Depletion of RLIP76 sensitizes lung cancer cells to doxorubicin

Mitogenic and drug-resistance mediating effects of PKCα require RLIP76

“…2A). Total RLIP76 protein normalized to total crude protein showed that RLIP76 represented ~0.5% of total protein in these MEFs, an amount in close agreement with that reported previously for other human cells [15]. Addition of increasing volumes of control liposomes (not containing purified RLIP76) caused no change in RLIP76 level, but addition of increasing amounts of RLIP76-liposomes caused an apparently saturable 4 to 7-fold increase in cellular RLIP76 protein in the RLIP76 +/+ MEFs.…”

“…Inhibition of RLIP76 would thus be predicted to increase intracellular pro-apoptotic/growth inhibitory alkenals, and conversely an increased level of activity or RLIP76 should lower this. Apoptosis triggered by RLIP76 inhibition or depletion [15,18] alone, as well as increased cellular proliferation caused by augmenting cellular RLIP76 [15] or by depleting 4HNE levels through increased expression of the 4HNE metabolizing GST isozymes,, strongly supports this model. Studies of alkenal levels in RLIP76-transfected cells as well as in RLIP76 −/− animal tissues confirm this model [14].…”

“…RLIP76-depletion caused a consistent ~70% decrease in surviving cells in both SCLC and NSCLC. We have previously shown that RLIP76-depletion causes apoptosis in both cell types [15,18]. Depletion of PKCα by siRNA also reduced cell growth, though the effect was more prominent in NSCLC as compared with SCLC.…”

“…Increased PKCα-mediated ☆ Abbreviations: RLIP76 (RALBP1), Ral-interacting protein; DOX, doxorubicin; SCLC, small cell lung cancer; NSCLC, non-small cell lung cancer; GSH, glutathione; GS-E, glutathione-electrophile conjugate; DNP-SG, dinitrophenyl S-glutathione; MRP, multidrug-resistance associated protein; PMA, phorbol ester (phorbol 12-myristate 13-acetate); PKC, protein-kinase-C. We have recently identified a new target of PKCα, RLIP76 (RALBP1). RLIP76 is a Ralbinding Rho-GAP protein (inhibitor of Rho-signaling) which we have shown to be the predominant cellular mechanism for ATP-dependent effux of glutathione-conjugate (GS-E) and chemotherapy drugs (such as DOX) [6][7][8][9][10][11][12][13][14][15][16][17][18]. Although it can function as a drugresistance transporter, the major physiological role of RLIP76 appears to be the regulation of intracellular levels of lipid-alkenals and alkenal-glutathione conjugates, the formation of which is an early and obligatory event in course of oxidant/radiant stress or signaling [8][9][10]19] The transport activity of RLIP76 functions to regulate cellular levels of glutathionyladducts of lipidoxidation derived reactive oxygen species which are known to exert direct effects in cell proliferation, differentiation, and apoptosis [8][9][10]19].…”

“…Cells were then washed with PBS, followed by 96 h incubation at 37 °C in medium before MTT assay [21]. Levels of RLIP76 protein in cells homogenates were measured by ELISA using anti-RLIP76 IgG as previously described [15]. …”