Effects And Potential Mechanism Of Paclitaxel Induced Senescence-associated Secretory Phenotype (SASP) On Proliferation Of Gastric Cancer Cells

Background:In china,gastric cancer is one of the most common gastrointestinal carcinoma,and more than 90%of patients are diagnosed with advanced gastric cancer.For such patients,surgery is often difficult to obtain a radical effect.In order to improve the rate of radical surgery,improve the resectability and prolong the survival time of patients,the clinical development of neoadjuvant chemotherapy for gastric cancer is of great significance.However,patients are individual to chemotherapy drugs.The tumor will form a retaliatory growth in some patients with drug resistance.What causes the tumor to show retaliatory growth?Numerous studies have shown that,apart from apoptosis and autophagy,senescence is an important form of response to chemotherapy-mediated DNA damage.After neoadjuvant chemotherapy,we found that a large part of gastric cancer cells became senescence in human body specimens.Therefore,in order to improve the clinical efficacy of chemotherapy,in-depth study of the important clinical phenomenon of chemotherapy-induced DNA damage-induced senescence is of great significance.Chemotherapy induced senescence produce two important effects on chemotherapy itself.Firstly,in an irreversible G0 period,senescent cells are extremely low reactivity to the radiotherapy and chemotherapy.Therefore,they are difficult to be completely eliminated and become an important source of resistance and relapse.Secondly,senescent cells also maintains an active physiological profile,one of the most important feature is the continuous secretion of a large number of bio-bioactive substances,such as IL-6,IL-8,IL-1α/β,MMPs and related growth factors,also known as senescence-associated secretory phenotype(SASP).Our department used paclitaxel-based combination regimen of neoadjuvant chemotherapy in patients with gastric cancer,we found that IL-6 expression was significantly higher in ineffective group of gastric cancer tissue than the effective group of gastric cancer tissue,indicating that chemotherapy-mediated gastric cancer tissue SASP-related factors IL-6 is negative to chemotherapy sensitivity.We know that SASP plays an important role in the drug resistance of neoadjuvant chemotherapy in gastric cancer.However,we do not know whether there is a certain relationship between SASP and tumor retaliatory growth.If there is a relationship between SASP and tumor retaliatory growth,then what mechanism make it happen?In recent years,studies have shown that steroid regulatory element binding protein(SREBPs)overexpression in a variety of tumors,suggesting that the rapid proliferation of tumor cells are closely related to rapid lipid synthesis.Inhibition of SREBPs expression can significantly inhibit tumor cell growth.SREBPs are transcription factor family members include SREBP1 and SREBP2.The former plays a major regulatory role in the synthesis of non-saturated fatty acids and triglycerides,while the latter plays a key role in regulating the homeostasis of cholesterol and is a major transcription factor that regulates lipid homeostasis.SREBPs regulate the expression of a variety of lipid synthesis related genes,as in human glioblastoma SREBP1 upregulate and promote of its downstream target genes ACC and FASN expression.In addition,in February last year,Ayano Kondo et al published a paper in Cell Reports pointed out that extracellular H~+enhances the progression of pancreatic cancer by up-regulating the expression of cholesterol-related enzyme genes such as IDI1,MSMO1 and INSIG1 by activating SREBP2.Therefore,we speculated that SASP may promote the proliferation of gastric cancer cells by activating SREBP2,thereby promoting tumor progression.Content:Based on the previous research of the department,we propose the following scientific assumptions:Neoadjuvant chemotherapy drug paclitaxel can induce gastric cancer cell senescence,and senescent cells promote tumor cell proliferation by secreting SASP to activate SREBP2.The specific study is divided into two parts:Firstly,the phenomenon that SASP promote gastric cancer cell proliferation.Secondly,the mechanism that SASP activate SREBP2 to promote cell proliferation.Methods:First,phenomenon verification.Methods in vitro experimental:The BGC823cells were divided into 3 groups:senescence-associated secretory phenotype conditioned medium group(group SASP-CM);normal tumor cell conditioned medium group(group CTR-CM);normal medium group(group NOR-CM).BGC823 cells were treated with PTX to induce senescence.After establishment of senescence which was approved by SA-β-gal staining,SASP-CM was collected.Concentrations of major SASP factors in SASP-CM were detected by ELISA.CCK8 assay and cell clone formation assay were used to detect the effect of SASP-CM on the proliferation of BGC823 cells.Cell cycle and apoptosis were detected by flow cytometry(FACS).In vivo experimental methods Nude mice subcutaneous tumor experiment points 2 groups.Experimental group:5 5-week-old nude male mice,each mouse was inoculated subcutaneously with 200μL mixed gastric cancer cells and normal tumor cells(5?10~7/mL).Control group:5 5-week-old nude male mice,each nude mouse was inoculated 200μL normal tumor cell suspension(5?10~7/mL),and tumor size was observed.Results:After treating BGC823 cell with 35nM PTX for 72h,a steady senescence model was established,which has the highest percentage of senescence.Concentrations of major SASP factors(ng/l)in SASP-CM were much higher than in CTR-CM,such as IL-6,IL-8,CXCL1,CCL2,INF-γ,IL-1β.The relative clone formation rate in SASP-CM group was higher than that in group CTR-CM,p<0.001,and group NOR-CM,p<0.05.The percentage of phase S cells in group SASP-CM was higher than that in group CTR-CM and NOR-CM,p<0.01,and the apoptosis rate was not significantly different.Conclusion SASP-CM promotes the proliferation of BGC823 cells.Experiment group tumor weight,volume was significantly higher than the control group in Nude mice subcutaneously tumor formation experience,p<0.01.Second,the mechanism of research:1.We cultured BC823 cells with SASP-CM and CTR-CM for 72h,then extracted total RNA for gene expression profiling and GO analysis based on differentially expressed genes.2.SREBP2 is a key transcription factor in the cholesterol synthesis pathway.Therefore,we verified the expression of SREBP2 and its target genes by qRT-PCR.SREBP2 activation was confirmed by immunofluorescence and WB.3.We silenced SREBP2 expression with siRNA and validated the interference effect using qRT-PCR,then SREBP2-silenced and non-silenced BC823 cells were cultured with SASP-CM and cck8 was used to detect cell proliferation activity.Results:1.By gene expression profiling,we found that the conditioned media of senescent cells can promote the expression of related enzymes in the pathway of cholesterol synthesis in BGC-823 cells;2.The expression of key transcription factors SREBP2 and its target genes IDI1,MSMO1 and INSIG1 were significantly up-regulated in cholesterol synthesis pathway.Activation and nuclear transfer of SREBP2 were induced by senescent cell conditioned media.3.Silence of SREBP2 by siRNA can reduce the ability of senescent cells conditioned media to promote the proliferation of GC-823 cells.Conclusion:Paclitaxel,a neoadjuvant chemotherapeutic drug,can induce gastric cancer cell senescence.Senescent tumor cells activate SREBP2 through the senescence related secretory phenotype and promote the proliferation of gasric cancer cells.