The Treatment Of Qianliexiaozheng Doses To Hormone Refractory Prostate Cancer And The Molecular Mechanisms Of Regulating The PI3K/AKT Pathway

1ObjectiveProstate cancer(PCa) is one of the most important cancer in men. In Asian countries such as China, although the incidence of PCa is lower than the European and American area, it increases significantly in China. In our country most PCa was found at the middle-late stage of the tumor and can not be cured at all. Although endocrine therapy to PCa is clearly effective at the beginning of the treatment, after a median time of14-30months, almost all PCa develop independence in prostate cancer for hormone (hormone independent PCa, HIPC). And the median surial in these patients is less than20months. For HIPC still there is not standard and effective treatment. And the application of traditional Chinese medicine treatment of HRPC has obtained the certain effect.Qianliexiaozheng Doses is a TCM dose created by Pro.Liu Youfang, which can treat advanced prostate cancer effectively. This formula is composed of Seven Herbs:Radix Astragali, Rhizoma Polygonati, coix seed, rhizoma curcumae, Rhizoma Bolbostemmae, Polyporus and Hedyotis diffusa. The function of this formula is tonifying kidney and spleen, detoxification, promoting blood circulation and removing dampnesshis. This formula pays attention to regulate spleen and stomach function, strengthening the body resistance to eliminate cancer pathogenic factors through replenishing the spleen Qi and kidney essence, clearing heat, removing dampness and promoting blood and Qi circulation. Qianliexiaozheng Doses has used more than10years of in clinic. By summing up the experiments of90cases’ treatment of advanced prostate cancer, Qianliexiaozheng Doses can be found that it can obviously alleviate the symptom such as dysuria, bad appetite, fatigue, prolong the development of androgen dependence to independence, improve the patient’s quality of life. The objective of This research is to explore Qianliexiaozheng Doses treating prostate cancer possible molecular mechanism, expound its scientific nature, and summarize and provide new ideas to mining traditional Chinese medicine experience of Pro.Liu Youfang through clinically observing Qianliexiaozheng Doses to35cases with hormone refractory prostate cancer and exploring the PI3K/Akt signal transduction pathway by containing medicine serum human prostate carcinoma PC3cells in vitro experiment. 2Methods2.1clinical observation72cases clinical cases, all which were treated with drug castration in the past, aged50to87years old, course of18～36months, were divided into two groups:top Qianliexiaozheng Doses combined with endocrine therapy group (treatment group) and western medicine treatment group (control group). Control group was treated with Castration:subcutaneous injection of goserelin acetate sustained-release depot3.6mg or intramuscular triptorelin acetate3.75mg, once every28-33days. Treatment group was treated with Qianliexiaozheng Doses plus Castration. Final treatment group were removed2cases (1case with radiotherapy,1case with chemotherapy). Control group were removed4cases (1case with radiotherapy,1case with chemotherapy,2cases loss of follow up). The final effective66cases were composed of35cases in the treatment group and31cases control group each group. The average age of treatment group and control group is77.2years and74.19years respectively. During6months of treatment, respectively, before treatment and1,3,6months after treatment patients quality of life was observed though scales:FACT-p (Version4) scale score Karnofsky score, symptoms rating scale of TCM. And Laboratory indexes also were tested: prostate specific antigen (PSA), testosterone (T) in serum, blood cell analysis of white blood cell count (WBC), hemoglobin (HB). Measurement data was analysed with mean t test, counting data was analysed with chi-square test and rank and inspection by SPSS19.0software.2.2In vitro experimentThis experiment selected human PC-3cells as the research object, used the method of serum pharmacology, and divided into low dose group (rat and human equivalent dose), middle dose group (ten times in the low dose group), high dose group (twenty times in the low dose group two), and saline group. Qianliexiaozheng Doses was fed to rats by gastric lavage method, drug-containing serum of rats prepared. Respectively using fetal bovine serum and high, medium and low doses of drug-containing serum of rats cultures prostate cancer PC-3cells after24hours. Using CCK8method measure cell proliferation and cell streaming technology measure the cell cycle. Cell invasion ability was detected by Transwell Chambers method. The protein expression of PI3K/Akt pathway, PTEN and PI3K, and Akt phosphorylation was detected by Western Blot method. The expression of PI3K/Akt pathway downstream molecular mTOR inhibitors, the nf-kappa B, PTEN gene mRNA were detected RT-PCR method. 3Results3.1clinical observationKarnofsky Performance Status Scale(KPS) and FACT-P (Version4) scale is currently accepted for clinical evaluation of patients with tumors of the index. In this study, prostate cancer patients quality of life was evaluated by KPS, FACT-P (Version4) scale score and TCM symptom score. Before treatment, type score, FACT-P (Version4) scale score and TCM symptom score of Patients in treatment group, had no significant differences compared with the control group, but after1months,3months,6months treatment compared with the control group, all scores in treatment group were better than control group, especially significant difference after treatment after6months.Laboratory index:after treatment for1months and3months, PSA remained stable in the treatment group, while the control group showed a rising trend. The two groups have significant difference. In the treatment group, after treatment for6months, PSA value rose again, but the trend of rising was slow compared with the control group with significant difference. The ovariectomized and antiandrogens treatment will inhibit the secretion of erythropoietin, lead to patients with hemoglobin decrease, so the hemoglobin (HB) were observed in the two groups before treatment. There was no difference in hemoglobin quantity before treatment, but after1month,3months,6months treatment, HB quantity decreased in control group, and below the normal value. HB quantity in treatment group remained stable, fluctuated in the normal line, and was significantly higher than the control group. Before and after1months,3months,6months treatment. the quantity of white blood cells had no difference in statistics in two groups, and also were in the range of normal value. After6months of treatment, serum testosterone was in castrated levels in two groups, while in the control group only2patients had mild abnormal liver function, resumed soon without treatment.3.2in vitro experimentThough cell proliferation assay (CCK-8method), different concentrations of Qianliexiaozheng Doses contained serum on prostate cancer cell line PC-3for24hours,48hours,72hours after symptom Decoction in rats, the inhibition of cell proliferation was showed. And inhibition was enhanced with increasing concentration of the drug. There was a significant difference between the high, middle Qianliexiaozheng Doses group and control group. Cultured rat PC-3cells in medicated serum for24hours, with the increase of drug concentration, PC-3cells detected by flow cytometry showed a G0/G1arrested phenomenon obviously, and arrested PC-3cells in G0/G1phase were increased, compared with S phase cells ratio decreased significantly. There was difference compared with the control group. Messured by Transwell assay, cultured PC-3cells by fetal bovine serum group and the Qianliexiaozheng Doses group after24hours, the invasion ability of PC-3cells and degradation of polycarbonate Matrigel8um through the aperture of the change. The result showed, in Qianliexiaozheng Doses middle, high dose group, invasion inhibition rate decreased more than30%. So Qianliexiaozheng Doses middle, high dose had the anti invasion effects, and can make PC-3cells decreased invasiveness. Prostaglandin Western blot method was used to detect serum containing different doses of elimination in cultured rat PC-soup The components of PI3K/Akt signaling pathway in3cells24hours after the cells. The result showed that cells p-Akt, p-mTOR, p-4E-BP1and p-p70S6K protein level and p-p65(NF-κB) protein levels in Qianliexiaozheng Doses high,middle,low group decreased, while the level of p-PTEN protein increased. P-Akt, p-4E-BP1and p-p70S6K protein level and NF-κB protein expression Qianliexiaozheng Doses high, middle, low group decreased, while p-PTEN protein expression increased. And there was statistical significantly difference compared with the control group. Mesured by RT-PCR, cultured rat PC-3cells by containing herbs serum after24h, the results of PTEN, mTOR RelA (NF-κB) gene expression in PC-3cells showed that mTOR gene and RelA (NF-κB) gene expression in high doses group was inhibited, and the expression of the PTEN gene was enhanced. And there was statistical significantly difference compared with the control group.4Conclusion:4.1clinical observation4.1.1Qianliexiaozheng Doses combined with western medicine can significantly improve life quality of HIPC patients, improve the card high ratings, and improve health in patients with advanced prostate cancer, emotional state, enable patients to return to family and society, maintain and improve survival of patients.4.1.2Qianliexiaozheng Doses combined western medicine therapy can significantly improve the symptoms of prostate cancer patients, such as fatigue, insomnia, constipation, dysuria, bloody urine, swelling and so on.4.1.3Qianliexiaozheng Doses combined western medicine therapy can delay the PSA growth.4.1.4Qianliexiaozheng Doses combined with western medicine treatment can improve patients’ peripheral blood hemoglobin.4.2In vitro experiment4.2.1Qianliexiaozheng Doses can increase the expression of PTEN gene and protein translation, block the excessive expression of Akt, thus suppress PI3K/Akt signal pathway, cell stagnation in G1period rise, G0/G1phase cell proportion increase, thus inhibiting cell value. Qianliexiaozheng Doses can inhibit the Akt, protein mTOR inhibitors and their downstream key protein4E-BP1and p70S6K activation, block Akt/mTOR pathway, PC-3cell proliferation inhibition.4.2.2Qianliexiaozheng Doses can inhibit the nuclear transcription factor NF-kB gene and protein expression, block PI3K/Akt/NF-kB signal shaft, restrain the PC-3lyse cells surrounding matrix to reduce its ability to invasion and metastasis. Qianliexiaozheng Doses can inhibit the elimination p70s6k/AKT/mTOR pathway of protein mTOR inhibitors, p70s6k protein activation, inhibition of actin filaments refactoring, and decrease cell migration ability.Through the above experiments can be concluded that Qianliexiaozheng Doses treatment of hormone refractory prostate cancer effect, its possible molecular mechanism is Qianliexiaozheng Doses by inhibiting prostate cancer cells PI3K/Akt pathway activation and expression of all levels of key proteins, inhibition of the appreciation of prostate cancer cells, promote cell apoptosis, and to reduce its ability to dissolve cells surrounding stroma and movement, and inhibit the metastasis. Of course, this research only from one aspect of the PI3K/Akt pathway has carried on the preliminary exploration, its specific mechanism still needs further research to reveal.