The Role And Mechanism Of Programmed Necrosis Induced By LCL161 In Reversing Drug Resistance Of Breast Cancer Cells

Objective: 1.To investigate the effect of SMAC analogue LCL161 on drug-resistant breast cancer cells under Caspase inhibition conditions;2.To explore the effects of SMAC analog LCL161 and the expression of drug-resistant cell death and its key protein expression induced by Caspase inhibition.Methods: 1.CCK-8 method was used to detect the effect of SMAC analog LCL161 on the survival rate of human breast cancer drug-resistant MCF-7/DDP under Caspase inhibition conditions.2.Colony formation assay to detect the effect of LCL161 on cell colony formation under Caspase inhibition conditions.3.Annexin V-FITC/PI double staining flow cytometry was used to detect the effect of LCL161 on mortality after acting on MCF-7/DDP cells for 24 h under Caspase inhibition.4.ATP detection kit detects changes in intracellular ATP levels in MCF-7/DDP cells treated with different combination drugs.Western blotting(West blotting)was used to detect the expression changes of necroptosis-related proteins(RIP1,RIP3,MLKL,p-MLKL),and to explore the potential mechanism of compounds to induce necrotic apoptosis.6.Immunofluorescence assay was used to detect the localization and expression of necroptosis-related proteins when MCF-7/DDP cells were treated with Caspase inhibitor.7.Electron microscopy test to observe the changes of submicroscopic structure of cells after drug treatment.8.After silencing the expression of RIP3 in MCF-7/DDP cells by si RNA,CCK-8 and flow cytometry were used to detect LCL161 and inhibit cell proliferation under Caspase inhibition conditions.Results: 1.LCL161 reduces the survival rate of cisplatin-resistant MCF-7/DDP in breast cancer The results showed that the cell death rate of MCF-7/DDP was positively correlated with the action time and concentration of LCL161,that is,LCL161 significantly enhanced the proliferation inhibition of MCF-7/DDP.At the same time,the number of cells gradually decreases,and the cell morphology gradually becomes rounded.The colony formation test showed that when different drug concentrations of LCL161 were applied to the cells,colony formation was significantly reduced as the drug concentration increased.2.LCL161 induces more severe cell death in breast cancer-resistant MCF-7/DDP cells under Caspase inhibition conditions The results showed that: by CCK-8 results analysis,when different concentrations of LCL161 were added to Caspase inhibitors,cell death rate increased;colony cloning experiments showed that LCL161 increased proliferation inhibition when Caspase inhibitor z-VAD-fmk was added.The number of colonies formed was significantly reduced.3.z-VAD-fmk enhances apoptosis of MCF-7/DDP cells induced by LCL161 The results showed that the caspase inhibitor z-VAD-fmk could not activate the apoptosis of MCF-7/DDP cells;LCL161 could induce apoptosis;when the Caspase inhibitor z-VAD-fmk was added,z-VAD-fmk Increased apoptosis induced by LCL161.4.LCL161 and z-VAD-fmk combine to reduce intracellular ATP levels The results showed that the combination of LCL161 and z-VAD-fmk significantly reduced ATP levels in MCF-7/DDP cells compared to LCL161 alone.5.RIP1 is a key protein that LCL161 induces necrotic apoptosis under the Caspase inhibition strip.The results showed that LCL161 induced the expression of RIP1 protein in MCF-7/DDP cells when Caspase inhibitor was used in combination.6.LCL161 and z-VAD-fmk combined to induce necrotic apoptosis of RIP3 under Caspase inhibition conditions The results showed that LCL161 induced the expression of necrotic apoptotic proteins RIP3,MLKL and p-MLKL in MCF-7/DDP cells when Caspase inhibitor was used.7.Immunofluorescence detected that LCL161 and z-VAD-fmk combined to induce necrotic apoptosis protein production in MCF-7/DDP cells Immunofluorescence results showed that the expression of cytoplasmic necrotic apoptotic proteins RIP1 and RIP3 in MCF-7/DDP was significantly increased when LCL161 and z-VAD-fmk were added.8.Electron microscopy showed necrotic apoptosis in cells after treatment with LCL161 and z-VAD-fmk Electron microscopy showed that after treatment with LCL161 and z-VAD-fmk,MCF-7/DDP cells showed cell death with necrotic apoptosis;MCF-7/DDP cells treated with cisplatin showed apoptosis.9.Using si RNA to interfere with RIP3 expression,protects LCL161 and z-VAD-fmk from inducing necrotic apoptosis in MCF-7 / DDP cells The results showed that the inhibition of proliferation of MCF-7/DDP cells by LCL161 and z-VAD-fmk was attenuated by silencing RIP3,and the necroptosis inhibitor Nec-1 was on MCF-7/DDP cells.The protection disappeared.Conclusions: 1.SMAC analog LCL161 can inhibit the proliferation of breast cancer resistant cells MCF-7/DDP.Under the condition of Caspase inhibition,LCL161 had better inhibitory effect on MCF-7/DDP cells.3.LCL161 and z-VAD-fmk inhibit cell proliferation by inducing necrotic apoptosis of MCF-7/DDP cells.4.LCL161 and z-VAD-fmk require RIP3 by inducing necrotic apoptosis in MCF-7/DDP cells.