Vitis rotundifolia Skin Extract

Fragile X‐associated tremor/ataxia syndrome (FXTAS) is a late‐onset neurological disorder that affects carriers of the alleles for the Fragile X gene, Fragile X Mental Retardation (FMR1) due to the CGG triplet repeat expansion within the 5’UTR of FMR1. Clinical symptoms that are likely to occur are progressive ataxia, intention tremor, intranuclear inclusions, and Parkinsonian‐like motor symptoms. The goal of this research is to assess the biological efficacy of Curcumin, Muscadine Grape Seed Extract (MSKE) on the neuroprotective capacity with respect to mitochondrial damage, and their ability to restore mitochondrial health in patient with FXTAS. To establish mitochondrial dysfunction, normal human cell lines and human‐induced pluripotent cells were treated with multiple concentrations of glucose/ glucose oxidase (GluOx) at 2,12,and 24 hour time points to induce varying intensities of oxidative stress. The degrees of oxidative stress were measured by apoptosis and mitochondrial reactive oxygen species (ROS) production. Curcumin and MSKE, compounds effective against oxidative damage in mitochondria, were used to rescue glucose oxidase induced oxidative damage in both cell lines. To test the ability of these drugs to restore mitochondrial health, cell viability and cellular superoxide production were assessed by propidium iodide and the MitoSox fluorescence assay, respectively. We anticipated that GluOx at varying concentrations and time points would proportionally increase levels of apoptosis and mitochondrial ROS, reflective of mitochondrial damage, with the most severe dysfunction occurring at a dose of 25 nM and the longest duration of 24‐hr exposure. Administration of MSKE in concentrations ranging from 10 ‐8 to 10 ‐5 M in half log increments, did not reverse the oxidative defects induced in the cell lines. However, curcumin concentrations increased cell viability at the 2, 12, and 24 hour time period. Results indicate that the research design should be modified by increasing concentration of both glucose and MSKE to provide a reliable test of the hypothesis. Although, the research design will be modified to better assessed the hypothesis, the overall research is translational in that it lends flexibility to testing therapeutics in neuronal FXTAS models and expands the discovery of mitochondrial markers for the syndrome.

Snail transcription factor can induce epithelial-mesenchymal transition (EMT), associated with decreased cell adhesion-associated molecules like E-cadherin, increased mesenchymal markers like vimentin, leading to increased motility, invasion and metastasis. Muscadine grape skin extract (MSKE) has been shown to inhibit prostate cancer cell growth and induce apoptosis without affecting normal prostate epithelial cells. We investigated novel molecular mechanisms by which Snail promotes EMT in prostate cancer cells via Reactive Oxygen Species (ROS) and whether it can be antagonized by MSKE.

ARCaP and LNCaP cells overexpressing Snail were utilized to examine levels of reactive oxygen species (ROS), specifically, superoxide, in vitro using Dihydroethidium (DHE) or HydroCy3 dyes. Mitosox staining was performed to determine whether the source of ROS was mitochondrial in origin. We also investigated the effect of Muscadine grape skin extract (MSKE) on EMT marker expression by western blot analysis. Migration and cell viability using MTS proliferation assay was performed following MSKE treatments.

Snail overexpression in ARCaP and LNCaP cells was associated with increased concentration of mitochondrial superoxide, in vitro. Interestingly, MSKE decreased superoxide levels in ARCaP and LNCaP cells. Additionally, MSKE and Superoxide Dismutase (SOD) reverted EMT as evidenced by decreased vimentin levels and re-induction of E-cadherin expression in ARCaP-Snail cells after 3 days, concomitant with reduced cell migration. MSKE also decreased Stat-3 activity in ARCaP-Snail cells.

This study shows that superoxide species may play a role in Snail transcription factor-mediated EMT. Therefore, therapeutic targeting of Snail with various antioxidants such as MSKE may prove beneficial in abrogating EMT and ROS-mediated tumor progression in human prostate cancer.