Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein 2028, South Africa.
Laser Research Centre, Faculty of Health Sciences, University of Johannesburg, P.O. Box 17011, Doornfontein 2028, South Africa.
J Photochem Photobiol B. 2020 Mar;204:111811. doi: 10.1016/j.jphotobiol.2020.111811. Epub 2020 Jan 28.
The development of multidrug resistance is often associated with the over-expression of P-glycoprotein (P-gp). This protein prevents drug accumulation and extrudes them out of the cell before they reach the intended target. The aim of this study was to develop an in vitro MCF-7 cell line with increased expression of P-gp and test the phototoxicity of a novel photoactivated zinc phthalocyanine tetrasulfonic acid (ZnPcS4) on these cells. The over-expressed P-gp MCF-7 cells (MCF-7/DOX) were developed from wildtype (WT) MCF-7 cells by a stepwise continuous exposure of the WT cells to different concentrations of Doxorubicin (DOX) (0.1 - 1 μM) over a period of 4 months. The P-gp expression was measured using flow cytometry, immunofluorescence and enzyme immunoassay. To verify whether zinc phthalocyanine-mediated photodynamic therapy (ZnPcS4 - PDT) is effective in MCF-7/DOX, we studied the subcellular localization, phototoxicity and nuclear damage. The flow cytometry result showed two distinct peaks of P-gp positive and negative expression in MCF-7/DOX cell population, which correlates with the ELISA-based assay (p˂0.001). The ME16C (Normal breast cells) was used as control. The localization studies showed that ZnPcS4 have greater affinity for lysosome than mitochondria. Phototoxicity results indicated that photoactivated zinc phthalocyanine decreased the cell proliferation and viability as the drug and laser light dosages increased to 16 μM and 20 J/cm respectively. PDT-induced cytotoxicity using lactose dehydrogenase (LDH) enzyme leakage as measure did not increase likewise. The ZnPcS4-induced PDT was less effective for MCF-7/DOX cells which could be attributed to decreased retention of ZnPcS4 in major cellular organelles due to the presence of increased drug efflux P-gp. The current findings suggest that, increased P-gp expression, a characteristic of multidrug resistance together with other related intrinsic mechanisms might contribute to render MCF-7/DOX cells less sensitive to ZnPcS4-induced phototoxicity.
多药耐药的发展通常与 P-糖蛋白（P-gp）的过度表达有关。这种蛋白质阻止药物积累，并在药物到达预期靶点之前将其排出细胞。本研究旨在开发一种 P-gp 表达增加的 MCF-7 细胞系，并测试新型光活化锌酞菁四磺酸（ZnPcS4）对这些细胞的光毒性。通过逐步连续暴露于不同浓度的阿霉素（DOX）（0.1-1 μM），将野生型（WT）MCF-7 细胞连续暴露于不同浓度的 DOX 4 个月，从而开发出过度表达 P-gp 的 MCF-7 细胞（MCF-7/DOX）。使用流式细胞术、免疫荧光和酶免疫测定法测量 P-gp 的表达。为了验证锌酞菁介导的光动力疗法（ZnPcS4-PDT）是否对 MCF-7/DOX 有效，我们研究了亚细胞定位、光毒性和核损伤。流式细胞术结果显示，MCF-7/DOX 细胞群体中存在两种明显的 P-gp 阳性和阴性表达峰，这与基于 ELISA 的测定结果相关（p˂0.001）。ME16C（正常乳腺细胞）用作对照。定位研究表明，ZnPcS4 对溶酶体的亲和力大于对线粒体的亲和力。光毒性结果表明，随着药物和激光光剂量分别增加到 16 μM 和 20 J/cm2，光活化锌酞菁降低了细胞增殖和活力。作为衡量标准的乳糖脱氢酶（LDH）酶渗漏诱导的 PDT 细胞毒性也没有相应增加。由于存在增加的药物外排 P-gp，导致 ZnPcS4 在主要细胞细胞器中的保留减少，因此 ZnPcS4 诱导的 PDT 对 MCF-7/DOX 细胞的效果较差。目前的研究结果表明，多药耐药的特征之一是 P-gp 表达增加，以及其他相关内在机制可能导致 MCF-7/DOX 细胞对 ZnPcS4 诱导的光毒性的敏感性降低。
