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结直肠癌是严重威胁人类生命健康的常见消化道恶性肿瘤[1]。我国结肠癌发病率呈逐年上升趋势,发病年龄逐渐年轻化,目前位居我国恶性肿瘤死因的第4位。手术治疗是有效地治疗结肠癌的方法,同时采取联合放疗、化疗等方法提升治疗效果,提高患者的生存率。但是,由于结肠癌具有对辐射较强的抗性,严重影响了放疗效果。因此,有效增加结肠癌的辐射敏感性,减少对正常组织的放射损伤,从而提高结肠癌患者的放疗效果和治愈率尤为重要。
褪黑素是由哺乳动物和人类大脑组织中的松果体合成与分泌的一种吲哚类物质,于1958年首次被发现[2]。褪黑素参与机体许多重要的生理进程,包括控制生物节律、抗氧化、抗炎、抗衰老、调节免疫和内分泌的功能[3-4]。大量文献报道,褪黑素对不同肿瘤具有抗瘤的活性,例如乳腺癌[5]、肺癌[6]、食管癌[7]、胰腺癌[8]以及前列腺癌[9]。褪黑素对不同的结肠癌类型也有抗瘤特性[10-11]。有研究结果表明,褪黑素有可能作为新型辐射增敏剂应用到临床[12-13]。但是,褪黑素联合放疗应用于结肠癌治疗的研究并不多,在放疗前使用褪黑素对结肠癌会产生什么效应并不明了。本研究通过观察褪黑素联合γ射线照射对体外结肠癌HCT 116细胞生长的影响,以及对荷瘤裸鼠的抑瘤效应,阐明褪黑素对结肠癌细胞辐射敏感性的影响,为以后临床上提高结肠癌的放疗效果提供实验依据。
褪黑素对人结肠癌细胞辐射敏感性的影响
Effects of melatonin on the radiosensitivity of human colon cancer cells
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摘要:
目的 分析褪黑素联合γ射线照射对体外和体内人结肠癌HCT 116细胞生长的影响,探讨褪黑素在人结肠癌HCT 116细胞辐射敏感性中的作用。 方法 将人结肠癌HCT 116细胞分为4组,即:空白对照组(不给予任何处理)、褪黑素组(给予褪黑素,给药浓度为1 mmol/L,给药时间为2 h)、照射组(接受6 Gy γ射线照射)及褪黑素+照射组(在照射前2 h给予褪黑素,给药浓度为1 mmol/L,然后接受6 Gy γ射线照射)。体外实验:人结肠癌HCT 116细胞分别进行2、4、6、8 Gy照射,采用克隆形成实验检测细胞的增殖能力;人结肠癌HCT 116细胞进行6 Gy照射,采用流式细胞术检测24 h后细胞周期以及24 h和48 h后细胞的凋亡;采用彗星实验检测2 h后细胞DNA的损伤。体内实验:将人结肠癌HCT 116细胞接种于裸鼠体内建立肿瘤模型,检测结肠癌瘤体体积和瘤体质量的变化并计算抑瘤率。两组间比较采用t检验。 结果 ①体外实验:照射前给予褪黑素处理的人结肠癌HCT 116 细胞的克隆形成数目明显少于对照组,差异有统计学意义(t=3.83,P=0.005);褪黑素+照射组停留在G2期的人结肠癌HCT 116细胞比例显著增加(53.04%±4.67%),与照射组(42.83%±7.10%)和褪黑素组(12.95%±0.96%)相比,差异均有统计学意义(t=2.94、20.66,P=0.017、P<0.01);褪黑素+照射组在处理后24 h和 48 h大量人结肠癌HCT 116细胞发生细胞凋亡,凋亡率分别达到(12.15±0.41)%和(30.57±1.91)%,与照射组(9.00%±0.70%、8.69%±0.71%)和褪黑素组(3.03%±0.42%、12.56%±0.89%)相比,差异均有统计学意义(t=7.46、17.75、29.12、14.80,均P<0.01);褪黑素+照射组HCT 116细胞的尾部DNA含量、尾长、尾矩和Olive尾矩均明显高于照射组(t=4.72、4.16、4.74、4.50,均P<0.01)和褪黑素组(t=20.27、22.80、13.81、18.85,均P<0.01),差异均有统计学意义。②体内实验:褪黑素+照射组结肠癌生长速度减慢,到处理后的第15天肿瘤体积明显小于照射组和褪黑素组,差异有统计学意义(t=3.51、2.72, P=0.006、P=0.021);褪黑素+照射组抑瘤率最高(54.7%±8.0%),远远高于照射组和褪黑素组(t=7.50、4.12,均P<0.01)。 结论 褪黑素联合辐射对人结肠癌细胞生长有显著的抑制效应,提高了细胞对γ射线辐射的敏感性。 Abstract:Objective To detect the effects of melatonin combined with γ-ray ionizing radiation on the proliferation of human colon cancer HCT 116 cells in vitro and in vivo and to explore the role of melatonin in regulating the radiosensitivity of HCT 116 cells. Methods The cohorts were divided into blank control group (HCT 116 cells were not given any treatment), melatonin group (HCT 116 cells were treated with 1 mmol/L melatonin for 2 h), radiation group (HCT 116 cells were exposed to 6 Gy γ-ray radiation), and melatonin+radiation group (HCT 116 cells were treated with 1 mmol/L melatonin for 2 h and then exposed to 6 Gy γ-ray radiation). In in vitro experiments, colony formation assay was used to detect cell proliferation after exposure to 0, 2, 4, 6, or 8 Gy radiation. Flow cytometry was applied to detect the cell cycle distribution at 24 h and cell apoptosis at 24 and 48 h after exposure to 6 Gy radiation. Comet assay was performed to detect DNA damage to cells 2 h after exposure to 6 Gy radiation. In in vivo experiments, the tumor-bearing nude mouse model was built by inoculating HCT 116 cells. The volume and inhibition ratio of tumor xenografts were examined. T-test was used for comparison between groups. Results ① In in vitro experiments, the colony number of HCT 116 cells treated with melatonin prior to radiation was significantly less than that of control cells (t=3.83, P=0.005). HCT 116 cells that were arrested at the G2 phase in the melatonin+radiation group (53.04%±4.67%) were increased, and significant differences were noted between the melatonin+radiation group and the radiation or melatonin group (t=2.940 and 20.660, P=0.017 and P<0.01, respectively). The cell apoptosis rate of HCT 116 cells in the melatonin+radiation group at 24 and 48 h after treatment was increased and reached (12.15±0.41)% and (30.57±1.91)%, respectively, which were markedly higher than those of the radiation (9.00%±0.70%, 8.69%±0.71%) or melatonin group (3.03%±0.42%, 12.56%±0.89%) (t=7.46, 17.75, 29.12, and 14.80, all P<0.01). The value of tail DNA, tail length, tail moment, and Olive tail moment in HCT 116 cells in the melatonin+radiation group were significantly higher than that in the radiation (t=4.72, 4.16, 4.74, 4.50, all P<0.01) or melatonin group (t=20.27, 22.80, 13.81, and 18.85, all P<0.01). ② In in vivo experiments, tumor xenografts in nude mice of the melatonin+radiation group grew slowly. The volume of tumor xenografts in the melatonin+radiation group at day 15 was significantly decreased compared with that in the radiation or melatonin group (t=3.51 and 2.72, P=0.006 and P=0.021, respectively). The inhibition ratio of xenografts in the melatonin+radiation group (54.7%±8.0%) was significantly higher than that in the radiation or melatonin group (t=7.50, 4.12, all P<0.01). Conclusion Melatonin combined with γ-ray radiation had obvious inhibition effect on colon cancer cells and increased the radiosensitivity of colon cancer cells. -
Key words:
- Colonic neoplasms /
- Radiation tolerance /
- Tumor-bearing nude mice /
- Melatonin
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[1] Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2018, 68(6): 394−424. DOI: 10.3322/caac.21492. [2] Alberti C. Melatonin: the first hormone isolated from the pineal body[J]. Farmaco Sci, 1958, 13(8): 604−605. [3] Su SC, Hsieh MJ, Yang WE, et al. Cancer metastasis: Mechanisms of inhibition by melatonin[J/OL]. J Pineal Res, 2017, 62(1): e12370 [2019-01-17]. https://onlinelibrary.wiley.com/doi/full/10.1111/jpi.12370. DOI: 10.1111/jpi.12370. [4] Singh M, Jadhav HR. Melatonin: functions and ligands[J]. Drug Discov Today, 2014, 19(9): 1410−1418. DOI: 10.1016/j.drudis.2014.04.014. [5] Alonso-González C, González A, Martínez-Campa C, et al. Melatonin enhancement of the radiosensitivity of human breast cancer cells is associated with the modulation of proteins involved in estrogen biosynthesis[J]. Cancer Lett, 2016, 370(1): 145−152. DOI: 10.1016/j.canlet.2015.10.015. [6] Zhou QY, Gui SY, Zhou Q, et al. Melatonin Inhibits the Migration of Human Lung Adenocarcinoma A549 Cell Lines Involving JNK/MAPK Pathway[J/OL]. PLoS One, 2014, 9(7): e101132 [2019-01-17]. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101132. DOI: 10.1371/journal.pone.0101132. [7] Lu YX, Chen DL, Wang DS, et al. Melatonin enhances sensitivity to fluorouracil in oesophageal squamous cell carcinoma through inhibition of Erk and Akt pathway[J/OL]. Cell Death Dis, 2016, 7(10): e2432 [2019-01-17]. https://www.nature.com/articles/cddis2016330. DOI: 10.1038/cddis.2016.330. [8] Ju HQ, Li H, Tian T, et al. Melatonin overcomes gemcitabine resistance in pancreatic ductal adenocarcinoma by abrogating nuclear factor-κB activation[J]. J Pineal Res, 2016, 60(1): 27−38. DOI: 10.1111/jpi.12285. [9] Tai SY, Huang SP, Bao BY, et al. Urinary melatonin-sulfate/cortisol ratio and the presence of prostate cancer: A case-control study[J/OL]. Sci Rep, 2016, 6: 29606 [2019-01-17]. https://www.nature.com/articles/srep29606. DOI: 10.1038/srep29606. [10] Fic M, Gomulkiewicz A, Grzegrzolka J, et al. The Impact of Melatonin on Colon Cancer Cells' Resistance to Doxorubicin in An In Vitro Study[J/OL]. Int J Mol Sci, 2017, 18(7): 1396[2019-01-17]. https://www.mdpi.com/1422-0067/18/7/1396. DOI: 10.3390/ijms18071396. [11] Bułdak RJ, Pilc-Gumuła K, Bułdak Ł, et al. Effects of ghrelin, leptin and melatonin on the levels of reactive oxygen species, antioxidant enzyme activity and viability of the HCT 116 human colorectal carcinoma cell line[J]. Mol Med Rep, 2015, 12(2): 2275−2282. DOI: 10.3892/mmr.2015.3599. [12] Onseng K, Johns NP, Khuayjarernpanishk T, et al. Beneficial Effects of Adjuvant Melatonin in Minimizing Oral Mucositis Complications in Head and Neck Cancer Patients Receiving Concurrent Chemoradiation[J]. J Altern Complement Med, 2017, 23(12): 957−963. DOI: 10.1089/acm.2017.0081. [13] Zou ZW, Liu T, Li Y, et al. Melatonin suppresses thyroid cancer growth and overcomes radioresistance via inhibition of p65 phosphorylation and induction of ROS[J]. Redox Biol, 2018, 16: 226−236. DOI: 10.1016/j.redox.2018.02.025. [14] Hirota Y, Masunaga S, Kondo N, et al. High linear-energy-transfer radiation can overcome radioresistance of glioma stem-like cells to low linear-energy-transfer radiation[J]. J Radiat Res, 2014, 55(1): 75−83. DOI: 10.1093/jrr/rrt095. [15] Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy[J]. Int J Radiat Oncol Biol Phys, 2004, 59(4): 928−942. DOI: 10.1016/j.ijrobp.2004.03.005. [16] Li W, Fan M, Chen Y, et al. Melatonin Induces Cell Apoptosis in AGS Cells Through the Activation of JNK and P38 MAPK and the Suppression of Nuclear Factor-Kappa B: A Novel Therapeutic Implication for Gastric Cancer[J]. Cell Physiol Biochem, 2015, 37(6): 2323−2338. DOI: 10.1159/000438587. [17] Wenzel U, Nickel A, Daniel H. Melatonin potentiates flavone-induced apoptosis in human colon cancer cells by increasing the level of glycolytic end products[J]. Int J Cancer, 2005, 116(2): 236−242. DOI: 10.1002/ijc.20837. [18] Kontek R, Nowicka H. The modulatory effect of melatonin on genotoxicity of irinotecan in healthy human lymphocytes and cancer cells[J]. Drug Chem Toxicol, 2013, 36(3): 335−342. DOI: 10.3109/01480545.2012.737805. [19] Alonso-González C, González A, Martínez-Campa C, et al. Melatonin sensitizes human breast cancer cells to ionizing radiation by downregulating proteins involved in double-strand DNA break repair[J]. J Pineal Res, 2015, 58(2): 189−197. DOI: 10.1111/jpi.12205.