葡萄籽原花青素对肺癌SPC-A-1细胞放射增敏作用的实验研究
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摘要
目的：
研究葡萄籽原花青素对肺腺癌SPC-A-1细胞X射线放疗的增敏作用。
方法：
1.应用MTT法和集落形成法研究原花青素的放疗增敏效应,单因素方差分析确定增敏剂量和增敏作用,单击多靶曲线拟合计算增敏比及相关参数。
2.流式细胞仪检测空白对照组(0Gy),单独X射线照射组(1Gy,5Gy)和药物增敏X射线组(100μg·mL-1原花青素1Gy,5Gy)细胞周期变化与凋亡
3.荧光定量PCR检测单独X射线照射组(1Gy)和不同浓度药物增敏组(50μg·mL-1,100μg·mL-1,1Gy)对细胞周期调控因子cyclin D1表达的影响。
结果：
1.MTT检测结果表明,单独应用原花青素对SPC-A-1的抑制作用较弱,IC50大于400 ug/ml;细胞药物处理后对X射线的敏感性增加,单因素方差分析表明药物和X射线有协同作用,50 ug/ml时表现出增敏效应；集落形成结果表明,100 ug/ml药物对X射线增敏作用明显,增敏比为2.56。
2.流式细胞术检测发现试验组细胞出现了明显的凋亡峰提示亚二倍体DNA含量的细胞数量明显增加,细胞凋亡率(28.4%)显著高于单独照射组(14.9%)及空白对照组(5.3%)。处理组与相应对照组分别比较差异有非常显著性意义(p<0.01)。实验结果说明,原花青素可以诱导细胞凋亡,并将细胞阻滞于G1期。
3.荧光定量PCR结果发现,原花青素50、100μg·mL-1作用于细胞48 h较对照组CyclinD1mRNA的表达分别下降0.189和0.426倍,表明原花青素可下调细胞CyclinD1mRNA的转录表达。
结论：
葡萄籽原花青素对肺腺癌SPC-A-1细胞有显著的放疗增敏作用。其机制可能是通过抑制细胞的增殖,G1期阻滞,诱导细胞凋亡,下调CyclinD1基因表达等而发挥抗肿瘤作用。
Objective:
To study the enhancement radiosensition of grape seed procyanidin extract for X-ray radiotherapy on lung cancer cell line SPC-A-1 in vitro and try to explore its mechanism.
Methods:
1.The enhancement of radiosensitising effect was detected by clone formation experiment and MTT methods. The radiosensitising dose of anthocyanidin was determined by single-factor analysis of variance. The ratio of sensitization and relevant parameters were calculated by the multi-target/single-hit model.
2.Flow cytometry (FCM)was used to measure cell cycle and apoptosis in radiotherapy group and increasing radiatesensitivity group.
3.Compared with control group, the expression of CyclinDl mRNA of a variety of concentration of the radiosensitising group analyzed by FPCR.
Results:
1.The results of MTT indicated that the inhibition effect on SPC-A-1 was slight when only using procyanidin, and the IC50 greater than 400 ug/ml. Though, after the lung cancer cell line was administrated by procyanidin, the sensitivity of X-ray radiotherapy was enhanced. The study showed that the procyanidin and X-ray has synergistic effects, the dose of 100 ug/ml has the significant sensitizing effect, and the ratio of sensitization was 2.56.
2.Flow cytometry analysis showed that procyanidin induced apoptosis of SPC-A-1 cells (The apoptotic rate were 28.4%,14.9%,5.3%, respectively.)and inhibited the proceeding of cell cycle in Gl period.
3.PCR showed that the CyclinD1 protein expression were down-regulated by administration of procyanidin.
Conclusion:
Grape seed procyanidin extract has the significant sensitizing effect on radiotherapy in lung adenocarcinoma cells SPC-A-1. GSEP inhibits the proliferation of malignant human Lung adenocarcinoma cancer cells and induces their apoptosis. Expression of CyclinD1 protein may be related to cell growth inhibition and the apoptosis mediated by GSEP in vitro.
研究葡萄籽原花青素对肺腺癌SPC-A-1细胞X射线放疗的增敏作用。
方法：
1.应用MTT法和集落形成法研究原花青素的放疗增敏效应,单因素方差分析确定增敏剂量和增敏作用,单击多靶曲线拟合计算增敏比及相关参数。
2.流式细胞仪检测空白对照组(0Gy),单独X射线照射组(1Gy,5Gy)和药物增敏X射线组(100μg·mL-1原花青素1Gy,5Gy)细胞周期变化与凋亡
3.荧光定量PCR检测单独X射线照射组(1Gy)和不同浓度药物增敏组(50μg·mL-1,100μg·mL-1,1Gy)对细胞周期调控因子cyclin D1表达的影响。
结果：
1.MTT检测结果表明,单独应用原花青素对SPC-A-1的抑制作用较弱,IC50大于400 ug/ml;细胞药物处理后对X射线的敏感性增加,单因素方差分析表明药物和X射线有协同作用,50 ug/ml时表现出增敏效应；集落形成结果表明,100 ug/ml药物对X射线增敏作用明显,增敏比为2.56。
2.流式细胞术检测发现试验组细胞出现了明显的凋亡峰提示亚二倍体DNA含量的细胞数量明显增加,细胞凋亡率(28.4%)显著高于单独照射组(14.9%)及空白对照组(5.3%)。处理组与相应对照组分别比较差异有非常显著性意义(p<0.01)。实验结果说明,原花青素可以诱导细胞凋亡,并将细胞阻滞于G1期。
3.荧光定量PCR结果发现,原花青素50、100μg·mL-1作用于细胞48 h较对照组CyclinD1mRNA的表达分别下降0.189和0.426倍,表明原花青素可下调细胞CyclinD1mRNA的转录表达。
结论：
葡萄籽原花青素对肺腺癌SPC-A-1细胞有显著的放疗增敏作用。其机制可能是通过抑制细胞的增殖,G1期阻滞,诱导细胞凋亡,下调CyclinD1基因表达等而发挥抗肿瘤作用。
Objective:
To study the enhancement radiosensition of grape seed procyanidin extract for X-ray radiotherapy on lung cancer cell line SPC-A-1 in vitro and try to explore its mechanism.
Methods:
1.The enhancement of radiosensitising effect was detected by clone formation experiment and MTT methods. The radiosensitising dose of anthocyanidin was determined by single-factor analysis of variance. The ratio of sensitization and relevant parameters were calculated by the multi-target/single-hit model.
2.Flow cytometry (FCM)was used to measure cell cycle and apoptosis in radiotherapy group and increasing radiatesensitivity group.
3.Compared with control group, the expression of CyclinDl mRNA of a variety of concentration of the radiosensitising group analyzed by FPCR.
Results:
1.The results of MTT indicated that the inhibition effect on SPC-A-1 was slight when only using procyanidin, and the IC50 greater than 400 ug/ml. Though, after the lung cancer cell line was administrated by procyanidin, the sensitivity of X-ray radiotherapy was enhanced. The study showed that the procyanidin and X-ray has synergistic effects, the dose of 100 ug/ml has the significant sensitizing effect, and the ratio of sensitization was 2.56.
2.Flow cytometry analysis showed that procyanidin induced apoptosis of SPC-A-1 cells (The apoptotic rate were 28.4%,14.9%,5.3%, respectively.)and inhibited the proceeding of cell cycle in Gl period.
3.PCR showed that the CyclinD1 protein expression were down-regulated by administration of procyanidin.
Conclusion:
Grape seed procyanidin extract has the significant sensitizing effect on radiotherapy in lung adenocarcinoma cells SPC-A-1. GSEP inhibits the proliferation of malignant human Lung adenocarcinoma cancer cells and induces their apoptosis. Expression of CyclinD1 protein may be related to cell growth inhibition and the apoptosis mediated by GSEP in vitro.
引文
1. Rosen EM, Fan S, Rockwell S, et al. The molecular and cellular basis of radiosensitivity:implications for understanding how normal tissues and tumors respond to therapeutic radiation[J]. Cancer Inrest.1999,17 (1):56-72.
2. Rosen Em, Fan S, Goldberg ID, et al. Biological basis of radiation sensitivity. Part 2:Cellwlar and molecular determinants of radiosensitivity[J]. Oncology (Williston Park).2000,14(5):741-757.
3.骆伟,黄荣清,骆传环,等.苯并杂环化合物的放射增敏研究进展[J].中华放射医学与防护杂志2004,24(2)：170-172.
4.王小萍,云松,张新良.放疗增敏剂甘氨双唑钠的二期临床研究[J].临床肿瘤学杂志.2003,8(1)：28-32.
5.朱靖蓉,王忠民,李瑾瑜,等.葡萄籽原花青素的分离纯化及其抗氧化活性研究[J].中国食物与营养.2007,4：34-37.
6. Packer L, Rinbach G, Virgili F. Antioxidant activity and biologic properties of a procyanidin-rich extract from p ine(Pinus maritim a) bark.pycnogenol [J]. Fre Rad Biol Med.1999,27(5P6):704-724.
7.李素梅,穆春兰.原花青素的研究进展[J].黑龙江医药.2006,19(3)：201-202.
8. Bagchi D, Bagchi M, Stohs S, et al. Cellular protection with proanthocy anidins derived from grape seeds[J]. Ann NYA cad Sci.2002,957(14):260-270.
9.陈小芬,汤为学,李龙江,等.姜黄素对人卵巢癌细胞株SKOV3的抗增殖作用[J].重庆医科大学学报.2005,30(2)：256-259.
10. Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochromec from mitochondria:a primary site for Bcl-2 regulationof apoptosis[J]. Science. 1997,275:1132-1136.
11. Sedlak TW, ltvai ZN, Yang E. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax[J]. Proc Natl Acad Sci USA. 1995,92:7834-7838.
12. Huyny TH, Teel RW. Selective induction of apoptosia in human mammary cancer cell[MCF-7] by Pycnogenol[J]. Anticancer Res.2000,20(4):2417.
13. Shih PH, Yeh CT, Yen GC. Efects of anthocyanidin on the inhibition of proliferation and induction of apoptosis in human gastric adenocarcinoma cells[J]. Food Chem Toxicol.2005,43(10):1557-'1566.
14. Ye X, Krohn RL, Liu W, et al. The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells[J]. Molecular and Cellular Biochemistry.1999,196:(1-2):99-108.
15. Megumi Yamagishi,Midori Natsume,Naomi Osakabe, et al. Chemoprevention of lung carciogenesis by cacao liquor proan thocyanidins in a male rat multi Organ carcinogenesis model. Cancer Letter.2003,191:49-57.
16. Antenask, Baliga MS, Katiyar SK. Grape seed proanthocyanidins induce apoptosis and inhibit metastasis of highly metastatic breast carcinoma cells]J]. Carcinogenesis.2006,27:1682-1691.
17. Meeran SM, Katiyar SK. Grape seed pmanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterafions in Cdki-Cdk-Cyclin cascade. and caspase-3 activation via loss of mitochondrial membrane potential[J]. Exp Dermatol.2007,16:405-415.
18. Meeran SM. Proanthocyanidins inhibit mitogenic and survival signalling in vitro and tumor growth in vivo[J]. Front Biosci.2008,13:887-897.
19. Mantena SK, Katiyar SK. Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes[J]. Free Radic Biol Med.2006,40(9):1603-1614.
20. Schuck A, Poremba C, Lanver C, et al. Radiationinduced changes of telomerase activity in a human Ewing xenograft tumor[J]. Strahlenther Onkol. 2002,178(12):701-708.
21.鲍永华,郭永臣.原花青素对SMMC-7721肿瘤细胞凋亡及其端粒酶活性的影响[J].西北农林科技大学学报.2007,35(1)：37-40.
22.亓玉琴,司君利,李文利,等.端粒酶抑制剂对小鼠胃癌放疗的增敏作用研究[J].肿瘤研究与临床.2003,15(6)：363-364.
23. Roberts JR, Allison DC, Donehower RC, et al. Development of ployploidization in taxol-resistant human leukemia cells in vitro[J]. Cancer Res.1990,50:710-716.
24. Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy[J]. Cancer.1994,73(8):2013-2016.
1.张冰若,劳业兴,苏薇薇.原花青素的研究现状及开发前景[J].中药材.2003,26(12)：905.
2.韩炯,李莹,刘新平,等.葡萄籽提取物原花青素诱导乳腺癌MCF-7细胞脱落凋亡[J].中草药.2003,34(8)：722-725.
3. Yange DR, JR., Sagar SM. Targeting angiogenesis with integrative cancer therapies[J]. Integr Cancer Ther.2006,5(1):9-29.
4. Katiyar SK. Matrix metalloproteinases in cancer metastasis; molecualr targets for prostate cancer prevention by green tea polphenols and grape seed proanthocyanidins[J]. Endocr Metab Immune Disord Drug Targets,2006(6): 17-24.
5. Chang C, Werb Z. The many faces of metalloproteases:cell growth, invasion, angiogenesis, and metastasis[J]. Trends cell.2001(11):37-43.
6. Engelbrecht AM, Mattheyse M, Ellis B, et al. Proanthocyanidin from grape seeds inactives the P13-kinase/PKB pathway and induces apoptosis in a colon cancer cell line[J].Cancer Lett.2007,258(1):144-153.
7.刘洁,庄乾元,张维怡.葡萄籽提取物原花青素对人膀胱癌BIU87细胞增殖和凋亡的影响[J].实用医学杂志.2009,25(18)：3000-3001.
8.刘洁,庄乾元,张维怡.丝裂霉素C联合原花青素对人膀胱癌BIU87细胞的生长抑制作用.医药导报.2009,28(12)：1556-1558.
9. Fraia A. Calhau C. Freitas V,et al Procyanidins as antioxidants and tumor cell growth modulators Agric[J].Food chem,2006; 54:2392-2397.
10. Mantena SK, Katiyar SK. Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes. Free Radic Biol Med,2006; 40(9): 1603-1614.
11. Agarwal C, Singh RP,Dhanalashhmi S. Anti-angiogenic efficacy of grape seed extract in endothelial cells[J]. Oncol Rep.2004(11):681-685.
12. Makarovskiy AN, Siryapom E, Hixson DC, et al. Survival of docetaxel-resistant prostate cancer cells in vitro depends on phenotype alterations and continuity of drug exposure[J].Mol Life Sci.2002,59(7):1198-1211.
13. Busserolles J, Gueux E, Balasinska B,et al. In vivo antioxidant activity of procyanidin-rich extracts from grape seed and pine(pinus maritima)bark inrats[J].Int J Vitam Nutr Res.2006,76(1):22-27.
14. Bagchi D,Garg A, Krohn RL. Protective effective of grape seed proanthocyanidins and seleted antioxidants against TPA-Induced hepatic and brain lipid peroxidation and DNA fragmentation, and peritoneal macrophage activation in mice[J]. Pharmac,1998,30(5):771-776.
15. Sharma SD, Katiyar SK. Ditary grape seed proanthocyanidin inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12[J]. Carcinogenesis,2006(1):95-102.
16. Sharma SD, Meeran SM, Katiyar SK. Dietary grape seed proanthocyanidins inhibit UVB-induced oxidative stress and activation of mitogen-activated protein kinases and nuclear factor-jB signaling in in vivo SKH-1 hairless mice[J].Mol Cancer Ther.2007(6):995-1005.
17. Meeran SM. Proanthocyanidins inhibit mitogenic and survival-signalling in vitro and tumor growth in vivo[J].Front Biosci.2008,13:887-987.
18. Mittal A, Elemets CA, Katiyar SK.Dietary feeding of proanthocyanidins from grape seeds prevents photocarcinogenesis in SKH-1 hairless mice:relationship to decreased fat and lipid peroxidation[J].Carcinogenesis.2003,24:1379-1388.
19. Nomoto H, Iigo M, Hamada H. et al. Chemoprevention of colorectal cancer by grape seed proanthocyanidin is accompanied by a decrease in proliferation and increase in apoptosis. Nutr Cancer,2004; 49(1):81-88.
20. Liu Jie, Zhuang Qianyuan, Zhang Weiyi,et al.Role of survivin in apoptosis induced by grape seed procyanidin extract in human bladder cancer BIU87 cells[J]. Chinese German Journal of Clinical Oncology.2009,8(7):420-425.
21. Agarwal C,Singh RP,Agarwal R.Grape seed extract induces apoptotic death of human prostate carcinoma DU-145 cells via caspases activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release[J]. Carcinogenesis.2002,23(11):1869-1876.
22.鲍永华,郭永臣.原花青素对SMMC-7721肿瘤细胞凋亡及其端粒酶活性的影响[J].西北农林科技大学学报,2007：35(1)37-40
2. Rosen Em, Fan S, Goldberg ID, et al. Biological basis of radiation sensitivity. Part 2:Cellwlar and molecular determinants of radiosensitivity[J]. Oncology (Williston Park).2000,14(5):741-757.
3.骆伟,黄荣清,骆传环,等.苯并杂环化合物的放射增敏研究进展[J].中华放射医学与防护杂志2004,24(2)：170-172.
4.王小萍,云松,张新良.放疗增敏剂甘氨双唑钠的二期临床研究[J].临床肿瘤学杂志.2003,8(1)：28-32.
5.朱靖蓉,王忠民,李瑾瑜,等.葡萄籽原花青素的分离纯化及其抗氧化活性研究[J].中国食物与营养.2007,4：34-37.
6. Packer L, Rinbach G, Virgili F. Antioxidant activity and biologic properties of a procyanidin-rich extract from p ine(Pinus maritim a) bark.pycnogenol [J]. Fre Rad Biol Med.1999,27(5P6):704-724.
7.李素梅,穆春兰.原花青素的研究进展[J].黑龙江医药.2006,19(3)：201-202.
8. Bagchi D, Bagchi M, Stohs S, et al. Cellular protection with proanthocy anidins derived from grape seeds[J]. Ann NYA cad Sci.2002,957(14):260-270.
9.陈小芬,汤为学,李龙江,等.姜黄素对人卵巢癌细胞株SKOV3的抗增殖作用[J].重庆医科大学学报.2005,30(2)：256-259.
10. Kluck RM, Bossy-Wetzel E, Green DR, et al. The release of cytochromec from mitochondria:a primary site for Bcl-2 regulationof apoptosis[J]. Science. 1997,275:1132-1136.
11. Sedlak TW, ltvai ZN, Yang E. Multiple Bcl-2 family members demonstrate selective dimerizations with Bax[J]. Proc Natl Acad Sci USA. 1995,92:7834-7838.
12. Huyny TH, Teel RW. Selective induction of apoptosia in human mammary cancer cell[MCF-7] by Pycnogenol[J]. Anticancer Res.2000,20(4):2417.
13. Shih PH, Yeh CT, Yen GC. Efects of anthocyanidin on the inhibition of proliferation and induction of apoptosis in human gastric adenocarcinoma cells[J]. Food Chem Toxicol.2005,43(10):1557-'1566.
14. Ye X, Krohn RL, Liu W, et al. The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells[J]. Molecular and Cellular Biochemistry.1999,196:(1-2):99-108.
15. Megumi Yamagishi,Midori Natsume,Naomi Osakabe, et al. Chemoprevention of lung carciogenesis by cacao liquor proan thocyanidins in a male rat multi Organ carcinogenesis model. Cancer Letter.2003,191:49-57.
16. Antenask, Baliga MS, Katiyar SK. Grape seed proanthocyanidins induce apoptosis and inhibit metastasis of highly metastatic breast carcinoma cells]J]. Carcinogenesis.2006,27:1682-1691.
17. Meeran SM, Katiyar SK. Grape seed pmanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterafions in Cdki-Cdk-Cyclin cascade. and caspase-3 activation via loss of mitochondrial membrane potential[J]. Exp Dermatol.2007,16:405-415.
18. Meeran SM. Proanthocyanidins inhibit mitogenic and survival signalling in vitro and tumor growth in vivo[J]. Front Biosci.2008,13:887-897.
19. Mantena SK, Katiyar SK. Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes[J]. Free Radic Biol Med.2006,40(9):1603-1614.
20. Schuck A, Poremba C, Lanver C, et al. Radiationinduced changes of telomerase activity in a human Ewing xenograft tumor[J]. Strahlenther Onkol. 2002,178(12):701-708.
21.鲍永华,郭永臣.原花青素对SMMC-7721肿瘤细胞凋亡及其端粒酶活性的影响[J].西北农林科技大学学报.2007,35(1)：37-40.
22.亓玉琴,司君利,李文利,等.端粒酶抑制剂对小鼠胃癌放疗的增敏作用研究[J].肿瘤研究与临床.2003,15(6)：363-364.
23. Roberts JR, Allison DC, Donehower RC, et al. Development of ployploidization in taxol-resistant human leukemia cells in vitro[J]. Cancer Res.1990,50:710-716.
24. Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy[J]. Cancer.1994,73(8):2013-2016.
1.张冰若,劳业兴,苏薇薇.原花青素的研究现状及开发前景[J].中药材.2003,26(12)：905.
2.韩炯,李莹,刘新平,等.葡萄籽提取物原花青素诱导乳腺癌MCF-7细胞脱落凋亡[J].中草药.2003,34(8)：722-725.
3. Yange DR, JR., Sagar SM. Targeting angiogenesis with integrative cancer therapies[J]. Integr Cancer Ther.2006,5(1):9-29.
4. Katiyar SK. Matrix metalloproteinases in cancer metastasis; molecualr targets for prostate cancer prevention by green tea polphenols and grape seed proanthocyanidins[J]. Endocr Metab Immune Disord Drug Targets,2006(6): 17-24.
5. Chang C, Werb Z. The many faces of metalloproteases:cell growth, invasion, angiogenesis, and metastasis[J]. Trends cell.2001(11):37-43.
6. Engelbrecht AM, Mattheyse M, Ellis B, et al. Proanthocyanidin from grape seeds inactives the P13-kinase/PKB pathway and induces apoptosis in a colon cancer cell line[J].Cancer Lett.2007,258(1):144-153.
7.刘洁,庄乾元,张维怡.葡萄籽提取物原花青素对人膀胱癌BIU87细胞增殖和凋亡的影响[J].实用医学杂志.2009,25(18)：3000-3001.
8.刘洁,庄乾元,张维怡.丝裂霉素C联合原花青素对人膀胱癌BIU87细胞的生长抑制作用.医药导报.2009,28(12)：1556-1558.
9. Fraia A. Calhau C. Freitas V,et al Procyanidins as antioxidants and tumor cell growth modulators Agric[J].Food chem,2006; 54:2392-2397.
10. Mantena SK, Katiyar SK. Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes. Free Radic Biol Med,2006; 40(9): 1603-1614.
11. Agarwal C, Singh RP,Dhanalashhmi S. Anti-angiogenic efficacy of grape seed extract in endothelial cells[J]. Oncol Rep.2004(11):681-685.
12. Makarovskiy AN, Siryapom E, Hixson DC, et al. Survival of docetaxel-resistant prostate cancer cells in vitro depends on phenotype alterations and continuity of drug exposure[J].Mol Life Sci.2002,59(7):1198-1211.
13. Busserolles J, Gueux E, Balasinska B,et al. In vivo antioxidant activity of procyanidin-rich extracts from grape seed and pine(pinus maritima)bark inrats[J].Int J Vitam Nutr Res.2006,76(1):22-27.
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