索拉非尼对高表达三磷酸腺苷结合转运蛋白G超家族成员2的鼻咽癌细胞杀伤敏感性的影响的研究

Effects of Sorafenib on the cytotoxic sensitivity of nasopharyngeal carcinoma cells with high expression of ATP-binding cassette superfamily G member 2(ABCG_2)

目的探讨索拉非尼提高高表达三磷酸腺苷(ATP)结合转运蛋白G超家族成员2(ABCG2)的人耐药鼻咽癌CNE2/DDP细胞(简写作ABCG2High细胞)对同种异体反应性自然杀伤(Allo-NK)细胞的杀伤敏感性及其相关机制。方法利用免疫磁珠技术分离ABCG2High CNE2/DDP和Allo-NK细胞,并设3个实验组:①处理组(经索拉非尼10ng/ml共孵育4h的ABCG2High CNE2/DDP细胞);②未处理组(常规培养的ABCG2High CNE2/DDP细胞);③对照组(常规培养的K562细胞)。流式细胞技术检测处理组和未处理组细胞的ABCG2表达率和5种NKG2D配体(MICA、MICB、ULBP1、ULBP2、ULBP3)的表达率。乳酸脱氢酶(LDH)释放测定法检测Allo-NK细胞对各实验组细胞的杀伤率。结果分离后的CNE2/DDP细胞的ABCG2表达率为91.40%±2.32%,分选出的CD3-CD16+CD56+(Allo-NK)细胞纯度大于90%。未处理组的ABCG2High CNE2/DDP细胞的NKG2D配体MICA、MICB、ULBP1、ULBP2、ULBP3表达率分别为2.92%±0.33%、4.27%±0.33%、5.80%±0.62%、11.10%±3.15%、7.75%±1.14%,而处理组明显升高(分别为10.38%±1.23%、10.68%±1.26%、11.62%±1.22%、43.24%±4.42%、11.91%±0.88%;P<0.05)。在效靶比为10∶1、20∶1时,Allo-NK细胞对未处理组靶细胞的杀伤率为15.32%±1.34%、27.26%±6.81%,而对处理组靶细胞杀伤率为27.75%±4.12%、43.17%±5.92%,两组相比差异有统计学意义(P<0.01)。结论索拉非尼通过诱导肿瘤细胞高表达NKG2D配体,使其对Allo-NK细胞的杀伤敏感性增强。

Objective To investigate the mechanisms and effects of Sorafenib on cytotoxic sensitivity of all, reactive natural killer （Allo-NK） cells against human multi-drug resistant nasopharyngeal carcinoma CNE2/DDP cells which expressing highly ATP-binding cassette superfamily G member 2 （ABCG2） （abbr. as ABCG^High CNE2/DDP cells）. Methods ABCG2^High CNE2/DDP and Allo-NK cells were isolated by magnetic bead technique. The target cells were divided into 3 groups： a） treated group （ABCG2^High CNE2/DDP cells incubated with 10 ng/ml sorafenib for 4h） ; b） untreated group （conventionally cultured ABCG2^High CNE2/DDP cells） ; and c） control group （conventionally cultured K562 cells）. Expression rates of ABCG2 in treated and untreated groups, and of five NKG2D-ligands （MICA, MICB, ULBP1, ULBP2, ULBP3） were evaluated by flow cytometry. The cytotoxic effects of NK cells against different groups of target ceils were detected with LDH releasing assay. Results Expression rate of ABCG2 in isolated CNE2/DDP cells was 91.40%±2. 32%. The pu rity of sorted CD3^-CD16^＋ CD66^＋ Allo-NK cells was 90% and higher. The expression rates of NKG2D-ligands （MICA, MICB, ULBP1, ULBP2 and ULBP3） in untreated group were 2. 92%±0. 33%, 4. 27%±0. 33%, 5. 80%±0. 62%, 11.10%±3. 15% and 7. 75%±1.14%, respectively, which were remarkablely higher than that in treated group （10. 38%±1.23%, 10. 68%±1.26%, 11.62%±1.22%, 43.24%±4. 42% and 11.91%±0. 88%, respectively, P〈0. 05）. The ratios of effectiveness / target cell （E/T） were 10：1 and 20：1, the killing rates of Allo-NK to the target cells of untreated group were 15.32 %±1.34 % and 27. 26 %±6. 81%, while of the treated group were 27. 75%±4. 12% and 43. 17%±5. 92%, respectively （P〈0. 01）. Conclusion Sorafenib may up-regulate the expression of NKG2D-ligands on ABCG2^High nasopharyngeal carcinoma cells, resulting in an increase in sensitivity to Allo-NK cells.