苦参碱对人前列腺癌PC-3M细胞作用的实验研究
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摘要
前列腺癌(prostate cancer,PCa)是一种严重威胁男性健康的恶性肿瘤。在美国前列腺癌的发病率已经超过肺癌,已成为危害男性健康的第一位肿瘤。其病死率仅次于肺癌,位居第2位。在我国随着人们预期寿命的不断延长和生活方式的改变,PCa的发病率和死亡率也有逐年上升的趋势,其发病率在男性泌尿、生殖系统恶性肿瘤中跃居第三位,且预后较差。
我国传统中药中有许多药物已证明具有抗肿瘤作用,对既能有效杀死肿瘤组织及细胞又能把药物对人的毒害作用降到最低程度的天然的抗肿瘤药物的研究就备受关注,苦参碱作为一类新型高效低毒的抗肿瘤活性成分,有着其广阔的应用前景。目前关于苦参碱对雄激素非依赖性前列腺癌(Androgen independent prostate cancer,AIPC)作用的研究比较少见。为了探讨苦参碱对AIPC的作用及其机制,我们选择AIPC细胞系PC-3M为研究对象,通过MTT法、Annexin V/PI双染法、SP法、Western blot、流式细胞仪(Flow cytometry,FCM)及激光共聚焦显微镜(laser scanning confocal microscope,LSCM)等方法观察了苦参碱对PC-3M细胞生长、凋亡调控及粘附、迁移作用的影响。结果显示:苦参碱在体外能明显抑制PC-3M细胞的生长、诱导细胞凋亡;亦能抑制PC-3M细胞的粘附、迁移能力。为进一步深入揭示苦参碱对AIPC的抗肿瘤机制奠定了理论基础,指导临床正确选择和合理使用,提供实验和理论依据,并为AIPC的临床治疗拓展了新的思路。
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 apoptosis
The 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 cells
Results 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 staining
Using 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 FCM
The 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 SP
It 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-PCR
It 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 cells
It 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 ELISA
It 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 SP
It 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-PCR
It 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 cells
It 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.
我国传统中药中有许多药物已证明具有抗肿瘤作用,对既能有效杀死肿瘤组织及细胞又能把药物对人的毒害作用降到最低程度的天然的抗肿瘤药物的研究就备受关注,苦参碱作为一类新型高效低毒的抗肿瘤活性成分,有着其广阔的应用前景。目前关于苦参碱对雄激素非依赖性前列腺癌(Androgen independent prostate cancer,AIPC)作用的研究比较少见。为了探讨苦参碱对AIPC的作用及其机制,我们选择AIPC细胞系PC-3M为研究对象,通过MTT法、Annexin V/PI双染法、SP法、Western blot、流式细胞仪(Flow cytometry,FCM)及激光共聚焦显微镜(laser scanning confocal microscope,LSCM)等方法观察了苦参碱对PC-3M细胞生长、凋亡调控及粘附、迁移作用的影响。结果显示:苦参碱在体外能明显抑制PC-3M细胞的生长、诱导细胞凋亡;亦能抑制PC-3M细胞的粘附、迁移能力。为进一步深入揭示苦参碱对AIPC的抗肿瘤机制奠定了理论基础,指导临床正确选择和合理使用,提供实验和理论依据,并为AIPC的临床治疗拓展了新的思路。
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 apoptosis
The 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 cells
Results 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 staining
Using 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 FCM
The 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 SP
It 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-PCR
It 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 cells
It 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 ELISA
It 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 SP
It 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-PCR
It 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 cells
It 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.
引文
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