| Prostate cancer (PCa) is the first incidence in malignant tumor of male in Euro-American nation, and the second leading cause of cancer death. The incidence rate of PCa shows rising tendency in China. Androgen-ablation therapy is the mainstay treatment for PCa. Unfortunately, however, eighty percent of PCa will become androgen independent within about eighteen months after androgen-ablation, and the cancer grow and worsen continuously. At last, the patient dies of this disease. Androgen independent prostate cancer (AIPC) becomes the most formidable problem in the management of PCa. At present, androgen receptor (AR) gene mutations /amplification and methylation are considered the principal cause. Although AR expression in some AIPC rised, but some investigation displayed that AR amplification rate was very low before and after androgen jerk, and mutated cell is a little of the cancer cell. In initial tumor there is AR amplification, but the tumor is also responsed to androgen ablation,meanwhile AR-ablation therapy cannot inverse androgen independence effectively. Therefore the rising of the activity of AR path lack available basis. AR lose or inactivation get concern gradually. There is huge difference between androgen-dependentent prostate cancer (ADPC) cell line LNCaP and AIPC cell line DU145 in AR and GR(Glucocorticoid receptor) expression.This phenomenon hints that cancer cell could continue to grow through GR pathway after AR lose or inactivation because of androgen-ablation.This may be an important reason of AIPC. HSP90 is an important molecular chaperone that can regulate and activate GR. The activation of GR pathway can spur the secretion of downstream inflammatory factor (IL-6 etc.) in great quantities, and IL-6 has androgen activity. These factors could also promote androgen-independent growth of PCa. We presumed: under the survival pressure, the cancer cells emerge "cell stress", assisted by HSP90, by expressing GR, to acquire new ability of growth fastly and continuously to grow on the absent of androgen. So, it has important significance to study the expression and function of GR, HSP90 and IL-6 in prostate cancer for exploring the occurrence mechanisms and treatment of AIPC. At the same time, we try to explore the drug that can regulate hormone balance, metabolism and immune to adjust the balance between AR and GR, to inhibit the abnormally activated GR pathway and its downstream inflammatory factors, in order to maintain or recover the sensitivity of cancer cell to androgen to make the androgen blockade get a long-term curative effect. The main research contents: 1. The serum cortisol level and plasma total cholesterol concentration were measured, Immunohistochemistry stain was used to investigate the expression of AR,GR,HSP90,IL-6 and VEGF protein in prostate specimens. The relationship among these factors and PCa, and the mechanisms of occurrence and progression of AIPC were explored. 2. The effect of ursolic acid, a traditional Chinese drug effective constituent, on human prostate cancer cell line LNCaP (ADPC) and DU145 (AIPC) was studied in vitro. 3. In order to explore the therapeutic effect and mechanisms of ursolic acid on ADPC and AIPC, two typical human prostate cancer cell line were used as the model, immunocytochemistry, RT-PCR and/ or Western blot were applied to examine the expression of AR,GR,HSP90,IL-6,VEGF and PSA in LNCaP and DU145 cell with and without ursolic acid treatment. The main results: 1. The serum cortisol and plasma cholesterol level increased gradually with the histology development from BPH to PCa in patients and the serum cortisol further increased in the stage of androgen independence. 2. AR expression was lower than in AIPC than in ADPC (49.6% and 57.9% respectively), but without statisticly significance, AR was lowering along with the tissue grade, but did not reach to statisticly difference in clinical PCa specimens. From normal prostate(NP),BPH to ADPC tissue, the GR expression presented a gradual descent trend, but in AIPC, GR expression increased significantly, and was closely related with pathological grade of PCa. 3. The expression of HSP90 increased significantly in AIPC tissue, and was closely related with the stage and grade of PCa. There was obviously positive correlation between GR and HSP90 expression. The expression of IL-6 increased significantly in AIPC and wasclosely related to clinical stage; the expression of VEGF was higher in PCa than in BPH and NP, but there was no statisticly increase in AIPC comparing to ADPC. 4. The concentration-growth curve of LNCaP cell presented clock shape in different concentration of R1881, and 0.1nM R1881 was the most suitable concentration. The ursolic acid inhibited growth of LNCaP cell in dosage-and time-dependent model in different concentration of R1881. The growth curve of LNCaP cell still presented the clock shape after treating with ursolic acid, but the top of the curve shifted right, the most suitable R1881 concentration rose more than tenfold. 5. The growth of DU145 cell was independent on androgen, lacking response to flutamide. Ursolic acid had dosage-and time-dependent inhibition effects on DU145. There was an exciting result that ursolic acid together with flutamide had stronger inhibitory effect than ursolic acid alone. 6. It was found by immunocytochemistry that AR expression lose, GR expression resurrected, HSP90 and IL-6 expression increased significantly in DU145 cell. These might be the internal cause of androgen independence. The expression of GR, HSP90 and IL-6 in DU145 cell treated by ursolic acid were further confirmed decreased by RT-PCR and western blot. The inhibitory effect of ursolic acid on DU145 was related with its lowering expression of GR, HSP90, IL-6 and VEGF. 7. The expression of HSP90 and IL-6mRNA and protein decreased with the rise of androgen concentration in LNCaP cell, this is not in accordance with the clock shape growth curve of LNCaP, indicating there is no direct relation between the expression variety of HSP90 and IL-6 and reaction of LNCaP to different androgen concentration. By treatment with ursolic acid, the expression of HSP90 and IL-6 decreased in different concentration of androgen in LNCaP. This is the important cause that the cell growth is suppressed and the growth curve declined. The main conclusions: 1. Cholesterol is a promoting agent for PCa, cortisol concentration is related with AIPC, AR has certain relation with pathological grade of PCa, GR is closely related with stage and grade, and IL-6 is closely related with clinical stage. The over expression of HSP90, IL-6 and VEGF promotes the occurence of PCa, and the over expression of GR, HSP90 and IL-6 improves the development and progression of AIPC.2. The androgen-ablation can result in AR lose or inactivation, then GR expression increases, and cause the expression of the downstream inflammatory factor HSP90, IL-6, VEGF to go up, this is an important mechanism of the occurrence of AIPC, the internal mechanism may be "cell stress". 3. Ursolic acid can improve the response of PCa cell to androgen, increasing the dependence of ADPC to androgen, inducing response of AIPC to androgen or androgen-blockade. The mechanism of that contributes to the capability of ursolic acid to lower the expression of HSP90 and IL-6 in LNCaP and to inhibit the expression of GR, HSP90 and IL-6 in DU145. 4. This research found that the increased activity of GR pathway after androgen-blockade is an important mechanism of AIPC occurrence. For the first time we found that ursolic acid improves the response of PCa to androgen, deferring or prohibiting the occurrence of AIPC. Ursolic acid also induces the responsibility of AIPC to androgen. This important discovery may develop a new therapy for PCa, especially for AIPC.
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