Klinik für Augenheilkunde, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, Berlin, Germany.
Department of Cell Morphology and Molecular Neurobiology, Faculty of Biology and Biotechnology, Ruhr University Bochum, Bochum, Germany.
Lab Invest. 2021 Jan;101(1):70-88. doi: 10.1038/s41374-020-00485-2. Epub 2020 Sep 18.
There are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca homeostasis. Accordingly, we determined if Asc induced intracellular Ca influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca, 1 mM Asc induced larger intracellular Ca transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La, 10 mM NAC, or 100 µM 2-APB, these Ca transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.
有迹象表明，作为辅助剂的药理学剂量抗坏血酸（Asc）可改善癌症的化学治疗管理。这种有利的结果源于其细胞毒性作用，这是由于氧化应激机制。由于细胞内 Ca 水平的调节有助于维持细胞活力，我们假设 Asc 的作用之一包括破坏细胞内 Ca 稳态的调节。因此，我们确定 Asc 是否通过激活百日咳敏感 Gi/o 偶联 GPCR 诱导细胞内 Ca 内流，进而在依托泊苷耐药和敏感的视网膜母细胞瘤（WERI-Rb1）肿瘤细胞中激活瞬时受体电位（TRP）通道。钙成像、全细胞膜片钳和实时定量 PCR（qRT-PCR）与使用台盼蓝细胞染料排除法测量 RB 细胞存活率同时进行。两种细胞系中 TRPM7 基因表达水平相似，而在依托泊苷耐药的 WERI-Rb1 细胞中，TRPV1、TRPM2、TRPA1、TRPC5、TRPV4 和 TRPM8 基因表达水平下调。在存在细胞外 Ca 的情况下，1 mM Asc 在依托泊苷耐药的 WERI-Rb1 中诱导更大的细胞内 Ca 瞬变，而在其依托泊苷敏感的对应物中则更小。用 100 μM CPZ、500 μM La、10 mM NAC 或 100 μM 2-APB，这些 Ca 瞬变明显减少。这些抑制剂也对 Asc 诱导的全细胞电流升高有相应的抑制作用。百日咳毒素（PTX）预孵育阻断 Ca 流入的增加。显微镜分析显示，在用 1 mM Asc 暴露 4 天后，两种 RB 细胞系的细胞活力几乎下降了 100%。综上所述，氧化介导的 Asc 诱导的 WERI-Rb1 细胞毒性的作用之一源于其促进 Gi/o 偶联 GPCR 通过 TRP 通道介导的细胞内 Ca 流入增加。因此，设计靶向 TRP 通道调节的药物可能是提高 RB 化学治疗效果的可行方法。此外，Asc 可作为癌症治疗的辅助剂。
