| BackgroundProstate cancer (Pca) was the most common male cancer in Western Europe and the United States, and the incidence rate of male malignancies in Western Europe in the second place, while the mortality of the male had become the first leading cause of death in patients who had malignant tumors. In recent years, the incidence of Pca showed an increasing trend. Pca patients had received endocrine therapy for 1 to 2 years (average median period of 18 months), and their disease often by androgen-sensitive prostate cancer (ADPC) gradually evolved into hormone-independent prostate cancer (AIPC), often resulting in patient's condition progress, symptoms get worse, a transfer, biochemical indicators showed elevated PSA. Second-line endocrine therapy was still used in the treatment of second-line endocrine response to AIPC patients. Pilepich MV [1] reported that second-line for AIPC patients with endocrine treatment should be performed: Including high-dose bicalutamide 200mg/d[16];adrenal androgen inhibitors of ammonia Rumi Special (ketoconazole 400mg orally three times a day); estrogen therapy: Diethylstilbestrol (1-3mg oral once every day) [2, 3] about 20%-80% of patients with improvement in symptoms, PSA decreased by 50% , and the period of effective times was only at 2 to 6 months, so looked for new therapeutic targets of AIPC patients with a new effective treatment program in recent years become a hot topic in the field of AIPC treatment.In recent years, the relationship between leptin and prostate cancer become a hot topic in urology research field. And the new research outcomes showed that: 1.Obesity was likely to cause prostate cancer [42]; 2. leptin levels was more associated with prostate cancer [4]; 3. Regulating of prostate cancer by leptin receptor signaling[7,8]; 4.The interaction between leptin and hormones would affect the progression of human prostate cancer. [9]; 5.The level of leptin would promote angiogenesis of prostate cancer progression.Leptin as a multifunctional cytokine, through different signal factor interactions regulate different signaling pathways in the body tissues in many physiological and pathological conditions play an important regulatory role.And studies had shown that it was high in tumor cells expression,such as: breast cancer, stomach cancer and prostate cancer. Pca patients received estrogen therapy due to estrogen stimulation of fat cells to promote the level of leptin in blood[10]; Androgen suppression of testosterone secretion and result in promotion of serum leptin levels [11] , The studies of jie Mei Ye [12]had shown that leptin levels would promot androgen-independent cells (PC-3/DU-145) proliferation. In summary, the occurrence of leptin played an important role in the development of AIPC, especially in receiving antiandrogen treatment failure patients;The leptin and its receptor as a target used in the treatment of AIPC and become the starting point of the experimental design and the final goal.The recombinant human leptin antagonist were used in the treatment of AIPC was the experimental innovation.Purpose:This study was designed to investigate:1.Recombinant human leptin antagonist in vitro DU-145 cell line (100ng/ml leptin induced )proliferation after 24h, and to explore the optimal concentration.2.Different concentration of Bicalutamide in vitro (100ng/ml leptin induced) after 24h the DU-145 cell line proliferation and to explore optimal concentration.3. Bicalutamide combination of recombinant human leptin antagonist on cell cycle and apoptosis of DU-145 cell line.Method:1.Using in vitro cell culture, selection of Shanghai Institute of Cell Library for hormone-independent prostate cancer cell line DU-145 cells as experimental seeds.2. In cultured prostate cancer DU-145 cells were observed and identified.3. Subculture, use growth phase of DU-145 cells were planted 96 wells board, after leptin (100ng/ml) induced 24h the randomized intervention,and it was divided into negative control group, leptin intervention group and intervention group, adding different concentrations of leptin antagonists and bicalutamide.And they were determined using the MTT cell activity in each group (OD), calculated rate of cell growth inhibition.4. Select growth phase DU-145 cell lines that stiumulated by leptin after 24h were randomly divided into intervention: negative control group, leptin treatment group, bicalutamide group, leptin antagonist bicalutamide group and joint leptin Antagonist group.And the intervention 24h, 48h were detected by flow cytometry after the above-mentioned group of DU-145 cell line cell cycle.5. The logarithmic growth phase DU-145 cell lines, leptin intervention 24h, were randomly divided into leptin intervention group, Bicalutamide group and combined leptin antagonist group.And added the corresponding drug intervention 24h underwent apoptosis rate by flow cytometry the changes.6. Experimental results were analyzed with SPSS 13.0 software for statistical. Results:1. Inverted microscope DU-145 cells morphological changed before and after intervention.2. MTT assay were found: 100ng/ml leptin in the intervention groups of cell proliferation activity was significantly better than the control group, and 50ng/ml recombinant human leptin antagonist group when the growth of DU-145 cells significantly inhibited .And cell growth inhibition rate was: 15.86%±0.8%, continue to increasing the dose of DU-145 did not inhibit the growth of cells.3. MTT results showed that:3.1 Different concentrations of bicalutamide group DU-145 cells were significantly inhibited , and DU-145 Cell growth inhibition rate were increasing with increasing bicalutamide doses gradually,and each group was significant difference (P < 0.01).3.2 Different time the rate of DU-145 cell growth inhibition was no significant difference (P> 0.05).4.Detection of the cell cycle by flow cytometry showed that:4.1 Leptin group of the DU-145 cells were increased compared with the control group G2 and S phase. The percentage of G1phase were significantly decreased, and different time (24h/48h) was no significant difference (P> 0.05).4.2 Leptin antagonist group was significantly different than Leptin group (P<0.05), DU-145 cells in G2 and S cell phase decreased significantly, especially in S cell phase;The percentage of G1cells were fraction increased,And the percentage of cell cycle was no significant difference at different time (24h/48h) (P> 0.05).4.3 Bicalutamide group were significantly different than Leptin group (P <0.05), The percentage of DU-145 cells in G2 phase significantly decreased than Leptin group, S phase no significant decreased; The percentage of G1 phase were increased with the increasing of time, and the percentage of G2 phase continued to declining.4.4 The change of pencentage of DU-145 cell phase were significantly different in the recorbinant leptin Antagonist united bicalutamide group than leptin and the control group (P <0.05);There was no significant difference in bicalutamide group in G1cell phase,and the rate of S phase was decreased; The change of G1 phase was no significant difference than it in the recorbinant leptin Antagonist group, and G2 phase was decreased; Different time periods (24h/48h) DU-145cell cycle were no significant difference (P> 0.05).5. Apoptosis measured by flow cytometry:Apoptosis test results showed that:It had significantly higher rate of apoptosis in the recombined leptin antagonist united bicalutamide group than the control group and bicalutamide group , and it was significant different (P <0.01); It showed more pronounced proapoptotic effect in the united group.Conclusion1.Recombinant human leptin antagonist inhibited the proliferation of DU-145 cells, and it was no compliance in time ; More DU-145 cells were arrested in G1 phase, and the rate of S phase was significantly decreased.2.Bicalutamide inhibited the proliferation of DU-145 cells significantly, and the DU-145 cells were arrested in G1 phase, and the rate of G2 phase was significantly decreased.3.Bicalutamide combination of recombinant human leptin antagonist inhibited the proliferation of DU-145 cells significantly . More cells were arrested in G1 phase, and promote apoptosis in DU-145 cells. |
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