亚硝酸盐调控人肝癌SMMC-7721细胞癌干细胞特性

Involvement of sodium nitrite in the regulation of cancer stem cell properties in human hepatoma cells

有证据表明肝癌含有一定数量的具有自我更新能力的癌干细胞亚群。然而,癌干细胞是如何被调控的还不清楚。肿瘤微环境中一些生物介质可以导致癌干细胞快速可逆性改变。本文旨在探讨,在肿瘤微环境中有较高含量,又是细胞关键的调控因子的亚硝酸盐是如何调控人肝癌干细胞样细胞表型的。用SMMC-7721细胞系无血清培养获得细胞球。流式细胞术检测细胞周期,体外克隆形成实验和球形成实验检测细胞自我更新能力,侵袭实验检测细胞侵袭能力,癌干细胞标志物检测采用流式细胞术和Westen blot方法,MTT检测细胞活力。皮下注射SMMC-7721球形成细胞建立裸鼠移植瘤模型,活性氧(ROS)检测试剂盒检测移植瘤组织内ROS水平,免疫荧光检测组织内缺氧有诱导因子-1α(HIF-1α)。结果显示,SMMC-7721细胞系来源的球细胞自我更新能力增强,干细胞标志物表达增加,耐药性增强,提示球形成细胞富集了较多的具有癌干细胞特征的细胞。用亚硝酸钠(150μmol·L^(-1))处理亲本细胞和球形成细胞后,G_0/G_1期细胞比例增加,肝癌干细胞标志物CD133、CD90和EpCAM表达增加,细胞耐药能力和侵袭能力增强。与亲本细胞相比,亚硝酸盐对球形成细胞的上述作用明显增强。体内实验结果也显示,亚硝酸盐促进球形成细胞移植瘤生长,组织内ROS水平下降,HIF-1α累积。结果表明,亚硝酸盐通过上调肝癌干细胞"干性"增强肝癌细胞侵袭能力。

Increasing evidence suggests that hepatocellular carcinomas（HCCs） are sustained by a distinct subpopulation of self-renewing cells known as cancer stem cells（CSC）. However, our understanding of their regulation is limited. Rapid reversible changes of CSC-like cells within tumors may result from the effect of biological mediators found in the tumor microenvironment. This paper aims to explore how nitrite, a key cellular modulator whose level is elevated in many tumors, affects CSC-like phenotypes of human hepatoma cells SMMC-7721 cells. The SMMC-7721 cell line was cultured under serum-free conditions to produce floating spheres. The distribution of cell cycle was analyzed by flow cytometry, the capability of cells self-renew was detected by colony-forming capabilities and spheroid-formation assay, the expression of stemness protein such as CD133, CD90 and Ep CAM were determined by flow cytometry and Western blot, cell invasion was analyzed by transwell assay, and viability of SMMC-7721 parental cells and spheroids cancer cells was determined by the 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide（MTT） assay. Xenograft tumor models were established by subcutaneously injecting SMMC-7721 spheroids cancer cells, the transplanted tumor tissue ROS levels was detected by reactive oxygen species（ROS） test kits, the expression of HIF-1α was observed by immunofluorescence. Our results showed that the SMMC-7721 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, SMMC-7721 parental cells and spheroids cancer cells were treated with 150 μmol·L-1 sodium nitrite for 6 days, compared with control cells, an increased accumulation of G0/G1 phase cells was observable in treatment cells. Indeed, our data demonstrated that in parent cells and spheres cells that were treated with sodium nitrite for different time, the cells＇ ability to chemoresistance and invasion, clone-forming efficiencies and the spheres forming ability were significantly higher than that of control cells. Exposure of sodium nitrite regulated CSC-like phenotype, indicated by increased expression of known CSC markers, CD133, CD90 and Ep CAM in the exposed parental cells, as well as in dormant spheroids cancer cells. Compared with the parent cells, the above effects of nitrite on the spheres cells were significantly enhanced. In vivo data also presented a more significant promotion of tumor xenograft growth from the nitrite treatment than from either of the control. Mechanistic analysis indicated that nitrite induced the upregulation of HIF-1α as well as the downregulation of ROS in the tumor microenvironment. These results suggest that nitrite increases the invasiveness of SMMC-7721 cells through up-regulation of tumor stemness.