Tv / O To 6-the Bg And Bcnu Combined With Inhibition Of Breast Cancer Cells In Mice

Background: Cancer has been the leading causes of death of many countries throughout the world. Mammary carcinoma is a kind of frequent malignant cancer which morbidity has increased obviously in our country and badly cured in clinical treatment in recent years. Therefore, looking for the potent therapeutic agents and sensitizing agents is the main tasks of oncologists. BCNU is widely used in clinical to treat the malignant cancer. In our studies, we find that it is effective to C-127. O~6-BG can sensitize tumor cells to the alkyling agents including BCNU that can make BCNU more potent to C-127 and prolonged its G2 phase arrest. But. its detailed process that how to induces those results are still not clear. Some researchers have recently discovered that tetrazolium violet could inhibit many kinds of tumors and could also make tumor cells sensitive to BCNU. But its function and mechanisms need more efforts to make distinct. Lots of therapeutic agents inhibit the proliferation of tumor cells through affecting their cell cycle and inducing apoptosis while sensitizing agents can enhance these effects by different manner. So, this kind of research can provide some theoretic basis to tumor biology and cancer chemotherapy.Objectives: To investigate the growth inhibition of C-127 cell lines in vitro in treatment of TV ,BCNU and O~6-BG in order to get more knowledge about the mechanism of how these therapeutic agents act on C-127 and provide some cell-level analysis for the therapy of the mammary carcinoma.Methods:Effect on the proliferation of C-127 Morphological observation under the electron fluoroscope, trypan blue exclusion assay and tetrazolium-based colorimetric assay (MTT assay) was used to analyze the cell proliferation of C-127 including the viability and the medium lethal concentration of 24 hours.Affection of C-127 apoptosis Membrane blabbing and apoptotic bodies was observed by morphologic assay using fluorescence microscope. Changes in cellnucleus were detected using H33258 staining through which we could observe the typical phenomena of apoptosis. The integrality of membrane was examined by LDH assay.Determination of p53 protein in C-127 The expression of p53 was determined by immunofluorescence assay using laser scanning confocal microscope.Cell cycle analysis Flow cytometry was used to detect the changes about the cell cycle of C-127 after treated with different drugs in vitro.Detection of the activity of GSTs in C-127 After treated with different drugs, C-127 was broken up by ultrasonic. Then, its content of GSTs was determined by colorimetric assay.Results:1. Observed by phase microscope, the configuration of C-127 had not visible changes after being treated with BCNU or O6-BG only. But when these two drugs were used together, the number of C-127 was decreased obviously and apoptosis or necrosis was observed. When treated with TV alone or TV+BCNU, the characteristic of apoptosis was presented such as cell prominences tailed off, particle and vacuole increased and cell nucleus enlarged.2.The results of trypan blue exclusion assay and MTT assay show: The percentage inhibition of C-127 after 24h treated with different drugs is 26.5%(BCNU),35.5%(TV), 17.5%(O6-BG),58.5% (BCNU+TV) and 54.5% (BCNU + O6-BG) respectively. Combination of these agents can inhibit the growth of C-127 more effectively.3. Observed by the fluorescence microscope, the phenomenon of apoptosis is not obvious when BCNU or O6-BG is used alone, but parts of cells take on apoptosis or necrosis when these two drugs are used together. With the treatment of TV, the main form of cell death regards apoptosis as principle. The chromatin in apoptotic cell nucleus assembles and turns to the edge, or break into crumb's section.4. We measured the content of LDH in the culture liquid and found that: The content of LDH with the treatment of TV and BCNU is low compared with 06-BG and BCNU, but the difference is not obvious {P>0.05) whereas the difference is rather obvious compared with the BCNU alone.5. The result of the activity test of enzyme: The activity of GSTs is lower in TV and BCNU section than in 06- BG and BCNU section, but there is no obvious difference.6. The immune fluorescence dying test of p53 reveals: The p53 expresses the quantity to compare the BCNU and O6-BGs in the TV section in high. Increase when the p53 expression quantity for consociation to use medicine compare the one to use the medicine, the TV section contain obvious difference (P<0.05), the 06-BG section difference is not obvious (P>0.05).7. The cell cycle of C-127 under different circumstance was examined by FACs: With the combination of TV and BCNU the cell cycle of C-127 is arrested in the Gl/ S phase, whereas the cell cycle is arrested in the G2/M phase with 06-BG and BCNU.Conclusion:1. Both TV and O6- BG have the synergetic effect to BCNU when act on C127, but the mechanisms are different.2. TV can cause C-127 not only apoptosis, but also can kill tumor cells through other paths such as necrosis when it is in combination with BCNU.3. The result of the activity test of the GSTs shows: This enzyme has less relationship with the synergetic effect of TV and BCNU. Nor have 06- BG and BCNU.4. TV can induce cell apoptosis by raising the expression of p53. The synergetic effect of O6- BG has no direct relation with p53 expressions.5. TV can induce the cell cycle of C-127 arrested in Gl/ S phase while 06-BG can also prolong the function of BCNU to C-127.