Synergistic cytotoxicity of radiation and oncolytic Lister strain vaccinia in (V600D/E)BRAF mutant melanoma depends on JNK and TNF-α signaling.

Melanoma is an aggressive skin cancer that carries an extremely poor prognosis when local invasion, nodal spread or systemic metastasis has occurred. Recent advances in melanoma biology have revealed that RAS-RAF-MEK-ERK signaling has a pivotal role in governing disease progression and treatment resistance. Proof-of-concept clinical studies have shown that direct BRAF inhibition yields impressive responses in advanced disease but these are short-lived as treatment resistance rapidly emerges. Therefore, there is a pressing need to develop new targeted strategies for BRAF mutant melanoma. As such, oncolytic viruses represent a promising cancer-specific approach with significant activity in melanoma. This study investigated interactions between genetically-modified vaccinia virus (GLV-1h68) and radiotherapy in melanoma cell lines with BRAF mutant, Ras mutant or wild-type genotype. Preclinical studies revealed that GLV-1h68 combined with radiotherapy significantly increased cytotoxicity and apoptosis relative to either single agent in (V600D)BRAF/(V600E)BRAF mutant melanoma in vitro and in vivo. The mechanism of enhanced cytotoxicity with GLV-1h68/radiation (RT) was independent of viral replication and due to attenuation of JNK, p38 and ERK MAPK phosphorylation specifically in BRAF mutant cells. Further studies showed that JNK pathway inhibition sensitized BRAF mutant cells to GLV-1h68-mediated cell death, mimicking the effect of RT. GLV-1h68 infection activated MAPK signaling in (V600D)BRAF/(V600E)BRAF mutant cell lines and this was associated with TNF-α secretion which, in turn, provided a prosurvival signal. Combination GLV-1h68/RT (or GLV-1h68/JNK inhibition) caused abrogation of TNF-α secretion. These data provide a strong rationale for combining GLV-1h68 with irradiation in (V600D/E)BRAF mutant tumors.

Oncolytic vaccinia virus in combination with radiation shows synergistic antitumor efficacy in pancreatic cancer.

Combining oncolytic viruses with conventional therapy such as radiation is an innovative option for pancreatic cancer. We demonstrated that combination of GLV-1h151 and radiation yielded a synergistic cytotoxic effect, with the greatest effect achieved in the AsPC-1cell line. Combination treatment significantly increased apoptosis compared with either single treatment or the control group. In mice bearing human pancreatic tumor xenografts, combination treatment resulted in significantly enhanced inhibition of tumor growth. No evidence of toxicity was observed in mice. These results indicate that the combination of GLV-1h151 and radiation has great potential for translation into clinic practice.

Dual function of troglitazone in ICAM-1 gene expression in human vascular endothelium.

Our previous work has shown that troglitazone (an antidiabetic, thiazolidione drug and a synthetic ligand for peroxisome proliferator-activated receptor gamma, PPARgamma) stimulated basal level of intercellular adhesion molecule-1 (ICAM-1) protein expression in the absence of cytokine stimulation in human vascular endothelial cells. In this study, we examine the molecular mechanism of troglitazone on the basal and TNFalpha-induced ICAM-1 gene expression. Activation of transcription factors, NF-kappaB and AP-1 proteins, known to regulate ICAM-1 gene expression upon external stimulators, was examined. In human vascular endothelial cells (ECV304 cells), troglitazone inhibited TNFalpha-induced ICAM-1 gene expression by suppressing NF-kappaB/DNA binding activity, NF-kappaB transcriptional responses, c-Fos mRNA and protein levels via a ligand-dependent, PPARgamma-activated manner. In contrast, both troglitazone (at 10 microM) and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2), at 15 microM), a natural ligand for PPARgamma, induce c-Jun phosphorylation by activation of c-Jun N-terminal kinase (JNK) through a posttranslational regulation of c-Jun activity, therefore increasing AP-1/DNA binding activity and transcriptional responses as results of increasing basal ICAM-1 gene expression. These findings suggest dual function of troglitazone in the modulation of both basal and stimulated ICAM-1gene expression in human vascular endothelial cells.

PPARgamma agonists enhance human vascular endothelial adhesiveness by increasing ICAM-1 expression.

Early atherosclerotic lesions are characterized by increased monocyte adhesion to the overlying endothelium. Oxidized LDL (oxLDL) stimulates the adhesion of human monocytes to endothelial cells, in part, by increasing expression of ICAM-1. However, the cellular role of oxLDL in endothelial adhesiveness is not well understood. The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor superfamily, is expressed in vascular endothelial cells. Whether it can be activated by a synthetic ligand, troglitazone, as well as by natural ligands, oxLDL and its lipid components (i.e., 9- and 13-HODE), has not yet been explored. This study was undertaken to determine whether PPARgamma is expressed in ECV304 human vascular endothelial cells and if so to define the biological effects of its activation by these agonists. Our results demonstrate that PPARgamma mRNA is expressed in ECV304 cells, and transfected cells with a PPARE luciferase construct respond to these agonists. In addition, ligand-dependent PPARgamma activation increased ICAM-1 protein expression and enhanced adherence of monocytes to ECV304 cells by two- to threefold. These findings suggest that the PPARgamma signaling pathway might contribute to the atherogenicity of oxLDL in vascular endothelial cells.

Estimation of quasi-straightforward propagating light in tissues.

We have developed a controlled Monte Carlo (CMC) method to calculate the time-dependent transmittance of light through a thick tissue, especially for evaluation of the contribution from early arriving photons. Quasi-straightforward propagating trajectories are favoured according to a selection mechanism, so adequate trajectories of interest can reach the detector, improving the statistics dramatically. Simulations were conducted for tissue models with a thickness of 3-5 cm, and with optical properties similar to human breast tissue. Temporal profiles of early transmittance were obtained with satisfactory convergence. In addition, comparison was made with the conventional Monte Carlo approach to verify our scheme when applied to cases of optically thin tissues.

Enhanced sensitivity of pancreatic islets from preobese 2-week-old ob/ob mice to neurohormonal stimulation of insulin secretion.

Insulin secretion from perifused islets of preobese, 2-week-old, genetically obese (ob/ob) mice and their lean littermates was examined to identify early-onset abnormalities in regulation of insulin secretion by ob/ob mice. The ob/ob mice were slightly hyperinsulinemic (+20%) and hypoglycemic (-12%) at 2 weeks of age. Pancreatic islet size, DNA content, and insulin content were similar in ob/ob and lean mice. The responsiveness of islets to glucose, as determined by 20 mM glucose-induced insulin secretion, and the sensitivity of islets to glucose, as determined by the glucose threshold for insulin secretion, were unaffected by phenotype, but two insulin secretagogues that potentiate glucose-induced insulin secretion via activation of the phospholipase-C signal transduction pathway (i.e. acetylcholine, and cholecystokinin) were more effective in stimulating insulin secretion from islets of ob/ob mice than from islets of lean mice. Both responsiveness and sensitivity to acetylcholine and cholecystokinin potentiation of glucose-induced insulin secretion were enhanced in islets from ob/ob mice. Further, glucose-dependent insulinotropic polypeptide, which stimulates glucose-induced insulin secretion via activation of adenylate cyclase, interacted with acetylcholine to further augment differences in insulin secretion between islets from ob/ob and lean mice. The signal transduction pathway common to acetylcholine and cholecystokinin, and cross-talk between this pathway and the glucose-dependent insulinotropic polypeptide signal transduction pathway are loci for early-onset defects in control of insulin secretion from islets of ob/ob mice.