SKOV3 Ovarian Cancer Cells Research Articles

Cucurbitacin I induces cancer cell death through the endoplasmic reticulum stress pathway.

Endoplasmic reticulum stress (ERS) is usually involved in tumor development and progression, and anticancer agents have recently been recognized to induce ERS. Cucurbitacin-I showed a potent anticancer action by inducing apoptosis through the inhibition of signal transducer and activator of transcription 3 pathway and triggering autophagic cell death. It is not known whether ERS mediates the cancer cell death induced by cucurbitacin-I. Here, we investigated the role of ERS in cucurbitacin-I-treated SKOV3 ovarian cancer cells and PANC-1 pancreatic cancer cells. We confirmed that cucurbitacin-I caused cell death and stirred excessive ERS levels by activating inositol requiring enzyme 1α (IRE1α) and protein kinase R-like endoplasmic reticulum kinase (PERK), as well as PERK downstream factors, includingIRE1α and C/EBP homologous protein, but not activating transcription factor 6 (ATF6α) pathway, which was in parallel with the increased Bax and caspase-12-dependent ERS-associated apoptosis, autophagy and autophagy flux levels and caspase-independent nonapoptotic cell death. Furthermore, 4-phenylbutyrate, an ERS inhibitor, suppressed cucurbitacin-I-induced apoptosis, autophagy, autophagy flux, and autophagic cell death. Simultaneously, there are positive correlations among ERS and cucurbitacin-I-induced reactive oxygen speciesand Ca 2+ . Our results suggested that cucurbitacin-I-induced cancer cell death through the excessive ERS and CHOP-Bax and caspase-12-dependent ERS-associated apoptosis, as well as ERS-dependent autophagy, autophagy flux, and caspase-independent nonapoptotic cell death. These novel signaling insights may be useful for developing new, effective anticancer strategies in oncotherapy.

Marsdenia Tenacissima Extract Inhibits Proliferation and Promotes Apoptosis in Human Ovarian Cancer Cells.

BACKGROUND Marsdenia tenacissima extract (MTE) is a traditional Chinese medicine that can be effectively used against various cancers. However, to the best of our knowledge, its role in ovarian cancer is not known. This study investigated the effects of MTE on human ovarian cancer SKOV3 cells. MATERIAL AND METHODS The viability and cell cycle of SKOV3 cells were assessed using the cell counting kit-8 (CCK-8) and propidium Iodide (PI) staining kit, respectively. Cell apoptosis and mitochondrial membrane potential (MMP) were detected by flow cytometry. The expression levels of proliferation-related and apoptosis-related factors were tested by quantitative real-time PCR (qRT-PCR) and Western blot assays, respectively. RESULTS We found that MTE markedly reduced the viability of SKOV3 cells in dose-dependent and time-dependent manners. MTE induced cell cycle arrest by downregulating the levels of cyclin D1and cyclin B1. MTE (10, 20, and 40 mg/mL) markedly increased apoptosis rates (2.77±0.6%, 4.95±0.97%, and 12.16±0.69%, respectively), and enhanced the loss of MMP. MTE obviously downregulated the expression of B cell lymphoma-2 (Bcl-2) and upregulated the expression levels of fibroblast-associated (Fas), Fas ligand (FasL), cleaved cysteinyl aspartate-specific proteinas-3 (caspase-3), and Bcl-2-associated X protein (Bax) compared to the control group. In addition, the expressions of phosphorylated mammalian target of rapamycin (p-mTOR), phosphorylated protein kinase B (p-AKT), and phosphorylated-phosphatidylinositol 3 kinase (p-PI3K) were decreased by MTE. CONCLUSIONS MTE inhibited proliferation and induced apoptosis of SKOV3 cells. The depression of the PI3K/AKT/mTOR pathway may augment the protective effect of MTE. Thus, MTE might be expected to be a new drug for curing ovarian cancer.

Silencing the expression of MTDH increases the radiation sensitivity of SKOV3 ovarian cancer cells and reduces their proliferation and metastasis.

Ovarian cancer has a high mortality rate among women worldwide. Radiotherapy is considered an effective method of ovarian cancer treatment, however, radioresistance presents a challenge. It is necessary to develop techniques that can increase radiosensitivity in ovarian cancer, and gene therapy is a promising option. The aim of the present study was to investigate the effects of metadherin (MTDH) silencing on the radiosensitivity of ovarian cancer. Ovarian cancer tissues (n=273) and normal ovarian tissues (n=277) were used, as were SKOV3 ovarian cancer cells and the immortalized human ovarian epidermal HOSEpiC cell line. MTT, Transwell and wound-healing assays were performed to assess the proliferation, invasion and migration abilities of the SKOV3 cells. Colony-forming assays and flow cytometry were applied to detect the radiosensitivity and apoptosis of the SKOV3 cells. Nude mouse xenograft models were established to evaluate the effect of MTDH gene silencing on tumor growth and the efficacy of radiotherapy. Ovarian cancer, in tissues and cells, was demonstrated to have a high level of MTDH. Additionally, MTDH silencing was found to significantly inhibit proliferation, migration and invasion, and induce apoptosis in SKOV3 cells, and it was suggested that MTDH depletion significantly increased the sensitivity of the SKOV3 cells to X-ray radiation. MTDH silencing enhanced radiosensitivity and delayed tumor growth in the nude mouse xenograft model. Collectively, the results obtained in the present study suggest the potential role of MTDH silencing as a technique for ameliorating radioresistance in ovarian cancer. The present study provides a promising experimental basis for the improvement of ovarian cancer radiotherapy treatment.

Construction and characterization of the recombinant immunotoxin RTA-4D5-KDEL targeting HER2/neu-positive cancer cells and locating the endoplasmic reticulum.

The specific targeting of immunotoxins enables their wide application in cancer therapy. The A-chain of the ricin protein (RTA) is an N-glycosidase that catalyzes the removal of adenine from the 28S rRNA, preventing protein translation and leading to cell death. Ricin is highly toxic but can only exert its toxic effects from within the cytoplasm. In this study, we linked the anti-HER2 single-chain variable fragment 4D5 scFv and the endoplasmic reticulum-targeting peptide KDEL to the C-terminal of the RTA to construct immunotoxin RTA-4D5-KDEL. In vitro experiments showed that the anticancer effect of RTA-4D5-KDEL towards ovarian cancer cells SKOV-3 increased 440-fold and 28-fold relative to RTA and RTA-4D5, respectively. RTA-4D5-KDEL had a strong inhibitory effect on HER2-overexpressing SKOV-3 cells and caused little damage to normal HEK-293 cells and H460 lung cancer cells. Immunofluorescence experiments showed that the immunotoxin RTA-4D5 could specifically bind to SKOV-3 cells, but not to normal cells HEK-293. The immunotoxin RTA-4D5-KDEL could rapidly localize the recombinant protein to the endoplasmic reticulum. These results suggest that the recombinant immunotoxin RTA-4D5-KDEL has a strong inhibitory effect on ovarian cancer cells that overexpress HER2 but little harm to the normal cells.

Alpinetin inhibits proliferation and migration of ovarian cancer cells via suppression of STAT3 signaling.

Natural bioactive components are increasingly being applied in cancer research. Alpinetin is a type of natural flavonoid primarily derived from Alpiniakatsumadai Hayata, which exhibits anti‑bacterial and anti‑inflammatory properties. Therefore, it may possess anticancer potential and be employed therapeutically for different diseases. The aim of the present study was to investigate the anticancer effects of alpinetin on the SKOV3 ovarian cancer cell line. The effect of alpinetin treatment on SKOV3 cell proliferation, apoptosis, spheroid and colony formation were measured using Cell Counting kit‑8, cell apoptosis, 3D spheroid and colony formation assays, respectively. Analysis of the cell cycle was performed using flow cytometry. Western blot analysis was used to determine the protein expression levels of B‑cell lymphoma (Bcl)‑2, Bcl‑2‑associated X protein, cleaved caspase‑3, cleaved poly (ADP‑ribose) polymerase (PARP), cyclin D1, cyclin‑dependent kinase (CDK)4, CDK6, signal transducer and activator of transcription (STAT) 3, phosphorylated (p)‑STAT3, c‑myc, survivin, tissue inhibitor of metalloproteinase (TIMP)‑1, TIMP‑2, matrix metalloproteinase (MMP)‑2 and MMP‑9. In addition, a wound healing assay was used to determine cancer cell migration. The results revealed alpinetin suppressed cell viability and induced apoptosis of SKOV3 cells in a dose‑ and time‑dependent manner, and cells were arrested in the G1 phase. Alpinetin treatment upregulated protein expression levels of Bax, cleaved caspase‑3 and PARP, and downregulated protein expression levels of Bcl‑2, cyclin D1, CDK4 and CDK6. Alpinetin also inhibited cell migration, through increased protein expression levels of TIMP‑1 and TIMP‑2, and decreased protein expression levels of MMP‑2 and MMP‑9. Alpinetin also significantly suppressed colony and spheroid formation by SKOV3 cells. In addition, the STAT3 pathway was suppressed as demonstrated by downregulation of p‑STAT3 and reduced expression of downstream factors, including c‑myc and survivin. Overall, these results indicated that alpinetin may have anticancer effects on human ovarian cancer by inhibiting the STAT3 signaling pathway.

Girinimbine Inhibits the Proliferation of Human Ovarian Cancer Cells In Vitro via the Phosphatidylinositol-3-Kinase (PI3K)/Akt and the Mammalian Target of Rapamycin (mTOR) and Wnt/β-Catenin Signaling Pathways.

BackgroundWorldwide, ovarian cancer is increasing in prevalence and has a high mortality rate. Girinimbine is a carbazole alkaloid isolated from Murraya koenigii (the curry tree) and is used in Chinese herbal medicine. The aim of this study was to evaluate the effects of girinimbine on cell proliferation, cell migration, and apoptosis in human ovarian cancer cells in vitro.Material/MethodsA human ovarian cancer cell line panel, which included SKOV3 cells and the SV40 immortalized normal human ovarian cell line, were treated with increasing doses of girinimbine. Cell proliferation was evaluated using the MTT assay. Confocal immunofluorescence using 4′,6-diamidino-2-phenylindole (DAPI), Annexin-V, and propidium iodide (PI) were used to measure cell apoptosis. Cell migration and invasion were determined by transwell assays. Protein expression was determined by Western blot.ResultsGirinimbine inhibited SKOV3 ovarian cancer cell proliferation in a dose-dependent manner. The half maximal inhibitory concentration (IC50) of grinimbine was 15 μM for the SKOV3 cells, and 120 μM for the SV40 cells. Grinimbine treatment resulted in apoptosis of SKOV3 cells, from 2.2% in untreated cells to 58.8% at a dose of 30 μM, which was associated with an increase in the Bax/Bcl-2 ratio. Girinimbine inhibited cell migration and invasion of the SKOV3 cancer cells in vitro and inhibited the PI3K/AKT/mTOR and Wnt/β-catenin signaling pathways.ConclusionsGirinimbine, a carbazole alkaloid used in Chinese herbal medicine, inhibited the proliferation and cell migration of human ovarian cancer cells in vitro, in a dose-dependent manner, via the PI3K/Akt/mTOR and Wnt/β-catenin signaling pathways.

Abstract B49: The role of LATS kinases in regulation of CDK4/6 in ovarian cancer

Abstract High-grade ovarian carcinoma (HGSOC) is the most lethal gynecologic cancer with little improvement in survival over the last decade. Identification of the “druggable” cancer-driving pathways could facilitate the development of effective treatments for HGSOC patients. Oncogenomic analyses of HGSOC tumors revealed widespread genome alterations but only a few recurrent mutations that could be targeted by either approved or experimental therapeutics. We observed copy number losses of genes encoding LATS1 and LATS2 tumor suppressor protein kinases in approximately 60% of HGSOC tumors analyzed by TCGA study. Loss of LATS kinases could contribute to cancer pathogenesis because of their important role in the Hippo tumor suppressor pathway that is known to control cell invasion, migration, and proliferation though inhibition of the oncogenic transcription factors YAP and TAZ. Here, we describe an alternative mechanism by which LATS kinases can control cell proliferation and discuss the implications of this finding for HGSOC treatment. In previous studies, LATS2 kinase was shown to promote growth suppressor function of retinoblastoma family. LATS2 phosphorylation of DYRK1A protein kinase promoted assembly of the transcriptional repressor DREAM. DREAM assembles during G0/G1 when RB-like p130 binds E2F4, DP1 and a stable complex of five proteins, including RBBP4, LIN9, LIN37, LIN52, and LIN54 (MuvB core), and inhibits the expression of more than 800 cell cycle-regulated genes. Here, we report that LATS1 and LATS2 function redundantly in the regulation of DREAM in SKOV3 ovarian cancer cells and in the nontransformed fallopian tube epithelial (FTE) cells. Downregulation of LATS1 and LATS2 expression resulted in increased proliferation and promoted transformed phenotype of these cells, suggesting that loss of these genes could contribute to ovarian cancer aggressiveness. Interestingly, we observed that depletion of both LATS kinases in these cells resulted in markedly increased phosphorylation of the retinoblastoma family proteins pRB and p130. This phosphorylation was likely mediated by CDK4/6 kinases because it was completely abolished by treatment of cells with the highly selective CDK4/6 inhibitor, PD-0332991 (palbociclib). We investigated the mechanism of CDK4/6 activation, and found that downregulation of LATS kinases in SKOV3 or FTE cells resulted in a markedly increased expression of cyclin D1 at the protein, but not at the mRNA level. In a recent study, loss of the CDK4/6 inhibitor, p16, correlated with increased sensitivity of ovarian cancer cell lines to palbociclib. Therefore, we tested whether aberrant activation of CDK4/6 in LATS1/2-depleted cells could similarly predispose ovarian cancer cells to palbociclib growth inhibition. Indeed, downregulation of LATS kinases in SKOV3 cells resulted in 3-fold decrease of palbociclib IC50 compared to control cell lines. Importantly, clinical trials using palbociclib and analogous drugs in several types of cancer revealed a wide range of outcomes, emphasizing significance of research that will help to predict tumor sensitivity. Currently, there are no HGSOC-specific clinical trials using palbociclib. Our findings reported here support the concept that frequent genetic losses of LATS1 and LATS2 kinases in HGSOC could provide a rationale for using CDK4/6 inhibitors, such as palbociclib, for treatment of patients suffering from this disease. Citation Format: Siddharth Saini, Fatmata Sesay, Larisa Litovchick. The role of LATS kinases in regulation of CDK4/6 in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B49.

Abstract B04: The bromodomain inhibitor INCB054329 enhances olaparib response in ovarian cancer cells by reducing homologous recombination efficiency

Abstract Background: Lack of effective treatment options for high-grade serous ovarian cancers (HGSOC) retaining functional homologous recombination (HR) DNA repair is a significant clinical problem. HR-proficient HGSOC tumors have poorer clinical outcomes and show relative resistance to DNA-damaging poly ADP ribose polymerase inhibitors (PARPi). We and others have shown that using epigenetic drugs to reduce HR efficiency in HR-proficient HGSOC sensitizes these cancer cells to DNA-damaging agents. One mechanism by which these drugs reduce HR efficiency is by transcriptional downregulation of HR pathway components. An emerging class of epigenetic mediators of protumorigenic transcription is the bromodomain (BRD) family of proteins, and BRD inhibitors (BRDi) have shown promising preclinical antitumor efficacy. However, it is unknown whether BRDi sensitize HR-proficient HGSOC to DNA-damaging agents. Objective: To test the hypothesis that BRDi decrease efficiency of HR DNA repair in HR-proficient ovarian cancer cells, thereby sensitizing them to PARPi. Methods: Cultured HR-proficient ovarian cancer cell lines, OVCAR-3, OVCAR-4, and SKOV-3, were treated with 0.01% DMSO vehicle, the PARPi olaparib (Astra Zeneca), the BRDi INCB054329 (Incyte Corporation), or the INCB054329/olaparib combination for 24-72 hours. Drug effects on HR repair efficiency were quantified by IF assays measuring formation of RAD51 foci after cisplatin-induced DNA damage, and GFP expression in cells co-transfected with I-Sce1 endonuclease and the DRGFP reporter plasmid. Sulforhodamine B (SRB) assays assessed cell growth. High-content immunofluorescence (IF) assays measured drug effects on adherent cell number, apoptosis (cleaved caspase-3), DNA damage (pH2AX), and cell cycle indices. Protein levels of the key HR component BRCA1, pH2AX and cleaved PARP were assessed by Western blot. Nude mice injected subcutaneously with SKOV-3 cells were treated by oral gavage with vehicle, INCB054329, olaparib, and combined INCB054329/olaparib for 19 days. Tumor volume was measured by serial caliper measurements. Harvested tumors were assayed for expression levels of HR components, and markers of proliferation, apoptosis, and DNA damage by Western blot and immunohistochemistry. Statistical significance (p<0.05) was determined by Student’s t test. Results: INCB054329 significantly reduced HR efficiency alone and in combination with olaparib in cultured cells. Consistent with reduced HR repair, the INCB054329/olaparib combination reduced levels of BRCA1 and increased levels of pH2AX, cleaved caspase-3, and cleaved PARP, compared to either drug alone. Moreover, the combination of INCB054329 and olaparib synergistically reduced cell growth following isobologram analyses of SRB experiments. Consistent with these results, cell cycle analyses demonstrated that the drug combination cooperatively reduced the number of adherent cells and cells in S phase, and induced a G0/G1 arrest. Similar drug effects were observed in vivo. Combined INCB054329/olaparib treatment inhibited tumor volume at an earlier time point than either drug alone (day 14 of treatment), and significantly reduced both tumor volume and weight at sacrifice compared to single-drug treatment. In tumors treated with the INCB054329/olaparib combination, we confirmed cooperative downregulation of BRCA1 and the proliferation marker PCNA, and cooperative upregulation of the apoptosis markers cleaved PARP and Bax, the cell cycle inhibitor p21, and pH2AX. Conclusions: Our results suggest that BRDi reduce efficiency of HR repair in HR-proficient ovarian cancer cells, sensitizing them to PARPi therapy. These findings have important implications for expanding the use of PARPi in HR-proficient HGSOC through rational combinations with epigenetic drugs such as BRDi that target the HR pathway. This has the potential to benefit a substantial number of women with this devastating disease. Citation Format: Andrew J. Wilson, Matthew Stubbs, Phillip Liu, Bruce Ruggeri, Dineo Khabele. The bromodomain inhibitor INCB054329 enhances olaparib response in ovarian cancer cells by reducing homologous recombination efficiency. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B04.

A hierarchical assembly strategy to engineer dextran-enveloped polyurethane nanopolyplexes for robust ovarian cancer gene therapy

A hierarchical assembly strategy is herein investigated to generate bio-responsive, dextran-enveloped, bioreducible polyurethane nanopolyplexes for nonviral gene therapy against ovarian tumor. Initially, a group of poly(urethane amine)s were designed and characterized for in vitro gene transfection. The polyurethane containing 1,4-bis(3-aminopropyl)piperazine residue (PUBAP) could induce the best in vitro transfection efficacy against SKOV-3 or A2780 ovarian cancer cells. Next, dextran-enveloped PUBAP polyplexes (e-polyplexes) were constructed by a hierarchical assembly procedure involving gene neutralization with PUBAP and subsequent gene condensation with a cationic dextran (SSDP800). Such dextran comprised dextran (15 kDa) as the main chain and multiple disulfide-linked branched polyethylenimine (BPEI) oligomers as the side grafts. Additionally, folate-dextran-enveloped PUBAP polyplexes (FA-e-polyplexes) were fabricated by folate-modified SSDP800. These nanoscale-enveloped polyplexes elicited an improved colloidal stability against salt ions and negatively charged heparin, efficient endosomal escaping, and bioreduction-triggered intracellular gene release. In vitro transfection against SKOV-3 cells illustrated that FA-e-polyplexes exerted higher transfection efficiency in the serum than e-polyplexes and 25 kDa BPEI-polyplexes. In vivo, FA-e-polyplexes yielded higher transgene expression level than e-polyplexes in an SKOV-3 tumor-bearing nude mouse model. In the tumor gene therapy with a small hairpin RNA silencing vascular endothelial growth factor, FA-e-polyplexes afforded higher tumor growth inhibition than polyplexes of folate-PEGylated PUBAP and 25 kDa linear polyethylenimine as positive controls. Importantly, such gene therapy had minor toxic effects on the health of the mouse. This work highlights a practical hierarchical assembly method to construct innovative enveloped polyurethane nanopolyplexes enabling robust ovarian cancer gene therapy. Statement of SignificanceIt is indispensable to rationally update binary cationic polyplexes into ternary polyplexes for vigorous tumor gene therapy. In this work, we have confirmed that a hierarchical assembly strategy, by using initial gene neutralization and subsequent gene condensation, is facile and effective to promote cationic polyurethane polyplexes into ternary folate-dextran-enveloped polyurethane polyplexes with a relatively high gene-loading capacity. The enveloped polyplex system enables more efficient gene transfection than the PEGylated polyplex counterpart in ovarian cancer in vitro and in vivo, thereby affording robust ovarian cancer gene therapy. The development of innovative enveloped polyplexes may be a new direction for a non-viral gene delivery system.

Downregulation of high mobility group protein box-1 resensitizes ovarian cancer cells to carboplatin.

Ovarian cancer, one of the most common types of cancer, has the highest mortality among all gynecological malignancies. The development of acquired drug resistance is the leading cause of chemotherapy failure. To study the mechanism underlying drug resistance in ovarian cancer, a drug-resistant ovarian cancer SKOV3 cell line was developed using the chemotherapeutic agent carboplatin (SKOV3-Carb) in the present study. It has been reported that high-mobility group protein box-1 (HMGB1) is associated with the chemoresistance of tumor cells. Therefore, the probable involvement of HMGB1 in the development of carboplatin resistance in ovarian cancer SKOV3 cells was investigated. HMGB1 has been reported to be overexpressed in carboplatin-resistant SKOV3-Carb cells compared with control SKOV3 cells. Subsequently, the expression of HMGB1 was silenced by small interference RNA technology. Reverse transcription-quantitative polymerase chain reaction and western blot analysis indicated that mRNA and protein expression levels of HMGB1 were significantly inhibited in HMGB1-silenced cells. Cell proliferation and apoptosis analyses were performed to evaluate the effect of HMGB1 silencing on resistant ovarian cancer cells. An MTT assay revealed that the proliferation of HMGB1-silenced SKOV3 and SKOV3-Carb cells were decreased compared with the proliferation of non-silenced control cells. Additionally, HMGB1 protein expression levels in SKOV3 cells, but not in SKOV3-Carb cells, were decreased in response to carboplatin treatment. Annexin V-fluorescein isothiocyanate/propidium iodide staining demonstrated that HMGB1 silencing enhanced the effects of carboplatin in inducing the apoptosis of SKOV3-Carb cells relative to HMGB1 non-silenced control cells. The results of the present study suggested that HMGB1 may be involved in the development of carboplatin resistance in ovarian cancer SKOV3 cells and that HMGB1 silencing may induce the sensitization of carboplatin-resistant ovarian cancer cells to carboplatin. Therefore, HMGB1 may be considered as a potent therapeutic target for increasing the sensitivity of ovarian cancer cells to carboplatin in order to improve the treatment and prognosis of ovarian cancer.

Abstract LB-048: c-Kit enhances ovarian cancer stemness via upregulating phospho-PHB in the lipid raft domain of the plasma membrane

Abstract Phosphorylation of prohibitin (PHB) at threonine 258 (phospho-PHBT258) and tyrosine 259 (phospho-PHBY259) in human cancer-cell membranes is associated with an increase in the invasiveness and metastasis of cancer cells. Phospho-PHBY259 can regulate phospho-PHBT258 but not vice versa. Here, we found that stably overexpressing c-Kit in SKOV3 ovarian cancer cells (SKOV3_c-Kit) increased phospho-PHBY259, phospho-PHBT258 and PHB levels in the lipid raft domain of the plasma membrane. On the other hand, inhibition of c-Kit expression decreased PHB expression in SKOV3_c-Kit and Kuramochi ovarian cancer cells. Immunoprecipitation analysis showed that c-Kit interacts with PHB. SKOV3_c-Kit cells or SKOV3 cells overexpressing PHB in the lipid raft had higher levels of Notch3, NICD3 (Notch3 intracellular domain) and ABCG2 protein expression as compared to wild-type SKOV3 cells. Moreover, generation of SKOV3-G4 cells through serial passage of SKOV3 cells into nude mice ovary 4 times, revealed that the levels of c-Kit, Notch3, NICD3, phospho-PHBY259, phospho-PHBT258 and PHB in the lipid raft domain of SKOV3-G4 were significantly higher than wild-type SKOV3 cells. The SKOV3-G4 cells formed spheroids with stem cell-like properties such as higher expression of stem cell markers (Oct4, Nanog, SOX2). In addition, SKOV3-G4 cells exhibited more clonogenic potential and migration/invasion ability than wild-type SKOV3. These results suggest that c-Kit interacts with PHB in the lipid raft domain to phosphorylate PHB to increase invasiveness and stemness in ovarian cancer. Citation Format: Shu-Mei Y. Liang, Chia-Hsun Fang, Pu-Yuan Chang, Chi-Ming Liang. c-Kit enhances ovarian cancer stemness via upregulating phospho-PHB in the lipid raft domain of the plasma membrane [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-048.

Abstract 373: Bioluminescent non-lytic metabolite and cytotoxicity assays for cancer and immunotherapy research

Abstract The growing interest in understanding metabolic adaptations that occur in cancer and immune cells in the tumor microenvironment and the advances made towards developing effective immunotherapies have increased the need for sensitive and efficient methods for studying cell metabolism and viability. We have developed bioluminescent assays for monitoring cell metabolism and cytotoxicity during cell growth and in response to environmental conditions and treatments. The plate-based assays employ coupled enzyme reactions and bioluminescent NADH detection chemistry to detect metabolites and enzymes. We used the assays in a non-lytic format, analyzing samples of cell culture medium and leaving the cells intact for additional measurements or multiplexing with other assays. In this way, changes occurring in a single well of cells were followed over time by collecting medium at multiple time points. We used assays for glucose, lactate, glutamine and glutamate detection to obtain information about the key metabolic pathways, glycolysis and glutaminolysis, and metabolically profile cancer cell lines. When we compared the metabolism of two ovarian cancer cell lines, OVCAR-3 and SKOV-3, the rates for glucose consumption and lactate secretion were similar, but the SKOV-3 cells consumed more glutamine and secreted 20-fold more glutamate. Glutamate secretion by the OVCAR-3 cells was low, only 5-fold higher than background controls. Analysis of the metabolites in cell lysates further supported the conclusion that the two cell lines differed in glutamine metabolism. The metabolite detection assays were also used to monitor metabolic pathway reprogramming in response to the environment. The induction of glycolysis is an early, critical step in the activation of T cells, and results in increased lactate production. We investigated the requirements for T-cell activation by measuring lactate levels over time and observed robust activation only in the presence of glutamine and anti-CD3/anti-CD28 costimulatory signals. In addition to metabolite levels, cell culture medium was used to assess cytotoxicity by following lactate dehydrogenase enzyme (LDH) release into cell culture medium. We used this assay to study cancer cell cytotoxicity induced by immunotherapeutic treatments, including antibody-drug conjugates (ADC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The high sensitivity of the assay permitted the use of fewer cells, in 2D and 3D cultures, and the earlier detection of small changes (10 lysed cells in medium lacking serum). These sensitive bioluminescent assays require small amounts of sample and minimal sample processing to facilitate (1) identification of unique cancer cell metabolic signatures and potential therapeutic targets, (2) studies of connections between metabolism and effective immune cell function, and (3) the evaluation of cytotoxic immunotherapies. Citation Format: Donna M. Leippe, Natasha Karassina, Michael P. Valley, James J. Cali, Jolanta Vidugiriene. Bioluminescent non-lytic metabolite and cytotoxicity assays for cancer and immunotherapy research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 373.

Abstract 495: Leptin facilitates ovarian cancer metastasis through miR-28-3p and matrix metalloproteinase-1 in the omental tumor microenvironment

Abstract Advanced stage serous ovarian cancers spread beyond the pelvis and preferentially metastasize to the omentum, which is mainly composed of adipose tissue, suggesting that the omental microenvironment is a favorable niche for ovarian cancer cells. By comparing the transcriptome profiles of microdissected ovarian cancer tissues from the primary ovarian site and from the omental site, we identified matrix metalloproteinase-1 (MMP-1) as one of the most significant genes that was up-regulated in the omental site compared with that in the primary site. To identify mediators secreted by the omental microenvironment that up-regulated MMP-1 in the omental site tumors, transcriptome profiling was performed on microdissected ovarian tumor associated adipose tissue in the omental site and the ovarian stromal tissue in the primary ovarian tumor site. We identified leptin as one of the top differentially expressed genes in ovarian tumor associated adipose tissue in the omental site, suggesting that it might up-regulate MMP-1 in the omental ovarian cancer. To further delineate the role of MMP-1 in mediating the effect of leptin on ovarian cancer cell metastasis, ovarian cancer SKOV3 and OVCA433 cells were treated with leptin. The results showed that leptin increased MMP-1 mRNA and protein expression as assessed by quantitative RT-PCR and Western blot analyses, respectively. Furthermore, we demonstrated that leptin induced both the cell migration and invasion potential of SKOV3 and OVCA433 cells, and the effects were abrogated by transfecting cells with MMP-1 siRNAs. In addition, using Ion Torrent next generation sequencing, we found that miR-28-3p is down-regulated in leptin-treated SKOV3 and OVCA433 cells compared to the untreated cells, suggesting that it may play a role in mediating the effect of leptin on MMP-1 expression and the subsequent ovarian cancer cell motility and invasion potential. In conclusion, leptin secreted by the adipocytes in ovarian cancer associated omental microenvironment may be important in facilitating the ovarian cancer cell metastasis through the miR-28-3p/MMP-1 pathway. Citation Format: Chi Lam Au Yeung, Ngai Na Co, Tsz-Lun Yeung, Samuel C. Mok. Leptin facilitates ovarian cancer metastasis through miR-28-3p and matrix metalloproteinase-1 in the omental tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 495.

WITHDRAWN: 3,3′-Diindolylmethane suppresses ovarian cancer cell viability and metastasis and enhances chemotherapy sensitivity via STAT3 and Akt signaling in vitro and in vivo

Signal transducer and activator of transcription-3 (STAT3) protein is constitutively activated in ovarian cancer. The purpose of this study was to investigate the effects of 3,3'-diindolylmethane (DIM) on the regulation of STAT3 signaling and ovarian cancer cell viability, invasion, and sensitivity to chemotherapy. Ovarian cancer SKOV3 and A2780 cell lines were treated with various concentrations of DIM for different periods of time for assessment of cell viability as well as gene expression before and after knockdown of STAT3 expression using STAT3 shRNA. DIM treatment potently suppressed the viabilities of ovarian cancer cells. Consequently, DIM inhibited xenograft growth in nude mice. In addition, at the gene level, DIM inhibited phosphorylation of STAT3 and AKT proteins and expression of their downstream proteins. Moreover, knockdown of STAT3 expression significantly enhanced DIM antitumor activity and cisplatin sensitivity. Their combination suppressed the protein expression of survivin, Bcl-2, Mcl-1, HIF-1α, VEGF, and MMPs, but activated caspase-3. Taken together, the antitumor activity of DIM is via inhibition of the STAT3 and Akt signaling pathways. The combination of STAT3 knockdown with DIM treatment could be further evaluated as a therapeutic strategy for the treatment of advanced ovarian cancer.

Abstract 1776: High-affinity anti-galectin-3 antibodies targeting oncogenic properties in serous ovarian cancer

Abstract Objective: Galectin-3 (LGALS3) regulates the interaction of surface proteins with the extracellular membrane domain and mediates a signal cascade leading to invasion, oncogene activation and growth. We targeted LGALS3 cancer promotion by developing high-affinity anti-galectin-3 antibodies directed either at the carbohydrate recognition domain (CRD) of the galectin-3 carboxyl-terminus (to block sugar binding) or at the N-terminus (NT) (to inhibit N-terminal mediated oligomerization of galectin-3) in order to prevent formation of the cell surface galectin lattice and suppress the oncogenic effects of the glycoprotein. Methods: Two distinct types of murine monoclonal antibodies (MAb) were generated by creating hybridomas from mice immunized either with a fusion protein derived from the 128-amino acid LGALS3 carboxyl-terminus fused to a human IgG1-Fc backbone followed by immunization with a recombinant human LGALS3 protein, or the mice were further immunized with a 25-amino acid peptide derived from the LGALS3-N-terminus conjugated to KLH. The resulting antibodies were confirmed to recognize either the LGALS3 CRD or NT by ELISA. Functional assays (Matrigel invasion, inhibition of laminin binding, and oncogene expression) and cell surface expression by FACS analysis were utilized to confirm MAb binding and activity. Mice bearing A2780 and SKOV3 ovarian cancer cell lines were treated with purified MAb to determine the effects of MAb on tumor growth in vivo. Data were analyzed for statistical significance using Student's t-test. Results: Initial screening yielded two candidate antibodies, of which one (named 14D11.2D2) had superior binding to galectin-3 by ELISA and Surface Plasmon Resonance (SPR). The antibody dissociation constant was 14.6 nM by SPR. At a concentration as low as 150 nM, the 14D11.2D2 MAb decreased galectin-3 binding to laminin by 36.6% compared to untreated control. In a Matrigel invasion assay, the anti-CRD MAb significantly inhibited invasion by MUC16 expressing SKOV3 cells and by wild-type MUC16 positive OVCAR3 cells. MUC16 expressing A2780 cells showed a statistically nonsignificant decrease in invasion. In vivo experiments with MUC16 expressing A2780 or SKOV3 tumors showed a significant inhibition of tumor growth in mice treated with anti-CRD MAb. Six candidate antibodies directed against the LGALS3-N-terminus have been identified. Characterization of two NT antibodies (1F5.D4 and 7D4.A1) is under way via in in vitro and in vivo experiments. Conclusion: High-affinity CRD and NT anti-galectin-3 antibodies inhibit MUC16-induced cellular invasion and growth. Anti-LGALS3 MAbs hold therapeutic potential for inhibition of glycosylation-dependent invasion. Citation Format: Dharmarao Thapi, Marina Stasenko, Thomas White, Sven J. Walderich, Noah Feit, Frances Weis-Garcia, David R. Spriggs. High-affinity anti-galectin-3 antibodies targeting oncogenic properties in serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1776.

Abstract 4424: Micro-RNA mediated deregulation of hHB-EGF in ovarian cancer

Abstract Background: Aberrant expression of microRNAs (miRNAs) has been implicated in human carcinogenesis where they function as oncogenes or tumor suppressor genes. Overexpression of HB-EGF, member of the epidermal growth factor (EGF) family, plays pivotal role in ovarian carcinogenesis. However the molecular mechanisms underlying miRNA(s) mediated aberrant function of HB-EGF in ovarian carcinoma remain unresolved. Here, we investigated the miRNA(s) mediated deregulation of HB-EGF in human ovarian carcinogenesis. Methods: Web-based bioinformatics algorithms were used to identify candidate miRNAs with putative binding sites on target hHB-EGF mRNA. Expression of miRNAs and mRNA were checked by real-time PCR whereas protein expression was investigated by western blotting assay. Luciferase reporter assay was carried out to examine miR-27a target on 3´UTR of hHB-EGF mRNA. Changes in the oncogenic effects of HB-EGF were studied after gain/loss -of-function of the miR-27a using soft agar colony formation assay and 3D collagen culture. In vivo tumor growth potential of miR-27a was measured in BALB/c nude mice. Results: By using web-based bioinformatics algorithms 11 miRNAs ((miR-27a/b, -29a/b/c, -96, -128, -132, -182,-194, -212) having putative binding sites on the 3′UTR of hHB-EGF mRNA have been selected for further analysis. Among the putative candidates miR-27a showed maximum reciprocal expression with hHB-EGF and thus we selected miR-27a as promising candidate for hHB-EGF regulation. Overexpression of miR-27a significantly inhibited the hHB-EGF expression in SKOV3 cells by almost 50% and miR-27a inhibitors (anti-miR-27a) upregulated the expression of endogenous hHB-EGF mRNA whereas miR-27a overexpression decreased hHB-EGF protein level in MCAS cells suggesting that miR-27a modulate endogenous hHB-EGF expression both at mRNA and protein level. Luciferase reporter assay revealed that miR-27a directly targets 3´UTR of hHB-EGF mRNA at the predicted site. Ectopic expression of miR-27a suppresses in-vitro growth of SKOV3 ovarian cancer cells. In addition, in-vivo tumorigenecity of SKOV3 cells was also inhibited by miR-27a treatment. Conclusion: The current study has identified novel miR-27a that negatively regulates hHB-EGF expression by directly targeting 3`UTR of its mRNA. This miR-27a might act as oncosupressor in human epithelial ovarian cancer. Citation Format: Md. Mustafizur Rahman, Razia Sultana, Ryo Iwamoto, Eisuke Mekada. Micro-RNA mediated deregulation of hHB-EGF in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4424.

Erythrocyte-Derived Optical Nanoprobes Doped with Indocyanine Green-Bound Albumin: Material Characteristics and Evaluation for Cancer Cell Imaging.

Nanosize structures activated by near-infrared (NIR) photoexcitation can provide an optical platform for the image-guided removal of small tumor nodules. We have engineered nanoparticles derived from erythrocytes that can be doped with NIR fluorophore indocyanine green (ICG). We refer to these constructs as NIR erythrocyte-derived transducers (NETs). The objective of this study was to determine if ICG-bound albumin (IbA), as the doping material, could enhance the fluorescence emission of NETs, and evaluate the capability of these nanoprobes in imaging cancer cells. Erythrocytes were isolated from bovine whole blood and depleted of hemoglobin to form erythrocyte ghosts (EGs). EGs were then extruded through nanosize porous membranes in the presence of 10-100 μm ICG or Iba (1:1 molar ratio) to form ICG- or IbA-doped NETs. The resulting nanosize constructs were characterized for their diameters, zeta-potentials, absorption, and fluorescence emission spectra. We used fluorescence microscopic imaging to evaluate the capability of the constructs in imaging SKOV3 ovarian cancer cells. Based on dynamic light-scattering measurements, ICG- and IbA-doped NETs had similar diameter distributions (Z-average diameter of 236 and 238 nm, respectively) in phosphate-buffered saline supplemented with 10% fetal bovine serum, which remained nearly constant over the course of 2 h at 37 °C. Despite a much-lower loading efficiency of IbA (∼0.7-8%) as compared to ICG (10-45%), the integrated normalized fluorescence emission of IbA-NETs was 2- to 6-fold higher than ICG-doped NETs. IbA-NETs also demonstrated an enhanced capability in fluorescence imaging of SKOV3 ovarian cancer cells, and can serve as potentially effective nanoprobes for the fluorescence imaging of cancerous cells.

Anti-cancer effects of Kaempferia parviflora on ovarian cancer SKOV3 cells

BackgroundKaempferia parviflora (KP) is an herb found in the north of Thailand and used as a folk medicine for improving vitality. Current reports have shown the anti-cancer activities of KP. However, the anti-cancer effects of KP on highly aggressive ovarian cancer have not been investigated. Therefore, we determined the effects of KP on cell proliferation, migration, and cell death in SKOV3 cells.MethodsOvarian cancer cell line, SKOV3 was used to investigate the anti-cancer effect of KP extract. Cell viability, cell proliferation, MMP activity, cell migration, and invasion were measured by MTT assay, cell counting, gelatin zymography, wound healing assay, and Transwell migration and invasion assays, respectively. Cell death was determined by trypan blue exclusion test, AnnexinV/PI with flow cytometry, and nuclear staining. The level of ERK and AKT phosphorylation, and caspase-3, caspase-7, caspase-9 was investigated by western blot analysis.ResultsKP extract was cytotoxic to SKOV3 cells when the concentration was increased, and this effect could still be observed even though EGF was present. Besides, the cell doubling time was significantly prolonged in the cells treated with KP. Moreover, KP strongly suppressed cell proliferation, cell migration and invasion. These consequences may be associated with the ability of KP in inhibiting the activity of MMP-2 and MMP-9 assayed by gelatin zymography. Moreover, KP at high concentrations could induce SKOV3 cell apoptosis demonstrated by AnnexinV/PI staining and flow cytometry. Consistently, nuclear labelling of cells treated with KP extract showed DNA fragmentation and deformity. The induction of caspase-3, caspase-7, and caspase-9 indicates that KP induces cell death through the intrinsic apoptotic pathway. The antitumor activities of KP might be regulated through PI3K/AKT and MAPK pathways since the phosphorylation of AKT and ERK1/2 was reduced.ConclusionsThe inhibitory effects of KP in cell proliferation, cell migration and invasion together with apoptotic cell death induction in SKOV3 cells suggest that KP has a potential to be a new candidate for ovarian cancer chemotherapeutic agent.

Effects of cigarette smoke extracts on apoptosis and oxidative stress in two models of ovarian cancer in vitro

Cigarette smoke contains thousands of harmful components, many of which exert toxic effects on reproductive organs, including the ovaries, and their development. In this study, we investigated the effects of cigarette smoke extract (CSE) on cell proliferation, apoptosis and oxidative stress in the SKOV3 and OVCAR3 ovarian cancer cell lines. A water soluble tetrazolium (WST) assay revealed that the cell proliferation of both ovarian cells was significantly inhibited by three types of CSE in a concentration-dependent manner. Western blot analysis showed that the inhibition of cell proliferation was related to regulation of cell cycle-related protein expression. To determine the effects of cigarette smoke on the death of ovarian cells, we conducted a TUNEL assay, which revealed a time-dependent increase in TUNEL-stained cells in response to CSE exposure. Moreover, the protein expression of the Bcl-2 family signaling pathway observed upon Western blot analysis were consistent with the results of the TUNEL assay. We also examined the reactive oxygen species (ROS) generation in two ovarian cell lines using DCF-DA (5(6)-Carboxy-2′,7′-dichlorofluorescein diacetate) assay and ROS-Glo™ H2O2 assay to investigate the oxidative stress caused by CSE. Both ROS detection assays demonstrated an increase in the amount of ROS by CSE in ovarian cells, which was consistent with the results observed for the Keap1-Nrf2 pathway upon Western blot analysis. Taken together, these results suggest that CSE may induce cell proliferation inhibition and oxidative stress in ovarian cells.

PO-186 Generation of the GLI1, GLI2 and GLI3 knock-out ovarian cancer cell lines using the CRISPR/Cas9

Introduction Hedgehog signalling pathway is a developmental signalling pathway which is dormant in most adult differentiated tissues, but aberrantly activated in various tumours. In ovarian tumours it can be activated in a canonical way, by the SHH ligand, or the non-canonical way, by upregulation of the GLI transcription factors. Expression of GLI1 is usually associated with tumour progression in a clinical setting, but GLI2 and GLI3 also play a role by modifying the activity of GLI1 and transcription of their common transcriptional targets. In ovarian cancer, GLI3 protein is expressed in the full-length activator form, and not the shortened repressor form which is the predominant form for GLI3 protein. Material and methods To study the roles of the three GLI proteins in detail, we used the CRISPR/Cas9 guided gene editing to generate knock-out lines for each of the three GLI proteins. The sgRNA were designed using the online tool at crispr.mit.edu web site, and cloned into the pX330 vector expressing the sgRNA and the Cas9 protein. The sgRNA were designed to target the region close to the ATG site of each of the three GLI proteins, and after Cas9 nuclease activity the double stranded DNA break should be repaired by non-homologous end joining, which generates indels and leads to frameshift. Frameshift close to the ATG often leads to early termination of the protein, generating a knock-out. The SKOV3 ovarian cancer cell line was transfected with each of the vectors, the cells were split to the density of a single cell/well in a 96-well plate, and cell lines were expanded from single cells to obtain homogenous lines. Results and discussions SKOV3 ovarian cancer cell lines with knock outs in each of the three GLI proteins, GLI1, GLI2 and GLI3 were generated. The knock-out was confirmed by qRT-PCR and Western blot, and DNA was sequenced to show the exact genetic modification of the lines leading to the knock-out. The modified cell lines showed differences in cell morphology in relation to the original SKOV3 cell line immediately after expansion from the single cell. Conclusion The Hedgehog signalling pathway plays an important role in ovarian cancer. Cells harbouring knock out for the effector proteins of the Hedgehog signalling pathway, GLI1, GLI2 and GLI3 show distinct changes in cell morphology and viability. The Hedgehog signalling pathway, and more specifically GLI proteins, may be a good potential therapeutic target in management of ovarian cancer.