Autophagy In Ovarian Cancer Cells Research Articles

Autophagy-independent enhancing effects of Beclin 1 on cytotoxicity of ovarian cancer cells mediated by proteasome inhibitors

BackgroundThe ubiquitin-proteasome system and macroautophagy (hereafter referred to autophagy) are two complementary pathways for protein degradation. Emerging evidence suggests that proteasome inhibition might be a promising approach for tumor therapy. Accumulating data suggest that autophagy is activated as a compensatory mechanism upon proteasome activity is impaired.MethodAutophagy activation was measured using acridine orange staining and LC3 transition. Cell viability and apoptosis were measured using MTT assay and flow cytometry, respectively. Beclin 1 expression vectors or shRNA against Beclin 1 (shBeclin 1) were transfected to investigate the role of Beclin 1 in autophagy activation and cytotoxicity of ovarian cancer cells induced by proteasome inhibitors.ResultsProteasome inhibitors suppressed proliferation and induced autophagy in ovarian cancer cells. Neither phosphoinositide 3-kinase (PI3K) inhibitors nor shRNA against Beclin 1 could abolish the formation of acidic vacuoles and the processing of LC3 induced by proteasome inhibitors. Moreover, Beclin 1 overexpression enhanced anti-proliferative effects of proteasome inhibitors in ovarian cancer cells.ConclusionsFor the first time, the current study demonstrated that proteasome inhibitors induced PI3K and Beclin 1-independent autophagy in ovarian cancer cells. In addition, this study revealed autophagy-independent tumor suppressive effects of Beclin 1 in ovarian cancer cells.

Regulation of macroautophagy in ovarian cancer cells in vitro and in vivo by controlling Glucose regulatory protein 78 and AMPK

In this study we show that diindolylmethane (DIM) induces autophagy in ovarian cancer cells by regulating endoplasmic reticulum (ER) stress and AMPK. Treatment of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations of DIM for 24 hours resulted in a concentration dependent induction of autophagy as measured by flowcytometry. Electron microscopy confirmed the presence of autophagosomes in DIM treated cells. Western blot analysis showed that DIM treatment increased the expression of LC3B, a hall mark of autophagy as well as p62 and Atg 12 proteins that are accumulated during autophagy. Autophagy inhibitors bafilomycin or chloroquine inhibited DIM induced autophagy. Furthermore, DIM treatment significantly increased the expression of ER stress regulators such as Grp78, IRE1 and GADD153. Cycloheximide or ER stress inhibitor mithramycin not only blocked ER stress proteins that were activated by DIM but also autophagy. Silencing Grp78 or GADD 153 significantly blocked the expression of LC3B and p62 indicating that autophagy in our model is mediated by ER stress. Knocking out LC3B inhibited DIM induced autophagy. DIM treatment increased the cytosolic calcium levels which lead to the activation of AMPK in our model. Chelating cytosolic calcium with BAPT-AM abrogated not only the phosphorylation of AMPK but also prevented DIM induced autophagy. Inhibiting AMPK by a chemical inhibitor or siRNA blocked the induction of LC3B or p62, indicating that DIM mediated autophagy requires activation of AMPK. Oral administration of DIM significantly suppressed SKOV-3 tumor xenografts in nude mice. Activation of ER stress and autophagy were observed in the tumors of DIM treated mice. Taken together, these results suggest that induction of autophagy by DIM in ovarian cancer cells was associated with ER stress and AMPK activation.

Abstract 4889: ERa-targeted therapy in ovarian cancer cells using novel Estradiol-Platinum(II) hybrids

Abstract Since several cancers in women are estrogen-dependent due to the presence and overexpression of estrogen receptor (ER), we sought to determine the possibility to use ER as a target for directed chemotherapy and have designed unique molecules capable of doing so. We have recently reported the synthesis of a new family of E2-platinum(II) hybrids. Previous study revealed high efficiency of these novel compounds compared to cisplatin towards breast cancer cells which were also selective towards ER+ breast cancer xenografts in nude mice. In the present study, we have investigated, in vitro and in vivo, the antitumor activity of one of the best hybrid VP-128 using ovarian cancer cell (Ovcar-3, Skov-3, A2780 and A2780CP) and investigated its mode of action and its ability to induce apoptosis and autophagy. Annexin V/PI staining using flow cytometry revealed an improved efficiency of VP-128 compared to cisplatin to induce apoptosis of ovarian cancer cells. We also transiently transfected A2780 cells with ERα gene to compare the hybrid in a similar genetic background. The presence of ER in A2780 increased apoptosis induced by VP-128 but not by cisplatin. VP-128 activity was also stronger compared to cisplatin in presence or absence of ER suggesting that the hybrid can act through different mechanisms of action. In vivo, using ovarian cancer cell xenografts in nude mice, we also found that VP-128 had improved antitumour activity compared to cisplatin, and was more specific and selective towards hormone-dependent ER+ than ER- xenografts. To examine VP-128 mechanism of action, we decided to study its ability to induce apoptosis and autophagy. We found that VP-128 induced apoptosis via caspase-dependant (caspase-9; -3 and PARP cleavage) mechanisms but also caspase-independent mechanisms such as AIF translocation to the nucleus, as revealed by Western blot of cytosolic/nuclear cell extracts and immunofluorescence microscopy in A2780(ER-). Conversely, AIF subcellular localization was not modified by VP-128 in Ovcar-3 ER+ cells. Following 8-12h of treatment with VP-128, increased formation of acidic vesicular organelles in cancer cells was observed suggesting the importance of autophagy in this process. We then found by Western blot that LC3B-I was converted to LC3B-II following VP-128 treatment suggesting formation of autophagosome. We then investigated pathways directly (mTor) or indirectly (AKT) possibly involved in the regulation of autophagy. VP-128 decreased the level of both phosphorylated/active mTor and AKT in cancer cells favouring autophagy induction. These results show that VP-128 can not only induce apoptosis but also induce autophagy in ovarian cancer cells. Altogether, this study also highlight the therapeutic value of VP-128 for the treatment of hormone-dependent ovarian cancers, and provide preliminary proof-of-concept for efficient targeting of ERα by E2-Pt(II) linked chemotherapeutic hybrids. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4889. doi:1538-7445.AM2012-4889

The different regulatory effects of p53 status on multidrug resistance are determined by autophagy in ovarian cancer cells

PurposeMultidrug resistance (MDR) has become an obstacle for chemotherapy of cancer. p53 is reported to participate in the regulation of MDR, but the association between p53 status and MDR are complicated and conditional. It has been verified that apoptosis is not the only mechanism for MDR regulation by p53, the roles of autophagy in MDR is less studied. Patients and methodsHuman ovarian carcinoma cell lines SKOV3 and multidrug resistant phenotype SKVCR cells were used and wild-type p53 (wt p53) and mutant 175H constructs were introduced into cells to establish cell models with different p53 status by gene engineering, the sensitivity to vincristine (VCR), cisplatin (DDP), pirarubicin (THP) and etoposide (VP-16) were detected by MTT assay, Western blot and quantitative real-time PCR were used to detect the expression of protein and mRNA, especially, monodansylcadaverine (MDC) staining was used for autophagy rate, Hoechst 33342/propidium iodide (PI) were used to assess apoptosis and necrosis. ResultsSKVCR cells induced by VCR shown overexpression of P-glycoprotein (P-gp) and MDR, and also displayed an enhanced autophagy compared with parental SKOV3. Wt p53 and 175H has no influence on drug sensitivity in SKOV3, while both sensitized SKVCR cells to VCR, THP and VP-16, especially 175H. The introduction of wt p53-induced apoptosis only, while 175H trigged autophagic cell death, necrosis and apoptosis so as to reverse the MDR. ConclusionThe enhancement of autophagy in MDR cells allows to survive during chemotherapy stress, autophagy plays important role in wt p53 and mutant p53-immediated MDR. The different influence of p53 status on drug sensitivity hint the individual treatment strategies based on p53 status in patients.

Abstract 3704: Diindolylmethane induces ER stress mediated autophagy in ovarian cancer cells

Abstract Endoplasmic reticulum (ER) is responsible for biosynthesis, folding assembly and modification of various soluble and membrane proteins. ER stress is the imbalance in protein folding resulting in the accumulation of misfolded proteins in ER lumen. In our study, we observed that Diindolylmethane (DIM), a compound present in cruciferous vegetables induces ER stress in ovarian cancer cells. Treatment of SKOV-3, OVCAR-3 and TOV-21G ovarian cancer cells with varying concentrations of DIM for 24hours resulted in the upregulation of Bip, a molecular chaperone. Bip or Grp78 binds to IRE1α, ATF6 and PERK under normal condition, however, during ER stress, Bip dissociates and releases all the three proteins. DIM treatment also significantly increased the expression of IRE1α, ER oxidase (ERO1) and CHOP. CHOP translocate to nucleus and initiate apoptotic stimuli in cells undergoing ER stress. Moreover, DIM induced ER stress also lead to the activation of eIF2α by phosphorylation. Activation of eIF2α inhibits protein translation to avoid further protein accumulation in ER. The release of calcium by DIM treatment in ovarian cancer cells was evaluated by Accuri C6 flowcytometer. The advantage with Accuri C6 is that calcium levels can be monitored without interruption. DIM treatment increased the cytosolic calcium levels in SKOV3 cells. Increase in cytosolic calcium is known to cause ER stress. In addition, our flowcytometry results indicate that DIM induces autophagy in all the ovarian cancer cells in a concentration dependent manner. Exposure of SKOV-3, OVCAR-3 and TOV-21G cells to DIM upregulated the expression of LC3B, a potent indicator of autophagy. DIM induced autophagy was blocked by bafilomycin, an autophagy inhibitor. Treatment of cells with mithramycin (ER stress inhibitor) blocked ER stress proteins activated by DIM. Interestingly, we also observed that blocking DIM-induced ER stress by mithramycin blocked autophagy in ovarian cancer cells. Taken together these results suggest that induction of autophagy in ovarian cancer cells was associated with ER stress caused by DIM. Further mechanistic studies are in progress. [Supported in part by R01 grants CA106953 and CA129038 (to S.K.S) awarded by the National Cancer Institute]. 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 3704. doi:10.1158/1538-7445.AM2011-3704

Abstract 88: Targeting Bcl-XL enhances arsenic-induced cell death in ovarian cancer cells

Abstract Autophagy is an evolutionary conserved process in which long-lived proteins and damaged organelles are digested under physiological conditions including starvation. Autophagy has also been shown to act as a programmed cell death mechanism (type II PCD) and considered as caspase independent cell death. Several studies showed that autophagy has been triggered by anti-cancer treatments, such as chemo and hormonal therapy and radiotherapy and growth factor deprivation, function either as a protective survival pathway or cellular death mechanism. Arsenic trioxide (ATO), which is a FDA approved drug and used as a treatment in acute promyelocytic leukemia, has been reported to induce autophagic and/or apoptotic cell death in leukemia, glioblastoma and also ovarian cancer cells, we have previously found that ATO induces autophagy in ovarian cancer cells. Here, we investigated the role of antiapoptotic protein Bcl-Xl, which inhibits autophagy by binding beclin-1, an essential autophagy promoting protein, in arsenic induced apoptosis and determined the effects of ATO/Bcl-XL RNA interference combination therapy in ovarian cancer. We found that ATO induces dose dependant growth inhibition in MTS proliferation assay. ATO treatment (2-4 uM) induced apoptosis in A2780 (40-50 %) and SKOV3 (20-25 %) cells, detected by annexin V staining and flow cytometric (FACS) analysis and by caspase 9 and PARP cleavage (Western blotting). ATO induced marked autophagy in A2780 (60-70 %) and in SKOV3 (15-20 %) as indicated by LC3-II, a marker for autophagy, formation autophagic vacuoles detected by acridine orange staining and FACS and by transmission electron microscopy. ATO induced Bcl-XL protein expression in ovarian cancer cells. We also found expression of high levels of Bcl-XL in all ovarian cancer cell lines including drug (i.e., cisplatin and paclitaxel) resistant cells. Inhibition of Bcl-XL expression by Bcl-XL siRNA induced growth inhibition (40-60 %), apoptosis (40-60 %) and autophagy (50-60% in A2780 and 15-25 % in SKOV3 ovarian cancer cell line). Furthermore, combination of ATO with Bcl-XL siRNA led to 90 % cell death and 70 % apoptosis in both A2780 and SKOV3 cells. Overall, our results suggest that combination of ATO and Bcl-XL induced predominantly apoptosis, although each of them induced autophagy. In conclusion we provided first evidence that inhibition of Bcl-XL induces autophagy in ovarian cancer cells and sensitizes them to ATO that may be a promising treatment regimen for ovarian cancer patients. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 88.

Abstract 3666: Follicle-stimulating hormone inhibits apoptosis and autophagy in ovarian cancer cells by activating survivin and Rab25

Abstract Follicle-stimulating hormone (FSH) has been demonstrated to increase the risk of ovarian malignancy, but the mechanisms remains unclear. Our previous results showed that FSH increases the expression of VEGF through activation of survivin, which is mediated by the activation of the PI3K/AKT signaling pathway. We reported, herein, that FSH promoted ovarian cancer cells proliferation and prevented apoptosis in vitro and in vivo. FSH up-regulated the expression of survivin and down-regulated the expression of programmed cell death 6 (PDCD6) and death receptor 5 (DR5), two molecules required for apoptosis induction. Further, we have defined that the decreased expression of DR5, but not PDCD6, was mediated by survivin. We have established animal xenografts tissue arrays, the tumors from the groups with FSH treatment had higher expression of survivin and lower expression of DR5 comparing with the tumors from untreated groups, further confirming the interaction of FSH-survivin-DR5. In addition, we have demonstrated that FSH does not only inhibit apoptosis, but also inhibit autophagy. FSH inhibits autophagy by activating mTOR pathway in ovarian cancer cells under different stress conditions, including serum starvation. By Reverse Phase Protein Arrays (RPPA) analysis and western blot confirmation, we found that FSH stimulation increases the level of Rab25 in multiple ovarian cancer cells in a dose dependent manner. Rab25, a small GTPase, is found to be associated with the progression of ovarian tumor. Rab25, belongs to Rab11 supfamily, was shown to involve in endosomal recycling. To study the role of FSH-Rab25 interaction in regulating autophagy, we knocked down Rab25 expression in ES-2, Hey and SKOv3 ovarian cancer cell lines by siRNA, and examined the effects of FSH on autophagy. Rab25 knockdown increased the starvation induced autophagy through the inhibition of pAkt, while FSH administration could partly reverse the effects of Rab25 knockdown, due to the increase of Rab25 expression stimulated by FSH. Our findings indicated that Rab25 is one of the molecules regulated by FSH in ovarian cancer cells that has a novel role as a regulator in autophagy. Taken together, we have demonstrated that FSH promotes ovarian cancer development through regulating expression of survivin, Rab25, PDCD6 and DR5, which in turn regulates cell proliferation, apoptosis as well as autophagy. Further understanding the molecular mechanisms of FSH in the process of ovarian carcinogenesis and development will ultimately help developing an effective cancer prevention method, and find a novel targeted therapy for ovarian cancer. 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 3666.

Abstract 4679: Integrin and FAK signaling regulate ARHI-induced autophagy

Abstract Autophagy selectively degrades cellular proteins, protein aggregates and organelles, providing energy in a nutrient deprived environment. We have reported that re-expression of ARHI, a maternally imprinted Ras-related tumor suppressor gene that is down regulated in more than 60% of ovarian cancers, induces autophagy. Persistence for ARHI-induced autophagy for several days is lethal for ovarian cancer cells in culture. In xenografts, however, ARHI-induced autophagy does not kill ovarian cancer cells, but rather is required to maintain a dormant state in the absence of a vascular supply. When ARHI is downregulated in vivo, previously dormant xenografts grow promptly and outgrowth of cancers is inhibited by treatment of dormant cancer cells with chloroquine. Growth factors (IGF-1, IL-8, VEGF) and ECM components (fibronectin) in the cancer microenvironment can rescue ovarian cancer cells from ARHI-induced autophagic death in cell culture. Rescue was associated with partial reversal of ARHI-mediated inhibition of PI3K signaling and AKT and mTOR activity. Consequently, we have hypothesized that both autophagy and survival factors (IGF-1, fibronectin) are required to maintain dormancy, where autophagy provides energy for avascular xenografts and survival factors prevent autophagic death by partially reversing inhibition of PI3K signaling. Here, we show that re-expression of ARHI in SKOv3 ovarian cancer cells inhibited integrin β1 expression and decreased FAK activation, stimulated by fibronectin and growth factors (IGF-1). Consistent with the importance of ECM, addition of fibronectin, a receptor for αvβ1 integrin, induced FAK activity and diminished ARHI-induced autophagy; whereas addition of neutralizing antibody against integrin β1 decreased FAK and Akt activities, enhancing autophagy. Autophagy induced by ARHI is dramatically reduced in cells that overexpress FAK and knockdown of FAK by RNA interference enhances ARHI-induced autophagy in ovarian cancer cells cultured on fibronectin. Our results suggest that the ECM and growth factors in the tumor microenvironment function to coordinate the activities of integrin β1, FAK and Akt to regulate ARHI-mediated autophagy, contributing to the survival of dormant 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 4679.