The inhibition of chloride intracellular channel 1 enhances Ca(2+) and reactive oxygen species signaling in A549 human lung cancer cells

Chloride intracellular channel 1 (CLIC1) is a promising therapeutic target in cancer due to its intrinsic characteristics; it is overexpressed in specific tumor types and its localization changes from cytosolic to surface membrane depending on activities and cell cycle progression. Ca(2+) and reactive oxygen species (ROS) are critical signaling molecules that modulate diverse cellular functions, including cell death. In this study, we investigated the function of CLIC1 in Ca(2+) and ROS signaling in A549 human lung cancer cells. Depletion of CLIC1 via shRNAs in A549 cells increased DNA double-strand breaks both under control conditions and under treatment with the putative anticancer agent chelerythrine, accompanied by a concomitant increase in the p-JNK level. CLIC1 knockdown greatly increased basal ROS levels, an effect prevented by BAPTA-AM, an intracellular calcium chelator. Intracellular Ca(2+) measurements clearly showed that CLIC1 knockdown significantly increased chelerythrine-induced Ca(2+) signaling as well as the basal Ca(2+) level in A549 cells compared to these levels in control cells. Suppression of extracellular Ca(2+) restored the basal Ca(2+) level in CLIC1-knockdown A549 cells relative to that in control cells, implying that CLIC1 regulates [Ca(2+)](i) through Ca(2+) entry across the plasma membrane. Consistent with this finding, the L-type Ca(2+) channel (LTCC) blocker nifedipine reduced the basal Ca(2+) level in CLIC1 knockdown cells to that in control cells. Taken together, our results demonstrate that CLIC1 knockdown induces an increase in the intracellular Ca(2+) level via LTCC, which then triggers excessive ROS production and consequent JNK activation. Thus, CLIC1 is a key regulator of Ca(2+) signaling in the control of cancer cell survival.