4-hydroxy-2-nonenal and Carcinoma--Non-Small-Cell-Lung

Multidrug-resistant proteins (MRPs) are members of the ATP-binding cassette superfamily that facilitate detoxification by transporting toxic compounds, including chemotherapeutic drugs, out of cells. Chemotherapy, radiation, and other xenobiotic stresses have been shown to increase levels of select MRPs, although the underlying mechanism remains largely unknown. Additionally, MRP3 is suspected of playing a role in the drug resistance of non-small-cell lung carcinoma (NSCLC). Analysis of the MRP3 promoter revealed the presence of multiple putative electrophile-responsive elements (EpREs), sequences that suggest possible regulation of this gene by Nrf2, the key transcription factor that binds to EpRE. The goal of this investigation was to determine whether MRP3 induction was dependent upon the transcription factor Nrf2. Keap1, a key regulator of Nrf2, sequesters Nrf2 in the cytoplasm, preventing entry into the nucleus. The electrophilic lipid peroxidation product 4-hydroxy-2-nonenal (HNE) has been shown to modify Keap1, allowing Nrf2 to enter the nucleus. We found that HNE up-regulated MRP3 mRNA and protein levels in cell lines with wild-type Keap1 (the human bronchial epithelial cell line HBE1 and the NSCLC cell line H358), but not in the Keap1-mutant NSCLC cell lines (A549 and H460). Cell lines with mutant Keap1 had constitutively higher MRP3 that was not increased by HNE treatment. In HBE1 cells, silencing of Nrf2 with siRNA inhibited induction of MRP3 by HNE. Finally, we found that silencing Nrf2 also increased the toxicity of cisplatin in H358 cells. The combined results therefore support the hypothesis that MRP3 induction by HNE involves Nrf2 activation.

Topics: Aldehydes; Base Sequence; Bronchi; Carcinoma, Non-Small-Cell Lung; Cisplatin; Epithelium; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lung Neoplasms; Molecular Sequence Data; Multidrug Resistance-Associated Proteins; NF-E2-Related Factor 2; Promoter Regions, Genetic; RNA, Messenger; RNA, Small Interfering; Tumor Cells, Cultured; Up-Regulation

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.

Topics: Adenosine Triphosphatases; Aldehydes; Amino Acid Sequence; Antineoplastic Agents; ATP-Binding Cassette Transporters; Biological Transport; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Carrier Proteins; Cross Reactions; Doxorubicin; Drug Resistance, Neoplasm; Glutathione; GTPase-Activating Proteins; HL-60 Cells; Humans; Immunoglobulin G; K562 Cells; Lung Neoplasms; Molecular Sequence Data; Neoplasm Proteins; Protein Processing, Post-Translational; Trypsin; Tumor Cells, Cultured; U937 Cells