P-glycoprotein down-regulates expression of breast cancer resistance protein in a drug-free state.

This study investigated whether P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are linked in terms of expression. RT-PCR and Western blot analyses showed that the lung cancer cell line SK-MES-1/WT expressed BCRP. In a drug-free state, BCRP expression was significantly down-regulated in doxorubicin-resistant SK-MES-1/DX1000 cells overexpressing Pgp. Pharmacological inhibitors (PSC833 or verapamil) or siRNA for Pgp inhibited the down-regulation of BCRP, which was confirmed by confocal microscopy. PSC833 induced the phosphorylation of c-Jun NH2-terminal kinase (JNK) and c-Jun, while the JNK inhibitor SP600125 inhibited this effect. Dominant negative c-Jun decreased the expression of BCRP, but increased that of Pgp. These results indicate that Pgp down-regulates BCRP expression in a drug-free state in which JNK/c-Jun is involved.

Discoidin domain receptor 1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages.

Nitric oxide (NO) is an important regulator of immune responses. Effects of cytokines, such as tumor necrosis factor (TNF)-alpha or IFN-gamma, and bacterial products, such as lipopolysaccharide, on macrophage NO production have been well documented; however, the role of the extracellular matrix proteins, including collagen, in this process remains unclear. We previously reported that discoidin domain receptor 1 (DDR1), a nonintegrin collagen receptor, was expressed in human macrophages, and its activation facilitated their differentiation as well as cytokine/chemokine production. Here, we examined the role for DDR1 in collagen-induced NO production using the murine macrophage cell line J774 cells that endogenously express DDR1. Activation of J774 cells with collagen induced the expression of inducible NO synthase (iNOS) and NO production. Inhibition of DDR1, but not beta1-integrins, abolished collagen-induced iNOS and NO production. Activation of J774 cells with collagen-activated nuclear factor-kappaB, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK) and a pharmacological inhibitor of each signaling molecule significantly reduced collagen-induced NO production. Thus, we have demonstrated, for the first time, that the interaction of DDR1 with collagen induces iNOS expression and subsequent NO synthesis in J774 cells through activation of NF-kappaB, p38 MAPK, and JNK and suggest that intervention of DDR1 signaling in macrophages may be useful in controlling inflammatory diseases in which NO plays a critical role.

Balance of NF-kappaB and p38 MAPK is a determinant of radiosensitivity of the AML-2 and its doxorubicin-resistant cell lines.

This study investigated radioresistance mechanisms in the doxorubicin-resistant acute myelogenous leukemia (AML)-2/DX100. AML-2/DX100 also showed resistance to radiation. AML-2/DX100 characterized by down-regulated catalase expression was supersensitive to exogenous hydrogen peroxide whereas they increased defense mechanisms against endogenous reactive oxygen species (ROS) as compared with AML-2/WT. In AML-2/WT, radiation increased Bax expression and its translocation to mitochondria but had little effect on translocation of Bcl-2 and consequently induced the release of cytochrome c from the mitochondria with the subsequent caspase-3 activation. On the contrary, in AML-2/DX100, radiation neither increased Bax expression nor its translocation to mitochondria while it increased Bcl-2 translocation to mitochondria. A specific p38 MAPK inhibitor SB203580 increased radioresistance in AML-2/WT but little in AML-2/DX100. It inhibited radiation-induced Bax translocation in AML-2/WT but not in AML-2/DX100, indicating that p38 MAPK is working after irradiation in AML-2/WT but not in AML-2/DX100. Electrophoretic mobility shift assay and Western blot analysis revealed that NF-kappaB in AML-2/DX100 was more activated with degradation of cytosolic IkappaBalpha than was that of AML-2/WT. cDNA microarray showed that Bfl-1/A1 and granzyme H in AML-2/DX100 were highly up-regulated (6.21-fold) and down-regulated (6.49-fold), respectively, as compared with each of AML-2/WT, which were confirmed by RT-PCR assay. Taken together, these results indicate that radioresistance mechanisms of AML-2/DX100 could be related to alterations in ROS-scavenging activity, in mitochondrial translocation of Bax and Bcl-2, and in expression of pro-apoptotic (granzyme H) and anti-apoptotic (Bfl-1/A1) genes. It has been shown that balance of p38 MAPK and NF-kappaB signals is a determinant in radiosensitivity of AML-2/WT and AML-2/DX100.

Elevated reactive oxygen species but not glutathione regulate mercury resistance to AML-2/DX100 cells.

The multidrug resistance-associated protein (MRP1) mediates cellular efflux of various xenobiotics and cellular resistance to heavy metals. Previously we reported that MRP1 mediates resistance to mercury exposure and possible mechanism mediating MRP1 expression after mercury exposure. This study was designed to investigate the role of reactive oxygen species (ROS) and glutathione on the resistance of AML-2/DX100 cells to mercuric chloride. The MRP1 overexpressing cells (AML-2/DX100) cells showed less scavenging activity to ROS induced by mercury while no difference in the basal glutathione levels between AML-2/WT and AML-2/DX100 cells. Mercury induced the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) but not c-jun-N-terminal kinase in AML-2/DX100 cells. The specific inhibitor for p38 MAPK and ERK, and antioxidant decreased the production of MRP1 and therefore resistance of AML-2/DX100 cells against mercury exposure. These results suggest that induction of ROS and downstream p38 MAPK and ERK were involved in the resistance of cells to mercury by expression MRP1 in AML-2/DX100 cells.

Mercury induces multidrug resistance-associated protein gene through p38 mitogen-activated protein kinase.

The multidrug resistance-associated protein (MRP1) belongs to a drug efflux membrane pump that confers multidrug resistance to the cells. The MRP1 mediates the cellular efflux of various xenobiotics including heavy metals and mediates cellular resistance to heavy metals. Mercury is a well-known health hazard and an environmental contaminant. Recently, information about the uptake of the heavy metals such as mercury has been suggested. However, little is known regarding molecular mechanisms of exporting mercury. This study was designed to determine if mercury could be extruded by MRP1 in acute myeloid leukemia cells (AML-2). The MRP-1-overexpressing AML-2/DX100 cells showed a higher resistance to mercury than AML-2/WT. Probenecid, which is a specific MRP1 inhibitor, decreased the resistance to mercury. Exposing the AML-2 cells to mercury-induced MRP1 gene expression and production without altering the MRP1 activity. Mercury activated p38 mitogen-activated protein kinase (MAPK) and SB 203580, a specific p38 MAPK inhibitor, blocked the mercury-induced MRP1 production. These results suggest that MRP1 can control mercury and p38 MAPK mediates the mercury-induced MRP1 gene expression.