双氢青蒿素对人口腔鳞癌细胞KBV200多药耐药的影响

doi:10.19439/j.sjos.2019.06.006

摘要/Abstract

摘要： 目的检测双氢青蒿素（dihydroartemisinin,DHA）对人口腔鳞癌细胞KBV200多药耐药的作用,并探讨其与ROS-MAPK通路的关系。方法利用MTT法检测不同浓度DHA对KB、KBV200细胞增殖的影响,顺铂（cisplatin,DDP）、长春新碱（vincristine,VCR）、阿霉素（adriamycin,ADM）、依托泊苷（etoposide,VP-16）对KB、KBV200细胞的增殖抑制率及加入DHA后的变化;分别联合ROS诱导剂3-AT、ERK抑制剂UO126、JNK抑制剂SP600125、p38MAPK抑制剂SB203580,观察干预前、后的逆转差异。利用流式细胞术检测各组细胞内ROS的荧光强度,采用SPSS 17.0软件包对数据进行统计学分析。结果与KB细胞相比,KBV200细胞对VCR、VP-16、ADM药物的耐药性显著提高（P<0.05）,10、20、30 μg/mL DHA作用后,KBV200细胞对VCR、VP-16、ADM的IC₅₀显著降低,呈浓度依赖性（P<0.05）。与KB组细胞相比,KBV200组细胞ROS荧光强度显著降低（P<0.05）;DHA处理后,ROS荧光强度显著增高,VCR、VP-16、ADM IC50 显著降低（P<0.05）,与ROS促进剂效果一致。与KB细胞相比,KBV200细胞p-ERK/ERK、p-JNK/JNK、p-p38MAPK/p38MAPK水平显著升高（P<0.05）;DHA处理后,p-ERK/ERK、p-JNK/JNK、p-p38MAPK/p38MAPK水平显著降低,VCR、VP-16、ADM IC₅₀ 显著增高（P<0.05）;加入ERK抑制剂UO126、JNK抑制剂SP600125、p38MAPK抑制剂SB203580后,KBV200细胞VCR、VP-16、ADM IC₅₀ 进一步降低。结论双氢青蒿素可能通过促进ROS生成、抑制MAPK通路而逆转KBV2001细胞的多药耐药性。

关键词: 口腔鳞癌, 双氢青蒿素, ROS, MAPK通路, 多药耐药

Abstract: PURPOSE: To investigate the effect of dihydroartemisinin (DHA) on multidrug resistance of human oral squamous cell carcinoma cell line KBV200 and to explore its relationship with ROS-MAPK pathway. METHODS: MTT assay was used to detect the effects of different concentrations of DHA on proliferation of KB and KBV200 cells. MTT assay was used to detect the inhibition rates of cisplatin (DDP), vincristine (VCR), adriamycin (ADM), etoposide (VP-16) on proliferations of KB and KBV200 cells and the changes after DHA addition, combined with ROS inducer 3-AT, ERK inhibitor UO126, JNK inhibitor SP600125, and p38MAPK inhibitor SB203580, respectively, the difference of reversal before and after intervention was observed; the fluorescence intensity of ROS was measured by flow cytometry. SPSS 17.0 software package was used to analyze the data. RESULTS: Compared with KB cells, drug resistances of KBV200 cells to VCR, VP-16 and ADM were significantly increased(P<0.05), after 10, 20, and 30 μg/mL DHA treatment; the IC₅₀ of KBV200 cells to VCR, VP-16 and ADM was decreased significantly in a concentration-dependent manner(P<0.05). Compared with KB cells, ROS fluorescence intensity of KBV200 cells decreased(P<0.05); after DHA treatment, ROS fluorescence intensity increased significantly, IC₅₀ of VCR, VP-16, ADM decreased significantly(P<0.05), which was consistent with ROS promoter effect. Compared with KB cells, the levels of p-ERK/ERK, p-JNK/JNK, p-p38MAPK/p38MAPK in KBV200 cells increased significantly (P<0.05); after DHA treatment, the levels of p-ERK/ERK, p-JNK/JNK, p-p38MAPK/p38MAPK decreased significantly, IC₅₀ of VCR, VP-16, and ADM increased significantly(P<0.05); after adding ERK inhibitor UO126, JNK inhibitor SP600125, and p38MAPK inhibitor SB203580, IC₅₀ of VCR, VP-16, and ADM to KBV200 cells were further reduced. CONCLUSIONS: Dihydroartemisinin may reverse multidrug resistance of KBV2001 cells by promoting ROS production and inhibiting MAPK pathway.

Key words: Oral squamous cell carcinoma, Dihydroartemisinin, ROS, MAPK pathway, Multidrug resistance

中图分类号:

R739.8

刘族志, 赵永兴, 林建能, 周国平. 双氢青蒿素对人口腔鳞癌细胞KBV200多药耐药的影响[J]. 上海口腔医学, 2019, 28(6): 586-590.

LIU Zu-zhi, ZHAO Yong-xing, LIN Jian-neng, ZHOU Guo-ping. Effect of dihydroartemisinin on multidrug resistance of human oral squamous cell carcinoma cell line KBV200 by regulating ROS-MAPK pathway[J]. Shanghai Journal of Stomatology, 2019, 28(6): 586-590.

参考文献

[1] 陈芬, 陈林林. 口腔疣状癌、口腔鳞癌与癌基因关系的研究进展[J]. 实用临床医学, 2016, 17(1): 92-93.
[2] 韩影, 苏彤, 林璐, 等. 口腔癌对侧颈淋巴结转移的临床研究[J]. 口腔医学研究, 2016, 32(2): 154-157.
[3] 王雪阳, 周昱川, 陈青立,等. Hippo信号通路相关蛋白YAP, Mst 1, Lats 1/2在口腔鳞癌中的表达及临床意义[J]. 口腔医学研究, 2017, 33(4): 400-403.
[4] Amaratunga C, Lim P, Suon S, et al.Dihydroartemisinin-piperaquine resistance in plasmodium falciparum malaria in Cambodia: a multisite prospective cohort study[J]. Lancet Infect Dis, 2016, 16(3): 357-365.
[5] Xi G, Wang M, Sun B, et al.Targeting autophagy augments the activity of DHA-E3 to overcome p-gp mediated multi-drug resistance[J]. Biomed Pharmacother, 2016, 84(1): 1610-1616.
[6] 张斌. 口腔鳞状细胞癌的表观遗传学研究现状和进展[J]. 口腔颌面外科杂志, 2016, 26(2): 77-82.
[7] Hientz K, Mohr A, Bhaktaguha D, et al.The role of p53 in cancer drug resistance and targeted chemotherapy[J]. Oncotarget, 2017, 8(5): 8921-8946.
[8] Allimuthu DT.Role of reactive oxygen species (ROS) in therapeutics and drug resistance in cancer and bacteria[J]. J Med Chem, 2017, 60(8): 3221-3240.
[9] Okon IS, Coughlan KA, Zhang M, et al.Gefitinib-mediated reactive oxygen species (ROS) instigates mitochondrial dysfunction and drug resistance in lung cancer cells[J]. J Med Chem, 2015, 290(14): 9101-9110.
[10] Mak PY, Mak DH, Mu H, et al.Apoptosis repressor with caspase recruitment domain is regulated by MAPK/PI3K and confers drug resistance and survival advantage to AML[J]. Apoptosis, 2014, 19(4): 698-707.
[11] Zhang C, Wang J, Zhang J, et al.Mechanism-guided design and synthesis of a mitochondria-targeting artemisinin analogue with enhanced anticancer activity[J]. Angew Chem Int Ed Engl, 2016, 128(44): 13974-13978.
[12] Gil J P, Krishna S. pfmdr1 (Plasmodium falciparum multidrug drug resistance gene 1): a pivotal factor in malaria resistance to artemisinin combination therapies[J]. Expert Rev Anti Infect Ther, 2017, 15(4): 527-543.
[13] Ding Y, Wang H, Niu J, et al.Induction of ROS overload by alantolactone prompts oxidative DNA damage and apoptosis in colorectal cancer cells[J]. Int J Mol Sci, 2016, 17(4): 558.
[14] Sung B, Prasad S, Ravindran J, et al.Capsazepine, a TRPV1 antagonist, sensitizes colorectal cancer cells to apoptosis by TRAIL through ROS-JNK-CHOP-mediated upregulation of death receptors[J]. Free Radic Biol Med, 2012, 53(10): 1977-1987.
[15] Yang J, Li TZ, Xu GH, et al.Low-concentration capsaicin promotes colorectal cancer metastasis by triggering ROS production and modulating Akt/mTOR and STAT-3 pathways[J]. Neoplasma, 2013, 60(4): 364-372.
[16] Fiorini C.Regulation of miR-23b expression and its dual role on ROS production and tumour development[J]. Cancer Lett, 2014, 349(2): 107-113.
[17] 杨丽娜, 杨宇琦, 杨恭, 等. Aurora A调控活性氧对卵巢癌顺铂耐药的影响[J]. 中国癌症杂志, 2018, 28(3): 184-190.
[18] Li D, Liang Z, Huang J, et al.Effect of multidrug resistance 1/P-glycoprotein on the hypoxia-induced multidrug resistance of human laryngeal cancer cells[J]. Oncol Lett, 2016, 12(2): 1569-1574.
[19] Wang P, Chen D, Ma H, et al.LncRNA SNHG12 contributes to multidrug resistance through activating the MAPK/Slug pathway by sponging miR-181a in non-small cell lung cancer[J]. Oncotarget, 2017, 8(48): 84086-84101.
[20] Fang R, Shen J, Shi H, et al.Novel mechanisms and approaches to overcome multidrug resistance in the treatment of ovarian cancer[J]. Biochim Biophys Acta, 2016, 1866(2): 266-275.