Movassaghian Sara, Xie Yuran, Hildebrandt Claudia, Rosati Rayna, Li Ying, Kim Na Hyung, Conti Denise S, da Rocha Sandro R P, Yang Zeng-Quan, Merkel Olivia M
Department of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University , Detroit, Michigan 48201, United States.
Department of Oncology, Karmanos Cancer Institute, Wayne State University , Detroit, Michigan 48201, United States.
Mol Pharm. 2016 Aug 1;13(8):2605-21. doi: 10.1021/acs.molpharmaceut.5b00948. Epub 2016 Jun 27.
Basal-like breast cancer (BLBC) accounts for the most aggressive types of breast cancer, marked by high rates of relapse and poor prognoses and with no effective clinical therapy yet. Therefore, investigation of new targets and treatment strategies is more than necessary. Here, we identified a receptor that can be targeted in BLBC for efficient and specific siRNA mediated gene knockdown of therapeutically relevant genes such as the histone demethylase GASC1, which is involved in multiple signaling pathways leading to tumorigenesis. Breast cancer and healthy breast cell lines were compared regarding transferrin receptor (TfR) expression via flow cytometry and transferrin binding assays. Nanobioconjugates made of low molecular weight polyethylenimine (LMW-PEI) and transferrin (Tf) were synthesized to contain a bioreducible disulfide bond. siRNA complexation was characterized by condensation assays and dynamic light scattering. Cytotoxicity, transfection efficiency, and the targeting specificity of the conjugates were investigated in TfR positive and negative healthy breast and breast cancer cell lines by flow cytometry, confocal microscopy, RT-PCR, and Western blot. Breast cancer cell lines revealed a significantly higher TfR expression than healthy breast cells. The conjugates efficiently condensed siRNA into particles with 45 nm size at low polymer concentrations, showed no apparent toxicity on different breast cancer cell lines, and had significantly greater transfection and gene knockdown activity on mRNA and protein levels than PEI/siRNA leading to targeted and therapeutic growth inhibition post GASC1 knockdown. The synthesized nanobioconjugates improved the efficiency of gene transfer and targeting specificity in transferrin receptor positive cells but not in cells with basal receptor expression. Therefore, these materials in combination with our newly identified siRNA sequences are promising candidates for therapeutic targeting of hard-to-treat BLBC and are currently further investigated regarding in vivo targeting efficacy and biocompatibility.
基底样乳腺癌（BLBC）是最具侵袭性的乳腺癌类型，其特点是复发率高、预后差，且目前尚无有效的临床治疗方法。因此，研究新的靶点和治疗策略十分必要。在此，我们鉴定出一种受体，可在BLBC中作为靶点，通过小干扰RNA（siRNA）高效、特异性地介导对治疗相关基因（如参与多条导致肿瘤发生信号通路的组蛋白去甲基化酶GASC1）的基因敲除。通过流式细胞术和转铁蛋白结合试验，比较了乳腺癌细胞系和健康乳腺细胞系中转铁蛋白受体（TfR）的表达情况。合成了由低分子量聚乙烯亚胺（LMW-PEI）和转铁蛋白（Tf）组成的纳米生物共轭物，其含有可生物还原的二硫键。通过凝聚试验和动态光散射对siRNA复合情况进行了表征。通过流式细胞术、共聚焦显微镜、逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法，在TfR阳性和阴性的健康乳腺及乳腺癌细胞系中研究了共轭物的细胞毒性、转染效率和靶向特异性。乳腺癌细胞系显示出比健康乳腺细胞显著更高的TfR表达。共轭物在低聚合物浓度下能有效地将siRNA凝聚成大小为45纳米的颗粒，对不同乳腺癌细胞系无明显毒性，且在mRNA和蛋白质水平上比聚乙烯亚胺/siRNA具有显著更高的转染和基因敲除活性，导致GASC1敲除后实现靶向性治疗生长抑制。合成的纳米生物共轭物提高了转铁蛋白受体阳性细胞中的基因转移效率和靶向特异性，但在具有基底受体表达的细胞中未提高。因此，这些材料与我们新鉴定的siRNA序列相结合，有望成为治疗难治性BLBC的候选药物，目前正在进一步研究其体内靶向疗效和生物相容性。
