Experimental Study On The Mechanism Of PEITC-induced Cell Death In Osteosarcoma And Its Inhibitory Effect On Osteosarcoma

Isothiocyanates（ITCs）,a group of small-molecule substances with a sulfhydryl structure,are widely found in cruciferous plants,such as radishes,broccoli,cabbages,etc.Extensive researches have showed that ITCs exhibt antitumor activity.β-phenethyl isothiocyanate（PEITC）is one of the most widely studied ITCs.PEITC exerts tumor suppressive effect by combining with glutathione（GSH）to form PEITC-GSH conjugate to decrease the level of GSH and generate reactive oxygen species（ROS）accumulation,thereby,inducing apoptosis,cell cycle arrest,and survival pathways inhibition.A large number of studies have shown that the anti-tumor effect of PEITC involves all major stages of tumor growth such as tumor initiation,promotion and development.Iron is one of the essential elements of the human body and participates in a variety of physiological and pathological activities,such as oxygen transport,DNA biosynthesis,ATP energy metabolism,tumorigenesis and development,neurodegenerative diseases,and iron-deficiency anemia.Under physiological conditions,the redox state of cells is in a dynamic equilibrium.By catalyzing the Fenton reaction,labile iron improves the oxidative stress level.Active free radicals can attack DNA molecules,proteins,lipids,and cause oxidative damage to cells in severe cases.A large number of studies have proved that"iron addiction"and sustained high levels of redox state exist in tumor cells.Therefore,iron metabolism and oxidative stress have become a new breakthrough points in tumor research.As a natural antitumor product that has entered phase II clinical trials,PEITC is highly safe and is being valued by more and more researchers.This thesis will take osteosarcoma as the research object,and choose PEITC as the research drug.On the one hand,its effect on the viability and proliferation of osteosarcoma cells was evaluated;on the other hand,it is the first time to investigate the effect of PEITC on the intracellular redox levels of osteosarcoma cells by studying the regulation of iron metabolism.In this study,at the cellular level,we studied the effects of PEITC on cellular vability,iron metabolism,and redox status of osteosarcoma cells（K7M2 murine osteoscarcoma cells and 4human osteosarcoma cell lines MNNG/HOS cells,U-2 OS cells,MG-63 cells,143B cells）.At the animal level,we investigated the effects of PEITC on the basic physiological indicators,histopathology and the tumor-inhibitory activity on syngeneic orthotopic osteosarcoma model and heterotopic xenograft osteosarcoma models in BALB/c mice and BALB/c-Nude immunodeficient mice,respectively.The results of experiments at cellular level showed that PEITC significantly reduced cell viability and inhibited cell proliferation in a concentration-dependent maner in osteosarcoma cells.PEITC caused cell cycle arrest in G₂/M phase and reduced the expression levels of cell cycle regulatory proteins,such as cdc2,cyclin D1 and cyclin D3.PEITC disrupted the mitochondrial membrane potential,regulated the expressions of Bcl2 and Bax,and activated the caspase cascade reaction to cleave its substrate PARP,causing mitochondria-mediated apoptosis.PEITC promoted the formation of acidic vesicles and regulated the autophagy-related protein like Beclin1,LC3Ⅱ,and p62 which activating the autophagy process.PEITC altered iron metabolism by up-regulating the expression of Tf R1 and inhibiting the expression of FPN,which resulted in the accumulation of iron in osteosarcoma cells.PEITC disrupted the redox balance and caused oxidative stress by depleting GSH,further inhibiting GPx4 expression,and leading to cytosolic ROS generation and lipid ROS accumulation,triggering ferroptosis.Furthermore,our studied found that PEITC induced ROS-dependent proliferation inhibition,MAPK signaling activation,apoptosis,autophage and ferroptosis in osteosarcoma cells.The results of experiments at animal level showed that compared with the low-dose control group,the weight difference of the mice in PEITC treatment group is not significant.In syngeneic orthotopic osteosarcoma model,administration of 60 mg/kg PEITC inhibited the growth of mouse osteosarcoma,and compared with the group,the difference was significant.In the model of heterotopic xenograft osteosarcoma,the volume and weight of osteosarcoma treated with 30 mg/kg PEITC were lower than those in the control group.Histopathological results showed that after PEITC treatment,there was no significant organ-related toxicity,and bone-like depositions were seen in the group with high-dose of PEITC.In addition,treatment with PEITC inhibited tumor cell proliferation,activated the expression of Caspase and LC3Ⅱ,increased Tf R1 expression,inhibited FPN expression,inhibited GPx4 expression,and activated MAPKs signaling pathways in tumor tissues.In summary,these findings suggested that the effect of PEITC on the redox status of osteosarcoma cells mediates its regulation of tumor cell death,and inhibits the growth of osteosarcoma to some extent,which will help to understand its underlying impact mechanism and provide some insights for the development of new strategies for tumor treatment in the future.