Sanguinarine induces apoptosis in A549 human lung cancer cells primarily via cellular glutathione depletion.

Sanguinarine is a plant-derived benzophenanthridine alkaloid and has been shown to possess anti-tumor activities against various cancer cells. In this study, we investigated whether sanguinarine induces apoptosis in A549 human lung cancer cells. Treatment of A549 cells with sanguinarine induced apoptosis in a dose- and time-dependent manner. Treatment with sanguinarine led to activation of caspases and MAPKs as well as increased MKP-1 expression. Importantly, pretreatment with z-VAD-fmk, a pan caspase inhibitor suppressed the sanguinarine-induced apoptosis in A549 cells. Moreover, pretreatment with NAC, a sulfhydryl group-containing reducing agent strongly suppressed the apoptotic response and caspase activation to sanguinarine. However, the sanguinarine-mediated cytotoxicity in A549 cells was not protected by pharmacological inhibition of MAPKs or MKP-1 siRNA-mediated knockdown of MKP-1. These results collectively suggest that sanguinarine induces apoptosis in A549 cells through cellular glutathione depletion and the subsequent caspase activation.

Cadmium specifically induces MKP-1 expression via the glutathione depletion-mediated p38 MAPK activation in C6 glioma cells.

Cadmium is a toxic heavy metal and an environmental pollutant. Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a negative regulator of the family of MAPK. In this study, we investigated the effect of heavy metals on MKP-1 expression in C6 rat glioma cells. Cadmium treatment induced MKP-1 at both protein and mRNA levels while cobalt or manganese treatment did not, suggesting the specificity. Cadmium treatment also depleted intracellular GSH and activated p38 MAPK, JNKs, and AKT. Profoundly, pretreatment with thiol-containing compounds NAC or GSH, but not vitamin E, blocked GSH depletion, 38 MAPK activation and MKP-1 expression by cadmium. Moreover, pharmacological inhibition of p38 MAPK by SB203580 suppressed the cadmium-induced MKP-1. Collectively, these results demonstrate that cadmium specifically induces MKP-1 by transcriptional up-regulation in C6 cells in a mechanism associated with the glutathione depletion-dependent p38 MAPK activation.

Leptomycin B, a metabolite of Streptomyces, inhibits the expression of inducible nitric oxide synthase in BV2 microglial cells.

Overexpression of inducible nitric oxide synthase (iNOS) and the resultant overproduction of NO has been implicated in neuronal inflammatory diseases. Leptomycin B (LMB), a metabolite of Streptomyces, has been identified as a specific inhibitor of CRM1 nuclear export receptor. In this study, we evaluated the effect of LMB on lipopolysaccharide (LPS)-induced iNOS expression in BV2 cells, a murine microglial cells and the associated mechanisms. LMB strongly inhibited LPS-induced iNOS protein and mRNA expressions in BV2 cells in which 10 ng/ml of LMB (18 nM) was sufficient to greatly down-regulate iNOS by LPS, suggesting the potency of LMB to inhibit iNOS. The data of iNOS promoter-driven luciferase assay further suggested that the LMB inhibitory effect was in part due to inhibition of iNOS transcription. However, LPS-induced activation of various intracellular signaling proteins, such as nuclear factor-kappaB (NF-kappaB), extracellular signal-regulated kinases (ERKs), p38s, and c-Jun N-terminal kinases (JNKs), whose activations are known to be important for iNOS expression by LPS in BV2 cells, were not affected in the presence of LMB. Together, these results suggest that LMB inhibits iNOS expression in response to LPS in BV2 microglia, and the inhibition seems to be associated with blockage of CRM1-mediated iNOS mRNA nuclear export and also in part transcriptional down-regulation of iNOS, but not through modulation of NF-kappaB and the mitogen-activated protein kinase signaling pathways.

Muscular cell proliferative and protective effects of N-acetylcysteine by modulating activity of extracellular signal-regulated protein kinase.

N-acetylcysteine (NAC), an antioxidant and a precursor of glutathione, is currently in clinical use for various pathological conditions. No data is available as to the relationship between NAC and muscular cell proliferation or muscular degenerative disease. In this study, we assessed the effect of NAC on growth of L6 myoblasts, a rat skeletal muscle cell line, under normal or bupivacaine-treated condition. Of interest, under normal growth conditions, NAC treatment concentration-dependently increased viability, cell number, and DNA incorporation of L6 cells. Remarkably, NAC treatment for 12 to 24 h led to increased phosphorylation of ERKs, a family of mitogen-activated protein kinase known to involve in cell proliferation, in L6 cells, and specific inhibition of ERKs by PD98059, a selective inhibitor of ERKs, greatly abolished the ability of NAC to increase the number of L6 cells. More importantly, pretreatment with NAC effectively blocked decrease in the number and ERKs phosphorylation in L6 cells induced by the exposure of bupivacaine, a local anesthetic with myotoxicity. These results collectively suggest that NAC has muscular cell proliferative and protective effects and the effects by NAC appear to be, in part, mediated via increase in ERKs activation.

Inhibitory modulation of ATP-sensitive potassium channels by gallate-ester moiety of (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenolic substance found in green tea, is well recognized to be beneficial for human health. However, it is still controversial as to what dose of this compound is indeed good for human health. Though some recent studies have interestingly reported various beneficial effects of EGCG in cell culture system, however, plasma levels of EGCG attainable by oral regular intake in humans are normally in nanomolar range. However, potential side effects of EGCG when administered parenterally at higher concentration have not been thoroughly tested. Here, we evaluated the effect of EGCG on ATP-sensitive potassium (K(ATP)) channels expressed in Xenopus oocytes. EGCG inhibited the activity of the Kir6.2/SUR1 and Kir6.2DeltaC36 channels with IC(50) of 142+/-37 and 19.9+/-1.7microM, respectively. Inhibition of EGCG was also observed in Kir6.2/SUR2A or Kir6.2/SUR2B channels. Notably, (-)-epicatechin-3-gallate (ECG), another major polyphenolic substance in green tea, was found to reduce the channel activity with greater potency than EGCG. In contrast to EGCG and ECG, which have the gallic acid-ester moiety in their own structures, (-)-epigallocatechin and (-)-epicatechin exhibited very weak inhibition of the K(ATP) channel. Collectively, these results suggest that the gallate-ester moiety of epicatechins may be critical for inhibiting the K(ATP) channel activity via the pore-forming subunit Kir6.2 and this may be a possible mechanism by which green tea extracts or EGCG may cause unexpected side effects at micromolar plasma level.