Hyperthermia and associated changes in membrane fluidity potentiate P2X7 activation to promote tumor cell death.

Extracellular ATP (eATP) accumulation within the tumor microenvironment (TME) has the potential to activate purinergic signaling. The eATP evoked signaling effects bolster antitumor immune responses while exerting direct cytotoxicity on tumor cells and vascular endothelial cells, mediated at least in part through P2X7 receptors. Approaches to augment purinergic signaling in TME e.g. by ectonucleotidase CD39 blockade, and/or boosting P2X7 functional responses, might be used as immunomodulatory therapies in cancer treatment. In this study, we delineated the translatable strategy of hyperthermia to demonstrate impacts on P2X7 responsiveness to eATP. Hyperthermia (40°C) was noted to enhance eATP-mediated cytotoxicity on MCA38 colon cancer cells. Increased membrane fluidity induced by hyperthermia boosted P2X7 functionality, potentiating pore opening and modulating downstream AKT/PRAS40/mTOR signaling events. When combined with cisplatin or mitomycin C, hyperthermia and eATP together markedly potentiate cancer cell death. Our data indicate that clinically tolerable hyperthermia with modulated P2X7-purinergic signaling will boost efficacy of conventional cancer treatments.

P2X7 integrates PI3K/AKT and AMPK-PRAS40-mTOR signaling pathways to mediate tumor cell death.

BACKGROUND: Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death. CONCLUSIONS: Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.

CD39 modulates hematopoietic stem cell recruitment and promotes liver regeneration in mice and humans after partial hepatectomy.

OBJECTIVE: To study molecular mechanisms involved in hematopoietic stem cell (HSC) mobilization after liver resection and determine impacts on liver regeneration. BACKGROUND: Extracellular nucleotide-mediated cell signaling has been shown to boost liver regeneration. Ectonucleotidases of the CD39 family are expressed by bone marrow-derived cells, and purinergic mechanisms might also impact mobilization and functions of HSC after liver injury. METHODS: Partial hepatectomy was performed in C57BL/6 wild-type, Cd39 ectonucleotidase-null mice and in chimeric mice after transplantation of wild-type or Cd39-null bone marrow. Bone marrow-derived HSCs were purified by fluorescence-activated cell sorting and administered after hepatectomy. Chemotactic studies were performed to examine effects of purinergic receptor agonists and antagonists in vitro. Mobilization of human HSCs and expression of CD39 were examined and linked to the extent of resection and liver tests. RESULTS: Subsets of HSCs expressing Cd39 are preferentially mobilized after partial hepatectomy. Chemotactic responses of HSCs are increased by CD39-dependent adenosine triphosphate hydrolysis and adenosine signaling via A2A receptors in vitro. Mobilized Cd39 HSCs boost liver regeneration, potentially limiting interleukin 1ß signaling. In clinical studies, mobilized human HSCs also express CD39 at high levels. Mobilization of HSCs correlates directly with the restoration of liver volume and function after partial hepatectomy. CONCLUSIONS: We demonstrate CD39 to be a novel HSC marker that defines a functionally distinct stem cell subset in mice and humans. HSCs are mobilized after liver resection, limit inflammation, and boost regeneration in a CD39-dependent manner. These observations have implications for monitoring and indicate future therapeutic avenues.

Percutaneous transhepatic variceal embolization combined with endoscopic ligation for the prevention of variceal rebleeding.

OBJECTIVE: To compare the efficiency of percutaneous transhepatic variceal embolization (PTVE) plus endoscopic variceal ligation (EVL) with EVL alone in the treatment of esophageal variceal bleeding. METHODS: Cirrhotic patients with recent esophageal variceal bleeding from January 2007 to December 2011 were collected and assigned to PTVE + EVL (N = 41) or EVL (N = 47) groups. We performed chart reviews and prospective follow-up to determine variceal rebleeding, recurrence of varices and survival. RESULTS: During the follow-up period, recurrence of esophageal varices (EV) occurred in 8 patients (19.5%) in the PTVE + EVL group and 23 (48.9%) in the EVL group (P = 0.004). The time to recurrence of EV was 9.2 ± 2.7 months and 4.9 ± 2.1 months, respectively. Three patients (7.3%) in the PTVE + EVL group and 12 (25.5%) in the EVL group experienced rebleeding from all sources (P = 0.023). One patient (2.4%) in the PTVE + EVL group and 7 (14.9%) in the EVL group experienced rebleeding from EV (P = 0.024). Multivariate Cox analysis indicated that the treatment method was the only predictor of rebleeding. There was no significant difference in the survival rate between the two groups. CONCLUSION: With adequate and permanent obliteration of EV and their feeding veins, the combination of PTVE with cyanoacrylate and EVL is more effective than EVL alone in the prevention and treatment of EV recurrence and rebleeding.

Disordered purinergic signaling and abnormal cellular metabolism are associated with development of liver cancer in Cd39/ENTPD1 null mice.

UNLABELLED: Liver cancer is associated with chronic inflammation, which is linked to immune dysregulation, disordered metabolism, and aberrant cell proliferation. Nucleoside triphosphate diphosphohydrolase-1; (CD39/ENTPD1) is an ectonucleotidase that regulates extracellular nucleotide/nucleoside concentrations by scavenging nucleotides to ultimately generate adenosine. These properties inhibit antitumor immune responses and promote angiogenesis, being permissive for the growth of transplanted tumors. Here we show that Cd39 deletion promotes development of both induced and spontaneous autochthonous liver cancer in mice. Loss of Cd39 results in higher concentrations of extracellular nucleotides, which stimulate proliferation of hepatocytes, abrogate autophagy, and disrupt glycolytic metabolism. Constitutive activation of Ras-mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR)-S6K1 pathways occurs in both quiescent Cd39 null hepatocytes in vitro and liver tissues in vivo. Exogenous adenosine 5'-triphosphate (ATP) boosts these signaling pathways, whereas rapamycin inhibits such aberrant responses in hepatocytes. CONCLUSION: Deletion of Cd39 and resulting changes in disordered purinergic signaling perturb hepatocellular metabolic/proliferative responses, paradoxically resulting in malignant transformation. These findings might impact adjunctive therapies for cancer. Our studies indicate that the biology of autochthonous and transplanted tumors is quite distinct.

Vascular CD39/ENTPD1 directly promotes tumor cell growth by scavenging extracellular adenosine triphosphate.

Extracellular adenosine triphosphate (ATP) is known to boost immune responses in the tumor microenvironment but might also contribute directly to cancer cell death. CD39/ENTPD1 is the dominant ectonucleotidase expressed by endothelial cells and regulatory T cells and catalyzes the sequential hydrolysis of ATP to AMP that is further degraded to adenosine by CD73/ecto-5'-nucleotidase. We have previously shown that deletion of Cd39 results in decreased growth of transplanted tumors in mice, as a result of both defective angiogenesis and heightened innate immune responses (secondary to loss of adenosinergic immune suppression). Whether alterations in local extracellular ATP and adenosine levels as a result of CD39 bioactivity directly affect tumor growth and cytotoxicity has not been investigated to date. We show here that extracellular ATP exerts antitumor activity by directly inhibiting cell proliferation and promoting cancer cell death. ATP-induced antiproliferative effects and cell death are, in large part, mediated through P2X(7) receptor signaling. Tumors in Cd39 null mice exhibit increased necrosis in association with P2X(7) expression. We further demonstrate that exogenous soluble NTPDase, or CD39 expression by cocultured liver sinusoidal endothelial cells, stimulates tumor cell proliferation and limits cell death triggered by extracellular ATP. Collectively, our findings indicate that local expression of CD39 directly promotes tumor cell growth by scavenging extracellular ATP. Pharmacological or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy in cancer management.

A facile template approach to high-nuclearity silver(I) alkynyl clusters.

Peanut clusters: Anion templates are used in a facile approach for the synthesis of high-nuclearity silver clusters. The cluster nuclearity can be controlled by adjusting the size of the templating anions and by using different alkynyl ligands. The largest silver alkynyl cluster, which consists of 35 silver(I) centers in the shape of a peanut, has been prepared by using chromate anions as templates (see picture).

High-nuclearity silver clusters templated by carbonates generated from atmospheric carbon dioxide fixation.

Two high-nuclearity silver clusters have been prepared by reacting AgC(2)Bu(t) with AgBF(4) (or AgOTf) in the presence of TMEDA in air. The reaction takes up atmospheric CO(2) to give CO(3)(2-), which functions as a template for the formation of the cage compounds, whose nuclearities depend on the particular counteranions employed.

Snowman-like silver alkynyl cluster consolidated by templating chloride and peripheral trifluoroacetates.

A novel nonadecanuclear silver double cage was isolated, in which a square antiprism and a pentacapped pentagonal prism templated by a chloride ion share a tetragonal face to form a snowman-like cluster that is held together by bridging alkynyl groups and trifluoroacetates.

[Effects of interferon-gamma and tumor necrosis factor-alpha on the fertilizing capacity of human sperm and their mechanisms].

OBJECTIVE: To investigate the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-gamma) on the sperm acrosin activity and the rate of acrosome reaction and to probe into their mechanisms. METHODS: Thirty-six nearly normal semen samples were treated with IFN-gamma and/or TNF-alpha after isolated by 75% Percoll. The sperm acrosin activity was tested by the method of BAEE/ADH Unity, the rate of acrosome reaction observed by Triple-stain technique, the NO concentration measured by HPLC and the activities of Na+ -K+ -ATPase, Ca2+ -ATPase and SOD assayed by kit method. RESULTS: Both IFN-gamma and TNF-gamma could decrease sperm acrosin activity and acrosome reaction (P < 0.05 or P < 0.01). TNF-alpha showed stronger inhibiting effect, IFN-gamma markedly reduced the activities of Na+ -K+ -ATPase, Ca2+ -ATPase and SOD in sperm (P < 0.01), and their synergistic action was weaker. However TNF-alpha produced hardly any effect on Na+ -K+ -ATPase and Ca2+ -ATPase. The NO concentration in sperm was significantly increased by IFN-gamma and/or TNF-alpha (P < 0.01). CONCLUSION: IFN-gamma and TNF-alpha have some inhibiting effect on sperm acrosin activity and the rate of acrosome reaction, which could be attributed to their influence on the activities of Na+ -K+ -ATPase, Ca2+ -ATPase and SOD, the NO concentration and so on.

Role of polyamines in myocardial ischemia/reperfusion injury and their interactions with nitric oxide.

Polyamines (putrescine, spermidine, and spermine) are present in all higher eukaryotic cells and are essential for cell growth, differentiation and apoptosis. Sharing common precursor with polyamines, nitric oxide (NO) is associated with myocardial ischemia/reperfusion injury by the generation of peroxynitrite. Although polyamines have been implicated in tissue ischemia injury, their metabolism and interactions with NO in myocardial ischemia/reperfusion injury have not been fully understood. In our experiment, when Langendorff perfused rat hearts were subjected to 40 min ischemia without reperfusion, both ornithine decarboxylase (ODC) and Spermidine/spermine N(1)-acetyltransferase (SSAT) activities were up-regulated and putrescine accumulated. While after reperfusion, ODC activity decreased and SSAT activity increased, resulting in putrescine accumulation and decreased spermidine and spermine. Meanwhile NO content was increased. In addition, sodium nitroprusside (SNP, a NO donor) decreased ODC activity in cardiac tissue homogenate but increased SSAT activity in a dose-dependent manner. Pre-treatment of isolated heart with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME, an inhibitor of NO synthase) increased ODC activity. Exogenous spermine (1 mM) administration prior to ischemia prevented spermine decrease, reduced cardiac myocyte necrosis and apoptosis, and promoted the recovery of cardiac function after ischemia/reperfusion. These results suggest that acute heart ischemia activates myocardial polyamine stress response characterized by increased ODC and SSAT activities and accumulation of putrescine. Ischemia/reperfusion disturbs polyamine metabolism, and the loss of spermine might be associated with NO increase and thereby influences myocardial cell viability. Exogenous spermine may protect the hearts from myocardial ischemia/reperfusion injury.

[The changing trends of expression of VEGF and its relationship with the levels of NO in rat corpus luteum of pseudopregnant].

AIM: The expression of VEGF in rat ovaries corpus luteum and its expression pattern were observed to investigate the effect of VEGF on luteal formation and regression. METHODS: The model of immature rat of pseudopregnant was established using pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), the expression of VEGF in corpus luteum was detected by immunohistochemistry, the levels of VEGF in corpus luteum was measured by ELISA, and the levels of NO in corpus luteum was measured by chemistry assay. RESULTS: VEGF expressed weakly in rat corpus luteum on the day 1, and enhanced gradually from day 3 to day 5, then went up to the peak on the day 7, and maintained to day 9. On the day 11, the expression of VEGF began to decrease. The levels of VEGF were similar to the expression of VEGF. The levels of NO appeared like double wave. The levels increased gradually from day 1 to day 5, and peaked on the day 7, then decreased on the day 9, while lightly increased on the day 11, and showed significant increase and reached the highest on the day 13, then decreased the lowest on the day 15. CONCLUSION: There is a intimate temporal relationship between the expression of VEGF and angiogenesis in corpus luteum, VEGF may play a role in luteum formation by improving angiogenesis mediated by NO, NO may play a role in luteum regression as a luteolytic during the late luteal phase.

[Effect of gamma-aminobutyric acid on the sperm acrosin activity].

OBJECTIVES: To investigate the effect of gamma-aminobutyric acid (GABA) on the sperm acrosin activity in normal men and positive antisperm antibody (AsAb) men. METHODS: Sperm acrosin activity was detected by BAEE/ADH method. RESULTS: GABA could increase the sperm acrosin activity in normal and AsAb positive patients (P < 0.01). The results also indicated that GABA significantly increased Na(+)-K(+)-ATPase activity (P < 0.01), Ca(2+)-ATPase activity (P < 0.05) and SOD activity (P < 0.01) of sperm. CONCLUSIONS: The results demonstrated that GABA could influence the sperm acrosin activity.