RESUMO
Clear cell adenocarcinoma of the ovary has a poor prognosis due to chemoresistance and early metastasis to the lymph nodes. It also can result in endometriosis and is the second most frequent type of ovarian cancer in Japan. Serous adenocarcinoma of the ovary is another common epithelial cancer tissue subtype in Japan, and it is highly sensitive to chemotherapy. In the current study, we examined the differential expression of genes in these types of ovarian cancer and tried to analyze their functions, especially as they relate to chemoresistance. We used differential display to compare clear cell carcinoma and serous adenocarcinoma of the ovary. We identified sperm protein 17 (SP17) as a candidate gene related to the chemoresistance of clear cell carcinoma. Its differential expression was confirmed by real-time polymerase chain reaction. Because the function of the SP17 gene in ovarian cancer is not known, we examined the effect of small interfering RNA targeting the SP17 gene on the chemoresistance and proliferation of ES-2 ovarian cancer cells to paclitaxel, currently the most effective treatment for ovarian cancer. We found that this treatment decreased the chemoresistance of these cells to paclitaxel. Our results strongly suggest that SP17 plays a role in the resistance of clear cell carcinoma to chemotherapy without influencing their ability to proliferate.
Assuntos
Adenocarcinoma de Células Claras/genética , Adenocarcinoma de Células Claras/patologia , Antígenos de Superfície/genética , Proteínas de Transporte/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Adenocarcinoma de Células Claras/metabolismo , Antígenos de Superfície/metabolismo , Proteínas de Ligação a Calmodulina , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Membrana , Neoplasias Ovarianas/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The natural antioxidant gallic acid (GA) was isolated from fruits of a medicinal Indonesian plant, Phaleria macrocarpa (Scheff.) Boerl. The structure was identified on the basis of spectroscopic analysis and comparison with authentic compound. GA demonstrated a significant inhibition of cell proliferation in a series of cancer cell lines and induced apoptosis in esophageal cancer cells (TE-2) but not in non-cancerous cells (CHEK-1). Observation of the molecular mechanism of apoptosis showed that GA up-regulated the pro-apoptosis protein, Bax, and induced caspase-cascade activity in cancer cells. On the other hand, GA down-regulated anti-apoptosis proteins such as Bcl-2 and Xiap. In addition, GA also induced down-regulation of the survival Akt/mTOR pathway. In non-cancerous cells, we observed delayed expression of pro-apoptosis related proteins. Our results suggest that GA might be a potential anticancer compound. However, in depth in vivo studies are needed to elucidate the exact mechanism.
Assuntos
Antineoplásicos/farmacologia , Ácido Gálico/farmacologia , Regulação Neoplásica da Expressão Gênica , Extratos Vegetais/farmacologia , Plantas/metabolismo , Apoptose , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Fragmentação do DNA , Citometria de Fluxo , Humanos , Modelos Químicos , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Proteína X Associada a bcl-2/biossínteseRESUMO
There is growing evidence that Rho proteins are deregulated by overexpression in tumours; and according to some reports, this correlates with disease progression. Our previous clinical study had demonstrated a correlation between RhoA expression and tumour progression in oesophageal squamous cell carcinoma (ESCC). These findings prompted us to study, using nude mice, pathological roles of Rho proteins in human ESCC cells. Western blot analysis in ESCC cell lines, in addition to cell proliferation and in vitro migration assays, were performed to observe the malignant potential of RhoA and RhoC in untransfected and transfected cells. Constitutively active RhoA, RhoC and dominant negative RhoA (dnRhoA) proteins were transfected to ESCC (TE-1 and TE-2) cells. The stably transfected cells were injected into nude mice, and the growth and metastasis of these cells to the lungs were analysed. Tumour tissues were then examined using immunohistochemical methods for proteins Ki-67 (MIB-1), FAK, MMP-1, MMP-9 and TIMP-3. Protein levels of RhoA and RhoC in ESCC cell lines were visualised by Western blotting, and showed highest expression in TE-2 cells. Results from the migration assay illustrated that both RhoA and RhoC play a role in migration of ESCC cells. In TE-2 transfected cells, RhoC showed greater migration compared to RhoA. By using an experimental metastasis model in nude mice, RhoA was found to promote more tumour growth than RhoC, whereas RhoC induced lung metastasis in comparison to RhoA. Ki-67 labelling index was used to evaluate the proliferation potential of tumour tissue inoculated from nude mice. In TE-2 cells RhoA gave a proliferation capacity of 24.8+/-0.5, which was significantly higher than those of TE-2 RhoC 10+/-0.4 (P<0.01). Strong immunoreactivity for FAK, MMP-1 and MMP-9 proteins was present in all tumour cells. By contrast, loss of TIMP-3 expression was observed in all tumour cells. In conclusion, our results indicate that pro-oncogenic Rho proteins are involved in promoting tumour growth, cell migration and metastasis in human ESCC cells in nude mice. The results from this study suggest that active Rho proteins may induce a transforming effect that leads to a malignant phenotype.
Assuntos
Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Esofágicas/patologia , Proteínas rho de Ligação ao GTP/farmacologia , Proteína rhoA de Ligação ao GTP/farmacologia , Animais , Western Blotting , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Transplante de Neoplasias , Proteínas ras , Proteína de Ligação a GTP rhoCRESUMO
Paclitaxel, a potent anti-neoplastic agent, has been found to be effective against several tumours, including cervical cancer. However, the exact mechanism underlying the cytotoxic effects of pacitaxel, especially in the survival-signalling pathway, is poorly understood. The aim of this study was to investigate the molecular pathway of the cytotoxic effect of paclitaxel in human cervical cancer cell lines. Four human cervical cancer cell lines were treated for 24 h with various concentration of paclitaxel, and the sensitivity was analysed by an MTT assay. The cell cycle progression and sub-G1 population were analysed by flow cytometry. Apoptosis was further measured by DNA fragmentation and microscope examination. The protein expression was determined by Western blot analysis. Our results showed that HeLa cells demonstrated the highest sensitivity to paclitaxel, whereas CaSki cells showed the lowest. In cervical cancer cells, paclitaxel induced apoptosis through an intrinsic pathway with prior G2/M arrest. In addition, we showed that paclitaxel downregulated the phosphorylation of Akt in both HeLa and CaSki cells. Interestingly, in CaSki cells, which were more suggestive of a resistant phenotype, paclitaxel induced the activation of mTOR as a downstream target of Akt. Pre-treatment with rapamycin inhibited activation of mTOR signalling and significantly enhanced the sensitivity of CaSki cells to paclitaxel by increasing apoptotic cell death. This effect was mediated, at least partly, through caspase activation. Overall, paclitaxel exerts its anti-tumour effects on cervical cancer cells by inducing apoptosis through intrinsic pathway, and rapamycin targeted to mTOR can sensitise paclitaxel-resistant cervical cancer cells.