The Study In The Apoptosis By Way Of Mitochondrium Pathway Of Human Esophageal Carcinoma Cell Induced By Oridonin

Objective: To study the anti-tumor effect in vitro and mechanism of the apoptosis of human esophageal carcinoma cell induced by oridonin(ORI). Methods: After being treated by ORI in different density during different time, we detected the rates of the growth inhibition of human esophageal carcinoma cell line SHEEC by Methyl Thiazolyl Tetrazolium(MTT) assay; Observe the change of the ultramicrostructure of apoptosis under transmission electron microscope, and the change of mitochondrium especially; Using a kind of double-dye assay of in situ end-labeling/iodinate pyridina(TUNEL/PI), the rates of the apoptotic cells and the phrase distribution of cell cycle were detected by flow cytometry; Similarly, the mitochondria membrane potential(Δψm) was detected by flow cytometry after being dyed with rhodaminel23(Rho123); Furthermore, we detected the expression of the apoptosis-associated protein by Western blot. Also the expression of the related genes were detected by using RT-PCR. Results: On the aspect of the anti-tumor effect, ORI could inhibit the cell growth of SHEEC conspicuously. The inhibition ratio reached 78.7% with ORI in a density of 64μg/ml after 24 hours. If the time lasted to 48 hours, the inhibition ratio would be 89.5%, the same, the rate was 91.7% after 72 hours; Having been treated by ORI in lot densities of 8, 16, 32μg/ml in 24 hours, we could see the chromatin clumps gathered around the inside edge of the caryotheca obviously in most cell nucleus, that is apoptotic phenomenon; The apoptosis ratio was 9.3%, 21.1% and 47.7% according to flow cytometry. At equal pace, an apoptotic peak which tended evidentlty displayed on the phrase distribution of cell cycle. And S stage raised gradually at the same time. The following results is attributed to mechanism of the apoptosis. The morphous of mitochondrium changed ahead of cell nucleus under transmission electron microscope. After 8 hours, the internal structure of mitochondrium disappeared, and the typical apoptotic morphous of cell nucleus could be seen; Following a time of 24 hours treatment with ORI in lot densitiesof 8, 16, 32μg/ml, the proportion of those cells definited Rho(—) was 3.2%, 9.8% and 45.7%to part. Besides, we found that the expression of Cytochrome C(CytC) and Caspase-9 appeared a kind of tendency of concentration dependent up regulation; While the time of the treatment by ORI in a density of 32μg/ml lasted from 1h to 8h, the expression of those genes related to apoptotic regulation seemed change obviously, including some up regulation, as well as, some down regulation. Conclusion: ORI probably belongs to cell cycle specific agents(CCSA). It could inhibited the growth of human esophageal carcinoma cell obviously. ORI could induce apoptosis on the human esophageal carcinoma cell efficiently. The mechanism concerned with starting mitochondrium apoptotic pathway.