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RALBP1 (RLIP76) is the major transporter of doxorubicin (DOX) in lung cancer cells, and that the difference in sensitivity of small cell lung cancer (SCLC) cells to DOX is due to differential phosphorylation by PKCalpha. Our recent studies have suggested that RALBP1 present in MCF-7 breast cancer cells has significantly lower specific activity for transport of DOX than wild-type recombinant protein, and its level of expression is significantly lower than that in lung cancer cells. In the present study, we have explored whether or not this is a generalized phenomenon for breast cancer, and have compared the relative contributions of RALBP1 and the ABC-family transporter, ABCG2 to total DOX transport activities in two SCLC (H1417 and H1618), two non-small cell lung cancer (NSCLC) (H358 and H520), and three breast cancer (T-47D, MDA-MB231, and MCF-7) cell lines. Results of these studies show lower protein expression and specific activity of RALBP1 in all three breast cancer cell lines as compared with lung cancer cell lines. Furthermore, we demonstrate that RALBP1 contributes only a minor fraction of DOX transport activity in breast cancer cell lines, suggesting that greater DOX sensitivity of breast cancer may be related to lower RALBP1 transporter activity and that the transport mechanisms involved in multidrug resistance of lung and breast cancer are distinct.

Doxorubicin (DOX) transport activity of Ral-interacting protein (RLIP76) in non-small cell lung cancer (NSCLC) is approximately twice that of in small cell lung cancer (SCLC). Since protein-kinase-C (PKC)alpha mediated phosphorylation of RLIP76 causes doubling of the specific activity of RLIP76, and NSCLC cells are known to have greater PKCalpha activity, we examined the contribution of PKC mediated phosphorylation of RLIP76 towards intrinsic DOX-resistance in human NSCLC. Expression of a deletion mutant RLIP76(delPKCalpha-sites) followed by depletion of the wild-type RLIP76 using a siRNA targeted at one of the deleted regions resulted in generation of cells expressing only the mutant protein, which could not be phosphorylated by PKCalpha. DOX-transport activity of the mutant RLIP76 purified from NSCLC and SCLC was similar and comparable to that of RLIP76 purified from the wild-type SCLC. However, this activity was significantly lower than that of RLIP76 purified from the wild-type NSCLC. After siRNA mediated depletion of PKCalpha, DOX-transport activities of RLIP76 purified from SCLC and NSCLC were indistinguishable. Depletion of PKCalpha inhibited the growth of NSCLC more than SCLC cells (70+/-3% vs. 43+/-5%, respectively). PKCalpha-depletion lowered the IC(50) of NSCLC cell lines for DOX to the same level as that observed for SCLC. RLIP76(-/-) mouse embryonic fibroblasts (MEFs) were significantly more sensitive to DOX as compared with RLIP76(+/+) MEFs (IC(50) 25 vs. 125nM, respectively). However, PKCalpha-depletion did not affect DOX-cytotoxicity towards RLIP76(-/-) MEFs, as opposed to RLIP76(+/+) MEFs which were sensitized by 2.2-fold. These results demonstrate that RLIP76 is a primary determinant of DOX-resistance, and that PKCalpha mediated accumulation defect and DOX-resistance in NSCLC is primarily due to differential phosphorylation of RLIP76 in SCLC and NSCLC.

In deletion mutant analyses of potential phosphorylation sites in RLIP76, we identified T297 and S509 as targets for phosphorylation by PKCalpha. Phosphorylation at T297 increased doxorubicin (DOX)-transport activity approximately 2-fold for RLIP76 purified from recombinant source, or from three small (H69, H1417, H1618) and three non-small cell, one each derived from H226 (squamous), H358 (bronchio alveolar), and H1395 (adenocarcinoma) lung cancer cell lines. T297 phosphorylation conferred sensitivity to tryptic digestion at R293. The specific activity for DOX-transport by RLIP76 purified from non-small cell, which was primarily in the phosphorylated form, was approximately twice that in small cell lung cancer cell lines. These finding offer a novel explanation for the observed intrinsic differences in sensitivity to DOX between non-small cell and small cell lung cancer cell lines.

Ral-interacting protein (RLIP76) (RALBP1) is an anti-apoptotic non-ABC glutathione (GSH)-conjugate transporter involved in receptor-ligand endocytosis, as well as in multispecific drug transport and resistance. Partial inhibition of RLIP76 using antibodies in the absence of chemotherapy drug causes apoptosis in multiple small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines and in the presence of doxorubicin (DOX), marked synergy is observed. These findings indicated that RLIP76 should be a good target for cancer cell killing; its down-regulation would promote apoptosis through both drug-dependent and drug-independent effects. To examine the effect of complete and specific RLIP76 depletion on apoptosis, we tested the effects of RLIP76 siRNA in a number of lung cancer cell lines. Growth inhibition and apoptosis was observed in all cases upon RLIP76 depletion. Consistent with these findings, augmenting cellular RLIP76 through transfection or liposomal protein delivery conferred resistance to apoptosis mediated by either DOX or 4-hydroxynonenal (4-HNE). Taken together, our results show that RLIP76 is rational and promising new target for lung cancer therapy.

RLIP76 (RALBP1) is a Ral-binding nucleotidase which functions as an energy-dependent transporter for glutathione (GSH)-conjugates as well as structurally unrelated xenobiotics. Partner of RALBP1 (POB1), also referred to as REPS2, was identified as the human RLIP76-binding protein, which contains a coiled-coil C-terminal region that binds with the RLIP76. Recent studies show that over-expression of POB1 in prostate cancer cells induces apoptosis. In present studies, we have purified POB1 and one of its deletion mutants POB1(1-512) (lacking the RLIP76-binding domain), and examined their effect on the transport activity of RLIP76. Both doxorubicin and a model GSH-conjugate, dinitrophenyl-S-glutathione (DNP-SG), transport were inhibited by POB1 in a concentration-dependent manner but not by POB1(1-512), lacking RLIP76-binding site. Liposomal delivery of recombinant POB1 to H358 (NSCLC) cancer cells caused apoptosis in a concentration-dependent manner, whereas the POB1 mutant deficient in RLIP76-binding site did not exert this effect. Augmentation of cellular POB1 resulted in increased intracellular DOX-accumulation as well as decreased rate of efflux from cells. These results show for the first time that POB1 can regulate the transport function of RLIP76 and are consistent with our previous studies showing that inhibition of RLIP76 induces apoptosis in cancer cells through the accumulation of endogenously formed GSH-conjugates.

ATP-dependent transport of doxorubicin (DOX) by recombinant human RLIP76 has been demonstrated previously in reconstituted proteoliposomes. In the preceding communication, we demonstrated that the ATPase activity of RLIP76 was 2-fold higher in NSCLC as compared with SCLC, and it correlated with their inherent DOX resistance. Present studies were performed to determine whether greater ATPase activity of RLIP76 in NSCLC translated into greater RLIP76 mediated DOX transport, and to determine the overall contribution of RLIP76 toward total DOX transport. Consistent with the greater RLIP76 ATPase activity in NSCLC, DOX transport in artificial proteoliposomes reconstituted with purified RLIP76 from NSCLC was 1.8-fold greater than in SCLC. Anti-RLIP76 antibodies completely abrogated DOX transport in these RLIP76 proteoliposomes, whereas anti-MRP or anti-Pgp antibodies had no effect on transport. DOX transport studies in crude membrane vesicles from SCLC and NSCLC also showed a 2.1-fold higher rate of transport in NSCLC as compared with SCLC. Anti-RLIP76 IgG, which recognized only RLIP76 in crude extracts of both SCLC and NSCLC, inhibited 67+/-4% (n=12 cell lines) of total DOX transport in crude membrane vesicles from both SCLC and NSCLC. Inhibition of DOX transport by anti-MRP and anti-Pgp antibody was 35+/-7% (n=12) and 2+/-0.3% (n=12), respectively. The mixture of the three antibodies inhibited DOX transport by 95+/-3% (n=12). These studies demonstrate that DOX transport activity of RLIP76 is significantly greater in NSCLC as compared with SCLC, and that RLIP76 is the major DOX transporter in lung cancer cell lines.

RLIP76 (ral-binding protein, RalBP1) is a non-ABC multi-specific transporter of amphiphilic chemotherapeutic drugs such as doxorubicin (DOX) and glutathione-electrophile conjugates. In the present studies, we used polyclonal rabbit anti-human RLIP76 IgG to inhibit RLIP76 function for determining the role of RLIP76 in DOX resistance of NSCLC cells. Western blot analyses and immunohistochemistry studies showed no recognition of other protein in crude NSCLC cell homogenates by anti-RLIP76, confirming the specificity of anti-RLIP76 IgG. In immunohistochemistry and flow cytometry studies, these antibodies recognized RLIP76 domain(s) on the cell surface. Cells coated with anti-RLIP76 IgG accumulated significantly greater DOX than cells coated with pre-immune IgG. Synergy was calculated using the Chou-Talalay median effect analysis. Herceptin was the positive control, and pre-immune IgG and Rituxan (anti-CD20) were negative controls. The interaction of anti-RLIP76 IgG and DOX were markedly synergistic (CI 0.36+/-0.27). Lesser synergy was observed between Herceptin and DOX (CI 0.75+/-0.49). Interaction between Herceptin and anti-RLIP76 was only additive (CI 1.12+/-0.5). Human IgG, Rituxan, and rabbit pre-immune IgG controls had no effect on DOX toxicity. DNA-laddering confirmed that DOX triggered apoptosis. Anti-RLIP76 IgG alone as well as Herceptin alone also triggered apoptosis in all 6 NSCLC cell lines. Anti-RLIP76 IgG and Herceptin were shown to increase DOX accumulation in NSCLC. These results demonstrated that specific inhibition of the transport function of RLIP by anti-RLIP76 IgG can trigger apoptosis and synergistically increase DOX cytotoxicity in NSCLC.

RLIP76 functions as an ATP-dependent transporter of amphiphilic chemotherapeutic drugs such as doxorubicin (DOX, adriamycin), as well as of glutathione-conjugates of endogenous electrophilic toxins such as 4-hydroxynonenal (4HNE). RLIP76 couples transport and ATP-hydrolysis with a 1:1 stoichiometry, making the ATPase activity of RLIP76 an excellent surrogate for its transport activity. Present studies were performed to determine the relationship of the RLIP76 ATPase activity with DOX and 4HNE resistance in a panel of 13 native human lung cancer cell lines. RLIP76 was purified from each cell line and homogeneity demonstrated by SDS-PAGE and amino acid composition analysis. Anti-RLIP76 antibodies were shown by Ouchterlony double immunodiffusion tests to be non-cross-reactive with any other proteins including P-glycoprotein (Pgp) or multidrug resistance associated protein (MRP). These antibodies completely immunoprecipitated ATPase activity of purified RLIP76 fractions, further confirming homogeneity of purified RLIP76. RLIP76 ATPase purified from NSCLC cell lines was about 2-fold more active than that from SCLC in the absence of the stimulator dinitrophenyl S-glutathione (206+/-47, n=7 vs. 94+/-22, n=6, nmol/min/mg protein, respectively), or in its presence (340+/-60, n=7 vs. 186+/-32, n=6, nmol/min/mg; p<0.01). Partial tryptic digest revealed a 44 kDa internal fragment of RLIP76 beginning at Thr-294 in NSCLC cell lines. This fragment was absent from all SCLC, suggesting the possibility that the activity of RLIP76 in SCLC and NSCLC is differentially regulated through post-translational modifications. Taken together, these findings suggest that RLIP76 activity is a general determinant of 4HNE and DOX resistance, and that its activity contributes to the drug-resistant phenotype of NSCLC.