小剂量三氧化二砷诱导肝癌干细胞分化并提高抗癌药物敏感性的机制研究

Hepatocellular carcinoma (HCC) cells are resistant to most anti-cancer drugs. Differentiation therapy is to use an appropriate agent which can result in induction of terminal differentiation of cancer stem cells (CSCs) and reversing malignant phenotypes of tumor as well as its drug sensitivity enhancement. Arsenic trioxide (ATO) has been successfully applied in differentiation induction therapy for acute promyelocytic leukemia. However, the therapeutic effect of ATO in HCC is poor with severe side effects. Our previous work revealed that after treatment with low dose ATO, the proportion of CD133+ cells dropped dramatically from 69% to 12% in HCC cells. Low dose ATO combined with cisplatin significantly suppressed proliferation of Hep3B cells. These results indicated the differentiation induction potential of ATO in HCC. In this study, we aim to further investigate the differentiation induction capacity of ATO in HCC by stemness related genes detection, sphere and clone formation assays and to explore the mechanism of ATO differentiation induction for HCC cells using gene microarray technology. We will also assess the sensitivity of HCC cells to conventional chemotherapy and sorafenib with and without ATO in vitro and in vivo. This study will further clarify the mechanism of ATO differentiation induction for HCC, and might provide theoretical and experimental basis for the novel therapeutic strategy, the combination of low dose ATO with anti-tumor drug in HCC treatment.

肝癌对大多数抗癌药物不敏感。诱导分化治疗可通过药物诱导肿瘤干细胞分化并逆转恶性表型，增强肿瘤药物敏感性。三氧化二砷（ATO）在白血病治疗中取得了巨大成功，但在肝癌治疗中效果不佳。我们前期研究用小剂量ATO作用Hep3B肝癌细胞，其干细胞表面标记CD133阳性率从69%减至12%；同时增强了顺铂抑制肝癌生长的能力。我们推测小剂量ATO可通过诱导肝癌干细胞分化提高肝癌的药物敏感性。本课题拟从干细胞标志物表达、肿瘤克隆形成及成球能力等方面探索小剂量ATO诱导肝癌干细胞分化的能力，比较ATO处理前后肿瘤干细胞分化相关基因及信号通路改变，并进行体内外验证；另外，通过肝癌患者来源的移植瘤PDTX模型探讨ATO诱导分化后肝癌对常规化疗药物及索拉非尼的增敏作用。本研究将进一步阐明ATO诱导肝癌干细胞分化的分子机制，为小剂量ATO联合抗癌药物治疗肝癌奠定理论和实验基础，为肝癌药物治疗提供新的思路。

肝癌对当前大多数常规化疗方案不敏感。因此，在晚期肝癌的治疗中，探寻新的药物治疗方案始终是一项长远的课题。肿瘤诱导分化疗法是应用化学药物诱导肿瘤细胞分化并逆转其增殖、浸润、转移等恶性表型，使其成为正常或接近正常细胞即再分化或肿瘤逆转，让其“改邪归正”，从而达到治疗的目的。三氧化二砷（ATO）在白血病的诱导分化治疗获得了成果，而其在实体肿瘤包括肝癌中的诱导分化效果极其机制尚不明确。.本课题中，我们从以下几个方面探讨了小剂量ATO对肝癌干细胞的诱导分化效果，并对其诱导分化机制进行了初步探索。1）ATO诱导分化剂量的确定及其对细胞功能的影响。2）ATO对肝癌干细胞标志物表达的影响。3）ATO对肿瘤干细胞相关基因表达的影响。4）ATO对肝癌干细胞自我更新，体外体内成瘤功能的影响。5）ATO对常规化疗药物的增敏效果观察。6）基于mRNA表达谱芯片的机制探索与进一步验证。.结果表明，小剂量（3μM）ATO并未促进细胞凋亡，但抑制了细胞增殖与侵袭。该剂量ATO显著下调了CD133，CK19，ALDH等干细胞表面标志物以及多个干细胞相关基因的表达。另外，肝癌干细胞的体内体外自我更新及成瘤效果在其作用下均显著减弱，提示ATO在体内体外对肝癌干细胞的诱导分化作用。在联合常规化疗药物的体内体外实验中，研究发现，小剂量ATO联合氟尿嘧啶及顺铂的联合使用可以达到比化疗药单用更好的疗效，并可降低化疗药物使用剂量。机制研究表明，ATO的诱导分化作用与LIF/JAK/STAT以及NFκB通路的联合抑制有关。.综上所述，本课题从干细胞相关标志物的表达、体外肿瘤成球及克隆形成能力，体内成瘤能力等角度明确了小剂量ATO诱导肝癌干细胞分化的能力，通过基因芯片技术比较了小剂量ATO处理前后的基因水平分子改变，发现ATO通过联合抑制LIF/JAK/STAT以及NFκB通路诱导细胞分化的潜在机制。该研究为小剂量ATO诱导分化治疗方案提供临床转化提供理论及实验基础，有望为当前百花齐放的肝癌药物治疗锦上添花，通过常规药物用新的方案改善肝癌的整体预后。