磁性微泡共载顺铂前药Pt(IV)与谷氨酰胺转运体SLC1A5-siRNA逆转基底样三阴性乳腺癌对顺铂耐药的研究

Most triple-negative breast cancers (TNBCs) belong to the basal-like subtype (BL-TNBC) and are highly sensitive to cisplatin. However, due to enhanced DNA damage repair, increased drug inactivation and less accumulation of intracellular drugs, tumors are easily resistant to cisplatin. Our previous work showed that inhibiting SLC1A5, a glutamine transporter, downregulated the expressions of DNA damage repair proteins and the content of glutathione, the important component of inactivation of cisplatin, and then enhanced the efficacy of cisplatin on BL-TNBC drug-resistant cells, suggesting that SLC1A5 may be an effective molecular target for reversing the resistance of BL-TNBC to cisplatin. Existent evidences reveal that using magnetic microbubbles for drug loading increases drug accumulation in tumor cells and improves anti-tumor efficacy with the help of magnetic targeting and ultrasound exposure. Therefore, we propose to use Fe3O4 and lipid microbubbles to construct magnetic microbubbles for loading cisplatin-based prodrug Pt(IV) and SLC1A5-siRNA (Pt(IV)-siSLC1A5-Fe3O4-MBs) in response to the key factors that causing cisplatin resistance. We will evaluate whether this complex combined with magnetic targeting and ultrasound exposure can effectively overcome the resistance of BL-TNBC to cisplatin through detecting tumor proliferation, apoptosis, DNA damage and other indicators on in vitro and in vivo modes. This study will provide experimental evidence for the exploration of strategies targeting cisplatin resistance in order to improve the application of cisplatin in the treatment of BL-TNBC.

大多数三阴性乳腺癌属基底样亚型（BL-TNBC），对顺铂高度敏感。但由于DNA损伤修复增强、药物失活及细胞内药物积累减少等因素，肿瘤极易对顺铂耐药。我们的前期工作表明抑制谷氨酰胺转运体SLC1A5可下调细胞DNA损伤修复蛋白的表达与促顺铂失活重要分子谷胱甘肽的含量，进而提高顺铂对BL-TNBC耐药细胞疗效，提示SLC1A5可能是逆转BL-TNBC对顺铂耐药的有效靶点。磁性微泡载药联合磁靶向与超声靶向辐照可增加细胞内药物积累，提高抗癌疗效。据此，我们拟针对引起顺铂耐药的因素，用Fe3O4、脂质微泡构建共载顺铂前药Pt(IV)与SLC1A5-siRNA的磁性微泡（Pt(IV)-siSLC1A5-Fe3O4-MBs），联合磁靶向与超声靶向辐照，在体内外模型上检测肿瘤增殖、凋亡、DNA损伤等指标，评价该复合物能否有效逆转BL-TNBC对顺铂的抵抗，为改善顺铂在BL-TNBC的临床应用提供实验依据。

大多数三阴性乳腺癌属基底样亚型（BL-TNBC），对顺铂高度敏感。但由于DNA损伤修复增强、药物失活及细胞内药物积累减少等因素，肿瘤极易对顺铂耐药。探索如何逆转BL-TNBC对顺铂的耐药性，以改善BL-TNBC患者的生存获益，是富有现实意义的科学研究方向。谷氨酰胺（GLN）是肿瘤细胞代谢的重要营养物质，研究表明抑制GLN转运体SLC1A5可特异抑制BL-TNBC的GLN摄取及肿瘤增殖。我们前期已发现SLC1A5参与调控肿瘤细胞的DNA损伤修复，并影响抗肿瘤治疗的疗效。我们通过TCGA数据库分析发现：乳腺癌组织中SLC1A5 mRNA表达较高的患者，其生存预后较差；TNBC中SLC1A5 mRNA水平高于正常组织及非-TNBC的乳腺癌组织中的SLC1A5 mRNA水平；BL-TNBC组织中SLC1A5 mRNA与多种DNA损伤修复相关因子的mRNA表达水平存在正相关关系。其中ERCC1是顺铂诱导DNA损伤后参与核苷酸切除修复的限速蛋白之一，我们进一步实验结果显示抑制SLC1A5可下调ERCC1的表达水平，这提示在顺铂诱导DNA损伤后SLC1A5可能参与调节DNA损伤修复活动。另外，谷胱甘肽（GSH）与顺铂结合可使顺铂无法入细胞核与DNA结合，是导致顺铂失活的重要原因。GLN是合成GSH的重要来源，我们实验结果表明：抑制BL-TNBC顺铂耐药细胞株的SLC1A5表达，可显著降低细胞内GSH的水平，这提示抑制SLC1A5很可能有助于减少细胞内顺铂的失活。我们进一步实验结果显示：抑制SLC1A5表达后，BL-TNBC顺铂耐药细胞株对顺铂的敏感性有所恢复。据此，我们推测：SLC1A5可影响BL-TNBC对顺铂的耐药，其机制可能是通过调控ERCC1介导的DNA损伤修复及细胞内的GSH水平。SLC1A5可能是逆转BL-TNBC对顺铂抵抗的潜在有效分子靶点。此外，磁性微泡载药联合磁靶向与超声靶向辐照可增加细胞内药物积累，提高抗癌疗效。据此，我们针对引起顺铂耐药的因素，用Fe3O4、脂质微泡构建共载顺铂前药Pt(IV)与SLC1A5-siRNA的磁性微泡（Pt(IV)-siSLC1A5-Fe3O4-MBs），联合磁靶向与超声靶向辐照，在体内外模型上检测肿瘤增殖、凋亡、DNA损伤等指标，评价该体系克服BL-TNBC对顺铂耐药的效果，为改善顺铂在BL-TNBC的临床应用提供实验依据。