| Nowadays cancer has become the most serious disease which is harmful to the health of human being.Prostate cancer is one of the most commonly malignant tumors found in male urinary system.In the United States, its incidence rate has come to the first place of the tumors happened in males,while fatality rate ranked second to pulmonary cancer. In our country the incidence of prostate cancer is lower than Europe , the United States and other countries, but with our population aging and changes in diet and others, which increase the incidence of growing trend. Patients with prostate cancer are generally no obvious clinical symptoms, so most patients diagnosed during the first shift have already lost the opportunity for surgery and combined therapy. Since Huggins proposed the theory of androgen-dependent prostate cancer, the endocrine therapy of prostate cancer has become the classic method of treatment,but the vast majority of prostate cancer patients receiving endocrine therapy for 1-2 years will be transformed into androgen independent prostate cancer, which shorten the survival time of these patients.It's threating the health and the quality of life of middle aged men,but there is still a lack of effective treatment in the androgen-independent prostate cancer,so it's urgent to find new treatments.Matrine(MAT) is one of the major alkaloids in traditional Chinese medicine ,Lightyellow Sophora Root, found in the roots of Sophora belong to the quinolizine Ring piperidine class.Its Molecular skeleton contains two piperidine rings Quinolizine miscellaneous body,its chemi- cal formula C15H24N2O, molecular weight is 248.36. MAT has a variety of pharmacological effect,for example: prevention and control hepatitis,anti-arrhythmia,anti-liver fibrosis, anti-bacterial anti-inflamma- tory,anti-pyretic and analgesic,expectorant asth ma,cardiac diuretic, anti- tumor and so on. U.S. National Cancer Institute announced it as the third generation of cancer chemopre -vention drug.In vitro and in vivo ,tests have confirmed that MAT exactly plays an important part on the anti-tumor activity in a variety of tumors,meanwhile it also has the effect on anti-tumor invasion and metastasis. Now MAT has aroused extensive attention on tumor prevention and treatment among the scholars at home and abroad,and become a research hotspot.Now the study on how MAT effect on androgen-independent prostate cancer is so less that this research explores the MAT on androgen independent prostate cancer and its mechanism by selecting androgen-independent prostate cancer PC-3M cells as the object.1,Effect of MAT on the growth of prostate cancer PC-3M cells inhi- bittion and apoptosisThe packet due to concentration of MAT into 0.50mg/ml, 1.0mg/ml, 2.0mg/ml 3 Ge treatment group and normal control group(Without drug MAT). Different groups dealing with PC-3M cells at different times, then observe cell morphology through inverted microscope; MTT assay using inhibition of cell proliferation; by flow cytometry (FCM) to detect the apoptosis rate; Immunohistochemical SP method to detect bcl-2, bax, caspase-3 protein expression levels in cells; RT-PCR to detect the expression level of bcl-2, bax gene; western blot to detect of caspase-3 protein expression.The results show:(1) Observation through inverted microscope The cells in the control group grow adhere the wall,polygon nal,cytoplasm plump,growth stretch,blended into pieces by adjacent ones. The different concentrations of MAT treatment, with increasing concentration and time extension, the cells begin to be round, size reduction, nuclear condensation , nuclear color deepened, refractive index increased, and exfoliated cells gradually increased suspending in culture medium,among which the cells in group 2mg/ml MAT 24h were most obvious,with the lest number while the most exfoliated ones.(2) Using MTT method measure the inhibition of MAT on PC-3M cellsResults showed that after the 12-48h effect of different concentra- tions of MAT, the PC-3M cells'growth were inhibited to varying degrees, with concentration-and time-dependent characteristics,of which the 4mg/ml MAT group has highest inhibitory rate in 48h. The cell growth inhibition rate of cancer cells which were in the groups with different concentration of MAT treatment at each time has significantly differences (P <0.01)compared with the control group.(3) Apoptosis of PC-3M cell detecting by Annexin V / FITC and PI double stainingUsing confocal laser microscopy to detect the apoptosis rate of PC-3M cell,the results show that:the apoptosis rates of PC-3M cells in the negative control group,0.5mg/ml MAT group,1.0mg/ml MAT group, 2.0mg/ml MAT group dealt with 24 h were (2.97±0.35)%,(6.24±0.27)%,(13.65±0.49)%,(24.88±0.68)%;compared the apoptosis rate in each MAT group with the control group by statistical analysis,it shows significant difference(P<0.01).(4) Determination of PC-3M cell apoptosis rate after treatment with drugs by FCMThe results showed that apoptosis rate in the control group and MAT group were:the control group (2.0%), 0.5mg/ml MAT group (11.8%) , 1.0mg/ml MAT group (18.7%), 2.0mg/ml MAT group (20.3%), in a concentration-dependent relationship, has significantly differences (P <0.01) compared with the control group.(5) PC-3M cells bcl-2, bax, caspase-3 protein content detected by SPIt has found that with the increase of concentration of matrine, bcl-2 protein expression was lower,while bax, caspase - 3 protein expression increased in a dose and time dependent relationship, compared with control group the differences were significant (P <0.01).(6) Detection of matrine on PC-3M cells, bcl-2 and bax gene transcri- ption results by RT-PCRIt showed that: PC-3M cells treated by different concentrations of matrine after 24h, with the increase of the concentration of matrine , bcl-2 gene expression decreased while the amount of bax gene expression increased. Bcl-2/gadph integral value of the ratio of optical density with increasing the concentration of matrine showed decreasing trend (P <0.01), while bax / gadph optical density ratio of integral value increases gradually with the concentration of matrine an increasing tendency (P <0.01).(7) Detection caspase-3 protein expression in different concentration of matrin on PC-3M cellsIt showed that: caspase-3 expression in their cells may be increased in matrine treatment group ,showing dose-dependent relationship, the concentration of group and normal control group also showing significant difference (P <0.01).(8) PC-3M cells PSA protein content detected by ELISAIt has found that with the increase of concentration of matrine, PSA protein expression was lower in a dose and time dependent relationship, compared with control group the differences were significant (P <0.01). Our results indicate that in vitro and in tivo,matrine can inhibit the growth of human androgen-independent prostate cancer cell line PC-3M, induce apoptosis.It's related to the reducted protein expression of bcl-2, and the increase of bax, caspase-3 protein expression, but the effect of the specific mechanism needs further study.2,Experimental study on the inhibition of prostate cancer PC-3M cell adhesion invasion by MAT(1) The impact on the ability of matrine inhibiting prostate cancer PC-3M cell adhension by scratches experiment:Observation results showed that prostate cancer PC-3M cell adhension is significantly inhibited by MAT.Compared with the normal control group, there is a significant difference (P <0.01).(2) The impact on the ability of matrine inhibiting prostate cancer PC-3M cell migration by scratches experiment:Observation results showed that prostate cancer PC-3M cell migration is significantly inhibited by MAT.Compared with the normal control group, there is a significant difference (P <0.01).(3) PC-3M cells OPN protein content detected by SPIt has found that with the increase of concentration of matrine, OPN protein expression was lower. and time dependent relationship, compared with control group the differences were significant (P <0.01).(4) Detection of matrine on PC-3M cells, OPN,MMP-2 and MMP-9 gene transcription results by RT-PCRIt showed that: PC-3M cells treated by different concentrations of matrine after 24h, with the increase of the concentration of matrine , OPN,MMP-2 and MMP-9 gene expression all decreased. OPN,MMP-2 and MMP-9/gadph integral value of the ratio of optical density with increasing the concentration of matrine showed decreasing trend (P <0.01).(5) Detection MMP-2 and MMP-9 protein expression in different concentration of matrin on PC-3M cellsIt showed that: MMP-2 and MMP-9 protein expression in their cells may be reduced in matrine treatment group ,showing dose dependent relationship, the concentration of group and normal control group also showing significant difference ( P <0.01).Our results indicate that MAT palys a prominent role on inhibiting PC-3M cell transplant and metastasis process,and its mechanism may be related to inhibiting cell apoptosis and down-regulate the expression of OPN,MMP-2 and MMP-9 mRNA, but there is still a lack of more experimental data in vivo to support the feasibility of clinical treatment of prostate cancer. Relevant tests need to be consummated to further explore the optimum therapeutic concentrations and the best plan combined with other treatment modalities. |
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