ritonavir and Colonic-Neoplasms

The protease inhibitor ritonavir is an integral part of current antiretroviral therapy targeting human immunodeficiency virus. Recent studies demonstrate that ritonavir induces apoptotic cell death with high efficiency in lymphoblastoid cell lines. Moreover, ritonavir can suppress activation of the transcription factor nuclear factor-kappaB and is an inhibitor of interleukin-1beta and tumor necrosis factor-alpha production in peripheral blood mononuclear cells. Thus, ritonavir appears to have anti-inflammatory properties. In the present study, we investigated in DLD-1 colon carcinoma cell effects of ritonavir on apoptotic cell death and expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme that may be critically involved in the modulation of colonic inflammation. Compared to unstimulated control, ritonavir resulted in a moderate increase in the rate of apoptotic cell death as observed after 20 h of incubation. Notably, ritonavir potently synergized with the short-chain fatty acid butyrate for induction of caspase-3-dependent apoptosis in DLD-1 cells. Ritonavir enhanced mRNA and protein expression of HO-1 in DLD-1 cells. Ritonavir-induced HO-1 protein was suppressed by SB203580 or SB202190 and preceded by immediate upregulation of cellular c-Fos and c-Jun protein levels. This process was associated with induction of activator protein-1 as detected by electrophoretic mobility shift analysis. The present data suggest that ritonavir has the potential to curb colon carcinogenesis by reducing cell growth via mechanisms that include apoptosis and by simultaneously modulating colonic inflammation via induction of anti-inflammatory HO-1.

Topics: Apoptosis; Butyrates; Caspase 3; Caspases; Cell Line, Tumor; Colonic Neoplasms; DNA; DNA Fragmentation; Drug Synergism; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Glyceraldehyde-3-Phosphate Dehydrogenases; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; HIV Protease Inhibitors; Humans; Immunoblotting; Membrane Proteins; Nuclease Protection Assays; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction; Ritonavir; RNA, Messenger

The present study characterized the response of P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP1) to chronic ritonavir (RIT) exposure by assessing increases in P-gp and MRP1 protein expression and activity. LS-180V intestinal carcinoma cells were exposed for 3 days to 1-100 microM RIT concurrently with controls. P-gp and MRP1 protein was quantified by Western blot analysis. Cell accumulation assays, using the P-gp substrate rhodamine 123 (RH123), the P-gp/MRP1 substrate doxorubicin (DOX), and the MRP substrate carboxyfluorescein (CBF), were performed as a measure of transporter activity. RIT strongly induced P-gp and MRP1 expression (maximum 6-fold and 3-fold increases, respectively) in a concentration-dependent fashion. Following extended exposure to RIT (> 10 microM), cells accumulated < 50% of the RH123 and DOX compared with controls, whereas accumulation of CBF was decreased by 30% at 30 microM. Differences in cell accumulation of RH123 could be eliminated with verapamil (100 microM; a P-gp inhibitor), whereas decreased DOX cell accumulation was only partially reversed by verapamil. Indomethacin (100 microM; an MRP1 inhibitor) had no significant effect on RH123 or DOX accumulation, suggesting limited MRP1-mediated activity. Thus, RIT induced protein expression of P-gp and MRP1 and increased cellular drug exclusion of RH123, DOX, and CBF. Similar in vivo phenomena may occur during anti-HIV drug therapy, explaining potential decrements in therapeutic efficacy due to decreases in bioavailability or alterations in drug distribution.

Topics: Adenocarcinoma; ATP Binding Cassette Transporter, Subfamily B, Member 1; Colonic Neoplasms; DNA-Binding Proteins; Dose-Response Relationship, Drug; HIV Protease Inhibitors; Humans; Membrane Transport Proteins; Multidrug Resistance-Associated Proteins; MutS Homolog 3 Protein; Ritonavir; Tumor Cells, Cultured

1. Chronic use of Saint John's wort (SJW) has been shown to lower the bioavailability for a variety of co-administered drugs including indinavir, cyclosporin, and digoxin. Decreases in intestinal absorption through induction of the multidrug resistance transporter, P-glycoprotein (P-gp), may explain decreased bioavailability. 2. The present study characterized the response of P-gp to chronic and acute exposure of SJW and hypericin (HYP, a presumed active moiety within SJW) in an in vitro system. Experiments were performed with 3 to 300 microg ml(-1) of methanol-extracted SJW and 0.03 to 3 microM HYP, representing low to high estimates of intestinal concentrations. 3. In induction experiments, LS-180 intestinal carcinoma cells were exposed for 3 days to SJW, HYP, vehicle or a positive control (ritonavir). P-gp was quantified using Western blot analysis. P-gp expression was strongly induced by SJW (400% increase at 300 microg ml(-1)) and by HYP (700% at 3 microM) in a dose-dependent fashion. Cells chronically treated with SJW had decreased accumulation of rhodamine 123, a P-gp substrate, that was reversed with acute verapamil, a P-gp inhibitor. Fluorescence microscopy of intact cells validated these findings. In Caco-2 cell monolayers, SJW and HYP caused moderate inhibition of P-gp-attributed transport at the maximum concentrations tested. 4. SJW and HYP significantly induced P-gp expression at low, clinically relevant concentrations. Similar effects occurring in vivo may explain the decreased bioavailability of P-gp substrate drugs when co-administered with SJW.

Topics: Adenocarcinoma; Anthracenes; Antidepressive Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Biological Transport, Active; Blotting, Western; Caco-2 Cells; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Interactions; Drug Resistance, Multiple; Humans; Hypericum; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Perylene; Protein Kinase C; Rhodamine 123; Ritonavir; Time Factors; Tumor Cells, Cultured; Verapamil

Topics: Adenocarcinoma; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carbamates; Cell Line, Transformed; Colonic Neoplasms; Drug Resistance, Multiple; Furans; Gene Expression Regulation, Neoplastic; HIV Protease Inhibitors; Humans; Indinavir; Ivermectin; Nelfinavir; Ritonavir; Saquinavir; Sulfonamides; Tumor Cells, Cultured; Verapamil; Vinblastine