Effect of 2-Deoxyglucose on Colorectal Cancer Cell Lines

The third leading cause of cancer deaths in the U.S. is colon cancer. The major disadvantage of cancer chemotherapy is its non-selective toxicity to healthy cells at the therapeutic doses. A possible target selective for cancer cells is their dependence on glycolysis for cellular energy. 2-deoxyglucose (2-DG) is a glycolytic inhibitor that has been shown to be safe in both animals and humans. The molecular mechanisms for the anticancer effect of 2-DG cannot be explained solely by its glycolytic inhibition. In this manuscript we studied the effect of 2-DG on colon cancer cells and its possible molecular mechanism. Colon cancer cells are more susceptible to 2-DG treatment than other cancerous and non-cancerous cell lines tested. The colon cancer cells tested are SW620, SW480 and GC3/C1. In cell cycle analysis studied using propidium iodide staining of DNA followed by flow-cytometry, 2-DG induced cell cycle arrest at G0/G1 phase in SW 620 cells. 2-DG also modified the expression of various cell cycle proteins such as p21, p53, and cyclins as measured through Western Blotting. In addition to cell cycle arrest, 2-DG also induced apoptosis through activation of Caspase 3. Complementing 5-Fluorouracil (5- FU) treatment of colon cancer cells with 2-DG significantly enhanced the efficiency of 5- FU treatment up to 3.5 fold. This study showed a new molecular mechanisms for 2-DG that could be used to design novel combination therapies with other known chemotherapeutic agents for colon cancer. The addition of a well-tolerated molecule like 2- DG increases the efficiency of 5-FU, thus reducing the patient’s cumulative exposure to 5- FU. This may lead to fewer dose dependent side effects and better patient outcomes

Evaluating Student Performance and Perception of a Workshop Integrating Pharmacy Practice and a Pharmaceutics Lab

  Objective: Evaluating Student Performance and Perception of a Workshop Integrating Pharmacy Practice and a Pharmaceutics Lab Innovation: Common methods for curricular integration are often time and faculty-intensive. An innovative approach to integration was developed and utilized in an introductory compounding workshop. Faculty members collaborated with a compounding pharmacist to design and facilitate a pharmaceutics workshop for first-year pharmacy students.  The workshop was composed of four major sections, an introduction to pharmaceutical compounding and the regulations surrounding manufacturing and sterility, a case discussion involving a pediatric patient and the need to develop an appropriate drug delivery system, a short review of pharmaceutical calculations and labeling requirements, and then an introduction to logistics and active learning in a lab setting. Critical Analysis: After taking part in the workshop, students indicated a significantly higher comfort level going into the pharmaceutics lab (3.48±0.83 to 4.04±0.70) and in the compounding process (3.06±0.83 to 3.71±0.80). Their views of the clinical application of the lab and the need to use knowledge gained from other courses in the lab were also significantly improved (4.36±0.68 to 4.61±0.49 and 3.71±0.77 to 4.26±0.74, respectively). In addition, their perceptions of how they will utilize the skills developed as a practicing pharmacist, and their feelings towards the safety procedures involved in compounding, were also positively affected (3.96±0.87 to 4.45±0.59 and 3.28±0.92 to 3.91±0.72, respectively). Finally, students’ average quiz score in Spring 2016, when the workshop was instituted, significantly increased from Spring 2015 (90.154±4.98 versus 85.89±10.87, respectively). Article Type:  Not

The Flavonoid Metabolite 2,4,6-Trihydroxybenzoic Acid Is a CDK Inhibitor and an Anti-Proliferative Agent: A Potential Role in Cancer Prevention

Flavonoids have emerged as promising compounds capable of preventing colorectal cancer (CRC) due to their anti-oxidant and anti-inflammatory properties. It is hypothesized that the metabolites of flavonoids are primarily responsible for the observed anti-cancer effects owing to the unstable nature of the parent compounds and their degradation by colonic microflora. In this study, we investigated the ability of one metabolite, 2,4,6-trihydroxybenzoic acid (2,4,6-THBA) to inhibit Cyclin Dependent Kinase (CDK) activity and cancer cell proliferation. Using in vitro kinase assays, we demonstrated that 2,4,6-THBA dose-dependently inhibited CDKs 1, 2 and 4 and in silico studies identified key amino acids involved in these interactions. Interestingly, no significant CDK inhibition was observed with the structurally related compounds 3,4,5-trihydroxybenzoic acid (3,4,5-THBA) and phloroglucinol, suggesting that orientation of the functional groups and specific amino acid interactions may play a role in inhibition. We showed that cellular uptake of 2,4,6-THBA required the expression of functional SLC5A8, a monocarboxylic acid transporter. Consistent with this, in cells expressing functional SLC5A8, 2,4,6-THBA induced CDK inhibitory proteins p21Cip1 and p27Kip1 and inhibited cell proliferation. These findings, for the first time, suggest that the flavonoid metabolite 2,4,6-THBA may mediate its effects through a CDK- and SLC5A8-dependent pathway contributing to the prevention of CRC

Development of Soluble Inulin Microparticles as a Potent and Safe Vaccine Adjuvant and Delivery System

The goal of the present study is to develop a potent and safe vaccine adjuvant that can also stabilize vaccine formulations during lyophilization and storage. Inulin is a safe plant polysaccharide, and in its water soluble isoform, it is known to stabilize protein formulations during storage. However, soluble inulins have never been shown to stimulate the immune system. In this study, for the first time, we showed that water soluble inulins could be developed into vaccine adjuvants by formulating as antigen encapsulated microparticles. A method was developed to prepare soluble inulin microparticles (sIMs) with high encapsulation efficiency (∼75%) and loading (∼75 μg/mg) of the antigen. When immunized in mice, sIMs have generated robust Th2-type antibody titers (IgG1: 500,000) compared to unadjuvanted antigens (IgG1: 17,500) or alum adjuvanted antigens (IgG1: 80,000). <i>In vitro</i> assays showed that a higher proportion of antigen presenting cells (APC’s) have taken up the antigen when presented in sIMs versus in solution (99 % vs 22 %). In addition, the amount of antigen taken up per cell has also been enhanced by more than 25 times when antigen was presented in sIMs. Efficient uptake of the antigen by APCs through sIMS was attributed to the observed enhancement in the immune response by antigen loaded sIMs. The sIMs neither caused any granuloma/tissue damage at the injection site in mice nor were they toxic to the APC’s in cell culture. In conclusion, the current study has developed a safe, soluble inulin based vaccine adjuvant and delivery system

Inhibition of chaperone activity is a shared property of several Cu,Zn-superoxide dismutase mutants that cause amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron degeneration, paralysis, and death. Mutant Cu,Zn-superoxide dismutase (SOD1) causes a subset of ALS by an unidentified toxic property. Increasing evidence suggests that chaperone dysfunction plays a role in motor neuron degeneration in ALS. To investigate the relationship between mutant SOD1 expression and chaperone dysfunction, we measured chaperone function in central nervous system tissue lysates from normal mice and transgenic mice expressing human SOD1 variants. We observed a significant decrease in chaperone activity in tissues from mice expressing ALS-linked mutant SOD1 but not control mice expressing human wild type SOD1. This decrease was detected only in the spinal cord, became apparent by 60 days of age (before the onset of muscle weakness and significant motor neuron loss), and persisted throughout the late stages. In addition, this impairment of chaperone activity occurred only in cytosolic but not in mitochondrial and nuclear fractions. Furthermore, multiple recombinant human SOD1 mutants with differing biochemical and biophysical properties inhibited chaperone function in a cell-free extract of normal mouse spinal cords. Thus, mutant SOD1 proteins may impair chaperone function independent of gene expression in vivo, and this inhibition may be a shared property of ALS-linked mutant SOD1 proteins

S-nitrosothiol depletion in amyotrophic lateral sclerosis

Recent data suggest that either excessive or deficient levels of protein S-nitrosylation may contribute to disease. Disruption of S-nitrosothiol (SNO) homeostasis may result not only from altered nitric oxide (NO) synthase activity but also from alterations in the activity of denitrosylases that remove NO groups. A subset of patients with familial amyotrophic lateral sclerosis (ALS) have mutations in superoxide dismutase 1 (SOD1) that increase the denitrosylase activity of SOD1. Here, we show that the increased denitrosylase activity of SOD1 mutants leads to an aberrant decrease in intracellular protein and peptide S-nitrosylation in cell and animal models of ALS. Deficient S-nitrosylation is particularly prominent in the mitochondria of cells expressing SOD1 mutants. Our results suggest that SNO depletion disrupts the function and/or subcellular localization of proteins that are regulated by S-nitrosylation such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thereby contributes to ALS pathogenesis. Repletion of intracellular SNO levels with SNO donor compounds rescues cells from mutant SOD1-induced death. These results suggest that aberrant depletion of intracellular SNOs contributes to motor neuron death in ALS, and raises the possibility that deficient S-nitrosylation is a general mechanism of disease pathogenesis. SNO donor compounds may provide new therapeutic options for diseases such as ALS that are associated with deficient S-nitrosylation