Primary Study On Tumor Cell Autophagy And Tumor Cell Drug Resistance Mechanism

Autophagy also known as type II cell death, is a kind of programmed cell death, which is characterized in autophagosome aggregation. Autophagosome is a double-membraned structure that swallows organelles and cytoplasm and fuses with lysosomes to form autolysosome. The cellular components swallowed by autophagosome were degraded by the lysosomal enzyme. This process has an important role in the removal of damaged organelles and abnormal proteins. Cells suffered from excessive self-digestion will go to death without morphological features of apoptosis Autophagy signaling pathway will be activated by environment factors, such as hunger, high temperature, oxidative stress and hormone stimulation, or intracellular stress, such as damaged organelles, misfunctional protein aggregation and microbial attack.Autophagy is closely related with tumor. However, its effect on cancer seems to be bifunctional. It has been reported to act as inhibition or promotion factor of cancer. Long time oi starvation, hypoxia, radiotherapy or chemotherapy of tumor cells leads to the increase of damaged intracellular organelles, protein oxidation and aggregation of misfolded proteins. The damaged intracellular organelles and abnormal proteins will be degraded in the pathway of autophagy, and the products will be reused to build new structures. Meanwhile moderate autophagy avoids the cells into apoptosis and maintains the tumor cells survival. The resistance of radiotherapy and chemotherapy can often be seen in the clinic, which reduces the effect of treatment. However, excessive autophagy can lead to autophagic cell death or apotosis in tumor cells. Therefore, studying the influence of antophagy on tumor cell proliferation, death and apoptosis, will provide theoretical basis for eliminating the protective effect of autophagy on tumor cells and exploring antitumor drugs through the intervention of autophagy. It will provide experimental basis for clinical use of existing autophagy inducers or inhibitors as sensitive drugs of radiotherapy and chemotherapy. Thus it has important theoretical significance and social benefit.In this study, we detected the autophagy in the common solid tumor as laryngeal, hypopharyngeal carcinoma and breast cancer using the content of the specific autophagy protein LC3as an indicator. The relationship between autophagy and the expression of heat shock protein HSP70and HSP90were also studied. LC3was expressed in three cancer tissues (25specimens detected), indicating that in tumor cells autophagy are universal. The analysis of9cases of hypopharyngeal cancer specimens showed that the expression of LC3in the center part of tumor is significantly higher than the normal mucosal tissue. HSP70was widely expressed in the tested samples, and the expression of HSP70in the normal mucosa and the edge of carcinoma was higher than the center of cancer tissue. HSP90was highly expressed in the center of laryngeal and hypopharyngeal carcinoma tissue, while it was not expressed or had very low expression level in the normal tissue. Among14cases of breast cancer samples, HSP90was highly expressed in12samples; meanwhile. In9cases of hypopharyngeal cancer samples, it can be observed that there is some correlation between HSP90and LC3.The inner part of solid tumors which is often ischemia, hypoxia, and lack of sufficient supply of the growth factor are easy to undergo tumor cell autophagy. We studied the influence of hypoxia and serum-free culture on A549cells autophagy, simulating the in vivo environment of solid tumors. The results showed that after7hours cell culture by serum-free and nitrogen, the floating and dead cells gradually increased. The autophagy was enhanced in the cells not shedding by digestion of intracellular material and organelles, which might maintain cell survival. This result was similar to the performance of in vivo tumor cells.We studied the effect of clinically used anti-cancer chemotherapeutic agent5-flurouracil on autophagy of A549cells by detecting the amount of LC3and the ratio of the LC3-â…¡/LC3-â… , the acridine orange staining of acidic vesicles, and electron microscopy of antophagic vacuoles formation. We found that low concentrations(l-10mg/L) of5-flurouracil treatment can induce autophagy, while high concentrations(>10mg/L) of5-flurouracil treatment caused cell death in A549cells. Further studies have shown that the5-flurouracil together with macrophage inhibitor3-MA was more potent to inhibit cell proliferation than5-flurouracil alone, indicating that inhibition of autophagy may enhance the effect of antitumor of5-flurouracil to some extent. By comparing the acidic membrane bubble formation time and collaboration efficiency, we preliminary found that within48hours of5-flurouracil treatment on A549cells, the amount of acidic vesicle formation was up to the most, and in this time, the synergistic effect of3-MA on5-flurouracil was the strongest, showing that inhibition of autophagy can increase the killing effect of5-flurouracil on A549cells. In addition, low concentrations of5-flurouracil in the process of inducing A549cells autophagy did not disturb the expression of HSP70and HSP90.