The Study Of Nrf2 On Endometrial Serous Carcinogenesis And Chemoresistant Mechanism

Endometrial cancer is one of the most common gynecological malignancies, which has an increased morbidity annually. According to the distinction in histopathology, pathogenesis and biological behavior, endometrial cancer has been divided into two major categories:type?and type?. Endometrial serous carcinoma (ESC), the prototype of type?, bears a significantly worse prognosis and high mortality compared with type?cancer, which were attributed to its rapid developmental, lack of effective early diagnostic markers, highly aggressive nature as well as high incidence of chemoresistance.Based on the clinical and pathological evidences, Zheng et al raised that EmGD is the precursor lesion of ESC. p53 signature cells have also been found which occured before EmGD lesion. Many molecular events, such as p53 mutation, HER2/neu overexpression, participate and promote the process of ESC.Erythroid-E2-related factor 2 (Nrf2), a significant nuclear transcription factor, can maintain intracellular redox homeostasis through regulating the transcription of a series of target genes and remove toxicants, carcinogens as well as drugs. So Nrf2 confers protections against many chronic diseases and cancers. Recent studies show that overexpression of Nrf2 has a close connection with carcinogenesis and is one of the promoting factors for chemoresistance.Based on the above studies, we have studied the mechanism of Nrf2 on endometrial serous carcinogenesis and chemoresistance: Part?The Study of Nrf2 Expression in Endometrial Serous Carcinomas and Its PrecancersBackground and Objective:As the prototype of type?endometrial carcinoma, ESC bears a significantly worse prognosis compared with type?cancer, which may due to lack of effect diagnostic markers as well as inadequately defined carcinogenesis. Nrf2 is an important nuclear transcription factor maintaining intracellular redox homeostasis. It has been found that Nrf2 overexpressed in tumor tissues and cell lines and played important role in the initial stage of ESC. Based on these studies, we supposed that consistent activation and over-expression of Nrf2 may be a promoting factor for endometrial serous carcinogenesis. In this study, we evaluated the protein level of Nrf2 in endometrial specimens including benign endometrium, endometrial cancers and their precursor lesions. Then we analyzed the Nrf2 expression differentiation among different endometrial specimens trying to see whether Nrf2 can be a novel diagnostic marker of ESC and meanwhile enrich our understanding to pathogenesis of ESC.Materials and Methods:We tested the Nrf2 level in 231 endometrial specimens using immunohistochemistry (IHC), including which there were benign endometrium (n=28), precursor lesions (n=81), endometrial cancers (n=122). Western blot was used to verify the expression status of Nrf2 with 20 pieces of frozen endometrial tissues and endometrial cancer cell lines (Ishikawa and SPEC-2).Results:Nrf2 was positive in 68%(28/41) ESCs, which was more than 6% in endometrioid carcinomas (EEC) (p<0.001) and 13% in endometrial clear cell carcinomas (ECCC) (p=0.001). Among endometrial precursor lesions, the positive rates in endometrial serous glandular dysplasia (EmGD) and endometrial serous intraepithelial carcinoma (EIC) were 40% and 44%, which both had significantly differences comparing with precursor lesions of EEC and CCC.Conclusions:Over-expression of Nrf2 was closely related to the carcinogenesis and development of ESC. Alteration of Nrf2 expression may represent one of the early molecular events in ESC carcinogenesis. Therefore, overexpression of Nrf2 may be used as a diagnostic marker for ESC in clinical.Part?The Study of Nrf2 in Relation to Chemo-resistance of Endometrial Serous CarcinomaBackground and Objective:Although ESC morbidity accounts for only 10%-15% endometrial cancer cases, it bears a disproportional mortality due to its low early diagnosis rate, rapid developmental process and chemoresistance. Therefore, improving the chemosensitivity of ESC has been a key component related to therapeutic effect and prognosis. It has shown that overexpression of Nrf2 helps to increase cancerous chemo-resistance and the chemosensitivity tends to vary inversely with the Nrf2 level, which has been proved in breast cancer, lung cancer and ovarian cancer cell lines. In this study, we explored effect of Nrf2 variation on chemo-resistance of ESC by in vitro experiments, and further verified it in animal model.Materials and Methods:1. The mRNA and protein levels of Nrf2 as well as its downstream genes were tested in Ishikawa and SPEC-2 cell lines (represent type?and?endometrial cancer) using real-time PCR and western blot. MTT was used to evaluate the sensitivities of these two cell lines towards cisplatin and paclitaxel with different concentrations.2. Nrf2 expression was down-regulated in Ishikawa and SPEC-2 cells by transient transfection of Nrf2 siRNA. MTT was used to detect the sensitivity variations in these two cell lines towards cisplatin and paclitaxel.3. Keap1-CBD was stably incorporated into SPEC-2 cell using retrovirus system. Real-time PCR and western blot tested the mRNA and protein levels of Nrf2 and its downstream genes in empty vector group and Keap1+group. MTT tested the cell viabilities of these two groups under cisplatin treatment.4. SPEC-2-derived stable cell lines, with stable incorporation of Keapl-CBD or empty vector, were subcutaneously transplanted into immuno-deficient mice. Cisplatin was injected into abdominal cavity periodically and tumor sizes were recorded. Dissected tumor tissues were used for IHC and frozen fresh tissues were used for western to test the protein levels of Nrf2 and downstream genes. Tumor sizes, weight and Nrf2 level were compared among groups.Results:1. SPEC-2 cells express a high protein level of Nrf2 than Ishikawa cells, which is consistent with the mRNA levels of Nrf2 downstream genes. The viability of SPEC-2 cells was higher than Ishikawa cells at the same concentration of cisplatin or palcitaxel.2. The SPEC-2 viability decreases under cisplatin or paclitaxel after Nrf2 level was downregulated using transient transfection, the descending level of which was more significant than Ishikawa cells. The MTT results were the same in SPEC-2 stably expressing Keap1 when cisplatin was used.3. The tumor sizes and weights of Keapl+group were lower than empty vector group. The weights difference before and after cisplatin treatment was bigger in Keap1+group than empty vector group. IHC and western blot methods showed that Nrf2 level of empty vector group slightly increased after cisplatin treatment, while it decreased in Keap1+group.Conclusions:Overexpression of Nrf2 is a significant promoting factor for chemoresistance of ESC. Nrf2 inhibition may be a novel and effect approach for ESC biological therapy.