| Background:Ovarian cancer is one of the common malignant tumors of female reproductive system and is the leading killer of female reproductive system malignant tumors.At present,cytoreductive surgery in combination with cisplatin-based chemotherapy is still considered as the basic therapeutic scheme for ovarian cancer.In the scheme,cisplatin can effectively inhibit the growth of unresectable or distantly metastatic tumor cells in gynecological surgery,and plays a key role in improving the prognosis of patients.However,the emergence of chemoresistance restricts the therapeutic effect of cisplatin and seriously affects the 5-year survival rate of ovarian cancer patients.Therefore,to solve the problem of cisplatin resistance has always been the difficulty and focus in the clinical treatment of ovarian cancer.According to the theory of traditional Chinese medicine,Arnebiae Radix has the functions of invigorating Qi,dispersing stasis,and promoting blood circulation,which coincides with the pathogenesis of ovarian cancer,such as lack of righteousness,blood stasis,and qi stagnation.Modern medical studies have shown that the extract of Arnebiae Radix has extensive anti-tumor effects in addition to its anti-microbial,anti-inflammatory,anti-allergic and hepatoprotective effects.Shikonin,as a naphthoquinone compound extracted and isolated from Arnebiae Radix,has shown great potential anti-tumor activity.Our previous study found that shikonin combined with cisplatin could inhibit the growth of cisplatin-resistant ovarian cancer cells.In addition,we also found that the level of ferroptosis key factor Fe2+ increased after the cells treated with the two drugs,suggesting that shikonin combined with cisplatin may play the above role through ferroptosis pathway.Therefore,based on these studies and our previous results,this paper mainly focuses on the effect and mechanism of shikonin combined with cisplatin overcome cisplatin-resistant in ovarian cancer through ferroptosis pathway.Objective:This study will explore the effect and mechanism of shikonin combined with cisplatin overcome cisplatin-resistant in ovarian cancer by means of molecular biology,experimental zoology and other experimental means.(1)Establishment of cisplatin-resistant ovarian cancer cell lines.(2)To clarify the effect of shikonin combined with cisplatin in inhibiting cisplatin-resistant ovarian cancer cells.(3)To clarify the mechanism of shikonin combined with cisplatin inducing ferroptosis in cisplatin-resistant ovarian cancer cell.Methods:(1)Cisplatin-resistant cell lines were induced and established by stepwise dose escalation method,and the resistance index of cisplatin-resistant cell lines and their parent cell lines was compared by CCK8 assay.(2)CCK8 assay was used to investigated the effect of shikonin on cisplatin sensitivity of different cisplatin-resistant ovarian cancer cell lines.(3)The synergistic effect of shikonin and cisplatin was investigated in the synergistic experiment,and the combination index corresponding to each drug concentration was calculated.(4)Clonal formation experiment was conducted to investigate the inhibitory effect of shikonin combined with cisplatin on cisplatin-resistant ovarian cancer cells.(5)Nude mice transplanted tumor model was used to investigate the anti-tumor effect of the two drugs on cisplatin-resistant ovarian cancer.(6)Biochemical indexes ALT,AST,CRE and BUN were detected to evaluate possible toxicity of the combination of the two drugs.(7)Q-Exactive&Orbitrap Exploris 480 dual-mass spectrometry platform combined with DDA&PRM dualtechnology was used to explore the possible mechanism of the combination of the two drugs overcoming cisplatin-resistant in ovarian cancer.(8)The effect of the combination of the two drugs on the levels of ROS and lipid peroxides in cisplatinresistant ovarian cancer cells were investigated using ROS and lipid peroxides fluorescence probes combined with flow cytometry.(9)The effect of the combination of the two drugs on the level of Fe2+ was evaluated by using fluorescence probe combined with laser confocal microscopy.(10)Ferroptosis inhibitor Fer-1,and iron chelating agent DFO were used to investigate whether the combination of the two drugs inducing ferroptosis through iron accumulation.(11)Ferroptosis related proteins were detected by Western Blot and immunohistochemistry assay.(12)Inhibitor ZnPP and siRNA assay were used to investigate whether the combination of the two drugs inducing cell ferroptosis through HMOX1.Results:(1)Cisplatin-resistant ovarian cancer cell lines A2780/DDP,OVCAR4/DDP and SKOV3/DDP were established by stepwise dose escalation method,and the resistance index was greater than 3 compared with the parental cell lines.(2)Low concentrations of shikonin could increase the sensitivity of cisplatin-resistant ovarian cancer cells to cisplatin.(3)At a certain concentration,shikonin and cisplatin showed synergistic effects,and the results were similar in different cisplatin-resistant cells.(4)In vivo results showed that 0.8 mg/kg shikonin combined with 3 mg/kg cisplatin could overcome cisplatin-resistant in ovarian cancer.(5)Results of proteomic experiments indicated that shikonin combined with cisplatin overcoming cisplatin-resistant in ovarian cancer through the ferroptosis pathway.(6)Results of targeted proteomics indicated that the combination of the two drugs might induce ferroptosis through iron metabolism pathway.(7)The levels of lipid peroxidation,ROS,Fe2+ and total ferric ions in A2780/DDP were significantly increased by treatment with the combination of the two drugs,while GPX4 protein level was decreased.(8)The effect of the combination of the two drugs on cell ferroptosis was inhibited by Fer-1 and DFO.(9)The combination of the two drugs could significantly increase the transcription and expression of genes related to iron metabolism in A2780/DDP.(10)Inhibition or interference HMOX1 in A2780/DDP could inhibit the ferroptosis induced by the combination of the two drugs.(11)siRNA interference with HMOX1 significantly reduced the level of Fe2+ accumulated by the combination of the two drugs.Conclusion:(1)In vitro and in vivo experiments showed that shikonin combined with cisplatin could overcome cisplatin-resistant in ovarian cancer.(2)Proteomic experiments showed that the combination of the two drugs might induce ferroptosis by up-regulating the expression of iron metabolism-related proteins,and thus exert the above effects(3)Molecular biology experiments showed that the combination of the two drugs could induce ferroptosis by promoting Fe2+ accumulation via upregulation of HMOX1. |
PDF Full Text Request