Effects And Its Preliminary Mechanism Of SIRT1 Subcellular Location On Cell Apoptosis Of Colorectal Carcinoma In Hypoxic Environment

As one of the most common malignant tumors,colorectal carcinoma has become the third most frequently occurring malignancies in the world.Due to the lack of early diagnosis and efficient screening methods,more than 50%cases of patients have developed to advanced stage at the time of diagnosis,resulting in a dismal prognosis and the third highest fatality rate in the world.Although with the continuous development of surgery technology,chemotherapeutic and targeted drugs,the prognosis of patients with colorectal carcinoma has improved,the median survival time of patients is merely around 18 to 22 months,from the perspective of long-term survival.Therefore,it is important to explore the molecular mechanisms of colorectal carcinoma.Colorectal carcinoma,as a solid tumor,is characterized by the decrease of blood and oxygen supply within tumor due to the abnormality of vascular structure and function.Meanwhile,the increase of oxygen consumption,which is caused by the rapid proliferation of tumor cells,usually leads to the formation of hypoxic tumor microenvironment.This microenvironment plays a crucial role in tumor biology.In order to adapt to hypoxia,tumor cells have to adjust their biological behaviors through various adaptive changes,resulting in malignant transformation.As a kind of deacetylase,silencing information regulator（SIRT1）mainly regulates acetylation of histone and non-histone through nicotinamide adenine dinucleotide（NAD）.Recent studies suggest that SIRT1 not only plays a key role in many biological processes,such as cell proliferation,differentiation,apoptosis,aging,and stress,but also in tumorigenesis and progression.However,there is still controversy as to whether SIRT1 is a tumor suppressor or a tumor promoter.One possible reason for this contradictory result is SIRT1 subcellular localization.It is well known that SIRT1 can shuttle dynamically between the cytoplasm and the nucleus.This shuttle ability is regulated by its two nuclear localization sequences（NLSs）and two nuclear export sequences（NESs）,respectively.Both nuclear SIRT1 and cytoplasmic SIRT1 have the function of deacetylation.SIRT1 subcellular localization may affect tumorigenesis and progression by regulating different signaling pathways.Hence,in the current study,we explore whether SIRT1 subcellular localization has alterations under hypoxic treatment,and whether this alteration affects SIRT1 regulating apoptosis in colorectal carcinoma HCT116 and SW480 cells.The first part of studies:In order to explore the alterations of SIRT1 subcellular localization in colorectal carcinoma cells with hypoxic treatment,we firstly collected tumor tissues from 217 cases of the patients with colorectal carcinoma and performed SIRT1immunohistochemical staining.The results of Kaplan-Meier survival analysis showed that there was statistical significance between the expression levels of cytoplasmic SIRT1 and disease-free survival duration（P=0.007）.The patients with higher expression level of cytoplasmic SIRT1 had longer disease-free survival duration than those with lower expression level.Next,the total,cytoplasmic and nuclear proteins from HCT116 and SW480 cells with hypoxic treatment for 6 h,12 h,and 24 h,were extracted and analyzed by western blot,respectively.The results showed that hypoxia inhibited the total,cytoplasmic,and nuclear protein levels of SIRT1.With the prolonged time of hypoxic treatment,the expression level of nuclear SIRT1 increased gradually,along with gradually decreased cytoplasmic SIRT1.The immunofluorescence staining of SIRT1 also confirmed that the protein levels of nuclear SIRT1 gradually increased with prolonged time of hypoxic exposure.Finally,the concentration gradient of LY294002（a PI3K inhibitor,also a nonspecific SIRT1 nuclear localization inhibitor）was used to treat SW480 cells.The results of western blot analysis revealed that LY294002 did not affect SIRT1 expression level in the tumor cells under normoxic condition.However,with 30μM or 50μM LY294002 and hypoxic treatment,the expression levels of cytoplasmic SIRT1 significantly increased（P（27）0.05）,and nuclear SIRT1 significantly decreased（P（27）0.01）,along with significantly down-regulated protein levels of SIRT1,Akt,and p-Akt in total proteins.The immunofluorescence staining further confirmed that fluorescence intensity of SIRT1 was decreased.The positive fluorescence staining of SIRT1 was mainly in the cytoplasm,after HCT116 cells were treated by 30μM LY294002 and hypoxia 24 h.The results of the first part of studies indicate that hypoxia inhibits the expression level of SIRT1 protein in colorectal carcinoma cells and induces SIRT1 nuclear translocation,thus enriching SIRT1 in the nucleus.LY294002 prevents the cytoplasmic SIRT1 from entering the nucleus,and partially reverse the nuclear enrichment of SIRT1 caused by hypoxia.The second part of studies:In order to explore whether the alteration of SIRT1subcellular localization in hypoxic environment affects apoptosis in colorectal carcinoma,both HCT116 and SW480 cells were transfected using the lentiviral vectors encoding over-expressing wild-type SIRT1 or SIRT1 with mutant NLSs(SIRT1^NLSmt),respectively.The HCT116 and SW480 cells with over-expressing wild-type SIRT1 cells（SIRT1 cells）or over-expressing SIRT1^NLSmt(SIRT1^NLSmt cells)were obtained after puromycin sorted.The results of western blot analysis showed that there was wild-type SIRT1 in the nucleus and cytoplasm.The mutations in the NLSs sequence led to SIRT1^NLSmtenrichment in the cytoplasm.This results support that the NLSs sequence is a key factor related to SIRT1subcellular localization.Next,the results of Flow cytometry apoptosis detection and CCK-8 cytotoxicity experiments showed that SIRT1 cells had lower tolerance to hypoxia and higher increased apoptosis than those of SIRT1^NLSmt cells.Given that p53（K382）is a target of SIRT1 deacetylation,we firstly analyzed p53,Ac-p53（K382）in total proteins from HCT116 and SW480 stably transfected cells with hypoxic treatment for 24 h by western blot analysis,respectively.The expression levels of p53 in SIRT1 cells were significantly higher than those of SIRT1^NLSmt cells.The expression levels of Ac-p53（K382）were significantly lower than those of SIRT1^NLSmt cells.Then,we analyzed p53 and Ac-p53（K382）in cytoplasmic or nuclear protein.The results showed that the p53 protein was located in both cytoplasm and nucleus（mainly the nucleus）under normoxic condition.After hypoxic treatment,p53 protein was merely located in the nucleus and its expression level was significantly increased.However,Ac-p53（K382）showed a completely different alteration to p53.Compare with HCT116 or SW480 stably transfected cells under normoxic treatment,after hypoxic treatment,the expression levels of Bax,caspase-3 and cleaved caspase-3 significantly decreased,and Bcl-2 significantly increased.Finally,LY294002 were used and the above experiments were repeated,the results showed that both HCT116 and SW480 cells with LY294002 and hypoxic treatment had decreased p53,Bax,caspase-3 and cleaved caspase-3,and increased Ac-p53（K382）and Bcl-2.The results of the second part of studies suggest that SIRT1 subcellular localization may affect the acetylation status of p53（K382）.The deacetylation modification of p53（K382）by SIRT1 may be mainly located in the nucleus.The nuclear SIRT1 regulates p53function by p53（K382）deacetylation,thereby promotes apoptosis caused by hypoxia and leads cells to be sensitive to hypoxic treatment.The reason of cytoplasmic SIRT1 resistant to hypoxic treatment may relate to lack of p53（K382）deacetylation.In summary,it is the first time to clarify that hypoxia promotes nuclear translocation in colorectal carcinoma cells.SIRT1 subcellular localization may affect hypoxia-induced cell apoptosis through the p53 pathway in colorectal carcinoma.These findings will enrich the roles of SIRT1 in the tumor progression and provide a possible pathway for the clinical treatment of colorectal carcinoma.