| Cervical cancer is one of theriomas in women’s reproductive system. About 300,000 women with cervical cancer die annually around the world. The incidence of cervical cancer in China ranks second in the world. The therapy methods often used for cervical cancer are surgery, radiotherapy and chemotherapy, respectively. Among them, radiotherapy was an important method because of its broad clinical indications. Radiotherapy is effective for almost all of cervical cancer in the different stages,particularly for patients who are in advanced cervical cancers or who could not be cured surgically. However, because of the heterogeneity of individual conditions and tumors, the response to radiotherapy varies with patients. Recurrence and metastasis probably occur for about 30-50% of patients with cervical cancer after 3-5 years. It was reported that radiation resistance has become one of the most important reasons leading to failure of cure. However, up to now, the mechanism of radiation resistance hasn’t been expounded. Therefore, if the mechanism of radiation resistance can be clarifyied, the results not only provide theoretical basis for individual treatment but also provide new therapeutic targets to reverse the radiation resistance of cervical cancer, which must be of both important theoretical significance and the practical application value.The theory of cancer stem cells(CSC) thinks that tumors tend to harbor a subset of cell populations. CSCs show three features such as self-renewal, multipotency, and expression of stem cell markers. Some studies have found that CSCs exist in many kinds of tumors including cervical cancer. However, up to now, no specific markers were found in cervical cancer stem cells. The studies on CSC surface markers and how to separate and identify CSC of cervical cancer have been paid more attention.Epithelial–mesenchymal transition(EMT) is a process in which epithelial cells undergo remarkable morphological changes characterized by a transition from epithelial to mesenchymal phenotype, leading to increased motility and invasion. EMT includes loss of epithelial cell–cell junctions, actin cytoskeletal reorganization, and upregulation of mesenchymal molecular markers such as fibronectin, vimentin, and N-cadherin. It plays an important role in the process of embryonic development in which stem cells migrate to the appropriate parts of the body and develop into organs. Cancer stem cells could experience from mesenchymal to epithelial transition(MET) and rebuild cell structure and then formed metastatic tumor. Morphology of cell changes to cambiform phenotype and the ability of migration is enhanced. It has been reposed that EMT played an important role in development and progression, invasion and migration of tumor. Many factors regulated EMT. Research found that molecular and phenotypic associations exist between radioresistance and the acquisition of EMT-like cancer cell phenotypes, and CSCs play major roles in metastasis and tumor resistance to radiation therapy. Based on the information above, EMT induced by tumor radioresistance might be related to CSCs.About one third of cervical cancer patients often experience local relapse and progression to metastatic disease, largely because of its high resistance to radiotherapy. In this study, we obtained CD44+/CD24+-expressing cervical cancer cells and studied whether the CD44+/CD24+-expressing cervical cancer cells acquired characteristics of CSCs and underwent EMT. Based on the analysis above and our preliminary experiments, we investigated the radiation resistance of CD44+/CD24+-expressing cervical cancer cells, whether the CD44+/CD24+-expressing cervical cancer cells acquired characteristics of CSCs and the relationship between CSC and relapse, migration of cervical cancer. The aim of the paper is at revealling the mechanism of radiation resistance and providing theoretical basis for individual treatment. The main contents are as follows. PartⅠ The cancer stem cells characteristics of CD44+/CD24+-expressing cervical cancer cellsObjective:The aim is to investigate the characteristics of radiation resistance of cervical cancer and reveal the reason leading to relapse and migration of cervical cancer.Methods : Cervical cancer cell lines(Siha) were cultured with radiotherapy to obtain resistant subpopulations. Colony-formation tests and tumor xenografts tests were used to evaluate ‘‘stemness’ ’ in resistant cells. Stem cell markers were studied using fluorescence-activated cell sorting analyses. Migration and invasiveness were assessed by Transwell tests. Gene and protein expression was determined by RT-PCR and immunoblotting.Continuous variables are expressed as mean ± standard deviation(SD). Differences between groups were determined by Student’s t-test and χ2 analysis where appropriate; P<0.05 was considered statistically significant.Results:Radiation-resistant Siha cells and CD44+/CD24+-expressing Siha cells expressed more antiapoptotic protein Bcl-2, apoptosis-inhibitory protein survivin, and stem cell markers, were more tumorigenic in vitro and in vivo, and showed phenotypic and molecular changes consistent with EMT. They were also more invasive and migratory.The results showed that there was statistical difference between the two groups(P<0.05).Conclusions: We found radiation-resistant cervical cancer cells and CD44+/CD24+-expressing cervical cancer cells to be similar to CSCs and to undergo EMT, suggesting that radiation resistance-induced EMT is linked to CSC generation. Part ⅡResearch of radioresistance mechanism of CD44+/CD24+-expressing cervical cancer cellsObjective: To investigate whether CD44+/CD24+-expressing cervical cancer cells resist radiotherphy and to analyze its mechanism.Methods: Cervical cancer cell lines(Siha) were cultured in intro; Stem cell markers were studied using fluorescence-activated cell sorting analyses; Colony-formation tests were used to evaluate the sensitivity of CD44+/CD24+-expressing cervical cancer cells to radiation therapy. Cellular apoptosis and its rate were determinated by Hochest33258, electronmicroscopy, DNA “ladder” pattern and FCM. Gene expression was determined by RT-PCR.Results: After radiaotherphy, the expression rates of the SihaCD44+/CD24+ cells were significant elevated; Colony formation ratio of SihaCD44+/CD24+ cells was higher than that of Siha cells(P < 0.05); Both the apoptotic body and the specific DNA “ladder” pattern for apoptotic cells were not found in the SihaCD44+/CD24+ cells; SihaCD44+/CD24+ cells haven’t present characteristic morphological changes of apoptosis under electronmieroscope; The expression of bcl-2, survivin and oct4 mRNA was increased in SihaCD44+/CD24+ cells.Conclusions: The CD44+/CD24+-expressing cervical cancer cells resist apoptosis induced by radiotherapy. Part Ⅲ Expression of the stem cell gene Oct-4 in cervical cancer and its clinical significanceObjective: To explore the expression of Oct-4 genes and evaluate its relationship between stem cell gene expression and clinical and pathological characteristics and prognosis of cervical cancer.Methods: From May 2009 to April 2010, 50 paraffin samples of cervical cancer tissues were obtained,and the expression of Oct-4 was examined by PT-PCR and immunohistochemical staining. Another 20 normal cervical squamous tissues were served as controls. The clinicopathological data of 50 patients with cervical cancer were retrospectively analysed, and their relationship with the expression of Oct-4 was analysed.Results: The expression of Oct-4 gene was significantly higher in cervical cancer tissues than in normal cervical squamous tissues(P<0.01). In contrast to Oct-4 protein negative expression in normal cervical squamous tissues, 56% of cervical cancer cases are all showed positive expression. There is significant difference between cervical normal tissue and cervical cancer tissue(P<0.01). Oct-4 protein expression was not associated with age but was significantly associated with differentiation, recurrence and metastasis of cervical cancer(P<0.05).Conclusions: The expression of Oct-4 in cervical cancer is increased and is downregulated during cell differentiation. Abnormal expression of Oct-4 protein plays an important role in radioresistance of cervical cancer. |
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