Lijun Tan

Resveratrol-induced Autophagocytosis in Ovarian Cancer Cells

Resveratrol (3,5,4-trihydroxystilbene), a natural phytoalexin present in grapes, nuts, and red wine, has antineoplastic activities. Several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo. In the present study, the response of ovarian cancer cells to resveratrol is explored. Resveratrol inhibited growth and induced death in a panel of five human ovarian carcinoma cell lines. The response was associated with mitochondrial release of cytochrome c, formation of the apoptosome complex, and caspase activation. Surprisingly, even with these molecular features of apoptosis, analysis of resveratrol-treated cells by light and electron microscopy revealed morphology and ultrastructural changes indicative of autophagocytic, rather than apoptotic, death. This suggests that resveratrol can induce cell death through two distinct pathways. Consistent with resveratrol’s ability to kill cells via nonapoptotic processes, cells transfected to express high levels of the antiapoptotic proteins Bcl-xL and Bcl-2 are equally sensitive as control cells to resveratrol. Together, these findings show that resveratrol induces cell death in ovarian cancer cells through a mechanism distinct from apoptosis, therefore suggesting that it may provide leverage to treat ovarian cancer that is chemoresistant on the basis of ineffective apoptosis.

Glucose deprivation activates AMPK and induces cell death through modulation of Akt in ovarian cancer cells.

OBJECTIVES Upregulation of glycolysis has been demonstrated in multiple tumor types. Glucose deprivation results in diminished intracellular ATP; this is counteracted by AMPK activation during energy deficiency to restore ATP levels. We sought to determine whether glucose deprivation could induce cytotoxicity in ovarian cancer cells through activation of AMPK, and whether AMPK activators could mimic glucose deprivation induced cytotoxicity. METHODS Sensitivity to 2DG induced cytotoxicity and glucose deprivation was determined in a panel of ovarian cancer cells. Cellular growth rate, rate of glucose uptake, and response to glucose deprivation were determined. Expression of Glut-1, HIF1-α, AMPK and Akt was determined by immunoblotting. RESULTS Incubation of ovarian cancer cells with glucose-free media, 2-DG and AMPK activators resulted in cell death. The glycolytic phenotype of ovarian cancer cells was present in both normoxic and hypoxic conditions, and did not correlate with HIF1-α expression levels. Sensitivity to glucose deprivation was independent of growth rate, rate of glucose uptake, and appeared to be dependent upon constitutive activation of Akt. Glucose deprivation resulted in activation of AMPK and inhibition of Akt phosphorylation. Treatment with AMPK activators resulted in AMPK activation, Akt inhibition, and induced cell death in ovarian cancer cells. CONCLUSIONS Ovarian cancer cells are glycolytic as compared to normal, untransformed cells, and are sensitive to glucose deprivation. Because ovarian cancer cells are dependent upon glucose for growth and survival, treatment with AMPK activators that mimic glucose deprivation may result in broad clinical benefits to ovarian cancer patients.

Trichostatin A restores Apaf-1 function in chemoresistant ovarian cancer cells

Chemoresistance is the major factor limiting long‐term treatment success in patients with epithelial ovarian cancers. Most cytotoxic drugs kill cells through apoptosis; therefore, defective execution of apoptotic pathways results in a drug‐resistant phenotype in many tumor types.

Resveratrol inhibits ovarian tumor growth in an in vivo mouse model

Resveratrol inhibits the growth of ovarian carcinoma cells in vitro through the inhibition of glucose metabolism and the induction of both autophagy and apoptosis. In the current study, we investigated the metabolic and therapeutic effects of resveratrol in vivo.

Abstract 2479: Bz-423: A novel benzodiazephine mitochondrial-targeted drug, cytotoxic to human ovarian cancer cells.

Objectives: Bz-423 is a chemically synthesized 1,4-benzodiazepine with cytotoxic activity in several cell types. We sought to determine the anti-neoplastic potential and mechanism of action in human ovarian cancer cells. Methods: A total of 12 human ovarian cancer cell lines were used. Cultured cells were treated with Bz-423 or vehicle control in dose-response and time-course experiments. Viability was determined on the basis of plasma membrane integrity using flow cytometry. The mechanism leading to death in these cells was characterized ultrastructurally using electron micrscopy and by determining changes in reaction oxygen species, glutathione, mitochondrial function, caspase and Bak activation. Cytochrome c release from mitochondria was analyzed by immunofluorescence. Apoptosome complex formation was monitored by gel filtration chromatography. Results: Bz-423 induced apoptosis in all ovarian cell lines at low micromolar concentrations by generating an intracellular superoxide signal from the mitochondria. The apoptotic response was associated with the release of cytochrome c from the mitochondria, collapse of the mitochondrial transmembrane gradient, and formation of the apoptosome complex containing Apaf-1 and caspase-9. The release of cytochrome c and a decrease in mitochondria glutathione within 2 hours of treatment was identified by ultrastructural evidence of mitochondrial swelling. Furthermore, Bak activation was necessary for apoptosis. Bz-423 effectively induced apoptosis in chemoresistant ovarian cancer cells overexpressing Bcl-2 and Bcl-xL. Conclusion: Bz-423 is a novel compound that effectively kills human ovarian cancer cells, including cells that are resistant to conventional chemotherapies such as Cisplatinum. This compound has cytotoxic qualities that are unique among benzodiazepines. The mechanism of action involves both proximal and direct effects on the cell mitochondria that lead to apoptosis. Bz-423 is a novel compound that may have broad clinical impact in the treatment of ovarian cancer patients. Citation Format: Gong He, Lijun Tan, Weimin Wang, Anthony E. Boitano, Natalie A. Vandeven, Anthony W. Opipari, J Rebecca Liu. Bz-423: A novel benzodiazephine mitochondrial-targeted drug, cytotoxic to human ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2479. doi:10.1158/1538-7445.AM2013-2479 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 1586: Oral administration of HIV-protease inhibitors reduces ovarian tumor growth in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL INTRODUCTION: Ritonavir boosted lopinavir (LPV/r) is an FDA approved HIV-protease inhibitor combination that is a well tolerated and preferred treatment for HIV patients. Ritonavir is a potent irreversible inhibitor of cytochrome P450 isoform 3A (CYP3A) which can substantially increase bioavailability of lopinavir in vivo. This combination of lopinavir and ritonavir has been shown to increase the efficacy of reducing viral load in AIDS patients. HIV patients taking antiretroviral protease inhibitors such as LPV/r have a lower incidence of HIV associated malignancies such as non-Hodgkins lymphoma, cervical cancer and Kaposis sarcoma, leading to the hypothesis that these drugs have antineoplastic activity. Given the need for novel treatment approaches in ovarian cancer, we are investigating the antineoplastic effects of LPV/r, in vivo. METHODS: A xenograph model of ovarian cancer is used to assess the antineoplastic effect of LPV/r in vivo. Oral doses of LPV/r dissolved in corn oil are administered to mice over the course of 2-3 weeks. Tumor area and mouse weight are monitored. After 2-3 weeks tumors are harvested and analyzed. RESULTS: Preliminary results indicate that LPV/r administered orally in combination with a high fat diet inhibits tumor growth in vivo without inducing weight loss. In contrast, the dose of intraperitoneal cisplatin required to attain a similar antineoplastic effect resulted in significant weight loss and morbidity. CONCLUSIONS: Because protease inhibitors can induce both apoptotic and non-apoptotic ovarian cancer cell death in vitro and in vivo, these drugs may circumvent chemoresistance due to alterations in apoptotic response. Our data suggests that LPV/r may also have clinical application in the treatment of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2011-1586

Abstract 4143: Bz-423, a novel benzodiazpine, inhibits PI3 kinase signaling in ovarian cancer cells

Objective: Bz-423 is a 1,4-benzodiazepine with immunomodulatory and cytotoxic properties. The effect of Bz-423 on survival signaling in lymphoid neoplasms predicts activity against similar pathways in ovarian cancer cells. Having previously established the cytotoxic activity of Bz-423 against ovarian cancer, we now characterize the mechanism of action. Methods: Human ovarian cancer cell lines were maintained in culture. Cells were treated with Bz-423 or vehicle control. Cell survival and proliferation were evaluated, along with signaling mechanisms including the PI3-kinase-Akt-mTOR axis. DNA degradation (apoptotic fraction), mitochondrial function and caspase activation were evaluated by flow cytometry. Cytochrome c release and protein kinase activation were analyzed by immunoblotting. Apoptosome complex formation was monitored by gel filtration chromatography. Cellular ultrastructure was determined by electron microscopy (EM). Results: Growth inhibition and apoptosis were observed in all ovarian cell lines following treatment with Bz-423. Caspase-9 and −3 cleavage, along with cytochrome c release were seen, consistent with activation of the intrinsic pathway of apoptosis. Early mitochondrial changes identified by ultrastructural evidence of mitochondrial swelling and the accumulation of superoxide within 1 hr of treatment were seen. Bz-423 induced cell death was associated with inhibition of Akt activation through a oxidant dependent mechanism. Antioxidants blocked Bz-423-induced signaling. Conclusions: Bz-423 is a novel benzodiazepine being developed as a mitochondria targeted therapy, and displays anti-neoplastic activity against ovarian cancer cells in vitro. Results show that by acting on mitochondrial complex V, this compound inhibits PI3-kinase signaling, including the tumor survival factor Akt. PI3-kinase signaling is increasingly recognized as a survival and growth promoting mechanism in ovarian cancer. Since Akt inhibition by Bz-423 is dependent upon cellular bioenergetics and anti-oxidant balance, strategies employing this compound offer a selective new approach to target ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4143.