Syntheses and discovery of a novel class of cinnamic hydroxamates as histone deacetylase inhibitors by multimodality molecular imaging in living subjects.

Histone deacetylases (HDAC) that regulate gene expression are being explored as cancer therapeutic targets. In this study, we focused on HDAC6 based on its ability to inhibit cancerous Hsp90 chaperone activities by disrupting Hsp90/p23 interactions. To identify novel HDAC6 inhibitors, we used a dual-luciferase reporter system in cell culture and living mice by bioluminescence imaging (BLI). On the basis of existing knowledge, a library of hydrazone compounds was generated for screening by coupling cinnamic hydroxamates with aldehydes and ketones. Potency and selectivity were determined by in vitro HDAC profiling assays, with further evaluation to inhibit Hsp90(α/ß)/p23 interactions by BLI. In this manner, we identified compound 1A12 as a dose-dependent inhibitor of Hsp90(α/ß)/p23 interactions, UKE-1 myeloid cell proliferation, p21(waf1) upregulation, and acetylated histone H3 levels. 1A12 was efficacious in tumor xenografts expressing Hsp90(α)/p23 reporters relative to carrier control-treated mice as determined by BLI. Small animal (18)F-FDG PET/CT imaging on the same cohort showed that 1A12 also inhibited glucose metabolism relative to control subjects. Ex vivo analyses of tumor lysates showed that 1A12 administration upregulated acetylated-H3 by approximately 3.5-fold. Taken together, our results describe the discovery and initial preclinical validation of a novel selective HDAC inhibitor.

Proteomic identification of pterostilbene-mediated anticancer activities in HepG2 cells.

In the present study, we attempt to shed light on the underlying molecular mechanism of the anticancer activity of pterostilbene (PTS) in HepG2 cells through the proteomic approach. PTS was found to induce apoptosis by altering the expression of apoptotic genes and the G2/M phase of cell cycle arrest. Further, the 2-DE map showed the expression of 72 differentially regulated proteins in PTS-treated HepG2 cells, of which 8 spots with >2 fold up- or down-regulated level were identified by MALDI-TOF analysis, which has a regulatory role in apoptosis. These findings for the first time offer valuable insights into the mechanism of apoptotis by PTS in HepG2 cells.

Quercetin ameliorates tunicamycin-induced endoplasmic reticulum stress in endothelial cells.

OBJECTIVE: Endothelial dysfunction highlights that it is a potential contributor in the pathogenesis of vascular complications arising from endoplasmic reticulum stress (ER stress) and has been emerging as a main causative factor in vascular failure. Here, we hypothesize that the natural flavonoid, quercetin plays an effective role in reducing ER stress in human umbilical vein endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells were pre-treated with different concentrations of quercetin (0-100 µm) before inducing ER stress using tunicamycin (TUN) (0.75 µg/ml); cytotoxicity was assessed by MTT assay. Expression levels of ER stress responsive genes, antioxidant enzymes and apoptotic markers were assessed by qRT-PCR, while roles of caspase-3 and PARP cleavage were measured by western blot analysis. RESULTS: Quercetin pre-treatment at 25 and 50 µm had a cytoprotective effect on cells against TUN-induced toxicity. Quercetin administration modulated expression level of ER stress genes coding for glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP), and antioxidant enzymes such as superoxide dismutase and catalase, along with free radical generation assessed by malondialdehyde assay. Induction of apoptosis was prevented with reduction in expression level of Bax, and concomitant increase in Bcl-2 levels, thus proving its potential against ER stress. CONCLUSION: The current study indicates that quercetin modulated stress responsive genes GRP78 and CHOP, helping endothelial cells prevent TUN-induced ER stress.

Dual-therapeutic reporter genes fusion for enhanced cancer gene therapy and imaging.

Two of the successful gene-directed enzyme prodrug therapies include herpes simplex virus-thymidine kinase (HSV1-TK) enzyme-ganciclovir prodrug and the Escherichia coli nitroreductase (NTR) enzyme-CB1954 prodrug strategies; these enzyme-prodrug combinations produce activated cytotoxic metabolites of the prodrugs capable of tumor cell death by inhibiting DNA synthesis and killing quiescent cells, respectively. Both these strategies also affect significant bystander cell killing of neighboring tumor cells that do not express these enzymes. We have developed a dual-combination gene strategy, where we identified HSV1-TK and NTR fused in a particular orientation can effectively kill tumor cells when the tumor cells are treated with a fusion HSV1-TK-NTR gene- along with a prodrug combination of GCV and CB1954. In order to determine whether the dual-system demonstrate superior therapeutic efficacy than either HSV1-TK or NTR systems alone, we conducted both in vitro and in vivo tumor xenograft studies using triple negative SUM159 breast cancer cells, by evaluating the efficacy of cell death by apoptosis and necrosis upon treatment with the dual HSV1-TK genes-GCV-CB1954 prodrugs system, and compared the efficiency to HSV1-TK-GCV and NTR-CB1954. Our cell-based studies, tumor regression studies in xenograft mice, histological analyses of treated tumors and bystander studies indicate that the dual HSV1-TK-NTR-prodrug system is two times more efficient even with half the doses of both prodrugs than the respective single gene-prodrug system, as evidenced by enhanced apoptosis and necrosis of tumor cells in vitro in culture and xenograft of tumor tissues in animals.

Noninvasive optical imaging of nitroreductase gene-directed enzyme prodrug therapy system in living animals.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising and emerging strategy that attempts to limit the systemic toxicity inherent to cancer chemotherapy by means of tumor-targeted delivery and expression of an exogenous gene whose product converts nontoxic prodrug(s) into activated cytotoxic agent(s). The bacterial nitroreductase (NTR) enzyme, coupled with its substrate prodrug 5-(azaridin-1-yl)-2,4-dinitrobenzamide (CB1954), is a promising GDEPT strategy that has reached clinical trials. However, no strategy exists to visually monitor and quantitatively evaluate the therapeutic efficacy of NTR/CB1954 prodrug therapy in cells and imaging in living animals. As the success of any GDEPT is dependent upon the efficiency of transgene expression in vivo, we developed a safe, sensitive and reproducible noninvasive imaging method to monitor NTR transgene expression that would allow quantitative assessment of both therapeutic efficacy and diagnostic outcome of NTR/CB1954 prodrug therapy in the future. Here, we investigate the use of a novel fluorescent imaging dye CytoCy5S (a Cy5-labeled quenched substrate of NTR enzyme) on various cancer cell lines in vitro and in NTR-transfected tumor-bearing animals in vivo. CytoCy5S-labeled cells become fluorescent at 'red-shifted' wavelengths (638 nm) when reduced by cellular NTR enzyme and remains trapped within the cells for extended periods of time. The conversion and entrapment was dynamically recorded using a time-lapsed microscopy. Systemic and intratumoral delivery of CytoCy5S to NTR-expressing tumors in animals indicated steady and reproducible signals even 16 h after delivery (P<0.001). This is the first study to address visual monitoring and quantitative evaluation of NTR activity in small animals using CytoCy5S, and establishes the capability of NTR to function as an imageable reporter gene.

Potent, tumor-specific gene expression in an orthotopic hepatoma rat model using a Survivin-targeted, amplifiable adenoviral vector.

Ideal cancer gene therapies should have high tumor specificity and efficacy, and allow systemic administration to target metastases. We recently developed a bi-directional, two-step transcriptional amplification (TSTA) system driven by the tumor-specific Survivin promoter (pSurv) to amplify the correlated expression of both the reporter gene firefly luciferase (FL) and therapeutic gene tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we compare the specificity and potency of an adenovirus carrying this system (Ad-pSurv-TSTA-TRAIL-FL) to a nonspecific vector (Ad-pCMV-FL) in an orthotopic hepatocellular carcinoma (HCC) rat model after systemic administration. At 24 h after injection of Ad-pCMV-FL, bioluminescence imaging revealed a trend (P=0.30) towards greater FL expression in liver versus tumor. In striking contrast, Ad-pSurv-TSTA-TRAIL-FL showed increased FL activity within the tumor compared with the liver (P<0.01), a strong trend towards reduced liver expression compared with Ad-pCMV-FL (P=0.07), and importantly, similar FL levels within tumor compared with Ad-pCMV-FL (P=0.32). Hence, this vector shows potent, tumor-specific transgene expression even after extensive liver transduction and may be of significant value in avoiding hepatotoxicity in HCC patients. Future studies will explore the benefits of tumor-specific TRAIL expression in this model, the potential to target metastases and the extension of this vector for the treatment of other Survivin-positive tumors is warranted.

Molecular imaging of phosphorylation events for drug development.

PURPOSE: Protein phosphorylation mediated by protein kinases controls numerous cellular processes. A genetically encoded, generalizable split firefly luciferase (FL)-assisted complementation system was developed for noninvasive monitoring phosphorylation events and efficacies of kinase inhibitors in cell culture and in small living subjects by optical bioluminescence imaging. PROCEDURES: An Akt sensor (AST) was constructed to monitor Akt phosphorylation and the effect of different PI-3K and Akt inhibitors. Specificity of AST was determined using a non-phosphorylable mutant sensor containing an alanine substitution (ASA). RESULTS: The PI-3K inhibitor LY294002 and Akt kinase inhibitor perifosine led to temporal- and dose-dependent increases in complemented FL activities in 293T human kidney cancer cells stably expressing AST (293T/AST) but not in 293T/ASA cells. Inhibition of endogenous Akt phosphorylation and kinase activities by perifosine also correlated with increase in complemented FL activities in 293T/AST cells but not in 293T/ASA cells. Treatment of nude mice bearing 293T/AST xenografts with perifosine led to a 2-fold increase in complemented FL activities compared to that of 293T/ASA xenografts. Our system was used to screen a small chemical library for novel modulators of Akt kinase activity. CONCLUSION: This generalizable approach for noninvasive monitoring of phosphorylation events will accelerate the discovery and validation of novel kinase inhibitors and modulators of phosphorylation events.

RIP1 links inflammatory and growth factor signaling pathways by regulating expression of the EGFR.

There is considerable interest in understanding how inflammatory responses influence cell proliferation and cancer. In this study, we show that the receptor-interacting protein (RIP1), a critical mediator of inflammation and stress-induced NF-kappaB activation, regulates the expression of the epidermal growth factor receptor (EGFR). Mouse embryo fibroblasts (MEFs) derived from RIP1 knockout mice express very high levels of the EGFR. Reconstitution of RIP1(-/-) MEFs with RIP1 results in a lowering of EGFR levels. RIP1 influences EGFR at the mRNA level by regulating the EGFR promoter. Expression of RIP1 inhibits the EGFR promoter. RIP1 downregulates EGFR expression by interfering with the function of Sp1, which is a key activator of EGFR transcription. RIP1 suppresses Sp1 activity and overexpression of Sp1 reverses RIP1-mediated repression of the EGFR promoter. RIP1 is present both in the cytoplasm and in the nucleus. RIP1 coimmunoprecipitates with Sp1 in vivo and binds directly to Sp1 in vitro. A RIP1 mutant lacking the death domain fails to suppress Sp1 activity and the EGFR promoter, suggesting a critical role for the RIP1 death domain in EGFR regulation. Thus, our study identifies a new link between inflammatory and growth factor signaling pathways mediated by RIP1 and provides insight into the mechanism used by RIP1 to regulate EGFR levels.

MicroPET imaging of Cre-loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene.

Site-specific recombination tools such as the Cre-loxP system are used to create animal models where conditional gene deletion/activation studies are required. In the current proof of principle study, we have demonstrated that a PET reporter gene (PRG), the herpes simplex virus type 1 thymidine kinase (HSV1-tk), can be made to remain silent and can be activated by Cre-loxP-mediated recombination in cell culture and in living mice. An adenovirus carrying a silent HSV1-tk was tail-vein injected (1 x 10(9) PFU) in six transgenic mice that express Cre recombinase in their liver (Cre+) and in four control mice (Cre-). The liver-specific expression of the PRG in Cre+ mice was detected in the microPET following injection of the reporter probe, 9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ([(18)F]-FHBG). The [(18)F]-FHBG accumulation in the liver in terms of percent-injected dose per gram of tissue was 7.72+/-1.13 for the Cre+ mice and 0.10+/-0.02 for the Cre- mice (P<0.05) 48 h after adenoviral injection. These results were further validated by quantitative RT-PCR, western blotting and by in vitro assays for herpes simplex virus type 1 thymidine kinase enzyme activity. Thus by using the Cre-loxP system it is possible to modulate a PRG and noninvasively monitor the extent of Cre-loxP-mediated gene activation by imaging in a microPET scanner.

Monitoring protein-protein interactions using split synthetic renilla luciferase protein-fragment-assisted complementation.

In this study we developed an inducible synthetic renilla luciferase protein-fragment-assisted complementation-based bioluminescence assay to quantitatively measure real time protein-protein interactions in mammalian cells. We identified suitable sites to generate fragments of N and C portions of the protein that yield significant recovered activity through complementation. We validate complementation-based activation of split synthetic renilla luciferase protein driven by the interaction of two strongly interacting proteins, MyoD and Id, in five different cell lines utilizing transient transfection studies. The expression level of the system was also modulated by tumor necrosis factor alpha through NFkappaB-promoter/enhancer elements used to drive expression of the N portion of synthetic renilla luciferase reporter gene. This new system should help in studying protein-protein interactions and when used with other split reporters (e.g., split firefly luciferase) should help to monitor different components of an intracellular network.

Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies.

In this study we have developed bioluminescence-imaging strategies to noninvasively and quantitatively image protein-protein interactions in living mice by using a cooled charge-coupled device camera and split reporter technology. We validate both complementation and intein-mediated reconstitution of split firefly luciferase proteins driven by the interaction of two strongly interacting proteins, MyoD and Id. We use transient transfection of cells and image MyoD-Id interaction after induction of gene expression in cell culture and in cells implanted into living mice. Techniques to study protein-protein interactions in living subjects will allow the study of cellular networks, including signal transduction pathways, as well as development and optimization of pharmaceuticals for modulating protein-protein interactions.

Noninvasive quantitative imaging of protein-protein interactions in living subjects.

We are developing methods to image molecular and cellular events in living subjects. In this study, we validate imaging of protein-protein interactions in living mice by using bioluminescent optical imaging. We use the well studied yeast two-hybrid system adapted for mammalian cells and modify it to be inducible. We employ the NF-kappaB promoter to drive expression of two fusion proteins (VP16-MyoD and GAL4-ID). We modulate the NF-kappaB promoter through tumor necrosis factor alpha. Firefly luciferase reporter gene expression is driven by the interaction of MyoD and ID through a transcriptional activation strategy. We demonstrate the ability to detect this induced protein-protein interaction in cell culture and image this induced interaction in living mice by using transiently transfected cells. The current approach will be a valuable and potentially generalizable tool to noninvasively and quantitatively image protein-protein interactions in living subjects. The approaches validated should have important implications for the study of protein-protein interactions in cells maintained in their natural in vivo environment as well as for the in vivo evaluation of new pharmaceuticals targeted to modulate protein-protein interactions.