Comparative In Vitro Toxicology of Novel Cytoprotective Short-Chain Naphthoquinones.

Short-chain quinones (SCQs) have been identified as potential drug candidates against mitochondrial dysfunction, which largely depends on the reversible redox characteristics of the active quinone core. We recently identified 11 naphthoquinone derivatives, 1-11, from a library of SCQs that demonstrated enhanced cytoprotection and improved metabolic stability compared to the clinically used benzoquinone idebenone. Since the toxicity properties of our promising SCQs were unknown, this study developed multiplex methods and generated detailed toxicity profiles from 11 endpoint measurements using the human hepatocarcinoma cell line HepG2. Overall, the toxicity profiles were largely comparable across different assays, with simple standard assays showing increased sensitivity compared to commercial toxicity assays. Within the 11 naphthoquinones tested, the L-phenylalanine derivative 4 consistently demonstrated the lowest toxicity across all assays. The results of this study not only provide useful information about the toxicity features of SCQs but will also enable the progression of the most promising drug candidates towards their clinical use.

Amide linked redox-active naphthoquinones for the treatment of mitochondrial dysfunction.

Naphthoquinones have been investigated as potential therapeutic molecules for neurodegenerative disorders, which is largely based on their anti-oxidative potential. However, a theoretical framework for the pleiotropic protective effects of naphthoquinone derivatives is largely missing. We synthesized a library of novel short chain 2,3-disubstituted naphthoquinone derivatives and measured their redox characteristics to identify a potential connection with their biological activity. Using two cell lines with different reducing potential, the compounds were tested for their inherent toxicity, acute rescue of ATP levels and cytoprotective activity. For the first time, a structure-activity-relationship for naphthoquinones has been established. Our results clearly demonstrate that it is the group on the alkyl side chain and not solely the redox characteristics of the naphthoquinone unit or lipophilicity that determines the extent of cytoprotection by individual compounds. From this, we developed a number of amide containing naphthoquinones with superior activity in ATP rescue and cell viability models compared to the clinically used benzoquinone idebenone.

Pathway Analysis of Fucoidan Activity Using a Yeast Gene Deletion Library Screen.

Fucoidan, the sulfated fucose-rich polysaccharide derived from brown macroalgae, was reported to display some anti-cancer effects in in vitro and in vivo models that included apoptosis and cell cycle arrest. The proposed mechanisms of action involve enhanced immune surveillance and direct pro-apoptotic effects via the activation of cell signaling pathways that remain largely uncharacterized. This study aimed to identify cellular pathways influenced by fucoidan using an unbiased genetic approach to generate additional insights into the anti-cancer effects of fucoidan. Drug⁻gene interactions of Undaria pinnatifida fucoidan were assessed by a systematic screen of the entire set of 4,733 halpoid Saccharomyces cerevsiae gene deletion strains. Some of the findings were confirmed using cell cycle analysis and DNA damage detection in non-immortalized human dermal fibroblasts and colon cancer cells. The yeast deletion library screen and subsequent pathway and interactome analysis identified global effects of fucoidan on a wide range of eukaryotic cellular processes, including RNA metabolism, protein synthesis, sorting, targeting and transport, carbohydrate metabolism, mitochondrial maintenance, cell cycle regulation, and DNA damage repair-related pathways. Fucoidan also reduced clonogenic survival, induced DNA damage and G1-arrest in colon cancer cells, while these effects were not observed in non-immortalized human fibroblasts. Our results demonstrate global effects of fucoidan in diverse cellular processes in eukaryotic cells and further our understanding about the inhibitory effect of Undaria pinnatifida fucoidan on the growth of human cancer cells.

Cost-effective three-dimensional printing of visibly transparent microchips within minutes.

One-step fabrication of transparent three-dimensional (3D) microfluidic to millifluidic devices was demonstrated using a commercial 3D printer costing $2300 with 500 mL of clear resin for $138. It employs dynamic mask projection stereolithography, allowing fast concept-to-chip time. The fully automated system allows fabrication of models of up to 43 mm × 27 mm × 180 mm (x × y × z) at printing speeds of 20 mm/h in height regardless of the design complexity. The minimal cross sectional area of 250 µm was achieved for monolithic microchannels and 200 µm for positive structures (templates for soft lithography). The colorless resin's good light transmittance (>60% transmission at wavelengths of >430 nm) allows for on-chip optical detection, while the electrically insulating material allows electrophoretic separations. To demonstrate its applicability in microfluidics, the printer was used for the fabrication of a micromixer, a gradient generator, a droplet extractor, and a device for isotachophoresis. The mixing and gradient formation units were incorporated into a device for analysis of nitrate in tap water with standard addition as a single run and multiple depth detection cells to provide an extended linear range.

Cytotoxic T cell adoptive immunotherapy as a treatment for nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). We assess the safety and tolerability of adoptive transfer of autologous cytotoxic T lymphocytes (CTLs) specific for the EBV latent membrane protein (LMP) in a patient with recurrent NPC. After infusion, the majority of pulmonary lesions were no longer evident, although the primary tumor did not regress.

Novel approach to the formulation of an Epstein-Barr virus antigen-based nasopharyngeal carcinoma vaccine.

Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.

Optimization of LMP-specific CTL expansion for potential adoptive immunotherapy in NPC patients.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV) positive in all undifferentiated cases, expressing the latency II phenotype of latent membrane proteins (LMPs) 1 and 2, in addition to EBV nuclear antigen (EBNA) 1. Several studies have attempted to treat NPC with EBV-specific cytotoxic T lymphocyte (CTL) with a partial response. To improve this therapy, there is a need to expand CTL targeted to the latency II antigens of EBV, rather than the immunodominant EBV nuclear antigens 3-6 peptides typically expanded by lymphoblastoid cells. In order to maximize the expansion of LMP-specific CTL in vitro for use in adoptive immunotherapy of nasopharyngeal carcinoma patients, we used lymphoblastoid cell lines coated with synthetic peptides corresponding to CTL determinants from the LMP proteins. We investigated several issues pertaining to the expansion of an immunologically weak CTL response, including peptide and interleukin-2 concentration, and screening assays for selecting the optimal peptide for use in expansion of LMP-specific CTL. Although screening of ex vivo peripheral blood mononuclear cells did not prove to be useful in the selection of an LMP peptide for use in CTL cultures, the peptide and interleukin-2 concentrations were critical for the maximum expansion of CTL. Therefore, it is imperative that stimulation protocols are optimized for the expansion of LMP-specific CTL.