Fmoc-D-homophenylalanine

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca2+ levels ([Ca2+]i) and growth in PC3 human prostate cancer cells was examined by using fura-2 as a fluorescent Ca2+ indicator and WST-1 as a fluorescent growth dye. NPC-14686 at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 100 microM. NPC-14686-induced Ca2+ influx was confirmed by Mn2+ quench of fura-2 fluorescence. The Ca2+ signal was also reduced by removing extracellular Ca2+. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ nearly abolished 200 microM NPC-14686-induced Ca2+ release; and conversely pretreatment with NPC-14686 completely inhibited thapsigargin-induced Ca2+ release. The Ca2+ release induced by 200 microM NPC-14686 was not affected by inhibiting phospholipase C with 2 microM U73122. Overnight treatment with 1-500 microM NPC-14686 decreased cell viability in a concentration-dependent manner. These findings suggest that in human PC3 prostate cancer cells, NPC-14686 increases [Ca2+]i by evoking extracellular Ca2+ influx and releasing intracellular Ca2+ from the endoplasmic reticulum via a phospholiase C-independent manner. NPC-14686 may be cytotoxic to prostate cancer cells.

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in HA22/VGH human hepatoma cells was explored by using fura-2 as a fluorescent Ca(2+) indicator. NPC-14686 at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. The Ca(2+) signal was reduced by removing extracellular Ca(2+) or by 10 microM nifedipine and was not changed by verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) abolished 200 microM NPC-14686-induced Ca(2+) release; and conversely pretreatment with NPC-14686 abolished thapsigargin-induced Ca(2+) release. The Ca(2+) release induced by 200 microM NPC-14686 was not changed by inhibiting phospholipase C with 2 microM U73122. Together, the results suggest that in human hepatoma cells, NPC-14686 induced a [Ca(2+)](i) increase by causing store Ca(2+) release from the endoplasmic reticulum in an phospholipase C-independent manner, and by inducing nifedipine-sensitive Ca(2+) influx.

The effect of the anti-inflammatory compound NPC-14686 on intracellular Ca²⁺ concentration ([Ca²⁺](i)) and viability in OC2 human oral cancer cells was investigated. The Ca²⁺-sensitive fluorescent probe fura-2 was used to examine [Ca²⁺](i). NPC-14686 induced [Ca²⁺](i) rises in a concentration-dependent fashion. The effect was reduced approximately by 10% by removing extracellular Ca²⁺. NPC-14686- elicited Ca²⁺ signal was decreased by nifedipine, econazole, SKF96365, and GF109203X. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished NPC-14686-induced [Ca²⁺](i) rises. Conversely, pretreatment with NPC-14686 abolished thapsigargin or BHQ-induced [Ca²⁺](i) rises. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²⁺](i) rises. At 20-100 μM, NPC-14686 inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid-acetoxymethyl ester (BAPTA/AM). NPC-14686 between 20 μM and 40 μM also induced apoptosis. Collectively, in OC2 cells, NPC-14686 induced [Ca²⁺](i) rises by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-regulated store-operated Ca²⁺ channels. NPC-14686 also caused Ca²⁺-independent apoptosis.