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The effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability is largely unknown. This study examined whether melittin alters Ca(2+) levels and causes Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 75% by removing extracellular Ca(2+). The melittin-induced Ca(2+) influx was also implicated by melittin-caused Mn(2+) influx. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), melittin-induced Ca(2+) release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca(2+) release. At concentrations of 0.5-20 microM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 microM melittin was nearly completely reversed by prechelating cytosolic Ca(2+) with BAPTA. Melittin at 0.5-2 microM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca(2+)](i) rise by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, melittin can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.

PGs (prostaglandins) are synthesized through the cyclo-oxygenase (COX-1 and -2) pathway in a variety of cells in response to various physiological stimuli. All cells require at least one pathway for apoptosis, and mitochondrial play a central role in regulation of apoptosis. In a previous study, incubation of A549 cells with NS-398 (a COX-2-specific inhibitor) induced apoptosis and inhibited cell proliferation, and the concentrations of different PGs between various cellular compartments were found to be changed. To determine whether PG receptors are involved in this regulation, Western-blot analyses were performed specific for PGE(2) (EP receptors) and PGF(2alpha) (FP receptor) receptors, which were expressed in A549 cells. Western-blot analysis revealed that mitochondria that were isolated from A549 cells expressed EP receptors (EP2, EP3 and EP4), whereas FP receptors were undetectable. EP receptors (EP1, EP3 and EP4) and FP receptors were detected from A549 cell membrane. These results suggest that the change of PG production in A549-cells-induced cancer cell apoptosis might be related to the different expressions of EP and FP receptors in cell and mitochondrial membrane.

Cyclooxygenases (COXs) catalyze the synthesis of prostaglandins (PGs) from arachidonic acid. Overexpression of COX-2 is frequently found in human cancers and is suggested to play an important role in tumorigenesis. Recent studies indicated that COX-2 inhibitors exert potent anti-cancer effects on a number of cancers. Interestingly, some COX-2 inhibitors potently induce apoptosis, while other COX-2 inhibitors primarily induce growth inhibition. Therefore, there is a variability in the effects that different COX-2 inhibitors have on cancer cells. In this study, we demonstrated that induction of apoptosis of high COX-2-expressing A549 lung cancer cells by a specific COX-2 inhibitor NS398 was observed in cells cultured under serum-free condition. However, this drug induced G1 growth arrest rather than apoptosis in A549 cells maintained in 10% serum medium. Conversely, low COX-2-expressing H226 lung cancer cells were resistant to NS398-induced apoptosis under both serum-free and serum-containing conditions. Moreover, our results showed that NS398-induced apoptosis is associated with activation of caspase-3, a cysteine protease that plays a crucial role in the execution phase of apoptosis. These results suggest that the cytotoxic effect of COX-2 inhibitors on cancer cells may be influenced by extracellular environments and the anti-cancer action of these inhibitors in vivo needs careful evaluation. Additionally, a correlation between the level of COX-2 expression and the extent of apoptosis induced by COX-2 inhibitors was found.

Our previous work has shown that a number of sphingolipid metabolites including sphingosine, sphinganine, and other long-chain bases potently induced apoptosis in human hepatoma cells. In this study, we examined the possibility that sphingosine may trigger apoptosis in human hepatoma cells via inhibition of anti-apoptotic pathways. We investigated the effect of sphingosine on AKT kinase, a serine/threonine kinase which was found to protect cells from apoptosis induced by a variety of extracellular stresses. Our results indicated that sphingosine inhibited basal and serum-stimulated AKT kinase activity in a dose-dependent manner in hepatoma cells. Additionally, sphingosine-induced inhibition of AKT kinase was correlated with induction of apoptosis in these cells. Pretreatment of insulin, a potent stimulator of AKT kinase, partially reversed the inhibition of AKT kinase by sphingosine and counteracted the apoptotic action of this sphingolipid. Expression of activated AKT kinase partially protected cells from sphingosine-induced apoptosis, whereas expression of kinase-dead AKT kinase had no effect. The molecular mechanism by which AKT kinase suppressed the apoptotic action of sphingosine was investigated. Our results showed that increased release of cytochrome C from mitochondria and subsequent activation of caspase-3 were detected in sphingosine-treated hepatoma cells. On the contrary, expression of activated AKT kinase in Hep3B cells attenuated cytochrome C release and caspase-3 activation induced by sphingosine. Taken together, these findings suggest that suppression of AKT kinase is one of the mechanisms by which sphingosine induces apoptosis in hepatoma cells and activation of AKT kinase may inhibit sphingosine-induced apoptosis by blocking a step upstream of cytochrome C release and caspase-3 activation., (Copyright 2001 Wiley-Liss, Inc.)

Sphingosine and other long-chain bases (including sphinganine, dimethylsphingosine and stearylamine), but not octylamine (a short-chain analogue of sphinganine), induced apoptosis in Hep3B hepatoma cells. Because both D- and L-erythrosphingosine and stearylamine exert potent apoptotic effects on Hep3B cells, it is possible that these long-chain bases may activate apoptosis by inhibiting protein kinase C (PKC) activity. However, pretreatment with the PKC activator PMA could not rescue cells from apoptosis triggered by long-chain bases. Therefore the involvement of PKC in this apoptotic process requires further characterization. We also investigated whether these long-chain bases might be metabolized into ceramide in order to elicit their apoptotic action. We found that long-chain bases acted independently of ceramide in the induction of apoptosis, since addition of fumonisin B1, a fungal agent which effectively inhibits ceramide synthesis from sphingosine, did not protect against apoptosis. Additionally, we found that sphingosine-induced apoptosis was accompanied by activation of caspases. The functional role of caspases in this apoptotic process was examined by using specific caspase inhibitors. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone, which exhibits a broad specificity for caspase-family proteases, effectively blocked sphingosine-induced apoptosis. Furthermore, our results indicate that caspase-3-like proteases, but not caspase-1, are activated during apoptosis triggered by sphingosine. Enhancement of caspase-3-like activity and cleavage of poly(ADP-ribose) polymerase, an in vivo substrate for caspase-3, was clearly demonstrated in sphingosine-treated Hep3B cells. Considered together, these results suggest that caspase-3-like proteases participate in apoptotic cell death induced by sphingosine.

Induction of apoptosis in Hep3B hepatoma cells by transforming growth factor beta 1 (TGF-beta 1) was accompanied by the activation of interleukin-1-beta-converting-enzyme-like proteases, which have recently been renamed as caspases. The caspase inhibitor ZVAD-FMK, which has a broader specificity for caspase family proteases, blocked TGF-beta 1-induced apoptosis in a concentration-dependent manner. The caspases in this apoptotic process were further characterized by using a more specific caspase inhibitor, DEVD-FMK, for CPP32-like (caspase-3-like) proteases. Our results demonstrated that CPP32-like proteases were activated during apoptosis triggered by TGF-beta 1. Enhancement of CPP32-like activity was clearly detected in TGF-beta 1-treated Hep3B cells. Furthermore, cleavage of poly(ADP-ribose) polymerase, an in vivo substrate for CPP32, in these cells was confirmed by immunoblotting. Thus, we suggest that CPP32-like proteases participate in apoptotic cell death induced by TGF-beta 1.

The effect of melitlin on cytosolic free Ca2+ concentration ([Ca2+]i) and viability is largely unknown. This study examined whether melittin alters Ca2+ levels and causes Ca2+-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 75% by removing extracellular Ca2+. The melittin-induced Ca2+ influx was also implicated by melittin-caused Mn2+ influx. After pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), melittin-induced Ca2+ release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca2+ release. At concentrations of 0.5-20 μM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 μM melittin was nearly completely reversed by prechelating cytosolic Ca2+ with BAPTA. Melittin at 0.5-2 μM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MIDCK cells, melittin induced a [Ca2+]i rise by causing Ca2+ release from endoplasmic reticulum and Ca2+ influx from extracellular space. Furthermore, melittin can cause Ca2+-dependent cytotoxicity in a concentration-dependent manner. [ABSTRACT FROM AUTHOR]