Melatonin downregulates TRPC6, impairing store-operated calcium entry in triple-negative breast cancer cells

Isaac Jardin,Raquel Diez-Bello,Debora Falcon,Sandra Alvarado,Sergio Regodon,Gines M Salido,Tarik Smani,Juan A Rosado

doi:10.1074/jbc.ra120.015769

Abstract

Melatonin has been reported to induce effective reduction in growth and development in a variety of tumors, including breast cancer. In triple-negative breast cancer (TNBC) cells, melatonin attenuates a variety of cancer features, such as tumor growth and apoptosis resistance, through a number of still poorly characterized mechanisms. One biological process that is important for TNBC cells is store-operated Ca2+ entry (SOCE), which is modulated by TRPC6 expression and function. We wondered whether melatonin might intersect with this pathway as part of its anticancer activity. We show that melatonin, in the nanomolar range, significantly attenuates TNBC MDA-MB-231 cell viability, proliferation, and migration in a time- and concentration-dependent manner, without having any effect on nontumoral breast epithelial MCF10A cells. Pretreatment with different concentrations of melatonin significantly reduced SOCE in MDA-MB-231 cells without altering Ca2+ release from the intracellular stores. By contrast, SOCE in MCF10A cells was unaffected by melatonin. In the TNBC MDA-MB-468 cell line, melatonin not only attenuated viability, migration, and SOCE, but also reduced TRPC6 expression in a time- and concentration-dependent manner, without altering expression or function of the Ca2+ channel Orai1. The expression of exogenous TRPC6 overcame the effect of melatonin on SOCE and cell proliferation, and silencing or inhibition of TRPC6 impaired the inhibitory effect of melatonin on SOCE. These findings indicate that TRPC6 downregulation might be involved in melatonin's inhibitory effects on Ca2+ influx and the maintenance of cancer hallmarks and point toward a novel antitumoral mechanism of melatonin in TNBC cells.

Highlights

Breast cancer is one of the most common tumors and the major cause of female cancer deaths in the Western world [1]
We have explored the effect on cell viability of the treatment of triple-negative breast cancer (TNBC) MDA-MB-231 cells and nontumoral breast epithelial MCF10A cells with melatonin by using the cell-permeant dye calcein and propidium iodide
Pretreatment with 100 nM melatonin for 168 h (p = 0.0004; Dunnett’s test) or with 1000 nM melatonin for 72 h (p = 0.0008) and 168 h (p = 0.0002) significantly enhanced propidium iodide staining (Fig. 1; n = 6–8). These findings indicate that melatonin reduces MDA-MB-231 cell viability in a time- and concentration-dependent manner without affecting the viability of MCF10A cells

Summary

Introduction

Breast cancer is one of the most common tumors and the major cause of female cancer deaths in the Western world [1]. Pretreatment of MDA-MB-231 cells with melatonin, exclusively for 72 or 168 h, significantly attenuated TG-induced Ca2+ influx in a concentration-dependent manner, without having any significant effect on Ca2+ release from the intracellular stores (Fig. 3, B, D, F, and H; n = 50 cells/day/5–7 days).

Results

Conclusion