The Study Of Mitochondrial Signal Mechanism Induced Apoptosis By Don In Human Colon Cancer Cells

Deoxynivalenol (DON), also known as vomitoxin, is a pathogenic mycotoxins produced by Fusarium fungi. Reports have shown that it has strong cytototic, significant toxicity to prokaryotic cells and eukaryotic cells. DON may inhibit DNA, RNA and protein synthesis, mitochondrial function and induce apoptosis. Currently, DON-induced apoptosis mainly focused on the immune mechanism, but the molecular mechanism of mitochondrial pathway has few reports. To address this issue, we studied the DON sensitive cells-human colon cancer cells (HT-29) to investigate the morphology change by DON under different concentrations; to indicate the key apoptosis protein in mitochondrial pathway:Cytochrome C,Caspase-9 and Caspase-3 expression induced by DON at different concentrations and different times; and to illustrate the important regulator of tumor apoptosis gene Bcl-2 family expression, the key regulators of Bcl-2, Bax and Bid, induced by DON under the different concentrations of HT-29 cells in vitro. The aim of this research is to investigate the DON induced apoptosis in cultured cells, especially the morphology change, key regulator of proteins and factors in the mitochondrial apoptosis pathway and provide evidence of the molecular mechanism of cell toxicity induced DON.Test I:The test was processed the logarithmic phase of HT-29 cells with different concentrations of DON (DON final concentrations were 125,250,500,1000,2000 ng mL-1), with a separate control group. Cells were incubated for 24h. Then we observed the morphological changes under optical microscope and transmission electron microscope. The results showed that the HT-29 cells were induced apoptosis by different concentrations of DON. Under the optical microscope, with the increasing concentration of DON, HT-29 cells decreased significantly, suggesting that DON can inhibit the growth of HT-29 cells. Under the transmission electron microscopy, in the control cells, the nuclei and organelles, surrounding microvilli, chromatin aggregation, ridge in mitochondria were clearly observed. The five test cells were the typical morphological changes of apoptosis, mainly for the nuclear shrinkage, fragmentation, chromatin aggregation and close to the nuclear membrane, fewer and even disappeared microvilli, with some apoptotic bodies, mitochondrial swelling of organelles, vacuolization.TestⅡ:The test was processed the logarithmic phase of HT-29 cells with different concentrations of DON (DON final concentrations were 125,250,500,1000,2000 ng mL-1), with a separate control group. Cells were incubated for 24h. Then the total protein was extracted and isolated by SDS-PAGE. Western blotting assay was applied to analyze the relative expression of Cyt C,Caspase-9,Caspase-3 and internal reference proteinβ-actin. The results showed that after 24 h incubation with different concentrations of DON in HT-29 cells, the relative expression of the key apoptosis protein Cyt C,Caspase-9,Caspase-3 achieved the peak at the concentration of 1000,500,1000 ng·mL-1 respectively. The result was the most significantly difference compared with that of control (p< 0.01). DON induced expression of the protein persists increasing from concentration of 125 to 500 ng·mL-1. With the concentrations higher than 500 ng·mL-1-1000 ng·mL-1, the relative expression of Caspase-3 was reduced with the increase of the toxic concentrations even though Caspase-3 expression decreased at 2000 ng·mL-1. Cyt C expression level gradually increased at 125-1000 ng mL-1, and a slight decrease at 2000 ng·mL-1.Meanwhile, the test was processed the logarithmic phase of HT-29 cells with DON 500 ng·mL-1, cells were incubated for 0,3,6,12,24,48 h. Then the total protein was extracted and isolated by SDS-PAGE. Western blotting assay was applied to analyze the relative expression of Cyt C,Caspase-9,Caspase-3 and internal reference proteinβ-actin. The results showed that with the increase treatment time of DON, the apoptosis protein Caspase-9 and Caspase-3 achieved the peak after 12 h and significantly different compared with the control group(P<0.01). Along with the increasing contribuation time,24 and 48 h, their both relative expression gradually decreased. However, the expression of Caspase-9 was slightly lower than the control group, while the expression of Caspase-3 was higher than the control group. The expression of apoptosis protein Cyto C achieved peak at 6 h and it was significantly different compared with the control group (P< 0.01). Along with the increasing contribution time, the relative expression decreased, and at 24,48 h, the expression was significantly lower than the control group. TestⅢ:The test was processed the logarithmic phase of HT-29 cells with different concentrations of DON (DON final concentrations 125,250,500,1000,2000 ng·ml-1), with a separate control group. Cells were incubated for 24 h. Then applied the real-time PCR method to detect the change of Bcl-2,Bax,Bid and internal reference GAPDH genes. The results showed the relative expression of Bcl-2 and Bax achieved the peak at the concentration of DON 500,1000 ng·mL-1 respectively. With the increase concentration of DON, the relative expression increased gradually and then decreased after the peak compared with the control group. Bax, with the DON concentration (125,250,500 ng·mL-1), its expression decreased slightly. While in 1000,2000 ng·mL-1, it expressed increased, and reached the peak. This kind of changes was contrary with Bcl-2. However, the relative expression of Bid displayed decreased tendency along with the increasing concentrations of DON and reached the minimum. The Bcl-2 and Bax expression ratio showed the maximum at 500 ng·mL-1. Then with increasing of DON concentration, the ratio of high dose was lower than low dose.