The Role Of Autophagic Cell Death In Colon Cancer Cell Chemotherapy

Conlon cancer is one of the malignant tumor, its global morbility is increasing year after year. In our contry, the morbility is higher in the southeast costal area. The mechanism of conlon cancer induction has not been well understood, environmental factors and genetic factors might be the main causions.Programmed cell death(PCD)is important for cancer cells to support malignant growth. Thus, understanding the mechanisms of programmed cell death and designing specific therapeutic approaches to induce cell death in cancer cells are critical for disease treatment. There are two morphologically distinctive forms of programmed cell death, apoptosis and autophagic cell death.Apoptosis(type I PCD) is a cell intrinsic suicide mechanism, which regulated by various cellular signaling pathways. Defective apoptotic cell death can result in autoimmune diseases and tumorigenesis, whereas excessive apoptosis is often associated with neurodegenerative diseases.Autophagic cell death(type II PCD) is another important physiological cell death process. This type of cell death is characterized by massive degradation of cellular contents, including essential organelles such as mitochondria, through a membrane vesicle autophagosome, then fuse to lysosomes to become autolysosomes. Autophagy is believed to have an important role in tumor development. When baseline levels were compared, the amount of autophagic degradation in cancer cells was less than that of their normal counterparts.Many Researchs shows that apoptosis and autophagy may be interconnected in some settings, and even can be simultaneously regulated by the same trigger resulting in different cellular outcomes in some cases. Depending on the cellular context and stimulus, autophagy may be indispensable for apoptosis by preceding and further turning on apoptosis. In other settings, autophagy may rather antagonize/delay apoptosis, inhibition of autophagy can inhance cell sensitivity to apoptotic signals, and there are also examples where the two processes may be mutually exclusive acting as backup mechanisms of each other.Bax and PUMA are critical apoptosis regulators, deficiency in Bax and PUMA or expression of Bcl-2 blocks apoptosis, and this failure to execute apoptotic cell death impacts development and promotes tumorigenesis. When apoptotic cell death is deficient, other ways of cell death are critical for cancer therapy.In recent years, the function of autophagy in human diseases, including neurodegenerative diseases﹑cancer﹑immunity and defense has been progressively appreciated based on the increasing understanding of the diverse biological functions of autophagy.The role of autophagy in cell death and cell survival is one particularly important and highly debatable aspect among the various functions of autophagy studied.Traditional cancer therapy is targeting on cell apoptosis, but it is well established that the inhibition of apoptosis is a critical event in tumorigenesis. Elimination of cancer cells might not only occur via apoptosis but could also be achieved by other forms of cell death such as autophagic cell death. So if we can induce autophagic cell death in apoptosis defective cancer cells, it may bring a new therapeutic strategy for cancer.Part I The effect of apoptotic pathway defection on colon cancer cells chemosensitivityIn this part, WT HCT116 cells and knocked out (KO) HCT116 cells were treated with 5-FU(0, 10, 20, 40μg/ml) for 24 h, cell viability was measured by WST-8 assay and trypan blue uptake, we found that 5-FU treatment could result in growth inhibition, the death rates are dose-dependent, and amost the same in every dose between WT cells and KO cells. After 5-FU treatment, cells were collected for apoptosis analysis, and found that the apoptotic rate of WT cells was much higher than KO cells. The obove results indicated that there may be another way of cell death in KO cells, such as autophagic cell death.Part II The role of autophagic cell death in apoptotic defective colon cancer cells with chemotherapeutic agent treatment(1) 5-FU treatment result in autophagy induction in KO colon cancer cells Then, we identificated autophagy induction in cells after 5-FU treatment, under electron microscopy detection, we could found that many membrane vesicles contain cellular components in KO cells, but no obvious autophagosomes in WT cells.To further confirm this autophagosome induction, cells were transfected with GFP-LC3 plasmid, LC3 (microtubule-associated protein-1 light chain-3) is the mammalian homolog of the yeast Atg8 protein, which located in autophagosome membrane. After 5-FU treatment, there were obvious punctate fluorescences in KO cells, whereas diffused fluorescence in WT cells. PI3K-Akt-mTOR signaling pathway is a negative regulation mechanism of autophagy induction, inhibition of mTOR has been consistently associated with indicators of autophagy in cancer cells. We measured mTOR activity by western blot analysis of its well-known substrate p70s6k, In contrast to WT cells, p70s6k activity was suppressed in KO cells. This result indicates that autophagic signaling was activated in KO cells. Taken together, these results further support the hypothesis that autophagy was induced in Bax and PUMA deficient HCT116 cells.(2) Inhibition of autophagy decreased chemosebsitivity in KO cellsAutophagy is a highly conserved pathway. Under certain circumstances, it seems that autophagy may serve as a pathway to cell death. In this part, we used 3-MA (3-Methyladenine) to block autophagy. Under electron microscopy, there were fewer or even no autophagic vesicles in cells treated with 5-FU plus 3-MA compared with cells treated with 5-FU alone, and punctuate fluorescence was disappeared in 5-FU treated cells in the presence of 3-MA. Cell morphous change was detected under phase contrast microscope and cell viability was measured by trypan blue uptake, and found that the cell death rate was decreased when treated with 5-FU plus 3-MA in KO cells. So we can conclude that 5-FU could induce autophagic cell death, as an alternative cell death pathway in apoptosis defective cells to achieve growth inhibition.Totally, we get the conclusions:1. 5-FU treatment can inhibit WT and KO HCT116 colon cancer cells growth, and the inhibition rates are almost the same;2. Chemotherapeutic agent 5-FU could induce autophagic cell death in KO colon cancer cells, as an alternative cell death pathway to achieve growth inhibition.