The effects of cardamonin on lipopolysaccharide-induced inflammatory protein production and MAP kinase and NFkappaB signalling pathways in monocytes/macrophages.

BACKGROUND AND PURPOSE: In this study we examined the effect of the natural product cardamonin, upon lipopolysaccharide (LPS)-induced inflammatory gene expression in order to attempt to pinpoint the mechanism of action. EXPERIMENTAL APPROACHES: Cardamonin was isolated from the Greek plant A. absinthium L. Its effects were assessed on LPS-induced nitrite release and iNOS and COX-2 protein expression in two macrophage cell lines. Western blotting was used to investigate its effects on phosphorylation of the mitogen activated protein (MAP) kinases, ERK, JNK and p38 MAP kinase, and activation of the NFkappaB pathway, at the level of IkappaBalpha degradation and phosphorylation of NFkappaB. Also its effects on NFkappaB and GAS/GAF-DNA binding were assessed by EMSA. KEY RESULTS: Cardamonin concentration-dependently inhibited both NO release and iNOS expression but had no effect on COX-2 expression. It did not affect phosphorylation of the MAP kinases, degradation of IkappaBalpha or phosphorylation of NFkappaB. However, it inhibited NFkappaB DNA-binding in both LPS-stimulated cells and nuclear extracts of the cells (in vitro). It also inhibited IFNgamma-stimulated iNOS induction and GAS/GAF-DNA binding. CONCLUSIONS AND IMPLICATIONS: These results show that the inhibitory effect of cardamonin on LPS-induced iNOS induction is not mediated via effects on the initial activation of the NFkappaB or MAP kinase pathways but is due to a direct effect on transcription factor binding to DNA. However, although some selectivity in cardamonin's action is implicated by its inability to affect COX-2 expression, its exact mechanism(s) of action has yet to be identified.

Nilotinib concentration in cell lines and primary CD34(+) chronic myeloid leukemia cells is not mediated by active uptake or efflux by major drug transporters.

Imatinib mesylate and nilotinib are highly effective at eradicating the majority of chronic myeloid leukemia (CML) cells; however, neither agent induces apoptosis of primitive CML CD34(+) cells. One possible explanation is that CD34(+) cells do not accumulate sufficient intracellular drug levels because of either inadequate active uptake or increased efflux. To determine the interaction of nilotinib with major clinically implicated drug transporters, we analyzed their interactions with MDR1 (ABCB1), MRP1 (ABCC1), ABCG2 (BCRP) and human organic cation transporter (hOCT)1 in CML cell lines and primitive (CD34(+)) primary CML cells. Nilotinib is neither dependent on active import by hOCT1, nor effluxed through the ATP-binding cassette transporters analyzed. Indeed, we found nilotinib to be an inhibitor of hOCT1, MDR1 and ABCG2. The efflux transporters MDR1, MRP1 and ABCG2 are expressed on CML CD34(+) cells at 13.5, 108 and 291% of control, respectively, although hOCT1 expression was absent; however, inhibition of efflux transporter activity did not potentiate the effect of nilotinib on apoptosis, Bcr-Abl inhibition or CML CD34(+) cell proliferation. Therefore, we have found no evidence for either active uptake of nilotinib through hOCT1 or efflux through MDR1, MRP1 or ABCG2, and it is therefore unlikely that these transporters will have any effect on the clinical response to this drug.