The Research On Tumor Suppression Effect And The Mechanism Of 2- Methoxy Estradiol On Malignant Melanoma B16 Tumor-bearing Mice

Objective:Malignant melanoma is a malignant tumor of high malignant degree,with melanin cell differentiation high invasive and proliferation,and it can produce heparin and fibroblast growth factor complex and so on to make a lot of neovascularization inside the tumor, and also make tumor cell prone to lymph node metastases and blood line transfer then with the poor prognosis. As one of the highest rates of malignant tumors,the morbidity of malignant melanoma increased year by year.The main treatment is a kind of comprehensive treatment,which includes surgery therapy,radiation therapy, chemotherapy,targeted therapy and immune therapy, etc,In spite of this,the overall treatment effect is still not ideal,the extension of the survival time is not obvious.2-methoxy estradiol(2-ME) is a kind of steroidal hormones compounds,and With effects of anti-angiogenesis and anti-tumor.2-ME showed significantly inhibit the growth of tumor in a wide variety of tumor-burdened animal model experiments.So far,there is no research report about the tumor suppression effect of 2-ME to malignant melanoma tumors in-vitro or in vivo studies.The early stage of the study shows that 2-ME has inhibitory effect on cell proliferation to malignant melanoma B16 tumor-bearing mice.This effect is time- dose dependent relationship, and the cell cycle G0/G1 blocked,cell apoptosis rate increased after 24 h.2-ME can obviously reduce the proto-oncogene(c-myc) expression,improve the expression of the tumor suppressor genes(gadd45b). This time, we continue to study the effect of 2-ME on malignant melanoma cells of proliferation and apoptosis through the establishment of animal models.To further discuss the possible mechanism through the changes in related protein expression,to provide new treatments and corresponding theory basis for malignant melanoma.Methods:We divided 12 female BALB/c mice randomly into 2 groups, each group had 6 mice.we established melanoma transplantation in mice tumor models, every mouse was vaccinated with the mouse melanoma B16 cells suspension in the right side of the back subcutaneous,each one got the number of cells were 1×106.We began to inject drugs through the tail intravenous when most of the mice were tumor-burdened.The experimental group were injected with 2-ME 50mg/kg, every other day at a time,the control group given saline injections,the same dose to experimental group, every two days measured the size of tumor of the two groups,and injected the last time at the 21 st day,the mice were put to death at the 22 nd day.after death, we took out the tumor tissue, lung, liver, spleen tissue. Measured the size and the weight of each tumor,calculated the tumor inhibition ratios based on volumes and the tumor inhibition ratios based on weights. Observed the changes of major organs and tumor tissue cells using the microscope.detected the protein of c-myc and Gadd45 b using immunohistochemical method.Analysised the data using spss 19.0 statistical software.Results:1 2-ME can inhibit the tumor growth in mice.Compared to two groups of the weight of the tumor at the 22 nd day, there’s statistical significance between the two groups(P<0.01).The tumor inhibition ratios based on weights was 62.8%.The volume of tumor in mice:no statistical difference was found between the two groups at the 7th day(P>0.05), on the contrary,the rest contrast were all had statistical significance(P<0.01).The tumor inhibition ratios based on volumes was 66.9%.the tumor growth curve showed that the tumor growth in the experimental group was slower than in the control group.2 The impact of 2-ME to cell morphology of the tumor cells and the Important organ cells: In the tumor tissue of control group,a lot of large size tumor cells closely packed together and had significant nuclear atypia,part of the tumor cells arranged like the nests.Compared with the control group, the tumor tissue of experimental group showed that the cell density was sparse and more mesenchyma, part of the nucleus showed nuclear pyknosis and nuclear cracking,and so on.it also showed a large number of necrosis and apoptosis of cells,part of the region could see the infiltration of inflammatory cells.Numbers of the cells containing pigment particles were more than in the control group.It showed that, in the experimental group,the growth of the tumor cells were restrained,and the form of the tumor cells were damaged. In the experimental group,we found no tumor metastases in lung, liver and spleen,and the tissue was complete,but in the control group,there were a small amount of pulmonary metastases in 3 mice.3 The results detected by IHC of the expression of c-myc, Gadd45-b.The positive expression of protein of c-myc is in nucleus,the color is tan.we can see the positive expression of c-myc in both of the two groups,and the expression in control group is more than in the experimental group.The positive expression of protein of Gadd45 b is in cytoplasm,the color is tan, distribution is focal or diffuse. The positive expression of c-myc is existed in both of the two groups, the expression in experimental group is more than in control group.Conclusions:1 2-ME has significant inhibitory effect on the growth of transplan- tation tumor of B16 tumor-bearing mice.2 2-ME can reduce the viscera metastases of transplantation tumor of B 16 tumor-bearing mice.3 2-ME has no obvious damage to other important organs when it redu- cing the viscera metastases of transplantation tumor of B16 tumor-bearing mice.This suggested that 2-ME has small side effects, and the selectivity is strong.4 2-ME can inhibit the proliferation and promote the apoptosis of mali- gnant melanoma.The likely mechanism is that 2-ME can raise the expression of Gadd45 b protein,reduce the expression of c-myc protein,then cause the cell apoptosis process.