Study On The Mechanism Of Guben Qingyuan Prescription Combined With Androgen Deprivation Therapy In The Control Of Castration-resistant Prostate Cancer From AR Pathway And Tumor Stem Cells

Backgroud:Prostate cancer is a common male malignant tumor.Global cancer statistics show that prostate cancer accounts for 26%of male malignant tumors,ranking first.In recent years,prostate cancer has increased rapidly in my country.In 2022,the National Cancer Center reported that prostate cancer had become the fastest growing male malignant tumor,ranking sixth in incidence.Castration Resistant Prostate Cancer(CRPC)is a difficult point in the treatment of prostate cancer and the focus of research.Androgen sensitivity is the most prominent biological feature of prostate cancer,so androgen deprivation therapy(ADT)is the cornerstone of treatment for patients with intermediate and high-risk prostate cancer,but after 18-30 months of treatment,most patients will turn to CRPC.Without aggressive treatment,the overall survival of patients is less than one year.Even with chemotherapy,new endocrine therapy,and targeted therapy,tumor progression cannot be controlled,thus becoming a bottleneck restricting the improvement of the efficacy of prostate cancer.Traditional Chinese medicine has unique advantages in the prevention and treatment of CRPC.Previous studies have shown that Sophora flavescens,turmeric and their active ingredients have a certain inhibitory effect on CRPC.Under the guidance of traditional Chinese medicine theory,combined with the progress of modern pharmacology research,the Guben Qingyuan Prescription(composed of Sophora flavescens,turmeric,ginseng,Huangjing,and Ganoderma)was formed.The inhibitory effect on the CRPC was better than that of the disassembled prescription Fuzheng Chinese medicine group and the detoxification Chinese medicine group.Based on this,we took CRPC as the entry point and Guben Qingyuan prescription as the intervention to observe the effect and mechanism of Guben Qingyuan prescription combined with ADT in inhibiting CRPC.Objective:1.To verify that Guben Qingyuan prescription combined with ADT can control CRPC.2.To explore the mechanism of Guben Qingyuan prescription combined with ADT in controlling CRPC from the AR signaling pathway.3.To explore the mechanism of Guben Qingyuan prescription combined with ADT in the control of CRPC from cancer stem cells and its Wnt pathway.Methods:In this study,Guben Qingyuan prescription(Sophora flavescens,turmeric,ginseng,Huangjing,Ganoderma lucidum)was used as the intervention,combined with ADT,and five groups were set up:normal saline group,ADT group,low,medium and high doses of Guben Qingyuan prescription combined with ADT group.The 22RV1 cells were selected and a nude mouse xenograft model was established.CCK8 method was used to detect the inhibitory effect of Guben Qingyuan prescription combined with ADT on 22RV1 cells in vitro.To observe the tumor inhibition of Guben Qingyuan prescription combined combined with ADT on tumor-bearing mice.RT-PCR and Western blotting were used to detect the expressions of AR,AR-V7 and PSA in tumor tissues.Prostate cancer stem cells were sorted by flow cytometry,and their tumor stem cell properties were verified by colony-forming unit assays.To observe the tumor inhibition of Guben Qingyuan prescription combined with ADT on prostate cancer stem cell-bearing mice,and to detect the expressions of Wnt and β-catenin by Western blotting.Results:1.After 48h and 72h of intervention in 22RV1 cells in each treatment group,the inhibition rate of middle and high doses of Guben Qingyuan prescription combined with ADT group was significantly increased,and there was a statistical difference compared with ADT group(P<0.05).The tumor inhibition experiment showed that the body weight of the tumor-bearing mice in the normal saline group,the ADT group,and the low-dose Guben Qingyuan prescription combined with ADT group was significantly lower than that before treatment(P<0.05).There was no significant difference in body weight after treatment between the middle and high doses of Guben Qingyuan prescription combined with ADT group(P>0.05).From the 8th day of administration,the changes of tumor volume in the middle and high doses of traditional Chinese medicine+ADT group were significantly lower than those in the normal saline group(P<0.05).The testosterone level of tumor-bearing mice in each treatment group was significantly lower than that in the normal saline group(P<0.05),but there was no statistical difference between the Guben Qingyuan prescription combined with ADT group and the ADT group at each dose(P>0.05).Guben Qingyuan prescription combined with ADT has an inhibitory effect on CRPC tumor-bearing mice.The tumor inhibition rates of low,medium and high dose groups were 27.95%,46.71%and 44.46%,respectively.The inhibitory effect of ADT group was significantly higher than that of ADT group(P<0.05).2.Compared with the ADT group,the mRNA and protein expression levels of AR,AR-V7 and PSA in the middle and high doses of Guben Qingyuan prescription combined with ADT group were significantly down-regulated(P<0.05).3.Prostate cancer stem cells were sorted by flow cytometry,accounting for 2.56%before sorting and 99.95%after sorting,indicating that high-purity prostate cancer stem cells were sorted.The colony-forming unit assays showed that the cloning efficiency of prostate cancer stem cells was 13.25%,which was significantly higher than that of ordinary prostate cancer PC-3 cells,which was 8.18%,and the difference was statistically significant(P<0.05).The tumor weight of the tumor-bearing mice inoculated with prostate cancer stem cells was significantly higher than that of the normal prostate cancer PC-3 cell tumor-bearing mice,and there was a statistical difference between the two groups(P<0.05).4.The tumor inhibition rates of low,medium and high doses of Guben Qingyuan prescription combined with ADT on prostate cancer stem cell tumor-bearing mice were 24.58%,36.67%,and 40.24%,respectively,which were statistically comparable to the ADT group alone.Further studies showed that low,medium and high doses of Guben Qingyuan prescription combined with androgen deprivation therapy could down-regulate the protein expression levels of Wnt and β-catenin,and there was a statistical difference compared with the ADT group(P<0.05).Conclusion:1.Guben Qingyuan prescription combined with ADT can control castration-resistant prostate cancer.2.Guben Qingyuan prescription combined with ADT to control CRPC is related to the regulation of AR signaling pathway.3.Guben Qingyuan prescription combined with ADT to control CRPC is related to the regulation of cancer stem cells and Wnt pathway.