Expression Of CYP17A1 And The Mechanism Of Chaetomugilin H Induced Apoptosis In Human Glioma Cells

Background:Glioma is a malignant nervous system tumor that accounts for 30%of central nervous system tumors and 80%of all intracranial malignancies.At present,most of the treatment methods for glioma are surgery combined with chemotherapy and radiotherapy.Despite comprehensive treatment,the prognosis of glioma is still poor,and postoperative recurrence is inevitable,which is closely related to the phenomenon of chemotherapy tolerance in glioma.It is urgent to develop new glioma sensitive chemotherapeutic drugs and new glioma non-invasive diagnosis,disease monitoring and prognosis evaluation methods.CYP17A1(Cytochrome P450 17A1),located on chromosome 10q24.3,is a membrane-bound bi-functional monooxygenase belonging to the CYP450 enzyme superfamily.The expression products of the CYP17A1 gene are involved in two key reactions that catalyze the conversion of androgen precursors to androgen-like DHEA(dehydroepiandrosterone)and androstendione.This mainly involves the involvement of NADPH-hemoprotein reductase,which is regulated by cellular cAMP levels.Overexpression of CYP17A1 can increase androgen production.In addition,excess androgens can increase the risk of malignancies such as prostate and pancreatic cancer.In recent years,some studies on sex differences in malignant tumors have suggested that changes in sex hormone expression pathways may be involved in the process of cancer development.Epidemiological studies have shown that the incidence of glioma is significantly higher in men than in women.In addition,the researchers found that a culture medium containing androgens promoted the proliferation of glioma cells.The glioblastoma chemotherapy drug Temozolomide usually causes apoptosis by inducing DNA damage and the production of reactive oxygen species.It is believed that DHEA can prevent such apoptosis because it regulates a variety of antioxidant enzymes through unknown signaling pathways.Since this mechanism requires CYP17A1 activity to produce DHEA,inhibition of CYP17A1 may be an option to achieve chemotherapy sensitization of gliomas.Although CYP17A1 is a candidate molecular target for glioma therapy,the development of new drugs targeting it has not been sufficient.The known CYP17A1 inhibitor chloramphenicol,due to its bone marrow suppression and blood toxicity and other side effects,has greatly limited its application in human and clinical.In recent years,the study of metabolites of endophytic bacteria has opened up new ideas for the development of new chemotherapy drugs.More and more studies on the secondary metabolites produced by endophytic bacteria show that many of them are similar to the active components of the host,so they are becoming important sources of natural products.At present,the search for drugs from endophytic bacteria has gradually become a research hotspot.In the early stage of this study,Epicoccum nigrum metabolite Chaetomugilin H,obtained from endophytic fungi of ginseng,can inhibit the proliferation of glioma cells and is predicted to be a CYP17A1 inhibitor through drug target database.Objective:This study attempted to analyze the relationship between the expression level of CYP17A1 and clinical grade of glioma,explore its relationship with clinical treatment resistance of glioma,and investigate whether Chaetomugilin H,as a CYP17A1 inhibitor,induces apoptosis of glioma cells through mitochondria.Lay a theoretical foundation for the development of Chaetomugilin H as an antitumor drug.Method:(1)TCGA database was used to analyze the relationship between the expression level of CYP17A1 and the survival time of glioma patients.(2)Immunohistochemical staining was used to detect the expression of CYP17A1 in glioma tumors of different pathological grades,so as to explore the relationship between CYP17A1 expression and glioma grade.(3)Using CYP17A1 inhibitors(chloramphenicol),Western Blotting was used to detect GRP78 and ATF4 to evaluate the occurrence of ER stress;DRP1 and MFN1 were detected to evaluate mitochondrial dynamic changes;autophagy related protein p62 was detected to evaluate autophagy.(4)CYP17A1 inhibitor(chloramphenicol)was used to detect apoptotic proteins by TUNEL staining,Flow Cytometry,Annexin-V/PI double staining and protein immunoimprinting.(5)The endophytic fungi of ginseng were identified by giant colony culture,small AGAR block culture and ITS sequence comparison.The endophytic bacteria of ginseng were identified by 16S sequence comparison.(6)The ethyl acetate extract of endophytic fungus Epicoccum nigrum was isolated and purified by fermentation culture and silica gel column chromatography.(7)MTT assay was used to detect the inhibition of endophytic metabolites on glioma cells,and mass spectrometry was used to identify the structural formula and molecular weight of the compound Chaetomugilin H.(8)Swiss Target Prediction was used to predict drug targets of compound Chaetomugilin H,and the development of inhibitors of existing target proteins was analyzed.(9)STING network was used to predict the interacting proteins of CYP17A1.(10)Chaetomugilin H alone or combined with CYP17A1 inhibitor(chloramphenicol)was applied,GRP78 and ATF4 were detected by Western Blotting to evaluate the occurrence of endoplasmic reticulum stress,and DRP1 and MFN1 were detected to evaluate the mitochondrial dynamic changes.Autophagy was evaluated by detecting autophagy associated protein p62.(11)Chaetomugilin H alone or combined with CYP17A1 inhibitor(chloramphenicol),apoptotic proteins were detected by TUNEL staining,Flow Cytometry,Annexin-V/PI double staining and protein immunoimprinting.(12)glioma allograft tumor model was established in nude mice and treated with intraperitoneal injection of Chaetomugilin H to verify the anti-glioma effect of Chaetomugilin H in experimental animals;The TUNEL staining of allograft tumor and the injury of important organs after treatment were detected.Results:1.Using TCGA database analysis,we found that the expression level of CYP17A1 was significantly negatively correlated with the survival of glioma patients.2.Immunohistochemical staining results showed that CYP17A1 protein was expressed in the cytoplasm of glioma samples,and the expression level was significantly higher than that of normal brain tissue samples,and the expression level of CYP17A1 was positively correlated with the pathological grade of glioma,the higher the expression level of samples with higher grade.3.CYP17A1 inhibitors can induce apoptosis of human glioma U87 cells.(1)CYP17A1 inhibitor(chloramphenicol)increased the expression of endoplasmic reticulum stress marker protein in glioma cells,indicating that the apoptosis of human glioma U87 cells induced by chloramphenicol was related to the endoplasmic reticulum stress pathway.(2)Chloramphenicol induced reduced mitochondrial fusion and increased mitochondrial division in human glioma cells.(3)Chloramphenicol can up-regulate the autophagy of human glioma U87 cells.4.There are 26 kinds of interacting proteins of CYP17A1 predicted by STING network.There are currently 37 inhibitors that have been developed to target CYP17A1.5.The target of ginseng endophytic fungal metabolite Chaetomugilin H is predicted to be CYP17A1 inhibitor.(1)27 strains of endophytic fungi and 18 strains of endophytic bacteria were isolated and cultured from different parts of ginseng.After isolation and purification,9 compounds were obtained,of which compound 1 had the ability to significantly inhibit the proliferation of human glioma cells,and was identified as Chaetomugilin H according to the structure comparison references.(2)Chaetomugilin H significantly inhibited the viability of glioma U87 and SHG44 cell lines,and induced apoptosis.(3)Determine the molecular structure formula and molecular weight of Chaetomugilin H.(4)Drug target prediction of compound Chaetomugilin H as a possible inhibitor of CYP17A1.6.Chaetomugilin H can induce apoptosis of human glioma U87 cells(1)The expression of endoplasmic reticulum stress marker protein was increased in glioma cells when Chaetomugilin H was applied alone,indicating that the apoptosis of human glioma U87 cells induced by Chaetomugilin H was related to the endoplasmic reticulum stress pathway.(2)Chaetomugilin H impels mitochondrial function and induces mitochondrial pathway apoptosis in human glioma cells by reducing membrane potential in tumor cells.(3)Chaetomugilin H caused decreased mitochondrial fusion and increased mitochondrial division.(4)Chaetomugilin H can activate autophagy in human glioma U87 cells.7.The mechanism of apoptosis induced by Chaetomugilin H and chloramphenicol combined with temozolomide was compared and verified.(1)Chaetomugilin H or chloramphenicol combined with temozolomide significantly increased the expression of endoplasmic reticulum stress marker protein in glioma cells.(2)Chaetomugilin H or chloramphenicol combined with temozolomide,respectively,reduced mitochondrial fusion and increased mitochondrial division in glioma cells.(3)Chaetomugilin H or chloramphenicol combined with temozolomide significantly activated the autophagy of human glioma U87 cells.8.Chaetomugilin H has the effect of inhibiting tumor growth in vivo.Conclusions:1.The expression level of CYP17A1 protein in human glioma samples was significantly higher than that in normal brain tissue samples,and the expression level of CYP17A1 was positively correlated with the pathological grade of glioma.TCGA database analysis showed that the expression level of CYP17A1 was significantly negatively correlated with the survival of glioma patients.2.CYP17A1 inhibitors can induce apoptosis of glioma cells,suggesting that CYP17A1 is expected to be a marker for glioma therapy.3.Chaetomugilin H,a secondary metabolite of Epicoccum nigrum endophytic fungus,can inhibit the proliferation of glioma cells.Drug target prediction and protein expression indicate that Chaetomugilin H may be an inhibitor of CYP17A1.4.Chaetomugilin H can cause endoplasmic reticulum stress and disrupt mitochondrial homeostasis of human glioma U87 cells,and then activate autophagy of U87 cells to induce apoptosis,suggesting that further exploration of CYP17A1-related pathways may provide new clues for the diagnosis and treatment of glioma.5.In vivo experiments have confirmed that Chaetomugilin H has definite anti-tumor effect and no obvious core organs side effects.