| IntroductionColorcetal cancer is one of the most common malignant gastrointestinal cancers. Recent investigations have shown that colorectal cancer ranks the fourth among the most common cancers in China and the incidence has been on the rise in the past years. Therefore, the study of the mechanisms of its initiation, progression, differentiation and apoptosis has been an urgent and practical subject in tumor research in China.Numerous epidemiological and experimental studies in vivo and in vitro have identified that a high - fibre is associated with diet a decreased incidence and growth of colorectal cancer. Butyrate, a four -carbon shot - chain fatty acid, is known as a product of bacterial fiber fermentation within the colon. It has shown that sodium butyrate ( NaB) can inhibit the growth of colorectal carcinoma cells under the physiological concentration, playing an important role in prevention of cancer. Other reports pointed out that NaB can induce apoptosis of human ovarian carcinoma and endometrial carcinoma as well as gastric cancer, improving the differentiation of human hepatocarcinoma and brain glioblastoma, making reversion of malignant phenotype of human high - metastatic lung cancer and so on. However, the molecular mechanisms are not known by now. Present studies have demonstrated that NaB triggered some genes mainly after histone hyperacetylation,finally reducing tumor proliferation and inducing its apoptosis. Most studies focused only on cell cycle proteins while there were few reports about caspase - 3 protein that is a pivotal one in cell apoptotic pathways. Hence we treated human colorectal cancer cell line CCL -187 in vitro on the basis above in order to study the cell arrest and apoptosis caused by NaB, at the same time determining the expression of caspase - 3 with Western Blot assay. We hope that we can offer further evidences and theories for integrated apoptosis mechanism and contribute NaB to be a new potential anticancer drug used in clinic.Materials and MethodsWe detected the growth inhibition of human colorectal cancer cell CCL - 187 exposed to NaB in vitro. Collect cells in the logarithmic ?growth stage, inoculate them at the final concentration of 1+105cells/ mL, add NaB in medium after 24hr. Use ddH2O to dilute NaB up to 0.4 mol/L to storage at 4C. The final concentration of NaB was 20 mM, 40 mM, 60mM, and synchronously add equal volume of saline in control. Observe changes of Morphology and chromatin with inverted phase contrast microscope, fluorescence microscope, transmission electron microscope; detect the growth inhibition by MTT assay while quantitatively analyzing the cell cycle distribution and apoptosis by u-sing flowcytometry. Western Blot analysis and DNA fragment detection was carried out to determine the feature of molecular biology.ResultsThe rate of growth survival of human colorectal cancer cell CCL- 187 decreased from 94. 7% to34. 2% with 20mM NaB treatment and from 86. 2% to 12. 2% with 40mM NaB, exhibiting that NaB played an important role in growth inhibition of CCL - 187 cells and showing a time - dose dependent manner. Under the inverted phase contrast microscope, cell structures changed to be small and around with a 48 - hour treatment by 40mM NaB, but not by 20mM NaB; there were a lot of floating cells and cell debris in medium under the treatment of 60mM NaB. We also found that chromatin distributed in nucleus evenly in control, showing blue fluorescence, however, it was easy to find that numerously highly - aggregated chromatin in cells treated by NaB, showing bright blue fluorescence. Under the transmission electron microscope, we found apoptotic bodies-a typical phenomenon of apoptosis in cells treated by 40mM NaB, but not in control. The result of flowcytometry analysis indicated that there was a typical G0/G1 phase in control, while hypoploid peak observed in 20mM and 40mM NaB. Furthermore, the hypoploid peak in 40mM NaB was higher than that of 20mM one. When the concentration of NaB persisted up to 60mM, cells u... |
