Adenoviral p53 effects and cell-specific E7 protein-protein interactions of human cervical cancer cells

We investigated the time-course tumor growth suppression effects of recombinant adenovirus expressing p53 on human cervical cancer cells and cell-specific E7 protein-protein interactions in cell lysates using surface plasmon resonance (SPR) biosensor. Six HPV-infected human cervical cancer cell lines (HPV 16-positive cells, CaSki and SiHa cells; HPV 18-positive cells, HeLa and HeLaS3 cells; and HPV negative C33A and HT3 cells) were used. After infection with AdCMVp53, the cell-specific growth inhibition was studied in vitro and in vivo. Also, we produced the recombinant E7 oncoprotein of HPV 16 type and tested chip-based protein-protein interactions with each cell lysate. For each cervical cancer cell, differential cell growth inhibitions were shown via cell count assay and MTT assay. Note that the same trend in suppression levels was shown in CaSki, HeLa and in SiHa, HeLaS3, respectively. In contrast, infection with AdCMVLacZ showed increased cell growth in a manner similar to the negative control group. The levels of p53 protein were notably expressed in CaSki and HeLa more than in SiHa and HeLaS3 for 4 days. In contrast, p53 expression was continually maintained in C33A and HT3 for 6 days. After transfection AdCMVp53 into CaSki- and SiHa-xenografted nude mice, the size of tumor was remarkably decreased in SiHa cells as compared to AdCMVLacZ transfection. The SPR sensor surface was successfully modified with the recombinant E7 oncoprotein and showed cell-specific interactions between E7 and its target proteins from cell lysates. The anti-tumor effects were accomplished via differential role of p53-specific apoptotic cell death, which is dependent upon the cervical cancer cell line. Also, a molecular level understanding of cell-dependent protein interaction effects of recombinant E7 was shown.     

Higher expression and activity of metalloproteinases in human cervical carcinoma cell lines is associated with HPV presence.

Matrix metalloproteinases (MMPs) MMP-2, MMP-9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in 3 human invasive cervical carcinoma cell lines. Two HPV16-positive cell lines (SiHa and CaSki) and an HPV-negative cell line (C33A) were cultured either onto a type-I collagen gel, Matrigel, or plastic, to recreate their three-dimensional growth environment and evaluate the expression of these genes using quantitative real-time PCR. We also analyzed the gelatinolytic activity of MMP-2 and MMP-9 by zymography. We found that HPV (human papillomavirus)-positive cell lines express higher levels of MMP-2, MT1-MMP, and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with higher pro-MMP-2 activity. Furthermore, Matrigel substrate influences MMP-2 expression in both SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP-2, MT1-MMP, and TIMP-2, and that pro-MMP-2 activity is higher in HPV-positive than in HPV-negative cells.     

Role of E-cadherin in the induction of apoptosis of HPV16-positive CaSki cervical cancer cells during multicellular tumor spheroid formation

Multicellular tumor spheroids (MCTS) are three dimensional cell culture systems induced by suspension culture. MCTS are widely used in cancer research because of their similarity to solid tumors. CaSki cells are derived from a metastatic cervical cancer containing human papillomavirus 16 (HPV16). Cell death of CaSki cells in MCTS has been previously reported, and our model is used to better characterize the mechanisms of cell death of HPV16-positive keratinocytes. In this study, we found that apoptosis of CaSki cells was induced by suspension culture along with the formation of MCTS after 24 h of incubation. In suspended CaSki cells, monoclonal antibodies blocking E-cadherin function inhibited MCTS formation and suppressed suspension-induced apoptosis in a dose-dependent manner. Western blot for E-cadherin detected upregulation of the authentic 120 kDa band from MCTS of CaSki cells as well as a shorter 100 kDa band. Addition of EGF, whose receptor is known to form a complex with E-cadherin, abrogated apoptosis of suspended CaSki cells in a dose-dependent manner. These findings suggest that E-cadherin-dependent cell–cell contact, directly or indirectly, mediates the signal to undergo apoptosis of CaSki cells during MCTS formation, and thus provides new information on the role of E-cadherin in cervical cancer cell apoptosis.     

The coxsackie adenovirus receptor inhibits cancer cell migration

The coxsackie and adenovirus receptor (CAR) is a key factor in adenoviral cancer gene therapy. Reduced expression of CAR during progression of prostate and bladder cancer has been reported. In embryonic development and tissue differentiation, CAR is also differentially expressed. This study suggests a role of CAR expression in cell adhesion and cell motility of human cancer cells. Stable CAR-expressing clones from E-cadherin-deficient A2780 ovarian and CaSki cervical cancer cells with originally low and high CAR expression levels, respectively, were established. CAR reexpression in otherwise singularly growing A2780 parental cells resulted in formation of cell-cell contacts and aggregation in cell clusters. CAR overexpression in cell adhesion-forming CaSki cells did not result in morphological changes. Migration of the A2780 CAR clones was strongly reduced as characterized by using spread-off assays. Using migration chambers, formation of satellite colonies was reduced by 97% in CAR-expressing A2780 cell clones and by 23% in CAR-expressing CaSki cell clones. Parental A2780 and CaSki cells selected for high migratory ability by using migration chambers expressed endogenous CAR on lower levels associated with lower adenoviral transduction efficiency. Our data suggest CAR as a new inhibitory factor for cancer cell migration.     

RbAp48 is a critical mediator controlling the transforming activity of human papillomavirus type 16 in cervical cancer

Although human papillomavirus (HPV) infections are the primary cause of cervical cancer, the molecular mechanism by which HPV induces cervical cancer remains largely unclear. We used two-dimensional electrophoresis with mass spectrometry to study protein expression profiling between HPV16-positive cervical mucosa epithelial H8 cells and cervical cancer Caski cells to identify 18 differentially expressed proteins. Among them, retinoblastoma-binding protein 4 (RbAp48) was selected, and its differentiation expression was verified with both additional cervical cancer-derived cell lines and human tissues of cervical intraepithelial neoplasia and cervical cancer. Suppression of RbAp48 using small interfering RNA approach in H8 cells significantly stimulated cell proliferation and colony formation and inhibited senescence-like phenotype. Remarkably, H8 cells acquired transforming activity if RpAp48 was suppressed, because H8 cells stably transfected with RbAp48 small interfering RNA led to tumor formation in nude mice. In addition, overexpression of RbAp48 significantly inhibited cell growth and tumor formation. This RbAp48-mediated transformation of HPV16 is probably because of the regulation by RbAp48 of tumor suppressors retinoblastoma and p53, apoptosis-related enzymes caspase-3 and caspase-8, and oncogenic genes, including E6, E7, cyclin D1 (CCND1), and c-MYC. In brief, RbAp48, previously unknown in cervical carcinogenesis, was isolated in a global screen and identified as a critical mediator controlling the transforming activity of HPV16 in cervical cancer.     

Gene expression profiles of necrosis and apoptosis induced by 5-fluoro-2'-deoxyuridine

5-Fluoro-2'-deoxyuridine (FUdR), a potent anticancer agent, exerts its effects by inhibiting thymidylate synthase, an essential machinery for DNA synthesis in cell proliferation. Also, cell death is caused by FUdR, primarily due to an imbalance in the nucleotide pool resulting from this enzyme inhibition. We have investigated the cancer cell death induced by FUdR, focusing on its molecular mechanisms. Using mouse mammary tumor FM3A cell lines, the original clone F28-7 and its variant F28-7-A cells, we previously reported an interesting observation that FUdR induces a necrotic morphology in F28-7, but induces, in contrast, an apoptotic morphology in F28-7-A cells. In the present study, to understand the molecular mechanisms underlying these differential cell deaths, i.e., necrosis and apoptosis, we investigated the gene expression changes occurring in these processes. Using the cDNA microarray technology, we found 215 genes being expressed differentially in the necrosis and apoptosis. Further analysis revealed differences between these cell lines in terms of the expressions of both a cluster of heat shock protein (HSP)-related genes and a cluster of apoptosis-related genes. Notably, inhibition of HSP90 in F28-7 cells caused a shift from the FUdR-induced necrosis into apoptosis. These findings are expected to lead to a better understanding of this anticancer drug FUdR for its molecular mechanisms and also of the general biological issue, necrosis and apoptosis.     

A major constituent of green tea,EGCG, inhibits the growth of a human cervical cancer cell line,CaSki cells,through apoptosis,G(1) arrest,and regulation of gene expression

A constituent of green tea, (-)-epigallocatechin-3-gallate (EGCG) has been known to possess antiproliferative properties. In this study, we investigated the anticancer effects of EGCG in human papillomavirus (HPV)-16 associated cervical cancer cell line, CaSki cells. The growth inhibitory mechanism(s) and regulation of gene expression by EGCG were also evaluated. EGCG showed growth inhibitory effects in CaSki cells in a dose-dependent fashion, with an inhibitory dose (ID)(50) of approximately 35 microM. When CaSki cells were further tested for EGCG-induced apoptosis, apoptotic cells were significantly observed after 24 h at 100 microM EGCG. In contrast, an insignificant induction of apoptotic cells was observed at 35 microM EGCG. However, cell cycles at the G1 phase were arrested at 35 microM EGCG, suggesting that cell cycle arrests might precede apoptosis. When CaSki cells were tested for their gene expression using 384 cDNA microarray, an alteration in the gene expression was observed by EGCG treatment. EGCG downregulated the expression of 16 genes over time more than twofold. In contrast, EGCG upregulated the expression of four genes more than twofold, suggesting a possible gene regulatory role of EGCG. This data supports that EGCG can inhibit cervical cancer cell growth through induction of apoptosis and cell cycle arrest as well as regulation of gene expression in vitro. Furthermore, in vivo antitumor effects of EGCG were also observed. Thus, EGCG likely provides an additional option for a new and potential drug approach for cervical cancer patients.