Antitumor Effects And Mechanism Of Butein Combined With Erastin In The Treatment Of Osteosarcoma

Objective:The main purpose of this study was to investigate the effects of ferroptosis inducer erastin and butein on the proliferation and cell death of oste-osarcoma（OS）cells,and to explore the possible mechanism of butein and eras-tin inhibiting osteosarcoma cells in vitro and the antitumor effect in vivo.Methods:Human OS cells were cultured in vitro.When the cell density reached about 70%,butein,erastin,reactive oxygen species（ROS）inhibitor,fer-roptosis inhibitor,endoplasmic reticulum stress（ER stress）inhibitor,MAPK pathway inhibitor and si RNA were used to intervene the cells.The viability of OS cells was detected by cell counting kit-8（CCK8）;Flow cytometry was used to evaluate the cell cycle;The invasion ability of OS cells was evaluated by transwell experiment;Apoptosis was investigated by mitochondrial membrane potential（MMP）staining,Hoechst 33258 staining,flow cytometry and caspase3/8/9 viability;Cell cycle related proteins,cell invasion related proteins,apopto-sis related proteins,oxidative stress related proteins,autophagy related proteins,cell ferroptosis related proteins,ER stress related proteins,and MAPK pathway related proteins were detected by western blot.The changes of ROS level in OS cells were evaluated by fluorescence microscope.The level of lipid peroxidation was evaluated by malondialdehyde（MDA）detection and lipid peroxidation flu-orescence probe C11-bodipy 581/595 detection;Glutathione（GSH）test kit was used to detect the changes of GSH level and evaluate the antioxidant capacity of cells;Ferrous ion(Fe²⁺)colorimetric test box is used to detect the level of iron ion;The changes of acid lysosomes after combined treatment were observed by fluorescence microscope;The autophagy bodies were observed by transmission electron microscope in the combined treatment group;The OS cells were in-fected with GFP-m RFP-LC3 lentivirus and autophagy was observed by laser confocal microscope.The effect of subcutaneous heterotopic implantation on the growth of osteosarcoma in nude mice was observed.24 BALB/C male nude mice（4-6weeks old,16-20g）were selected and 100μL cold PBS with 143B cell（density1x10⁷ cells/ml）were transplanted subcutaneously under the armpit of nude mice.When the axillary tumor of nude mice was visible to the naked eye,the mice were assigned to four groups randomly:control group（DMSO group）,butein treatment group（10mg/kg）,erastin group（10mg/kg）,butein（10mg/kg）and eras-tin（10mg/kg）combined treatment group,with 6 rats in each group.The drug was administered every other day by intraperitoneal injection for 10 times.The nude mice body weight/tumor volume in every group were assessed（0.5×short diameter²×length diameter）.After the treatment,the functions of liver and kidney were examinated by eyeball blood collection,and then all nude mice were killed.Subcutaneous osteosarcoma tissues were carefully removed.Heart,liver,spleen,lung and kidney tissues were gathered and examinated by H&E method,and the cell proliferation and apoptosis of osteosarcoma tissues were detected by Ki67and TUNEL immunofluorescence.ROS was detected by fluorescence micro-scope;The changes of lipid peroxidation level were evaluated by using MDA detection kit;GSH level were examinated using GSH detection kit;The effect of butein combined with erastin on the protein expression levels of chop,p-JNK,GPX4 was detected by immunohistochemistry.Results:1 CCK8 results showed that butein and erastin could inhibit the viability of OS cells in a dose and time-dependent manner;Erastin could signif-icantly enhance the inhibition of butein on the viability of OS cells;2.The results of flow cytometry and WB showed that the combination of butein and erastin could cause G2/M phase arrest of OS cells;3.Transwell and WB results showed that the combination of butein and erastin could inhibit the invasion of OS cells;4.The results of MMP,Hoechst 33258 staining,flow cytometry apoptosis,caspase 3/8/9 viability and the expression level of apoptosis related proteins showed that the combination of butein and erastin could induce apoptosis of OS cells;5.ROS detection,HO-1 protein expression level detection and GSH detec-tion showed that the combination of butein and erastin could induce oxidative stress in OS cells;6.Lysotraker fluorescence detection,transmission electron microscopy and autophagy related protein expression showed that the combina-tion of butein and erastin could cause autophagy in OS cells;7.The detection of iron ion level,lipid peroxidation level and the expression level of iron metabolism related proteins showed that the combination of butein and erastin could cause ferroptosis in OS cells;8.Deferoxamine Mesylate（DFOM）can in-hibit the increase of apoptosis rate and autophagy of OS cells induced by com-bined use;9.Inhibition of autophagy can reduce the inhibition of combined use on OS cell viability and alleviate the death of OS cells caused by combination;10.NAC can reduce the inhibition of combined use on OS cell viability and al-leviate the death of OS cells caused by combination;11.The combination of butein and erastin can activate endoplasmic reticulum stress,inhibit the viability of OS cells and promote cell death.12.Butein combined with erastin can activate JNK pathway,inhibit OS cell viability and promote cell death.13.The tumor volume of the control group was larger than that of butein and combined treat-ment group,and the volume of combined treatment group was smaller than that of butein alone group;There was no significant difference in body weight among groups;HE staining showed that both butein and combined treatment groups had obvious necrosis.Immunofluorescence showed that the expression of Ki67 in butein and combined treatment group was lower than that in control group,and the fluorescence intensity of TUNEL was higher than that in control group;The levels of ROS and MDA increased and the level of GSH decreased in the com-bined treatment group of butein and erastin;The expression level of chop and p-JNK protein increased and the expression level of GPX4 protein decreased;There was no significant difference in liver and kidney function indexes and his-tology of important organs among the groups.Conclusion:1.Erastin can significantly enhance butein’s inhibition of oste-osarcoma cell viability,inhibition of invasion and promotion of apoptosis2.Butein combined with erastin can inhibit the viability of osteosarcoma cells through ROS-endoplasmic reticulum stress pathway,ROS-MAPK pathway,and induce a variety of death forms of osteosarcoma cells,including apoptosis,au-tophagy and ferroptosis;3.Butein can inhibit the tumor growth of osteosarcoma bearing mice,and erastin can enhance the antitumor effect of butein in vivo;4.The combination of butein and erastin can inhibit tumor in vivo through oxida-tive stress,endoplasmic reticulum stress,MAPK pathway and iron metabolism pathway;In addition,Butein and the combination of butein and erastin were rel-atively safe in vivo,and there was no obvious toxicity.