| Objective:Tumor microenvironment plays an indispensable role in the occurrence,progression and treatment resistance of pancreatic cancer.The purpose of this study was to explore the effect and specific mechanism of high lactate on ferroptosis resistance caused by lipid droplets accumulation in pancreatic cancer.Our research would to provide a more complete theoretical basis for the treatment of tumor microenvironment.Methods:(1)The content of lipid droplets in cells was labeled with BODIPY 493/503 lipophilic fluorescent probe to explore the effect of exogenous lactate on lipid droplet accumulation in pancreatic cancer cells.(2)The LDHA inhibitor sodium oxamate was used to inhibit glycolysis and reduce the production of endogenous lactate.BODIPY 493/503 was used to detect the content of intracellular lipid droplets.(3)Western blot was used to detect the expression of key protein of lipid droplets PLIN2 under the treatment of exogenous lactate.(4)After the treatment of SOAT1 inhibitor—avasimibe,which is used to inhibit lipid droplets accumulation and exogenous lactate production in pancreatic cancer cells,the content of lipid droplets in PANC-1 and PATU8988 t cell lines was detected by BODIPY 493/503,and the expression of PLIN2 was detected by western blot.(5)Monocarboxylate transporter 1(MCT1)antagonist AZD3965 was used to inhibit the entrance of exogenous lactate into PANC-1 and PATU8988 t.After the treatment of exogenous lactate,the content of lipid droplets in PANC-1 and PATU8988 t was detected,and the expression of PLIN2 was detected by western blot.(6)CCK8,MDA and BODIPY-C11581/591 staining were employed to explore the effect of exogenous lactate on the ferroptosis resistance of pancreatic cancer cells induced by erastin for24 hours,the protein expression of GPX4,ACSL3,ACSL4 and PLIN2 was detected by western blot,and the content of lipid droplets in PANC-1 and PATU8988 t was detected with BODIPY493/503.(7)After avasimibe treatment,the effect of exogenous lactate on ferroptosis of pancreatic cancer cells induced by erastin for 24 hours was detected by CCK8 and BODIPY-C11581/591,and the expression of GPX4 protein was detected by western blot.(8)Immunofluorescence and Western blot were used to detect the changes of intracellular SOAT1 expression in PANC-1 treated with exogenous lactate.(9)After SOAT1 was inhibited by avasimibe,the expression of PLIN2 in PANC-1 was detected by western blot.(10)AZD3965 was employed to inhibit exogenous lactate entering PANC-1,western blot was used to detect the change of SOAT1 expression.Results:(1)After treatment of exogenous lactate,the content of lipid droplets in PANC-1 and PATU8988 t increased significantly.(2)After stimulation with sodium oxamate,production of endogenous lactate was inhibited in PANC-1 cells,the effect of exogenous lactate on the content of intracellular lipid droplets was not significantly different from that of the group without sodium oxamate treatment.(3)In the exogenous lactate-treated PANC-1 cells,the expression level of PLIN2 increased,which was the key protein of lipid droplets.(4)After avasimibe treatment,the intracellular lipid droplets content of PANC-1 and PATU8988 t decreased,and the intracellular lipid droplets content did not increase significantly after the stimulation by exogenous lactate.Similarly,there was no significant change in PLIN2 expression.(5)AZD3965 inhibited the exogenous lactate passage into PANC-1 and PATU8988 t,it could not promote the accumulation of intracellular lipid droplets and the increase of PLIN2 expression.(6)The results of CCK8,MDA and BODIPY-C11581/591 showed that lactate could save erastin-induced ferroptosis in PANC-1.Western blot showed that the expression of ACSL3 in Bx PC-3 increased,while in PANC-1,the expression of ACSL4 decreased and GPX4 increased after lactate treatment.However,after joining AZD3965,the above results are not valid.(7)After the treatment of avasimibe,the content of lipid droplets in PANC-1 decreased.CCK8 and BODIPY-C11 581/591 showed that lactate could not save erastin-induced ferroptosis,while Western blot results showed the expression of GPX4 decreased.(8)The results of IF and western blot implied that the expression of SOAT1 in PANC-1cells increased after the treatment of exogenous lactate.(9)The results of Western blot demonstrated that the expression of PLIN2 in PANC-1decreased after SOAT1 was inhibited by avasimibe.(10)Western blot proved that exogenous lactate could not increase the expression of SOAT1 in cells when AZD3965 was used to inhibit the entry of exogenous lactate into PANC-1.Conclusions:(1)L-lactate promotes lipid droplets accumulation in pancreatic cancer.(2)Accumulation of lipid droplets in pancreatic cancer induced by L-lactate can lead to ferroptosis resistance.(3)L-lactate promotes the increase of lipid droplets in pancreatic cancer cells by stimulating the expression of SOAT1.(4)The effect of L-lactate on lipid droplets in pancreatic cancer cells depends on MCT1. |
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