诱导肿瘤细胞分化通过MLCK及其介导的紧密连接蛋白结构和功能的变化抑制其侵袭、转移及机制的研究

The recurrence and metastasis is one of failure reasons in the treatment of malignant tumores. The differentiation of tumor cells induced by all-trans retinoic acid (ATRA) increase the sensitivity of tumor cells to radiotherapy and chemotherapy, reduce the recurrence and metastasis, but the exact mechanism is unclear. During their invasion and metastasis, abnormal changes of adhesion and movement capacity in tumor cell are important factors. Cell-cell adhesion and motorial ability, respectively, are closely related with structure and function of cytoskeletal proteins and expression and activity of myosin light chain kinase(MLCK). In our pre-experiments, the A549 and HepG2 cells were treated with ATRA, and found that the proliferation was significantly inhibited, the differentiation was induced, while MLCK expression and MLC phosphorylation was significantly reduced, colony formation in soft agar and migration were decreased，the level of tight junction protein occludin in cellular menbrance was increased significantly；MLCK inhibitor ML-7 inhibited A549 cell migration, and increased cell menbrane occludin. It was suggested that ATRA-induced differentiation of tumor cells may be mediated by changes of MLCK and MLCK-dependent structure and function of tight junction proteins in inhibition of the invasion and metastasis. In this study, the A549, HepG2 cells will be differentiated by ATRA, and to investigate that whether the invasion, migration and metastasis of tumor cells in the differentiation process are inhibited through MLCK changes and MLCK-dependent tight junction remodeling by regulation of Rho/ROCK, MAPK and RNA interfere and other experiments.

复发和转移是导致恶性肿瘤治疗失败的主要原因之一。诱导分化可明显增加肿瘤细胞对放、化疗的敏感性，降低复发和转移，但确切机制尚不清楚。在侵袭、转移过程中，肿瘤细胞间粘附和运动能力出现异常改变，分别与细胞紧密连接蛋白结构是否异常和MLCK表达、活性等密切相关。我们先前研究表明分化诱导剂ATRA明显抑制A549和HepG2增殖、促进其分化，软琼脂克隆形成和迁移能力下降，MLCK表达和MLC磷酸化显著降低；MLCK抑制剂ML-7可抑制A549细胞迁移，同时胞膜紧密连接蛋白occludin含量显著增加，提示ATRA诱导肿瘤细胞的分化可能通过MLCK及其介导的紧密连接蛋白结构和功能的变化抑制其侵袭、转移。本研究将用ATRA诱导A549、HepG2细胞分化，通过Rho/ROCK、MAPK调控和RNA干扰等实验，阐明诱导肿瘤细胞分化是否通过MLCK表达和活性变化及其介导的紧密连接重塑抑制侵袭、转移及调控机制

本研究用ATRA处理肿瘤细胞，观察其对肿瘤细胞的分化、侵袭和迁移等影响，并探讨其作用机制。结果表明：ATRA可抑制多种肿瘤细胞增殖，诱导其分化、侵袭和迁移。ATRA通过与维甲酸受体结合，下调cyclinD1和上调p21表达抑制RKO等肿瘤细胞的增殖；通过结合不同的维甲酸受体后，上调p53的转录、翻译和ASPP1的表达水平，以及下调MDM2和iASPP表达，来调控下游凋亡和周期相关蛋白，进而引起G0/G1期阻滞和肿瘤细胞凋亡。在诱导肿瘤细胞分化过程中，ATRA与相应受体结合后可抑制ERK/MAPK、Rho/ROCK、RARβ/MLCK等多条信号转导通路，下调MLCK的表达和活性，MLC磷酸化下降而细胞运动降低；增加细胞间紧密连接蛋白的表达和定位，最终降低细胞运动性增加细胞间粘附而抑制肿瘤细胞的侵袭和迁移。乳腺癌细胞MCF-7经小剂量γ射线照射后生成的MCF-7 FIR细胞 更具有干细胞特性，器侵袭性、成瘤性强、转移性增强、对常规药物及放射线不敏感。ATRA通过抑制MCF-7 FIR细胞的过度增殖、促进细胞凋亡、抑制其迁移、转移和成瘤能力、诱导干细胞化的MCF-7 细胞分化，抑制DNA的损伤修复等机制来发挥抗肿瘤作用。.研究过程中发现诱导肿瘤细胞分化所用的ATRA剂量较大，且剂量达到较大时才出现对信号转导通路的抑制和影响MLCK的表达和MLC磷酸化及其紧密连接蛋白的表达和重塑。较高剂量ATRA若应用于整体，易产生维甲酸的副作用，甚至出现维甲酸综合征。为此，本课题组和本校药学院合作，以ATRA为先导化合物，通过对其碳链末端极性基团进行结构修饰，合成了一系列全新的维甲酸衍生物，经体外药效学筛选发现 4-氨基-2-三氟甲基苯基维甲酸酯（4-amino-2-trifluoromethyl- phenyl retinate，ATPR）具有较强的抗肿瘤增殖和诱导分化活性，申请获得自主知识产权（中国专利号：专利ZL 200810110794.1；美国专利号：US8,110,703B2）。初步研究表明ATPR的IC50明显低于ATRA；抑制肿瘤细胞增殖、诱导肿瘤细胞分化、软琼脂克隆形成和迁移的能力明显强于ATRA等，为进一步研究和寻找低毒、高效的ATRA衍生物打下良好的基础。