PA-1 Ovarian Cancer Cells Research Articles

HY251, a Novel Decahydrocyclopenta[a]indene Analog, Induces Apoptosis via tBid-Mediated Intrinsic Pathway in Human Ovarian Cancer PA-1 Cells

We previously isolated a novel compound, HY251, with the molecular structure of 3-propyl-2-vinyl-1,2,3,3a,3b,6,7,7a,8,8adecahydrocyclopenta[ a]indene-3,3a,7a,8a-tetraol from the roots of Aralia continentalis. The current study was designed to evaluate the detailed molecular mechanisms underlying the apoptotic induction by HY251 in human ovarian cancer PA-1 cells. TUNEL assay and Western blot analyses revealed an appreciable apoptotic induction in PA-1 cells treated with 60 μM of HY251 for 24 h. This apoptotic induction was associated with caspase-8-dependent Bid cleavage, which in turn resulted in the formation of pro-apoptotic truncated Bid (tBid), and activation of caspase-9 and -3, as well as the cleavage of poly(ADP-ribose) polymerase (PARP). Moreover, we found that this death event was also associated with the significant upregulation and activation of the p53 tumor-suppressor protein through phosphorylation at Ser15. Therefore, we suggest that HY251 may be a potent cancer chemotherapeutic candidate for the treatment of ovarian cancer.

Anti-cancer effect of bee venom toxin and melittin in ovarian cancer cells through induction of death receptors and inhibition of JAK2/STAT3 pathway

We investigated whether bee venom and melittin, a major component of bee venom, inhibit cell growth through enhancement of death receptor expressions in the human ovarian cancer cells, SKOV3 and PA-1. Bee venom (1–5 μg/ml) and melittin (0.5–2 μg/ml) inhibited the growth of SKOV3 and PA-1 ovarian cancer cells by the induction of apoptotic cell death in a dose dependent manner. Consistent with apoptotic cell death, expression of death receptor (DR) 3 and DR6 was increased in both cancer cells, but expression of DR4 was increased only in PA-1 cells. Expression of DR downstream pro-apoptotic proteins including caspase-3, 8, and Bax was concomitantly increased, but the phosphorylation of JAK2 and STAT3 and the expression of Bcl-2 were inhibited by treatment with bee venom and melittin in SKOV3 and PA-1 cells. Expression of cleaved caspase-3 was increased in SKOV3, but cleaved caspase-8 was increased in PA-1 cells. Moreover, deletion of DR3, DR4, and DR6 by small interfering RNA significantly reversed bee venom and melittin-induced cell growth inhibitory effect as well as down regulation of STAT3 by bee venom and melittin in SKOV3 and PA-1 ovarian cancer cell. These results suggest that bee venom and melittin induce apoptotic cell death in ovarian cancer cells through enhancement of DR3, DR4, and DR6 expression and inhibition of STAT3 pathway.

Resveratrol Suppresses Growth of Human Ovarian Cancer Cells in Culture and in a Murine Xenograft Model: Eukaryotic Elongation Factor 1A2 as a Potential Target

The eukaryotic elongation factor 1A2 (eEF1A2) is known to retain oncogenic potential and is recognized as a novel target for cancer prevention and therapy. Resveratrol (trans-3,4',5-trihydroxystilbene), a phytoalexin present in grapes, has been reported to possess chemopreventive and chemotherapeutic activities. In the present study, we examined the growth-inhibitory effects of resveratrol in human ovarian cancer PA-1 cells, considering eEF1A2 as a potential molecular target. Pretreatment with resveratrol attenuated proliferation of serum-starved PA-1 cells stimulated with insulin or serum. Resveratrol also activated caspase-9, -7, and -3 and induced apoptosis in PA-1 cells in the presence of insulin or serum. Insulin or serum stimulation of PA-1 cells resulted in the marked induction of eEF1A2, which was suppressed by pretreatment with resveratrol. Moreover, resveratrol inhibited insulin- or serum-induced soft-agar colony formation in eEF1A2-transfected NIH3T3 cells. An antibody array directed to assess the phosphorylation of protein kinases revealed that treatment with insulin or serum induced the phosphorylation of Akt in PA-1 cells. Pharmacologic inhibition of Akt with LY294002 abrogated insulin- or serum-induced eEF1A2 expression and increased the caspase-3 activity. In another experiment, i.p. administration of resveratrol retarded the growth of PA-1 cell xenograft and the expression of eEF1A2 in athymic nude mice in association with decreased bromodeoxyuridine positivity, reduced expression of proliferating cell nuclear antigen, increased the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase-3 staining, and diminished CD31 positivity. Taken together, eEF1A2 may be considered as a potential molecular target for the antiproliferative effects of resveratrol in PA-1 ovarian cancer cells.

Induction of apoptosis in PA-1 ovarian cancer cells by vitamin K2 is associated with an increase in the level of TR3/Nur77 and its accumulation in mitochondria and nuclei

We examined the growth-inhibitory and apoptosis-inducing effects of vitamin K(2) (VK(2); menaquinone-4) on various lines of human ovarian cancer cells to study the mechanism of induction of apoptosis by VK(2). Cell proliferation was determined by XTT method, and apoptotic cells were detected by Hoechst staining. TR3, also known as Nur77 and NGFI-B, was detected by immunoblotting and immunofluorescence analysis. Role of TR3 on induction of apoptosis was examined by a siRNA experiment. We found that PA-1 cells were the most sensitive to VK(2) (IC(50) = 5.0 +/- 0.7 microM), while SK-OV-3 cells were resistant to VK(2). Immunoblotting and immunofluorescence analyses indicated that levels of TR3 were elevated in cell lysates 48 h after the start of treatment with 30 microM VK(2). In the VK(2)-treated cells, TR3 accumulated at significant levels in mitochondria, as well as in the nuclei of PA-1 cells. No similar changes were observed in SK-OV-3 cells under the same conditions. Treatment of PA-1 cells with small interfering RNA (siRNA) directed against TR3, and with cycloheximide or SP600125 (an inhibitor of c-jun N-terminal kinase; JNK), separately, inhibited the VK(2)-induced synthesis of TR3 and apoptosis. From these results, we can conclude that an increase in the synthesis of TR3 and the accumulation of TR3 in mitochondria and in nuclei might be involved in the induction of apoptosis by VK(2) and that the synthesis of TR3 might be regulated through a JNK signaling pathway.

Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy

Carrier Cell–mediated Delivery of a Replication-competent Adenovirus for Cancer Gene Therapy

Maspin Expression Is Characteristic for Cisplatin-Sensitive Ovarian Cancer Cells and for Ovarian Cancer Cases of Longer Survival Rates

High cytoplasmic expression of maspin was described in ovarian cancers of shorter survival rates. Until now, no relationship has been described between expression of maspin and sensitivity to cisplatin in ovarian cancers. This study aimed at examining the relationship between expression of maspin, detected by immunohistochemistry and clinical response to cisplatin in ovarian cancer cases as well as the in vitro sensitivity to cisplatin of 11 ovarian cancer cell lines. The analyzes were performed on 73 samples of ovarian cancer and on A2780P, A2780RCIS, CAOV-3, EFO 21, EFO 27, ES-2, Mdah 2774, OAW 42, OVCAR-3, PA-1, and SKOV-3 ovarian cancer cells. Cytoplasmic maspin expression in studied cells significantly correlated with cisplatin sensitivity. A significantly shorter overall survival and progression-free survival was associated with lower cytoplasmic maspin expression at first-look laparotomies and nuclear maspin expression and secondary cytoreductions. Higher nuclear maspin at first-look laparotomies expression was specific for cases of complete response. In the study, the elevated expression of maspin was shown to be typical for cisplatin-sensitive ovarian cancers.

Schedule-Dependent Interactions Between Pemetrexed and Cisplatin in Human Carcinoma Cell Lines In Vitro

The combination of pemetrexed and cisplatin shows good clinical activity against mesothelioma and lung cancer. In order to study the potential cellular basis for this, and provide leads as to how to optimize the combination, we studied the schedule-dependent cytotoxic effects of pemetrexed and cisplatin against four human cancer cell lines in vitro. Tumor cells were incubated with pemetrexed and cisplatin for 24 h at various schedules. The combination effects after 5 days were analyzed by the isobologram method. Both simultaneous exposure to pemetrexed and cisplatin for 24 h and sequential exposure to cisplatin for 24 h followed by pemetrexed for 24 h produced antagonistic effects in human lung cancer A549, breast cancer MCF7, and ovarian cancer PA1 cells and additive effects in colon cancer WiDr cells. Pemetrexed for 24 h followed by cisplatin for 24 h produced synergistic effects in MCF7 cells, additive/synergistic effects in A549 and PA1 cells, and additive effects in WiDr cells. Cell cycle analysis of MCF7 and PA1 cells supported these findings. Our results suggest that the simultaneous clinical administration of pemetrexed and cisplatin may be suboptimal. The optimal schedule of pemetrexed in combination with cisplatin at the cellular level is the sequential administration of pemetrexed followed by cisplatin and this schedule is worthy of clinical investigations.

529. Oncolytic Virotherapy Employing flt-1 Driven mda-7/IL-24 Gene Delivery

Top of pageAbstract Vascular-targeted anticancer therapy using conditionally replicating adenoviruses (CRAd) is an attractive strategy for cancer treatment. For specific retargeting of CRAd, selective transcription of viral genes utilizing tumor-specific promoters has been developed. CRAd targeting using insertion of the tumor-specific binding RGD ligand in the Ad fiber has shown increased infectivity. A cancer growth-suppressing gene, mda-7/IL-24, has demonstrated profound and selective anticancer activity. The central hypothesis of this study was that selective transcriptional and transductional targeting of CRAd encoding the mda-7/IL-24 gene using flt-1 promoter for specific E1a expression and RDG-4C modification of the Ad fiber-knob region can increase efficacy via direct killing of tumor endothelial cells and flt-1-positive cancer cells. Specific mda-7/IL-24 overexpression should enhance the anti-tumor efficacy of CRAd oncolytic virotherapy alone. The flt-1 promoter-driven firefly luciferase expression was determined using replication-defective Adflt-Luc and AdCMV-Luc vectors on a panel of human glioma, breast, ovarian and prostate cancer cells and human or mouse endothelial cells. The cytotoxicity of cancer and endothelial cells to CRAd infection was analyzed using the crystal violet inclusion assay. The CRAd progeny production was determined using real time PCR and a standard 50% tissue culture infectious dose assay. Tumor xenograft models were used to determinate the therapeutic efficacy of oncolytic virotherapy to treat human glioma, breast, ovarian and prostate tumors. Specificity of flt-1 promoter for human and mouse endothelial cells and U251MG glioma, PA-1 ovarian, LCC6 breast, and DU145 prostate cancer cells was demonstrated. We have developed a CRAd using the flt-1 promoter element for specific E1a gene expression, encoding the mda-7/IL-24 gene (CRAdRGDflt-mda-7) and a CDCGRDCFC-containing peptide in the HI loop of the fiber knob domain. The cancer cells demonstrated different levels of cytotoxicity after CRAdRGDflt-mda-7 infection. There was a significant decrease in viability of U251MG, PA-1, LCC6, and DU145 cells infected with CRAdRGDflt-mda-7. In vivo therapy studies demonstrated that intratumoral injection of CRAdRGDflt-mda-7 produced significant inhibition of subcutaneous LCC6 breast, OVCAR-3 ovarian, and DU145 prostate cancer xenografts and delay of D54MG human glioma tumor growth in athymic nude mice. These results suggest that selective targeting of oncolytic virotherapy with enhanced potency via mda-7/IL-24 pro-apoptotic/anti-angiogenic gene expression would be a superior strategy for treatment of human cancer.

Insulin-like growth factor binding protein 2 promotes ovarian cancer cell invasion

BackgroundInsulin-like growth factor binding protein 2 (IGFBP2) is overexpressed in ovarian malignant tissues and in the serum and cystic fluid of ovarian cancer patients, suggesting an important role of IGFBP2 in the biology of ovarian cancer. The purpose of this study was to assess the role of increased IGFBP2 in ovarian cancer cells.ResultsUsing western blotting and tissue microarray analyses, we showed that IGFBP2 was frequently overexpressed in ovarian carcinomas compared with normal ovarian tissues. Furthermore, IGFBP2 was significantly overexpressed in invasive serous ovarian carcinomas compared with borderline serous ovarian tumors. To test whether increased IGFBP2 contributes to the highly invasive nature of ovarian cancer cells, we generated IGFBP2-overexpressing cells from an SKOV3 ovarian cancer cell line, which has a very low level of endogenous IGFBP2. A Matrigel invasion assay showed that these IGFBP2-overexpressing cells were more invasive than the control cells. We then designed small interference RNA (siRNA) molecules that attenuated IGFBP2 expression in PA-1 ovarian cancer cells, which have a high level of endogenous IGFBP2. The Matrigel invasion assay showed that the attenuation of IGFBP2 expression indeed decreased the invasiveness of PA-1 cells.ConclusionsWe therefore showed that IGFBP2 enhances the invasion capacity of ovarian cancer cells. Blockage of IGFBP2 may thus constitute a viable strategy for targeted cancer therapy.

IG20, in contrast to DENN-SV, (MADD splice variants) suppresses tumor cell survival, and enhances their susceptibility to apoptosis and cancer drugs.

We identified seven putative splice variants of the human IG20 gene. Four variants namely, IG20, MADD, IG20-SV2 and DENN-SV are expressed in human tissues. While DENN-SV is constitutively expressed in all tissues, expression of IG20 appears to be regulated. Interestingly, overexpression of DENN-SV enhanced cell replication and resistance to treatments with TNFalpha, vinblastine, etoposide and gamma-radiation. In contrast, IG20 expression suppressed cell replication and increased susceptibility to the above treatments. Moreover, cells that were resistant and susceptible to TNFalpha-induced apoptosis exclusively expressed endogenous DENN-SV and IG20, respectively. When PA-1 ovarian cancer cells that are devoid of endogenous IG20 variant, but express higher levels of DENN-SV, were transfected with IG20, they showed reduced cell proliferation and increased susceptibility to apoptosis induced by TNFalpha, TRAIL and gamma-radiation. This indicated that overexpression of IG20 can override endogenous DENN-SV function. CrmA reversed the effects of IG20, but not DENN-SV. In contrast, dominant-negative-I-kappa B reversed the effects of DENN-SV, but not IG20, and showed that DENN-SV most likely exerted its effects through NFkappaB activation. Together, our data show that IG20 gene can play a novel and significant role in regulating cell proliferation, survival and death through alternative mRNA splicing.

Genome-scale analysis of resveratrol-induced gene expression profile in human ovarian cancer cells using a cDNA microarray

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural compound found in large quantities, most notably in grapes and red wine, which has been shown to have anti-inflammatory, chemopreventive and anti-angiogenic effects. We examined whether resveratrol has any effect on growth and gene expression in the human ovarian cancer PA-1 cells. We show that resveratrol inhibits cell growth and induces apoptosis in PA-1 human ovarian cancer cells. We also investigated the effect of resveratrol on changes of global gene expression during resveratrol-induced growth inhibition and apoptosis in PA-1 cells using a human cDNA microarray with 7,448 sequence-verified clones. Out of the 7,448 genes screened, 118 genes were founded to be affected in their expression levels by more than 2-fold after 24-h treatment with 50 micro M resveratrol. Resveratrol treatment of PA-1 cells at the final concentration of 50 micro M for 6, 12, 24 and 48 h and gene expression patterns were analyzed by microarray. Clustering of the genes modulated more than 2-fold at three of the above times points divided the genes into 2 groups. Within these groups, there were specific subgroups of genes whose expressions were substantially changed at the specified time points. One of the most highly up-regulated genes found in this study was NAD(P)H quinone oxidoreductase 1(NQO-1), which has recently been shown to be involved in p53 regulation. Although the precise roles of genes whose expression levels were found to fluctuate after resveratrol treatment remain to be elucidated, we hope that the new view of gene expression in human ovarian cancer cells following resveratrol exposure, as offered by this study, provides clues for the mechanism of resveratrol action.

Schedule-dependent interactions between paclitaxel and etoposide in human carcinoma cell lines in vitro.

Clinical studies of paclitaxel in combination with etoposide against solid tumors have been carried out. The combination schedules used in these studies are different. We studied the cytotoxic effects of paclitaxel with etoposide against four human cancer cell lines in vitro to determine the optimal schedule of this combination at the cellular level. Cells were exposed simultaneously to paclitaxel and to etoposide for 24 h or sequentially to one drug for 24 h followed by the other for 24 h, after which they were incubated in drug-free medium for 4 and 3 days, respectively. Cell growth inhibition was determined by an MTT reduction assay. The effects of drug combinations at concentrations producing 80% inhibition (IC(80)) were analyzed by the isobologram method of Steel and Peckham. The cytotoxic effect of paclitaxel and etoposide was cell line- and schedule-dependent. Simultaneous exposure to paclitaxel and etoposide for 24 h produced additive effects in the lung cancer cell line A549 and ovarian cancer PA1 cells, and antagonistic effects in the breast cancer cell line MCF7 and colon cancer WIDr cells. Sequential exposures to paclitaxel followed by etoposide and vice versa produced additive effects in all four cell lines. These results suggest that maximum cytotoxic effects can be obtained with sequential administration, but not simultaneous administration, of paclitaxel and etoposide. These findings may have important clinical implications for this combination.

Involvement of caspase family proteases in FPT inhibitor III-induced apoptosis in human ovarian cancer cells.

We have previously demonstrated that a new farnesyltransferase inhibitor, FPT inhibitor III, triggers apoptosis in human ovarian cancer cells. Here, we report that induction of apoptotic cell death in PA-1 ovarian cancer cells by FPT inhibitor III was accompanied by the activation of interleukin-1 #-converting enzyme (ICE)-like proteases, which have recently been renamed as caspases. The caspase inhibitor, ZVAD-FMK, which inhibits a number of caspase family proteases, blocked FPT inhibitor III-induced apoptotic cell death in a dose-dependent manner. Additionally, cleavage of poly(ADP-ribose) polymerase, an identified in vivo substrate for caspase family proteases, in FPT inhibitor III-treated cells was confirmed by immunoblotting. Our results suggest that the caspase family proteases are involved in the induction of apoptosis triggered by FPT inhibitor III.

Antisense Oligodeoxynucleotides Targeted against Different Regions of CyclinD1 mRNA May Exert Different Inhibitory Effects on Cell Growth and Gene Expression

The alteration of cyclin D1 gene expression is a common feature of malignancies of diverse histogenesis. Our recent results showed that over expression of cyclin D1 protein is frequently found in human ovarian cancer. In this study, we investigated the effect of cyclin D1 antisense oligodeoxynucleotides on cell growth and gene expression in PA-1 ovarian cancer cells. Our results demonstrated that cyclin D1 antisense oligodeoxynucleotides indeed functioned as an antiproliferative agent. More strikingly, we found that different cyclin D1 antisense oligodeoxynucleotides exert different efficacy on the inhibition of cell growth and gene expression. We suggest that selection and characterization of high affinity oligodeoxynucleotides is strictly required before the application of antisense technology.

Schedule-dependent interaction between paclitaxel and doxorubicin in human cancer cell lines in vitro.

The schedule-dependent interaction of paclitaxel and doxorubicin was evaluated in four human cancer cell lines. The cells were exposed simultaneously or sequentially to the two agents for 24 h, and were then incubated in drug-free medium for 4 and 3 days, respectively. The cell growth inhibitions were determined by the MTT assay. The cytotoxic interactions at the IC80 level were evaluated by the isobologram method of Steel and Peckham. In non-small cell lung cancer A549, breast cancer MCF7 and colon cancer WiDr cells, antagonistic effects were observed for the paclitaxel and doxorubicin combination on simultaneous exposure to the two agents and on sequential exposure to doxorubicin followed by paclitaxel, while additive effects were observed for the combination on sequential exposure to paclitaxel followed by doxorubicin. In ovarian cancer PA1 cells, additive effects were observed for all schedules. These findings suggest that sequential administration of paclitaxel followed by doxorubicin may be the most suitable sequence, while the simultaneous administration of the two agents and the sequential administration of doxorubicin followed by paclitaxel may result in less tumour cell kill than anticipated. Further preclinical and clinical studies are required to elucidate the relationship between paclitaxel and doxorubicin with regard to both antitumour activity and toxicity.