Role And Mechanism Of Low-Dose Chemotherapy-induced DNA Damage Response And Autophagy In Senescence And Apoptosis In Human Colorectal Cancer Cells

Cellular senescence is a specialized form of irreversible growth arrest that ischaracterized by the enlarged and flattened morphology and the alteration of geneexpression pattern and chromatin structure. Due to the persistent functions oftelomerase in tumor cells, tumor cells were believed until recently to have lost thecapacity to undergo senescence. Unlike replicative senescence, premature senescencein independent of telomere shortening or cell cycle distribution. It is believed thatbecause of this characteristic, premature senescence plays a vital role in tumorsuppression and is increasingly recognized as a possible outcome for the treatment ofhuman tumorsHowever, senescence does not mean death, and some problems arise fromsenescence-inducing therapies should be considered:1) senescence-associatedsecretory phenotypes components secreted by senescent tumor cells might stimulatethe malignant phenotypes of nearby tumor cell;2) a noticeable possibility exists thatone day the senescent cells could come into active again as long as they are notcleared by the system;3) senescent tumor cells are resistant to cell death. Therefore,how to find a chemotherapeutic approach that specifically switches senescent tumorcells into apoptotic seems to have great potential value in cancer therapy.Autophagy, a lysosome-dependent degradation pathway that is activated by avariety of stress such as starvation and oxidative stress, regulates cell fate byselectively degrading long-lived proteins and damaged organelles and maintainingcellular homeostasis and genomic integrity. More recently, accumulated evidencesuggested that autophagy was implicated in cellular senescence. Increased autophagicvacuoles and senescence-associated β-galactosidase activity was observed in agingfibroblasts. The up-regulation of autophagy-related genes in the process of oncogene-induced senescence was found and blockade to autophagy delayed the senescencephenotype. Thus, it remains unclear whether autophagy can affect the induction ofsenescence by regulating the DNA damage response.In order to determine the role and mechanism of autophagy in low-dosecamptothecin-induced senescence in human colorectal cancer cells, we carry out thesubject from the following two aspects: First, the effect of DNA damage responseinduced by low-dose camptothecin on senescence in colorectal cancer cells; Second,the effect of autophagy on low-dose camptothecin-induced senescence and apoptosis in colorectal cancer cells.Part1The effect of DNA damage response induced by low-dosecamptothecin on senescence in colorectal cancer cellsIn this study, we observed the induction of senescence by low-dose CPT. wedetected the senescence phenotype in colorectal cancer cells treated with low-doseCPT. We found enlarged and flattened cell morphology and increased time-dependentSA-β-gal activity. The results of real-time PCR and Western blotting indicated that theexpression of p21was greatly induced by low-dose CPT. To investigate the activationof DNA damage signaling pathway by low-dose CPT, several key proteins of DDRwere detected by Immunofluorescence staining and Western blotting. The expressionof γ-H2AX, ATM (Ser1981), Chk2(Thr68) and p53was increased over time as aresult of CPT. To further confirm this point, a highly specific ATM inhibitor KU55933was used to confirm the role of DDR signaling in low-dose CPT-induced senescence.In the presence of KU55933, senescence cells induced by low-dose CPT weredramatically decreased. Taken together, these results suggest that low-dose CPTinduces senescence through DDR signaling in colorectal cancer cells.Part2The effect of autophagy on low-dose camptothecin-inducedsenescence and apoptosis in colorectal cancer cellsIn this study, we first observed the induction of autophagy by low-dose CPT. ThemRNA of ATG5, ATG7and Beclin1was increased over the time. And low-dose CPTcaused distinct decrease of p62in a time-dependent manner. Concentrated LC3inautophagic vacuoles is considered as the activation of autophagy. Thus, CRC cellswere transfected with adenovirus GFP-LC3. Time-dependent increase of GFP-LC3puncta was shown after CPT treatment. Furthermore, electron microscopy analysisshowed more autophagosomes concentrated in cells exposed to CPT for72h. Todetermine the mechanism underlie the autophagy induced by low-dose CPT, weanalyzed the expression of AMPK, TSC2, mTOR. Low-dose CPT up-regulated thephosphorylation of AMPK and TSC2while down-regulated the phosphorylation ofmTOR over time. This suggests low-dose CPT may induce autophagy via the AMPK-TSC2-mTOR pathway in a time-dependent manner in colorectal cancer cells.We next observed the induction of senescence after autophagy inhibition byautophagic inhibitior or small interfering RNA. SA-β-gal staining showed thatsenescent cells were dramatically decreased when autophagy was blocked. To furtherverify the impact of autophagy on senescence, we silenced BECN1by small interfering RNA. The expression of BECN1was assessed to confirm the transientknockdown of BECN1by siBECN1. As with pharmacological inhibition of autophagy,senescence phenotype was also significantly attenuated in cells transfected withsiBECN1. We wondered whether the inhibition of autophagy down-regulated thelevels of p53and p21, thus promoted cancer cells to death. We detected the expressionof p21and its upstream regulator p53. The CPT treatment combined with autophagyinhibition by pharmacological approach significantly reduced p53and p21expression,and specific silence of Beclin1by siRNA has the similar effect. These resultsindicated that autophagy can affect the induction of senescence by regulating theDNA damage response.We observed the the induction of apoptosis after autophagy inhibition byautophagic inhibitior. As demonstrated by CCK-8, CPT treatment caused less than10%cell death, whereas approximately50%cell death was detected in cells exposedto CPT with either3-MA or CQ. What’s more, a significantly increased amount ofapoptosis caused by the combined treatment was confirmed by FCM analysis ofAnnexin V-FITC/PI dual staining. Caspase3, the executioner of apoptosis, isgenerally considered a primary hallmark of apoptosis. Compared with CPT treatmentalone, the increased expression of active caspase3was observed in cells exposed toCPT with either3-MA or CQ. These data confirm a critical role for autophagy indetermining the destiny of senescent tumor cells, and suggest that autophagy is apotential novel target to improve therapy efficiency of conventionalchemotherapeutics.Conclusions:1. Low-dose CPT induces senescence response through ATM activation of theChk2-p53-p21pathway in human colorectal cancer cells.2. Low-dose CPT treatment induces autophagy via the AMPK-TSC2-mTORpathway in human colorectal cancer cells.3. Beclin1-mediated autophagy modulates the survival of senescent tumor cellsthrough p53/p21pathway. Once autophagy was inhibited, p21decreased and nolonger sustained senescence, and finally cell death occurred.