Role Of LSD1 On The Anti-tumor Effect Of Doxorubicin And PD-L1 In Immunodetection Sites And Its Underlying Mechanism

Histone lysine demethylase(LSD1),an epigenetic enzyme,is highly expressed in various tumors such as gastric cancer and lung cancer.Many studies have been reported that LSD1 specific inhibitors can inhibit cancer cell proliferation,regulate gene transcriptional inhibition or activation and play an important role in DNA damage repair.The purpose of this study was to explore the role of inhibiting the activity of LSD1 on the anti-tumor of doxorubicin effects and it's molecular mechanisms in gastric cancer cells,which is a DNA damaging drug.On the other hand,PD1 is an inhibitory receptor mainly expressed on activated T cells.Binding of its ligand PD-L1 can significantly inhibit the activation and proliferation of T cells,so that tumor cells can escape from the body's immune monitoring and killing.The abnormal expression of PD-L1 and LSD1 is present in non-small cell lung cancer cells(NSCLCS).Antibodies targeting PD-1 or PD-L1 have been exhibited good effects in NSCLCS.Therefore,we also explored whether the transcriptional expression of PD-L1 is affected by the apparent regulation of LSD1 in NSCLCS.The main research contents and results of this topic are as follows:Part I: Inhibition of LSD1 activity enhances the antitumor effects of DNA damaging drug of doxorubicin and its molecular mechanisms.1.MTT assay showed that the proliferation of MGC-803 cells was inhibited by GSK-LSD1 2HCL,C26 and SP2509.This shows that inhibition of LSD1 activity can inhibit the proliferation of MGC-803 cells.2.Flow cytometry analysis of the cell cycle results showed that the irreversible inhibitor of GSK-LSD1 2HCL and SP2509 of LSD1 in MGC-803 and SGC-7901 cells did not affect the cell cycle progression.The irreversible inhibitor of C26,which is designed by our group has a certain degree of cell cycle arrest.Further targeting of siRNA in MGC-803 cells reduced the expression of LSD1,and the results did not affect the cell cycle progression,indicating that inhibition of LSD1 does not affect the progression of gastric cancer cells.3.The results of the combination of LSD1 inhibitor 2-PCPA with DNA damage drugs,such as doxorubicin(DOX),5-Fu and cisplatin showed that combination group could inhibit the cells proliferation more clearly than the single useing group.Especially,the combination of 2-PCPA and low-dose DOX has a better effect.4.The combination of GSK-LSD1 2HCL and doxorubicin(DOX),a specific inhibitor of LSD1,showed that both of the MGC-803 and HGC-27 cells can upregulate the pro-apoptotic protein Cleaved-PARP,Cleaved-Caspase3 and BAX.While down-regulating the expression of the anti-apoptotic protein BCL-2.Further targeting of siRNA in MGC-803 cells reduced the expression of LSD1 and was combined with DOX.The results still showed that the expression of the pro-apoptotic protein Cleaved-PARP increased in the combination group compared with the singleuseing group,suggesting that inhibit the activity of LSD1 may enhances DOX's ability to promote apoptosis by activating mitochondria-mediated endogenous apoptotic pathways.5.The combination of GSK-LSD1 2HCL,a specific inhibitor of LSD1,and doxorubicin(DOX)on the whole animal level showed that tumor volume and tumor weight were significantly reduced in the combined group compared with the single useing group,and the tissue was use to extract protein.Western blot showed that the expression of the pro-apoptotic protein increased in the combination group than in the single useing group.The results indicated that inhibition of LSD1 activity in vivo can also enhance the antitumor activity of DOX.Part II: The molecular mechanism of LSD1 regulating PD-L1 expression in tumor microenvironment and the effect of PD-L1 on the non-immunological function of tumor cells.1.The results of flow cytometry and Western blot showed that PD-L1 was expressed in NSCLCS.The expression of PD-L1 in H1975 cells was significantly higher than in other cells,and the expression of LSD1 was significantly higher in H1975 cells than in other cells.The results suggested that there may be a certain correlation between the expression of PD-L1 and the expression of LSD1 in nonsmall cell lung cancer cells.2.Inhibition the activity of LSD1 significantly reduced PD-L1 expression in H1975 cells in a dose-dependent and time-dependent manner.After silencing LSD1 with siRNA,the expression of PD-L1 protein was significantly reduced.After preupregulation of PD-L1 expression with IFN?,inhibition the activity of LSD1 can still decrease the expression of PD-L1 in a dose-dependent and time-dependent manner.It is suggested that inhibition the activity of LSD1 can significantly down-regulate the expression of PD-L1 and is not affected by the tumor microenvironment.3.Regardless of the presence or absence of IFN?,inhibition of LSD1 activity significantly reduced the mRNA levels of PD-L1 in H1975 cells.The results also show that inhibit of the activity of LSD1 can significantly reduce the expression of IFNGR1 and the mRNA levels of IFNGR1,regardless of the presence or absence of IFN?.Further studies found that inhibit the activity of LSD1 can also inhibit the activation of interferon receptor-mediated signaling pathways and reduce the protein expression of p-JAK2 and p-JAK3.In addition,it was also found that inhibit the activity of LSD1 can promote the expression of P53 and further inhibit the expression of PD-L1.4.Lentivirus-transfected H1975 cells with knocked-down PD-L1 were less likely to undergo EMT than undeleted cells,and cell proliferation,migration,and scratch repair capability were less.Inhibited the activity of LSD1 was in H1975 cells,the EMT process of cells was also less prone to occur,and scratch repair capability of cells was also reduced.In summary,inhibiting the activity of LSD1 can decrease the proliferation of gastric cancer cells and increase the sensitivity of tumor cells to DOX.In addition,LSD1 regulates the expression of PD-L1 and tumor cell-associated non-immune functions in H1975 cells by modulating interferon receptor IFNGR1 and interferon receptor-mediated signaling and P53.