Effect Of Autophagy On Colon Cells Induced By Rapamycin And It’s Possible Mechanisms

Objective:To observe the effects of rapamycin induced autophagy on the invasion, migration of colon cancer cells and investigate the possible mechanisms.Methods1. The autophagy vesicles were detected by monodansylcadaverin (MDC) fluorescence.2. Autophagy level and the level of ROS were detected using flow cytometry.3. The ultrastructure was observed using transmission electron microscope (TEM).4. The level of LC3was detected by western blot.5. The invasion of colon cells was measured by Boyden Chamber. Cell wound healing assay was used to observe the migration of colon cells.6. Using RT-PCR to analyse the gene expression of Beclin1and Bcl-2.7. The expression of Beclin1、Bcl-2and Caspase3protein on tissue was detected by immunohistochem ical.Resultsl.The SW480cells were purified with autophagic vesicle under rapamycin environment and autophagy level also increased (P<0.05)2.The number of SW480moving into the second floor was increased under the rapamycin environment (P<0.01),while the migration is also improved (P<0.05)3. The production of ROS increased in rapamycin group. NAC could inhibit the occurrence of autophagy.4. RT-PCR data showed that the expression of Beclin1and Bcl-2mRNA were higher in the rapamycin group than in the control group.(P<0.05)5. The positive expressions of Beclin1in normal colorectal mucosa, adenomas and colorectal adenocarcinoma were11.76%,21.05%and68.92%(P<0.01). The positive expressions of Bcl-2in normal colorectal mucosa, adenomas and colorectal adenocarcinoma were5.88%-15.79%and63.51%(P<0.01). The positive expressions of Caspase3in normal colorectal mucosa, adenomas and colorectal adenocarcinoma were94.12%,73.68%and58.11%(P<0.05). There was a stable positive relationship between the Beclin1and Bcl-2(rs=0.583,P<0.01), a negative relationship between Beclin1and caspase3(rs=-0.511, P<0.01), Bcl-2and caspase3(rs=-0.530,P<0.01).6. In colorectal adenocarcinoma, the expression of Beclin1was related to lymphatic metastasis, cancer cell differentiation and Dukes stage (P<0.05). The expression of Bcl-2was related to cancer cell differentiation and Dukes stage (P<().05). The expression of Caspase3was related to cancer cell differentiation (.P<0.05).Conclusions1. Rapamycin can induce autophagy on SW480cell line, and both of the invasion and migration of SW480cells can inhance with the increase of autophagy. It is implied that autophagy can protect SW480cells.2. Rapamycin can improve the produce of ROS in SW480cells, while the scavenger of ROS can inhibit autophagy. The results indicate that the level of autophagy induced by rapamycin may cause by the increase of ROS first.3.The expression of Beclin1、Bcl-2and Caspase3in normal colorectal mucosa, adenomas and colorectal adenocarcinoma tissue suggest that:autophagy related protein Beclin1, apoptosis-related protein Bcl-2and Caspase3participate in the occurrence and development of colorectal cancer. Autophagy activity increased; the more lower the apoptosis ability, the more higher the escape from apoptosis of tumor cells.4. The expression of Beclin1, Bcl-2and Caspase3were related to cancer cell differentiation, and the expression of Beclin1was also related to lymphatic metastasis and Duke’s stage. These results may be helpful in understanding and judging the progression of colorectal cancer.