Section of Pathology, Department of Morphological Biology, Fukuoka Dental College, Fukuoka 8140193, Japan; Oral Medicine Research Center, Fukuoka Dental College, Fukuoka 8140193, Japan.
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University, Fukuoka 8128582, Japan.
Eur J Pharmacol. 2021 Mar 15;895:173881. doi: 10.1016/j.ejphar.2021.173881. Epub 2021 Jan 19.
Volume-regulated anion channels (VRACs), expressed in various cells, play an important role in cell volume regulation. Despite being physiologically defined almost half a century ago, only the molecular candidates of VRAC, TMEM16A, LRRC8A, and bestrophin-1 (BEST1), are known. Here, we aimed to explore the functional significance of VRAC in, HST-1, an oral squamous cell carcinoma (OSCC) cell line.
Cell proliferation assays, RT-PCR, Western blot, and flow cytometry were used to estimate changes in gene expression and cell proliferation. Ion channel activity was recorded using the patch-clamp technique. Specific genes were knocked-down by siRNA assays.
VRAC, identified as a hypotonicity-induced current, was highly functional and associated with the proliferation of HST-1 cells but not of HaCaT (a normal keratinocyte) cells. The pharmacological profile of VRAC in HST-1 was similar to that reported previously. DCPIB, a specific VRAC inhibitor, completely inhibited VRAC and proliferation of HST-1 cells, eventually leading to apoptosis. VRAC in HST-1 was attenuated by the knockdown of TMEM16A and LRRC8A, while knockdown of BEST1 affected cell proliferation. In situ proximity ligation assay showed that TMEM16A and LRRC8A co-localized under isotonic conditions (300 mOsM) but were separated under hypotonic conditions (250 mOsM) on the plasma membrane.
We have found that VRAC acts to regulate the proliferation of human metastatic OSCC cells and the composition of VRAC may involve in the interactions between TMEM16A and LRRC8A in HST-1 cells.
容积调节阴离子通道（VRAC）在各种细胞中表达，在细胞体积调节中发挥重要作用。尽管 VRAC 在近半个世纪前就已经在生理学上被定义，但目前只知道 VRAC 的分子候选物 TMEM16A、LRRC8A 和 bestrophin-1（BEST1）。在这里，我们旨在探讨 VRAC 在口腔鳞状细胞癌（OSCC）细胞系 HST-1 中的功能意义。
使用细胞增殖测定、RT-PCR、Western blot 和流式细胞术来估计基因表达和细胞增殖的变化。使用膜片钳技术记录离子通道活性。通过 siRNA 测定敲低特定基因。
VRAC 被鉴定为低渗诱导电流，在 HST-1 细胞的增殖中高度活跃且相关，但在 HaCaT（正常角质形成细胞）细胞中则没有。HST-1 中 VRAC 的药理学特征与之前报道的相似。DCPIB，一种特异性 VRAC 抑制剂，完全抑制了 HST-1 细胞中的 VRAC 和增殖，最终导致细胞凋亡。TMEM16A 和 LRRC8A 的敲低减弱了 HST-1 中的 VRAC，而 BEST1 的敲低则影响了细胞增殖。原位接近连接测定显示，TMEM16A 和 LRRC8A 在等渗条件（300 mOsM）下在质膜上共定位，但在低渗条件（250 mOsM）下则分离。
我们发现 VRAC 作用于调节人转移性 OSCC 细胞的增殖，而 VRAC 的组成可能涉及 HST-1 细胞中 TMEM16A 和 LRRC8A 之间的相互作用。
