Inhibition Of Proliferation And Promotion Of Apoptosis Of GIST-T1 Cells By The Down-regulation Of C-KIT Expression

Gastrointestinal stromal tumor（GIST）is the most common mesenchymal tumor of the gastrointestinal tract,and it is thought to originate from Cajal interstitial cells or their progenitor cell.It can be divided into c-KIT mutation,PDGFRA mutation and wild type-GIST type.Among them,c-KIT mutation is the most common type,accounting for about 80%of the total cases,and the immunohistochemical manifestations is CD117 positive.The preferred treatment for GIST is R0 resection,with the resection margin 2～3 cm from the tumor.For unresectable localized GIST,surgical treatment can be performed after preoperative molecular targeted drug therapy to shrink the tumor.Imatinib（IM）,a small molecule selective tyrosine kinase inhibitor,is a first-line agent for GIST therapy.It can combine with c-KIT and PDGFRA and prevent its downstream oncogene activation by inhibiting tyrosine kinase.IM can be used as an adjuvant to surgery in high-risk cases as well as the main treatment for GIST metastatic disease.GIST with c-KIT exon 11 mutation is sensitive to IM treatment,and exon 9 mutation is rare.Patients with PDGFRA D842V mutation and wt-GIST often develop primary drug resistance within half a year of first-line treatment.However,even with early sensitivity to IM treatment,secondary IM resistance with disease progression is observed in half of patients within 2 years.It may be related to a new mutation in exon 13 or 17 of c-KIT.Sunitinib,a second-line drug,can inhibit VEGFR in vitro in addition to targeting c-KIT and PDGFRA receptors,and has an excellent therapeutic effect on GIST with c-KIT gene exon9 mutation.However,for patients with c-KIT exon 11 and PDGFRA mutant,after the failure of first-line treatment,increasing the dose of IM or switching to sunitinib can not achieve satisfactory results.The multi-target kinase inhibitor regofinib can inhibit PDGFRA,KIT,VEGFR,Tie-2,RET,FGFR,etc.It is a third-line treatment for GIST after the failure of IM and sunitinib,and can be used for the third-line treatment of c-KIT exon 17 mutation.In addition,after first-and second-line resistance,alternative drugs such as nilotinib and sorafenib can be used,but routine use is not recommended.However,although above targeted drugs have shown initial effects in the treatment of GIST,the occurrence of drug resistance is inevitable.And the high drug price also brings heavy economic burden to patients.Therefore,this study is expected to find a cheap and efficient drug that may replace expensive imported molecular targeted drugs for clinical treatment.Toosendanin（TSN）,a tetracyclic triterpenoid,was used in the treatment of parasitic diseases in the early stage.It has been shown to inhibit the growth of a variety of human tumor cells,such as leukemia,glioblastoma,liver cancer,pancreatic cancer,Ewing’s sarcoma,osteosarcoma,colorectal cancer and so on.The mechanism of broad-spectrum anticancer ability of TSN is widely involved.However,searching domestic and foreign literatures,it is not clear whether TSN has an effect on survival of GIST cells.Therefore,this study selected GIST-T1 cell line as the research object,using CCK-8,Hoechst 33342/PI staining,flow cytometry,qRT-PCR and Western Blot and other experimental techniques,in order to explore the effects of TSN on the growth and apoptosis of GIST cells,and preliminary exploration of its mechanism.PART I:Effects of TSN on proliferation and apoptosis of GIST-T1 cells.Objective:To investigate the effects of TSN on proliferation and apoptosis of GIST-T1cells.Methods:The experimental groups were divided into 0,15,17,20,22,25 and 30 nmol/L TSN,and cultured at 37℃and 5%CO₂ for 48h.The optical density values at 450nm in each group were measured by CCK-8,and the inhibition rate of cell proliferation was calculated.The half-inhibition concentration IC₅₀ was calculated by SPSS 20.0.The same method was used to detect the proliferation inhibition rate of 72h,and the concentration was set as 0,5,7,10,14,16,18,and 20 nmol/L.Subsequent experiments were divided into 0,15 and 30nmol/L TSN groups according to 48h CCK-8 results and 50nmol/L Imatinib（IM）as positive control.The effects of 48h TSN on apoptosis and cell cycle of GIST-T1 cells were detected by flow cytometry and Hoechst 33342/PI fluorescence double staining.Results:TSN could inhibite the proliferation of GIST-T1 cells at 48h or 72h,compared with the control group.When the dose of TSN was increased in a certain concentration range,the proliferation inhibition of GIST-T1 cells was also enhanced.The 48h IC₅₀ was 18.75nmol/L by SPSS and the IC₅₀ at 72h was 17.93 nmol/L.Flow cytometry results showed that compared with the control group,the drug group increased the level of apoptosis,but there was no statistically significant difference between the 15,30 nmol/L TSN and 50 nmol/L IM groups.Hoechst 33342/PI double staining confirmed this result.Microscopic results of TSN and IM groups showed early apoptotic cells with dense chromatin and strong blue fluorescence.Cell cycle results showed that compared with the control group,the number of cells in S phase increased in the 15 nmol/L TSN group,but the difference was not statistically significant.30nmol/L TSN could induce S-phase arrest of cell cycle.Conclusion:TSN could inhibit the proliferation and promote the apoptosis of GIST-T1cells.PART 2:The effect of TSN on c-kit expression in GIST-T1 cellsObjective:To investigate the effect of TSN on c-KIT expression in GIST-T1 cellsMethods:GIST-T1 cells were treated with 0,15,30 nmol/L TSN and 50 nmol/L IM for48h in vitro,then the expression of c-KIT mRNA and its related proteins were detected by qRT-PCR and Western Blot.Results:The qRT-PCR results showed that TSN could down-regulate c-KIT mRNA expression,but the inhibition effect of 15nmol/L TSN was not as good as that of 30 nmol/L TSN and IM group,and the difference between 30 nmol/L TSN and IM group was not statistically significant.Western Blot results showed that TSN significantly inhibited c-KIT protein expression,and the inhibition effect of 30 nmol/L TSN was stronger than that of 15nmol/L TSN.Compared with the control group,TSN significantly inhibited p-c-KIT protein expression,and the inhibition effect of 30 nmol/L TSN was better than that of the IM group.Conclusion:TSN may inhibit proliferation and promotes apoptosis of GIST-T1 cells by inhibiting c-KIT expression.PART 3:The effect of TSN on PI3K/AKT/mTOR expression in GIST-T1 cellsObjective:To explore the effect of TSN on PI3K/AKT/mTOR signaling pathwayMethods:The groups were the same as the second part.The mRNA expressions of PI3K,AKT and mTOR were detected by qRT-PCR,and the protein expressions of PI3K,AKT,mTOR and p-PI3K,p-AKT and p-mTOR were detected by Western Blot.Results:Compared with the control group,the mRNA levels of PI3K,AKT and mTOR were significantly inhibited,and the inhibition effect of 15 nmol/L TSN was better than that of the IM group.Western Blot results showed that compared with the control group,the expression of PI3K,AKT,mTOR and its phosphorylated form proteins were significantly inhibited.Among them,15 nmol/L TSN could significantly inhibit the expression of PI3K,mTOR,p-PI3K,p-mTOR and p-AKT,and the inhibition of PI3K,mTOR,p-PI3K and p-AKT was significantly better than that of IM group.30 nmol/L TSN significantly inhibited the expression of AKT.Conclusion:TSN may inhibit the activation of PI3K/AKT/mTOR signaling pathway in GIST-T1 cells.In summary,TSN could inhibit proliferation and promote apoptosis of GIST-T1 cells by down-regulating c-KIT expression,which may play a role through PI3K/AKT/mTOR pathway.