| Gastric cancer is a common tumor of the digestive system worldwide,with a 5-year survival rate 30-65% and a serious risk to human health.However,although the mortality of gastric cancer patients in Asia has been high worldwide in the past decades,there is still no drug with better efficacy and less toxic side effects.Cinobufagin is a water-decocted extract of the dried epidermis of Bufo chinensis or Bufo melitensis,which has the effects of clearing heat,detoxifying,swelling and pain relief.In recent years,its anti-gastric cancer effect has attracted people’s attention.It can exert its anti-gastric cancer activity through different ways,but it still cannot fully explain the anti-cancer drug efficacy of cinobufagin injection and the important role of ferroptosis related to fatal lipid peroxidation in cancer biology.Therefore,this study investigated the inhibitory effect of cinobufacin injection on the proliferation of gastric cancer cells from the perspective of ferroptosis,and further explored the possible mechanism of cinobufacin injection promoting ferroptosis.Objective:To study the inhibitory effect of cinobufacin injection on gastric cancer in vitro and in vivo,and to explore the anti-gastric cancer effect and molecular mechanism of cinobufacin injection from the ferroptosis and lipid metabolism pathway.Methods:Firstly,the inhibition effect of cinobufagin injection on gastric cancer in vitro and in vivo was observed.MGC-803 xenograft tumor was constructed in nude mice to observe the changes in tumor size,H&E staining and immunohistochemical results of the transplanted tumor to explore the inhibitory effect of cinobufagin injection on gastric cancer in nude mice.The inhibitory effect of cinobufacin injection on the proliferation of different gastric cancer cell lines was observed by MTT assay in vitro and the appropriate concentration for subsequent research was screened.The morphological changes of gastric cancer cells were observed by inverted microscope.After treatment with different cell death inhibitors,whether cinobufacin injection could promote the occurrence of ferroptosis of gastric cancer cells was preliminarily determined.The changes of mitochondria in gastric cancer cells,an indicator of ferroptosis,were observed by transmission electron microscope.The levels of MDA,GSH,and SOD were detected by corresponding detection kits.The changes of lipid and cytoplasmic ROS levels were analyzed by flow cytometry,and the changes of Fe ion content were detected by ultraviolet spectrophotometer.The expression changes of ferroptosis characteristic proteins such as GPX4,COX2,FTH1 and lipid metabolism-related proteins such as ACSL4,LPCAT3 and ALOX15 in gastric cancer cells and tumors were detected by Western blot.In order to further determine whether cinobufagin injection promotes ferroptosis in gastric cancer by regulating ACSL4-centered lipid metabolism,the expression of ACSL4 protein in cells was inhibited by pharmacological inhibition and gene silencing.The changes in cell viability and lipid metabolism-related protein changes after cinobufagin injection were detected.Results:1.Cinobufacin injection inhibits proliferation of gastric cancer cells in vitro and in vivoCinobufacin injection can inhibit the growth of gastric cancer MKN-45,SGC-7901 and MGC-803 cells,and the inhibitory effect on gastric cancer SGC-7901 and MGC-803 cells is more significant,but the sensitivity to gastric cancer MKN-45 cells is low.Cinobufacin injection inhibited gastric cancer SGC-7901 and MGC-803 cells in a timedose dependent manner.Cinobufacin injection group showed poor cell adhesion,cell shrinkage and lower refractive index compared to the control group,with IC50 values of6.387 mg/m L and 5.761 mg/m L,respectively.Cinobufacin injection at low,medium and high doses could inhibit the growth of MGC-803 subcutaneous transplanted tumor weight and volume in nude mice compared with the control group(P<0.05 or P<0.01),and alleviate the weight loss caused by the presence of gastric cancer in nude mice.The anti-tumor effect of cinobufacin injection in the middle and high dose groups was equivalent or better than that of the positive control drug 5-Fu.H&E staining showed that the nucleus of the control tumor cells was intact and the cells were densely arranged.In the gastric cancer tissues of each dose group of cinobufacin injection,the staining of gastric cancer cells became lighter,the obvious cracks appeared between the cells,and the nuclei appeared nuclear pyknosis and nuclear fragmentation.Immunohistochemistry showed that cinobufacin injection could inhibit the expression of Ki-67 protein in gastric cancer.2.Cinobufacin injection can induce ferroptosis in gastric cancer cellsThe inhibitory effect of cinobufacin injection on gastric cancer SGC-7901 and MGC-803 cells could be partially offset by autophagy inhibitor CQ,apoptosis inhibitor Z-VAD-FMK,ferroptosis inhibitor Ferrostin-1 and iron chelator DFO.The offsetting effect of ferroptosis inhibitor Ferrostin-1 and iron chelator DFO was more significant.The results of transmission electron microscopy showed that the outer membrane of mitochondria in SGC-7901 and MGC-803 cells was broken,smaller and darker stained after the treatment of cinobufacin injection.After the treatment of ferroptosis inhibitor Ferrostin-1,the mitochondrial volume became slightly larger and the staining became lighter.Compared with the control cells,the contents of MDA,Fe,cytoplasmic and lipid ROS in SGC-7901 and MGC-803 cells increased(P<0.05 or P<0.01),while the contents of GSH and SOD decreased(P<0.05 or P<0.01),and these effects could be antagonized by Ferrostin-1.Immunohistochemistry and Western blot staining showed that,compared to control,the expression of ferroptosis marker GPX4 and FTH1 proteins in gastric cancer cells and tumors in the low-,medium-and high-dose cinobufacin injection groups was down-regulated,while the expression of COX2 protein was upregulated(P<0.05 or P<0.01).3.Cinobufacin injection induces ferroptosis by regulating ACSL4-dominated lipid metabolismThe results of immunohistochemistry and Western blot showed that the expressions of ACSL4,LPCAT3 and ALOX15 proteins,which were closely related to ferroptosis and lipid metabolism,were up-regulated in gastric cancer cells and tumors in the low-,medium-and high-dose cinobufacin injection groups as compared with the control group(P<0.05 or P<0.01).The inhibitory effect of cinobufacin injection on MGC-803 cells could be partially reversed by the combination of ACSL4 pharmacological inhibitor RSG with non-cytotoxic dose of cinobufacin injection,which was similar to the inhibition of ferroptosis on Ferrostin-1.The results of Western blot showed that the expression of ACSL4 protein in MGC-803 cells was down-regulated after RSG treatment and could be partially antagonized by ferroptosis inhibition Ferrostin-1(P<0.05 or P<0.01).Si-ACSL4 successfully silenced ACSL4 protein in gastric cancer MGC-803 cells,but after cinobufacin injection,ACSL4 protein was significantly expressed(P<0.05).Conclusions:1.Cinobufacin injection can inhibit the proliferation of gastric cancer cells in vitro and in vivo;2.Cinobufacin injection can promote the ferroptosis of gastric cancer cells,thereby inhibiting the proliferation of gastric cancer in vitro and in vivo;3.Cinobufacin injection regulates ferroptosis in gastric cancer by regulating ACSL4-mediated lipid metabolism. |
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