Repurposing live attenuated trivalent MMR vaccine as cost-effective cancer immunotherapy.

It has long been known that oncolytic viruses wield their therapeutic capability by priming an inflammatory state within the tumor and activating the tumor immune microenvironment, resulting in a multifaceted antitumor immune response. Vaccine-derived viruses, such as measles and mumps, have demonstrated promising potential for treating human cancer in animal models and clinical trials. However, the extensive cost of manufacturing current oncolytic viral products makes them far out of reach for most patients. Here by analyzing the impact of intratumoral (IT) administrations of the trivalent live attenuated measles, mumps, and rubella viruses (MMR) vaccine, we unveil the cellular and molecular basis of MMR-induced anti-cancer activity. Strikingly, we found that IT delivery of low doses of MMR correlates with tumor control and improved survival in murine hepatocellular cancer and colorectal cancer models via increased tumor infiltration of CD8+ granzyme B+ T-cells and decreased macrophages. Moreover, our data indicate that MMR activates key cellular effectors of the host's innate and adaptive antitumor immunity, culminating in an immunologically coordinated cancer cell death. These findings warrant further work on the potential for MMR to be repurposed as safe and cost-effective cancer immunotherapy to impact cancer patients globally.

Effects of Gamma-Tocotrienol on Intestinal Injury in a GI-Specific Acute Radiation Syndrome Model in Nonhuman Primate.

The gastrointestinal (GI) system is highly susceptible to irradiation. Currently, there is no Food and Drug Administration (FDA)-approved medical countermeasures for GI radiation injury. The vitamin E analog gamma-tocotrienol (GT3) is a promising radioprotector in mice and nonhuman primates (NHP). We evaluated GT3-mediated GI recovery in total-body irradiated (TBI) NHPs. Sixteen rhesus macaques were divided into two groups; eight received vehicle and eight GT3 24 h prior to 12 Gy TBI. Proximal jejunum was assessed for structural injuries and crypt survival on day 4 and 7. Apoptotic cell death and crypt cell proliferation were assessed with TUNEL and Ki-67 immunostaining. Irradiation induced significant shortening of the villi and reduced mucosal surface area. GT3 induced an increase in crypt depth at day 7, suggesting that more stem cells survived and proliferated after irradiation. GT3 did not influence crypt survival after irradiation. GT3 treatment caused a significant decline in TUNEL-positive cells at both day 4 (p < 0.03) and 7 (p < 0.0003). Importantly, GT3 induced a significant increase in Ki-67-positive cells at day 7 (p < 0.05). These data suggest that GT3 has radioprotective function in intestinal epithelial and crypt cells. GT3 should be further explored as a prophylactic medical countermeasure for radiation-induced GI injury.

Antimelanoma activities of chimeric thiazole-androstenone derivatives.

The discovery of chimeric anti-melanoma agents is reported. These molecules are potent growth suppressors of melanoma cells in vitro with growth inhibition of 50% (GI50) values as low as 1.32 µM. Compounds were more toxic to melanoma cells in vitro than commonly used anti-melanoma agent dacarbazine as measured by TUNEL assay. They induced both caspase-independent apoptosis evident by colocalization of TUNEL with endonuclease G (EndoG) and caspase-mediated apoptosis measured by colocalization of TUNEL with caspase-activated DNase (CAD). In addition, compounds 3 and 5 strongly induced oxidative injury to melanoma cells as measured by TUNEL colocalization with heme oxygenase-1 (HO1). Dacarbazine induced only caspase-independent apoptosis, which may explain why it is less cytotoxic to melanoma cells than compounds 3, 4 and 5.

DNase I Induces Other Endonucleases in Kidney Tubular Epithelial Cells by Its DNA-Degrading Activity.

Endonuclease-mediated DNA fragmentation is both an immediate cause and a result of apoptosis and of all other types of irreversible cell death after injury. It is produced by nine enzymes including DNase I, DNase 2, their homologs, caspase-activated DNase (CAD) and endonuclease G (EndoG). The endonucleases act simultaneously during cell death; however, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most abundant of endonucleases, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK-52E cells were transfected with the DNase I gene or its inactive mutant in a pECFP expression vector, while control cells were transfected with the empty vector. mRNA expression of all nine endonucleases was studied using real-time RT-PCR; DNA strand breaks in endonuclease genes were determined by PCR and protein expression of the enzymes was measured by Western blotting and quantitative immunocytochemistry. Our data showed that DNase I, but not its inactive mutant, induces all other endonucleases at varying time periods after transfection, causes DNA breaks in endonuclease genes, and elevates protein expression of several endonucleases. This is the first evidence that endonucleases seem to be induced by the DNA-degrading activity of DNase I.

Fractionated radiation suppresses Kruppel-like factor 2 pathway to a greater extent than by single exposure to the same total dose.

Kruppel-like factor 2 (KLF2) is a positive transcriptional regulator of several endothelial protective molecules, including thrombomodulin (TM), a surface receptor, and endothelial nitric oxide synthase (eNOS), an enzyme that generates nitric oxide (NO). Loss of TM and eNOS causes endothelial dysfunction, which results in suppressed generation of activated protein C (APC) by TM-thrombin complex and in upregulation of intercellular adhesion molecule 1 (ICAM-1). Mechanistic studies revealed that activation of extracellular signal-regulated kinase 5 (ERK5) via upregulation of myocyte enhancer factor 2 (MEF2) induces KLF2 expression. Radiation causes endothelial dysfunction, but no study has investigated radiation's effects on the KLF2 pathway. Because fractionated radiation is routinely used during cancer radiotherapy, we decided to delineate the effects of radiation dose fractionation on the KLF2 signaling cascade at early time points (up to 24 h). We exposed human primary endothelial cells to radiation as a series of fractionated or as a single exposure, with the same total dose delivered to each group. We measured the expression and activity of critical members of the KLF2 pathway at subsequent time points, and determined whether pharmacological upregulation of KLF2 can reverse the radiation effects. Compared to single exposure, fractionated radiation profoundly suppressed KLF2, TM, and eNOS levels, subdued APC generation, declined KLF2 binding ability to TM and eNOS promoters, enhanced ICAM-1 expression, and decreased expression of upstream regulators of KLF2 (ERK5 and MEF2). Pharmacological inhibitors of the mevalonate pathway prevented fractionated-radiation-induced suppression of KLF2, TM, and eNOS expression. Finally, fractionated irradiation to thoracic region more profoundly suppressed KLF2 and enhanced ICAM-1 expression than single exposure in the lung at 24 h. These data clearly indicate that radiation dose fractionation plays a critical role in modulating levels of KLF2, its upstream regulators, and its downstream target molecules in endothelial cells. Our findings will provide important insights for selecting fractionated regimens during radiotherapy and for developing strategies to alleviate radiotherapy-induced toxicity to healthy tissues.

Gamma-Tocotrienol Protects the Intestine from Radiation Potentially by Accelerating Mesenchymal Immune Cell Recovery.

Natural antioxidant gamma-tocotrienol (GT3), a vitamin E family member, provides intestinal radiation protection. We seek to understand whether this protection is mediated via mucosal epithelial stem cells or sub-mucosal mesenchymal immune cells. Vehicle- or GT3-treated male CD2F1 mice were exposed to total body irradiation (TBI). Cell death was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Villus height and crypt depth were measured with computer-assisted software in tissue sections. Functional activity was determined with an intestinal permeability assay. Immune cell recovery was measured with immunohistochemistry and Western blot, and the regeneration of intestinal crypts was assessed with ex vivo organoid culture. A single dose of GT3 (200 mg/kg body weight (bwt)) administered 24 h before TBI suppressed cell death, prevented a decrease in villus height, increased crypt depth, attenuated intestinal permeability, and upregulated occludin level in the intestine compared to the vehicle treated group. GT3 accelerated mesenchymal immune cell recovery after irradiation, but it did not promote ex vivo organoid formation and failed to enhance the expression of stem cell markers. Finally, GT3 significantly upregulated protein kinase B or AKT phosphorylation after TBI. Pretreatment with GT3 attenuates TBI-induced structural and functional damage to the intestine, potentially by facilitating intestinal immune cell recovery. Thus, GT3 could be used as an intestinal radioprotector.

Mechanism of graphene-induced cytotoxicity: Role of endonucleases.

Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright © 2017 John Wiley & Sons, Ltd.