Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
J Physiol Pharmacol. 2020 Oct;71(5). doi: 10.26402/jpp.2020.5.08. Epub 2021 Jan 16.
Cigarette smoking is a major risk factor for pulmonary diseases, including chronic obstructive pulmonary disease (COPD) and cancer. Cigarette smoke is reported to contain over 4,000 chemical compounds. Therefore, it needs to study the effects of cigarette smoke extract (CSE) administration on intracellular calcium concentration. In this study, we investigated how CSE influences intracellular calcium concentration in human lung adenocarcinoma A549 cells. The CSE concentrations used (0.4, 2, 3%) did not influence cell viability. However, at these CSE concentrations, calcium influx transient receptor potential vanilloid 4 (TRPV4) and transient receptor potential vanilloid 6 (TRPV6) proteins significantly increased, whereas calcium efflux sodium-calcium exchanger (NCX1) and plasma membrane Ca ATPase (PMCA1) proteins significantly decreased from those of the control cells. The 3% CSE treatment produced an intracellular calcium concentration higher than that of the control treatment through methods of co-transfection of pGP-CMV-GCaMP6f/CMV-R-GECO1.2 and Rhod-4 Assay. CSE induced concentration-dependent increments in hypoxia-inducible factor (HIF)-1α and HIF-2α protein levels. Moreover, phosphorylation of ERK and Akt was induced by CSE treatment. Also, mitochondrial marker B-cell lymphoma 2 (Bcl-2) protein level decreased and Bcl-2-associated X (Bax) protein level increased following CSE treatment. Also, endoplasmic reticulum (ER) stress markers BiP, CHOP, p-SAPK, and p-eIF2α levels were increased by CSE treatment. These results suggest that CSE may increase the concentration of intracellular calcium, thus increasing mitochondrial and ER stress.
吸烟是肺部疾病的主要危险因素，包括慢性阻塞性肺疾病（COPD）和癌症。据报道，香烟烟雾中含有超过 4000 种化学物质。因此，需要研究香烟烟雾提取物（CSE）给药对细胞内钙浓度的影响。在这项研究中，我们研究了 CSE 如何影响人肺腺癌细胞 A549 细胞内的钙浓度。使用的 CSE 浓度（0.4、2、3%）不会影响细胞活力。然而，在这些 CSE 浓度下，钙内流瞬时受体电位香草酸 4（TRPV4）和瞬时受体电位香草酸 6（TRPV6）蛋白显著增加，而钙外排钠钙交换蛋白（NCX1）和质膜 Ca ATP 酶（PMCA1）蛋白则显著减少从对照细胞。3%的 CSE 处理通过共转染 pGP-CMV-GCaMP6f/CMV-R-GECO1.2 和 Rhod-4 测定法，产生比对照处理更高的细胞内钙浓度。CSE 诱导缺氧诱导因子（HIF）-1α和 HIF-2α蛋白水平呈浓度依赖性增加。此外，CSE 处理诱导 ERK 和 Akt 磷酸化。此外，CSE 处理后，线粒体标记物 B 细胞淋巴瘤 2（Bcl-2）蛋白水平降低，Bcl-2 相关 X（Bax）蛋白水平升高。此外，内质网（ER）应激标志物 BiP、CHOP、p-SAPK 和 p-eIF2α 水平在 CSE 处理后增加。这些结果表明，CSE 可能增加细胞内钙浓度，从而增加线粒体和 ER 应激。
