Ca2+ movement modulates cell physiology [26]. Whether esculetin influences Ca2+ movement in PC3 cells is unknown. Esculetin provoked [Ca2+]i raises in cells dose-dependently at 25–100 µM. Esculetin provoked [Ca2+]i raises by emptying Ca2+ depots and provoking Ca2+ entry since removing external Ca2+ reduced 100 µM esculetin-provoked [Ca2+]i raises by 15%. This implicated Ca2+ influx and Ca2+ discharge occurred during 220 s of recording. It seems Ca2+ influx was via the store-operated Ca2+ channels because of the inhibition of esculetin-provoked [Ca2+]i raises by nifedipine, econazole and SKF96365. These reagents are regularly utilized as inhibitors of this Ca2+ entry [27–29]. Nevertheless, there are no specific inhibitors available [30, 31].
PKC modulators, nifedipine, SKF96365, and econazole inhibited 50% of the response-provoked by 100 µM esculetin. This implicates these modulators not only totally blocked Ca2+ influx, but also inhibited some portion of Ca2+ discharge from ER. The action of kinases is thought to couple with Ca2+ handling [32]. In the same vein, in PC3 cells, diindolylmethane- and resveratrol-provoked Ca2+ entry involved PKC-modulated store-operated Ca2+ entry [33, 34].
Regarding Ca2+ depository that participated in esculetin-provoked Ca2+ discharge, the thapsigargin-sensitive ER depots seem to be the main one. Because esculetin did not affect thapsigargin-provoked Ca2+ discharge, it appears that although both esculetin and thapsigargin discharged Ca2+ from ER, the thapsigagin-sensitive Ca2+ depository is much larger than that sensitive to esculetin. The data also depict Ca2+ discharge was through a PLC-associated route, given the discharge was completely suppressed when PLC was suppressed. Thus the results imply that esculetin evoked Ca2+ discharge from ER in a manner dependent on PLC. Our results implicate esculetin provoked death in PC3 cells at concentrations akin to that provoking [Ca2+]i raises.
I has been shown (7) that 75 µM esculetin caused apoptosis by 6.25% in PC3 cells incubated for 48 h in WST-1 whereas our data show that incubation of 70 µM esculetin overnight killed nearly 95% of PC3 cells. Thus it appears different methodology produced different results. The cytotoxic effect of esculetin on viability of PC3 cells is controversial. Esculetin was reported not to alter cell viability in PC3 cells (35).
Note that in [Ca2+]i experiments esculetin at 25–100 µM was not cytotoxic, whereas in cytotoxicity experiments, 20–70 µM esculetin killed cells dose-dependently. This was because [Ca2+]i assays were terminated within 20 min, and cytotoxicity assays were conducted after overnight incubation for WST-1 to act. This cytotoxic action seems to be dissociated of [Ca2+]i raises since BAPTA/AM incubation suppressed 100 µM esculetin-provoked [Ca2+]i raises, but did not reverse cytotoxicity (11). Cytotoxic action could be Ca2+-associated or -dissociated (10,36). In PC3 cells, Chang et al (10) suggested resveratrol provoked cell proliferation in a Ca2+-associated fashion. Conversely, Tsai et al (33) illustrated diindolylmethane provoked cell death in a Ca2+-dissociated fashion.
Collectively, in PC3 cells, esculetin provoked Ca2+ influx through PKC-modulated store-operated Ca2+ channels and Ca2+ discharge from ER in a PLC-associated manner. Esculetin provoked Ca2+-dissociated death. The effect of esculetin on Ca2+ signaling and death in other cells merits further investigation.