ABSTRACT
Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. Adhesion receptors of the beta1 integrin family are assumed to be involved in carcinogenesis, but it is not clear how they contribute to RCC progression. In an in vitro model, we evaluated growth and adhesion capacity of Caki-I and KTC-26 kidney carcinoma cell lines compared to normal renal proximal tubular epithelial cells (PTC). alpha1-alpha6beta1 integrin subunits in malignant and non-malignant cells were evaluated by Western blotting and RT-PCR, integrin surface expression was measured by flow cytometry and confocal microscopy. Additionally, tumor cells were allowed to re-differentiate in the presence of valproic acid (VPA) and dynamic alterations of the integrin profile were analyzed. Caki-I and KTC-26 were characterized by accelerated proliferation and adhesion to an endothelial cell monolayer, compared to PTC cells. The integrin beta1 repertoire in RCC cell lines was significantly different from that detected in PTC, and included down-regulated alpha2 and alpha6, but up-regulated alpha1, alpha3 and alpha5 proteins. VPA application reduced tumor malignancy which was evidenced by reduced cell growth and adhesion capacity. The reduction in tumor malignancy was paralleled by the integrin expression profile of renal tumor cells 'matching' the pattern seen in PTC. We assume that a sensitive integrin balance exists in normal renal epithelial cells, and that dysregulation of the 'physiological' receptor equipment drives these cells towards malignancy. VPA acted on all investigated integrin subtypes and restored the receptor pattern typical for non-malignant cells. Therefore, VPA may represent a novel therapeutic option in RCC treatment.
Subject(s)
Carcinoma, Renal Cell/metabolism , Cell Differentiation/drug effects , Cell Line, Tumor/drug effects , Enzyme Inhibitors/pharmacology , Integrin beta1/metabolism , Kidney Neoplasms/metabolism , Valproic Acid/pharmacology , Cell Adhesion/physiology , Cell Line, Tumor/physiology , Cells, Cultured , Disease Progression , Dose-Response Relationship, Drug , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Integrin alpha Chains/genetics , Integrin alpha Chains/metabolism , Integrin beta1/genetics , Kidney Tubules, Proximal/cytologyABSTRACT
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.
Subject(s)
Apoptosis/drug effects , Butyrates/pharmacology , Colonic Neoplasms/chemically induced , HIV Protease Inhibitors/pharmacology , Heme Oxygenase (Decyclizing)/biosynthesis , Ritonavir/pharmacology , Caspase 3 , Caspases/metabolism , Cell Line, Tumor , Colonic Neoplasms/enzymology , DNA/metabolism , DNA Fragmentation , Drug Synergism , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Glyceraldehyde-3-Phosphate Dehydrogenases/biosynthesis , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase-1 , Humans , Immunoblotting , Membrane Proteins , Nuclease Protection Assays , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Monocytes release interleukin-18 after activation by lipopolysaccharide/ATP. Since inflammatory conditions such as sepsis are characterized by augmented interleukin-18 in sera of patients, we sought to modulate lipopolysaccharide/ATP-induced interleukin-18 release by pharmacological means. Here we report that 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), an inhibitor of ATP-mediated cellular activation by the purinoreceptor subtype P(2x7), potently suppresses interleukin-18 release from peripheral blood mononuclear cells. Interleukin-18 liberation was likewise inhibited by glyburide, a modulator of ion transport and inhibitor of ATP-binding cassette transporter 1. The data presented herein indicate that by pharmacologically interfering with the process of cytokine secretion agents such as KN-62 or glyburide have the potential to curb overproduction of interleukin-18 in septic patients.
Subject(s)
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Adenosine Triphosphate/pharmacology , Glyburide/pharmacology , Interleukin-18/antagonists & inhibitors , Interleukin-18/metabolism , Lipopolysaccharides/pharmacology , Adenosine Triphosphate/antagonists & inhibitors , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lipopolysaccharides/antagonists & inhibitorsABSTRACT
The proinflammatory cytokine interleukin (IL)-18 appears to be involved in the pathogenesis of diseases associated with immunoactivation and inflammation. Consequently, blockage of IL-18 bioactivity by use of IL-18 binding protein (IL-18 BP) is likely a promising therapeutic concept. In the present study, we investigated immunomodulatory activities of IL-18 BPa:Fc in human whole blood cultures. We report that IL-18 BPa:Fc (200 ng/mL) significantly inhibited lipopolysaccharide (LPS, 10 ng/mL)/IL-12 (5 ng/mL)-induced release of interferon-gamma (IFNgamma) and matrix metalloproteinase-9 (MMP-9) from whole blood cultures of healthy donors. Notably, IL-18 BPa:Fc (200 ng/mL) further reinforced dexamethasone (5 nM)- or mycophenolic acid (2 microM)-mediated reduction of LPS/IL-12-induced IFNgamma production by an additional 50.5 or 49.9%, respectively. To investigate effects of IL-18 BP:Fc in the context of autoimmune diseases, experiments were performed with whole blood obtained from patients with systemic lupus erythematosus or Wegener's granulomatosis undergoing immunosuppressive therapy. After ex vivo stimulation with LPS (10 ng/mL), production of IFNgamma and MMP-9 was determined. Both mediators likely contribute to renal inflammation frequently seen in these diseases. In accord with the aforementioned data, LPS (10 ng/mL)-induced IFNgamma was significantly reduced by coincubation with IL-18 BPa:Fc at 200 ng/mL. IL-18 BPa:Fc also inhibited production of MMP-9. The present data demonstrate that IL-18 BPa:Fc has the potential to amplify anti-inflammatory actions of immunosuppressive drugs, and thus may prove to be a valuable novel pharmacological component in the treatment of human autoimmune diseases.
