CD44中和抗体对黑色素瘤B16细胞生长及细胞糖酵解的影响

Neutralizing anti-CD44 antibodies suppresses the growth of B16 cells and enhances AKT-mediated glycolytic metabolism in melanoma

目的 CD44在肿瘤的生长及糖酵解代谢中起重要作用。文中旨在探讨CD44中和性抗体对鼠黑色素瘤B16细胞生长及糖酵解代谢的影响。方法首先设置对照抗体(50μg/m L)和不同浓度CD44抗体(2、10、50μg/m L)处理B16细胞,24 h后通过免疫印迹检测AKT通路活化情况。随后设置对照抗体(50μg/m L)和CD44抗体(50μg/m L)平行试验使用4μmol/L API-2预处理细胞,处理细胞48 h后通过免疫荧光观察乳酸脱氢酶A(LDHA)表达,比色法检测细胞上清乳酸含量。最后设置对照抗体(50μg/m L)组、API-2(4μmol/L)处理组、CD44抗体(50μg/m L)组和联合处理组(API-2+CD44抗体),作用B16细胞96 h后,通过MTT法检测细胞增殖,AO/EB和Annexin V染色检测细胞死亡及凋亡。结果与对照抗体B16细胞p-AKT水平(1.00±0.25)相比,2、10、50μg/m L CD44抗体呈浓度依赖性增强[2.51±0.32,3.89±0.46,4.07±0.42,P<0.01],与对照抗体相比,50μg/m L CD44抗体作用后LDHA表达增强(2.13±0.24)倍。中和CD44后B16细胞LDHA荧光表达增强,且细胞上清乳酸含量升高,与对照抗体相比,CD44抗体(50μg/m L)96 h后细胞上清乳酸含量升高[(35.32±3.24)mmol/L vs(56.34±8.19)mmol/L,P<0.01]。联合使用AKT通路抑制剂API-2处理后,CD44抗体所致LDHA表达增强现象明显受到抑制,且对照抗体和CD44抗体组乳酸含量均下降;与对照组相比,CD44抗体细胞上清乳酸含量差异无统计学意义[(20.12±4.35)mmol/L vs(22.65±5.16)mmol/L,P>0.05]。MTT检测结果显示,与对照组细胞增殖率[(103±12.91)%]比较,API-2组[(84.87±19.35)%]、CD44抗体组[(71.35±16.23)%]和联合处理组[(41.16±9.15)%]均明显降低(P<0.05)。细胞凋亡检测显示,与对照抗体组凋亡率[(5.23±0.96)%]相比,API-2组[(13.65±4.27)%]、CD44抗体组[(19.21±3.53)%]和联合处理组[(43.21±7.87)%]均显著升高(P<0.01)。结论体外中和B16细胞CD44功能抑制细胞生长但促进AKT通路介导的糖酵解代谢,抑制AKT通路可增强CD44抗体对肿瘤细胞的杀伤作用。

Objective CD44,a cell surface glycoprotein,plays an important role in tumor growth and glycolysis. The aim of this study was to investigate the effects of neutralizing CD44 antibodies on the growth and glycolytic metabolism of B16 cells in melanoma in vitro. Methods B16 cells were treated with control antibodies（ 50μg/m L） or different concentrations of CD44 antibodies（ 2,10,and50 μg/m L） for 24 hours,followed by examination of the activation of the AKT pathway in the B16 cells by Western blot. Then the tumor cells were also treated with control antibodies（ 50 μg/m L） or CD44 antibodies（ 50μg/m L） after pretreated with API-2（ 4 μmol/L）in a parallel test. After 48 hours of treatment,the expression of lactate dehydrogenase A（ LDHA） in the B16 cells and the level of lactate in the culture supernatant were detected by immunofluorescence and colorimetry,respectively. Lastly,the B16 cells were treated with control antibodies（ 50μg/m L）,API-2（ 4 μmol/L）,CD44 antibodies（ 50μg/m L）,or API-2 ＋ CD44 antibodies for 96 hours,followed by measurement of the proliferation of the cells by MTT and their apoptosis by AO/EB and Annexin V staining. Results In comparison with the control antibody group,the level of AKT phosphorylation（ p-AKT） in the B16 cells showed a concentration-dependent increase in the 2,10,and 50 μg/m L CD44 antibody groups（ 1.00±0.25 vs 2.51± 0.32,3.89± 0.46,and 4.07± 0.42,P〈 0.01）,and the expression of LDHA was increased by（ 2.13±0.24） times,with the lactate level in the culture supernatant significantly elevated from（ 35.32±3.24） to（ 56.34±8.19） mmol/L（ P〈0.01） after 96 hours of treatment with 50 μg/m L CD44 antibodies. Treatment with API-2＋CD44 antibodies,however,suppressed the increase in the LDHA expression and reduced the level of lactate. Compared with the control antibody group,the proliferation rate of the B16 cells was markedly decreased in the API-2,CD44 antibody,and API-2＋CD44 antibody groups（ [103±12.91] vs [84.87±19.35],[71.35±16.23],and [41.16±9.15]%,P〈0.05）,while the apoptosis rate remarkably increased（ [5.23±0.96] vs [13.65±4.27],[19.21± 3.53],and [43.21± 7.87]%,P〈 0.01）. Conclusion Neutralizing the function of CD44 in the B16 cells in vitro can inhibit the growth of the cells and promote AKT-mediated glycolytic metabolism,while suppressing the AKT pathway may enhance the antitumor activity of the CD44 antibody.