Transcutaneous Carbon Dioxide Induces Mitochondrial Apoptosis and Suppresses Metastasis of Oral Squamous Cell Carcinoma In Vivo

Squamous cell carcinoma (SCC) is the main histological type of oral cancer. Its growth rate and incidence of metastasis to regional lymph nodes is influenced by various factors, including hypoxic conditions. We have previously reported that transcutaneous CO₂ induces mitochondrial apoptosis and decreases lung metastasis by reoxygenating sarcoma cells. However, previous studies have not determined the sequential mechanism by which transcutaneous CO₂ suppresses growth of epithelial tumors, including SCCs. Moreover, there is no report that transcutaneous CO₂ suppresses lymphogenous metastasis using human cell lines xenografts. In this study, we examined the effects of transcutaneous CO₂ on cancer apoptosis and lymphogenous metastasis using human SCC xenografts. Our results showed that transcutaneous CO₂ affects expressions of PGC-1α and TFAM and protein levels of cleavage products of caspase-3, caspase-9 and PARP, which relatives mitochondrial apoptosis. They also showed that transcutaneous CO₂ significantly inhibits SCC tumor growth and affects expressions of HIF-1α, VEGF, MMP-2 and MMP-9, which play essential roles in tumor angiogenesis, invasion and metastasis. In conclusion, transcutaneous CO₂ suppressed tumor growth, increased mitochondrial apoptosis and decreased the number of lymph node metastasis in human SCC by decreasing intra-tumoral hypoxia and suppressing metastatic potential with no observable effect in vivo. Our findings indicate that transcutaneous CO₂ could be a novel therapeutic tool for treating human SCC.     

Enhancement of mice susceptibility to infection with Listeria monocytogenes by the treatment of morphine

The effect of morphine on the susceptibility of BALB/c mice to diarrheagenic Escherichia coli, Shigella flexneri, Listeria monocytogenes, Salmonella Enteritidis, Yersinia enterocolitica, was examined via the intraperitoneal inoculation. Morphine treatment increased the susceptibility to S. Enteritidis and L. monocytogenes, resulting in bacteremia and central nervous system (CNS) invasion (for L. monocytogenes), while the infection with other bacteria did not show the systemic dissemination in the morphinetreated mice. Notably, L. monocytogenes infection caused 100% mortality with a mean survival time (MST) of 1.3 days in morphine-treated mice, but untreated mice did not die. The present data suggested that individuals using heroin or treated with morphine derivatives might be at high risk for listeriosis, especially those who are immunocompromised. Recent increasing consumption of morphine may propose the necessity for further epidemiological surveillance on infectious diseases.     

Synthesis and structure-activity relationship of tetrahydropyrazolopyrimidine derivatives--a novel structural class of potent calcium-sensing receptor antagonists

A series of novel tetrahydropyrazolopyrimidine derivatives containing an adamantyl group were synthesized and evaluated as potential calcium-sensing receptor (CaSR) antagonists. After chemical modification of 9a, which was identified as a hit compound in a random screening of CaSR antagonist assay, 7,7-dimethyl derivative 16c was found to be the most active compound of this new series (IC(50)=10nM). We report the synthesis of this series and their biological activities and structure-activity relationship.     

Design,synthesis and structure–activity relationships of 1,3,4-oxadiazole derivatives as novel inhibitors of glycogen synthase kinase-3β

Glycogen synthase kinase-3β (GSK-3β) is implicated in abnormal hyperphosphorylation of tau protein and its inhibitors are expected to be a promising therapeutic agents for the treatment of Alzheimer’s disease. Here we report design, synthesis and structure–activity relationships of a novel series of oxadiazole derivatives as GSK-3β inhibitors. Among these inhibitors, compound 20x showed highly selective and potent GSK-3β inhibitory activity in vitro and its binding mode was determined by obtaining the X-ray co-crystal structure of 20x and GSK-3β.     

1,4‐butanediyl‐bismethanethiosulfonate (BMTS) induces apoptosis through reactive oxygen species‐mediated mechanism

Although methane sulfonate compounds are widely used for the protein modification for their selectivity of thiol groups in proteins, their intracellular signaling events have not yet been clearly documented. This study demonstrated the methane sulfonate chemical 1,4‐butanediyl‐bismethanethiosulfonate (BMTS)‐induced cascades of signals that ultimately led to apoptosis of Jurkat cells. BMTS induced apoptosis through fragmentation of DNA, activation of caspase‐9 and caspase‐3, and downregulation of Bcl‐2 protein with reduction of mitochondrial membrane potential. Moreover, BMTS intensely and transiently induced intracellular reactive oxygen species (ROS) production and ROS produced by BMTS was mediated through mitochondria. We also found that a reducing agent dithiothreitol (DTT) and an anti‐oxidant N‐acetyl cysteine (NAC) inhibited BMTS‐mediated caspase‐9 and ‐3 activation, ROS production and induction of Annexin V/propidium iodide double positive cells, suggesting the involvement of ROS in the apoptosis process. Therefore, this study further extends our understanding on the basic mechanism of redox‐linked apoptosis induced by sulfhydryl‐reactive chemicals. J. Cell. Biochem. 108: 1059–1065, 2009. © 2009 Wiley‐Liss, Inc.     

Localization by scanning immunoelectron microscopy of triosephosphate isomerase,the molecules responsible for contact‐mediated killing of Cryptococcus,on the surface of Staphylococcus

In our previous studies, TPI were found to be the molecules responsible for contact‐killing of C. neoformans by S. aureus cells. Since TPI is a glycolytic protein that functions in the cytoplasm, evidence that TPI is present on the surface of S. aureus was required. In the present study, the presence of TPI on the cell surface of S. aureus was demonstrated by agglutination test and scanning immunoelectron microscopy. Furthermore, TPI was found to be present at a lower density than protein A/G molecules on the surface of S. aureus.     

A redox‐linked novel pathway for arsenic‐mediated RET tyrosine kinase activation

We examined the biochemical effects of arsenic on the activities of RET proto‐oncogene (c‐RET protein tyrosine kinases) and RET oncogene (RET‐MEN2A and RET‐PTC1 protein tyrosine kinases) products. Arsenic activated c‐RET kinase with promotion of disulfide bond‐mediated dimerization of c‐RET protein. Arsenic further activated RET‐MEN2A kinase, which was already 3‐ to 10‐fold augmented by genetic mutation compared with c‐RET kinase activity, with promotion of disulfide bond‐mediated dimerization of RET‐MEN2A protein (superactivation). Arsenic also increased extracellular domain‐deleted RET‐PTC1 kinase activity with promotion of disulfide bond‐mediated dimerization of RET‐PTC1 protein. Arsenic increased RET‐PTC1 kinase activity with cysteine 365 (C365) replaced by alanine with promotion of dimer formation but not with cysteine 376 (C376) replaced by alanine. Our results suggest that arsenic‐mediated regulation of RET kinase activity is dependent on conformational change of RET protein through modulation of a special cysteine sited at the intracellular domain in RET protein (relevant cysteine of C376 in RET‐PTC1 protein). Moreover, arsenic enhanced the activity of immunoprecipitated RET protein with increase in thiol‐dependent dimer formation. As arsenic (14.2 µM) was detected in the cells cultured with arsenic (100 µM), direct association between arsenic and RET in the cells might modulate dimer formation. Thus, we demonstrated a novel redox‐linked mechanism of activation of arsenic‐mediated RET proto‐oncogene and oncogene products. J. Cell. Biochem. 110: 399–407, 2010. © 2010 Wiley‐Liss, Inc.     

Agaritine purified from Agaricus blazei Murrill exerts anti-tumor activity against leukemic cells

Background

Mushrooms of the genus Agaricus are a common folk remedy against carcinoma. The active ingredients, polysaccharides and protein-polysaccharide complexes containing β-glucan, have been isolated and shown to have indirect tumor-suppressing activity via an immunological activation.

Methods

The diffusible fraction of a hot-water extract of Agaricus blazei Murrill (ABM) powder was fractionated by HPLC based on the anti-tumor activity against leukemic cells in vitro. The structure of the anti-tumor substance was determined by NMR and MS analyses.

Results

We purified a tumorcidal substance from the diffusible fraction of ABM and identified it as agaritine, β-N-(γ-l(+)-glutamyl)-4-(hydroxymethyl) phenylhydrazine, having a molecular mass of 267 Da. This compound inhibited the proliferation of leukemic cell lines such as U937, MOLT4, HL60 and K562 with IC₅₀ values of 2.7, 9.4, 13.0, and 16.0 μg/mL, respectively, but showed no significant effect on normal lymphatic cells at concentrations up to 40 μg/mL. Although agaritine has been suspected of having genotoxic or carcinogenic properties, agaritine did not activate the umu gene of Salmonella, which reacts to carcinogens.

General significance

The results indicate that agaritine from ABM has direct anti-tumor activity against leukemic tumor cells in vitro. This is in contrast to the carcinogenic activity previously ascribed to this compound. Our results also show that this activity is distinct from that of β-glucan, which indirectly suppresses proliferation of tumor cells.     

Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions

Adiponectin plays a central role as an antidiabetic and antiatherogenic adipokine. AdipoR1 and AdipoR2 serve as receptors for adiponectin in vitro, and their reduction in obesity seems to be correlated with reduced adiponectin sensitivity. Here we show that adenovirus-mediated expression of AdipoR1 and R2 in the liver of Lepr(-/-) mice increased AMP-activated protein kinase (AMPK) activation and peroxisome proliferator-activated receptor (PPAR)-alpha signaling pathways, respectively. Activation of AMPK reduced gluconeogenesis, whereas expression of the receptors in both cases increased fatty acid oxidation and lead to an amelioration of diabetes. Alternatively, targeted disruption of AdipoR1 resulted in the abrogation of adiponectin-induced AMPK activation, whereas that of AdipoR2 resulted in decreased activity of PPAR-alpha signaling pathways. Simultaneous disruption of both AdipoR1 and R2 abolished adiponectin binding and actions, resulting in increased tissue triglyceride content, inflammation and oxidative stress, and thus leading to insulin resistance and marked glucose intolerance. Therefore, AdipoR1 and R2 serve as the predominant receptors for adiponectin in vivo and play important roles in the regulation of glucose and lipid metabolism, inflammation and oxidative stress in vivo.