| Hepatocellular carcinoma is one of the most common and fatal malignancies in the world.Most patients are diagnosed at a late stage and are often treated with dose-dependent cytotoxic anti-tumor drugs such as cisplatin(DDP).Even though chemotherapy has been used for many years,drug resistance is still an important obstacle to it.Therefore,the combination of small molecule substances that can improve the sensitivity of chemotherapy may be an effective treatment plan to improve the sensitivity of liver cancer to DDP.Low molecular weight heparin(LMWH)is not only an anticoagulant,but also has a good effect in other aspects: inhibiting angiogenesis and metastasis of tumor cells;The gene expression of DDP resistance in A2780 ovarian cancer cells was inhibited by influencing Wnt signaling pathway.Autophagy is a form of suicide that is evolutionarily conserved and degrades excess intracellular material and organelles.Studies have shown that LMWH combined with chemotherapy drugs can increase autophagy and lead to the death of cancer cells.Then can LMWH combined with DDP regulate autophagy of liver cancer cells to increase the sensitivity of liver cancer to DDP?Objective:1.To investigate whether the combined action of DDP and LMWH can inhibit the proliferation of HCC cells;2.To investigate whether DDP and LMWH alone or in combination can enhance autophagy in Bel-7404 and SMMC-7721 cells;3.To investigate whether the single and combined effects of DDP and LMWH enhance autophagy through AMPK-ULK1-Beclin1 signaling pathway.Methods:The experiment was divided into four groups : control group,LMWH group,DDP group and LMWH combined with DDP group,namely L+D group.1.CCK-8 assay was used to detect the effects of DDP and LMWH on cell proliferation of BEL-7404 hepatoma cells.2.Fluorescent staining with MDC was used to detect autophagy in liver cancer cells of Bel-7404 and SMMC-7721 in four groups.3.The protein and m RNA expressions of autophagy protein LC3 and P62 in Bel-7404 and SMMC-7721 cells were detected by Western Blot and RT-PCR.4.The protein expression levels of AMPK,ULK1,Beclin1,p-AMPK,p-ULK1 in each group of BEL-7404 and SMMC-7721 hepatoma cells were detected by RT-PCR and Western Blot.Results:1.The results of CCK-8 assay showed that the proliferation capacity of BEL-7404 cells in DDP group and L+D group was decreased compared with the control group,and the decrease was more significant in the L+D group(P < 0.05).2.MDC autophagy staining results showed that the number of green fluorescent autophagosomes in L+D group of BEL-7404 and SMMC-7721 hepatoma cells was significantly increased compared with the other three groups(P < 0.05).3.Western Blot and RT-PCR detection of autophagy-related protein LC3 and P62expression: Compared with the other three groups,the LC3 protein and m RNA expression of BEL-7404 and SMMC-7721 liver cancer cells in L+D group was significantly increased,while the expression of P62 in L+D group was significantly decreased(P < 0.05).4.Western Blot were used to detect the expression of pathway proteins: there was no significant change in AMPK and ULK1 protein expression in the L+D group(P >0.05),but the protein expressions of p-AMPK /AMPK,p-ULK1 /ULK1 and Beclin1 were significantly increased(P < 0.05).Conclusions:1.The CCK-8 cytotoxicity test showed that LMWH could enhance the inhibitory effect of DDP on the proliferation of BEL-7404 hepatoma cells;2.DDP combined with LMWH significantly induced the enhancement of autophagy in BEL-7404 and SMMC-7721 cells;3.DDP combined with LMWH induced autophagy enhancement in hepatocellular carcinoma BEL-7404 and SMMC-7721 cells,which may be realized by activating AMPK-ULK1-Beclin1 signaling pathway. |
