OVCAR-3 Human Ovarian Cancer Cells Research Articles

Tumor Treating Fields (TTFields) induce homologous recombination deficiency in ovarian cancer cells, thus mitigating drug resistance.

Ovarian cancer is the leading cause of mortality among gynecological malignancies. Carboplatin and poly (ADP-ribose) polymerase inhibitors (PARPi) are often implemented in the treatment of ovarian cancer. Homologous recombination deficient (HRD) tumors demonstrate increased sensitivity to these treatments; however, many ovarian cancer patients are homologous recombination proficient (HRP). TTFields are non-invasive electric fields that induce an HRD-like phenotype in various cancer types. The current study aimed to investigate the impact of TTFields applied together with carboplatin or PARPi (olaparib or niraparib) in preclinical ovarian cancer models. A2780 (HRP), OVCAR3 (HRD), and A2780cis (platinum-resistant) human ovarian cancer cells were treated in vitro with TTFields (1 V/cm RMS, 200 kHz, 72 h), alone or with various drug concentrations. Treated cells were measured for cell count, colony formation, apoptosis, DNA damage, expression of DNA repair proteins, and cell cycle. In vivo, ID8-fLuc (HRP) ovarian cancer cells were inoculated intraperitoneally to C57BL/6 mice, which were then treated with either sham, TTFields (200 kHz), olaparib (50 mg/kg), or TTFields plus olaparib; over a period of four weeks. Tumor growth was analyzed using bioluminescent imaging at treatment cessation; and survival analysis was performed. The nature of TTFields-drug interaction was dependent on the drug's underlying mechanism of action and on the genetic background of the cells, with synergistic interactions between TTFields and carboplatin or PARPi seen in HRP and resistant cells. Treated cells demonstrated elevated levels of DNA damage, accompanied by G2/M arrest, and induction of an HRD-like phenotype. In the tumor-bearing mice, TTFields and olaparib co-treatment resulted in reduced tumor volume and a survival benefit relative to olaparib monotherapy and to control. By inducing an HRD-like phenotype, TTFields sensitize HRP and resistant ovarian cancer cells to treatment with carboplatin or PARPi, potentially mitigating a-priori and de novo drug resistance, a major limitation in ovarian cancer treatment.

Porphin e6 complex loaded with gold nanorod mesoporous silica enhances photodynamic therapy in ovarian cancer cells in vitro.

A growing amount of experimental evidence has proven that the application of gold nanorods (AuNRs) in photodynamic therapy (PDT) can significantly enhance its therapeutic efficacy. The aim of this study was to establish a protocol for investigating the effect of gold nanorods loaded with the photosensitizer chlorin e6 (Ce6) on photodynamic therapy in the OVCAR3 human ovarian cancer cell line in vitro and to determine whether the PDT effect was different from that of Ce6 alone. OVCAR3 cells were randomly divided into three groups: the control group, Ce6-PDT group, and AuNRs@SiO2@Ce6-PDT group. Cell viability was measured by MTT assay. The generation of reactive oxygen species (ROS) was measured by a fluorescence microplate reader. Cell apoptosis was detected by flow cytometry. The expression of apoptotic proteins was detected by immunofluorescence and western blotting. The results showed that compared with that of the Ce6-PDT group, the cell viability of the AuNRs@SiO2@Ce6-PDT group was significantly decreased (P < 0.05) in a dose-dependent manner, and ROS production increased significantly (P < 0.05). The flow cytometry results showed that the proportion of apoptotic cells in the AuNRs@SiO2@Ce6-PDT group was significantly higher than that in the Ce6-PDT group (P < 0.05). Immunofluorescence and western blot results showed that the protein expression levels of cleaved caspase-9, cleaved caspase-3, cleaved PARP, and Bax in the AuNRs@SiO2@Ce6-PDT-treated-OVCAR3 cells were higher than those in the Ce6-PDT-treated cells (P < 0.05), and the protein expression levels of caspase-3, caspase-9, PARP, and Bcl-2 were slightly lower than those in the Ce6-PDT group (P < 0.05). In summary, our results show that AuNRs@SiO2@Ce6-PDT has a significantly stronger effect on OVCAR3 cells than the effect of Ce6-PDT alone. The mechanism may be related to the expression of Bcl-2 family and caspase family in the mitochondrial pathway.

Cytotoxicity Evaluation of Unmodified Paddlewheel Dirhodium(II,II)-Acetate/-Formamidinate Complexes and Their Axially Modified Low-Valent Metallodendrimers.

Two diphenyl formamidine ligands, four dirhodium(II,II) complexes, and three axially modified low-valent dirhodium(II,II) metallodendrimers were synthesized and evaluated as anticancer agents against the A2780, A2780cis, and OVCAR-3 human ovarian cancer cell lines. The dirhodium(II,II) complexes show moderate cytotoxic activity in the tested tumor cell lines, with acetate and methyl-substituted formamidinate compounds displaying increased cytotoxicity that is relative to cisplatin in the A2780cis cisplatin resistant cell line. Additionally, methyl- and fluoro-substituted formamidinate complexes showed comparable and increased cytotoxic activity in the OVCAR-3 cell line when compared to cisplatin. The low-valent metallodendrimers show some activity, but a general decrease in cytotoxicity was observed when compared to the precursor complexes in all but one case, which is where the more active acetate-derived metallodendrimer showed a lower IC50 value in the OVCAR-3 cell line in comparison with the dirhodium(II,II) tetraacetate.

Study on the effect of 5-aminolevulinic acid-mediated photodynamic therapy combined with cisplatin on human ovarian cancer OVCAR-3 ​cells

PurposeThis article explores the effect of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) combined with cisplatin (CDDP) on the apoptosis of human ovarian cancer cells and the mechanism of action of the combination therapy. Materials and methodsHuman ovarian cancer OVCAR-3 ​cells were cultured in vitro and divided into 5-ALA/PDT group, CDDP group and combined treatment group (5-ALA/PDT combined with different concentrations of CDDP). After administration of the corresponding drugs, a CCK-8 assay was used to detect the inhibition rate of cell proliferation. After Rhodamine 123 staining, mitochondrial membrane potential changes were observed under fluorescence microscopy. The apoptosis rate and reactive oxygen species (ROS) content were detected by flow cytometry. Western blotting was used to detect protein expression. ResultsThe CCK-8 assay showed that CDDP in combination with 5-ALA/PDT significantly enhanced cytotoxicity compared to treatment with CDDP alone and that low doses of CDDP were sufficient to induce these combination effects. The mitochondrial membrane potential in each combination treatment group gradually decreased with increasing CDDP concentration, while the apoptosis rate and reactive oxygen species (ROS) content detected by flow cytometry gradually increased. Western blotting assay showed that the expression of bax, cleaved caspase-9, cleaved caspase-3, and cleaved PARP was increased, while the expression of bcl-2, caspase-9, caspase-3, and PARP was decreased, and the differences were statistically significant (P ​< ​0.05). ConclusionsIn summary, 5-ALA/PDT combined with CDDP can effectively inhibit cell proliferation and promote apoptosis, and this combination may induce apoptosis by activating the mitochondrial pathway.

Betulin terpenoid targets OVCAR-3 human ovarian carcinoma cells by inducing mitochondrial mediated apoptosis, G2/M phase cell cycle arrest, inhibition of cell migration and invasion and modulating mTOR/PI3K/AKT signalling pathway.

The main purpose of the current research work was to study in vitro anticancer effects of betulin in OVCAR-3 human ovarian carcinoma cells along with examining its effects on cellular apoptosis, cell cycle phase distribution, cell migration and invasion and mTOR/PI3K/AKT signalling pathway. The cell proliferation of OVCAR-3 cells at various doses of the drug was studied by CCK8 cell viability assay. Effects on cell apoptosis were studied by fluorescence microscopy and western blot. Effects on cell cycle were evaluated by flow cytometry and western blot. Transwell assays were used to study effects on cell migration and invasion. The results indicated that betulin led to significant reduction of OVCAR-3 cell viability in a dose-dependent as well as time dependent manner. Betulin also led to reduction in cell colonies. The anticancer effects of betulin were due to the induction of apoptosis which was seen by increased apoptotic cells with yellow and orange fluorescence. Betulin prompted mitochondrial apoptosis which was also associated with alteration in the apoptosis-related protein expression (Bax, Bad and Bcl-2 and Bcl-xL). The molecule also led to G2/M phase cell cycle arrest on OVACR-3 ovarian carcinoma cells. It was also observed that betulin could inhibit the migration and invasion of the ovarian cancer cells in a concentration-dependent manner. Betulin molecule also resulted in blocking of mTOR/PI3K/AKT signalling pathway. In conclusion, this study clearly indicates the anticancer effects of betulin natural product in OVCAR-3 human ovarian cancer cells are mediated via apoptosis induction, G2/M phase cell cycle arrest, cell migration and invasion inhibition and targeting of mTOR/PI3K/AKT signalling pathway.

Abstract 2820: A human Claudin-3 monoclonal antibody as a potential multimodal theranostic probe in ovarian cancer

Abstract Introduction: Claudin-3 (CLDN3), a tight junction protein, regulates cell-to-cell interactions in epithelial or endothelial cell sheets. During tumorigenesis, epithelial cells are transformed and tumor cells proliferate through out-of-plane division, resulting in external exposure of CLDN3. This alteration of CLDN3 expression is associated with cancer progression, correlating with malignancy in various carcinomas. Since CLDN3 is particularly overexpressed in most ovarian cancers and used as an effective diagnostic marker, we tested the possibility of using a CLDN3-specific antibody as a novel imaging probe. Materials and Methods: After reducing the CLDN3 specific antibody to expose the -SH group, click chemistry was used to conjugate radioactive isotope 111In or fluorescent protein FNR648. Human ovarian cancer OVCAR-3 cells and human glioblastoma (U87MG) cells were used as CLDN3 positive and negative cells, respectively. Flow cytometry was used to measure the binding of CLDN3 IgG1 monoclonal antibody to theses cell lines. To establish xenograft model, OVCAR-3 cells were injected subcutaneously into the mice. 111In-labeled CLDN3 antibody (370 kBq/50 μl) was administered intravenously to mice with. After 24 hours, organs including tumor were excised and measured with a γ-counter. Images were acquired with IVIS and SPECT/CT. Results: The labeling efficiency of NOTA-111In or antibody-NOTA-111In was 98.52% or 100%, respectively. FNR648 labeled CLDN3 antibody was bound to the cell surface of OVCAR-3 at 83.4% and to U87MG at 5.7%, respectively. In OVCAR-3 tumor xenografted mice, mice injected with CLDN3 antibody showed 2.5-fold higher tumor uptake (20.4 ± 7.4% ID/g) than mice injected with human IgG (8.8 ± 2.6% ID/g) at 24 hour p.i. The fluorescence signal of CLDN3 antibody peaked at 24 hour p.i. Conclusion: We successfully conjugated radioisotope and fluorescent protein using the CLDN3 specific antibody. Since the specific binding of CLDN3 antibodies to OVCAR-3 tumors has been validated in a mouse model and diagnostic radionuclides can be replaced with therapeutic radionuclides, this human monoclonal antibody could be used as a useful theranostic probe. Citation Format: Sera Oh, Hobin Yang, Ho Rim Oh, Chul-Hee Lee, Young-Hwa Kim, Gi Jeong Cheon, Keon Wook Kang, Young Kee Shin, Hyewon Youn. A human Claudin-3 monoclonal antibody as a potential multimodal theranostic probe in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2820.

Abstract 304: Peptide-mediated delivery of siRNAs targeting CSNK2A1 decreases migration of ovarian cancer cells in vitro

Abstract Background Ovarian cancer (OC) is among the most commonly diagnosed cancers and combined with the low responses to therapeutics, it has the highest mortality among gynecological cancers. When diagnosed at stage III and stage IV, survival rates remain below 30%. Over 90% of ovarian cancers are epithelial-based, resulting in proliferation with both local and distant metastasis. Common modes of treatment typically include surgical debulking and platinum-based chemotherapies. However, approximately 80% of confirmed late-stage OC patients will encounter recurrence, multidrug resistance, or metastasis. As a result, there is a need for more effective treatments by means of sustainable therapeutics. RNA interference (RNAi)- based therapies via short interfering RNA (siRNA) have shown promise a potential treatment method. However, siRNAs cannot readily enter the cell due to their large size and negative charge. Thus, siRNAs must be combined with a carrier that maintains the efficacy of the therapeutic by promoting membrane interaction, internalization, and release of siRNAs. Fusogenic peptides (FPs) serve as efficient carriers by complexing with siRNAs through electrostatic interactions and mediating endosomal escape to maintain bioactivity. Here, we investigate the use of a novel FP for the delivery of siRNAS targeting CSNK2A1 and characterize ovarian cancer cell response. Methods OVCAR3 human ovarian cancer cells were treated with fusogenic peptides complexed with either non-targeting siRNAs or siRNAs targeting CSNK2A1 at varying nitrogen to phosphorus (N:P) ratios and downstream cellular analysis was conducted. Cytotoxicity of both the fusogenic peptide vehicle alone and peptide/siRNA complexes was determined using MTS assays. The effect of the peptide/siRNA complex on ovarian cancer cell migration was measured using a scratch wound healing assays. Results The FP vehicle displayed no significant decreases in cell viability at higher N:P ratios. This result indicates that the FP vehicle is not cytotoxic to the cells and demonstrate the potential biocompatibility of the delivery system. In the scratch wound healing assay, percent wound healing was substantially decreased when ovarian cancer cells were treated with peptide complexed with siRNAs targeting CSNK2A1 in contrast to treatment with peptide complexed with non-targeting siRNAs. Higher N:P ratios displayed an overall greater decrease in wound healing. In future studies, we will examine whether peptide/siRNA complexes affect the invasiveness of ovarian cancer cells. Citation Format: Anthony Hazelton, Timothy Samec, Jessica Boulos, Serena Gilmore, Angela Alexander-Bryant. Peptide-mediated delivery of siRNAs targeting CSNK2A1 decreases migration of ovarian cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 304.

Abstract 281: pH-sensitive liposome for siRNA delivery to treat drug-resistant ovarian cancer

Abstract Introduction: With a 5-year survival rate of 47%, ovarian cancer is the 5th leading cause of death amongst women worldwide. Over 75% of patients experience recurrence after initial treatment, indicating a need for improved treatment options. Drug resistance is a major barrier hindering the success of current treatment methods. Our study analyzes the characteristics of a stimuli-sensitive liposomal delivery system for combatting drug resistance. Our delivery system will deliver bioactive siRNAs targeting genes related to drug resistance, cell proliferation, and apoptosis. In this study, we investigate the characteristics of the liposomes to determine particle size, surface charge, and ability to encapsulate/bind both siRNAs. We also begin to investigate the delivery potential of the pH-sensitive liposomal formulation in vitro using ovarian cancer cell lines. Methods: Empty and siRNA loaded cationic, pH-sensitive liposomes (CHEMS-LPs) were synthesized by the thin-film hydration method. Liposome size, zeta potential, and polydispersity index (PDI) were measured by dynamic light scattering (DLS). To measure siRNA encapsulation efficiency, fluorescently labeled siRNA was loaded into CHEMS-LPs and subjected to centrifugation to pellet the LPs. Fluorescence spectroscopy was used to detect siRNA in the supernatant. The toxicity of unloaded CHEMS-LPs was determined by an MTS assay using OVCAR3 (drug-sensitive) and OVCAR3-T40 (drug-resistant) human ovarian cancer cells. Results: The size and zeta potential of blank and siRNA-loaded CHEMS-LPs were 97.88 ± 2.39 nm and 29.0 ± 2.00 mV, and 80.78 ± 0.77​ nm and 13.1 ±1.66​ mV, respectively. The positively charged zeta potential confirms the cationic nature of our liposomes. The PDI demonstrated that the liposomes were unimodal and monodisperse with PDI values of less than 0.300 for each formulation. In addition, siRNA was successfully bound to CHEMS-LPs through electrostatic interaction with the cationic lipid layer, resulting in an encapsulation efficiency of 99.6% Conclusion: CHEMS-LPs are pH-sensitive, cationic, monodisperse liposomes able to encapsulate siRNAs in order to mediate delivery into ovarian cancer cells. Their stable structure, positive charge, and low cytotoxicity is promising for future studies, including delivery of bioactive siRNAs to stimulate downregulation of target genes related to drug resistance. Acknowledgements: This work was supported in part by the National Science Foundation EPSCoR Program under Award # OIA-1655740. We would like to thank George Duran from Stanford University for donating the OVCAR3-T40 cell line. Citation Format: Kharimat Lora Alatise, Samantha Gardner, Angela Alexander-Bryant. pH-sensitive liposome for siRNA delivery to treat drug-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 281.

Altered Long Non-coding RNAs Expression and Cytotoxic and Anti-proliferative Activity of Dendrosomal Nano-curcumin in Ovarian Cancer Cells

Curcumin is a polyphenol derived from the rhizome of turmeric, which has significant chemoprevention and chemotherapeutic effects. Long non-coding RNAs (lncRNAs) are longer than 200 nucleotides and play a crucial role in various biological processes. Since aberrant lncRNAs expression affects the development and progression of tumors, the present study was conducted to determine the cytotoxic potential of dendrosomal nano-curcumin (DNC) against ovarian cancer cell proliferation and apoptosis and altered expression of some lncRNAs involved in ovarian cancer. In vitro cytotoxic activity of DNC against OVCAR3 and SKOV3 human ovarian cancer cells and HFSF-PI3 human fibroblasts cells as a control was evaluated by MTT assay. Real-time PCR and western blot methods were used to determine the apoptosis-inducing effect of DNC through analyzing apoptosis marker genes and protein expression, respectively. We also checked the ovarian cancer cells apoptosis induced with change in expression levels of some lncRNAs. Our results revealed that DNC significantly decreased viability of ovarian cancer cells as compared to Cur. Moreover, DNC reduced the relative expression levels of HOTAIR and H19 and increased the expression of MEG3. Bcl2 expression was down-regulated in mRNA and protein level after DNC treatment, whereas knockdown of MEG3 reduced it. Our findings discovered the effect of DNC on growth inhibition as well as modulation of some lncRNAs in ovarian cancer cells. We also observed that MEG3 down-regulation can suppress the effect of DNC on down-regulation of Bcl-2 as an anti-apoptotic protein. This may suggest the role of MEG3 in DNC-induced apoptosis.

Study on the mechanism of photodynamic therapy mediated by 5-aminoketovalerate in human ovarian cancer cell line.

We aimed to investigate the mechanism and effect of photodynamic treatment mediated by 5-aminoketovalerate (5-ALA-PDT) on human ovarian cancer cells (OVCAR3 cells) and to provide a theoretical basis for the subsequent experimental step in vivo. Human ovarian cancer OVCAR3 cells were randomly divided into four groups: control group, laser irradiation alone group, photosensitizer alone group, and photodynamic treatment group. Alterations in cell morphology were observed with an inverted light microscope; cell viability was examined by CCK-8 assays. The ROS content and apoptosis rate were examined by flow cytometry analysis. Western blot was used to detect the expression of apoptosis-related proteins, such as caspase-3, Bax, and Bcl-2, and the expression of cleaved caspase-3 in live cells was detected by a cleaved caspase-3 assay kit. Inverted light microscopy showed alterations in cell morphology in different stages. Comparison with the three other groups indicated that tumor cell proliferation was significantly decreased in the photodynamic treatment group (P < 0.05). Flow cytometry analysis revealed that the content of ROS was higher in the photodynamic group than in the other three groups, and the apoptosis rate was higher in the photodynamic treatment group. The difference compared with the other three groups was statistically significant (P < 0.001). The western blot results indicated that the protein expression of Bcl-2 and caspase-3 was decreased in the photodynamic treatment group, and the protein expression level of Bax was increased (P < 0.05). The expression of cleaved caspase-3 was increased in the photodynamic treatment group compared with the other groups according to the data obtained with a microplate reader. Thus, our results demonstrated that the apoptosis and viability of OVCAR3 cells are altered in response to 5-ALA-PDT; however, no remarkable effects were observed in ovarian cancer cells treated with laser irradiation or photosensitizer alone. 5-ALA-PDT can significantly inhibit the growth of human ovarian cancer cells, and the mechanism of this effect is related to the tumor cell apoptosis mediated by the downregulation of Bcl-2 and caspase-3 and upregulation of Bax protein expression.

Anticancer Effects of Cordyceps Militaris Extract in Human Ovarian Cancer Cells via ADORA2B (P05-012-19)

ObjectivesThe study was aimed to determine anticancer effects of Cordyceps militaris extract (CME) and its major bioactive compound, cordycepin, in human ovarian cancer cells, and to identify their putative molecular mechanism mediated by adenosine receptors (ADORAs). MethodsCME was prepared in 50% ethanol solution. LC-MS was used for quantification and Q-TOF MS for qualifying bioactive compounds in CME. MTT assay was performed for cell viability in A2780, SKOV-3, TOV112D, and OVCAR-3 human ovarian cancer cell lines. cAMP response element (CRE)-luciferase reporter gene assays were used to determine whether antitumorigenic effect of CME/cordycepin is based on adenosine derivatives. Additionally, the involvement of ADORA signaling pathway was measured using with ADORA2A antagonist SCH 58261 and ADORA2B antagonist PSB 603. ResultsCordycepin concentrations of CME was 21.8%. CME was effective to reduce cell viability in A2780 and OVCAR-3 with IC50 115.2 μg/ml and 155.94 μg/ml respectively, while SKOV-3 and TOV112D were relatively resistant to CME. cAMP production was significantly increased by treatment with cordycepin and, lesser extent, with CME. Among the four types of ADORAs, ADORA2A and 2B showed relatively higher expression levels in ovarian cancer cells. The cAMP production by CME was ameliorated by PSB 603, not SCH 58261, treatment. ConclusionsCME and cordycepin have anticancer effects in human ovarian cancer cells via ADORA2B-cAMP pathway. Funding SourcesNRF of Korea (2017R1D1A1B03034936 & 22A20130012143) and Health Fellowship Foundation.

Low glucose and metformin-induced apoptosis of human ovarian cancer cells is connected to ASK1 via mitochondrial and endoplasmic reticulum stress-associated pathways

BackgroundMetformin, a first-line drug for type 2 diabetes, could induce apoptosis in cancer cells. However, the concentration of glucose affects the effect of metformin, especially low glucose in the culture medium can enhance the cytotoxicity of metformin on cancer cells. Since mitochondria and endoplasmic reticulum is vital for maintaining cell homeostasis, we speculate that low glucose and metformin-induced cell apoptosis may be associated with mitochondria and endoplasmic reticulum. ASK1, as apoptosis signaling regulating kinase 1, is associated with cell apoptosis and mitochondrial damage. This study was designed to investigate the functional significance of ASK1, mitochondria and endoplasmic reticulum and underlying mechanism in low glucose and metformin-induced cell apoptosis.MethodsAn MTT assay was used to evaluate cell viability in SKOV3, OVCAR3 and HO8910 human ovarian cancer cells. Cell apoptosis was analyzed by flow cytometry. The expression of ASK1 was inhibited using a specific pharmacological inhibitor or ASK1-siRNA. Immunofluorescence was used to detect mitochondrial damage and ER stress. Nude mouse xenograft models were given metformin or/and NQDI-1, and ASK1 expression was detected using immunoblotting. In addition, subcellular fractionation of mitochondria was performed to assay the internal connection between ASK1 and mitochondria.ResultsThe present study found that low glucose in culture medium enhanced the anticancer effect of metformin in human ovarian cancer cells. Utilization of a specific pharmacological inhibitor or ASK1-siRNA identified a potential role for ASK1 as an apoptotic protein in the regulation of low glucose and metformin-induced cell apoptosis via ASK1-mediated mitochondrial damage through the ASK1/Noxa pathway and via ER stress through the ROS/ASK1/JNK pathway. Moreover, ASK1 inhibition weakened the antitumor activity of metformin in vivo. Thus, mitochondrial damage and ER stress play a crucial role in low glucose–enhanced metformin cytotoxicity in human ovarian cancer cells.ConclusionsThese data suggested that low glucose and metformin induce cell apoptosis via ASK1-mediated mitochondrial damage and ER stress. These findings indicated that the effect of metformin in anticancer treatment may be related to cell culture conditions.

Molecular Targeting of Notch Signaling Pathway by DAPT in Human Ovarian Cancer: Possible Anti Metastatic Effects

Background:Ovarian cancer is one of the most important gynecological malignancies, causing significant mortality. Recently, there has been extensive attention to the involvement of signaling cascades in its initiation/progression. In this study, we focused on the possible role of Notch signal transduction in proliferation and metalloproteinase 2 and 9 function in human ovarian cancer OVCAR-3 cells.Methods:MTT proliferation assays were used to evaluate effects of a DAPT inhibitor on cell proliferation. For measurement of Hes-1 mRNA levels, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was applied following 48 h incubation with the inhibitor. In addition, metalloproteinase (MMPs) activity was assessed by zymography.Results:Inhibition of Notch signaling resulted in a significant reduction in OVCAR-3 cell proliferation. Additionally, DAPT treatment of cells significantly decreased Hes-1 mRNA levels (p < 0.05) as well as activity of MMP-2 and -9 (p < 0.05).Conclusion:Our results suggested that suppression of Notch signaling by a specific inhibitor can effectively decrease proliferation and the potential for metastasis of OVCAR-3 cells via a reduction in the activity of metalloproteinases 2 and 9. Thus, pharmacological targeting of the Notch signaling pathway could be a promising future treatment for ovarian cancer.

Orostachys japonicus induce p53-dependent cell cycle arrest through the MAPK signaling pathway in OVCAR-3 human ovarian cancer cells.

Orostachys japonicus (O. japonicus) is utilized as a traditional medicine for patients with various diseases. This study investigated the effect of the ethyl acetate fraction from O. japonicus extract (OJE) on the growth inhibition of OVCAR‐3 human ovarian cancer cells demonstrated to inhibit cell growth and arrest the cell cycle in OVCAR‐3 cells by blocking the sub‐G1 phase and decreasing cyclin E1/CDK2 expression. Cell cycle arrest was connected to the increased expression of the cell cycle regulating factors p53 and p21. Apoptosis was initiated through the intrinsic pathway by up‐regulating the expression of the Bcl‐2/Bax ratio and down‐regulating the expression of pro‐caspase‐3. Furthermore, OJE treatment elicited p‐p38 activation and p‐ERK1/2 inhibition. In conclusion, our results demonstrated that OJE reduced the growth of OVCAR‐3 human ovarian cancer cells mediated by arrest of the cell cycle and regulation of MAPK signaling pathways.

Abstract 1395: HDAC6 and DNMT inhibition affect immunogenicity of ovarian cancer cells: A rationale for combining epigenetic and immune therapy in ovarian cancer

Abstract Background: Therapies that activate the immune system to fight cancer have shown robust responses in solid tumors. However, most patients, including those with ovarian cancer, do not respond to these therapies alone. Drugs that inhibit epigenetic modifiers increase immune signaling from cancer cells. Epigenetic modifiers DNA methyltransferase inhibitors (DNMTi) and selective histone deacetylase inhibitors (HDACi),in particular selective HDAC6i, modulate immune-related pathways involved in anti-tumor immune responses. HDAC6i downregulate immunosuppressive ligands PD-L1 and PD-L2 via dephosphorylating pSTAT3 and upregulate tumor associated antigens (TAA) and antigen presentation machinery. Similarly, DNMTi activate anti-viral signaling via expression of Endogenous Retroviruses (ERVs) to trigger the type I interferon response, upregulate tumor antigen processing and presentation, and stimulate pro-inflammatory cytokines. The aim of our study is to test if the combination of epigenetic modulators Nexturastat A (Next A), a selective HDAC6i, and 5-azacytidine (AZA), a DNMTi, can be safely used to increase an immune response in ovarian cancer. We hypothesize that these drugs will enhance tumor immunity alone and when combined with immune checkpoint blockades targeting PD-1. Results: HDAC enzymes are differentially expressed in A2780, HEY, OVCAR3, SKOV3, and TYKNu human ovarian cancer cell lines. HDAC1 and HDAC2 proteins were similarly expressed in HEY and SKOV3 whereas HDAC6 was expressed at lower levels in HEY and TYKNu but at higher levels in SKOV3, OVCAR3, and A2780. As previously reported we believe this may be due to the presence of the chromatin modifier ARID1A mutation in the SKOV3 and A2780 cell lines. The upregulation of HDAC6 also correlated with a higher IC50 for NextA treatment in those particular cell lines. Further immunoblots showed that PD-L1, a marker of poor prognosis in ovarian cancer, decreased after treatment with NextA and even more in combination with AZA. Additionally, DNMT1, the known target of AZA, was decreased after treatment with AZA and NextA, both independently and in combination, a finding that has not been previously reported. Conclusions: As shown previously, HDAC6 enzyme levels are higher in cell lines with ARID1A mutations. DNMT1 was decreased after treatment with AZA, as expected, but surprisingly also after treatment with NextA. PD-L1 decreased after treatment with NextA and even more so when combined with AZA. We thus believe that combining these two epigenetic modifiers will lead to an additive effect on immune signaling through stimulation of antiviral signaling (DNMTi), which can upregulate the immunosuppressive ligand PD-L1, which is then reduced by HDAC6 inhibition. We are currently testing the combination of both epigenetic modifiers with anti-PD-1 in an immunocompetent mouse model of ovarian cancer. Citation Format: Aneil P. Srivastava, Sara M. Moufarrij, Melissa Hadley, Sarah Chisholm, Micael Lopez-Acevedo, Alejandro Villagra, Katherine B. Chiappinelli. HDAC6 and DNMT inhibition affect immunogenicity of ovarian cancer cells: A rationale for combining epigenetic and immune therapy 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 1395.

The protective role of melatonin in cadmium-induced proliferation of ovarian cancer cells.

Cadmium (Cd), a ubiquitous environmental and occupational pollutant, acts as a metalloestrogen to induce cell proliferation. It is suggested that Cd may also contribute to the development of estrogen-related cancers like ovarian cancer which is the most lethal cancer in women. Furthermore, it was shown that melatonin has antiproliferative effect on estradiol (E2)-induced proliferation. The aim of the present study was to evaluate whether melatonin inhibits Cd-induced proliferation in ovarian cancer cell lines and also whether Cd and melatonin can modulate estrogen receptor α (ERα) expression. OVCAR3 and SKOV3 human ovarian cancer cell lines were treated with CdCl2 (1-100 nM) and melatonin (1 μM) for 48 h. Cell proliferation evaluation was carried out by bromodeoxyuridine (BrdU) incorporation assay. ERα expression was detected by western blotting method 24 h after cell treatment. The results were demonstrated that Cd increased proliferation of ovarian cancer cell lines in a dose dependent manner. Melatonin inhibited Cd-induced proliferation of OVCAR3 and SKOV3 cell lines. Moreover, CdCl2 significantly increased ERα expression in both OVCAR3 and SKOV3 cell lines compared to control. Melatonin significantly inhibited Cd inducing effect on ERα expression of OVCAR3 and SKOV3 cell. In conclusion, due to the proliferative effect on ovarian cancer cell lines, Cd could play an important role in the etiology of ovarian cancer by inducing cells ERα expression. Furthermore, melatonin has the protective role on Cd-induced cell proliferation by inhibition of ERα expression.

Free fatty acid receptor (FFAR) agonists inhibit proliferation of human ovarian cancer cells

Many cellular actions of omega-3 fatty acids are mediated by two G protein-coupled receptors, FFA1 and FFA4, free fatty acid receptor (FFAR) family members that are activated by these dietary constituents. FFAR agonists inhibit proliferation of human prostate and breast cancer cells. Since omega-3 fatty acids can inhibit ovarian cancer cell growth, the current study tested the potential role of FFARs in the response. OVCAR3 and SKOV3 human ovarian cancer cell lines express mRNA for FFA1; FFA4 mRNA was detected at low levels in SKOV3 but not OVCAR3. Lysophosphatidic acid (LPA) and epidermal growth factor (EGF) stimulated proliferation of both cell lines; these responses were inhibited by eicosopentaneoic acid (EPA) and by GW9508, a synthetic FFAR agonist. The LPA antagonist Ki16425 also inhibited LPA- and EGF-induced proliferation; FFAR agonists had no further effect when added with Ki16425. The results suggest that FFARs are potential targets for ovarian cancer therapy.

Prodelphinidins isolated from Chinese bayberry leaves induces apoptosis via the p53-dependent signaling pathways in OVCAR-3 human ovarian cancer cells.

Chinese bayberry leaves are rich in prodelphinidins. Since the isolation and purification of prodelphinidins is difficult, the association between the degree of prodelphinidin polymerization and their anti-carcinogenic activity remains ambiguous. The cytotoxic and apoptotic activities of prodelphinidin Chinese bayberry leaf extracts (PCBLs), oligomeric proanthocyanidins (OPAs) and polymeric proanthocyanidins (PPAs), isolated by normal-phase preparative high-performance liquid chromatography were investigated in OVCAR-3 human ovarian cancer cells. The PCBLs, OPAs and PPAs inhibited cancer cell growth and induced apoptosis via the caspase-dependent pathway. Apoptosis was triggered through the intrinsic pathway by upregulating the expression of several B-cell lymphoma-2 (Bcl-2) family proapoptotic proteins, including p53-upregulated modulator of apoptosis (PUMA), Bcl-2-associated X protein and Bcl-2-associated agonist of cell death, and by downregulating the antiapoptotic protein Bcl-extra large. Apoptosis was also triggered through the extrinsic pathway via the upregulation of death receptor 5 (DR5) and Fas expression. In addition, OPAs and PPAs induced caspase-dependent apoptosis at least partially through the inhibition of the protein kinase B signaling pathway. The knockdown of p53 by specific small interfering RNA resulted in the depletion of p53, and inhibited the OPA and PPA treatment-induced increases in p53, which led to a decrease in the expression of p21, DR5, Fas, PUMA and phosphatase and tensin homolog proteins. These observations demonstrate that p53 is a mediator of OPA and PPA-induced apoptosis in OVCAR-3 cells. The PPAs exhibited stronger anti-proliferative and pro-apoptotic activities compared with OPAs and PCBLs. These results suggest that PCBLs, OPAs and PPAs may be useful for the treatment of ovarian cancer.

MiR-9 functions as a tumor inhibitor of cell proliferation in epithelial ovarian cancer through targeting the SDF-1/CXCR4 pathway

The current study aimed to investigate the potential role of miR-9 in the inhibition of ovarian cancer progression through the stromal cell-derived factor-1 (SDF-1)/ C-X-C chemokine receptor type 4 (CXCR4) pathway and to provide a theoretical basis for the diagnosis and treatment of ovarian cancer. Human ovarian cancer OVCAR-3 cells were transfected with miR-9 short hairpin RNA (shRNA). The effect of miR-9 on the mRNA expression levels of CXCR4 were analyzed using reverse transcription-quantitative polymerase chain reaction. The effects of miR-9 on OVCAR-3 cell proliferation, invasion and apoptotic ability were detected using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay, Matrigel method, and Annexin V-fluorescein isothiocyanate flow cytometry, respectively. In addition, expression levels of SDF-1/CXCR4 pathway associated proteins were determined by western blot analysis. mRNA expression levels of CXCR4 in OVCAR-3 cells transfected with miR-9 shRNA was significantly downregulated compared with the blank and control groups (P<0.05). Furthermore, compared with the two control groups, the current results revealed that miR-9 inhibited cell proliferation, suppressed invasive ability and induced cell apoptosis in OVCAR-3 cells (P<0.05). Finally, it was observed that miR-9 functioned as a tumor inhibitor through the SDF-1/CXCR4 pathway by suppressing the expression levels of extracellular signal-regulated kinase 1 (ERK1), ERK2 and matrix metalloproteinase-9 proteins. The present study suggested that miR-9 may function as a promising tumor inhibitor for ovarian cancer through targeting the SDF-1/CXCR4 pathway.

Expression of Inorganic Pyrophosphatase (PPA1) Correlates with Poor Prognosis of Epithelial Ovarian Cancer

Ovarian serous carcinoma (OSC) is the most common epithelial ovarian cancer. Inorganic pyrophosphatase (PPA1) catalyzes the hydrolysis of pyrophosphate to inorganic phosphate, thereby providing extra energy for metabolism. The significance of PPA1 in the prognosis of OSC has not been investigated. Our study aimed to explore the expression and predictive role of PPA1 in OSC progression. We screened the expression of PPA1 protein in OSC tissues from 139 patients by immunohistochemistry, and evaluated its correlation with clinicopathological characteristics. PPA1 was categorized as high expression in 58 OSC cases (41.7%), which was correlated with poor differentiation, positive lymph node (LN) metastasis and advanced FIGO (The International Federation of Gynecology and Obstetrics) stages. Univariate and multivariate analyses identified PPA1 as a novel independent prognostic biomarker in OSC patients; meanwhile, conventional factors such as LN status and FIGO stages also showed statistical significance. Moreover, the expression levels of PPA1 protein were higher in A2780 and OVCAR3 human ovarian cancer cell lines than those in normal ovarian surface epithelial cells. Using these ovarian cancer cell lines, we showed that PPA1 overexpression caused the decrease in the expression level of p53, the tumor suppressor, with the increase in β-catenin level, as determined by Western blot analysis. Conversely, knockdown of PPAI expression was associated with the increase of p53 level and the decreased of β-catenin level. Consistently, the proliferation and invasion capacities of ovarian cancer cells were enhanced upon PPA1 overexpression. In conclusion, PPA1 serves as a potential prognostic biomarker for patients with OSC.