The Study On Sodium Phenylbutyrate And Dimethylformamide Inhibiting Proliferation And Inducing Differentiation In Breast Carcinoma Cells

Breast carcinoma is one of the most common malignancies among the women in the world and the second most frequent cause of cancer-related death in the United State. In China, although the incidence rates of mammary cancer are relatively lower than those of western countries and United States of America, the phenomena of clinical observation in recent years suggest that in many cities of our country the mammary carcinoma have occupied the first place on the female malignant tumour lists. The metastasis and relapse of mammary carcinoma are the most common reasons which lead to the death of breast cancer patients.The combined therapy mainly consisted of operation technique, radiotherapy, chemotherapy and endocrine therapy galacticly raises the survival rates of breast carcinoma patients, but unfortunately there has been comparatively little change in mortality from recurrence and progression breast cancer.With the development of biotechnology, gene therapy has made great advance, but it is impossible to apply it to clinical treatment in large scales at present. Induction of differentiation is a kind of inversive phenomenon that various differentiation inducers can turn malignant tumor cells into normal cells or the approaching normal ones, which is a new route of oncotherapy and thought highly of by the researchers in recent years. It has the fundamental features that this therapeutic method would rather induce tumor cells to change from malignant cells to normal ones than kill them. Accordingly, induction of differentiation is regarded as a basical strategy in this project to explore oncotherapy of mammary cancer.With the advence of the theories and techniques of molecularbiology it has been considered that the reasons and pathogenesis of breast carcinoma are composed of multiple factors which seem to make contributions altogether to revoking the disorders of genes regulating cell proliferaiton and the procedure of apoptosis. Therefore, many researches presume that carcinogenesis and tumor progression are concerned with a series of progressive gene alterations which include the abnormal activation of oncogenes and the inactivation of anti-oncogenes.In the recently years, many researches have suggested that the differentiation agents can induce tumor cells to high differentiation and inhibit the invasion and recurrence of tumor cells to some extent, but it can not be achieved that tumor cells are completely induced to become normal ones and avoided their relapse. Furthermore, the combination use of different types of differentiation agents,which could gather the anticancer ability of differentiation agents by synergistic or complementary function, has become a new research tendency. Obviously, understanding the exact mechanism of each differentiation agents is the base of the combination use of them. This subject aims at investigating the functions of sodium phenylbutyrate and dimethylformamide inhibiting proliferation and inducing differentiation in breast carcinoma cells and studying the changed expression of C-myc and p53 gene protein and HOXA5 gene mRNA regulated by sodium phenylbutyrate(PB) and dimethylformamide(DMF) at the cellular level, subcellsular level, and molecular level in vitro, which can further identify molecular alterations in breast carcinoma and screen new diagnositic biomarkers and new treatment targets. This observation will provide valuable insight into the molecular basis of breast carcinoma, and can contribute to cure rates, prolongation of survival, reduction of local recurrence rates and enhanced quality of life in patients with advanced tumour diseases.The study on the effect and mechanisms of sodium phenylbutyrate and dimethylformamide inhibiting proliferation and inducing differentiation and the changed expression of C-myc and p53 gene protein and HOXA5 gene mRNA regulated by sodium phenylbutyrate(PB) and dimethylformamide(DMF) in breast carcinoma cells was performed in this subject. Analysis of our findings in a wider perspective is instructive from several aspects.1. In this article, we investigated the degree of expressions of oncogene C-myc,CerbB-2 and tumor-suppressor gene P53 in breast primary tumours. Meanwhile we also researched the states of expressions of ER and PR proteins. It suggested that C-myc and CerbB-2 protein were overexpressed in primary tumours, which were not relative to pathological stage and tissue grade. Our findings showed that mutant P53 was overexpressed in primary tumours, which had not signs relative to pathological stage and tissue grade. The results hinted that wild type P53 as a anti-oncogene was in a kind of low expression state and was down-regulated in primary tumours. ER and PR protein showed relatively high expression in in primary mammary cancers. This phenomenon telled us that ER and PR protein played an important role in occurrence and development of mammary cancers. The expression of C-myc protein showed a positive correlation with that of CerbB-2 and ER, which indicated that the causes and pathogenesis of breast carcinoma was multifactorial.2. The clinical applications of PB and DMF have a bright future. MCF-7 cells were cultured in vitro. Using MTT tetrazolium dye reduction assay, we showed the antitumoral and antiproliferative effects of PB and DMF on breast carcinoma MCF-7 cells. The studies had demonstrated dose-and time-dependent growth inhibition of MCF-7 cells induced by PB and DMF. The inhibitive rates of MCF-7 cells incubated with 0(control), 0.391mM, 0.781mM, 1.563mM and 3.125mMPB in culture medium for 24h-72h were 3.5%-20.8%, 22.3%-68.1%, 39.5-90.7%, respectively, and when MCF-7 cells were exposed to 3.125 mM for 72h, the rate of anti-proliferative was 90.7%. The inhibitive rates of MCF-7 cells incubated with 0(control), 0.5mM, 1.0mM, 2.0mM, 4.0mM and 8.0mM DMF in culture medium for 24-72h were 4.5-25.4%, 10.2-58.7%, 25.9-77.8%, respectively. When MCF-7 cells exposed to 8.0 mM DMF for 72h, the rate of antiproliferation was 77.8%.Our experimental results confirmed that the effects of PB inhibiting proliferation and inducing differentiation in breast carcinoma cells were superior to DMF because compared with DMF, PB had the advantages of relatively low dose medication, nice effects and long duration.3. Flow cytometery analysis demonstrated that prohibition of cell proliferation by PB and DMF was due to G0/G1 phase arrests. By the flow cytometery, we observed the changes of cell cycle phases of MCF-7 cells treated with PB and DMF. The findings revealed that both PB and DMF had the significant effects on the distribution of cell cycle phases. Compared with control group, MCF-7 cells stopping at G0/G1 increased, but the cell stopping at S1, G1/M phase reduced. The proportion of cell treated with 0.781 mM and 1.563 mM PB at G0/G1 was respectively 61.1±1.5% and 62.8±1.9%, compared with the 42.5±1.1% of control group, the differences were markedly showed. The propotrion of cell exposed to 0.5mM 1.0mM DMF at G0/G1 was respectively 69.6±1.5% and 71.1±1.4%, compared with the 42.5±1.1% of control group, the differences were significantly revealed. Our studies suggested that PB and DMF can notably suppressed the DNA synthesis and forbid cells turning into mitosis in MCF-7 cells, which indicated that PB and DMF can induce differentiation in breast carcinoma cells.4.PB and DMF can increase the differentitation degree of MCF-7 cells. The extent of tumour cell differentiation was assessed between the first to the third day after exposure to 1.563mM, 3.125mMPB and 0.5mM, 1.0mMDMF.Under the electron microscope, We saw that PB and DMF significantly changed the morphology of MCF-7 cells. The nucelus of MCF-7 cells showed extremely irregular shapes and the ratio between nucleus and cytoplasm increased before they were treated by PB and DMF. However, after treated, nucelus became round and the ratio between nucleus and cytoplasm decreased with large number of lipid droplets appearing in cytoplasm.The synapses of nucleus reduced, which indicated that nucleus surface area and the numbers of nucleopore had decreased. This change was in favour of genetic information transmitting in normal way. Morover, we also discover that a great quantity vicrovilli (MV) fell off from cell surfaces.These phenomena manifested that PB and DMF could improve the differentiation degree of breast carcinoma cells and futher make them change from malignant phenotypes to normal ones.5. The immunohistochemical methods were used in this experiment to investigate the levels of C-myc and p53 protein expression after treated by PB and the results were compared with those of control groups. Our findings demonstrated that the levels of C-myc protein expression in cytoplasm notably decreased when PB restrained cell proliferation of MCF-7 cells, and the decreasing degree was concerned with the concentrations of PB. The results of this expriment showed that, with the increases of concentrations of PB, C-myc positive expression ratio of MCF-7 cells reduced. For example, the proportion of C-myc positive expressions of MCF-7 cells treated by 0.78mM and 1.563mM were respectively 50.5±3.6% and 46.4±2.2%, compared with the 58.3±1.8% of control group, the differences were markedly showed. The proportion of p53 positive expression of MCF-7 cells treated by 0.781mM and 1.563mM were respectively 36.6±2.6% and 34.6±1.6%, compared with the 44.0±2.4% of control group, and the differences were significantly revealed. It can be seen that PB suppressed the transcription regulation functions of C-myc oncogene, thereby arrested the malignant proliferation of breast carcinoma cells, which suggested that the RNA synthesis of tumor cells had been restrained. On the other hand, it can be seen that PB also suppressed the transcription regulation functions of mutant p53 gene, accordingly arrested DNA duplication and mitotic.6. HOX genes, the master control gene of transcrioption process, respond to PB. We used RT-RCR mothods to explore the effect of PB and DMF on the expressions of HOXA5 mRNA. Our findings demonstrated that MCF-7 cells had the high-expressions of HOXA5 mRNA after treated by PB. The relative level of HOXA5 mRNA of MCF-7 cells treated by 0.781mM and 1.563mM PB were respectively 0.801±0.122 and 0.903±0.096, compared with the 0.460±0.065 of control group, the differences were markedly showed. This manifested that PB can upregulate the expressions of HOXA5 mRNA in MCF-7 cells. On the contrary, Our findings showed that MCF-7 cells had the low-expressions of HOXA5 mRNA after treated by DMF. The relative level of HOXA5 mRNA of MCF-7 cells treated by 0.5mM and 1.0mM DMF were respectively 0.474±0.057 and 0.484±0.061, compared with the 0.460±0.065 of control group, the differences were not significantly revealed.This manifested that PB can up-regulate the expression of HOXA5 mRNA in MCF-7 cells, however, DMF had not that function. Creative points of our studies:1. We first investigted the antiproliferation and inducing differentiation of PB in breast carcinoma cells in our country. The results that PB regulated the expression of C-myc and p53 gene protein were creative studies at home and abroad. We primarily confirmed that PB can regulate C-myc and p53 gene on the processes of carcinogenesis and develpoment of mammary cancers. Our studies identified the mechanism and mode of action of PB during antiproliferation and inducing differentiation.2 We first confirmed that DMF had the effect on antiproliferation and inducing differentiation in breast carcinoma cells, and its mechanism had been primarily probed in our studies. 3 Our results that PB regulated the mRNA expression of HOXA5 genes were creative studies at home and abroad.Both PB and DMF were effective differentiation agents, especially, PB as a new type of differentiation agent was consided to be the new strategy of mammary cancer treatment because it can induce tumour cell maturation and was free of cytotoxic and carcinogenic effects. Further exploring the mechanism of PB and DMF would be helpful to the combination use of anticarcinogen, improving the therapeutic effect of anticarcinogen, decreaing the cost of treatment, and reducing the toxic effects.Our studies provided a large number of theoretical and experimental evidences for the clinical application of PB and DMF.