A murine model of targeted infusion for intracranial tumors.

Historically, intra-arterial (IA) drug administration for malignant brain tumors including glioblastoma multiforme (GBM) was performed as an attempt to improve drug delivery. With the advent of percutaneous neuorovascular techniques and modern microcatheters, intracranial drug delivery is readily feasible; however, the question remains whether IA administration is safe and more effective compared to other delivery modalities such as intravenous (IV) or oral administrations. Preclinical large animal models allow for comparisons between treatment routes and to test novel agents, but can be expensive and difficult to generate large numbers and rapid results. Accordingly, we developed a murine model of IA drug delivery for GBM that is reproducible with clear readouts of tumor response and neurotoxicities. Herein, we describe a novel mouse model of IA drug delivery accessing the internal carotid artery to treat ipsilateral implanted GBM tumors that is consistent and reproducible with minimal experience. The intent of establishing this unique platform is to efficiently interrogate targeted anti-tumor agents that may be designed to take advantage of a directed, regional therapy approach for brain tumors.

Pterostilbene inhibits lung cancer through induction of apoptosis.

BACKGROUND: Lung cancer remains the leading cause of cancer mortality in the United States. Resveratrol is a potent antioxidant found in grapes that inhibits several types of cancer, including lung cancer. Herein, we investigated the effects of pterostilbene, an analog of resveratrol found in blueberries, on lung cancer, in vitro. We hypothesized that pterostilbene would inhibit lung cancer cell growth in vitro by a pro-apoptotic mechanism. METHODS: Two lung cancer cell lines (NCI-H460 and SK-MES-1) were cultured using standard techniques. Cells were treated with increasing doses of pterostilbene (10-100 microM). Cell viability was measured at 24, 48, and 72h using a MTT assay. Apo-ONE Caspase-3/7 assay was used to evaluate caspase activity. T-test and two-way ANOVA were used for statistical analysis. RESULTS: Pterostilbene significantly decreased cell viability in lung cancer cells in a concentration- and time-dependent manner (P<0.001). Concentrations greater than 20 microM of pterostilbene produced significant growth inhibition by 72h (P<0.001). Apoptosis and caspase-3/7 activity were significantly increased by pterostilbene treatment (P<0.05). CONCLUSIONS: Pterostilbene inhibits growth via apoptosis induction in vitro. Further in vitro mechanistic studies and in vivo experiments are warranted to determine the potential role for pterostilbene in lung cancer treatment or prevention.

Pterostilbene inhibits breast cancer in vitro through mitochondrial depolarization and induction of caspase-dependent apoptosis.

BACKGROUND: Epidemiologic studies suggest that diets high in fruits and vegetables reduce cancer risk. Resveratrol, a compound present in grapes, has been shown to inhibit a variety of primary tumors. Pterostilbene, an analogue of resveratrol found in blueberries, has both antioxidant and antiproliferative properties. We hypothesized that pterostilbene would induce apoptosis and inhibit breast cancer cell growth in vitro. METHODS: Breast cancer cells were treated with graduated doses of pterostilbene. Cell viability was measured by MTT assay. Apoptosis was evaluated via DNA fragmentation assay and TUNEL assay. Apo-ONE caspase-3/7 assay was used to evaluate caspase activity. Flow cytometry was used to evaluate mitochondrial depolarization, superoxide formation, and cell cycle. Student's t-test and two-way ANOVA with Bonferroni posttests were utilized for statistical analysis. RESULTS: Pterostilbene decreased breast cancer cell viability in a concentration- and time-dependent manner. Pterostilbene treatment increased caspase-3/7 activity and apoptosis in both cell lines. Caspase-3/7 inhibitors completely reversed pterostilbene's effects on cell viability. Pterostilbene treatment triggered mitochondrial depolarization, increased superoxide anion, and caused alteration in cell cycle. CONCLUSIONS: Pterostilbene treatment inhibits the growth of breast cancer in vitro through caspase-dependent apoptosis. Mitochondrial membrane depolarization and increased superoxide anion may contribute to the activation downstream effector caspases. Caspase inhibition leads to complete reversal of pterostilbene's effect on cell viability. Further in vitro mechanistic studies and in vivo experiments are warranted to determine its potential for the treatment of breast cancer.

Effects of pterostilbene on melanoma alone and in synergy with inositol hexaphosphate.

BACKGROUND: Pterostilbene and inositol-6-phosphate (IP6) have been shown to inhibit melanoma growth in vitro. However, pterostilbene's mechanism of action has not been clearly demonstrated. We aimed to further investigate the mechanism of action for pterostilbene and to determine whether combination treatment with IP6 produced synergistic growth inhibition. METHODS: Melanoma cells were treated with increasing doses of pterostilbene, IP6, or combinations thereof. Cell viability was measured at 24 hours, 48 hours, and 72 hours using a MTT assay. Caspase activity and vascular endothelial growth factor (VEGF) production were measured using enzyme-linked immunosorbent assay (ELISA). Analysis of variance (ANOVA) and t tests were used for statistical analysis. RESULTS: Pterostilbene inhibits melanoma growth in vitro in association with increased effector caspase activity. Combination treatment with inositol hexaphosphate produces synergistic growth inhibition, greater than either treatment alone. CONCLUSIONS: Pterostilbene produces caspase-dependent apoptosis in melanoma cell lines. Combination treatment with IP6 produces synergistic growth inhibition. Both compounds have significant potential for a therapeutic role in the treatment of melanoma.

Peptide YY mediates inhibition of tumor growth and inflammation.

Peptide YY (PYY) orchestrates the functions of the gut and the pancreas by regulating growth, digestion and absorption. In addition to its physiological role, PYY exhibits immune and antitrophic properties in the pancreas by decreasing cytokine and amylase release. Although the exact mechanism(s) of action are still incompletely understood, PYY interacts at the acinar level with numerous intracellular transcription factors. In addition to ameliorating pancreatic inflammation, novel synthetic analogs of PYY have been developed that are potent inhibitors of pancreatic cancer proliferation, in vitro and in vivo. Additionally, PYY and its analogs have been shown to inhibit the growth of breast, esophagus, and gastric cancer in vitro. We, herein, plan to review some of the methods employed in the laboratory while investigating the utility of PYY in the treatment of inflammation and cancer.

Direct transdifferentiation gives rise to the earliest new hair cells in regenerating avian auditory epithelium.

The avian auditory epithelium is capable of complete regeneration after hair cell (HC) loss. Most new HCs arise via cell division, but approximately one-third of new HCs arise via direct transdifferentiation (DT), in which supporting cells (SCs) alter their phenotype without dividing. In this study, we used synchronous, gentamicin-induced near-total HC loss in the basal end of the epithelium and continuous infusion of the cell division marker bromodeoxyuridine (BrdU) to identify the origin of each individual regenerating HC. Early new HCs were identified by immunolabeling for the HC-specific marker myosin-VIIa, and mitotic cells with BrdU immunolabeling. The first new HCs arising via DT appear 72-96 hr after gentamicin, 24-48 hr earlier than the first new mitotic HCs. After Day 6, however, most new HCs are mitotic. The "intermediate" morphology that has been suggested to be characteristic of DT is seen in HCs arising via both pathways. These findings suggest that DT is a simpler, more rapid process that produces the first new HCs, and that mitotic regeneration is somewhat slower but ultimately produces most new HCs. The identical morphology of regenerating HCs from both pathways suggests that once HC fate is established, all new HCs follow similar cellular processes during differentiation and reorganization into the regenerated epithelium.

Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo.

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.

REST mRNA expression in normal and regenerating avian auditory epithelium.

Hair cells (HCs) and supporting cells (SCs) in the auditory epithelium initially arise from a sheet of undifferentiated cells. Although much has been learned about the initial steps leading to the fate determination of HCs and SCs, respectively, little is known about what molecular events 'finalize' cell fate determination. We investigated the role of repressor element-1 (RE-1) silencing transcription factor (REST), whose inactivation is known to be a requirement for a cell to assume a neuronal identity. Here we show by in situ hybridization (ISH) that SCs express REST messenger RNA (mRNA) but sensory HCs lack detectable expression. Using a more sensitive reverse transcription-polymerase chain reaction assay, however, we detected the presence of a neuron-specific splice variant in the epithelium, suggesting that HCs express REST mRNA at levels too low to be detectable by ISH. In regenerating auditory epithelium, we found that REST mRNA was expressed and upregulated in all remaining cells in the damaged region of the epithelium, consistent with its expression pattern during development prior to neurogenesis. Surprisingly, REST mRNA was also upregulated in SCs in the apical, undamaged region of the epithelium, and readily detectable by ISH in the HCs in this region. This finding suggests that the grossly undamaged region of the epithelium is in fact biochemically altered towards a 'less developed' state. Our results indicate that REST inactivation is an important step in finalizing HC fate in the chick inner ear.