| Mucoepidermoid carcinoma is the most common salivary gland malignancy, representing up to 30% of all cases. It is well known that hormone and hormone-like substances(E2) play important roles in controlling cell differentiation and proliferation. E2 has been widely used in treating menopause syndrome, preventing atheroscalarosis, osteoporosis and Alzheima's disease. However, since E2 had carcinogenic effects, it was declared by FDA as a major carcinogen in 2002. It has been reported that estrogen receptor exists not only in target cell of mammary gland and womb,but in non-target organs such as liver, kiddny, colon, pancreas, oral mucosa and skin as well as. Though there have been many studies concerning the effects of E2 on breast cancer and endometrial cancer, reports about the relationship between E2 and mucoepidermoid carcinoma is scarce.In this study, the effects of E2 on the proliferation and metastasis of oral salivary mucoepidermoid carcinoma M3SP4 cells were examined both in vitro and in vivo by cell counting, MTT assay, flow cytometry, clonegenetic assay, tumor growth and metastasis assay in nude mice. Gene chip (cDNA microarray) was used to identify the differentialexpression genes. The expression of VEGF,c-erbB-2, Kit-67,16 in M3SP4 cells was examined by immunohistochemistry assay. Following results were obtained:1. In vitro study of the effect of E2 on M3SP4 cell proliferation1.1 The effects of E2 on M3SP4 cell proliferation were examined by cell counting. M3SP4,cells were exposed to E2 at different concentrations and with different duration. The proliferation rate increased with the extension of exposing time. The promoting effect of E2 on M3SP4 cell proliferation reached peak when the cells were exposed to E2 for 48 hr at 10-7mol/L. Compared with that of the control group, doubling time of M3SP4 cells in E2 group decreased by 10.8%.1.2 The effect of E2 on cell proliferation was also examined by MTT assay. No significant promoting effect was observed at 24 hours after E2 treatment. However, with the extension of E2 treatment, cell proliferation was increased and the peak result was observed at 120 hours. The proliferation rate of M3SP4 cells treated with 10-8, 10-7, 10-6, 10-5mol/L E2 increased by 39.1%> A6A%, 45.8%, 31.0% respectively compared with that of control group.1.3 The effect of E2 on M3SP4 cell cycle were examined by cell cytometry. It was observed that when the cells were treated with E2 at 10-7mol/L for 48 hours the proliferation index of M3SP4 cells was 49.7, S phase cell increased by 29.1%. It was also found that the effect of E2 on cell cycle was dose-dependent.1.4 The colony forming rate(%) of M3SP4 cells treated with 10-7mol/L E2 was 71.5 + 8.3, whereas that of the control group was 22 ±8.6. Compared with the control group, the colony forming rate of the E2 group was increased by 225%.1.5 Immunohistochemistry results indicated that VEGF, Kit-67, C-erBb-2 and P16 were expressed in M3SP4 cells. The expression of VEGF, Kit-67 and C-erBb-2 were enhanced, while the expression of P16 was greatly inhibited by E2 at the concentration of 10-7mol/L.2 In vivo study of the effects of E2 on the tumor growth and metastasis of mucoepidermoid carcinoma2.1 0.2ml of M3SP4 cell suspension (1.0×106 cells) was injected into the mandibular glands of 10 nude mice. The mice were randomly divided into 2 groups, each containing 5 mice. Each mouse in the treatment group was given 0.0052mg E2 daily for 4 weeks, where as the control mice received normal saline of the same volume. Both groups developed mucoepidermoid carcinoma at day 7. The average tumor weight of treatment group was 1.98±0.56g, which was 65% percent heavier than that of the control group's 1.20±0.44g at day 28.2.2 Lung metastasis in nude mice after tail vein injection:M3SP4 cells were injected into tail vein of nude mice. Lung metastasis was estimated by counting the metastatic nodes on lung surface 8 weeks after injection. The number of metastatic nodes was 78 + 7.8 in E2 group, which is 6 times of that of control group (11 ±3.4).3 Differential gene expression detected by gene chip182 differential expression genes were detected before and after E2 treatment. Among which, 91 were up-regulated and 91 were down-regulated. 6 were newly discovered gene fragments.In conclusion, E2 could promote the proliferation of M3SP4 cells as examined by in vitro study, the strongest effect was observed at a concentration of 10-7mol/L. It could also increase tumor growth and...
|
PDF Full Text Request