Expression of CD40 and growth-inhibitory activity of CD40 agonist in ovarian carcinoma cells

The CD40 receptor is a member of the tumour necrosis factor receptor family and is widely expressed on various cell types. The antitumour activity of CD40 agonist antibody has been observed in B-cell-derived malignancies, but its activity on ovarian cancer remains unclear. However, in this paper, we first confirmed that the anti-CD40 agonist antibody could inhibit the growth of ovarian cancer cells and induce apoptosis. This study investigated the expression of CD40 by ovarian carcinoma tissues and cell lines, at the same time, we evaluated the effect of a recombinant soluble human CD40L (rshCD40L) and an anti-CD40 agonist antibody on cell growth and apoptosis. Flow cytometry and immunohistochemistry assay demonstrated that CD40 was expressed on ovarian carcinoma cell lines and primary ovarian carcinoma cells derived from ascites, as well as on ovarian carcinoma tissues. The growth inhibition of rshCD40L and the anti-CD40 agonist antibody on ovarian carcinoma cells was examined by MTT assay, and the proportion of apoptotic tumour cells was analysed by flow cytometry and Hoechst staining. Our study showed that CD40 was expressed on all ovarian carcinoma cell lines and was examined in 86.2% (162/188) of ovarian cancer tissue samples, but not in normal ovarian tissues (n?=?20). Treatment with rshCD40L or anti-CD40 agonist antibody significantly inhibited ovarian carcinoma cell growth and induced apoptosis. Theses results suggest that CD40 is expressed on ovarian carcinoma cells, moreover, that rshCD40L and anti-CD40 agonist antibody have therapeutic potential to inhibit human ovarian cancer growth.     

IGF1R Axis Inhibition Restores Dendritic Cell Antitumor Response in Ovarian Cancer

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. The insulin-like growth factor (IGF) system plays a key role in regulating growth and invasiveness in several malignancies, including ovarian cancer. IGF1R targeting showed antiproliferative activity of EOC cells. However, clinical studies failed to show significant benefit. EOC cells suppress antitumor immune responses by inducing dendritic cell (DC) dysfunction. The IGF1 axis can regulate DC maturation.The current study evaluated involvement of the IGF1 axis in DC differentiation in EOC. Studies were conducted on EOC and on a human monocyte cell line. Tissue microarray analysis (TMA) was performed on 36 paraffin blocks from EOC patients. Expression of IGF1R, p53, Ki67, BRCA1, and DC markers was evaluated using immunohistochemistry. Co-culture of EOC cells with DC pretreated with IGF1R inhibitor blocked cancer cell migration. TMA demonstrated higher rate of IGF1R protein expression in patients with advanced (76.9%) as compared to early (40%) EOC. A negative correlation between IGF1R protein expression and the CD1c marker was found. These findings provide evidence that IGF1R axis inhibition could be a therapeutic strategy for ovarian cancer by restoring DC-mediated antitumor immunity.     

Organotypic culture of human ovarian surface epithelial cells: A potential model for ovarian carcinogenesis

Summary The objective of this work was to establish an in vitro multidimensional culture system for human ovarian surface epithelial (HOSE) cells as a model for ovarian carcinogenesis. The epithelial origin of cell outgrowth from cells obtained from the ovarian surface was confirmed by keratin staining. Two cultures from two different patients were established, HOSE-A and HOSE-B. Cultures were infected with a retrovirus expressing human papillomavirus genes E6 and E7 to extend their life span. HOSE cells were seeded onto collagen gels containing NIH3T3-J2 fibroblasts as feeder cells and grown to confluence submerged in growth medium. The collagen bed was then raised to the air-medium interface for 7 d (organotypic culture). Microscopically, fixed cultures revealed a single layer of flat cells growing on the collagen surface, reminiscent of HOSE cells in vivo. Infected HOSE-A and HOSE-B cells exhibited aberrant growth because they stratified. In addition, established ovarian cancer lines grown in this fashion stratified and showed malignant phenotypes. Thus, cells grown in organotypic culture resemble their in vivo counterparts, providing a basis for establishing a system to study growth, proliferation, differential gene expression, and perhaps malignant transformation of HOSE cells.     

Expression of the human fast-twitch skeletal muscle troponin I cDNA in a human ovarian carcinoma suppresses tumor growth

To explore the efficiency and mechanism of ovarian carcinoma gene therapy with the human fast-twitch skeletal muscle troponin I gene (Tnl-fast), Tnl-fast cDNA was transferred into human ovarian adenocarcinoma cell-line SK-OV-3. In vitro, the cell growth and cell cycle of Tnl-fast-, vector-, and mock-transfected cells were determined by MTT and flow cytometry assay, respectively. The conditioned media of Tnl-fast-, vector-, and mock-transfected SK-OV-3 cells were collected, and the cell proliferation inhibiting rates of human umbilical cord venous endothelial cells (HUVECs) by the three conditioned media were assayed. All the three cell lines were implanted into node mice, and the tumor growth, cell apoptosis, angiogenesis, and expression of Tnl-fast were observed or analyzed, respectively. In vitro, expression of Tnl-fast protein had no inhibiting effect on the growth of the dominant and stable transfectant cells, but endothelium, when compared with vector-transfected cells and nontransfected parental SK-OV-3 cells. Implantation of stable clone expressing Tnl-fast in the female BALB/c nude mice inhibits primary tumor growth by an average of 73%. The nude mice grafts expressing Tnl-fast exhibit a significant decrease of microvascular density, a higher rate of tumor cells apoptosis and a comparable proliferation rate as control. Our study, to our knowledge, shows the slowed down growth of the primary ovarian carcinoma, suggested that grafts were self-inhibitory by halting angiogenesis. Our data might also provide a novel useful strategy for cancer therapy by antiangiogenic gene therapy with a specific angiogenesis inhibitor Tnl-fast.     

Activation of BAD by therapeutic inhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor receptor

Novel cancer chemotherapeutics are required to induce apoptosis by activating pro-apoptotic proteins. Both epidermal growth factor (EGF) and insulin-like growth factor (IGF) provide potent survival stimuli in many epithelia, and activation of their receptors is commonly observed in solid human tumors. Here we demonstrate that blockade of the EGF receptor by a new drug in phase III clinical trails for cancer, ZD1839, potently induces apoptosis in mammary epithelial cell lines and primary cultures, as well as in a primary pleural effusion from a breast cancer patient. We identified the mechanism of apoptosis induction by ZD1839. We showed that it prevents cell survival by activating the pro-apoptotic protein BAD. Moreover, we demonstrate that IGF transactivates the EGF receptor and that ZD1839 blocks IGF-mediated phosphorylation of MAPK and BAD. Many cancer therapies kill tumor cells by inducing apoptosis as a consequence of targeting DNA; however, the threshold at which apoptosis can be triggered through DNA damage is often different from that in normal cells. Our results indicate that by targeting a growth factor-mediated survival signaling pathway, BAD phosphorylation can be manipulated therapeutically to induce apoptosis.     

Dihydroartemisinin induces apoptosis and sensitizes human ovarian cancer cells to carboplatin therapy

The present study was designed to determine the effects of artemisinin (ARS) and its derivatives on human ovarian cancer cells, to evaluate their potential as novel chemotherapeutic agents used alone or in combination with a conventional cancer chemotherapeutic agent, and to investigate their underlying mechanisms of action. Human ovarian cancer cells (A2780 and OVCAR-3), and immortalized non-tumourigenic human ovarian surface epithelial cells (IOSE144), were exposed to four ARS compounds for cytotoxicity testing. The in vitro and in vivo antitumour effects and possible underlying mechanisms of action of dihydroartemisinin (DHA), the most effective compound, were further determined in ovarian cancer cells. ARS compounds exerted potent cytotoxicity to human ovarian carcinoma cells, with minimal effects on non-tumourigenic ovarian surface epithelial (OSE) cells. DHA inhibited ovarian cancer cell growth when administered alone or in combination with carboplatin, presumably through the death receptor- and, mitochondrion-mediated caspase-dependent apoptotic pathway. These effects were also observed in in vivo ovarian A2780 and OVCAR-3 xenograft tumour models. In conclusion, ARS derivatives, particularly DHA, exhibit significant anticancer activity against ovarian cancer cells in vitro and in vivo , with minimal toxicity to non-tumourigenic human OSE cells, indicating that they may be promising therapeutic agents for ovarian cancer, either used alone or in combination with conventional chemotherapy.     

Atypical Protein Kinase C Zeta: Potential Player in Cell Survival and Cell Migration of Ovarian Cancer

Ovarian cancer is one of the most aggressive gynaecological cancers, thus understanding the different biological pathways involved in ovarian cancer progression is important in identifying potential therapeutic targets for the disease. The aim of this study was to investigate the potential roles of Protein Kinase C Zeta (PRKCZ) in ovarian cancer. The atypical protein kinase C isoform, PRKCZ, is involved in the control of various signalling processes including cell proliferation, cell survival, and cell motility, all of which are important for cancer development and progression. Herein, we observe a significant increase in cell survival upon PRKCZ over-expression in SKOV3 ovarian cancer cells; additionally, when the cells are treated with small interference RNA (siRNA) targeting PRKCZ, the motility of SKOV3 cells decreased. Furthermore, we demonstrate that over-expression of PRKCZ results in gene and/or protein expression alterations of insulin-like growth factor 1 receptor (IGF1R) and integrin beta 3 (ITGB3) in SKOV3 and OVCAR3 cells. Collectively, our study describes PRKCZ as a potential regulatory component of the IGF1R and ITGB3 pathways and suggests that it may play critical roles in ovarian tumourigenesis.     

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.     

Endothelin-1 is required during epithelial to mesenchymal transition in ovarian cancer progression

In a range of human cancers, tumorigenesis is promoted by activation of the endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis. ET-1 and ET(A)R are overexpressed in primary and metastatic ovarian carcinomas, and high levels of ET-1 are detectable in patient ascites, suggesting that ET-1 may promote tumor dissemination. Moreover, in these tumors, engagement of ET(A) receptor by ET-1 triggers tumor growth, survival, angiogenesis, and invasiveness. Thus, ET-1 enhances the secretion of matrix metalloproteinases, disrupts intercellular communications, and stimulates cell migration and invasion. Therefore, we investigated the role of the ET-1/ET(A)R autocrine axis in promoting epithelial to mesenchymal transition (EMT) in ovarian tumor cells, a key event in cancer metastasis, in which epithelial cells depolarize, disassemble cell-cell contacts, and adopt an invasive phenotype. Here, we examine the potential role of ET-1 in regulating cell morphology and behavior and epithelial and mesenchymal proteins employing an in vitro 3-D culture system. We found that in 3-D serum-free collagen I gel cultures, HEY and OVCA 433 ovarian carcinoma cells undergo fibroblast-like morphologic changes between 3 and 5 days of ET-1 treatment. In these cells, ET-1 induces loss of adherens and tight-junction protein expression, E-cadherin, beta-catenin, and zonula occludens-1, and gain of N-cadherin and vimentin expression. These results confirm the ability of ET-1 to promote EMT, a metastable process involving sustained loss of epithelial markers and gain of mesenchymal markers. Collectively, these findings provide evidence of a critical role for the ET-1/ET(A)R axis during distinct steps of ovarian carcinoma progression, thus underlining this axis as a potential target in the treatment of ovarian cancer.     

Combined application of camptothecin and the guanylate cyclase activator YC-1: Impact on cell death and apoptosis-related proteins in ovarian carcinoma cell lines

Camptothecin analogs and guanylate cyclase activator YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole] have been shown to induce apoptosis in cancer cells. However, the combined effect of camptothecin analogs and YC-1 on the viability of epithelial ovarian cancer cells remains uncertain. We assessed the combined effect of YC-1 on the camptothecin toxicity in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Camptothecin and YC-1 induced apoptosis in OVCAR-3 and SK-OV-3 cells in a dose- and time-dependent manner. Both compounds induced nuclear damage, decreased Bid and Bcl-2 protein levels, enhanced cytochrome c release, activated caspase-3 and upregulated tumor suppressor p53. Camptothecin decreased Bax protein levels, whereas YC-1 increased Bax levels. YC-1 enhanced the camptothecin-induced changes in the apoptotic protein levels and increased apoptotic effect of camptothecin on ovarian carcinoma cell lines. The results suggested that YC-1 may enhance a camptothecin toxicity against ovarian carcinoma cell lines by increasing activation of the caspase-8 and Bid pathway as well as activation of the mitochondria-mediated apoptotic pathway, leading to cytochrome c release and subsequent caspase-3 activation. Combination of camptothecin analogs and YC-1 may provide a therapeutic benefit against ovarian adenocarcinoma.     

BRCA1 regulates follistatin function in ovarian cancer and human ovarian surface epithelial cells

Follistatin (FST), a folliculogenesis regulating protein, is found in relatively high concentrations in female ovarian tissues. FST acts as an antagonist to Activin, which is often elevated in human ovarian carcinoma, and thus may serve as a potential target for therapeutic intervention against ovarian cancer. The breast cancer susceptibility gene 1 (BRCA1) is a known tumor suppressor gene in human breast cancer; however its role in ovarian cancer is not well understood. We performed microarray analysis on human ovarian carcinoma cell line SKOV3 that stably overexpress wild-type BRCA1 and compared with the corresponding empty vector-transfected clones. We found that stable expression of BRCA1 not only stimulates FST secretion but also simultaneously inhibits Activin expression. To determine the physiological importance of this phenomenon, we further investigated the effect of cellular BRCA1 on the FST secretion in immortalized ovarian surface epithelial (IOSE) cells derived from either normal human ovaries or ovaries of an ovarian cancer patient carrying a mutation in BRCA1 gene. Knock-down of BRCA1 in normal IOSE cells demonstrates down-regulation of FST secretion along with the simultaneous up-regulation of Activin expression. Furthermore, knock-down of FST in IOSE cell lines as well as SKOV3 cell line showed significantly reduced cell proliferation and decreased cell migration when compared with the respective controls. Thus, these findings suggest a novel function for BRCA1 as a regulator of FST expression and function in human ovarian cells.     

Phototoxicity of Hemoporfin to ovarian cancer

Hematoporphyrin monomethyl ether (Hemoporfin) is a novel porphyrin-related photosensitizer. Photocytotoxic effect of Hemoporfin to ovarian cancer is still unclear. We used human epithelial ovarian carcinoma cell line SKOV3 and its xenograft model in nude mice to investigate the Hemoporfin-based photodynamic therapy (PDT) for ovarian cancer. The growth rates of SKOV3 cells were determined by MTT assays. Flow cytometry combined with dual Annexin V/PI staining was used to identify the death mode of the cells following PDT. We demonstrated that Hemoprofin-based PDT induced significant cell death via direct necrosis induction, and the photocytotoxity to SKOV3 cells is dose related. With SKOV3 xenograft model in nude mouse, we further demonstrated that Hemoporfin-based PDT is effective for controlling the tumor growth. Our results suggest that Hemoporfin is a promising novel photosensitizer for the treatment of ovarian cancer and merit further evaluation in the clinical practice.     

Estrogen stimulation of ovarian surface epithelial cell proliferation

Summary Ovarian cancer is the leading cause of gynecological cancer mortality, and 85–90% of this malignancy originates from the ovarian surface epithelium (OSE). The etiology of ovarian epithelial cancer is unknown but a role for estrogens has been suspected. However, the effect of estrogens on OSE cell proliferation remains to be determined. Using the rabbit model, our studies have demonstrated that 17β-estradiol stimulates OSE cell proliferation and the formation of a papillary ovarian surface morphology similar to that seen in human ovarian serous neoplasms of low malignant potential. Immunohistochemical staining of ovarian tissue sections with an antibody to the estrogen receptor α demonstrates its expression in both OSE cells and stromal interstitial cells. In primary ovarian cell cultures, the proliferative response of the epithelial cells to 17β-estradiol depends on the expression of the estrogen receptor α in the epithelial cells. However, when the epithelial cells are grown together with ovarian stromal cells, their proliferative response to this hormone is greatly enhanced, suggesting the involvement of stromal-epithelial interactions. These studies suggest a role for estrogens and the estrogen receptor α in OSE growth.     

ALDH1-High Ovarian Cancer Stem-Like Cells Can Be Isolated from Serous and Clear Cell Adenocarcinoma Cells,and ALDH1 High Expression Is Associated with Poor Prognosis

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have high tumorigenicity. Furthermore, CSCs/CICs are resistant to several cancer therapies, and CSCs/CICs are therefore thought to be responsible for cancer recurrence after treatment and distant metastasis. In epithelial ovarian cancer (EOC) cases, disease recurrence after chemotherapy is frequently observed, suggesting ovarian CSCs/CICs are involved. There are four major histological subtypes in EOC, and serous adenocarcinoma and clear cell adenocarcinoma are high-grade malignancies. We therefore analyzed ovarian CSCs/CICs from ovarian carcinoma cell lines (serous adenocarcinoma and clear cell adenocarcinoma) and primary ovarian cancer cells in this study. We isolated ovarian CSCs/CICs as an aldehyde dehydrogenase 1 high (ALDH1^high) population from 6 EOC cell lines (3 serous adenocarcinomas and 3 clear cell adenocarcinomas) by the ALDEFLUOR assay. ALDH1^high cells showed greater sphere-forming ability, higher tumorigenicity and greater invasive capability, indicating that ovarian CSCs/CICs are enriched in ALDH1^high cells. ALDH1^high cells could also be isolated from 8 of 11 primary ovarian carcinoma samples. Immunohistochemical staining revealed that higher ALDH1 expression levels in ovary cancer cases are related to poorer prognosis in both serous adenocarcinoma cases and clear cell adenocarcinoma cases. Taken together, the results indicate that ALDH1 is a marker for ovarian CSCs/CICs and that the expression level of ALDH1 might be a novel biomarker for prediction of poor prognosis.     

Parthenolide induces apoptosis by activating the mitochondrial and death receptor pathways and inhibits FAK-mediated cell invasion

The natural product parthenolide induces apoptosis in cancer cells. However, the mechanism of apoptosis in ovarian cancer cells exposed to parthenolide is not clear. In addition, it is unclear whether parthenolide-induced apoptosis is mediated by the formation of reactive oxygen species and the depletion of GSH contents, and the effect of parthenolide on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effects of parthenolide exposure on apoptosis, cell adhesion, and migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that parthenolide may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of parthenolide appears to be mediated by the formation of reactive oxygen species and the depletion of GSH. Parthenolide inhibited fetal bovine serum-induced cell adhesion and migration of OVCAR-3 cells, possibly through the suppression the focal adhesion kinase-dependent activation of cytoskeletal-associated components. Therefore, parthenolide might be beneficial in the treatment of epithelial ovarian adenocarcinoma and combination therapy.     

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.     

Upregulation of Fas in epithelial ovarian cancer reverses the development of resistance to Cisplatin

This study was to investigate the role of Fas in the development of Cisplatin-resistant ovarian cancer. On the cellular level, Fas expression was significantly reduced in Cisplatin resistant A2780 (A2780/CP) cells compared with A2780 cells. Fas silence with siRNA would promote tumor cell lines proliferation, facilitate tumor cell cycle transition of G1/S, prevent cell apoptosis, and promote cell migration. Expression of drug resistance gene was negatively correlated to Fas. In nude mice metastasis model of human ovarian carcinoma by subcutaneous transplantation, after Ad-Fas injected intratumorly, we found that upregulation of Fas could inhibit transplantation tumor tissue growth and reduce the expression of drug resistance gene. Our results indicated that upregulation of Fas in epithelial ovarian cancer reversed the development of resistance to Cisplatin. In conclusion, our findings suggested that Fas might act as a promising therapeutic target for improvement of the sensibility to Cisplatin in ovarian cancer. [BMB Reports 2015; 48(1): 30-35]