和厚朴酚对人结肠癌细胞放疗增敏的实验研究
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摘要
背景美国肿瘤协会统计结果表明,结肠癌在各类恶性肿瘤发病率及死亡率中均占第三位,放射治疗是结肠癌综合治疗中的重要组成部分,尤其是对于较晚期的结直肠癌,放射治疗的作用更显得重要,然而高剂量的放射治疗给患者带来严重并发症,某些类型结肠癌细胞对于放疗敏感性低,甚至发生放射抵抗。寻找一种理想的放疗增敏剂,既可以抑制肿瘤细胞生长,又能增加肿瘤细胞对放疗的敏感性,成为当前抗肿瘤研究的一个重要方向。DNA错配基因修复系统可以修复DNA复制和重组过程中出现的基因错配,而DNA错配基因修复系统的功能缺失会导致由DNA损伤所致的生长停滞。研究发现,人结肠癌细胞HCT116的hMLH1错配修复基因发生突变,导致微卫星不稳定性和错配基因修复缺失。和厚朴酚是从中国传统中药厚朴中分离出的联苯酚类化合物,研究表明,和厚朴酚对多种肿瘤具有生长抑制作用。
目的本课题旨在研究和验证和厚朴酚对人结肠癌HCT116细胞的放疗增敏作用,检验错配基因修复系统在和厚朴酚对人结肠癌HCT116细胞的放疗增敏的调控作用,观察和厚朴酚对错配基因修复正常的HCT116-CH3细胞和错配基因修复缺失的HCT116细胞的放疗增敏作用的差异。探讨hMLH1基因对于和厚朴酚在人结肠癌细胞放疗作用的可能调控机制。
实验方法首先通过联合应用不同浓度与剂量的和厚朴酚(10umol/ml,20umol/ml,30umol/ml,40umol/ml,50umol/ml)与γ-射线(0,2.5,5.0Gy)作用于人结肠癌细胞(HCT116、HCT116-CH2和HCT116-CH3细胞),分别作用24h、48h、72h,观察和厚朴酚对人结肠癌细胞的生长抑制和放疗增敏作用；平板细胞集落形成实验可进一步验证和厚朴酚作用的远期效果；采用流式细胞术分析和厚朴酚及γ-射线作用后细胞周期调控情况,Caspase3,7活性分析实验检测凋亡的发生,再通过Western Blotting蛋白印迹实验和RT-PCR检测检测凋亡相关蛋白表达水平及RNA复制水平的变化。
结果单用和厚朴酚或和厚朴酚联合γ-射线对三个细胞系的细胞增殖和克隆形成均有明显的抑制作用,且呈显著的剂量-,时间-依赖性。然而,抑制效果在hMLH1缺陷型的HCT116、HCT116-CH2细胞更为显著。单用和厚朴酚作用48小时的IC50是40umol/ml,单用Y-射线ID50需剂量为5Gy作用72小时,而联用和厚朴酚与γ-射线细胞增殖IC50为25umol/ml HNK+2.5Gy γ-射线作用48小时,和厚朴酚对人结肠癌细胞放疗增敏作用较明显。经25umol/ml HNK+2.5Gy γ-射线作用48小时,hMLHl缺陷型HCT116细胞与hMLHl完整型HCT116-CH3细胞的增值分数分别是41.55%和68.02%；与hMLHl完整型HCT116-CH3细胞比较,联用和厚朴酚和Y-射线可以使hMLHl缺陷型HCT116细胞的Caspases系统激活与P-p53蛋白表达更为显著。在hMLHl完整型HCT116-CH3细胞组中P-p53蛋白表达的水平极低,而促凋亡基因Bax的表达水平比hMLHl缺陷型HCT116细胞显著升高,抑凋亡基因Bcl-2表达则显著下降。流式细胞术细胞周期分析发现和厚朴酚与Y-射线联合作用后,可诱导较高水平的细胞凋亡,在HCT116细胞组,凋亡现象发生更加显著。联合应用可以导致G0/G1期,G2/M期细胞阻滞,Western Blotting检测HCT116和HCT116-CH3细胞系均有cyclin Bl与Cyclin Dl的表达水平下降,而HCT116细胞作用后下降的更为明显,提示hMLHl的高水平表达能够降低和厚朴酚对人结肠癌细胞的放疗增敏作用。
结论1、和厚朴酚是一种非常有效的结肠癌细胞放疗增敏剂,尤其是对错配基因修复功能缺陷的结肠癌细胞效果更明显；2、和厚朴酚可以导致人结肠癌细胞发生G0/G1期阻滞；3、和厚朴酚联合Y-射线作用人结肠癌细胞凋亡增加；4、hMLHl gene可能通过抑制P53蛋白的功能活性来降低和厚朴酚对人结肠癌细胞的放疗增敏性。
Background:Both of the morbidity and mortality of colorectal cancer were in the third place among all kinds of malignant tumors in USA. Radiation therapy plays an important role in the complicated treatment of colorectal cancers, especially for those late-stage patients. However, high dose radiation will lead to some deadly side effects to those patients, some colorectal cancer cells are not sensitive to radiation therapy, and some cancer cells show the capability of radiation resistance. It is necessary to find an ideal radiosensitizer, which can not only inhibit the growth of colorectal cancer cells but also increase the sensitivity to radiation therapy. Mismatch repair is required for correcting any mismatches that are created during replication and recombination, and a defective mismatch repair system contributes to DNA damage-induced growth arrest. The colorectal cancer cell line HCT116is known to have a mutation in the hMLHl mismatch repair gene resulting in microsatellite instability and defective mismatch repair. Honokiol is a biphenolic compound that has been used in traditional Chinese medicine for treating various ailments including cancer.
Objective:This study was designed to test the hypothesis that honokiol enhances the radiosensitivity of cancer cells with mismatch repair defect (HCT116) compared with those that are mismatch repair proficient (HCT116-CH3), and to explore the possible mechanism of hMLHl gene in adjusting rediosensitizing effect of honokiol in human colon cancer cells.
Methods:Three human colon cancer cell lines HCT116cell, HCT116-CH2cell and HCT116-CH3cell were treated with the combination of different concentrations of honokiol(10umol/ml,20umol/ml,30umol/ml,40umol/ml,50umol/ml)and different dose of γ-irradiation(0,2.5,5.OGy) for different time points(24h,48h,72h), detecting honokiol's growth inhibition and radiosensitizing effects on human colon cancer cells. Cell colony experiement was used to test honokiol's long-time effects. With the same treatment, cells were collected and cell cycles were analyzed with Flow cytometry analysis. Caspase3,7assay kit help to detect the apoptosis happened the combination of honokiol and γ-irradiation. The protein and mRNA variation of apoptosis related gene, such as p53, Bax and Bcl-2, were detected with western blotting and RT-PCR.
Results:First, Cell proliferation data indicated that the combination of honokiol and/or γ-irradiation treatment could lead to time-and dose-dependent inhibition of cell proliferation and colony formation in all three cell lines, the effect was more pronounced in hMLHl deficient HCT116and HCT116-CH2cells. Only treated with honokiol, the IC50of48hours is40umol/ml, and ID50the only treated with Y-irradiation was5Gy for72hours. While treated with the combination of honokiol and γ-irradiation, IC50were25umol/ml and2.5Gy for48hours, which indicated honokiol greatly increased the radiosensitivity of human colon cancer cells. With the same treatment, the proliferation inhibition of hMLHl deficient HCT116(41.55%) was more significant than that in hMLHl proficient HCT116-CH3cell (68.02%). Compared with HCT116-CH3cells, similarly, the combination of honokiol and Y irradiation treatment induced higher levels of apoptosis (caspase3activation, P-p53expression) in the HCT116cells. There were significantly lower amounts of phosphorylated p53in the HCT116-CH3cells, while there were much higher level of proapoptotic gene Bax and lower level of antiapoptotic gene Bcl-2expression. Cell cycle analyses revealed higher levels of dead cells in the HCT116cells than in the HCT116-CH3cell. The combination treatment reduced expression of cyclin B1and D1in both cell lines, which suggested that the high levels of hMLH1may reduce the radiosensitivity of human colon cancer cells. These data demonstrated that honokiol may be highly effective in radiosensitizing colorectal cancer cells, especially in those with a mismatch repair defect. hMLH1gene may reduce Honokiol's radiosensitizing effect by inhibit the activation of p53protein in colorectal cancer cells.
Conclusion:1. Honokiol is an effective radiosensitizer for human colon cancer cells, especially for mismatch repair defect colon cancer cells.2. Honokiol can lead to the G0/G1phase arrest of human colon cancer cells.3. The combination of honokiol and γ-irradiation can increase the apoptosis of human colon cancer cells.4. hMLHl gene may reduce honokiol's radiosensitizing effect on human colon cancer cells by inhibiting the activity of P53.
目的本课题旨在研究和验证和厚朴酚对人结肠癌HCT116细胞的放疗增敏作用,检验错配基因修复系统在和厚朴酚对人结肠癌HCT116细胞的放疗增敏的调控作用,观察和厚朴酚对错配基因修复正常的HCT116-CH3细胞和错配基因修复缺失的HCT116细胞的放疗增敏作用的差异。探讨hMLH1基因对于和厚朴酚在人结肠癌细胞放疗作用的可能调控机制。
实验方法首先通过联合应用不同浓度与剂量的和厚朴酚(10umol/ml,20umol/ml,30umol/ml,40umol/ml,50umol/ml)与γ-射线(0,2.5,5.0Gy)作用于人结肠癌细胞(HCT116、HCT116-CH2和HCT116-CH3细胞),分别作用24h、48h、72h,观察和厚朴酚对人结肠癌细胞的生长抑制和放疗增敏作用；平板细胞集落形成实验可进一步验证和厚朴酚作用的远期效果；采用流式细胞术分析和厚朴酚及γ-射线作用后细胞周期调控情况,Caspase3,7活性分析实验检测凋亡的发生,再通过Western Blotting蛋白印迹实验和RT-PCR检测检测凋亡相关蛋白表达水平及RNA复制水平的变化。
结果单用和厚朴酚或和厚朴酚联合γ-射线对三个细胞系的细胞增殖和克隆形成均有明显的抑制作用,且呈显著的剂量-,时间-依赖性。然而,抑制效果在hMLH1缺陷型的HCT116、HCT116-CH2细胞更为显著。单用和厚朴酚作用48小时的IC50是40umol/ml,单用Y-射线ID50需剂量为5Gy作用72小时,而联用和厚朴酚与γ-射线细胞增殖IC50为25umol/ml HNK+2.5Gy γ-射线作用48小时,和厚朴酚对人结肠癌细胞放疗增敏作用较明显。经25umol/ml HNK+2.5Gy γ-射线作用48小时,hMLHl缺陷型HCT116细胞与hMLHl完整型HCT116-CH3细胞的增值分数分别是41.55%和68.02%；与hMLHl完整型HCT116-CH3细胞比较,联用和厚朴酚和Y-射线可以使hMLHl缺陷型HCT116细胞的Caspases系统激活与P-p53蛋白表达更为显著。在hMLHl完整型HCT116-CH3细胞组中P-p53蛋白表达的水平极低,而促凋亡基因Bax的表达水平比hMLHl缺陷型HCT116细胞显著升高,抑凋亡基因Bcl-2表达则显著下降。流式细胞术细胞周期分析发现和厚朴酚与Y-射线联合作用后,可诱导较高水平的细胞凋亡,在HCT116细胞组,凋亡现象发生更加显著。联合应用可以导致G0/G1期,G2/M期细胞阻滞,Western Blotting检测HCT116和HCT116-CH3细胞系均有cyclin Bl与Cyclin Dl的表达水平下降,而HCT116细胞作用后下降的更为明显,提示hMLHl的高水平表达能够降低和厚朴酚对人结肠癌细胞的放疗增敏作用。
结论1、和厚朴酚是一种非常有效的结肠癌细胞放疗增敏剂,尤其是对错配基因修复功能缺陷的结肠癌细胞效果更明显；2、和厚朴酚可以导致人结肠癌细胞发生G0/G1期阻滞；3、和厚朴酚联合Y-射线作用人结肠癌细胞凋亡增加；4、hMLHl gene可能通过抑制P53蛋白的功能活性来降低和厚朴酚对人结肠癌细胞的放疗增敏性。
Background:Both of the morbidity and mortality of colorectal cancer were in the third place among all kinds of malignant tumors in USA. Radiation therapy plays an important role in the complicated treatment of colorectal cancers, especially for those late-stage patients. However, high dose radiation will lead to some deadly side effects to those patients, some colorectal cancer cells are not sensitive to radiation therapy, and some cancer cells show the capability of radiation resistance. It is necessary to find an ideal radiosensitizer, which can not only inhibit the growth of colorectal cancer cells but also increase the sensitivity to radiation therapy. Mismatch repair is required for correcting any mismatches that are created during replication and recombination, and a defective mismatch repair system contributes to DNA damage-induced growth arrest. The colorectal cancer cell line HCT116is known to have a mutation in the hMLHl mismatch repair gene resulting in microsatellite instability and defective mismatch repair. Honokiol is a biphenolic compound that has been used in traditional Chinese medicine for treating various ailments including cancer.
Objective:This study was designed to test the hypothesis that honokiol enhances the radiosensitivity of cancer cells with mismatch repair defect (HCT116) compared with those that are mismatch repair proficient (HCT116-CH3), and to explore the possible mechanism of hMLHl gene in adjusting rediosensitizing effect of honokiol in human colon cancer cells.
Methods:Three human colon cancer cell lines HCT116cell, HCT116-CH2cell and HCT116-CH3cell were treated with the combination of different concentrations of honokiol(10umol/ml,20umol/ml,30umol/ml,40umol/ml,50umol/ml)and different dose of γ-irradiation(0,2.5,5.OGy) for different time points(24h,48h,72h), detecting honokiol's growth inhibition and radiosensitizing effects on human colon cancer cells. Cell colony experiement was used to test honokiol's long-time effects. With the same treatment, cells were collected and cell cycles were analyzed with Flow cytometry analysis. Caspase3,7assay kit help to detect the apoptosis happened the combination of honokiol and γ-irradiation. The protein and mRNA variation of apoptosis related gene, such as p53, Bax and Bcl-2, were detected with western blotting and RT-PCR.
Results:First, Cell proliferation data indicated that the combination of honokiol and/or γ-irradiation treatment could lead to time-and dose-dependent inhibition of cell proliferation and colony formation in all three cell lines, the effect was more pronounced in hMLHl deficient HCT116and HCT116-CH2cells. Only treated with honokiol, the IC50of48hours is40umol/ml, and ID50the only treated with Y-irradiation was5Gy for72hours. While treated with the combination of honokiol and γ-irradiation, IC50were25umol/ml and2.5Gy for48hours, which indicated honokiol greatly increased the radiosensitivity of human colon cancer cells. With the same treatment, the proliferation inhibition of hMLHl deficient HCT116(41.55%) was more significant than that in hMLHl proficient HCT116-CH3cell (68.02%). Compared with HCT116-CH3cells, similarly, the combination of honokiol and Y irradiation treatment induced higher levels of apoptosis (caspase3activation, P-p53expression) in the HCT116cells. There were significantly lower amounts of phosphorylated p53in the HCT116-CH3cells, while there were much higher level of proapoptotic gene Bax and lower level of antiapoptotic gene Bcl-2expression. Cell cycle analyses revealed higher levels of dead cells in the HCT116cells than in the HCT116-CH3cell. The combination treatment reduced expression of cyclin B1and D1in both cell lines, which suggested that the high levels of hMLH1may reduce the radiosensitivity of human colon cancer cells. These data demonstrated that honokiol may be highly effective in radiosensitizing colorectal cancer cells, especially in those with a mismatch repair defect. hMLH1gene may reduce Honokiol's radiosensitizing effect by inhibit the activation of p53protein in colorectal cancer cells.
Conclusion:1. Honokiol is an effective radiosensitizer for human colon cancer cells, especially for mismatch repair defect colon cancer cells.2. Honokiol can lead to the G0/G1phase arrest of human colon cancer cells.3. The combination of honokiol and γ-irradiation can increase the apoptosis of human colon cancer cells.4. hMLHl gene may reduce honokiol's radiosensitizing effect on human colon cancer cells by inhibiting the activity of P53.
引文
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43. Deng, G. Chen, A. Hong, J. et al. Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res,1999. 59(9):p.2029-2033.
44. Nystrom-Lahti. M. Parsons, R. Sistonen, P.et al., Mismatch repair genes on chromosomes 2p and 3p account for a major share of hereditary nonpolyposis colorectal cancer families evaluable by linkage. Am J Hum Genet,1994.55(4):p.659-665.
45. Miyata, H. Doki. Y. Shiozaki, H.et al. CDC25B and p53 are independently implicated in radiation sensitivity for human esophageal cancers. Clin Cancer Res,2000.6(12):p. 4859-4865.
46. Dietrich, J.B. Apoptosis and anti-apoptosis genes in the Bcl-2 family. Arch Physiol Biochem,1997.105(2):p.125-135.
47. Diaz, J.L. Oltersdorf, T. Home, W. et al. A common binding site mediates heterodimerization and homo dimerization of Bcl-2 family members. J Biol Chem,1997. 272(17):p.11350-5.
48. Suzuki, M..Youle. R.J. Tjandra. N. Structure of Box:coregulation of dimer formation and intracellular localization. Cell,2000.103(4):p.645-654.
49. Reed, J.C. Tsujimoto, Y. Alpers, J. D. et al. Regulation of bcl-2 proto-oncogene expression during normal human lymphocyte proliferation. Science,1987.236(4806):p. 1295-1299.
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52. Konoshima, T. Kozuka, M. Tokuda, H. et al. Studies on inhibitors of skin tumor promotion, IX. Neolignans from Magnolia officinalis. J Nat Prod,1991.54(3):p. 816-822.
53. Wang, Y. Z. Yang. X. Zhao. Honokiol induces paraptosis and apoptosis and exhibits schedule-dependent synergy in combination with imatinib in human leukemia cells. Toxicol Mech Methods,2010.20(5):p.234-241.
54. Shigemura, K. Arbiser, J.L. Sun, S.Y. et al. Honokiol, a natural plant product, inhibits the bone metastatic growth of human prostate cancer cells. Cancer,2007.109(7):p. 1279-1289.
55. Battle, T.E. Arbiser.J.. Frank. D.A. The natural product honokiol induces caspase-dependent apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells. Blood,2005.106(2):p.690-697.
56. Li, L. Han, W. Gu, Y. et al. Honokiol induces a necrotic cell death through the mitochondrial permeability transition pore. Cancer Res,2007.67(10):p.4894-4903.
57. Xu, D. Q. Lu. X.Hu. Down-regulation of P-glycoprotein expression in MDR breast cancer cell MCF-7/ADR by honokiol. Cancer Lett,2006.243(2):p.274-280.
58. Wolf, I. O'Kelly, J. Wakimoto, N. et al. Honokiol, a natural biphenyl, inhibits in vitro and in vivo growth of breast cancer through induction of apoptosis and cell cycle arrest. Int J Oncol,2007.30(6):p.1529-1537.
59. Hou, W. Chen, L. Yang, G. et al., Synergistic antitumor effects of liposomal honokiol combined with adriamycin in breast cancer models. Phytother Res,2008.22(8):p. 1125-1132.
60. Garcia, A. Zheng, Y. Zhao, C. et al. Honokiol suppresses survival signals mediated by Ras-dependent phospholipase D activity in human cancer cells. Clin Cancer Res,2008. 14(13):p.4267-4274.
61. Park, E.J. Min, H. Y. Chung, H. J. et al., Down-regulation of c-Src/EGFR-mediated signaling activation is involved in the honokiol-induced cell cycle arrest and apoptosis in MDA-MB-231 human breast cancer cells. Cancer Lett,2009.277(2):p.133-140.
62. Raja, S.M. Chen, S. Yue, P. et al. The natural product honokiol preferentially inhibits cellular FLICE-inhibitory protein and augments death receptor-induced apoptosis. Mol Cancer Ther,2008.7(7):p.2212-2223.
63. Jiang, Q.Q. Fan, L. Y. Yang, G. L.et al., Improved therapeutic effectiveness by combining liposomal honokiol with cisplatin in lung cancer model. BMC Cancer,2008.8:p.242.
64. Wen, J. Fu, A. F. Chen, L. J. et al. Liposomal honokiol inhibits VEGF-D-induced lymphangiogenesis and metastasis in xenograft tumor model. Int J Cancer,2009.124(11): p.2709-2718.
65. Lee, A.S. The glucose-regulated proteins:stress induction and clinical applications. Trends Biochem Sci,2001.26(8):p.504-510.
66. Little, E. Ramakrishnan, M.Roy, B. et al., The glucose-regulated proteins (GRP78 and GRP94):functions, gene regulation, and applications. Crit Rev Eukaryot Gene Expr, 1994.4(1):p.1-18.
67. Little, E. A.S. Lee. Generation of a mammalian cell line deficient in glucose-regulated protein stress induction through targeted ribozyme driven by a stress-inducible promoter. J Biol Chem,1995.270(16):p.9526-9534.
68. Kubota, H. Suzuki, T. Lu, J.et al., Increased expression of GRP94 protein is associated with decreased sensitivity to X-rays in cervical cancer cell lines. Int J Radiat Biol,2005. 81(9):p.701-709.
69. Sheu, M.L. S.H. Liu. K.H. Lan. Honokiol induces calpain-mediated glucose-regulated protein-94 cleavage and apoptosis in human gastric cancer cells and reduces tumor growth. PLoS One,2007.2(10):p.1096.
70. Liu, S.H. Shen, C. C. Yi, Y. C.et al. Honokiol inhibits gastric tumourigenesis by activation of 15-lipoxygenase-l and consequent inhibition of peroxisome proliferator-activated receptor-gamma and COX-2-dependent signals. Br J Pharmacol, 2010.160(8):p.1963-1972.
71. Hahm, E.R. Arlotti, J. A. Marynowski, S. W. et al. Honokiol, a constituent of oriental medicinal herb magnolia officinalis, inhibits growth of PC-3 xenografts in vivo in association with apoptosis induction. Clin Cancer Res,2008.14(4):p.1248-1257.
72. Yuan, Z. Liu, H. Yan, F..et al. Improved therapeutic efficacy against murine carcinoma by combining honokiol with gene therapy of PNAS-4, a novel pro-apoptotic gene. Cancer Sci,2009.100(9):p.1757-1766.