Val16A SOD2 Polymorphism Promotes Epithelial-Mesenchymal Transition Antagonized by Muscadine Grape Skin Extract in Prostate Cancer Cells.

Epithelial-mesenchymal transition (EMT), a key event in cancer metastasis, allows polarized epithelial cells to assume mesenchymal morphologies, enhancing invasiveness and migration, and can be induced by reactive oxygen species (ROS). Val16A (Ala) SOD2 polymorphism has been associated with increased prostate cancer (PCa) risk. We hypothesized that SOD2 Ala single nucleotide polymorphism (SNP) may promote EMT. We analyzed SOD2 expression and genotype in various prostate cell lines. Stable overexpression of Ala-SOD2 or Val-SOD2 allele was performed in Lymph Node Carcinoma of the Prostate (LNCaP) cells followed by analysis of intracellular ROS and EMT marker protein expression. Treatments were performed with muscadine grape skin extract (MSKE) antioxidant, with or without addition of H2O2 to provide further oxidative stress. Furthermore, MTS cell proliferation, cell migration, and apoptosis assays were completed. The results showed that SOD2 expression did not correlate with tumor aggressiveness nor SOD2 genotype. We demonstrated that the Ala-SOD2 allele was associated with marked induction of EMT indicated by higher Snail and vimentin, lower E-cadherin, and increased cell migration, when compared to Val-SOD2 allele or Neo control cells. Ala-SOD2 SNP cells exhibited increased levels of total ROS and superoxide and were more sensitive to co-treatment with H2O2 and MSKE, which led to reduced cell growth and increased apoptosis. Additionally, MSKE inhibited Ala-SOD2 SNP-mediated EMT. Our data indicates that treatment with a combination of H2O2-generative drugs, such as certain chemotherapeutics and antioxidants such as MSKE that targets superoxide, hold promising therapeutic potential to halt PCa progression in the future.

Larrea tridentata Extract Mitigates Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells.

Creosote bush (Larrea tridentata; LT) leaves extracts were tested for their potential efficacy to mitigate cellular oxidative stress on human SH-SY5Y cells. Here, the differential nuclear staining assay, a bioimager system, and flow cytometric protocols, concurrently with several specific chemicals, were used to measure the percentage of cell viability and several facets implicated in the cytoprotective mechanism of LT extracts. Initially, three LT extracts, prepared with different solvents, ethanol, ethanol:water (e/w), and water, were tested for their capacity to rescue the viability of cells undergoing aggressive H2O2-induced oxidative stress. Results indicate that the LT extract prepared with a mixture of ethanol:water (LT-e/w; 60:40% v/v) displayed the most effective cytoprotection rescue activity. Interestingly, by investigating the LT-e/w mechanism of action, it was found that LT-e/w extract decreases the levels of H2O2-provoked reactive oxidative species (ROS) accumulation, mitochondrial depolarization, phosphatidylserine externalization, caspase-3/7 activation, and poly (ADP-ribose) polymerase (PARP) cleavage significantly, which are hallmarks of apoptosis. Thus, out of the three LT extracts tested, our findings highlight that the LT-e/w extract was the most effective protective reagent on SH-SY5Y cells undergoing oxidative stress in vitro, functioning as a natural anti-apoptotic extract. These findings warrant further LT-e/w extract examination in a holistic context.

CCAAT-displacement protein/cut homeobox transcription factor (CUX1) represses estrogen receptor-alpha (ER-α) in triple-negative breast cancer cells and can be antagonized by muscadine grape skin extract (MSKE).

Triple-Negative Breast Cancers (TNBCs) are the most difficult to treat subtype of breast cancer and are often associated with high nuclear expression of Snail and Cathepsin L (Cat L) protease. We have previously shown that Snail can increase Cat L expression/activity in prostate and breast cancer cells. This study investigated the role of CUX1 (a downstream substrate of Cat L) in TNBC. We showed that Cat L and CUX1 were highly expressed in TNBC patient tissue/cell lines, as compared to ER-positive samples, using cBioportal data and western blot/zymography analyses. Additionally, luciferase reporter and chromatin immunoprecipitation assays showed that CUX1 directly bound to estrogen receptor-alpha (ER-α) promoter in MDA-MB-468, a representative TNBC cell line, and that CUX1 siRNA could restore ER-α transcription and protein expression. Furthermore, Snail and CUX1 expression in various TNBC cell lines was inhibited by muscadine grape skin extract (MSKE, a natural grape product rich in anthocyanins) or Cat L inhibitor (Z-FY-CHO) leading to decreased cell invasion and migration. MSKE decreased cell viability and increased expression of apoptotic markers in MDA-MB-468 cells, with no effect on non-tumorigenic MCF10A cells. MSKE also decreased CUX1 binding to ER-α promoter and restored ER-α expression in TNBC cells, while both MSKE and CUX1 siRNA restored sensitivity to estradiol and 4-hydoxytamoxifen as shown by increased cell viability. Therefore, CUX1 activated by Snail-Cat L signaling may contribute to TNBC via ER-α repression, and may be a viable target for TNBC using natural products such as MSKE that targets cancer and not normal cells.

Epithelial-Mesenchymal Transition (EMT) and Prostate Cancer.

Typically the normal epithelial cells are a single layer, held tightly by adherent proteins that prevent the mobilization of the cells from the monolayer sheet. During prostate cancer progression, the epithelial cells can undergo epithelial-mesenchymal transition or EMT, characterized by morphological changes in their phenotype from cuboidal to spindle-shaped. This is associated with biochemical changes in which epithelial cell markers such as E-cadherin and occludins are down-regulated, which leads to loss of cell-cell adhesion, while mesenchymal markers such as vimentin and N-cadherin are up-regulated, thereby allowing the cells to migrate or metastasize to different organs. The EMT transition can be regulated directly and indirectly by multiple molecular mechanisms including growth factors and cytokines such as transforming growth factor-beta (TGF-ß), epidermal growth factor (EGF) and insulin-like growth factor (IGF), and signaling pathways such as mitogen-activated protein kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3K). This signaling subsequently induces expression of various transcription factors like Snail, Twist, Zeb1/2, that are also known as master regulators of EMT. Various markers associated with EMT have been reported in prostate cancer patient tissue as well as a possible association with health disparities. There has been consideration to therapeutically target EMT in prostate cancer patients by targeting the EMT signaling pathways.