IADR Abstract Archives
TMEM16A and LRRC8 Regulate Proliferation of Human Tongue Cancer Cells
Objectives: Volume-regulated anion channel (VRAC) is known to have a crucial role in the proliferation of cancer cells. Molecular nature of VRAC has been investigated for over 20 years and TMEM16A, LRRC8A and bestrophin1 are assumed to be involved, albeit controversial. In the present study, we investigated features of VRAC using a human tongue squamous cell carcinoma (SCC) cell line, HST-1, and found that interaction of TMEM16A and LRRC8A regulate VRAC activities and cell proliferation.
Methods: HST-1 cells were grown in DMEM containing 10 % fetal bovine serum. Osmolarity of medium was controlled by adding water or mannitol. RT-PCR, Western blot, a single channel electrophysiology by patch-clamp technique, RNA interference (RNAi) and In situ proximity ligation assay (PLA) to detect intrinsic protein-protein interaction, were performed.
Results: TMEM16A, LRRC8A and bestrophin1 were all detected in HST-1 by RT-PCR and Western blot analysis. Hypotonic (250 mOsM) solution activated VRAC current as assessed by patch-clamp experiment, and the cell proliferation was up-regulated by 20 %. Both were inhibited by DCPIB at 30 µM, a selective inhibitor of VRAC. We then generated HST-1 cells deficient of either TMEM16A, LRRC8A or bestrophin1 molecule by RNAi. Deficiency of TMEM16A showed significant decrease of VRAC current density and suppression of growth rate, while knock-down of LRRC8A produced constitutive activation of VRAC current even in an isotonic (300 mOsM) condition. In contrast, deficiency of bestrophin1 showed no significant changes in VRAC current and growth rate. In addition, PLA revealed that TMEM16A and LRRC8A were associated in an isotonic or a hypertonic (350 mOsM) condition, but detached in a hypotonic solution.
Conclusions: These results suggest that TMEM16A is a key regulator of VRAC activities and cell proliferation, and LRRC8A is inhibitory to TMEM16A activities by a direct interaction.
Methods: HST-1 cells were grown in DMEM containing 10 % fetal bovine serum. Osmolarity of medium was controlled by adding water or mannitol. RT-PCR, Western blot, a single channel electrophysiology by patch-clamp technique, RNA interference (RNAi) and In situ proximity ligation assay (PLA) to detect intrinsic protein-protein interaction, were performed.
Results: TMEM16A, LRRC8A and bestrophin1 were all detected in HST-1 by RT-PCR and Western blot analysis. Hypotonic (250 mOsM) solution activated VRAC current as assessed by patch-clamp experiment, and the cell proliferation was up-regulated by 20 %. Both were inhibited by DCPIB at 30 µM, a selective inhibitor of VRAC. We then generated HST-1 cells deficient of either TMEM16A, LRRC8A or bestrophin1 molecule by RNAi. Deficiency of TMEM16A showed significant decrease of VRAC current density and suppression of growth rate, while knock-down of LRRC8A produced constitutive activation of VRAC current even in an isotonic (300 mOsM) condition. In contrast, deficiency of bestrophin1 showed no significant changes in VRAC current and growth rate. In addition, PLA revealed that TMEM16A and LRRC8A were associated in an isotonic or a hypertonic (350 mOsM) condition, but detached in a hypotonic solution.
Conclusions: These results suggest that TMEM16A is a key regulator of VRAC activities and cell proliferation, and LRRC8A is inhibitory to TMEM16A activities by a direct interaction.