Carboxyamidotriazole-induced inhibition of mitochondrial calcium import blocks capacitative calcium entry and cell proliferation in HEK-293 cells

Abstract

Blocking calcium entry may prevent normal and pathological cell proliferation. There is evidence suggesting that molecules such as carboxyamidotriazole, widely used in anti-cancer therapy based on its ability to block calcium entry in nonexcitable cells, also have antiproliferative properties. We found that carboxyamidotriazole and the capacitative calcium entry blocker 2-aminoethoxydiphenyl borate inhibited proliferation in HEK-293 cells with IC50 values of 1.6 and 50 μM, respectively. Capacitative calcium entry is activated as a result of intracellular calcium store depletion. However, non-capacitative calcium entry pathways exist that are independent of store depletion and are activated by arachidonic acid and diacylglycerol, generated subsequent to G protein coupled receptor stimulation. We found that carboxyamidotriazole completely inhibited the capacitative calcium entry and had no effect on the amplitude of arachidonic-acid-activated non-capacitative calcium entry. However, investigation of the effects of carboxyamidotriazole on mitochondrial calcium dynamics induced by carbachol, capacitative calcium entry and exogenously set calcium loads in intact and digitonin-permeabilized cells revealed that carboxyamidotriazole inhibited both calcium entry and mitochondrial calcium uptake in a time-dependent manner. Mitochondrial inner-membrane potential was altered by carboxyamidotriazole treatment, suggesting that carboxyamidotriazole antagonizes mitochondrial calcium import and thus local calcium clearance, which is crucial for the maintenance of capacitative calcium entry.

Arachidonic Acid–Induced Ca2+ Entry Is Involved in Early Steps of Tumor Angiogenesis

“…This is of particular interest considering that CAI is under phase I and phase II clinical trials for the treatment of solid tumors (22,(24)(25)(26)(27). CAI is a nonselective blocker of nonvoltage-gated Ca 2+ channels and it has been shown to inhibit both SOCE and NSOCE (17,(66)(67)(68). In particular, we previously showed that CAI blocks AAinduced NSOCE in normal endothelial cells (17).…”

Calcium signalling and cancer cell growth

“…Conversely, in cells with de-energized mitochondria and reduced mitochondrial Ca 2+ uptake, the elevated [Ca 2+ ] i maintains storeoperated channels closed, consistent with the Ca 2+ -dependent inactivation of I CRAC (Hoth, et al, 2000, Parekh, 1998. As mitochondria have the potential to regulate intracellular Ca 2+ signalling and determine the transcriptional potential of a cell (Hoth, et al, 2000), targeting mitochondria has recently been exploited to control I CRAC -mediated Ca 2+ influx and associated cell proliferation (Enfissi, et al, 2004, Holmuhamedov, et al, 2002, Mignen, et al, 2005, Nunez, et al, 2006. Drugs such as carboxyamidotriazole (CAI) or salicylate, which inhibit mitochondrial calcium import (Mignen, et al, 2005, Nunez, et al, 2006, or diazoxide, which displays a predilection for the inner mitochondrial membrane (Holmuhamedov, et al, 2002), can arrest cell cycle progression and block cell proliferation.…”

Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

“…For mitochondrial calcium analysis, the fluorescence of Rhod-2 was recorded during consecutive 500-ms pulses at 550/590 nm excitation/emission. Rhod-2 fluorescence was analyzed by selecting regions of interest where MitoTracker Green and Rhod-2 co-localized (43). Fluorescence intensity at each time (F t ) was calculated after correcting for background and differential bleaching.…”