Inhibition of p38 pathway leads to OA-like changes in a rat animal model

Objectives The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is involved in a variety of inflammatory responses, including cytokine generation, cell differentiation proliferation and apoptosis. Here, we examined the effects of systemic p38 MAPK inhibition on cartilage cells and osteoarthritis (OA) disease progression by both in vitro and in vivo approaches. Methods p38 kinase activity was evaluated in normal and OA cartilage cells by measuring the amount of phosphorylated protein. To examine the function of p38 signaling pathway in vitro, normal chondrocytes were isolated and differentiated in the presence or absence of p38 inhibitor; SB203580 and analysed for chondrogenic phenotype. Effect of systemic p38 MAPK inhibition in normal and OA (induced by menisectomy) rats were analysed by treating animals with vehicle alone (DMS0) or p38 inhibitor (SB203580). Damage to the femur and tibial plateau was evaluated by modified Mankin score, histology and immunohistochemistry. Results Our in vitro studies have revealed that a down-regulation of chondrogenic and increase of hypertrophic gene expression occurs in the normal chondrocytes, when p38 is neutralized by a pharmacological inhibitor. We further observed that the basal levels of p38 phosphorylation were decreased in OA chondrocytes compared with normal chondrocytes. These findings together indicate the importance of this pathway in the regulation of cartilage physiology and its relevance to OA pathogenesis. At in vivo level, systematic administration of a specific p38 MAPK inhibitor, SB203580, continuously for over a month led to a significant loss of proteoglycan; aggrecan and cartilage thickness. On the other hand, SB203580 treated normal rats showed a significant increase in TUNEL positive cells, cartilage hypertrophy markers such as Type 10 collagen, Runt-related transcription factor and Matrix metalloproteinase-13 and substantially induced OA like phenotypic changes in the normal rats. In addition, menisectomy induced OA rat models that were treated with p38 inhibitor showed aggravation of cartilage damage. Conclusions In summary, this study has provided evidence that the component of the p38 MAPK pathway is important to maintain the cartilage health and its inhibition can lead to severe cartilage degenerative changes. The observations in this study highlight the possibility of using activators of the p38 pathway as an alternative approach in the treatment of OA.

Brivanib alaninate, a dual inhibitor of vascular endothelial growth factor receptor and fibroblast growth factor receptor tyrosine kinases, induces growth inhibition in mouse models of human hepatocellular carcinoma

PURPOSE: Hreceptor (VEGFR) and FGF receptor (FGFR) signaling pathways. EXPERIMENTAL DESIGN: Six different s.c. patient-derived HCC xenografts were implanted into mice. Tumor growth was evaluated in mice treated with brivanib compared with control. The effects of brivanib on apoptosis and cell proliferation were evaluated by immunohistochemistry. The SK-HEP1 and HepG2 cells were used to investigate the effects of brivanib on the VEGFR-2 and FGFR-1 signaling pathways in vitro. Western blotting was used to determine changes in proteins in these xenografts and cell lines. RESULTS: Brivanib significantly suppressed tumor growth in five of six xenograft lines. Furthermore, brivanib-induced growth inhibition was associated with a decrease in phosphorylated VEGFR-2 at Tyr(1054/1059), increased apoptosis, reduced microvessel density, inhibition of cell proliferation, and down-regulation of cell cycle regulators. The levels of FGFR-1 and FGFR-2 expression in these xenograft lines were positively correlated with its sensitivity to brivanib-induced growth inhibition. In VEGF-stimulated and basic FGF stimulated SK-HEP1 cells, brivanib significantly inhibited VEGFR-2, FGFR-1, extracellular signal-regulated kinase 1/2, and Akt phosphorylation. CONCLUSION: This study provides a strong rationale for clinical investigation of brivanib in patients with HCC.

p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Notch and NOXA-related pathways in melanoma cells

Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch expression are associated with various malignancies in which Notch may function as an oncogene or less commonly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small molecule gamma-secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-Nle-CHO, which can block processing and activation of all four different Notch receptors, we identified a specific apoptotic vulnerability in melanoma cells. GSI triggers apoptosis in melanoma cells, but only G2/M growth arrest in melanocytes without subsequent cell death. Moreover, GSI treatment induced a pro-apoptotic BH3-only protein, NOXA, in melanoma cells but not in normal melanocytes. The use of GSI to induce NOXA induction overcomes the apoptotic resistance of melanoma cells, which commonly express numerous cell survival proteins such as Mcl-1, Bcl-2, and survivin. Taken together, these results highlight the concept of synthetic lethality in which exposure to GSI, in combination with melanoma cells overexpressing activated Notch receptors, has lethal consequences, producing selective killing of melanoma cells, while sparing normal melanocytes. By identifying signaling pathways that contribute to the transformation of melanoma cells (e.g. Notch signaling), and anti-cancer agents that achieve tumor selectivity (e.g., GSI-induced NOXA), this experimental approach provides a useful framework for future therapeutic strategies in cutaneous oncology.

Effects of varied ionic calcium and phosphate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells in vitro

Background and Objective: A number of bone filling materials containing calcium (Ca++) and phosphate (P) ions have been used in the repair of periodontal bone defects; however, the effect that local release of Ca++ and P ions have on biological reactions is not fully understood. In this study, we investigated the effects of various levels of Ca++ and P ions on the proliferation, osteogenic differentiation, and mineralization of human periodontal ligament cells (hPDLCs). Materials and Methods: hPDLCs were obtained using an explant culture method. Defined concentrations and ratios of ionic Ca++ to inorganic P were added to standard culture and osteogenic induction media. The ability of hPDLCs to proliferate in these growth media was assayed using the Cell Counting Kit-8 (CCK-8). Cell apoptosis was evaluated by FITC-Annexin V/PI double staining method. Osteogenic differentiation and mineralization were investigated by morphological observations, alkaline phosphatase (ALP) activity, and Alizarin red S/von Kossa staining. The mRNA expression of osteogenic related markers was analyzed using a reverse transcriptase polymerase chain reaction (RT-PCR). Results: Within the ranges of Ca++ and P ions concentrations tested, we observed that increased concentrations of Ca++ and P ions enhanced cell proliferation and formation of mineralized matrix nodules; whereas ALP activity was reduced. The RT-PCR results showed that elevated concentrations of Ca++ and P ions led to a general increase of Runx2 mRNA expression and decreased ALP mRNA expression, but gave no clear trend on OCN mRNA levels. Conclusion: The concentrations and ratios of Ca++ and P ions could significantly influence proliferation, differentiation, and mineralization of hPDLCs. Within the range of concentrations tested, we found that the combination of 9.0 mM Ca++ ions and 4.5 mM P ions were the optimum concentrations for proliferation, differentiation, and mineralization in hPDLCs.

Clusterin facilitates COMMD1 and I-kB degradation to enhance NF-kB activity in prostate cancer cells

Secretory clusterin (sCLU) is a stress-activated, cytoprotective chaperone that confers broad-spectrum cancer treatment resistance, and its targeted inhibitor (OGX-011) is currently in phase II trials for prostate, lung, and breast cancer. However, the molecular mechanisms by which sCLU inhibits treatment-induced apoptosis in prostate cancer remain incompletely defined. We report that sCLU increases NF-κB nuclear translocation and transcriptional activity by serving as a ubiquitin-binding protein that enhances COMMD1 and I-κB proteasomal degradation by interacting with members of the SCF-βTrCP E3 ligase family. Knockdown of sCLU in prostate cancer cells stabilizes COMMD1 and I-κB, thereby sequestrating NF-κB in the cytoplasm and decreasing NF-κB transcriptional activity. Comparative microarray profiling of sCLU-overexpressing and sCLU-knockdown prostate cancer cells confirmed that the expression of many NF-κB–regulated genes positively correlates with sCLU levels. We propose that elevated levels of sCLU promote prostate cancer cell survival by facilitating degradation of COMMD1 and I-κB, thereby activating the canonical NF-κB pathway.

Clinical strategies for the alleviation of contractures from a predictive mathematical model of dermal repair

Hypertrophic scars arise when there is an overproduction of collagen during wound healing. These are often associated with poor regulation of the rate of programmed cell death(apoptosis) of the cells synthesizing the collagen or by an exuberant inflammatory response that prolongs collagen production and increases wound contraction. Severe contractures that occur, for example, after a deep burn can cause loss of function especially if the wound is over a joint such as the elbow or knee. Recently, we have developed a morphoelastic mathematical model for dermal repair that incorporates the chemical, cellular and mechanical aspects of dermal wound healing. Using this model, we examine pathological scarring in dermal repair by first assuming a smaller than usual apoptotic rate for myofibroblasts, and then considering a prolonged inflammatory response, in an attempt to determine a possible optimal intervention strategy to promote normal repair, or terminate the fibrotic scarring response. Our model predicts that in both cases it is best to apply the intervention strategy early in the wound healing response. Further, the earlier an intervention is made, the less aggressive the intervention required. Finally, if intervention is conducted at a late time during healing, a significant intervention is required; however, there is a threshold concentration of the drug or therapy applied, above which minimal further improvement to wound repair is obtained.