由Cx32/Cx26组成的间隙连接细胞间通讯降低光动力疗法的光毒性：一种“见义勇为”效应。

Decreased phototoxicity of photodynamic therapy by Cx32/Cx26-composed GJIC: A "Good Samaritan" effect.

作者信息

Wu Deng-Pan, Ding Chun-Hui, Bai Li-Ru, Zhou Yan, Yang Si-Man, Zhang Fan, Huang Jin-Lan

机构信息

Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Pharmacy School of Xuzhou Medical University, Xuzhou City 221004, Jiangsu Province, P. R. China.

Department of Pharmacology, Pharmacy School of Xuzhou Medical University, Xuzhou City 221004, Jiangsu Province, P. R. China.

出版信息

Lasers Surg Med. 2019 Mar;51(3):301-308. doi: 10.1002/lsm.23044. Epub 2019 Jan 7.

DOI:10.1002/lsm.23044

PMID:30615224

Abstract

BACKGROUND AND OBJECTIVE

Photodynamic therapy (PDT) has been widely used to treat malignant tumors. Our previous studies indicated that connexin (Cx) 32- and Cx26-composed gap junctional intercellular communication (GJIC) could improve the phototoxicity of PDT. However, the role of heterotypic Cx32/Cx26-formed GJIC in PDT phototoxicity is still unknown. Thus, the present study was aimed to investigate the effect of Cx32/Cx26-formed GJIC on PDT efficacy.

METHODS

CCK8 assay was used to detect cell survival after PDT. Western blot assay was utilized to detect Cx32/Cx26 expression. "Parachute" dye-coupling assay was performed to measure the function of GJ channels. The intracellular Ca concentrations were determined using flow cytometer. ELISA assay was performed to detect the intracellular levels of PGE and cAMP.

RESULTS

The present study demonstrates there is a Cx32/Cx26-formed GJIC-dependent reduction of phototoxicity when cells were exposure to low concentration of Photofrin. Such a protective action is missing at low cell density due to the lack of GJ coupling. Under high-cell density condition, where there is opportunity for the cells to contact each other and form GJ, suppressing Cx32/Cx26-formed GJIC by either inhibiting the expression of Cx32/Cx26 or pretreating with GJ channel inhibitor augments PDT phototoxicity after cells were treated with at 2.5 µg/ml Photofrin. The above results suggest that at low Photofrin concentration, the presence of Cx32/Cx26-formed GJIC may decrease the phototoxicity of PDT, leading to the insensitivity of malignant cells to PDT treatment. The GJIC-mediated PDT insensitivity was associated with Ca and prostaglandin E (PGE ) signaling pathways.

CONCLUSION

The present study provides a cautionary note that for tumors expressing Cx32/Cx26, the presence of Cx32/Cx26-composed GJIC may cause the resistance of tumor cells to PDT. Oppositely, treatment strategies designed to downregulate the expression of Cx32/Cx26 or restrain the function of Cx32/Cx26-mediated GJIC may increase the sensitivity of malignant cell to PDT. Lasers Surg. Med. 51:301-308, 2019. © 2019 Wiley Periodicals, Inc.

摘要

背景与目的

光动力疗法（PDT）已被广泛用于治疗恶性肿瘤。我们之前的研究表明，由连接蛋白（Cx）32和Cx26组成的间隙连接细胞间通讯（GJIC）可提高PDT的光毒性。然而，异型Cx32/Cx26形成的GJIC在PDT光毒性中的作用仍不清楚。因此，本研究旨在探讨Cx32/Cx26形成的GJIC对PDT疗效的影响。

方法

采用CCK8法检测PDT后细胞存活率。利用蛋白质免疫印迹法检测Cx32/Cx26表达。进行“降落伞”染料偶联试验以测量GJ通道的功能。使用流式细胞仪测定细胞内钙浓度。采用酶联免疫吸附测定法检测细胞内前列腺素E（PGE）和环磷酸腺苷（cAMP）水平。

结果

本研究表明，当细胞暴露于低浓度的血卟啉时，存在一种依赖Cx32/Cx26形成的GJIC的光毒性降低现象。由于缺乏GJ偶联，在低细胞密度时这种保护作用不存在。在高细胞密度条件下，细胞有机会相互接触并形成GJ，通过抑制Cx32/Cx26的表达或用GJ通道抑制剂预处理来抑制Cx32/Cx26形成的GJIC，在用2.5μg/ml血卟啉处理细胞后可增强PDT光毒性。上述结果表明，在低血卟啉浓度下，Cx32/Cx26形成的GJIC的存在可能会降低PDT的光毒性，导致恶性细胞对PDT治疗不敏感。GJIC介导的PDT不敏感性与钙和前列腺素E（PGE）信号通路有关。