Fucoidan Induces ROS-Dependent Apoptosis in 5637 Human Bladder Cancer Cells by Downregulating Telomerase Activity via Inactivation of the PI3K/Akt Signaling Pathway.

Preclinical Research Fucoidan, a sulfated polysaccharide, is a compound found in various species of seaweed that has anti-viral, anti-bacterial, anti-oxidant, anti-inflammatory, and immunomodulatory activities; however, the underlying relationship between apoptosis and anti-telomerase activity has not been investigated. Here, we report that fucoidan-induced apoptosis in 5637 human bladder cancer cells was associated with an increase in the Bax/Bcl-2 ratio, the dissipation of the mitochondrial membrane potential (MMP, Δψm), and cytosolic release of cytochrome c from the mitochondria. Under the same experimental conditions, fucoidan-treatment decreased hTERT (human telomerase reverse transcriptase) expression and the transcription factors, c-myc and Sp1. This was accompanied by decreased telomerase activity. Fucoidan-treatment also suppressed activation of the PI3K/Akt signaling pathway. Inhibition of PI3K/Akt signaling enhanced fucoidan-induced apoptosis and anti-telomerase activity. Meanwhile, fucoidan treatment increased the generation of intracellular ROS, whereas the over-elimination of ROS by N-acetylcysteine, an anti-oxidant, attenuated fucoidan-induced apoptosis, inhibition of hTERT, c-myc, and Sp1 expression, and reversed fucoidan-induced inactivation of the PI3K/Akt signaling pathway. Collectively, these data indicate that the induction of apoptosis and the inhibition of telomerase activity by fucoidan are mediated via ROS-dependent inactivation of the PI3K/Akt pathway. Drug Dev Res 78 : 37-48, 2017. © 2016 Wiley Periodicals, Inc.

Protective role of V-set and immunoglobulin domain-containing 4 expressed on kupffer cells during immune-mediated liver injury by inducing tolerance of liver T- and natural killer T-cells.

UNLABELLED: V-set and Ig domain-containing 4 (VSIG4, CRIg, or Z39Ig), a newly identified B7-related cosignaling molecule, is a complement receptor and a coinhibitory ligand that negatively regulates T-cell immunity. Despite its exclusive expression on liver Kupffer cells (KCs) that play key roles in liver tolerance, the physiological role of VSIG4 in liver tolerance remains undefined. Mice lacking VSIG4 had poor survival rates and severe liver pathology in a concanavalin A (ConA)-induced hepatitis (CIH) model, which could be prevented by adoptive transfer of VSIG4(+) KCs. The absence of VSIG4 rendered endogenous liver T- and natural killer T (NKT)-cells more responsive to antigen-specific stimulation and impaired tolerance induction in those cells against their cognate antigens. T-cell costimulation with VSIG4.Ig suppressed Th1-, Th2-, and Th17-type cytokine production and arrested the cell cycle at the G(0) /G(1) phase but did not induce apoptosis in vitro. VSIG4-mediated tolerance induction and cell-cycle arrest were further supported by down-regulation of G(1) phase-specific Cdk2, Cdk4, and Cdk6, and up-regulation of tolerance-inducing p27(KIP-1) in VSIG4.Ig-stimulated T-cells. Administration of soluble VSIG4.Ig to wildtype mice prevented CIH development and prolonged the survival of mice with established CIH. CONCLUSION: Collectively, our results suggest that VSIG4(+) KCs play a critical role in the induction and maintenance of liver T- and NKT-cell tolerance, and that modulation of the VSIG4 pathway using a VSIG4.Ig fusion protein may provide useful immunological therapies against immune-mediated liver injury including autoimmune hepatitis.

Gamma-aminobutyric acid (GABA)-mediated bone formation and its implications for anti-osteoporosis strategies: Exploring the relation between GABA and GABA receptors.

Osteoporosis is a significant global health concern, linked to reduced bone density and an increased fracture risk, with effective treatments still lacking. This study explored the potential of gamma-aminobutyric acid (GABA) and its receptors as a novel approach to promote osteogenesis and address osteoporosis. GABA concentrations up to 10 mM were well-tolerated by MC3T3-E1 preosteoblast, stimulating osteoblast differentiation and mineralization in a concentration- and time-dependent manner. In vivo experiments with zebrafish larvae demonstrated the ability of GABA to improve vertebral formation and enhanced bone density, indicating the potential therapeutic value for osteoporosis. Notably, GABA countered the adverse effects of prednisolone on vertebral formation, bone density, and osteogenic gene expression in zebrafish larvae, suggesting a promising therapeutic solution to counteract corticosteroid-induced osteoporosis. Moreover, our study highlighted the involvement of GABA receptors in mediating the observed osteogenic effects. By using GABAA, GABAB, and GABAC receptor antagonists, we demonstrated that blocking these receptors attenuated GABA-induced osteoblast differentiation and vertebral formation in both MC3T3-E1 cells and zebrafish larvae, underscoring the importance of GABA receptor interactions in promoting bone formation. In conclusion, these findings underscore the osteogenic potential of GABA and its ability to mitigate the detrimental effects of corticosteroids on bone health. Targeting GABA and its receptors could be a promising strategy for the development of novel therapeutic interventions to address osteoporosis. However, further investigations are warranted to fully elucidate the underlying molecular mechanism of GABA and its clinical applications in treating osteoporosis.

Acertannin attenuates LPS-induced inflammation by interrupting the binding of LPS to the TLR4/MD2 complex and activating Nrf2-mediated HO-1 activation.

Acertannin (ACTN) is a polyphenol known for its powerful anticancer and antioxidant effects. However, its anti-inflammatory effects have not been investigated at the molecular levels. Therefore, to evaluate anti-inflammatory effects of ACTN and its signaling pathway, the expression of proinflammatory markers was measured in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Molecular docking predicted the binding site of ACTN to the TLR4/MD2 complex. Moreover, in LPS-microinjected zebrafish, we investigated whether ACTN reduces nitric oxide and reactive oxygen species (ROS) production. ACTN significantly attenuated LPS-induced proinflammatory cytokines and mediators by inhibiting nuclear factor-kappa B (NF-κB) activation. ACTN also reduced LPS-induced ROS production and activated nuclear factor E2-related factor 2 and heme oxygenase-1 (HO-1). In addition, zinc protoporphyrin, an HO-1 inhibitor, markedly abolished the anti-inflammatory and antioxidant effects of ACTN in LPS-stimulated zebrafish larvae. Moreover, molecular docking predictions verified that ACTN forms a conventional hydrogen bond with LYS91 in myeloid differentiation factor-2 (MD2) and interrupts LPS binding to the Toll-like receptor 4 (TLR4)/MD2 complex. In addition, ACTN forms many non-covalent bonds, such as π-π stacking, π-alkyl, unfavorable donor-donor, and van der Waals interactions, with the TLR4/MD2 complex. Furthermore, the binding of ACTN to the TLR4/MD2 complex inhibited the recruitment of intracellular adaptor proteins, including myeloid differentiation primary response 88 and interleukin-1 receptor-associated kinase 4, and consequently attenuated NF-κB-mediated inflammatory responses. The conclusion of this study is that ACTN is a potent anti-inflammatory agent in LPS-mediated inflammation, such as endotoxemia.

In vitro and in vivo anti-tumor efficacy of krill oil against bladder cancer: Involvement of tumor-associated angiogenic vasculature.

Krill oil (KO) obtained from Euphausia superba is nutrient-rich and has a positive effect on human health. Here, we explored the efficacy of KO in inhibiting tumor progression and tumor vascularization. KO (100-300 µg/mL) repressed the proliferation of bladder tumor cell lines MBT-2 and T24. Treatment of cells with KO raised cell-cycle arrest at G0/G1-phase via modulation of positive regulators and negative regulators in bladder cancer cells. KO treatment regulated phosphorylation of proteins involved in PI3K/AKT and MAPK signaling pathways, including ERK, JNK, and p38 MAPK. Additionally, KO hampered the invasion and migration of both cell lines via reduction of MMP-9 expression levels by disrupting transcriptional binding of Sp-1, AP-1, and NF-κB motifs. Moreover, in animal studies, KO (150-300 mg/kg) significantly decreased tumor growth in xenograft mice bearing T24 tumor cells. No significant toxic effects were observed in acute toxicity tests, including biological analysis and H&E staining. The reduced level of CD31 expression in KO-treated tumor tissues prompted us to investigate the effect of KO on tumor angiogenesis. KO (5-40 µg/mL) treatment impeded VEGF-induced capillary tube formation and proliferation by inhibiting VEGFR2-modulated eNOS/AKT/ERK1/2 signaling axis in HUVECs. Treatment of HUVECs with KO inhibited VEGF-stimulated migration and invasion by reducing MMP-2 expression level. VEGF-driven sprouting capacity of neo-microvessels was suppressed in the presence of KO (20-40 µg/mL), as determined via an ex vivo aortic ring assay. Our results indicated that KO can regulate both tumor growth and tumor-associated angiogenesis via a novel mechanism. Thus, KO may be a promising antitumor candidate, potentially useful to prevent or treat bladder cancer.

Antiproliferative activity of fucoidan was associated with the induction of apoptosis and autophagy in AGS human gastric cancer cells.

Fucoidan, a sulfated polysaccharide purified from brown algae, possesses a variety of pharmacologic effects, including antiinflammatory, antioxidant, and anticancer properties; however, the underlying action mechanisms are not completely understood. This study investigated the possible mechanisms through which fucoidan exerts its antiproliferative action in cultured AGS human gastric adenocarcinoma cells. We found that fucoidan effectively inhibits the growth of AGS cells by inducing autophagy, as well as apoptosis. Apoptosis by fucoidan treatment was associated with the downregulation of antiapoptotic Bcl-2 and Bcl-xL expression, loss of mitochondrial membrane potential, activation of caspases, and concomitant degradation of poly-(ADP-ribose) polymerase protein. In addition, the morphological study indicated a characteristic finding of autophagy, such as the formation of autophagosomes in fucoidan-treated AGS cells. Furthermore, markers of autophagy, namely, the conversion of microtubule-associated protein light chain 3 (LC3)-I to LC3-II and increased beclin-1 accumulation, were observed. Overall, the present data suggest that fucoidan induces apoptotic and autophagic cell death, and both apoptotic and autophagic mechanisms contribute to the fucoidan-induced AGS cell death.

Sensitization of vascular smooth muscle cell to TNF-alpha-mediated death in the presence of palmitate.

Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-alpha-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokine did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-alpha-mediated nuclear factor kappa B (NF-kappaB) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-kappaB subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-kappaB predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-alpha-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular consequences of such patients with elevated levels of FFAs as diabetics and obese individuals via the triggering of receptor-mediated death pathways of VSMC.