Apoptosis In OVCAR-3 Cells Research Articles

Effects of rosmarinic acid and doxorubicine on an ovarian adenocarsinoma cell line (OVCAR3) via the EGFR pathway.

We aimed to reveal the effects of rosmarinic acid (RA), which has come to the forefront with its antitumor and antioxidant properties in many studies recently in the ovarian adenocarcinoma cell line, on the epidermal growth factor receptor (EFGR) signaling pathway in the presence of doxorubicin (DOX). Ovarian adenocarcinoma cell line (OVCAR3) and human skin keratinocyte cell line human skin keratinocyte cell line (HaCaT) were used as control. (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was applied to determine the effect of RA and DOX on the proliferation of OVCAR3 and HaCaT cells. Bcl2 expression and epidermal growth factor receptor (EGFR) and western blot analysis were performed to determine the expression levels of the markers. It was determined that RA (IC50 = 437.6 μM) and DOX (IC50 = 0.08 μM) have the ability to inhibit the proliferation of OVCAR3 cells and induce apoptosis in a 72-hour time and dose-dependent manner. Western blot showed that the expression level of Bcl-2 and EGFR in OVCAR3 cells was down-regulated by RA and DOX. Apoptosis in OVCAR3 cells can potentially be induced by RA via the EGFR pathway, and RA may be a potent agent for cancer therapy.

Targeting Ovarian Cancer with Chalcone Derivatives: Cytotoxicity and Apoptosis Induction in HGSOC Cells

Ovarian cancer ranks as the eighth most prevalent form of cancer in women across the globe and stands as the third most frequent gynecological cancer, following cervical and endometrial cancers. Given its resistance to standard chemotherapy and high recurrence rates, there is an urgent imperative to discover novel compounds with potential as chemotherapeutic agents for treating ovarian cancer. Chalcones exhibit a wide array of biological properties, with a particular focus on their anti-cancer activities. In this research, we documented the synthesis and in vitro study of a small library of chalcone derivatives designed for use against high-grade serous ovarian cancer (HGSOC) cell lines, specifically OVCAR-3, OVSAHO, and KURAMOCHI. Our findings revealed that three of these compounds exhibited cytotoxic and anti-proliferative effects against all the tested HGSOC cell lines, achieving IC50 concentrations lower than 25 µM. Further investigations disclosed that these chalcones prompted an increase in the subG1 phase cell cycle and induced apoptosis in OVCAR-3 cells. In summary, our study underscores the potential of chalcones as promising agents for the treatment of ovarian cancer.

Targeting and neutralizing human epididymis protein 4 by novel nanobodies to suppress ovarian cancer cells and attenuate cisplatin resistance

Human epididymis protein 4 (HE4) is a glycoprotein secreted by epithelial ovarian cancer (EOC) cells and is a novel and specific biomarker for diagnosing and prognosing EOC. Previous studies have shown that overexpression of HE4 is correlated with EOC tumorigenesis and chemoresistance. However, less has been reported regarding the direct effect of the secreted HE4 protein as an autocrine factor in EOC cells. Here, we investigated the molecular mechanism of the secretory form of HE4 on the growth of EOC cells by applying nanobodies with a targeted interaction of free HE4. Three anti-HE4 nanobodies were selected from an immune library by phage display. HE4 secreted by serum-free cultured OVCAR3 cells increased and was effectively neutralized by anti-HE4 nanobodies, which inhibited cell viability. Treatment with the anti-HE4 nanobody 1G8 decreased Bcl-2 expression and increased BAX, cleaved PARP, and p53 levels, resulting in apoptosis of OVCAR3 cells. Moreover, 1G8 significantly improved the cisplatin response of OVCAR3 cells. Our data suggest that secretory HE4 played a novel pro-survival autocrine role and was a target of the anti-HE4 nanobody to improve the therapeutic effects of cisplatin-based chemotherapy.

Down-regulation of long non-coding RNA antisense non-coding RNA in the INK4 locus suppresses OVCAR-3 cells proliferation and induction of apoptosis by Wnt/β-catenin.

Ovarian cancer is a lethal gynecological malignancy. Long non-coding RNA antisense non-coding RNA in the INK4 locus (lncRNA ANRIL) was reported to have a critical role in cancer advancement. The ANRIL-mediated oncogenic underlying molecular mechanisms are not fully understood in ovarian cancer. We aimed to study ANRIL silencing effects on the proliferation and apoptosis of OVCAR-3 cells. The ANRIL was Knockdown by transfection of OVCAR-3 cells with si-RNA against ANRIL. MTT assay and cell death ELISA kit were used to evaluate cellular proliferation and apoptosis. The expression levels of ANRIL, pro-and anti-apoptotic genes were assessed using q-RT-PCR. Western blotting was used to assess Wnt/β-catenin signalling pathway. ANRIL down-regulating in OVCAR-3 cell lines resulted in significant inhibition of cellular proliferation, apoptosis induction, as well as suppression of cellular invasion. Besides, knockdown of ANRIL led to pro-apoptotic genes up-regulation, Bad and Bax and anti-apoptotic genes down-regulation, Bid and Bcl-2. More importantly, we observed that ANRIL inhibition suppressed the vital components expression of the Wnt/β-catenin cascade. Our findings showed that down-regulation of lncRNA ANRIL resulted in the effective suppression of OVCAR-3 cell proliferation and invasion and induction of apoptosis by preventing Wnt/β-catenin signal transduction.

1-Bromopropane-induced apoptosis in OVCAR-3 cells via oxidative stress and inactivation of Nrf2.

The bromoalkane, 1-bromopropane (1-BP), may damage the reproductive system though oxidative stress, while the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) plays an important role in regulating intracellular antioxidant levels against oxidative stress. This study explored the role of oxidative stress and the Nrf2 signaling pathway in mediating the reproductive toxicity of 1-BP using the ovarian carcinoma cell line OVCAR-3 as an in vitro model of the human ovary. OVCAR-3 cells were treated with 1, 5, 10 and 15 mM 1-BP. After 24 h, the cellular reactive oxygen species and malondialdehyde concentrations significantly increased, while the superoxide dismutase activity decreased; translocation of Nrf2 from the cytosol to the nucleus as well as downstream protein expression of Nrf2-regulated genes heme oxygenase-1 and Bcl-2 was inhibited. Apoptosis was also observed, accompanied by increased caspase-3 and caspase-9 activity. The antioxidant vitamin C alleviated 1-BP-induced apoptosis by inhibiting caspase activity activating the Nrf2 signaling pathway. These findings suggested that 1-BP induced oxidative stress and apoptosis in OVCAR-3 cells through inactivation of Nrf2 signaling.

Galectin-3 regulates chemotherapy sensitivity in epithelial ovarian carcinoma via regulating mitochondrial function.

Galectin-3 (Gal-3) is a multifunctional carbohydrate-binding protein associated with cell migration, cell proliferation, cell adhesion, and cell-cell interaction in tumor cells. It has been implied to be involved in the tumor progression and chemoresistance of epithelial ovarian cancer (EOC). However, it is unclear whether the Gal-3-mediated regulation on the EOC chemosensitivity is associated with hypoxia or mitochondrial dysfunction. In the present study, we examined the regulation by Gal-3 overexpression on cisplatin-sensitivity or cisplatin-resistance in EOC cells in vitro. We manipulated Gal-3 via plasmid transfection and RNA interference in the cisplatin-resistant EOC cells, and re-evaluated the sensitivity of the cisplatin-resistant EOC cells to cisplatin, with CCK-8 assay, colony forming assay, apoptosis analysis and mitochondrial function examination. Results demonstrated that galectin-3 overexpression downregulated the cisplatin sensitivity in EOC OVCAR-3 cell clone, resulting in an upregulated growth and a reduced apoptosis in the cisplatin-treated OVCAR-3 cells. On the other hand, the Gal-3 knockdown with Gal-3-specific siRNA transfection aggravated cisplatin-induced apoptosis in OVCAR-3 cells. In conclusion, Galectin-3 reduces the sensitivity of ovarian cancer cells to cisplatin via regulating cisplatin-induced mitochondrial dysfunction. Galectin-3 knockdown inhibits the chemo-resistance of EOC cells. It implies that Galectin-3 might be a potential target to overcome the chemo-resistance in EOC cells.

Piperidine nitroxide Tempol enhances cisplatin-induced apoptosis in ovarian cancer cells.

A nitroxide radical, Tempol (Tempol, TPL), is usually used as an antioxidative agent clinically, whereas the mechanism underlying its pro-oxidative effect has not been thoroughly investigated. The present study investigated the pro-oxidative effect of TPL on the inhibition of cellular proliferation and its role in enhancing the effect of anticancer drug cisplatin (DDP) on the induction of apoptosis in ovarian cancer cells. Cell viability and proliferation were evaluated by MTT assay. Cell apoptosis was analyzed by flow cytometry (FCM) following staining with Annexin V/propidium iodide. Western blot analysis was performed to determine the expression levels of anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and pro-apoptotic protein Bcl-2-associated X protein (Bax), and the Bcl-2:Bax expression ratio. Cellular reactive oxygen species (ROS) were labeled with dichlorofluorescin-diacetate and analyzed by FCM. The results revealed that cell viabilities of OVCAR3 and SKOV3 cells were decreased by TPL in dose-dependent manner at concentrations of 2 to 10 mM after 48 h incubation. The cell proliferation rates of OVCAR3 and SKOV3 cells were suppressed by TPL at lower toxic concentrations of 1.5 and 1 mM, respectively, compared with the control group. The MTT assay indicated that the combination therapy significantly inhibited the cell proliferation of OVCAR3 cells compared with treatment with DDP alone. FCM demonstrated that the combination treatment increased the proportion of early apoptotic cells in OVCAR3 cells compared with single DDP treatment. Western blot analysis revealed that the combination treatment markedly decreased the Bcl-2:Bax expression ratio compared with treatment with DDP alone. Detection of cellular ROS expression levels demonstrated that the combination therapy significantly increased cellular ROS generation compared with the DDP-only therapy. These data indicated that TPL increased the effect of DDP on inducing apoptosis in OVCAR3 cells.

Response of OVCAR-3 Cells to Cisplatin and Hyperthermia: Does Hyperthermia Really Matter?

Hyperthermic intraperitoneal chemotherapy (HIPEC) is proposed as a promising treatment method, but fundamental information about the contribution of hyperthermia to intraperitoneal chemotherapy is lacking. The purpose of this study was to investigate the cytotoxic effect of hyperthermia and cisplatin on OVCAR-3 cells in vitro. Imitating the typical clinical conditions of HIPEC, OVCAR-3 cells were exposed to hyperthermia and cisplatin for 1 h. MTT viability test, flow cytometric analysis, and real-time cell and isobologram analysis were performed. Hyperthermia up to 42°C did not significantly increase the effect of cisplatin regarding the viability and apoptosis of OVCAR-3 cells. Moreover, an antagonistic effect of hyperthermia and cisplatin was revealed. Our investigation of OVCAR-3 cells critically disputes the benefit of hyperthermia in ovarian cancer treatment. Further in vitro and in vivo research is essential for better understanding of the mechanisms of action of hyperthermia and its role in the treatment of epithelial ovarian cancer.

Geraniin suppresses ovarian cancer growth through inhibition of NF-κB activation and downregulation of Mcl-1 expression

This study investigated the anticancer effects of geraniin on ovarian cancer cells and the signaling pathways involved. Ovarian cancer cells were treated with different concentrations of geraniin for 48h and examined for viability, apoptosis, mitochondrial membrane depolarization, and gene expression. Xenograft tumor studies were performed to determine the anticancer activity of geraniin in vivo. Geraniin significantly decreased cancer cell viability in a concentration-dependent fashion. Geraniin significantly triggered apoptosis, which was accompanied by loss of mitochondrial membrane potential and increased cytochrome c release and caspsase-3 activity. Mechanistically, geraniin significantly downregulated Mcl-1 and impaired NF-κB p65 binding to the mcl-1 promoter. Overexpression of Mcl-1 significantly reversed geraniin-induced apoptosis in OVCAR3 cells. In addition, geraniin retarded ovarian cancer growth and reduced expression of phospho-p65 and Mcl-1. Collectively, geraniin elicits growth suppression in ovarian cancer through inhibition of NF-κB and Mcl-1 and may provide therapeutic benefits for this malignancy.

Abstract NTOC-095: THE SYNERGISTIC EFFECTS OF CARBOPLATIN AND PF–477736, A SMALL MOLECULE CHECKPOINT KINASE 1 INHIBITOR ON A PLATINUM RESISTANT OVARIAN CANCER CELL LINE

Abstract PURPOSE: To investigate the effects of PF-477736, a small molecular checkpoint kinase 1 (Chk1) inhibitor with and without carboplatin on cell apoptosis and proliferation in a p53-mutated platinum resistant ovarian cancer cell line. METHODS: OVCAR3 cells were treated with PF-477736 alone and in combination with carboplatin. Western blot analyses were performed to assess the protein expression of Chk1, phosphorylated Chk1, H2AX, and phosphorylated H2AX. Cell proliferation was evaluated with colony formation assays 10 days following treatment and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assays over the course of 5 days following treatment. Cell apoptosis was measured with flow cytometry after staining with FITC Annexin V and propidium iodide. Paired t-tests were performed for comparative analyses. RESULTS: PF-477736 decreased the expression of phosphorylated Chk1 on serine 249 within 24 hours following treatment as seen on Western blot assay. Total Chk1 expression was unchanged after treatment with PF-477736. The combination treatment of PF-477736 and carboplatin effectively decreased OVCAR3 cell proliferation. Colony formation assays revealed fewer colonies after dual treatment with PF-477736 and carboplatin compared to PF-477736 alone (average colony count 1.5 vs 13.6, p = 0.03) and compared to carboplatin alone (average colony count 1.5 vs 9.7, p < 0.001). MTT assays demonstrated a synergistic effect with PF-477736 and carboplatin: dual treated cells had lower mean dye absorbance values than PF-477736 alone (mean absorbance fold change 1.7 vs 3.2, p = 0.009) and carboplatin alone (mean absorbance fold change 1.7 vs 5.6, p < 0.001). OVCAR3 cells after treatment with PF-477736 and carboplatin had higher rates of apoptosis. On flow cytometry, a higher proportion of cells stained with the apoptotic markers Annexin V and propidium iodide after treatment with PF-477736 and carboplatin compared to PF-477736 alone (percentage of apoptotic cells 34.9% vs 13.0%, p = 0.004) and carboplatin alone (percentage of apoptotic cells 34.9% vs 16.0%, p = 0.008). In addition, western blot analysis of the cell lysates following dual treatment of PF-477736 and carboplatin showed increased expression of phosphorylated H2AX, a marker of double-stranded DNA damage while there was minimal expression with PF-477736 alone and carboplatin alone. CONCLUSIONS: Carboplatin and PF-477736, a small molecule Chk1 inhibitor, synergistically decreased cell proliferation and increased cell apoptosis in OVCAR3 cells, an in vitro model of platinum resistant ovarian cancer. PF-477736 may be a potential new therapeutic strategy for the resensitization of platinum resistant ovarian cancer. Citation Format: Jessica Lee MD, Stephanie V. Blank MD, Robert J. Schneider MD. THE SYNERGISTIC EFFECTS OF CARBOPLATIN AND PF–477736, A SMALL MOLECULE CHECKPOINT KINASE 1 INHIBITOR ON A PLATINUM RESISTANT OVARIAN CANCER CELL LINE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr NTOC-095.

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.

Mangiferin induces apoptosis in human ovarian adenocarcinoma OVCAR3 cells via the regulation of Notch3.

Ovarian cancer is the most lethal gynecological malignancy in the world. Our previous studies showed that mangiferin, purified from plant source, possessed anti-neoplasm effect on human lung adenocarcinoma A549 cells. This study aimed to determine the apoptosis-inducing effect of mangiferin on human ovarian carcinoma OVCAR3 cells. By in vitro studies, we found mangiferin significantly inhibited viability of OVCAR3 cells, and remarkably increased the sensitivity of OVCAR3 cells to cisplatin. In addition, the activation of caspase-dependent apoptosis was observed in mangiferin treated ovarian cancer cells. Importantly, we observed an obviously downregulated Notch expression after mangiferin treatment, indicating the crucial role of Notch in mangiferin mediated apoptosis. In contrast, overexpression of Notch3 abrogated the apoptosis-inducing efficacy of mangiferin, further demonstrating that mangiferin induced apoptosis via Notch pathway. Furthermore, OVCAR3 cell xenograft models revealed that mangiferin treatment inhibited tumor growth and expanded survival of tumor xenograft mice. Based on these results, we concluded that mangiferin could significantly inhibit the proliferation and induce apoptosis in OVCAR3 cells. Our study also suggested the anti-neoplasm effect of mangiferin might be via the regulation of Notch3. Taken together, by targeting cell apoptosis pathways and enhancing the response to cisplatin treatment, mangiferin may represent a potential new drug for the treatment of human 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.

MicroRNA-1284 Inhibits Cell Viability and Induces Apoptosis of Ovarian Cancer Cell Line OVCAR3.

Ovarian cancer is a malignancy with high mortality among women. Multiple reports show that microRNAs (miRs) act as regulators in ovarian cancer inhibition, while the role of miR-1284 in ovarian cancer is still unknown. This study aimed to investigate the effects of miR-1284 on ovarian cancer cells. Human ovarian cancer cell line OVCAR3 was cultured and transfected with miR-1284 mimics, inhibitors, or control. Viability and apoptosis of transfected cells were then determined by MTT assay, BrdU assay, and flow cytometry. Expression changes of p27, p21, and PI3K/Akt pathway-related proteins were measured by Western blot. Results showed that miR-1284 overexpression suppressed cell viability while increasing the apoptosis in OVCAR3 cells. Moreover, the expression level of p27 was upregulated by miR-1284 overexpression. Furthermore, miR-1284 overexpression and Akt inhibitor GSK690693 downregulated the levels of p-Akt and Bcl-2 while upregulating the levels of Bax and caspase 3. However, miR-1284 suppression attenuated the regulatory effects of GSK690693 on these proteins. In conclusion, miR-1284 could inhibit cell viability via regulating the expression of p27 and induce apoptosis via regulating the PI3K/Akt pathway in OVCAR3 cells.

All-trans retinoic acid and genistein induce cell apoptosis in OVCAR-3 cells by increasing the P14 tumor suppressor gene.

In this study, we evaluated the effects of all-trans retinoic acid (ATRA) alone or in combination with genistein (GEN) in p14 tumor suppressor gene and subsequent apoptosis of human ovarian carcinoma cells (OVCAR-3). The cells were treated with ATRA or GEN at concentrations of 50 and 25 μM respectively, either alone or in combination for 24 and 48 h. The cell viability was evaluated using 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. The percentage of cell apoptosis was determined using flow cytometry and p14 gene expression was measured using real time PCR. The MTT results showed that in both ATRA and GEN treated groups, the cell viabilityviability in group treated for 48 h was significantly lower than group treated for 24 h. The flow cytometry results showed that the percentage of apoptotic cells in groups that treated with ATRA and GEN in combination for 24 h and 48 h was significantly more than all other tested groups. The real time results showed that the mRNA level of p14 in cells treated with both drugs for 48 h was significantly higher than all other groups. In conclusion, we confirm that GEN in combination with ATRA is an effective strategy to up regulate the p14 tumor suppressor gene and induce cell apoptosis in OVCAR-3 cell line.

Myricetin inhibits proliferation of cisplatin-resistant cancer cells through a p53-dependent apoptotic pathway.

Cisplatin is a commonly used drug for cancer treatment by crosslinking DNA, leading to apoptosis of cancer cells, resistance to cisplatin treatment often occurs, leading to relapse. Therefore, there is a need for the development of more effective treatment strategies that can overcome chemoresistance. Myricetin is a flavonoid from fruits and vegetables, showing anticancer activity in various cancer cells. In this study, we found myricetin exhibited greater cytotoxicity than cisplatin in two cisplatin-resistant ovarian cancer cell lines, OVCAR-3 and A2780/CP70, and it was less cytotoxic to the normal ovarian cell line IOSE-364. Myricetin selectively induced apoptosis in both cisplatin-resistant cancer cell lines, but did not induce apoptosis in the normal ovarian cell line. It induced both Bcl-2 family-dependent intrinsic and DR5 dependent extrinsic apoptosis in OVCAR-3 cells. P53, a multifunctional tumor suppressor, regulated apoptosis in OVCAR-3 cells through a Bcl-2 family protein-dependent pathway. Myricetin did not induce cell cycle arrest in either ovarian cancer cell line. Because of its potency and selectivity against cisplatin-resistant cancer cells, myricetin could potentially be used to overcome cancer chemoresistance against platinum-based therapy.

Nutlin-3 preferentially sensitises wild-type p53-expressing cancer cells to DR5-selective TRAIL over rhTRAIL

Background:Tumour cell-selective activation of apoptosis by recombinant human TNF-related apoptosis-inducing ligand (rhTRAIL) is enhanced through co-activation of p53 by chemotherapeutic drugs. The novel anticancer agent nutlin-3 provides a promising alternative for p53 activation by disrupting the interaction between p53 and its negative feedback regulator MDM2.Methods:We examined whether nutlin-3 enhances apoptosis induction by rhTRAIL and the DR5-selective TRAIL variant D269H/E195R in wild-type p53-expressing ovarian, colon and lung cancer cell lines and in an ex vivo model of human ovarian cancer.Results:Nutlin-3 enhanced p53, p21, MDM2 and DR5 surface expression. Although nutlin-3 did not induce apoptosis, it preferentially enhanced D269H/E195R-induced apoptosis over rhTRAIL. Combination treatment potentiated the cleavage of caspases 8, 9, 3 and PARP. P53 and MDM2 siRNA experiments showed that this enhanced apoptotic effect was mediated by wild-type p53. Indeed, nutlin-3 did not enhance rhTRAIL-induced apoptosis in OVCAR-3 cells harbouring mutant p53. Addition of the chemotherapeutic drug cisplatin to the combination further increased p53 and DR5 levels and rhTRAIL- and D269H/E195R-induced apoptosis. As a proof of concept, we show that the combination of D269H/E195R, nutlin-3 and cisplatin induced massive apoptosis in ex vivo tissue slices of primary human ovarian cancers.Conclusion:Nutlin-3 is a potent enhancer of D269H/E195R-induced apoptosis in wild-type p53-expressing cancer cells. Addition of DNA-damaging agents such as cisplatin further enhances DR5-mediated apoptosis.

Activation of the PKC Pathway Stimulates Ovarian Cancer Cell Proliferation, Migration, and Expression of MMP7 and MMP101

Postmenopausal women are at a higher risk of ovarian cancer due, in part, to increased levels of gonadotropins such as luteinizing hormone (LH). Gonadotropins and other stimuli are capable of activating two pathways, PKA and PKC, that are altered in ovarian cancer. To determine the role of LH on ovarian cancer, we explored the effects of human chorionic gonadotropin (hCG), an LH mimic, and an activator of the PKC pathway, phorbol-12-myristate 13-acetate (PMA), on ovarian cancer cell-cycle kinetics and apoptosis in Ovcar3 cells. PMA treatment increased cells in the S phase of the cell cycle and initially increased apoptosis after 4 h before diminishing apoptosis after 8 h. Treatment of ovarian cancer cells with hCG had no effect on these parameters. The PKC pathway is known to differentially regulate matrix metalloproteinase (MMP) expression. Results showed that ovarian cancer cells treated with PMA increased MMP7 and MMP10 mRNA levels after 8 h of treatment, and expression remained high after 12 h before decreasing at 24 h. The mRNA expression of extracellular matrix metalloproteinase inducer (BSG), an activator of MMPs, was unaffected by PMA. Due to the role that MMPs play in migration, we investigated the effect of PMA activation of MMPs on ovarian cancer cell migration. The use of the MMP inhibitor GM6001 blocked the increased migratory effects of PMA on ovarian cancer cells. Together, these studies show that activating the PKC pathway causes significant changes in cell cycle kinetics and selective expression of MMPs that are involved in enhancing ovarian cancer cell proliferation and migration.

Deleted in liver cancer-1 and focal adhesion kinase genes are implicated in the growth and apoptosis of malignant OVCAR-3 cells

Objective To investigate the effects of deleted in liver cancer-1 (DLC-1) and focal adhesion kinase (FAK) on the growth and apoptosis of malignant OVCAR-3 cells.Methods DLC-1 expression plasmid,FAK specific small hairpin RNA (shRNA) expression plasmid,and FAK-shRNA combined DLC-1 expression plasmid were constructed and transfected into OVCAR-3 cells.Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were used to detect the expression of DLC-1 and FAK in OVCAR-3 cells,cell proliferation and apoptosis were examined by cell counting kit-8 (CCK-8) kit and flow cytometry,and the expression of phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) was measured by using Western blotting.Results While the DLC-1 expression increased or/and FAK knocked down,the growth of OVCAR-3 cells was obviously inhibited [(0.737 ± 0.094),P ＜0.05],but apoptosis was significantly induced [(15.21 ± 1.05) ％,P ＜ 0.05].In addition,the expression of p-ERK1/2 was notably decreased [(0.151 ±0.010),P ＜0.05].Conclusion DLC1 might be implicated in the growth and apoptosis of malignant tumor cells via its regulation on FAK and downstream ERK signaling. Key words: Deleted in liver cancer-1 ; Focal adhesion kinase; Small hairpin RNA

Anticancer role of MUC1 aptamer–miR-29b chimera in epithelial ovarian carcinoma cells through regulation of PTEN methylation

Ovarian cancer has a poor prognosis and advanced ovarian cancer lacks effective therapy. In this study, we seek to establish targeting therapy for ovarian cancer through tumor tissue-specific delivery of miRNA-29b to reexpress PTEN tumor-suppressor gene. A chimera (Chi-29b) was constructed to compose of a mucin 1 (MUC1) aptamer targeting tumor cell surface MUC1 protein and miR-29b inhibiting DNA methyltransferases' expression, subsequently reexpressing PTEN gene. The specificity and efficacy of the chimera delivery were analyzed in OVCAR-3 ovarian tumor cells, and the biological activities of the chimera were identified by the expression of its downstream molecules and cell apoptosis. We demonstrated that Chi-29b chimera can be specifically delivered into OVCAR-3 cells in a concentration-dependent manner. Dicer efficiently cleaved the Chi-29b chimera to release miR-29b. Chi-29b chimera downregulated Dnmt1, Dnmt3a, and Dnmt3b protein levels; induced hypomethylation in PTEN promoter; and upregulated PTEN mRNA and protein expression in OVCAR-3 cells. Importantly, Chi-29b chimera significantly induced apoptosis in OVCAR-3 cells. Our study indicated that Chi-29b chimera can effectively exert antitumor effect through specific delivery of miR-29b into OVCAR-3 tumor cells, subsequently reexpressing PTEN gene and inducing cell apoptosis.