Human Ovarian Cancer HO-8910 Cells Research Articles

A novel derivative of Genistein inhibits proliferation of ovarian cancer HO-8910 cells by regulating reactive oxygen species*

Abstract Objective To investigate the anticancer effect of a novel derivative of genistein (5-hydroxy-4′-nitro-7-propionyloxy-genistein, HNPG) on human ovarian cancer HO-8910 cells and its possible molecular mechanism. Methods HO-8910 cells were cultured in vitro, and the inhibitory effect of HNPG on proliferation was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The effect of HNPG on inducing apoptosis was examined using FCM with Annexin V-FITC and propidium iodide staining. The effect of HNPG on regulating reactive oxygen species (ROS) was measured using FCM with 2′,7′-di chlorodihydro-fluorescein diacetate staining. The effect of HNPG on modulating mitochondrial membrane potential (MMP) was determined using FCM with lipophilic cationic dye 2 (6 Amino 3 imino 3H xanthen 9 yl) benzoic acid methyl ester (Rh123) staining. The bioactivity of superoxide dismutase (SOD) and catalase (CAT) and the content of glutathione (GSH) and malondialdehyde (MDA) were detected using enzyme-linked immunosorbent assay. The related apoptotic proteins, including bcl-2, bax, cyt-c, and cleaved-caspase-3, were assessed using western blotting. Results HNPG exhibited dramatic antitumor activity against HO-8910 cells in vitro, inhibited proliferation, and induced apoptosis in a time- and dose-dependent manner. These effects were accompanied by reduced bioactivity of SOD and CAT, reduced GSH content, and enhanced MDA content. Simultaneously, the amount of ROS was increased and the level of MMP was reduced, along with upregulation of mitochondrial apoptosis pathway-related proteins, bax, cyt-c, and cleaved-caspase-3; bcl-2 protein was downregulated. Conclusion HNPG inhibited proliferation of human ovarian cancer HO-8910 cells in vitro, which might be related to decreased bioactivity of SOD and CAT. HNPG also reduced GSH content, which resulted in ROS accumulation in cells, damaged the integrity of mitochondrial membrane, and induced cell apoptosis.

Effects of EIF3B gene downregulation on apoptosis and proliferation of human ovarian cancer SKOV3 and HO-8910 cells

In order to investigate the role of EIF3B (eukaryotic translation initiation factor 3B, EIF3B) gene in the proliferation and apoptosis of ovarian cancer cells, a lentiviral vector system and shEIF3B lentiviral vector were constructed to transfect human ovarian cancer cells. SKOV3 and HO-8910 cells were used in this experiment. The cell growth was detected by Celigo cell counting assay, the apoptosis rate was measured by flow cytometry and the cell proliferation ability of lentivirus transfected cells was tested by MTT assay and clone formation assay. Results showed that the specific shRNA had a significant inhibitory effect on the expression of EIF3B gene. Compared with the negative control, the expression of EIF3B mRNA and protein in SKOV3 and HO-8910 cells were significantly inhibited in the knockdown group. Then the proliferation rate of each group was tested, we found that SKOV3 and HO-8910 cells in siRNA lentivirus infected group was significantly decreased. Same result was obtained from the clonogenic assay of which the colony formation of transfected cells was significantly inhibited compared with the control group. Further study showed that the proliferation inhibitory effect was associated with as increased apoptosis rate of SKOV3 and HO-8910 cells in EIF3B knockdown groups. All in all, inhibition of EIF3B gene expression significantly inhibit the proliferation of ovarian cancer cells and increase the apoptosis of ovarian cancer cells. These results provide a new basis for the study of the molecular mechanism of ovarian cancer development and provide new target gene for ovarian cancer treatment.

Delivery of Constitutively Active Mutant MKK6(E) With TAT-OSBP Induces Apoptosis in Human Ovarian Carcinoma HO8910 Cells.

Biologically active peptides and proteins are novel agents that show promise in the development of anticancer drugs. Their relatively low cell permeability and poor tumor selectivity, however, impede their widespread applicability. In this study, we evaluated the tumor selectivity, cellular internalization, and biological activity of a cell-permeable ovarian cancer cell-specific therapeutic protein consisting of TAT-OSBP and constitutively active MKK6(E), an upstream kinase of the p38 signaling pathway that mediates cellular apoptosis. OSBP, a 7-amino-acid peptide with high affinity for human ovarian cancer HO8910 cells, was conjugated to the cell-penetrating peptide (TAT) to form a tumor-selective peptide (TAT-OSBP), which was further conjugated with EGFP or MKK6(E). Flow cytometry and fluorescent microscopy were performed to evaluate the tumor-targeted penetration of TAT-OSBP-EGFP. The inhibitory effects of TAT-OSBP-MKK6(E) were determined by cell proliferation and apoptosis assays. The internalization efficiency of TAT-OSBP-EGFP was significantly higher than that of TAT-EGFP. TAT-OSBP-EGFP selectively penetrated HO8910 cells. TAT-OSBP-MKK6(E) fusion protein inhibited cancer cell growth to varying degrees, with the highest level of inhibition in HO8910 cells. Moreover, TAT-OSBP-MKK6(E) significantly induced apoptosis of HO8910 cells. However, there was no significant difference in apoptosis in the normal ovarian epithelial cells treated with either TAT-OSBP-MKK6(E) or TAT-MKK6(E). Our results demonstrate that TAT-OSBP-MKK6(E) is a novel artificially designed molecule, which induces apoptosis and selectively targets human ovarian carcinoma HO8910 cells. Our study provides novel insights that may aid in the development of a new generation of anticancer drugs.

TAT-OSBP-1-MKK6(E), a novel TAT-fusion protein with high selectivity for human ovarian cancer, exhibits anti-tumor activity.

To improve the selectivity of TAT-fusion proteins for targeted cancer therapy, we developed a novel TAT-based target-specific fusion protein, TAT-OSBP-1-MKK6(E), and evaluated its selectivity and anti-tumor activity in vitro and in vivo. The fusion protein containing TAT-OSBP-1-MKK6(E) has three functional domains: (1) the protein transduction domain of TAT, (2) the human ovarian cancer HO8910 cell-specific binding peptide (OSBP-1) and (3) the potential anti-tumor effector domain of MKK6(E). The transduction efficiency, selectivity, cytotoxicity and apoptotic effect of TAT-OSBP-1-MKK6(E) were examined using immunofluorescence, CCK8 assay and flow cytometry. The in vivo anti-tumor efficacy and target specificity of the fusion protein were evaluated using a nude mouse model with subcutaneous xenografts of human ovarian cancer HO8910 cells. Tumor-bearing mice were divided into three treatment groups that received tail vein injections of TAT-OSBP-1-MKK6(E), TAT-OSBP-1 or normal saline. Tumor growth inhibition was determined by tumor volume, weight and morphology. The distribution and apoptotic effect of TAT-OSBP-1-MKK6(E) were assessed by immunohistochemical staining and TUNEL assays. TAT-OSBP-1-MKK6(E) can be selectively internalized into human ovarian cancer HO8910 cells, rather than normal ovarian OSE cells. In vivo, the fusion protein was mainly expressed in the tumor xenograft, but not in ovary or liver tissues. As a result, TAT-OSBP-1-MKK6(E) significantly induced growth inhibition and apoptosis of tumor cells in vitro and in vivo, with limited effects in normal cells and tissues. TAT-OSBP-1-MKK6(E) treatment can selectively target HO8910 cells in vitro and in vivo, leading to growth inhibition and apoptosis of tumor cells. As such, TAT-OSBP-1-MKK6(E) may be a potential approach for ovarian cancer target therapy.

20(s)-ginsenoside Rg3 promotes apoptosis in human ovarian cancer HO-8910 cells through PI3K/Akt and XIAP pathways.

Ovarian cancer is a serious tumor which represents a great threat to women's health. Recently, researchers had found that 20(s)-ginsenoside Rg3 could inhibit growth of several cancer cell lines; however, the mechanism is not fully understood so far. In the present study, we found that 20(s)-ginsenoside Rg3 reduced cell viability and induced apoptosis in a dose- and time-dependent manner in the human ovarian cancer cells HO-8910. The induction of apoptosis was accompanied by downregulation of phosphatidylinositol 3-kinase (PI3K)/Akt family proteins and inhibitor of apoptosis protein (IAP) family proteins. 20(s)-ginsenoside Rg3 treatment resulted in activation of caspase-3 and -9, which may partly explain the anti-cancer activity of 20(s)-ginsenoside Rg3. Taken together, our study for the first time suggests that 20(s)-ginsenoside Rg3 is able to enhance apoptosis of HO-8910 cells, at least in part, through downregulation of PI3K/Akt and IAP family proteins. Moreover, the triggering of caspase-3 and -9 activation mediated apoptotic induction. Our data indicate that 20(s)-ginsenoside Rg3 is an effective apoptosis-inducing natural compound in ovarian cancer cells and may have a role in future therapies for ovarian cancer.

SGI-1776, an imidazo pyridazine compound, inhibits the proliferation of ovarian cancer cells by inactivating Pim-1.

To investigate the antitumor effect of SGI-1776 on human ovarian cancer HO-8910 cells and its molecular mechanism. HO-8910 cells were cultured in vitro, and the proliferation inhibitory effects of SGI- 1776 were determined by MTT assay and colony formation assay. The effect of SGI-1776 on the distribution of cell cycle phase was observed by flow cytometry with propidium iodide (PI) staining. The inhibition rate of migration and invasion were valued by transwell cell assay. Multiple molecular techniques, such as ELISA, Western blot, siRNA and cDNA transfection were used to explore the molecular mechanism. SGI-1776 presented dramatic anti-tumor activity against HO-8910 cells in vitro, inhibited the cells proliferation and colony formation, and attenuated the migration and invasion in a dosedependent manner, accompanied by cell cycle arrest in G1 phase. SGI-1776 caused the proliferation inhibition with concomitant decrease in Pim-1 kinase activity, down-regulated the expression of Pim-1 protein and and its downstream genes, such as CDK6, pCDK6, CDK4, pCDK4, CDK2 and pCDK2, and increased the expression of P21 and P27. Down-regulation expression of Pim-1 by siRNA followed SGI-1776 treatment resulted in enhanced cell proliferation inhibition rate and attenuated migration/invasion. Up-regulation of Pim-1 by cDNA transfection attenuated SGI- 1776-induced cell proliferation inhibition and its migration/invasion. Pim-1 mediates the biological effect of SGI-1776 in human ovarian cancer HO-8910 cells, suggesting Pim-1 might be a novel target for human ovarian cancer.

Aspidin BB, a phloroglucinol derivative, induces cell cycle arrest and apoptosis in human ovarian HO-8910 cells

Aspidin BB, an effective phloroglucinol derivative from Dryopteris fragrans (L.) Schott, has been previously reported to exert high biological activities. In this study, we analyzed the underlying mechanisms of aspidin BB on human ovarian cancer cell line, HO-8910. Aspidin BB significantly inhibited HO-8910 cell proliferation in a dose- and time-dependent manner. The IC50 values were 15.02, 25.79 and 68.81μM after 72, 48 and 24h treatment, respectively. Meanwhile, aspidin BB markedly induced apoptosis evidenced by characteristic apoptotic morphological changes, nuclear DNA fragmentation, annexin V-FITC/propidium iodide (PI) double staining and S peak. Western blot analysis showed that aspidin BB suppressed Bcl-2 expression and enhanced Bax expression to desintegrate the outer mitochondrial membrane, then caused cytochrome c release which led to the activation of effector caspase-3, and further cleaved the poly ADP-ribose polymerase (PARP) in the nucleus, finally induced cell apoptosis. Furthermore, aspidin BB provoked S phase arrest in HO-8910 cells with up-regulation of pRb, E2F1, CDK2, cyclin E and cyclin A proteins. Taken together, these findings support the conclusion that aspidin BB exhibits cytotoxicity towards human ovarian cancer HO-8910 cells through induction of apoptosis via mitochondrial pathway and arresting cell cycle progression in S phase.

Casticin combination with Cisplatin in sub-toxic concentration induced apoptosis of human ovarian cancer HO-8910 cells in vitro

The aim of the study was to investigate the effect of Casticin (CAS) combination with Cisplatin (DDP) in sub-toxic concentration on apoptosis of human ovarian cancer HO-8910 cells in vitro and unravel the associated mechanisms. Human ovarian cancer HO-8910 cells were cultured in vitro. The inhibitory effect of CAS combination with DDP in sub-toxic concentration on viability of human ovarian cancer HO-8910 cells was evaluated by the MTT assay. Morphological changes of cell apoptosis were detected by Hoechst 33258 staining assay. Cell apoptosis rate was analyzed by flow cytometry. The protein expression level was analyzed by Western blot. CAS in sub-toxic concentration and DDP in sub-toxic concentration could slightly inhibit Human ovarian cancer HO-8910 cells, but CAS combination with DDP in sub-toxic concentration significantly inhibited the growth of HO-8910 cells, and growth inhibition rate was increased drastically compared with the control group (P<0.01), and the inhibiting effect showed synergistic action. Human ovarian cancer HO-8910 cells showed the typical morphological changes of apoptosis and apoptosis rate markedly increased when they were exposed to CAS combination with DDP in sub-toxic concentration for 48 h. Western blot showed that the expression of bcl-2 protein was down-regulated and protein level of caspase-3 was activated by CAS combination with DDP in sub-toxic concentration. CAS combination with DDP in sub-toxic concentration could inhibit the cells growth and lead to cell apoptosis in human ovarian cancer HO-8910 cells. And the down-regulation of bcl-2 protein expression and activation of caspase-3 protein might contribute to CAS combination with DDP in sub-toxic concentration in human cancer HO-8910 cells.

The inhibitory effect of a polysaccharide from Codonopsis pilosula on tumor growth and metastasis in vitro

In this study, we prepared an acidic polysaccharide (CPPA) from the roots of Codonopsis pilosula. The effects of CPPA on tumor cell growth, invasion, and migration were examined in vitro. The CPPA not only induced a potent inhibitory effect on the invasion and migration potential of human epithelial ovarian cancer HO-8910 cells in vitro, evaluated by wound healing, transwell and cell adhesion assays, but also had an efficient anti-proliferation effect on tumor cells. Moreover, the CD44 expression on the HO-8910 cells was also attenuated by CPPA treatment. Therefore, our results indicate that CPPA may be a potential candidate compound for the prevention of tumor metastasis, presumably by inhibiting invasion, migration and adhesion of tumor cells, as well as the CD44 expression on the tumor cells.

Influence of human epithelial ovarian cancer HO-8910 cells with modified survivin gene on the cell cycle distribution and chemosensitivity

To study the influence of survivin mutant-T34A (survivin(T34A)) and survivin deletant-N-terminal 8 amino acids residues (survivin(N-8AA)) on the cell cycle distribution and chemosensitivity in human ovarian cancer HO-8910 cells for explorating the roles of modified survivin-mediated apoptosis induced by chemotherapeutic agents and possible signaling pathways involved. pcDNA3.1 plasmid contained wild-type, survivin(T34A) and survivin(N-8AA) genes were transfected into HO-8910 cells, respectively, the control groups were HO-8910 cells transfected with pcDNA3.1 plasmids. The expression of mRNA was examined by reverse transcription(RT) PCR and identified by DNA sequencing; the cell cycles were determined by flow cytometer analysis (FCM); the growth inhibitions rate of cisplatin (DDP), paclitaxel (PTX) and LY294002 on the transfected cells were determined using methyl thiazolyl tetrazolium (MTT) assay. (1) The RT-PCR procedures and genome sequences showed that the survivin mRNA were expressed stable in the transfected HO-8910 cells. (2) There was lower percent of G(0)/G(1) phase cells in SN-HO-8910 cells than that in PC-HO-8910 cells (44.72% vs. 49.64%, P < 0.05); while higher percentage of G(2)/M phase and S phase cells (1.06% and 54.22% vs. 0.56% and 49.80%, P < 0.05). There was lower the G(2)/M phase and S phase cells in M-HO-8910 cells 0.16% and 36.33%, than that in PC-HO-8910 cells (P < 0.05); while higher percentage of G(0)/G(1) phase cells (63.51%, P < 0.05). G(0)/G(1), G(2)/M and S phase cells in Sur-HO-8910 cells were 54.46%, 0.62% and 44.92%, and there were not significantly difference (P > 0.05), compared to those in PC-HO-8910 cells. (3) The inhibitory concentration (IC(50)) of DDP and PTX were higher in Sur-HO-8910 cells than those in control cells [(20.4 ± 6.1) vs. (14.4 ± 3.9) µmol/L, (36.7 ± 4.0) vs. (28.6 ± 3.6) µmol/L; all P < 0.05]. The IC(50) of DDP and LY294002 in SN-HO-8910 cells were lower than those in control cells [(7.6 ± 1.0) vs. (14.4 ± 3.9) µmol/L, (13.2 ± 4.0) vs. (41.0 ± 7.9) µmol/L; all P < 0.01]. The IC(50) of PTX [(37.9 ± 4.8) µmol/L] in SN-HO-8910 cells were higher than that in control cells (P < 0.05). The IC(50) of DDP in M-HO-8910 cells [(9.9 ± 1.2) µmol/L] were lower than that in control cells (P < 0.05), and the IC(50) of LY294002 in M-HO-8910 cells [(66.9 ± 4.8) µmol/L] higher than that in control cells (P < 0.01). The changes of cells cycle distribution caused by survivin(T34A) or survivin(N-8AA) enhanced the G(2)/M cell cycle-dependent chemosensitivity of PTX. Compared to survivin(T34A), survivin(N-8AA) preferentially to mediate the cytotoxicity of DDP and LY294002, suggesting that it may be related to the cell cycle-dependence of survivin function and to blockage of the formation of its active dimer.

Mouse trophoblastic cells exhibit a dominant invasiveness phenotype over cancer cells

Invasion process occurs both in mammalian embryo implantation during development and malignant cancer cell metastasis. We investigated the interactions between trophoblasts and metastatic cancer cells and found the phenomenon that mouse trophoblastic cells invaded the monolayer of malignant cancer cells in vitro and appeared the general trait of invasiveness to more than 30 types of malignant cancer cell lines which were derived from different histological origins and with different invasive or metastatic potential. We further investigated the cellular and molecular changes in the process of mouse trophoblastic cells invading human ovarian cancer HO-8910 cells. The results show that the invasion of trophoblastic cells lead HO-8910 cells near mouse embryo to apoptosis, and expression of cell-cycle-related protein cyclinD1 and Ki-67 mRNA were steadily remained both in mouse blastocysts and human ovarian cancer HO-8910 cells, which in part explain the proliferation activities of these cells. Our study also shows that expression of some proteins including MMP-9, FAK and Integrinαvβ3 was changeable in trophoblastic cells and HO-8910 cells in the process of blastocyst invasion, which suggested temporal expression of these molecules may involved in the invasive behavior of trophoblasts cells to cancer cells.

Genistein induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint pathways

Genistein is a major isoflavonoid in dietary soybean, commonly consumed in Asia. Genistein exerts inhibitory effects on the proliferation of various cancer cells and plays an important role in cancer prevention. However, the molecular and cellular mechanisms of genistein on human ovarian cancer cells are still little known. We show that exposure of human ovarian cancer HO-8910 cells to genistein induces DNA damage, and triggers G2/M phase arrest and apoptosis. Furthermore, we also found that checkpoint proteins ATM and ATR are phosphorylated and activated in the cells treated with genistein. It is also shown that genistein increases the phosphorylation and activation of Chk1 and Chk2, which results in the phosphorylation and inactivation of phosphatases Cdc25C and Cdc25A, and thereby the phosphorylation and inactivation of Cdc2 which arrests cells in G2/M phase. Moreover, genistein enhances the phosphorylation and activation of p53, while decreases the ratio of Bcl-2/Bax and Bcl-xL/Bax and the level of phosphorylated Akt, which result in cells undergoing apoptosis. These results demonstrate that genistein-activated ATM-Chk2-Cdc25 and ATR-Chk1-Cdc25 DNA damage checkpoint pathways can arrest ovarian cancer cells in G2/M phase, and induce apoptosis while the cellular DNA damage is too serious to be repaired. Thus, the antiproliferative, DNA damage-inducing and pro-apoptotic activities of genistein are probably responsible for its genotoxic effects on human ovarian cancer HO-8910 cells.

A New Flavanone Glycoside with Anti-proliferation Activity from the Root Bark of Morus alba

Aim To study the bioactive constituents from the root bark of Morus alba. Methods Compound 1 was separated and purified by silica gel chromatography and Sephadex LH-20 (MeOH). Its structural identification was based on physical properties and spectroscopic analysis (including 2D NMR). And it was tested for its anti-proliferation activity against HO-8910 cells. Results A novel flavanone glycoside, 5,2′-dihydroxyflavanone-7,4′-di- O- β- D-glucoside (steppogenin-7,4′-di- O- β- D-glucosiade), was isolated from the root bark of Morus alba. Its IC 50 of 48 h and 72 h are 3.68, 1.87 μmol·L −1, respectively. Conclusion Compound 1 is a new flavanone glycoside and it shows significant inhibiting activity against human ovarian cancer HO-8910 cells' proliferation.

Effect of 3'-azido-3'-deoxythymidine (AZT) on telomerase activity and proliferation of HO-8910 cell line of ovarian cancer

Objective: To study the effect of 3`-azido-3`-deoxythymidine (AZT) on telomerase activity and the proliferation of ovarian cancer cells in vitro. Methods: Telomerase activity was detected by enzyme linked immunosorbent assay (ELISA) in treated and untreated HO-8910 cells by AZT. The detection of cell viability was performed with 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide (MTT) assay and the ultrastructure of the cells was observed by electron microscopy. The apoptotic rate of the cells was measured by flow cytometry. Results: AZT significantly inhibited telomerase activity of HO-8910 cells, and the effect was both time- and dose-dependent. The HO-8910 cells treated at different concentrations of AZT showed a significant reduction of cell viability and morphological changes of apoptosis. The apoptotic peak was detected in the AZT treated cells and the apoptotic rate was 14.2%. Conclusion: AZT can effectively inhibit both telomerase activity and proliferation of human ovarian cancer HO-8910 cells in vitro, suggesting that AZT may be used in the clinic treatment of ovarian cancer.