| Part one:Cell cycle arrest induced by MPPa-PDT in breast cancerBreast cancer is one of women’s ten common malignant tumors. In recent years, the incidence of breast cancer increases year by year in our country, and there is also a trend of younger age of breast cancer. But the cause of breast cancer is not yet fully understood, there being no effective preventive measures and also failing to ideally control its recurrence and metastasis. Therefore, the high quality of scientific research results play a vital role in the treatment of patients with breast cancer.Photodynamic therapy is a tumor treatment technolgy, which is invented in the 1970s. Malignant tissue can selectively absorb photosensitizer and stimulate the photosensitizer with appropriate wavelengths of light. And the stimulated photosensitizer can convey the energy to oxygen molecular. By a series of energy conversion, finally tissue will produce oxygen with high reaction activity and lead to cell death.Photosensitizer can be injected by intravenous or intralocal injections. Tumor tissue can selectively absorb the photosensitizer. The majority of the photosensitizers in PDT studied at present have a heterocyclic ring structure similar to that of chlorophyll or hemoglobin. Pyropheophorbidea methyl ester (MPPa), which is a derivative of chlorophyll, is a novel photosensitizer.The responses of cells to external stimuli are various, involving cell proliferation, differentiation and death and their coordination. The two aspects of cells, proliferation and death, are the two distinct different ways, but there are some similarities in morphology and biochemistry. Cell cycle regulation is the key to the proliferation and apoptosis, which is a two-way switch. Different physical and chemical stimulation can cause different cell damage, and the ability to repair the damage to the cells is quite different in different periods.It is shown that apoptosis could occur in different cell cycle phases. There exists a linkage between cell cycle control mechanisms and apoptosis. Cell cycle checkpoints are to ensure the quality of chromosome distribution and DNA replication in cell cycle. This study shows that controlling checkpoints of the cell cycle could result in apoptosis.Objective:After photodynamic therapy of the MDA-MB-231 cells, we test the cell cycle arrest and the change of cell cycle related protein CHK2, P21 and CyclinD1, so that we can futher understand the mechanism of MPPa-PDT on MDA-MB-231 cells.Methods:1. We divided the MDA-MB-231 cells into four groups. In addition to the control group, the other three groups were given MPPa of the same concentration and different dose of light.2. We detected the cell cycle arrest through flow cytometry.3. We determined the expression of cell cycle related proteins CHK2, P21, CyclinD1 by Western blot.Results:1. As shown by the flow cytometry, the apoptosis rate was about 46% after PDT. The percentage of cells in G1 phase changed a little compared with the control, while the cells in S phase increased obviously and the cells in G2 phase decreased obviously after PDT.2. Western blot results revealed that the level of Cyclin D1 was decreased after PDT, and the expression of P21 Wafl/Cipl/Sdil and Chk2 showed a gradually increasing trend towards higher light dosage.Conclusion:1. The results of this study showed that the MPPa-PDT induced a cell cycle arrest is S phase.2. The MPPa-PDT inhibited the proliferation of MDA-MB-231 Cells.3. The cells were led to apoptosis by down regulating the expression of Cyclin D1 and up regulation of Chk2 and P21.Part two:Research for the treatment of prostate carcinoma by combining MPPa-PDT with HSVl-TK therapySuicide gene therapy is a tratement, which needs to transduce specific viral genes into tumor cells. Specific enzyme encoded by the gene can turn non-toxic or low toxic prodrugs into the toxic production in the process of metabolism of tumor cells, and thus tumor cells can be killed.Granciclovir is the derivative of guanine nucleotide. It can be turned into a single phosphorus GCV acid salt with the impact of HSV1-TK, and then it will quickly transform into diphosphate and triphosphates through toxic cell kinase, which is highly poisonous to the mammalian cells in the stage of proliferation.Glucose regulated protein 78 (GRP78) is highly expressed in many tissues including prostate cancer, breast cancer, gastric cancer, brain tumors and so on. GRP78 promoter can eliminate cancer by activating the expression of HSV1-TK.Methyl pyropheophorbide-a (MPPa) is the derivative of chlorophyll. This new type of photosensitizer is apt to concentrate in PC-3. It can kill cancer cells by photodynamic therapy and nearly takes no effection on normal cells.Objective:Our purpose is to combine the MPPa-PDT with HSV1-TK therapy for prostate carcinoma. Active GRP78 promoter enable HSV1-TK to be transfected into PC-3 and highly expressed. Thus, it can turn Ganciclovir into phosphate, which could inhibit the DNA synthesis of tumor cells and kill tumor cells. By combining the treatment with MPPa-PDT, we can determine whether HSV1-TK/GCV treatment with MPPa-PDT is an efficient way for prostate carcinoma.Methods:1. To gain the target gene, we searched the mRNA of HSV1-TK in the Gen-Bank, design the upstream and downstream primers, and do PCR amplification.2. We built HSV1-TK expression vector GV230-TK, and then take part of the recombinant plasmid for measuring it’s sequence to identify whether it is successfully reconstructed.3. The recombinant plasmid was transfected into PC-3 prostate cancer cells by lipofectamine 2000. After transfecting tumor cells, we could observed a bright green fluorescence under fluorescence microscope for plasmid GV230-TK containing GFP. The cell protein were collected for Western blot detection to demonstrate the successfull transfection and expression of HSV1-TK.4. Cells could be divided into four groups: HSV1-TK/GCV combining MPPa-PDT group, pure MPPa-PDT group, pure HSV1-TK/GCV treatment group and control group.5. We did CCK-8 experiments to determine the survival rates of the four groups.6. Flow cytometry instrument could help us reveal the cell cycle changes and the apoptosis of the treatment groups and the control group.Results:1. CCK-8 experiment results showed that the cells’survival rate of pure HSV1-TK/GCV treatment group is lower than the control group. The cells’ survival rate of pure MPPa-PDT group is lower than the control group. Moreover, the cells’survival rate of HSV1-TK/GCV treatment with MPPa-PDT were lower than other three groups.2. The results of flow cytometry instrument revealed that detect the cell cycle arrest happened in G2 phase and the apoptosis of the combination treatment group is more serve than pure HSV1-TK/GCV treatment group and pure MPPa-PDT group.Conclusion:HSV1-TK/GCV treatment with MPPa-PDT result in serve apoptosis of prostate carcinoma cells in contrast with pure HSV1-TK/GCV treatment group and pure MPPa-PDT group. Its mechanism may be related to proliferation inhibition by promoting apoptosis. HSV1-TK/GCV treatment with MPPa-PDT is an efficient way for the treatment of prostate carcinoma. |
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