Targeting p53 via JNK Pathway: A Novel Role of RITA for Apoptotic Signaling in Multiple Myeloma

The low frequency of p53 alterations e.g., mutations/deletions (∼10%) in multiple myeloma (MM) makes this tumor type an ideal candidate for p53-targeted therapies. RITA is a small molecule which can induce apoptosis in tumor cells by activating the p53 pathway. We previously showed that RITA strongly activates p53 while selectively inhibiting growth of MM cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy in MM. However, the molecular mechanisms underlying the pro-apoptotic effect of RITA are largely undefined. Gene expression analysis by microarray identified a significant number of differentially expressed genes associated with stress response including c-Jun N-terminal kinase (JNK) signaling pathway. By Western blot analysis we further confirmed that RITA induced activation of p53 in conjunction with up-regulation of phosphorylated ASK-1, MKK-4 and c-Jun. These results suggest that RITA induced the activation of JNK signaling. Chromatin immunoprecipitation (ChIP) analysis showed that activated c-Jun binds to the activator protein-1 (AP-1) binding site of the p53 promoter region. Disruption of the JNK signal pathway by small interfering RNA (siRNA) against JNK or JNK specific inhibitor, SP-600125 inhibited the activation of p53 and attenuated apoptosis induced by RITA in myeloma cells carrying wild type p53. On the other hand, p53 transcriptional inhibitor, PFT-α or p53 siRNA not only inhibited the activation of p53 transcriptional targets but also blocked the activation of c-Jun suggesting the presence of a positive feedback loop between p53 and JNK. In addition, RITA in combination with dexamethasone, known as a JNK activator, displays synergistic cytotoxic responses in MM cell lines and patient samples. Our study unveils a previously undescribed mechanism of RITA-induced p53-mediated apoptosis through JNK signaling pathway and provides the rationale for combination of p53 activating drugs with JNK activators in the treatment of MM

Targeting p53 by small molecules in hematological malignancies

Abstract p53 is a powerful tumor suppressor and is an attractive cancer therapeutic target. A breakthrough in cancer research came from the discovery of the drugs which are capable of reactivating p53 function. Most anti-cancer agents, from traditional chemo- and radiation therapies to more recently developed non-peptide small molecules exert their effects by enhancing the anti-proliferative activities of p53. Small molecules such as nutlin, RITA, and PRIMA-1 that can activate p53 have shown their anti-tumor effects in different types of hematological malignancies. Importantly, nutlin and PRIMA-1 have successfully reached the stage of phase I/II clinical trials in at least one type of hematological cancer. Thus, the pharmacological activation of p53 by these small molecules has a major clinical impact on prognostic use and targeted drug design. In the current review, we present the recent achievements in p53 research using small molecules in hematological malignancies. Anticancer activity of different classes of compounds targeting the p53 signaling pathway and their mechanism of action are discussed. In addition, we discuss how p53 tumor suppressor protein holds promise as a drug target for recent and future novel therapies in these diseases

A Hibernation-Like State for Transplantable Organs: Is Hydrogen Sulfide Therapy the Future of Organ Preservation?

Significance: Renal transplantation is the treatment of choice for end-stage renal disease, during which renal grafts from deceased donors are routinely cold stored to suppress metabolic demand and thereby limit ischemic injury. However, prolonged cold storage, followed by reperfusion, induces extensive tissue damage termed cold ischemia/reperfusion injury (IRI) and puts the graft at risk of both early and late rejection. Recent Advances: Deep hibernators constitute a natural model of coping with cold IRI as they regularly alternate between 4 degrees C and 37 degrees C. Recently, endogenous hydrogen sulfide (H2S), a gas with a characteristic rotten egg smell, has been implicated in organ protection in hibernation. Critical Issues: In renal transplantation, H2S also seems to confer cytoprotection by lowering metabolism, thereby creating a hibernation-like environment, and increasing preservation time while allowing cellular processes of preservation of homeostasis and tissue remodeling to take place, thus increasing renal graft survival. Future Directions: Although the underlying cellular and molecular mechanisms of organ protection during hibernation have not been fully explored, mammalian hibernation may offer a great clinical promise to safely cold store and reperfuse donor organs. In this review, we first discuss mammalian hibernation as a natural model of cold organ preservation with reference to the kidney and highlight the involvement of H2S during hibernation. Next, we present recent developments on the protective effects and mechanisms of exogenous and endogenous H2S in preclinical models of transplant IRI and evaluate the potential of H2S therapy in organ preservation as great promise for renal transplant recipients in the future

Formation of Vesicular Stomatitis Virus Pseudotypes Bearing Surface Proteins of Hepatitis B Virus

It has been difficult to propagate and titrate hepatitis B virus (HBV) in tissue culture. We examined whether vesicular stomatitis virus (VSV) pseudotypes bearing HBV surface (HBs) proteins infectious for human cell lines could be prepared. For this, expression plasmids for three surface proteins, L, M, and S, of HBV were made. 293T cells were then transfected with these plasmids either individually or in different combinations. 293T cells expressing HBs proteins were infected with VSVΔG*-G, a recombinant VSV expressing green fluorescent protein (GFP), to make VSV pseudotypes. Culture supernatants together with cells were harvested and sonicated for a short time. The infectivities of freshly harvested supernatants were determined by quantifying the number of cells expressing GFP after neutralization with anti-VSV serum and mouse monoclonal antibodies (MAbs) against HBs protein. Among 14 cell lines tested for susceptibility to HBV pseudotype samples, HepG2, JHH-7, and 293T cells were judged to be the most susceptible. Namely, the infectious units (IU) of the culture supernatant samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively, suggesting the presence of VSVΔG*(HBV) pseudotypes. This infectivity was inhibited by treatment with lactoferrin or dextran sulfate. Pretreatment of the cells with trypsin or tunicamycin inhibited plating of the pseudotype samples. The HBV pseudotypes can be used to analyze early steps of HBV infection, including the entry mechanism of HBV