scholarly journals Desialylation of Atg5 by sialidase (Neu2) enhances autophagosome formation to induce anchorage-dependent cell death in ovarian cancer cells

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Eswara Murali Satyavarapu ◽  
Shalini Nath ◽  
Chitra Mandal
Keyword(s):  
Ovarian Cancer ◽  
Cell Death ◽  
Cyclin B1 ◽  
Sialic Acids ◽  
Mtor Pathway ◽  
Parp Cleavage ◽  
Bafilomycin A1 ◽  
Autophagosome Formation ◽  
Dependent Cell ◽  
Induced Apoptosis

AbstractIncreased sialylation is one of the hallmarks of ovarian cancer (OC) but its relation with programmed cell death is not known. Here we explored the molecular interplay between autophagy, apoptosis/anoikis, and aberrant-expression of the PI3K-Akt/mTOR pathway in the context of sialidase. OC is accompanied by low expression of cytosolic sialidase (Neu2) and ~10-fold more α2,6- than α2,3-linked sialic acids found through qPCR, western blot, and flow cytometry. Interestingly, Neu2 overexpression cleaved α2,6- and α2,3-linked sialic acids and reduced cell viability. Several autophagy-related molecules like LC3B/Atg3/Atg5/Atg7/Atg12/Atg16L1/Beclin1 were upregulated upon Neu2 overexpression. Atg5, a crucial protein for autophagosome formation, was desialylated by overexpressed Neu2. Desialylated Atg5 now showed enhanced association both with Atg12 and Atg16L1 leading to more autophagosome formation. Neu2-overexpressing cells exhibited extrinsic pathway-mediated apoptosis as reflected the in activation of Fas/FasL/FADD/Bid/caspase 8/caspase 6/caspase 3/PARP cleavage. There was also increased Bax, reduced Bcl2, and several cell-cycle molecules (CDK2/CDK4/CDK6/cyclin-B1/cyclin-E). Inhibition of autophagy using bafilomycin A1 or Beclin1 siRNA leads to reversal of Neu2-induced apoptosis suggesting their possible relationship. Additionally, overexpressed Neu2 inhibited growth factor-mediated signaling molecules involved in the PI3K/Akt-mTOR pathway probably through their desialylation. Furthermore, overexpressed Neu2 inhibited epithelial (ZO-1/Claudin1), mesenchymal (snail/slug), and cell-adhesion (integrin-β3/focal-adhesion kinase) molecules suggesting anchorage-dependent cell death (anoikis). Such changes were absent in the presence of bafilomycin A1 indicating the involvement of autophagy in Neu2-induced anoikis. The physiological relevance of our in vitro observations was further confirmed in the OC xenograft model. Taken together, it is the first report demonstrating that Atg5 is a sialoglycoprotein having α2,6- and α2,3-linked sialic acids and its desialylation by overexpressed Neu2 leads to its activation for autophagosome formation, which induced apoptosis/anoikis in OC.

FTY720 Induces Apoptosis and Autophagy in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5103-5103
Author(s):  
Aijun Liao ◽  
Rong Hu ◽  
Qihui Zhao ◽  
Huihan Wang ◽  
Yingchun Li ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Cell Viability ◽  
Cell Line ◽  
Control Group ◽  
Bafilomycin A1 ◽  
Antiapoptotic Proteins ◽  
Dependent Cell ◽  
Induced Apoptosis ◽  
Dose Dependent

Abstract Abstract 5103 Despite recent treatment advances, multiple myeloma(MM) remains incurable. FTY720 has initially been used as an immunosuppressant and is promising in treating relapsing/remitting multiple sclerosis. FTY720 has also been studied in several hematological malignancies including MM, but there are no reports about autophagy induced by FTY720 in MM. Therefore, we assessed the efficacy of FTY720 on MM cell line U266 and investigated the associated mechanisms of cell death. First we examined whether or not FTY720 induce cell death in U266 cells. We treated U266 cells with different dosage of FTY720 and then cell viability was measured at 24 hours by CCK-8 assay. The results showed cell viability started to reduce at 5uM and reach to 15% at 20uM, suggesting FTY720 affects cell survival in MM cell line. We then measured cell viability at 2□ A6□ A24 hours at fix concentration of 20.0 mM, 50% cells were killed even at 2 hours as compared with control group. Cell apoptosis was also measured by flow cytometry(FCM). Apoptosis rate(%) increased in a dose-dependent manner after FTY720 treatment. The percent Annexin+/7AAD- cells in the control group remained constant 5% and 15% to 30% in the drug treated time course experiments. The cell viability and apoptosis induced by FTY720 showed a dose- and time-dependant manner. Z-VAD-fmk, a pancaspase inhibitor, could rescue cell death caused by FTY720 and exposure of U266 cells to different concentration of FTY720 induced cleavage of caspase-3. We conclude FTY720 can induce a caspase-dependent cell death in U266 cells. Mcl-1, survivin, bcl-2 and Bid are antiapoptotic proteins and degradation of these proteins are required for the induction of apoptosis. In our study, the expression of Mcl-1, survivin and bcl-2 were reduced after the treatment which confirmed FTY720 induced apoptosis by down-regulating the expression of antiapoptotic proteins. Consistent with this, cleavage of Bid was also increased after the FTY720 treatment. Autophagy is another cell death pathway characterized by intracellular degradation system that delivers cytoplasmic constitutions to the lysosome. Conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II is a marker of autophagosome degradation. We observed increasing amount of conversion from LC3-I to LC3-II after FTY720 treatment, suggesting that FTY720 could induce autophagy in U266 cells. This conversion is dose-dependent. Interestingly, LC3-II accumulated in the presence of Bafilomycin A1, an inhibitor of autophagosome-lysosome fusion and LC3-II degradation, in cells treated with FTY720, confirmed that autophagic flux was induced by FTY720. To understand the role of apoptosis and autophagy played in the FTY720 induced cell death, we examined cell viability and apoptosis after FTY720 treatment in the presence or absence of Bafilomycin A1. Cell viability was higher and apoptosis was lower in the presence of Bafilomycin A1 after FTY720 treatment, indicating that Bafilomycin A1 could rescue cell death and apoptosis caused by FTY720. The results suggested that autophagy and apoptosis synergized to induce MM cell death after FTY720 treatment. We found Bafilomycin A1 rescued FTY720 induced cell death is dependent on the accumulation of anti-apoptotic protein Mcl-1 and survivin, it is possible that autophagy helps to degrade the anti-apoptotic proteins in the lysosome and synergize the cell death induced by FTY720. To investigate the mediators of FTY720-induced apoptosis and autophagy, we examined reactive oxygen species (ROS), which plays an important role in regulating both apoptosis and autophagy. Potential antioxidants N-acetyl-L-cysteine (NAC) and Tiron both could rescue apoptosis induced by FTY720, suggesting that FTY720 induced apoptosis via the activation of ROS. Furthermore, both NAC and Tiron blocked the conversion of LC3-I to LC3-II, indicating that FTY720 leads to ROS production, which results in autophagosome development and autolysosomal degradation. These studies indicate autophagy induced by FTY720 contributes to cell death in U266 cells and suggest that the function of FTY720 maybe can benefit from autophagy enhancement. To this regard, the ability of FTY720 to induce autophagy-dependent cell death in U266 cells, if confirmed in vivo, may represent a relatively selective therapy for the treatment of MM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inhibition of WIP1 Phosphatase in Multiple Myeloma Overcomes Bortezomib Resistance and Promotes Cell Death Via ER Stress-Induced Apoptotic JNK/c-Jun Signaling and Downregulation of Inhibitors of Apoptosis Proteins (IAPs)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1366-1366
Author(s):  
Katia Beider ◽  
Evgenia Rosenberg ◽  
Valeria Voevoda ◽  
Hanna Bitner ◽  
Yaarit Sirovsky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Stress Response ◽  
Er Stress ◽  
Cell Lines ◽  
Potential Role ◽  
Cyclin B1 ◽  
Parp Cleavage ◽  
Induced Apoptosis

Abstract Acquired or de novo resistance to the traditional and novel anti-multiple myeloma (MM) agents remains a major treatment obstacle, therefore novel therapies are in need. Wild-type p53-induced phosphatase 1 (WIP1) is an oncogenic serine/threonine phosphatase implicated in silencing of cellular responses to genotoxic stress. WIP1 overexpression was documented in various solid cancers in correlation with aggressive features and poor prognosis. Thus, we studied WIP1 in MM addressing its potential role in mediating resistance and aggressive phenotype. Increased expression of WIP1 was detected in MM cell lines (n=8) and primary samples (n=18) at both mRNA and protein level as compared with normal PBMCs (n=5). Furthermore, a positive correlation between WIP1 and CXCR4 levels (p<0.02, R2=0.5) was revealed. The latter is a well-known oncogenic receptor in MM. WIP1 expression levels were significantly up-regulated following bortezomib (Bort) treatment. Using MM cell lines with acquired resistance to Bort (RPMI8226BortRes and CAGBortRes), a higher induction of WIP1 upon Bort exposure could be demonstrated, suggesting a possible role for WIP1 in the acquisition of MM drug resistance to proteasome inhibitors. WIP1 was also upregulated in MM cells cultured on human BM stroma (BMSC) known to protect the tumor cells from Bort-induced apoptosis, further supporting its function in mediating resistance. GSK2830371 (GSK), a novel allosteric inhibitor of WIP1, significantly suppressed MM cells proliferation (p<0.01) and induced apoptosis, as demonstrated by phosphatidylserine externalization, mitochondrial depolarization (ψm), caspase 3 and PARP cleavage, and DNA fragmentation. Moreover, combined treatment with GSK and Bort synergistically potentiated cell death in both Bort-sensitive and resistant MM cells and overcame BMSC protection (CI<0.5). The robust apoptosis induced by Bort/GSK treatment was accompanied by increased mitochondrial ROS accumulation, subsequent mitochondrial destabilization and extensive DNA damage. GSK treatment resulted in a reduction of WIP1 basal expression and abrogated WIP1 induction upon Bort treatment. Thus, we defined that GSK can regulate WIP1 expression in MM cells. To determine the molecular mechanism of Bort/GSK synergism we performed gene and protein expression analysis. Combination of both agents significantly reduced expression of anti-apoptotic proteins such as cIAP1, cIAP2, XIAP and Survivin. Previous studies indicate that maintaining IAPs expression is part of an adaptive unfolded protein response that promotes MM survival upon Bort-induced endoplasmic reticulum (ER) stress. Therefore, it is conceivable that targeting IAPs upon WIP1 inhibition may overcome protective responses, inducing unresolved ER stress and MM cell death. Indeed, we found that combination of Bort and GSK significantly enhanced ER stress, as indicated by increase in the pro-apoptotic factors ATF4, CHOP and GADD34. Concomitantly, mitosis-inducing factors Cyclin B1, CDK1 and PLK1 were prominently reduced upon Bort/GSK treatment. To assess the potential role of p53 activation in GSK-mediated effects, p53-stabilizing agents nutlin3a and PRIMA1 were applied in combination with WIP1 inhibition. We observed a significant (p<0.01) increase in the responsiveness of both p53WT and p53mut MM cells to GSK-mediated apoptosis. Consistently, combined GSK/Bort treatment upregulated p53 targets, including PUMA, NOXA, GADD45A and p21 genes. These data suggest that p53 may potentiate the WIP1 inhibition mediated stress induction. Finally, we assessed the signaling pathways that may be involved in WIP1 mediated cessation of stress response. GSK profoundly augmented Bort-induced phosphorylation of JNK and c-Jun, without affecting p38 phosphorylation. Accordingly, JNK inhibitor SP600125 successfully reverted both the apoptosis and the downregulation of IAPs induced by Bort/GSK treatment. Altogether, these results identify pro-apoptotic JNK/c-Jun signaling being preferential target of WIP1 in the process of dampening Bort-induced stress response. To conclude, we disclose the role of WIP1 in blunting stress response and promoting resistance to bortezomib. Collectively, WIP1 suppression prevents MM cell adaptation and recovery upon ER stress. These findings may provide the scientific basis for a novel combinatorial anti-myeloma therapy. Disclosures Peled: Cellect Biotherapeutics Ltd: Consultancy.

Daphnetin triggers ROS-induced cell death and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway in ovarian cancer

Phytomedicine ◽  
2021 ◽  
Vol 82 ◽  
pp. 153465
Author(s):  
Xiaoye Fan ◽  
Min Xie ◽  
Feijie Zhao ◽  
Jiajia Li ◽  
Changqing Fan ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Death ◽  
Mtor Pathway

Reactive oxygen species and caspase activation mediate silica-induced apoptosis in alveolar macrophages

2001 ◽  
Vol 280 (1) ◽  
pp. L10-L17 ◽  
Author(s):  
Han-Ming Shen ◽  
Zhuo Zhang ◽  
Qi-Feng Zhang ◽  
Choon-Nam Ong
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Alveolar Macrophages ◽  
Caspase 3 ◽  
Reactive Oxygen ◽  
Parp Cleavage ◽  
Oxygen Species ◽  
Caspase Activation ◽  
Caspase 9 ◽  
Induced Apoptosis

Alveolar macrophages (AMs) are the principal target cells of silica and occupy a key position in the pathogenesis of silica-related diseases. Silica has been found to induce apoptosis in AMs, whereas its underlying mechanisms involving the initiation and execution of apoptosis are largely unknown. The main objective of the present study was to examine the form of cell death caused by silica and the mechanisms involved. Silica-induced apoptosis in AMs was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and cell cycle/DNA content analysis. The elevated level of reactive oxygen species (ROS), caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in silica-treated AMs were also determined. The results showed that there was a temporal pattern of apoptotic events in silica-treated AMs, starting with ROS formation and followed by caspase-9 and caspase-3 activation, PARP cleavage, and DNA fragmentation. Silica-induced apoptosis was significantly attenuated by a caspase-3 inhibitor, N-acetyl-Asp-Glu-Val-Asp aldehyde, and ebselen, a potent antioxidant. These findings suggest that apoptosis is an important form of cell death caused by silica exposure in which the elevated ROS level that results from silica exposure may act as an initiator, leading to caspase activation and PARP cleavage to execute the apoptotic process.

scholarly journals Inhibition of Caspases Inhibits the Release of Apoptotic Bodies: Bcl-2 Inhibits the Initiation of Formation of Apoptotic Bodies in Chemotherapeutic Agent-induced Apoptosis

1999 ◽  
Vol 145 (1) ◽  
pp. 99-108 ◽  
Author(s):  
Jiandi Zhang ◽  
Mary C. Reedy ◽  
Yusuf A. Hannun ◽  
Lina M. Obeid
Keyword(s):  
Cell Death ◽  
Chemotherapeutic Agent ◽  
Chromatin Condensation ◽  
Membrane Lipid ◽  
Lymphoid Cells ◽  
Parp Cleavage ◽  
Apoptotic Bodies ◽  
Normal Morphology ◽  
Release Assay ◽  
Induced Apoptosis

During apoptosis, the cell actively dismantles itself and reduces cell size by the formation and pinching off of portions of cytoplasm and nucleus as “apoptotic bodies.” We have combined our previously established quantitative assay relating the amount of release of [3H]-membrane lipid to the degree of apoptosis with electron microscopy (EM) at a series of timepoints to study apoptosis of lymphoid cells exposed to vincristine or etoposide. We find that the [3H]-membrane lipid release assay correlates well with EM studies showing the formation and release of apoptotic bodies and cell death, and both processes are regulated in parallel by inducers or inhibitors of apoptosis. Overexpression of Bcl-2 or inhibition of caspases by DEVD inhibited equally well the activation of caspases as indicated by PARP cleavage. They also inhibited [3H]-membrane lipid release and release of apoptotic bodies. EM showed that cells overexpressing Bcl-2 displayed near-normal morphology and viability in response to vincristine or etoposide. In contrast, DEVD did not prevent cell death. Although DEVD inhibited the chromatin condensation, PARP cleavage, release of apoptotic bodies, and release of labeled lipid, DEVD-treated cells showed accumulation of heterogeneous vesicles trapped in the condensed cytoplasm. These results suggest that inhibition of caspases arrested the maturation and release of apoptotic bodies. Our results also imply that Bcl-2 regulates processes in addition to caspase activation.

scholarly journals Isoflavones Isolated from the Seeds of Millettia ferruginea Induced Apoptotic Cell Death in Human Ovarian Cancer Cells

Molecules ◽  
2020 ◽  
Vol 25 (1) ◽  
pp. 207 ◽  
Author(s):  
Yi-Yue Wang ◽  
Jun Hyeok Kwak ◽  
Kyung-Tae Lee ◽  
Tsegaye Deyou ◽  
Young Pyo Jang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Death ◽  
Cancer Cells ◽  
Caspase 3 ◽  
Apoptotic Cell ◽  
Apoptotic Cell Death ◽  
Ovarian Cancer Cells ◽  
Human Ovarian Cancer ◽  
Millettia Ferruginea ◽  
Induced Apoptosis

The seeds of Millettia ferruginea are used in fishing, pesticides, and folk medicine in Ethiopia. Here, the anti-cancer effects of isoflavones isolated from M. ferruginea were evaluated in human ovarian cancer cells. We found that isoflavone ferrugone and 6,7-dimethoxy-3’,4’-methylenedioxy-8-(3,3-dimethylallyl)isoflavone (DMI) had potent cytotoxic effects on human ovarian cancer cell A2780 and SKOV3. Ferrugone and DMI treatment increased the sub-G1 cell population in a dose-dependent manner in A2780 cells. The cytotoxic activity of ferrugone and DMI was associated with the induction of apoptosis, as shown by an increase in annexin V-positive cells. Z-VAD-fmk, a broad-spectrum caspase inhibitor, and z-DEVD-fmk, a caspase-3 inhibitor, significantly reversed both the ferrugone and DMI-induced apoptosis, suggesting that cell death stimulated by the isoflavones is mediated by caspase-3-dependent apoptosis. Additionally, ferrugone-induced apoptosis was found to be caspase-8-dependent, while DMI-induced apoptosis was caspase-9-dependent. Notably, DMI, but not ferrugone, increased the intracellular levels of reactive oxygen species (ROS), and antioxidant N-acetyl-L-cysteine (NAC) attenuated the pro-apoptotic activity of DMI. These data suggest that DMI induced apoptotic cell death through the intrinsic pathway via ROS production, while ferrugone stimulated the extrinsic pathway in human ovarian cancer cells.

Cell death provoked by loss of interleukin-3 signaling is independent of Bad, Bim, and PI3 kinase, but depends in part on Puma

Blood ◽  
2006 ◽  
Vol 108 (5) ◽  
pp. 1461-1468 ◽  
Author(s):  
Paul G. Ekert ◽  
Anissa M. Jabbour ◽  
Anand Manoharan ◽  
Jacki E. Heraud ◽  
Jai Yu ◽  
...  
Keyword(s):  
Cell Death ◽  
Family Members ◽  
Pi3 Kinase ◽  
Minor Role ◽  
Growth And Survival ◽  
Interleukin 3 ◽  
Dependent Cell ◽  
Induced Apoptosis ◽  
A Minor ◽  
Survival Signals

Growth and survival of hematopoietic cells is regulated by growth factors and cytokines, such as interleukin 3 (IL-3). When cytokine is removed, cells dependent on IL-3 kill themselves by a mechanism that is inhibited by overexpression of Bcl-2 and is likely to be mediated by proapoptotic Bcl-2 family members. Bad and Bim are 2 such BH3-only Bcl-2 family members that have been implicated as key initiators in apoptosis following growth factor withdrawal, particularly in IL-3-dependent cells. To test the role of Bad, Bim, and other proapoptotic Bcl-2 family members in IL-3 withdrawal-induced apoptosis, we generated IL-3-dependent cell lines from mice lacking the genes for Bad, Bim, Puma, both Bad and Bim, and both Bax and Bak. Surprisingly, Bad was not required for cell death following IL-3 withdrawal, suggesting changes to phosphorylation of Bad play only a minor role in apoptosis in this system. Deletion of Bim also had no effect, but cells lacking Puma survived and formed colonies when IL-3 was restored. Inhibition of the PI3 kinase pathway promoted apoptosis in the presence or absence of IL-3 and did not require Bad, Bim, or Puma, suggesting IL-3 receptor survival signals and PI3 kinase survival signals are independent.

scholarly journals Ginkgo biloba Prevents Oxidative Stress-Induced Apoptosis Blocking p53 Activation in Neuroblastoma Cells

Antioxidants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 279 ◽  
Author(s):  
Francesco Di Meo ◽  
Rossana Cuciniello ◽  
Sabrina Margarucci ◽  
Paolo Bergamo ◽  
Orsolina Petillo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Neurodegenerative Diseases ◽  
Ginkgo Biloba ◽  
Apoptotic Cell ◽  
Neuroblastoma Cells ◽  
Apoptotic Cell Death ◽  
Parp Cleavage ◽  
Egb 761 ◽  
Induced Apoptosis

Oxidative stress has been associated to neuronal cell loss in neurodegenerative diseases. Neurons are post-mitotic cells that are very sensitive to oxidative stress—especially considering their limited capacity to be replaced. Therefore, reduction of oxidative stress, and inhibiting apoptosis, will potentially prevent neurodegeneration. In this study, we investigated the neuroprotective effect of Ginkgo biloba extract (EGb 761) against H2O2 induced apoptosis in SK-N-BE neuroblastoma cells. We analysed the molecular signalling pathway involved in the apoptotic cell death. H2O2 induced an increased acetylation of p53 lysine 382, a reduction in mitochondrial membrane potential, an increased BAX/Bcl-2 ratio and consequently increased Poly (ADP-ribose) polymerase (PARP) cleavage. All these effects were blocked by EGb 761 treatment. Thus, EGb 761, acting as intracellular antioxidant, protects neuroblastoma cells against activation of p53 mediated pathway and intrinsic mitochondrial apoptosis. Our results suggest that EGb 761, protecting against oxidative-stress induced apoptotic cell death, could potentially be used as nutraceutical for the prevention and treatment of neurodegenerative diseases.

scholarly journals TNFR1-dependent cell death drives inflammation in Sharpin-deficient mice

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
James A Rickard ◽  
Holly Anderton ◽  
Nima Etemadi ◽  
Ueli Nachbur ◽  
Maurice Darding ◽  
...  
Keyword(s):  
Cell Death ◽  
Transcriptional Activity ◽  
Peyer's Patches ◽  
Peyer’S Patches ◽  
Inflammatory Phenotype ◽  
Dependent Cell ◽  
Perinatal Lethality ◽  
Induced Apoptosis ◽  
Deficient Mice

SHARPIN regulates immune signaling and contributes to full transcriptional activity and prevention of cell death in response to TNF in vitro. The inactivating mouse Sharpin cpdm mutation causes TNF-dependent multi-organ inflammation, characterized by dermatitis, liver inflammation, splenomegaly, and loss of Peyer's patches. TNF-dependent cell death has been proposed to cause the inflammatory phenotype and consistent with this we show Tnfr1, but not Tnfr2, deficiency suppresses the phenotype (and it does so more efficiently than Il1r1 loss). TNFR1-induced apoptosis can proceed through caspase-8 and BID, but reduction in or loss of these players generally did not suppress inflammation, although Casp8 heterozygosity significantly delayed dermatitis. Ripk3 or Mlkl deficiency partially ameliorated the multi-organ phenotype, and combined Ripk3 deletion and Casp8 heterozygosity almost completely suppressed it, even restoring Peyer's patches. Unexpectedly, Sharpin, Ripk3 and Casp8 triple deficiency caused perinatal lethality. These results provide unexpected insights into the developmental importance of SHARPIN.

Evaluation of GPER agonist G-1 combined with navitoclax in platinum-resistant and -sensitive ovarian cancer cells.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e13563-e13563
Author(s):  
Dennis C. DeSimone ◽  
Trung T. Nguyen ◽  
Eugen Brailiou ◽  
John C. Taylor ◽  
Gabriela Cristina Brailoiu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ovarian Cancer ◽  
Cancer Cells ◽  
Cell Cycle Progression ◽  
Parp Cleavage ◽  
Ovarian Cancer Cells ◽  
Cycle Progression ◽  
Platinum Based Chemotherapy ◽  
Induced Apoptosis ◽  
In Cells

e13563 Background: Most ovarian cancer patients are treated with platinum-based chemotherapy but eventually relapse with incurable disease. The G protein-coupled estrogen receptor GPER (GPR30) mediates Ca2+ mobilization in response to estrogen and G-1, a synthetic agonist. Large and sustained Ca2+ responses can lead to mitochondrial Ca2+ overload and apoptosis. Hence, we evaluated whether G-1 could induce apoptosis in cisplatin-sensitive A2780 and isogenic cisplatin–resistant CP70 (14-fold resistant), C30 (70-fold resistant) and C200 (157-fold resistant) human ovarian cancer cells. Bcl-2 and Bcl-xL protect mitochondria from Ca2+overload, and were overexpressed in these cisplatin-resistant cells; thus we also examined combining the Bcl-2 family inhibitor navitoclax with G-1. Methods: Cytoplasmic [Ca2+]c and mitochondrial [Ca2+]m were monitored using microscopy and fluorescent Ca2+ probes. Cell cycle, apoptosis and mitochondrial membrane potential (MMP) were assessed by flow cytometry of propidium iodide, Annexin V and DiIC1(5) -stained cells. The intracellular Ca2+ chelator BAPTA was used to block Ca2+mobilization. Results: Expression of the 53kDa GPER but not the 38 kDa isoform progressively increased with increasing cisplatin resistance. G-1 elicited sustained [Ca2+]c rises that correlated with 53 kDa GPER expression, followed by rises in [Ca2+]m. In all cells, 2.5 μM G-1 blocked cell cycle progression at G2/M, inhibited proliferation, and induced apoptosis (A2780 > C30 > CP70 ≥ C200). G-1 induced p53, caspase-3 and PARP cleavage, and MMP loss. BAPTA prevented G-1’s cell cycle and apoptotic effects in cells showing large Ca2+ mobilization responses but did not in cells with small Ca2+responses. Combining navitoclax with G-1 superadditively decreased cell viability and increased apoptosis. Conclusions: G-1 blocked cell cycle progression and induced apoptosis via a Ca2+-dependent pathway in cells expressing high 53 kDa GPER levels, but via a Ca2+-independent pathway in cells with low 53 kDa GPER expression. G-1 also interacted cooperatively with naviticlax. Therefore, G-1 plus navitoclax shows potential for therapeutic use in platinum-sensitive and -resistant ovarian cancer.

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