P25 Sulfide signalling potentiates apoptosis through the up-regulation of IP3 receptors type 1 and 2

Abstract

Inositol 1,4,5-triphosphate receptors (IP3R) are intracellular calcium channels localised mainly on the endoplasmic reticulum. Up to now, three types of IP3R were characterised – type 1 (IP3R1), type 2 (IP3R2) and type 3 (IP3R3), differing predominantly in their localisation. These channels are releasing calcium upon binding IP3to the channel protein. Calcium release through IP3R can result in the activation of inner (mitochondrial) pathway of apoptosis induction. Thus, modulation of these channels in cancer cells is of a special importance. Unravelling regulation and function of IP3R, especially under the pathological situations, can result in the development of new therapeutic strategies based on the IP3R’s activation and/or blocking. Aim of the present work was to investigate an interaction between the calcium and sulfide signalling pathways, particularly effects of the slow H2S release donor morpholin-4-ium-4-methoxyphenyl-(morpholino)-phosphino-dithioate (GYY4137) on the expression of (IP3R) with the possible impact on the apoptosis induction in HeLa cells. Gene expression, Western blot analysis, apoptosis determination by Annexin V-Fluos and drop in mitochondrial membrane potential by 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC1) and immunofluorescence were used to determine differences in control and GYY4137 treated HeLa cells. In HeLa cell line, GYY4137 (10 μmol L−1) up-regulated expression of the IP3R1 and IP3R2, but not IP3R3 on both, mRNA and protein levels. Concurrently, cytosolic calcium increased and reticular calcium was depleted in concentration-dependent manner, partially by the involvement of IP3R. Depletion of calcium from reticulum was accompanied by increase in endoplasmic reticulum (ER) stress markers, such as X-box, CHOP and ATF4, thus pointing to a development of ER stress due to GYY4137 treatment. Also, GYY4137 treatment of HeLa cells increased number of apoptotic cells. These results suggest an involvement of H2S in both IP3-induced calcium signalling and induction of apoptosis, possibly through the activation of ER stress.