诺米林对黑色素瘤B16F-10细胞增殖、侵袭和迁移能力的影响
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摘要
目的探讨诺米林对黑色素瘤B16F-10细胞增殖、侵袭和迁移能力的影响。方法 CCK-8法检测B16F-10细胞在0～500μmol/L浓度梯度诺米林处理下的细胞活力。B16F-10细胞分为4组:B16F-10对照组,10μmol/L诺米林组,20μmol/L诺米林组和50μmol/L诺米林组。CCK-8法检测细胞增殖,Transwell小室实验检测细胞侵袭,划痕实验检测细胞迁移,Western blotting检测细胞增殖核抗原-67 (Ki-67),血管内皮细胞生长因子(VEGF)和基质金属蛋白酶9 (MMP-9)的表达。结果 <50μmol/L诺米林不会对B16F-10细胞株产生明显的细胞毒性。诺米林组(10、20、50μmol/L)细胞增殖倍数从48 h开始低于对照组(P<0.01)。与对照组比较,诺米林组每个视野下的侵袭细胞数减少(P<0.01),其中对照组、10μmol/L诺米林组、20μmol/L诺米林组、50μmol/L诺米林组的侵袭细胞数分别为(122±15)、(88±20)、(70±13)和(23±9)个。诺米林组划痕愈合率低于对照组(P<0.01),其中对照组、10μmol/L诺米林组、20μmol/L诺米林组、50μmol/L诺米林组的划痕愈合率分别为(95.0±4.7)%、(84.3±7.0)%、(58.2±9.1)%和(36.0±5.9)%。与对照组比较,诺米林组Ki-67,VEGF和MMP-9的蛋白相对表达量降低(P<0.01)。结论诺米林具有抑制黑色素瘤细胞B16F-10的增殖、侵袭和迁移的作用。
Objective To explore the effect of normirin on the proliferation, invasion and migration of melanoma B16 F-10 cells.Methods The activity of B16 F-10 cells treated with 0-500 μmol/L concentration of normirin was detected by CCK-8. B16 F-10 cells were divided into four groups, including B16 F-10 control group, normirin(10 μmol/L) group, normirin(20 μmol/L) group and normirin(50 μmol/L) group. The cell proliferation was detected by CCK-8. Cell invasion was measured by transwell chamber test. Cell migration was analyzed by scratch assay. The expression of antigen identified by monoclonal antibody(Ki-67), vascular endothelial growth factor(VEGF) and matrix metalloprotein 9(MMP-9) was detected by Western blotting. Results The concentration of normirin less than 50 μmol/L had no obvious cytotoxicity on melanoma cells B16 F-10. The cell proliferation in normirin(10, 20, 50 μmol/L) group was lower than that of control group from 48 h(P<0.01). Compared with control group, the number of invasive cells per field in normirin groups decreased(P<0.01), and the invasive numbers of control group, normirin(10, 20, 50 μmol/L) groups were(122±15),(88±20),(70±13) and(23±9). The wound closure rate in normirin groups was lower than that of control group(P<0.01), and the rates of control, normirin(10, 20, 50 μmol/L) groups were(95.0±4.7)%,(84.3±7.0)%,(58.2±9.1)% and(36.0±5.9)%. Compared with control group, the relative protein levels of Ki-67, VEGF and MMP-9 in normirin(10, 20, 50 μmol/L) groups were reduced(P<0.01).Conclusion Normirin can inhibite the proliferation, invasion and migration of melanoma B16 F-10 cells.
Objective To explore the effect of normirin on the proliferation, invasion and migration of melanoma B16 F-10 cells.Methods The activity of B16 F-10 cells treated with 0-500 μmol/L concentration of normirin was detected by CCK-8. B16 F-10 cells were divided into four groups, including B16 F-10 control group, normirin(10 μmol/L) group, normirin(20 μmol/L) group and normirin(50 μmol/L) group. The cell proliferation was detected by CCK-8. Cell invasion was measured by transwell chamber test. Cell migration was analyzed by scratch assay. The expression of antigen identified by monoclonal antibody(Ki-67), vascular endothelial growth factor(VEGF) and matrix metalloprotein 9(MMP-9) was detected by Western blotting. Results The concentration of normirin less than 50 μmol/L had no obvious cytotoxicity on melanoma cells B16 F-10. The cell proliferation in normirin(10, 20, 50 μmol/L) group was lower than that of control group from 48 h(P<0.01). Compared with control group, the number of invasive cells per field in normirin groups decreased(P<0.01), and the invasive numbers of control group, normirin(10, 20, 50 μmol/L) groups were(122±15),(88±20),(70±13) and(23±9). The wound closure rate in normirin groups was lower than that of control group(P<0.01), and the rates of control, normirin(10, 20, 50 μmol/L) groups were(95.0±4.7)%,(84.3±7.0)%,(58.2±9.1)% and(36.0±5.9)%. Compared with control group, the relative protein levels of Ki-67, VEGF and MMP-9 in normirin(10, 20, 50 μmol/L) groups were reduced(P<0.01).Conclusion Normirin can inhibite the proliferation, invasion and migration of melanoma B16 F-10 cells.
引文
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[17] Li LT, Jiang G, Chen Q, et al. Ki67 is a promising molecular target in the diagnosis of cancer [J]. Mol Med Rep, 2015, 11(3):1566-1572.
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[20] 暴亚锋,刘飞飞,刘静,等,新型酪氨酸激酶抑制剂XCF-43b体外抗血管新生研究 [J]. 医学分子生物学杂志, 2017, 14(3): 130-136.
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[22] 毛小荣,度森荣,杨忠霞,等.PARP-1抑制剂对人肝癌细胞HepG2增殖和迁移的抑制作用 [J]. 医学分子生物学杂志, 2017, 14(4):210-216.
[23] Falzone L, Salemi R, Travali S, et al. MMP-9 overexpression is associated with intragenic hypermethylation of MMP-9 gene in melanoma [J]. Aging (Albany NY), 2016,8(5):933-944.
[24] Pratheeshkumar P, Raphael TJ, Kuttan G. Nomilin inhibits metastasis via induction of apoptosis and regulates the activation of transcription factors and the cytokine profile in B16F-10 cells [J]. Integr Cancer Ther, 2012, 11(1):48-60.
[2] Siegel R, Ma J, Zou Z, et al. Cancer statistics, 2014 [J]. CA Cancer J Clin, 2014,64(1):9-29.
[3] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017 [J]. CA Cancer J Clin, 2017,67(1):7-30.
[4] Corrie P, Hategan M, Fife K, et al. Management of melanoma [J]. Br Med Bull, 2014,111(1):149-162.
[5] AlQathama A, Prieto JM. Natural products with therapeutic potential in melanoma metastasis [J]. Nat Prod Rep, 2015,32(8):1170-1182.
[6] Turati F, Rossi M, Pelucchi C, et al. Fruit and vegetables and cancer risk: A review of southern european studies [J]. Br J Nutr, 2015,113 (Suppl 2):102-110.
[7] Xiang Y, Cao J, Luo F, et al. Simultaneous purification of limonin, nomilin and isoobacunoic acid from pomelo fruit (citrus grandis) segment membrane [J/OL]. J Food Sci, 2014[2018-04-10]. https://www.ncbi.nlm.nih.gov/pubmed/25212475.
[8] Kim J, Chakraborty S, Jayaprakasha GK, et al. Citrus nomilin down-regulates tnf-alpha-induced proliferation of aortic smooth muscle cells via apoptosis and inhibition of ikappab [J/OL]. Eur J Pharmacol, 2017[2018-04-15]. https://www.ncbi.nlm.nih.gov/pubmed/28551013.
[9] Maverakis E, Cornelius LA, Bowen GM, et al. Metastatic melanoma-a review of current and future treatment options [J]. Acta Derm Venereol, 2015,95(5):516-524.
[10] Li RH, Hou XY, Yang CS, et al. Temozolomide for treating malignant melanoma [J]. J Coll Physicians Surg Pak, 2015,25(9):680-688.
[11] Wilson MA, Schuchter LM. Chemotherapy for melanoma [J]. Cancer Treat Res, 2016,167(3):209-229.
[12] Boada A, Carrera C, Segura S, et al. Cutaneous toxicities of new treatments for melanoma [J]. Clin Transl Oncol, 2018,20(11):1373-1384.
[13] Ouyang L, Luo Y, Tian M, et al. Plant natural products: From traditional compounds to new emerging drugs in cancer therapy [J]. Cell Prolif, 2014,47(6):506-515.
[14] Raja Singh P, Arunkumar R, Sivakamasundari V, et al. Anti-proliferative and apoptosis inducing effect of nimbolide by altering molecules involved in apoptosis and igf signalling via pi3k/akt in prostate cancer (pc-3) cell line [J]. Cell Biochem Funct, 2014,32(3):217-228.
[15] Rahman A, Siddiqui SA, Jakhar R, et al. Growth inhibition of various human cancer cell lines by imperatorin and limonin from poncirus trifoliata rafin. Seeds [J]. Anticancer Agents Med Chem, 2015,15(2):236-241.
[16] Kim J, Jayaprakasha GK, Patil BS. Limonoids and their anti-proliferative and anti-aromatase properties in human breast cancer cells [J]. Food Funct, 2013,4(2):258-265.
[17] Li LT, Jiang G, Chen Q, et al. Ki67 is a promising molecular target in the diagnosis of cancer [J]. Mol Med Rep, 2015, 11(3):1566-1572.
[18] Robert J. Biology of cancer metastasis [J]. Bull Cancer, 2013, 100(4):333-342.
[19] Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors [J]. Nat Med, 2003, 9(6):669-676.
[20] 暴亚锋,刘飞飞,刘静,等,新型酪氨酸激酶抑制剂XCF-43b体外抗血管新生研究 [J]. 医学分子生物学杂志, 2017, 14(3): 130-136.
[21] Pratheeshkumar P, Kuttan G. Nomilin inhibits tumor-specific angiogenesis by downregulating VEGF, NO and proinflammatory cytokine profile and also by inhibiting the activation of MMP-2 and MMP-9 [J].Eur J Pharmacol, 2011, 668(3):450-458.
[22] 毛小荣,度森荣,杨忠霞,等.PARP-1抑制剂对人肝癌细胞HepG2增殖和迁移的抑制作用 [J]. 医学分子生物学杂志, 2017, 14(4):210-216.
[23] Falzone L, Salemi R, Travali S, et al. MMP-9 overexpression is associated with intragenic hypermethylation of MMP-9 gene in melanoma [J]. Aging (Albany NY), 2016,8(5):933-944.
[24] Pratheeshkumar P, Raphael TJ, Kuttan G. Nomilin inhibits metastasis via induction of apoptosis and regulates the activation of transcription factors and the cytokine profile in B16F-10 cells [J]. Integr Cancer Ther, 2012, 11(1):48-60.