title: Role of RLIP76 in lung cancer doxorubicin resistance: III. Anti-RLIP76 antibodies trigger apoptosis in lung cancer cells and synergistically increase doxorubicin cytotoxicity.
abstract: 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.
authors.affiliation: Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX 76019-0065, USA. sawasthi@uta.edu
authors.forename: Sanjay
authors.initials: S
authors.lastname: Awasthi
authors.forename: Sharad S
authors.initials: SS
authors.lastname: Singhal
authors.forename: Jyotsana
authors.initials: J
authors.lastname: Singhal
authors.forename: Yusong
authors.initials: Y
authors.lastname: Yang
authors.forename: Piotr
authors.initials: P
authors.lastname: Zimniak
authors.forename: Yogesh C
authors.initials: YC
authors.lastname: Awasthi
mesh_terms: D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001692:Biological Transport; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000378:metabolism; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004306:Dose-Response Relationship, Immunologic; D004317:Doxorubicin / Q000627:therapeutic use*; D019008:Drug Resistance, Neoplasm; D020690:GTPase-Activating Proteins; D005978:Glutathione / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D000069283:Rituximab; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D000068878:Trastuzumab
chemical_list: D018528:ATP-Binding Cassette Transporters; D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D058846:Antibodies, Monoclonal, Murine-Derived; D000970:Antineoplastic Agents; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D007074:Immunoglobulin G; C095637:RALBP1 protein, human; D013778:Tetrazolium Salts; D013844:Thiazoles; D000069283:Rituximab; D004317:Doxorubicin; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; C022616:thiazolyl blue; D005978:Glutathione; D000068878:Trastuzumab