维甲酸联合曲古抑素对甲状腺癌细胞的诱导分化作用

Combined effects of all-trans-retinoic acid and trichostatin A on the induction of differentiation of thyroid carcinoma cells

背景与目的:维甲酸诱导甲状腺癌分化的有效率约30%,但其毒副作用限制了临床应用。两种以上诱导剂联合应用于肿瘤的分化,临床已开展了Ⅰ～Ⅲ期试验。但对维甲酸和组蛋白脱乙酰基酶的抑制剂的联合应用,未有报道。本试验初步研究全反式维甲酸(retinoic acid,RA)联合曲古抑素(trichostain A,TSA)诱导人乳头状甲状腺癌细胞株(K1)和人滤泡状甲状腺癌细胞株(FTC-133)的分化,增强诱导作用的效果,同时降低单一药物的毒副作用,为临床前期试验提供理论依据。方法:RA联合TSA,分为4种浓度,分别为1组RA1×10-4mol/L+TSA1.65×10-7mol/L、2组RA1×10-4mol/L+TSA3.31×10-7mol/L、3组RA1×10-5mol/L+TSA1.65×10-7mol/L、4组RA1×10-5mol/L+TSA3.31×10-7mol/L,在3个时间点(12、24、48h)处理两种细胞后,采用苏木精-伊红染色后,观察细胞生长的数量、形态;采用氮蓝四唑盐法(methythiazoly ltetrazolium,MTT)计算细胞生存率(survival rate,SR),研究联合药物对细胞的增殖、抑制和毒性作用;采用电化学发光法测定两种细胞株培养液中,甲状腺球蛋白(thyroglobulin,Tg)的水平,研究联合药物对肿瘤细胞的诱导分化作用。结果:联合药物作用K1和FTC-133后,细胞形态趋于规则,细胞间隔增大,细胞核明显缩小。4种浓度、3个时间点SR的方差分析,K1的F值分别为:23.52、170.14,FTC-133的F值分别为:57.09、224.35(P=0.000,均<0.01),有统计学意义。SNK分析,SR由低到高的排列分别为:2组<1组<4组<3组。4种浓度、3个时间点Tg方差分析,K1的F值分别为:69.63、101.07,FTC-133的F值分别为:79.77、81.72(P=0.000,均<0.01),差异有统计学意义。LSD两两比较:K1和FTC-133的1组与3组比较,P分别为:0.06、0.2,>0.05,两者之间差异无统计学意义,其他组均有统计学意义。结论:低浓度RA联合低浓度TSA,既可以抑制K1和FTC-133增殖,降低药物毒性作用,又可以增强对肿瘤细胞的诱导分化。其可能的机制是,TSA作用于转录步骤的DNA前调节,RA可能作用于转录步骤的信号后续调节,两种作用可能形成增强作用。通过转录后的传导系统,达到了抑制肿瘤细胞增殖和增强诱导分化的作用。

Background and Objective：The effectiveness rate of all-trans-retinoic acid （RA） is only about 30% in the clinical application of inducing thyroid carcinoma differentiation. In addition,there are severe toxic side effects,which limit its clinical application. Phase Ⅰ-Ⅲ clinical studies have been conducted on the combined application of two or more kinds of inductors in tumors. Nevertheless,the combination of RA with histone deacetylase inhibitors is rarely reported. This article studied the effects of differentiation for papillary thyroid carcinoma and follicular thyroid carcinoma cell lines induced by RA combined with trichostatin A （TSA）,enhancing the effect of induction,while reducing the toxic side effects of a single drug,to provide a theoretical basis for preclinical trials. Methods：After incubation with RA combined with TSA,K1 and FTC-133 were grouped into Group 1 （RA 1×10^-4 mol/L plus TSA 1.65×10^-7 mol/L）,Group 2 （RA 1× 10^-4 mol/L plus TSA 3.31×10^-7 mol/L）,Group 3 （RA 1× 10^-5 mol/L plus TSA 1.65×10^-7 mol/L）,Group 4 （RA 1×10^-5 mol/L plus TSA 3.31×10^-7 mol/L） by four varied concentrations and three time points （12,24,and 48 h）. The cell proliferation,conformation,toxic effect,and induced differentiation on K1 and FTC-133 cell lines were studied microscopically with hematoxylin-eosin （HE） to observe cell quantity and morphology,methyl-thiazolyl-tetrazolium （MTT） to calculate cell survival rates,and electrochemiluminescence analysis to measure in vitro thyroglobulin （Tg） levels. Results：The research showed that K1 and FTC-133 cells had cell spacing increases,with an outer edge of smooth,nuclear chromatin condensation after RA combined TSA. Survival rates were assessed by an analysis of variance （ANOVA） by concentration and time point,F values of K1 were 23.52 and 170.14,and F values of FTC-133 were 57.09 and 224.35,respectively. There were significant differences for both cells （ P〈0.01）. The SNK analysis indicated that survival rates were in the order of Group 2〈Group 1〈Group 4〈Group 3. Tg was also assessed by ANOVA,F values of K1 were 69.63 and 101.07,and F values of FTC-133 were 79.77 and 81.72 （ P〈0.01）. Group 1 was compared with Group 3 of K1 and FTC-133 by the least significant difference （LSD） method,and there was no statistical difference between the two groups （ P=0.06,0.20,respectively,P〉0.05）,yet a significant difference was seen between the other groups. Conclusions：Lower concentrations of RA combined with lower concentrations of TSA have both inhibited cell proliferation,decreased toxicity of the drugs,and increased the effect of K1 and FTC-133 cell differentiation. The mechanism of action may be that TSA has pretranscription DNA regulation and that RA has posttranscriptional signal regulation to enhance the effects ofinhibited proliferation and differentiation of cells by transcription systems.