| Part Ⅰ Mechanism of Qinghao Zhimu Decoction in treating non- small-cell lung cancer by inducing ferroptosis and necroptosis of tumor cells Objective: Qinghao Biejia Decoction is a classic prescription for curing fever.It is composed of Artemisiae Annuae,Trionycis Carapax,Rehmannia Glutinosa,Anemarrhena Asphodeloides Bunge and Moutan Cortex.It has a history of about 220 years.In vitro and in vivo experiments showed that the active components of Artemisiae Annuae,such as artemisinin,artesunate,artemisinin acid and the activity of Anemarrhena Asphodeloides Bunge,such as Timosaponin AⅢ,showed the potential to inhibit the progression of cancer.According to the clinical experience of the supervisor,Artemisiae Annuae and Anemarrhena Asphodeloides Bunge are often used in the prescription of lung cancer patient.In this study,Qinghao Zhimu Decoction(QZD)is composed of Artemisiae Annuae and Anemarrhena Asphodeloides Bunge(1:1).The objective is to clarify the scientific connotation of QZD in the prevention and treatment of lung cancer,by observing the QZD on human non-smallcell lung cancer(NSCLC)cells and tumor bearing animal models of mouse NSCLC cells.Methods:(1)Human NSCLC cell lines(A549 and H1299)were treated with QZD,the cell activity was detected by CCK-8.(2)NSCLC cells were treated with QZD,and the clone formation were detected by crystal violet staining.(3)Flow cytometry was used to detect the cell cycle and apoptosis of NSCLC cells after QZD treatment.(4)After co-culture of QZD with inhibitors of apoptosis,autophagy,necroptosis and ferroptosis,the activity of NSCLC cells was detected by CCK-8.(5)After the treatment of QZD in NSCLC cells,WB was used to detect the expression levels of necroptosis and ferroptosis related proteins,and q RT-PCR was used to detect the m RNA expression levels of necroptosis and ferroptosis related genes.(6)LLC cells were injected subcutaneously into the right axilla of C57/BL6 J mice to established xenograft tumor model model.After 12 days of intervention with PBS and QZD,tumor volume and body weight of mice were measured every two days during the period.Results:(1)Compared with the control group,QZD inhibited the activity of NSCLC cells in a dose-(0.1,0.25,0.5,1,2 mg/m L)and time-(24,48,72 h)dependent manner(P< 0.05).(2)Compared with the control group,QZD significantly inhibited clonal formation of NSCLC cells.(3)Flow cytometry results of cell cycle showed that compared with the control group,QZD arrseted NSCLC cells in G2/M phase(P< 0.05).(4)Flow cytometry results of apoptosis results showed that QZD induced NSCLC cell death rather than apoptosis compared with the control group(P< 0.05).(4)Compared with the control group,necroptosis and ferroptosis inhibitor could reverse QZD-induced cell death(P< 0.05).(5)WB results showed that the expression of necroptosis pasitive regulatory proteins(RIPK3,MLKL),the expression of ferroptosis negative regulatory protein(SLC40A1,FTL,SLC7A11 and GPX4)was decreased,the expression of positive regulator of ferroptosis protein(TCRC)was increased in NSCLC cells increased after QZD treatment(P< 0.05).q RT-PCR results showed that the m RNA expression of necroptosis negative regulatory gene(Cas-8)was decreased,while the m RNA expression of necroptosis pasitive regulatory gene genes(RIPK1,RIPK3 and MLKL)was increased,the m RNA expression of ferroptosis negative regulatory genes(SLC40A1,SLC7A11 and GPX4)was decreased,while the m RNA expression of ferroptosis pasitive regulatory gene(TFRC)was increased in NSCLC cells increased after QZD treatment(P< 0.05).(6)After 12 days of QZD(50mg/kg)intervention,the tumor weight and volume of the QZD group were significantly lower than that of the control group(P< 0.05),but there was no difference in body weight between the two groups(P>0.05).Conclusion: QZD treats NSCLC by inducing necroptosis and ferroptosis of tumor cells.Part Ⅱ The mechanism of ferroptosis in non-small-cell lung cancer cells mediated by timosaponin AⅢ through HSP90Objective: Timosaponin AⅢ(Tim-AⅢ)is a steroid saponin derived from Anemarrhena asphodeloides Bunge,which has shown strong anticancer activity in a variety of cancers,especially breast cancer and liver cancer.However,the mechanism of Tim-AⅢ in the treatment of NSCLC remains unclear.The first part has explained the mechanism of QZD in the treatment of NSCLC,and the second part aims to further explain the mechanism ofTim-AⅢ,one of the effective components of QZD,in the treatment of NSCLC,so as to provide scientific basis for the further application of traditional Chinese medicine in the treatment of lung cancer.Methods:(1)The cell activity was detected by CCK-8 method after Tim-AⅢ treatment in NSCLC cells(H1299,A549,SPC-A1,LLC),and the IC50 value of Tim-AⅢ on the above cells was calculated.(2)Lipid ROS,iron,MDA and GSH levels were detected,mitochondrial morphology was observed by transmission electron microscope,ferroptosis related regulatory proteins were detected by WB after Tim-AⅢ treatment in NSCLC cells.(3)The interaction between HSP90 and GPX4 was investigated by Co-immunoprecipitation(Co-IP),and GPX4 ubiquitination was detected by Co-IP after Tim-AⅢ treatment in NSCLC cells.(4)The degradation of GPX4 by Tim-AⅢ-HSP90 complex was verified in vitro.(5)Stable NSCLC cell lines with low expression of HSP90 were established by sh RNA-HSP90,and the transfection efficiency was verified by WB.(6)H1299 cells were injected subcutaneously into the right axilla of BALB/c-nu/nu nude mice to establish H1299 human NSCLC subcutaneous tumor bearing animal model.LLC cells were injected subcutaneously into the right axilla of C57BL/6J mice to establish the subcutaneous tumor bearing animal model of LLC mouse NSCLC.LLC cells were injected into C57BL/6J mice through tail vein to establish lung metastatic tumor model.During the intervention of PBS,low dose and high dose Tim-AⅢ,tumor volume and body weight of mice were measured every two days.Results:(1)Tim-AⅢ inhibited the activity of NSCLC cells in a dose-(0.25,0.5,1,2,4 mg/m L)and time-(24,48,72 h)dependent manner compared with the control group(P< 0.05).The IC50 values of Tim-AⅢ against H1299 cells were 7.04 μmol/L at 24 h,1.55 μmol/L at 48 h and 2.68 μmol/L at 72 h.The IC50 values of Tim-AⅢ against A549 cells were 2.57 μmol/L at 24 h,2.16 μmol/L at 48 h and 0.99 μmol/L at 72 h.The IC50 values of TimAⅢ on LLC cells were 6.7 μmol/L for 24 h,3.02 μmol/L for 48 h and 3.78 μmol/L for 72 h.The IC50 values of Tim-AⅢ against SPC-A1 cells were 5.2 μmol/L at 24 h,1.89 μmol/L at 48 h,and 3.23 μmol/L at 72 h.(2)Compared with the control group,Tim-AⅢ caused ROS and iron accumulation,MDA increase and GSH depletion in NSCLC cells,and the differences were statistically significant(P< 0.05).Mitochondria became smaller and cristae decreased,and the expression of GPX4,a key enzyme that regulates ferroptosis,was decreased after Tim-AⅢ treatment in NSCLC cells.(3)HSP90 interacted with GPX4,resulting in the increase of GPX4 ubiquitination after Tim-AⅢ treatment in NSCLC cells.(4)Tim-AⅢ targeted HSP90 to form a complex that degraded GPX4.(5)The low expression model of HSP90 was successfully constructed,and low expression of HSP90 inhibited ferroptosis induced by Tim-AⅢ.(6)After different concentrations of Tim-AⅢ intervention in subcutaneous xenograft tumor model,the tumor volume and weight in lowdose and high-dose Tim-AⅢ groups were significantly lower than those in control group,and the results were statistically significant(P< 0.05).After different concentrations of TimAⅢ intervention in lung metastasis tumor model,lung weight in low-dose and high-dose Tim-AⅢ groups was significantly lower than that in control group(P< 0.05).Conclusions: Tim-AⅢ promotes NSCLC ferroptosis through targeting and facilitating HSP90 mediated GPX4 ubiquitination and degradation. |
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