Induced Inhibition And Apoptosis Of Colonic Carcinoma Cell Line HCT-8 By Oridonin

Background and objectionThe cell apoptosis, also named the programmed cell death, is a natural process controlled by a series of endogenous genes. The block of cell apoptosis is one of the important mechanisms as the same as the abnormality of cell proliferation in carcinogenesis. Therefore, the apoptosis of tumor cell may be a new area in tumor research. It would be a new hot spot to find some drugs which would induce some malignant cells apoptosis with high efficacy and low toxicity in tumor therapy.Oridonin is a kind of ent-kaurane diterpenoids isolated from the aerial parts of Rabdosia rubescens, and it has much more potent cytotoxic effects on many kinds of tumor cells. Further studies show that Orid, has exhibited DNA-damaging activity in assay which employed DNA-repair deficient and repair proficient yeast strains. In addition, there are data suggested that Orid. can restrain tumor cell growth by means of inhibiting sodium pump activity. Feng C W et al find that Orid. can inhibit the growth of human leukemia cell line HL-60 in vitro. Zhang C L et al report that Orid. has significant apoptotic effect on Hela cells in a dose- and time- dependent manner and deduce that the mechanism is associated with caspase pathway. Some researches support that Orid. can effectively inhibit the growth and proliferation of human lung cancer cell SPCA-1. The telomerase activity is remarkably decreased concurrentlywhen apoptosis occurred induced by Orid.. Orid, can influent the telomerase activity during the treatment of SPCA-1 cells hi vitro. The negative regulatory mechanism of telomerase activity may be caused by cell apoptosis. More and more studies show that Orid, would be a chemotherapy agent with high efficacy and low toxicity in tumor therapy.Colorectal carcinoma is one of the most malignant tumors. In recent years the incidence of the disease is rising rapidly, hi our country its incidence has increased from 10/105 to 30/1 05 comparing with that of thirty years ago. hi the range of malignant tumors it has raised from the N0.6 to the NO.3. Many clinic studies have suggested that Orid, can inhibit the growth of many kinds of tumor. But the inhibition in the growth of colorectal carcinoma has not been studied. In this paper, human colon cancer cell line, HCT-8 was cultured hi vitro and the cell growth and apoptosis was observed with the concentration approaching IC50 detected by MTT method. We investigated the effect of HCT-8 cell apoptosis was examined by immunochemistry and flow cytometry to probe the mechanism of cell apoptosis induced by Orid, to provide the theoretical basis for clinical medication.Materials and Methods1. Human colon cancer cell line, HCT-8 was chosen as the material. 2. MTT assay examined suppressive rate of cell growth dealt with different concentration of oridonin, and chose the appropriate concentration to induce the cells apoptosis for assessing the changes of the cell growth suppression. 3. HE dye was applied to observe the morphologic changes induced by oridonin. 4. The protein expression of cell apoptosis associated genes caspase3 and Survivin was checked with immunochemistry in the depend on dealing group (adding oridonin) and control group (without oridonin) after oridonin at the concentration of 5 g/ml was added for 48 hour. 5. The development of cell cycle and apoptosis change caused by different concentration groups were examined by flow cytometry. 6. With SPSS 10.0 software package, one-way ANOVA , Independent-samples T Test and Chi-Square test were used to analyze all experimental data, size of the test was P<0.05.Results1. MTT assay showed the adding different concentration of oridonin for 48 hours inhibited the cell growth and proliferation in some degree. The oridonin cytotoxicity was increased more obviously with the concentration of oridonin increasing gradually. Its degree was associated with dosages. 2. Morphology: After exposed to oridonin for 48h, colon cancer cells presented some morphologic feachers of apoptosis, including cell shrinkage, nuclear condensati...