Aims: To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca 2+ increase in androgen-independent prostate cancer (PCa) cells. Main methods: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca 2+ dynamics by the fluorescent Ca 2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. Key findings: EGCG inhibited DU145 and PC3 proliferation with IC 50 = 46 and 56 ?M, respectively, and induced dose-dependent peaks of internal Ca 2+ that were dependent on extracellular Ca 2+ . The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca 2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca 2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca 2+ influx acts as a trigger for Ca 2+ -induced Ca 2+ release, involving both ryanodine and IP 3 receptors. Different from EGCG, ATP caused a rapid Ca 2+ increase, which was independent of external Ca 2+ , but sensitive to U73122. Significance: EGCG induces an internal Ca 2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca 2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.
Epigallocatechin-3-gallate mobilizes intracellular Ca 2+ in prostate cancer cells through combined Ca 2+ entry and Ca 2+ -induced Ca 2+ release
Carla Marchetti;Paola Gavazzo;
2020
Abstract
Aims: To elucidate the mechanism by which (-)-epigallocatechin-3-gallate (EGCG) mediates intracellular Ca 2+ increase in androgen-independent prostate cancer (PCa) cells. Main methods: Following exposure to different doses of EGCG, viability of DU145 and PC3 PCa cells was evaluated by MTT assay and the intracellular Ca 2+ dynamics by the fluorescent Ca 2+ chelator Fura-2. The expression of different channels was investigated by qPCR analysis and sulfhydryl bonds by Ellman's assay. Key findings: EGCG inhibited DU145 and PC3 proliferation with IC 50 = 46 and 56 ?M, respectively, and induced dose-dependent peaks of internal Ca 2+ that were dependent on extracellular Ca 2+ . The expression of TRPC4 and TRPC6 channels was revealed by qPCR in PC3 cells, but lack of effect by modulators and blockers ruled out an exclusive role for these, as well as for voltage-dependent T-type Ca 2+ channels. Application of dithiothreitol and catalase and sulfhydryl (SH) measurements showed that EGCG-induced Ca 2+ rise depends on SH oxidation, while the effect of EGTA, dantrolene, and the PLC inhibitor U73122 suggested that EGCG-induced Ca 2+ influx acts as a trigger for Ca 2+ -induced Ca 2+ release, involving both ryanodine and IP 3 receptors. Different from EGCG, ATP caused a rapid Ca 2+ increase, which was independent of external Ca 2+ , but sensitive to U73122. Significance: EGCG induces an internal Ca 2+ increase in PCa cells by a multi-step mechanism. As dysregulation of cytosolic Ca 2+ is directly linked to apoptosis in PCa cells, these data confirm the possibility of using EGCG as a synergistic adjuvant in combined therapies for recalcitrant malignancies like androgen-independent PCa.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
