Raza Asif, Ghosh Siddhartha Sankar
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati, India.
Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati, India.
Biochem Insights. 2019 Jan 21;12:1178626418818182. doi: 10.1177/1178626418818182. eCollection 2019.
cytosine deaminase (CD) converts 5-fluorocytosine (5-FC), a prodrug, into 5-fluorouracil (5-FU), a chemotherapeutic drug. However, the poor binding affinity of CD towards 5-FC as compared to the natural substrate cytosine, limits its application towards a successful suicide gene therapy. Although F186W mutant was developed to enhance the effect of wild-type CD, still scope for its improvement remains to further minimize the dose-dependent cytotoxicity of the drugs. Hence, in this study, we employ the anti-tumour attribute of the gap junction forming protein connexin-43 (Cx43) in conjunction with CD or F186W mutant.
Lipofectamine was used to co-transfect CD/F186W-pVITRO2 and Cx43-pEGFP-N1 plasmids construct into MCF-7 cells. Comparative analysis of cell viability was observed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) and trypan blue-based assays. To further confirm the mode of cell death was apoptosis, propidium iodide and annexin V/7-aminoactinomycin D (7-AAD)-based apoptosis assays were performed.
Semi-quantitative polymerase chain reaction (PCR) confirmed the expression of both Cx43 and CD/F186W genes after transfection. Furthermore, cell viability assays revealed the enhanced activity of F186W-Cx43 compared with CD-Cx43 and F186W alone. The trend of the reduction in cell viability was also reflected in the flow cytometry-based apoptosis analyses. Overall, F186W-Cx43 combination demonstrated its superiority over the CD-Cx43 and F186W mutant alone.
The enhanced cytotoxic activity of F186W mutant was further amplified by gap junction protein Cx43.
胞嘧啶脱氨酶（CD）可将前体药物5-氟胞嘧啶（5-FC）转化为化疗药物5-氟尿嘧啶（5-FU）。然而，与天然底物胞嘧啶相比，CD对5-FC的结合亲和力较差，限制了其在成功的自杀基因治疗中的应用。尽管开发了F186W突变体以增强野生型CD的效果，但仍有改进空间，以进一步最小化药物的剂量依赖性细胞毒性。因此，在本研究中，我们将形成间隙连接的蛋白连接蛋白43（Cx43）的抗肿瘤特性与CD或F186W突变体结合使用。
使用脂质体将CD/F186W-pVITRO2和Cx43-pEGFP-N1质粒构建体共转染到MCF-7细胞中。使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）和基于台盼蓝的试验观察细胞活力的比较分析。为了进一步确认细胞死亡方式为凋亡，进行了基于碘化丙啶和膜联蛋白V/7-氨基放线菌素D（7-AAD）的凋亡试验。
半定量聚合酶链反应（PCR）证实转染后Cx43和CD/F186W基因均表达。此外，细胞活力试验显示F186W-Cx43的活性比单独的CD-Cx43和F186W增强。基于流式细胞术的凋亡分析也反映了细胞活力降低的趋势。总体而言，F186W-Cx43组合显示出优于单独的CD-Cx43和F186W突变体。
间隙连接蛋白Cx43进一步增强了F186W突变体的细胞毒活性。
