Modulation of expression of the nuclear receptor NR0B2 (small heterodimer partner 1) and its impact on proliferation of renal carcinoma cells.

Mammalian nuclear receptors (NRs) are transcription factors regulating the expression of target genes that play an important role in drug metabolism, transport, and cellular signaling pathways. The orphan and structurally unique receptor small heterodimer partner 1 (syn NR0B2) is not only known for its modulation of drug response, but has also been reported to be involved in hepatocellular carcinogenesis. Indeed, previous studies show that NR0B2 is downregulated in human hepatocellular carcinoma, suggesting that NR0B2 acts as a tumor suppressor via inhibition of cellular growth and activation of apoptosis in this tumor entity. The aim of our study was to elucidate whether NR0B2 may also play a role in other tumor entities. Comparing NR0B2 expression in renal cell carcinoma and adjacent nonmalignant transformed tissue revealed significant downregulation in vivo. Additionally, the impact of heterologous expression of NR0B2 on cell cycle progression and proliferation in cells of renal origin was characterized. Monitoring fluorescence intensity of resazurin turnover in RCC-EW cells revealed no significant differences in metabolic activity in the presence of NR0B2. However, there was a significant decrease of cellular proliferation in cells overexpressing this NR, and NR0B2 was more efficient than currently used antiproliferative agents. Furthermore, flow cytometry analysis showed that heterologous overexpression of NR0B2 significantly reduced the amount of cells passing the G1 phase, while on the other hand, more cells in S/G2 phase were detected. Taken together, our data suggest that downregulation of NR0B2 may also play a role in renal cell carcinoma development and progression.

Pimecrolimus increases the expression of interferon-inducible genes that modulate human coronary artery cells proliferation.

The pharmacodynamics of the loaded compounds defines clinical failure or success of a drug-eluting device. Various limus derivatives have entered clinics due to the observed positive outcome after stent implantation, which is explained by their antiproliferative activity resulting from inhibition of the cytosolic immunophilin FK506-binding protein 12. Although pimecrolimus also binds to this protein, pimecrolimus-eluting stents failed in clinics. However, despite its impact on T lymphocytes little is known about the pharmacodynamics of pimecrolimus in cultured human coronary artery cells. We were able to show that pimecrolimus exerts antiproliferative activity in human smooth muscle and endothelial cells. Furthermore in those cells pimecrolimus induced transcription of interferon-inducible genes which in part are known to modulate cell proliferation. Modulation of gene expression may be part of an interaction between calcineurin, the downstream target of the pimecrolimus/FK506-binding protein 12-complex, and the toll-like receptor 4. In accordance are our findings showing that silencing of toll-like receptor 4 by siRNA in A549 a lung carcinoma cell line reduced the activation of interferon-inducible genes upon pimecrolimus treatment in those cells. Based on our findings we hypothesize that calcineurin inhibition may induce the toll-like receptor 4 mediated activation of type I interferon signaling finally inducing the observed effect in endothelial and smooth muscle cells. The crosstalk of interferon and toll-like receptor signaling may be a molecular mechanism that contributed to the failure of pimecrolimus-eluting stents in humans.

Expression of OATP2B1 as determinant of drug effects in the microcompartment of the coronary artery.

Clinical success of coronary drug-eluting stents (DES) is hampered by simultaneous reduction of smooth muscle cell (HCASMC) and endothelial cell proliferation due to unspecific cytotoxicity of currently used compounds. Previous in vitro data showing SMC-specific inhibition of proliferation suggested that statins may be suitable candidates for DES. It was aim of this study to further investigate statins as DES drug candidates to identify mechanisms contributing to their cell-selectivity. In vitro proliferation assays comparing the influence of various statins on HCASMC and endothelial cells confirmed that atorvastatin exhibits HCASMC-specificity. Due to similar expression levels of the drug target HMG-CoA reductase in both cell types, cellular accumulation of atorvastatin was assessed, revealing enhanced uptake in HCASMC most likely driven by significant expression of OATP2B1, a known uptake transporter for atorvastatin. In accordance with the finding that endogenous OATP2B1 influenced cellular accumulation in HCASMC we used this transporter as a tool to identify teniposide as new DES candidate drug with HCASMC-specific effects. We describe OATP2B1 as a determinant of pharmacokinetics in the coronary artery. Indeed, endogenously expressed OATP2B1 significantly influences the uptake of substrate drugs, thereby governing cell specificity. Screening of candidate drugs for interaction with OATP2B1 may be used to promote SMC-specificity.

Cell-specific expression of uptake transporters--a potential approach for cardiovascular drug delivery devices.

Enhanced proliferation of human coronary artery smooth muscle cells (HCASMCs) and thereby formation of neointima is one of the factors contributing to failure of coronary stents. Even if the use of drug eluting stents (DES) and thereby the local delivery of cytotoxic compounds has significantly improved the clinical outcome, unselective cytotoxic effects are assumed to hamper clinical success. Novel pharmacological approaches are required to enhance cellular selectivity of locally delivered drugs. Cell specific overexpression of a drug transporter could be used to enhance cellular accumulation and therefore cell specificity. In the herein reported study we tested the possibility of cell specific transporter expression to enhance drug effects in HCASMCs. We generated adenoviral constructs to overexpress the organic cation transporter 1 (OCT1) under control of the promoter of SM22α, which had been previously reported as muscle cell specific gene. First the activity of the SM22α-promoter was assessed in various cell types supporting the notion of muscle cell specificity. Subsequently, the activity of the transporter was compared in infected HCAECs and HCASMCs revealing enhanced accumulation of substrate drugs in HCASMCs in presence of the SM22α-promoter. Testing the hypothesis that this kind of targeting might serve as a mechanism for cell-specific drug effects, we investigated the impact on paclitaxel treatment in HCASMC and HCAECs, showing significantly increased antiproliferative activity of this substrate drug on muscle cells. Taken together, our findings suggest that cell-specific expression of transport proteins serves as mechanism governing the uptake of cytotoxic compounds for a selective impact on targeted cells.