Glycitin Enhances The Therapeutic Effect Of Gemcitabine On Pancreatic Cancer Cells By Inhibiting Autophagy

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Study of glycitin on proliferation and apoptosis of human pancreatic cancer cells Objective To study the effects of glycitin on proliferation and apoptosis of human pancreatic cancer cells.Methods Cell proliferation activity was detected by CCK-8;cell clone formation ability was detected by Colony formation assay;Apoptosis was detected by Annexin V-FITC/PI double staining;Western blot was used to detect apoptosis-related protein(Cleaved PARP,Cleaved caspase3)expression level;The proapoptotic targets of Glycitin were detected by caspase inhibitors(Z-VAD-FMK,Z-IETD-FMK and Z-LEHD-FMK).Results Glycitin moderately inhibited the growth of human pancreatic cancer cell line(Panc-1,Mia PaCa-2),and the inhibitory effect increased with the increase of drug concentration and time;Glycitin inhibited cell clone formation;Glycitin could induce apoptosis of pancreatic cancer cells;and Western Blot results showed that Cleaved PARP and Cleaved caspase3 expression levels were up-regulated,suggesting that glycitin can induce the expression of apoptotic proteins;Meanwhile,glycitin-induced apoptosis is dependent on the activation of caspase9-related endogenous apoptotic pathway.Conclusion Glycitin inhibits proliferation of human pancreatic cancer cells in vitro and induces apoptosis,and this apoptosis is dependent on activation of the endogenous apoptotic pathway.Part ? Effect of Glycitin on autophagy of human pancreatic cancer cellsObjective To study the effect of glycitin on autophagy of human pancreatic cancer cells.Methods The expression level of autophagy-associated protein(P62/SQSTM1,LC3-II)was detected by Western Blot after glycitin alone or combined with autophagy blocker chloroquine(CQ).After adenovirus-carrying GFP-mRFP-LC3 was transfected into human pancreatic cancer cell Panc-1,it was treated with glycitin,and red and green spots in the cells and yellow spots after confocal observation were observed by laser confocal microscopy.Results Western Blot results showed that glycitin could induce the accumulation of autophagy-related proteins LC3-II and P62/SQSTM1 in two human pancreatic cancer cells,and the protein level gradually increased with the increase of drug concentration and time;but after combined with chloroquine glycitin failed to further increase the expression level of LC3-II in both cells.The results of laser confocal experiment showed that red spots were found in the cells after the positive control group rapamycin,and yellow spots appeared in the cells after treatment with CQ or glycitin.Conclusion Glycitin inhibits autophagy in human pancreatic cancer cells,and this inhibition is achieved by inhibiting the late maturation process of autophagy.Part ? Study of gemcitabine on autophagy in human pancreatic cancer cellsObjective To study the effect of gemcitabine on autophagy of human pancreatic cancer cells and its function.Methods After gemcitabine alone or in combination with chloroquine treated human pancreatic cancer cells(Panc-1,Mia Pa Ca-2),Western Blot was used to detect changes in autophagy-related protein LC3 and P62/SQSTM1 protein levels;Apoptosis of human pancreatic cancer cells treated with gemcitabine + chloroquine was detected by Annexe V-FITC/PI double staining;Western Blot was used to detect the expression of apoptosis-associated protein(Cleaved PARP,Cleaved caspase 3).si RNA was used to silence the autophagy key gene Atg5 in human pancreatic cancer cells(Panc-1,Mia Pa Ca-2),and then combined with gemcitabine to detect the changes of autophagy and apoptosis.Results Under the action of gemcitabine,the expression of autophagy-associated protein P62/SQSTM1 was decreased,while the expression of LC3-II was increased with the increase of drug concentration and time.After combined with chloroquine,the expression level of P62/ SQSTM1 increased,and the expression level of LC3-II increased more significantly.Compared with gemcitabine alone,the apoptosis increased with chloroquine(p<0.05).At the same time,the gene was silenced with Atg5 and then treated with gemcitabine,the level of LC3-II decreased,and the level of P62/SQSTM1 increased.At the same time,it significantly increased the apoptosis induced by gemcitabine and increased the expression level of Cleaved PARP and Cleaved caspase3 protein.Conclusion Gemcitabine induces autophagy in human pancreatic cancer cells,and this autophagy reduces the pro-apoptotic effect of gemcitabine on pancreatic cancer cells.Part ? Study on the effect of Glycitin combined with gemcitabine on human pancreatic cancer cellsObjective To study the effects of glycitin combined with gemcitabine on human pancreatic cancer cells.Methods Two human pancreatic cancer cells were treated with different concentrations of gemcitabine alone or in combination with fixed concentration of Glycitin.CCK-8 was used to detect cell viability;Cell clone formation ability was detected by Colony formation assay;Apoptosis was detected by Annexin V-FITC/PI double staining;Western Blot was used to detect autophagy-associated protein(P62/SQSTM1,LC3-II)and the expression level of apoptosis-related protein(Cleaved PARP,Cleaved caspase 3).After adenovirus-carrying GFP-mRFP-LC3 was transfected into human pancreatic cancer cell Panc-1,it was treated with DMSO,Glycitin,gemcitabine and gemcitabine + Glycitin,respectively.The red and green spots in the cells and yellow spots after confocal observation were observed by laser confocal microscopy.Results Compared with gemcitabine alone,glycitin combined with gemcitabine inhibited the proliferation of pancreatic cancer cells(p<0.05);and significantly reduced the colony forming ability of cells;Meanwhile,glycitin enhanced gemcitabine-induced LC3-II accumulation and reduced P62/SQSTM1 clearance in a manner similar to chloroquine;The results of laser confocal experiments further confirmed that glycitin may inhibit gemcitabine-induced autophagy and yellow spots appear in cells.Compared with gemcitabine alone,gemcitabine combined with glycitin enhanced the pro-apoptotic effect on pancreatic cancer cells(p<0.05),and the expression levels of apoptosis-related proteins Cleaved PARP and Cleaved caspase3 were also significantly increased.Conclusion Glycitin enhances the proliferation inhibition and pro-apoptotic effects of gemcitabine on pancreatic cancer cells by inhibiting gemcitabine-induced protective autophagy.