Anticancer Impact of Nitric Oxide (NO) and NO Combination with SMYD-3 Inhibitor on Breast Carcinomas.

Despite enormous advances in the detection and treatment of breast cancer, it still remains the leading cancer diagnosis and has the second highest mortality rate. Thus, breast cancer research is a high priority for academics and clinicians alike. Based on previous research indicating the potential of nitric oxide (NO) and SMYD-3 inhibition, this work sought to expand upon these concepts and combine the two approaches. Both NO (from S-Nitrosoglutathione (GSNO)), termed Group 1, and a combination therapeutic, inhibitor-4 (SMYD-3 inhibitor) plus NO (from GSNO), termed Group 2, were evaluated for their efficacy on breast carcinoma cell lines MCF7 and MDA-MB-231, and the normal MCF10A breast cell line, using cellular viability, colony formation capacity, cytotoxicity, and cellular apoptosis analysis. These results indicated that, in Group 1, breast carcinoma lines MCF7 and MDA-MB-231, cells experienced a moderate reduction in cellular viability (~20-25%), a large reduction in colony formation capacity (~80-90%), a moderate increase in the relative number of dead cells, and a moderate increase in cellular apoptosis. Group 2 was significantly more impactful, with a ~50% knockdown in cellular viability, a 100% reduction in colony formation capacity, a large increase in the relative number of dead cells, and a large increase in cellular apoptosis. Additionally, Group 2 induced a very small impact on the normal MCF10A cell line. Cumulatively, this work revealed the exciting impact of this combination therapeutic, indicating its potential for clinical application and further research.

Anticancer potential of nitric oxide (NO) in neuroblastoma treatment.

The most common extracranial solid tumor in childhood, paediatric neuroblastoma, is frequently diagnosed at advanced stages and identified as high risk. High risk neuroblastoma is aggressive and unpredictable, resulting in poor prognosis and only ∼40% five-year survival rates. Herein, nitric oxide (NO) delivered via the S-nitrosothiol, S-nitrosoglutathione (GSNO), is explored as an anticancer therapeutic in various neuroblastoma lines. After 24 h of treatment with GSNO, cell viability assays, as assessed by resazurin and MTT ((3-4,5-dimethylthiazol-2-yl)-2,5-diphyltetrazolium bromide), consistently identified a moderate, ∼13-29%, decrease in metabolic activity, colony formation assays revealed notably significant reduction of clonogenic activity, and cytotoxicity assays revealed a visibly significant reduction of total number of cells and live cells as well as an increase in number of dead cells in treated cells versus untreated cells. Thrillingly, RNA-sequence analysis provided highly valuable information regarding the differentially expressed genes in treated samples versus control samples as well as insight into the mechanism of action of NO as an anticancer therapeutic. Favorably, the collective results from these analyses exhibited tumoricidal, non-tumour promoting, and discriminatory characteristics, illuminating the feasibility and significance of NO as a cytotoxic adjuvant in neuroblastoma treatment.

Nitric Oxide as a Potential Adjuvant Therapeutic for Neuroblastoma: Effects of NO on Murine N2a Cells.

Neuroblastoma, the most common extracranial solid tumor in children, accounts for 15% of all pediatric cancer deaths. Pharmaceutical applications of S-Nitrosylation, which, under normal conditions is involved with a host of epigenetic and embryological development pathways, have exhibited great potential for use as adjuvant therapeutics in the clinical management of cancer. Herein, an evaluation of the impact of nitric oxide (NO) as a potent anticancer agent on murine neuroblastoma cells is presented. Excitingly cell viability, colony formation, and non-carcinogenic cell analysis illustrate the significance and practicality of NO as a cytotoxic anticancer therapeutic. Resazurin, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphyltetrazolium bromide) assays consistently displayed a moderate, ~20-25% reduction in cell viability after exposure to 1 mM S-Nitrosoglutathione (GSNO). A colony formation assay demonstrated that treated cells no longer exhibited colony formation capacity. Identically GSNO-treated Adult Human Dermal Fibroblasts (HDFa) exhibited no decrease in viability, indicating potential discrimination between neoplastic and normal cells. Collectively, our findings indicate a potential application for NO as an adjuvant therapeutic in the clinical management of neuroblastoma.

Nitric Oxide-Releasing Emulsion with Hyaluronic Acid and Vitamin E.

S-nitrosoglutathione (GSNO) is a naturally available S-nitrosothiol that can be incorporated into non-toxic formulations intended for topical use. The value of nitric oxide (NO) delivered topically relates to its well-studied physiological functions such as vasodilation, angiogenesis, cell proliferation and broad-spectrum antibacterial activity. Previously reported topical NO-releasing substrates include polymeric materials that exhibit non-toxic behaviors on dermal tissue such as polyethylene glycol. However, they do not serve as humectants nor provide vitamins to the skin. In this study, GSNO was added to an emulsion that was fortified with α-tocopheryl acetate (vitamin E) and hyaluronic acid. The average total NO content for the NO-releasing emulsion was 58 ± 8 µmol g-1 at 150 °C and the cumulative NO release over 53 h at physiological temperature (37.4 °C) was 46 ± 4 µmol g-1. The GSNO concentration in the lotion was optimized in order to reach a pH value similar to that of human skin (pH 5.5). The viscosity was analyzed using a rotational viscometer for the S-nitrosated and the non-nitrosated emulsions to obtain a material that can be readily spread on dermal tissue. The viscosity values obtained ranged from 7.88 ± 0.99 to 8.50 ± 0.36 Pa•s. Previous studies have determined that the viscosity maximum for lotions is 100 Pa•s. A low viscosity increases the diffusion coefficient of active ingredients to the skin given that they are inversely proportional as described by the Einstein-Smoluchowski equation. The effect of the S-nitrosated and non-nitrosated emulsions on adult human dermal fibroblasts (HDFs) was assessed in comparison to untreated HDFs using Colorimetric Cell Viability Kit I - WST- 8. The findings indicate that neither the S-nitrosated nor non-nitrosated emulsions induced cytotoxicity in HDFs.

Cell-Based Methods for Determination of Efficacy for Candidate Therapeutics in the Clinical Management of Cancer.

Determination of therapeutic efficacy is a major challenge in developing treatment options for cancer. Prior to in vivo studies, candidate therapeutics are evaluated using cell-based in vitro methods to assess their anti-cancer potential. This review describes the utility and limitations of evaluating therapeutic efficacy using human tumor-derived cell lines. Indicators for therapeutic efficacy using tumor-derived cell lines include cell viability, cell proliferation, colony formation, cytotoxicity, cytostasis, induction of apoptosis, and cell cycle arrest. Cell panel screens, 3D tumor spheroid models, drug-drug/drug-radiation combinatorial analysis, and invasion/migration assays reveal analogous in vitro information. In animal models, cellular assays can assess tumor micro-environment and therapeutic delivery. The utility of tumor-derived cell lines for efficacy determination is manifest in numerous commercially approved drugs that have been applied in clinical management of cancer. Studies reveal most tumor-derived cell lines preserve the genomic signature of the primary tumor source and cell line-based data is highly predictive of subsequent clinical studies. However, cell-based data often disregards natural system components, resulting in cell autonomous outcomes. While 3D cell culture platforms can counter such limitations, they require additional time and cost. Despite the limitations, cell-based methods remain essential in early stages of anti-cancer drug development.