as Bad, procaspase-9, and Forkhead transcription factors. Antiapoptotic NF-κB and progrowth mTOR signaling pathways are downstream targets of activated Akt/PBK. NF-κB family of transcription factors controls the expression of genes involved in immune, inflammatory and oncogenic responses. It also plays a critical role in resistance of cancer cells to anticancer therapies by protecting them from apoptosis (24). mTOR is a serine-threonine kinase, which controls cell growth, survival, ribogenesis and is a target for novel anticancer agents (25). PM inhibited each of these survival-promoting (antiapoptotic) signaling proteins in ovarian cancer cells. Others have also reported inhibition of some of these prosurvival signaling pathways by PM in certain tumor types (15,16).

NF-κB-regulated Bcl-2, Bcl-xL, c-IAP1 and survivin are major antiapoptotic proteins that are often overexpressed in cancer cells. The inhibition of NF-κB in ovarian cancer cells by PM (above) can be expected to decrease the expression of NF-κB-regulated antiapoptotic proteins. Indeed, levels of Bcl-2, Bcl-xL, c-IAP1 and survivin were significantly reduced in ovarian cancer cells treated with PM. Taken together, our data indicate that the inhibition of prosurvival signaling proteins (Akt, mTOR and NF-κB) and NF-κB-regulated antiapoptotic proteins (Bcl-2, Bcl-xL, c-IAP1 and survivin) is part of the mechanism by which PM induces apoptosis in ovarian cancer cells.

In conclusion, the present study demonstrated the antiproliferative effect of pristimerin in ovarian carcinoma cells, which was associated with induction of apoptosis. PM also compromised the integrity of mitochondria. We also identified Akt/NF-κB/mTOR signaling pathway and NF-κB-regulated antiapoptotic Bcl-2, Bcl-xL, c-IAP1 and survivin as potential targets mediating the proapoptotic activity of PM in ovarian carcinoma cells. Thus, pristimerin is a promising agent worthy of development for the treatment/prevention of ovarian cancer.

Acknowledgments

This work was supported by NIH grant IR01CA130948.

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