Molecular Mechanisms On The Sirtuin 1/2 Inhibition-induced Autophagy In Human Lung Cancer Cells

Research PurposeAccording to the World Cancer Report 2014 from World Health Organization (WHO), the cancer patients and death cases are disturbingly increased, among which nearly half of the cancer cases occurred in Asia, mostly in China. Cancer has become one of the major fatal human diseases. Chemotherapy is the principal means of cancer treatment, but the side effects and its complications bring great threat to tumor patients. Therefore, developing highly specific, lower side effects and more effective antitumor drugs to provide new anti-tumor targets and new tumor treatment strategies is particularly important.Sirtuins have been reported as longevity proteins and have emerged as a promising novel class of anti-cancer drug targets. The mammalian sirtuins (SIRT1-7) are class III deacetylase enzymes which need nicotinamide adenine dinucleotide-positive (NAD+) as their essential cofactor. Post-translational and epigenetic modulations have important roles in regulate protein activities. Furthermore, metabolic disorders may cause lots of disease, for example cancer. According to the present studies, sirtuins’ roles in modulating tumorigenesis were controversial. On the one hand, they may act as tumor suppressor genes. On the other hand, recently data showed that sirtuins had functions in promoting tumorigenesis. Therefore, lots of agents targeting sirtuins were synthesized. In our study, we used salermide, which is reverse amide of sirtinol and an inhibitor of SIRT1 and SIRT2, to explore the relevance between SIRT1/2 and tumorigenesis or tumor therapy.Our study focuses on the relationship between sirtuins and autophagy. Autophagy which primarily plays degradative role in eukaryote is important in maintaining organellar homeostasis and may be influenced by sirtuins. The mechanistic target of rapamycin (MTOR) is a conservative negative regulator of autophagy in organisms from yeast to human. However, some factors such as ATF4 regulate MTOR signaling pathway. We hope to explore the relationship between SIRT1/2 and the mechanism of autophagy. What’s more, we want to indicate, under the given circumstance, the relation between autophagy and apoptosis. On the base of the experimental data, we hope find a novel anti-tumor target and new anti-cancer thrapy.Research Method1. Detecting the EGFP-MAP1LC3 fluorescent puncta corresponding to protein aggregates in cells by point scanning laser confocal microscope to explore the relationship between SIRT1/2 and autophagy on image level.2. Using Western Blot to explore the relationship between SIRT1/2 and autophagy on protein level.3. Using autophagy inhibitors and ATG5, ATG7 siRNAs transfection to detect SIRT1/2 inhibition-induced autophagic flux.4. Using ATF4, DDIT4 siRNAs transfection and plasmid transfection to explore the relationship between ER Stress, MTOR signaling pathway and autophagy.5. Using co-immunoprecipitation to explore the relationship bwteen SIRT1, SIRT2 and ER Stress.6. Using co-immunoprecipitation to explore the combined domains of SIRT1/2 and HSPA5.7. Using Western Blot and flow cytometry to explore the relationship between SIRT1/2 inhibition-induced autophagy and apoptosis.Result1. salermide increased the fluorescent puncta corresponding to protein aggregates in human lung cancer cell line Calu-1-EGFP-MAP1LC3 and upregulated the expression of MAP1LC3B-Ⅱ in A549, Calu-1 and H1792.2. SIRT1/2 inhibition increased the fluorescent puncta corresponding to protein aggregates in human lung cancer cell line Calu-1-EGFP-MAP1LC3 and upregulated the expression of MAP1LC3B-Ⅱ in A549, Calu-1 and H1792.3. SIRT1/2 inhibition upregulated the expression of ATF4 and DDIT4, then inhibited MTOR signaling pathway and finally induced autophagy.4. HSPA5 was acetylated by SIRT1/2 inhibition, dissociated from endoplasmic reticulum receptor protein EIF2AK3 and finally triggered ER Stress.5. After inhibiting SIRT1/2 inhibition-mediated autophagy in cells, the cleavage of apoptosis marker proteins and the percentage of apoptosis cells were increased.Conclusion1. salermide induced autophagy in human Non-Small Cell Lung Cancer cells.2. SIRT1/2 inhibition induced autophagy in human Non-Small Cell Lung Cancer cells.3. SIRT1/2 inhibition induced autophagy through ATF4--DDIT4-MTOR signaling pathway.4. SIRT1/2 inhibition induced ER Stress via regulated the acetylation of HSPA5.5. The autophagy induced by SIRT1/2 inhibition protected cells from apoptosis.All in all, we used human lung cancer cell lines as models to demonstrate that when SIRT1/2 were inhibited in cells, the acetylation of molecular chaperone HSPA5 would increase and dissociated from endoplasmic reticulum receptor protein EIF2AK3, and finally triggered ER Stress. The activation of ER Stress upregulated the expression of ATF and DDIT4, then inhibited MTOR signaling pathway and induced autophagy in cells. At last, we found that SIRT1/2 inhibition induced autophagy protect cells from apoptosis.