The Role And Mechanisms Of SIRT1in The Malignant Behavior Of Gastric Cancer

Gastric cancer is the fourth most common cancer and the second leading causeof cancer-related deaths worldwide. Although the prevalence and death raterelated to gastric cancer have decreased worldwide, there are still933,293newcases and700,000deaths from gastric cancer every year, and the5-year survivalrate for advanced-stage gastric cancer is approximately20%－30%. Therefore, itis necessary to identify novel molecules involved in the development andprogression of gastric cancer.SIRT1(Silent mating type information regulation2homolog1) is anicotinamide adenine dinucleotide dependent histone deacetylase that belongs toclass III histone deacetylase. SIRT1deacetylates histones and many non-histoneproteins which are involved in a variety of biological processes, including cellgrowth, apoptosis, neuronal protection, adaptation to calorie restriction, organmetabolism and function, cell senescence, and tumorigenesis.Over the past decade, the function of SIRT1in tumorigenesis has been studied. However, it remains controversial whether SIRT1acts as a tumor promoter ortumor suppressor due to recent controversy over SIRT1regarding:1) itsexpression level in human cancers;2) its activity on tumor suppressors andoncoproteins;3) its effect on growth arrest, cell death, and DNA damage repair;and, finally,4) its long-term impact on lifespan and cancer risk. At present, therole and mechanism of SIRT1in the malignant behavior of gastric cancer stillunknown, so it is necessary to explore.【Aims】1．To detect the expression of SIRT1in a large number of gastric cancer patientsand the corresponding non-tumor mucosa tissues, and the possible clinicalsignificance between SIRT1expression and clinical features was also explored.2．To study the effect of SIRT1on the malignant behavior of gastric cancer.3．To explore the possible underlying mechanisms for the effect of SIRT1on themalignant behavior of gastric cancer.【Methods】1．A total of112human gastric adenocarcinoma tissue and the correspondingnon-tumor tissue were acquired from the Xijing Hospital of Digestive Diseases.Immunohistochemical staining was performed to detect the expression of SIRT1in112cases of primary gastric adenocarcinoma, corresponding adjacentnon-tumor tissues and38cases of metastatic lymph nodes. The possible clinicalsignificance between SIRT1expression and clinical features was analyzed.2．An additional24tumor tissue and the corresponding non-tumor mucosa tissuesamples were collected from patients with gastric adenocarcinoma and wereimmediately stored in liquid nitrogen after resection for real-time PCR analysis.Total RNA was isolated and cDNA was synthesized. Real-time PCR wasperformed with a SYBR Green Master Mix to examine the mRNA level of SIRT1.3．Immunohistochemical staining was performed to detect the expression of p53and DBC1, and the correlation between SIRT1expression and p53, DBC1expression were analyzed.4． Lentivirus expressing SIRT1-targeting shRNA and SIRT1were produced inHEK-293T cells by using the corresponding plasmid and the packaging plasmids.GES and SGC7901cells were infected with LV-shRNA-Control,LV-SIRT1-shRNA, LV-Control or LV-SIRT1, and stable transfectants werecollected. Western blotting were used to confirm the validation of lentivirus.5．10μM EX-527(SIRT1inhibitor) and200nM resveratrol (SIRT1activator)were administered to change the activity of SIRT1.6．MTT assay and colony formation assay were performed to examine the effectof SIRT1on the cell growth and cell proliferation in vitro.7．Tumorigenicity in nude mice and in vivo bioluminescence imaging wereapplied to examine the effect of SIRT1on the cell growth and cell proliferation invivo.8．Stable infected GES, stable infected SGC7901cells, EX-527or resveratroltreated GES and SGC7901cells were used for cell cycle analysis and cellapoptosis analysis. The percentages of cells in different cell cycle phases and thepercentages of apoptotic cells were identified with a FACS scanner.9．To confirm the results of cell cycle and cell apoptosis analysis, the expressionof cell cycle and cell apoptosis related proteins were examined by westernblotting.【Results】1．SIRT1expression was significantly reduced in gastric adenocarcinomaIHC was performed to detect the expression of SIRT1in112cases of primary gastric adenocarcinoma, corresponding adjacent non-tumor tissues and38casesof metastatic lymph nodes. To increase the reliability of our results, we chose twodifferent SIRT1antibodies (Santa Cruz and Upstate) for IHC, and we found theyhave similar staining patterns. The high expression (+++), moderate expression(++), low expression (+) and negative expression (－) of SIRT1were found in41,38,26and7cases of the adjacent non-tumor tissues, respectively. However, thehigh expression (+++), moderate expression (++), low expression (+) andnegative expression (－) of SIRT1were detected in3,4,24and81cases ofgastric adenocarcinoma, respectively. SIRT1was detected in93.7%of theadjacent non-tumor tissues which mainly localized in cytoplasm with a moderateto intense expression level. However, positive staining localized in the nucleiand/or cytoplasm was identified in27.7%of the gastric adenocarcinoma tissuesand10.5%of the metastatic lymph nodes. Statistical analysis revealed SIRT1expression was significantly reduced in gastric adenocarcinoma. Moreover, thechange of SIRT1staining score was associated with histologic type and TNMstage, but no significant correlation was found in sex, age, or metastasis.In addition，SIRT1mRNA expression was investigated in24cases of gastricadenocarcinoma and corresponding non-tumor mucosa tissues using quantitativereal-time PCR. Consistent with the IHC staining, the results showed that theSIRT1mRNA expression levels in gastric cancer tissues were significantly lowerthan that in corresponding non-tumor mucosa tissues.2．SIRT1expression was correlated with p53expression but not with DBC1p53is an important non-histone deacetylation target for SIRT1, so we detectedthe expression of p53in our tissue samples. We found p53expression wasdetected in30%of gastric adenocarcinoma. Immunohistochemical studyindicated the staining pattern of SIRT1(+) p53(+), SIRT1(+) p53(－), SIRT1(-) p53(+), and SIRT1(-) p53(-) were found in8,5,4and23cases of gastric cancer,respectively. Statistical analysis revealed a positive correlation between SIRT1expression and p53expression in gastric cancer (p=0.003).DBC1is a negative regulator of SIRT1, so we detected the expression ofDBC1in our tissue samples. We found DBC1expression was detected in77.4%of gastric adenocarcinoma. Immunohistochemical study indicated the stainingpattern of SIRT1(+) DBC1(+), SIRT1(+) DBC1(－), SIRT1(-) DBC1(+), andSIRT1(-) DBC1(-) were found in13,2,28and10cases of gastric cancer,respectively. Statistical analysis revealed no significant correlation betweenSIRT1expression and DBC1expression in gastric cancer (p>0.05).3．SIRT1inhibited cell growth and proliferation in vitroTo further explore the effect of SIRT1on the development and tumorigenesisof gastric cancer, we applied LV-shRNA-Control, LV-SIRT1-shRNA, LV-Controlor LV-SIRT1to infect GES and SGC7901cells. Western blotting confirmed amarked reduction of SIRT1by LV-SIRT1-shRNA infection and a significantover-expression of SIRT1by LV-SIRT1infection. Furthermore, to test the role ofendogenous SIRT1in regulating cell growth,10μM EX-527(SIRT1inhibitor)and200nM resveratrol (SIRT1activator) were administered to change theactivity of SIRT1.Compared with LV-shRNA-Control infected cells, LV-SIRT1-shRNA infectedcells showed a significantly increased rate of cell proliferation, whereas theover-expression of SIRT1by LV-SIRT1strongly inhibited cell proliferation inGES and SGC7901cells. Moreover,10μM EX-527(SIRT1inhibitor) promotedcell growth in both GES and SGC7901cells, but these results did not reachstatistical significance. However, when treated with200nM resveratrol (SIRT1activator), the proliferation rates of GES and SGC7901cells were significantly lower than those of control cells.Anchorage-independent growth is an important characteristic of malignanttumor cells. Therefore, colony and soft agar colony formation assays wereperformed to examine the effects of SIRT1on cell growth and proliferation. Thedown-regulation of SIRT1greatly increased colony formation, butover-expression of SIRT1significantly decreased colony formation. Furthermore,compared with control cells, LV-SIRT1-shRNA infected SGC7901cells producedsignificantly increased cell colonies in soft agar, but there was a substantialreduction of colony formation produced by LV-SIRT1infected SGC7901cells insoft agar.4．SIRT1inhibited tumor formation in nude miceWe further investigated the effect of SIRT1on tumorigenesis in vivo. Stableinfected SGC7901cells were collected to test the ability of tumor formation inxenograft animals. The stable infected cells were inoculated into female nudemice subcutaneously. SIRT1knockdown cells had higher luciferase signals andtumor volume than those infected with LV-shRNA-Control. Conversely,over-expression of SIRT1dramatically inhibited tumor formation in vivo, and theaverage luciferase signals and tumor weights were lower for LV-SIRT1infectedcells than those infected with LV-Control.5．SIRT1arrested cell cycle progression by regulating the expression of cell cyclerelated proteinsSustaining proliferation is an important hallmark of cancer. Thus, flowcytometry was performed to determine the effect of SIRT1on cell cycle. Afterserum deprivation for24h, compared with LV-shRNA-Control infected cells, theratio of G1distribution significantly decreased in LV-SIRT1-shRNA infectedcells(58.5±0.8%vs.41.9±0.6%for GES cells, and46.5±0.7%vs.37.8±0.6 %for SGC7901cells, p<0.05), whereas the ratio of G1distribution significantlyincreased in LV-SIRT1infected cells (57.6±1.0%vs.64.6±1.3%for GEScells, and45.1±1.2%vs.58.9±1.6%for SGC7901cells, p<0.05). Furthermore,cell cycle analysis was performed in GES and SGC7901cells treated with SIRT1inhibitor (EX-527) or activator (resveratrol). Compared with control cells,EX-527caused a significantly decreased percentage of the G1phase (59.8±0.9%vs.47.4±0.8%for GES cells, and55.3±1.2%vs.41.1±1.0%forSGC7901cells). However, resveratrol caused a significant increase in thepercentage of cells in the G1phase (59.8±0.9%vs.69.6±1.1%for GES cells,and55.3±1.2%vs.68.1±1.6%for SGC7901cells). These results indicate thatinhibition of cell proliferation by SIRT1was partially through arresting cell cycleprogression.To confirm the cell cycle arrest caused by SIRT1, we examined the expressionof cell cycle related proteins. Western blotting indicated that knockdown ofSIRT1up-regulated the expression level of Cyclin D1and Cdk4, withoutchanging the expression of Rb. However, up-regulation of SIRT1expression byLV-SIRT1reduced the expression levels of Cyclin D1and Cdk4.6．SIRT1induced cell apoptosis by targeting cell apoptosis related proteinsResistance to cell death is another important hallmark of cancer, and apoptosisserves as a natural barrier to cancer development. Cmpared with theLV-shRNA-Control infected cells, knockdown of SIRT1decreased the apoptoticindex of GES and SGC7901cells (7.6±0.7%vs.3.8±0.6%for GES cells, and9.8±0.8%vs.4.8±0.5%for SGC7901cells), while SIRT1over-expressionsignificantly increased the percentage of apoptotic cells (6.3±0.5%vs.16.5±1.0%for GES cells, and9.6±0.8%vs.21.6±2.1%for SGC7901cells).Moreover, we applied EX-527or resveratrol to modify the activity of SIRT1in GES and SGC7901cells. We observed that inhibition of SIRT1by EX-527suppressed cell apoptosis in GES cell line, while as activation of SIRT1byresveratrol promoted cell apoptosis in GES and SGC7901cells.To explore the underlying mechanism, we analyzed the protein expression ofthe apoptosis associated factors. When compared with LV-shRNA-Controlinfected cells, a significant increase in Bcl-2and a reduction in Bax expressionlevels were observed after LV-SIRT1-shRNA transduction. Conversely, asignificant increase in Bax expression and a reduction in Bcl-2expression wereobserved after LV-SIRT1infection.【Conclusion】Our data suggest that SIRT1is reduced in gastric adenocarcinoma. Our studydemonstrates that repression of SIRT1increases gastric cancer cell proliferationand in vivo tumorigenesis. In contrast, over-expression and activation of SIRT1suppresses gastric cancer cell growth and tumor formation. These findingstherefore identify SIRT1as a potential tumor suppressor in gastric cancer, andprovide a rationale for using SIRT1activators as therapeutic agents.