The Role Of Nutrient-free Induced Autophagy In Insensitivity Of Cholangiocarcinoma To Chemotherapy

Cholangiocarcinoma is one of the most common malignancies in the world and the incident rate is growing every year in our country. Right now, the most common way to cure the cholangiocarcinoma is undergoing the comprehensive treatment based on surgery, and also, surgery is the primary therapy of this kind of cancers. As the low degree of differentiation and the high degree of malignant, the recurrence rate after surgery is so high, together with the poor prognosis. More over, the advanced cancer patients has already lost the chance of operation, so the radiotherapy and chemotherapy became the main therapy. But in addition the cholangiocarcinoma is not sensitive to the radiotherapy and chemotherapy, so the treatment effect is poor and so does the prognosis. For these reasons, the study about the insensitivity of cholangiocarcinoma to chemotherapy have great importance to improve the treatment effect of cholangiocarcinoma and the patients survival rates.Although there are so many researches of drug-resistant mechanisms about cholangiocarcinoma, but it is still out of fully understanding. Recently, growing evidence show that autophagy was involved in tumor drug resistance.The form of autophagy is regulated precisely by cells. There are many gene products essential for autophagy and related pathways referred to as the ATG genes.The proteins encoded by the ATG genes were take part in the induction, the generation, the maturation, and the recycling of autophagy. On the other hand, several pathways are involved in the regulation of autophagy, such as the PI-3K-AKT-mTOR or the p38-MAPK pathways. The progress of autophagy is regulated by a series of complicated signals. And also, the deregulation of autophagy is related to many human diseases, including infections, cancer, neurodegeneration, aging, and heart disease. p53 is a well-known tumor suppressor genes and functional proteins, it plays an important role in the inhibition of tumor's occurrence and development. But only 50% of the gene mutation occurs in all the malignant tumors, the other mechanism is not clear. Some studies show that when apoptosis induced by p53 expression, the autophagy only found in the tumor cells without the occurrence of apoptosis. For these reasons, the escape from the apoptosis and the resistance to the chemotherapeutic drugs of cholangiocarcinoma cells in nutrient deprivation, most likely mediated by the autophagy and has the participation of p53 gene. To confirm this conjecture, the subject following the next two areas:first we make the study about the influence on the chemo-sensitivity of the cholangiocarcinoma cells in the shortage of nutrient; after that we make the study about mechanism of p53 participate in the insensitivity to the chemotherapy of the cholangiocarcinoma cells in nutrient deprivation.The cholangiocarcinoma cell lines (QBC) were cultured in the full-nutrient and nutrient-free media, and then were treated with chemotherapeutic agents (5FU and cisplatin) for 12h. The cell viability was measured by cck8 assay, and the cell morphological change was detected under phase contrast microscope and the apoptosis cells were stained with DAPI staining. The results showed that cells grown in nutrient-free media demonstrated more resistant to chemotherapeutic agents than that in full-nutrient media. It has been demonstrated that nutrient starvation is the natural inducer of autophagy in recent researches. As we know, the shortage of nutrient is a natural inducer of autophagy. Autophagy was also reported to be contributing to chemoresistance in tumor. Thus we want to investigate whether autophagy was invovled in cholangiocarcinoma cells and contributed to the insensitivity to chemotheraputic agents.The cholangiocarcinoma cell lines (QBC) were cultured in the full nutrient and nutrient free media. Electron microscope was performed to detect autophagysome. LC3-GFP plasmid was transfected into the cholangiocarcinoma cell lines, and the green dot was detected and measured. The results demonstrated that the expression of autophagy related gene was significant upregulated and the green dots were also increased during nutrient starvation which suggested that the cholangiocarcinoma cells showed more activated autophagy.Futher reseach suggested that the inhibition of autophagy increased cell death and chemosensitivity during nutrient starvation. The cholangiocarcinoma cell lines (QBC) were cultured in the full nutrient and nutrient free media. These tumor cells were pretreated with autophagy inhibitor(3MA) for 2h and then were incubated with chemotherapeutic agents (5FU and cisplatin) for another 12h. Cell viability was measured by cck8 assay, cell morphological change was detected under phase contrast microscope and apoptosis cell was stained with DAPI staining. The results showed that cells treated with autophagy inhibitor demonstrated more insensitivity to chemotherapeutic during nutrient starvation.Taken our data together, it suggested that autophagy was not only one of the survival mechnism of the cholangiocarcinoma cells during nutrient starvation, but also contributed to the chemotherapy resistance. But the mechnism of how autophagy contributed to chemotherapy resistance is largely unknown. It has been reported that p53 were connected with autophagy, thus we investigated the possible role of p53 and the autophagy in the cholangiocarcinoma cells.The cholangiocarcinoma cell lines (QBC) were pretreated with p53 inhibitor(PFT-a) for 1h, and then were cultured in the full nutrient and nutrient free media for another 24h. Electron microscope was performed to detect autophagysome. LC3-GFP plasmid was transfected into the cholangiocarcinoma cell lines, and the green dot was detected and measured. The results demonstrated that the autophagysome and green dots were decreased obviously during nutrient starvation when p53 was inhibited. To further investigate the role of p53 in autophagy activation, the cholangiocarcinoma cell lines (QBC) were cultured in the full nutrient and nutrient free media. Cells were pretreated with p53 inhibitor (PFT-a) for 1h or transfected with si-p53 plasmid, and then were incubated with chemotherapeutic agents (5FU and cisplatin) for another 24h. Cell viability was measured by cck8 assay, cell morphological change was detected under phase contrast microscope and apoptosis cell was stained with DAPI staining. The results showed that cells treated with p53 inhibitor demonstrated more insensitivity to chemotherapeutic during nutrient starvation. Thus, our results suggested that p53 were involved in chemotherapy resistance in the cholangiocarcinoma cells.To be concluded, our results suggested that:1,Nutrient starvation can activate autophagy in the cholangiocarcinoma cells, and autophagy contributes to chemoresistance; inhibtion of autophagy increased the senstivity to chemotherpeutic agents of the cholangiocarcinoma cells during nutrient starvation.2,P53 was invovled in autophagy activation of the cholangiocarcinoma cells during nutrient starvation. Inhibition of p53 increased the tumor cell death and chemosensitivity during nutrient starvation. Taken together, our researches make the conclusion that the activation of autophagy have taken part in the insensitivity to the chemotherapy of the cholangiocarcinoma cells, and also, P53 was invovled in autophagy activation of the cholangiocarcinoma cells during nutrient starvation, it probably be one of the mechanisms of chemotherapy resistance of cholangiocarcinoma cells.