Runx2 association with progression of prostate cancer in patients: mechanisms mediating bone osteolysis and osteoblastic metastatic lesions.

Runx2, a bone-specific transcriptional regulator, is abnormally expressed in highly metastatic prostate cancer cells. Here, we identified the functional activities of Runx2 in facilitating tumor growth and osteolysis. Our studies show that negligible Runx2 is found in normal prostate epithelial and non-metastatic LNCaP prostate cancer cells. In the intra-tibial metastasis model, high Runx2 levels are associated with development of large tumors, increased expression of metastasis-related genes (MMP9, MMP13, VEGF, Osteopontin) and secreted bone-resorbing factors (PTHrP, IL8) promoting osteolytic disease. Runx2 siRNA treatment of PC3 cells decreased cell migration and invasion through Matrigel in vitro, and in vivo shRunx2 expression in PC3 cells blocked their ability to survive in the bone microenvironment. Mechanisms of Runx2 function were identified in co-culture studies showing that PC3 cells promote osteoclastogenesis and inhibit osteoblast activity. The clinical significance of these findings is supported by human tissue microarray studies of prostate tumors at stages of cancer progression, in which Runx2 is expressed in both adenocarcinomas and metastatic tumors. Together these findings indicate that Runx2 is a key regulator of events associated with prostate cancer metastatic bone disease.

Indomethacin induces apoptosis via a MRP1-dependent mechanism in doxorubicin-resistant small-cell lung cancer cells overexpressing MRP1.

Small-cell lung cancers (SCLCs) initially respond to chemotherapy, but are often resistant at recurrence. The non-steroidal anti-inflammatory drug indomethacin is an inhibitor of multidrug resistance protein 1 (MRP1) function. The doxorubicin-resistant MRP1-overexpressing human SCLC cell line GLC(4)-Adr was highly sensitive for indomethacin compared with the parental doxorubicin-sensitive line GLC(4). The purpose of this study was to analyse the relationship between hypersensitivity to indomethacin and MRP1 overexpression. The experimental design involved analysis of the effect of MRP1 downregulation on indomethacin-induced cell survival and apoptosis in GLC(4)-Adr and GLC(4), using siRNA. In addition the effect of indomethacin on glutathione levels and mitochondrial membrane potential was investigated. Small interfering RNAs directed against MRP1 reduced MRP1 mRNA levels twofold and reduced efflux pump function of MRP1, which was reflected by a 1.8-fold higher accumulation of MRP1 substrate carboxyfluorescein, in si-MRP1 versus si-Luciferase-transfected GLC(4)-Adr cells. Multidrug resistance protein 1 downregulation decreased initial high apoptosis levels 2-fold in GLC(4)-Adr after indomethacin treatment for 24 h, and increased cell survival (IC(50)) from 22.8+/-2.6 to 30.4+/-5.1 microM following continuous indomethacin exposure. Multidrug resistance protein 1 downregulation had no effect on apoptosis in GLC(4) or on glutathione levels in both lines. Although indomethacin (20 microM) for 2 h decreased glutathione levels by 31.5% in GLC(4)-Adr, complete depletion of cellular glutathione by L-buthionine (S,R)-sulphoximine only resulted in a small increase in indomethacin-induced apoptosis in GLC(4)-Adr, demonstrating that a reduced cellular glutathione level is not the primary cause of indomethacin-induced apoptosis. Indomethacin exposure decreased mitochondrial membrane potential in GLC(4)-Adr cells, suggesting activation of the mitochondrial apoptosis pathway. Indomethacin induces apoptosis in a doxorubicin-resistant SCLC cell line through an MRP1-dependent mechanism. This may have implications for the treatment of patients with MRP1-overexpressing tumours.

Single expression of CD45RC and RT6 in correlation with T-helper 1 and T-helper 2 cytokine patterns in the rat.

Previous studies involving the function and development of peripheral T cells have proposed that, in the rat, CD4(+)CD45RC(+)RT6(-) and CD4(+)CD45RC(-)RT6(+) T-cell subsets may represent Th1 and Th2 cells, respectively. Here we tested this hypothesis directly by analyzing frequencies of IFN-gamma- and IL-4-producing cells in these two subpopulations using ELISPOT assays. We found that the CD4(+)CD45RC(-)RT6(+) subset showed higher numbers of IL-4-producing cells than the CD4(+)CD45RC(+)RT6(-) subset and, though less pronounced, that the latter demonstrated higher numbers of IFN-gamma producers. Therefore, we conclude that our results provide evidence for the existence of phenotypically defined Th1 and Th2 cells in the rat. This is supported by the finding that the ratios of IFN-gamma/IL-4 and CD45RC/RT6 correlated positively among various rat strains. Finally, rat strains susceptible to induction of a Th1-mediated autoimmune disease showed the highest CD45RC/RT6 ratio, whereas the reverse was true for strains susceptible to a Th2-mediated autoimmune disease.