目的 探究杜仲绿原酸（CGA）通过调控PI3K/AKT/Nrf-2通路，对视网膜母细胞瘤细胞系HXO-Rb44放射敏感性的增强作用。 方法 通过不同浓度的CGA处理HXO-Rb44细胞，检测IC10，作为后续实验中CGA浓度。将细胞分为Ctrl、Radio、CGA、Radio+CGA组，Radio组给予4 MV X射线照射24 h，CGA组使用CGA处理24 h，Radio+CGA组在CGA处理24 h后再给予4 MV X射线照射24 h，流式细胞术检测细胞周期与细胞凋亡，蛋白质印迹检测Ki67、依赖还原型辅酶I/II醌氧化还原酶I（NQO1）、活化型半胱天冬酶（cl-caspase-3）、硫氧还蛋白还原酶1（TrxR1）、活化磷脂酰肌醇3-激酶（p-PI3K）、蛋白激酶B（AKT）、p-AKT、核因子E2相关因子2（Nrf2）蛋白表达。进一步通过PI3K激活剂740Y-P处理细胞，检测细胞周期、凋亡、增殖和相关蛋白表达、PI3K/AKT/Nrf2通路蛋白表达。 结果 随着CGA浓度的增高，细胞的相对存活率降低，药物毒性呈浓度依赖性，IC10为81.59 μmol/L。与Ctrl组相比，Radio和CGA组G2/M期细胞比例显著升高，细胞凋亡比率显著升高（P<0.01）；与Radio组相比，Radio+CGA组G2/M期细胞比例和细胞凋亡比率进一步显著升高（P<0.01）。同时在蛋白水平上，与Ctrl组相比，Radio和CGA组Ki67、NQO1、TrxR1、p-PI3K、Nrf2蛋白表达及p-AKT/AKT比率显著降低（P<0.01）；放射和CGA联合作用进一步降低以上各指标（P<0.01）。此外，PI3K激活剂可逆转放射对细胞周期、增殖、凋亡以及PI3K/AKT/Nrf2通路的作用，CGA可恢复放射引起的上述各指标的变化（P<0.01）。 结论 CGA可增强HXO-Rb44细胞的放射敏感性，其作用机制与抑制PI3K/AKT/Nrf2通路相关。
Abstract
Objective To investigate the enhancement of eucommia chlorogenic acid(CGA) on the radiosensitivity of retinoblastoma cell line HXO-Rb44 by regulating PI3K/AKT/Nrf-2 pathway. Methods HXO-Rb44 cells were treated with different concentrations of CGA, then IC10 was detected as CGA concentration in subsequent experiments. Cells were divided into 4 groups: a Ctrl group, a Radio group, a CGA group and a Radio+CGA group. Radio group was disposed with 4 MV X-rays for 24 h, CGA group was processed with CGA for 24 h, and Radio+CGA group was disposed with 4 MV X-rays for 24 h after CGA treatment for 24 h. Flow cytometry was used to detect the cell cycle and cell apoptosis. The protein expressions of Ki67, NAD (P) H quinone oxidoreductase I (NQO1), cleaved-caspase-3 (cl-caspase-3), thioredoxin reductase-1 (TrxR1), activated phosphatidylinositol 3-kinase (PI3K), protein kinase B (AKT), p-AKT and nuclear factor-erythroid 2-related factor 2 (Nrf2) were determined by Western blot. Furthermore, PI3K activator 740Y-P was added to detect, the cell cycle, cell apoptosis, cell proliferation and apoptosis associated protein expressions, expressions of proteins in PI3K/AKT/Nrf2 pathway. Results With the increasing concentrations of CGA, the relative survival rates of cells were decreased, and the drug toxicity was concentration-dependent. The IC10 value was 81.59 μmol/L. Compared with Ctrl group, the cell population in G2/M period was significantly increased, and the cell apoptosis rates were increased in Radio group and CGA group (P<0.01). Compared with Radio group, the cell population in G2/M period and cell apoptosis rates were increased further in Radio+CGA group. Meanwhile, compared with Ctrl group, the protein expressions of Ki67, NQO1, TrxR1, p-PI3K, Nrf2 and p-AKT/AKT ratios in other groups were decreased significantly (P<0.01), and the combined effect of Radiation and CGA further reduced the above indicators in Radio+CGA group. Furthermore, PI3K activator could reverse the effects of radiation treatment on cell cycle, cell proliferation, cell apoptosis and PI3K/AKT/Nrf2 pathway. CGA could restore the changes of the above indicators caused by radiation (P<0.01). Conclusions CGA can enhance the radiosensitivity of retinoblastoma cell line HXO-Rb44, and the protective effect of CGA may be related to its inhibition of PI3K/AKT/Nrf2 pathway.
关键词
杜仲绿原酸 /
放射敏感性 /
视网膜母细胞瘤 /
PI3K/AKT/Nrf2通路
Key words
Eucommia chlorogenic acid; /
/
Radiosensitivity; /
/
Retinoblastoma; /
/
PI3K/AKT/Nrf2 pathway
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] Rao R, Honavar SG. Retinoblastoma[J]. Indian J Pediatr, 2017, 84(12): 937-944.
[2] Mendoza PR, Grossniklaus HE. Therapeutic options for retinoblastoma[J]. Cancer Control, 2016, 23(2): 99-109.
[3] Shields CL, Shields JA, Cater J, et al. Plaque radiotherapy for retinoblastoma: long-term tumor control and treatment complications in 208 tumors[J]. Ophthalmology, 2001, 108(11): 2116-2121.
[4] Chawla B, Singh R. Recent advances and challenges in the management of retinoblastoma[J]. Indian J Ophthalmol, 2017, 65(2): 133-139.
[5] 李慧敏, 牛东攀, 陈丹阳, 等. 响应面法优化大孔树脂纯化杜仲绿原酸工艺研究[J]. 食品研究与开发, 2018, 39(13): 40-46.
[6] Deka SJ, Gorai S, Manna D, et al. Evidence of PKC binding and translocation to explain the anticancer mechanism of chlorogenic acid in breast cancer cells[J]. Curr Mol Med, 2017, 17(1): 79-89.
[7] Yamagata K, Izawa Y, Onodera D, et al. Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells[J]. Mol Cell Biochem, 2018, 441(1-2): 9-19.
[8] Yan Y, Liu N, Hou N, et al. Chlorogenic acid inhibits hepatocellular carcinoma in vitro and in vivo[J]. J Nutr Biochem, 2017, 46: 68-73.
[9] 赵静滨, 何熹微, 姚素艳, 等. 绿原酸对视网膜细胞瘤细胞株HXO-RB44的生长抑制作用[J]. 西安交通大学学报（医学版）, 2016, 37(5): 734-738.
[10] 陆烨, 童剑萍. 视网膜母细胞瘤的发生机制及诊断和治疗进展[J]. 现代肿瘤医学, 2016, 24(6): 1007-1014.
（下转第655页）
（上接第649页）
[11] Eldebawy E, Parker W, Abdel Rahman W, et al. Dosimetric study of current treatment options for radiotherapy in retinoblastoma[J]. Int J Radiat Oncol Biol Phys, 2012, 82(3): e501-e505.
[12] Liu J, Hou M, Yuan T, et al. Enhanced cytotoxic effect of low doses of metformin combined with ionizing radiation on hepatoma cells via ATP deprivation and inhibition of DNA repair[J]. Oncol Rep, 2012, 28(4): 1406-1412.
[13] Moloudi K, Neshasteriz A, Hosseini A, et al. Synergistic effects of arsenic trioxide and radiation: triggering the intrinsic pathway of apoptosis[J]. Iran Biomed J, 2017, 21(5): 330-337.
[14]Juríková M, Danihel Ľ, Polák Š, et al. Ki67, PCNA and MCM proteins: Markers of proliferation in the diagnosis of breast cancer[J]. Acta Histochem, 2016, 118(5): 544-552.
[15] Huang KH, Fang WL, Li AF, et al. Caspase-3, a key apoptotic protein, as a prognostic marker in gastric cancer after curative surgery[J]. Int J Surg, 2018, 52: 258-263.
[16]Baskar R, Lee KA, Yeo R, et al. Cancer and radiation therapy: current advances and future directions[J]. Int J Med Sci, 2012, 9(3): 193-199.
[17]Zhang Y, Chen F, Tai G, et al. TIGAR knockdown radiosensitizes TrxR1-overexpressing glioma in vitro and in vivo via inhibiting Trx1 nuclear transport[J]. Sci Rep, 2017, 7: 42928.
[18]Oh ET, Park HJ. Implications of NQO1 in cancer therapy[J]. BMB Rep, 2015, 48(11): 609-617.
[19]Wang T, Hu P, Li B, et al. Role of Nrf2 signaling pathway in the radiation tolerance of patients with head and neck squamous cell carcinoma: an in vivo and in vitro study[J]. Onco Targets Ther, 2017, 10: 1809-1819.
[20] Tonelli C, Chio IIC, Tuveson DA. Transciptional regulation by Nrf2[J]. Antioxid Redox Signal, 2018, 29(17): 1727-1745.
[21] Kim J, Keum YS. NRF2, a key regulator of antioxidants with two faces towards cancer[J]. Oxid Med Cell Longev, 2016, 2016: 2746457.
[22] Wang Q, Ma J, Lu Y, et al. CDK20 interacts with KEAP1 to activate NRF2 and promotes radiochemoresistance in lung cancer cells[J]. Oncogene, 2017, 36(37): 5321-5330.
[23] Spangle JM, Roberts TM, Zhao JJ. The emerging role of PI3K/AKT-mediated epigenetic regulation in cancer[J]. Biochim Biophys Acta Rev Cancer, 2017, 1868(1): 123-131.
[24] Jiang XY, Zhu XS, Xu HY, et al. Diallyl trisulfide suppresses tumor growth through the attenuation of Nrf2/Akt and activation of p38/JNK and potentiates cisplatin efficacy in gastric cancer treatment[J]. Acta Pharmacol Sin, 2017, 38(7): 1048-1058.
京ICP备08002354号-3
