The phosphatidylinositol-3-kinase (PI3K) / protein kinase B (AKT) signal transduction pathway is commonly misregulated in lymphoma and associated with tumorigenesis and enhanced resistance to radiotherapy. Curcumin has been shown to inhibit the PI3K/AKT signal transduction pathway in several tumor models. In this study, we found that curcumin inhibits constitutive and radiation-induced expression of the PI3K/AKT pathway and its downstream regulator nuclear factor kappaB (NF-κB) in human Burkitt’s lymphoma, a high-grade non-Hodgkin’s lymphoma (NHL). We further demonstrated that the blockage of radiation-induced activation of the PI3K/AKT pathway and its downstream regulator NF-κB by either curcumin or specific PI3/AKT inhibitors (LY294002 for PI3K or SH-5 for AKT) enhance apoptosis in three human Burkitt’s lymphoma cell lines (Namalwa, Ramos, and Raji) that were treated with ionizing radiation. However, no synergic effect on radiation-induced apoptosis was found in the cells co-pretreated with curcumin combined with LY294002 or curcumin combined with SH-5. The results from this study suggest that curcumin might play an important role in radiotherapy of high-grade NHL through inhibition of the PI3K/AKT-dependent NF-κB pathway.
To clarify a role of substance P (SP) in an endogenous pain control mechanism involving the rat striatum, striatal SP release was measured over time by microdialysis following intraplantar injection of 0.4% formalin. A slow-onset but significant increase of SP and neurokinin 1 receptor (NK1R) internalization in the contralateral striatum were observed following the second phase of formalin-induced nociceptive behaviors. Moreover, 60 min after formalin injection, preprotachykinin-A, the SP mRNA, and the immediate early gene cFOS were upregulated in the contralateral striatum. Continuous infusion of SP into the striatum by reverse microdialysis attenuated formalin-induced second phase, but not the first phase, nociceptive behaviors, and hind paw mechanical allodynia. Moreover, these anti-nociceptive effects of SP were completely inhibited by co-treatment with the NK1R antagonist CP96345. Acute microinjection of SP, however, at a dose that was similar to the total dose of SP continuously infused into the striatum, did not affect formalin-induced nociceptive behaviors. These data indicate that striatal NK1R activation leads to pain suppression rather than facilitation. Furthermore, volume transmission of SP in the striatum appears to be indispensable in the mechanism of pain control. Modulation of striatal NK1Rs could prove to be a useful method of inducing analgesia.
Genipin, an active constituent of Gardenia fruit, has been reported to show an anti-tumor effect in several cancer cell systems. Here, we demonstrate how genipin exhibits a strong apoptotic cell death effect in human non–small-cell lung cancer H1299 cells. Genipin-mediated decrease in cell viability was observed through apoptosis as demonstrated by induction of a sub-G1 peak through flow cytometry, DNA fragmentation measured by TUNEL assay, and cleavage of poly ADP-ribose-polymerase. During genipin-induced apoptosis, the mitochondrial execution pathway was activated by caspase-9 and -3 activation as examined by a kinetic study, cytochrome c release, and a dose-dependent increase in Bax/Bcl-2 ratio. A search for the downstream pathway reveals that genipin-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2. SB203580, a p38MAPK inhibitor, markedly blocked the formation of TUNEL-positive apoptotic cells in genipin-treated cells. Besides, the interference of p38MAPK inhibited Bax expression and cytochrome c release. Altogether, our observations imply that genipin causes increased levels of Bax in response to p38MAPK signaling, which results in the initiation of mitochondrial death cascade, and therefore it holds promise as a potential chemotherapeutic agent for the treatment of H1299 cells.
Mosapride citrate hydrate (mosapride) has been known to act as a 5-HT4 agonist and to enhance gastric emptying. However, its mode of action, such as time course and dosage effect, on gastric emptying has not been clarified. This study aimed to clarify these points by the breath test using [1-13C]acetic acid in conscious rats. Mosapride significantly and dose-dependently enhanced the gastric emptying increased Cmax and AUC120 min at doses between 0.1 and 3 mg/kg. Pre-treatment with GR113808 (5-HT4 antagonist) significantly attenuated the enhancement of gastric emptying by mosapride. On the contrary, at a dose of 30 mg/kg, mosapride significantly inhibited the gastric emptying. The major metabolite (M1: 5-HT3 receptor antagonist) significantly inhibited gastric emptying at doses of 19.2 and 64.1 mg/kg (equimolar to 30 and 100 mg/kg of mosapride, respectively), suggesting that the inhibitory effect by mosapride may be caused at least in part by the 5-HT3 receptor antagonistic effect of M1. These findings show that mosapride has dual role on the gastric emptying and may support the usefulness of mosapride for the therapy of postprandial distress syndrome such as early satiation and postprandial fullness.
Osteoclasts (OCLs) are multinucleated bone-resorbing cells that are differentiated by stimulation with receptor activator of nuclear factor kappa-B ligand (RANKL) and macrophage colony-stimulating factor. We recently demonstrated that regulation of heme-oxygenase 1 (HO-1), a stress-induced cytoprotective enzyme, also functions in OCL differentiation. In this study, we investigated effects of fisetin, a natural bioactive flavonoid that has been reported to induce HO-1 expression, on the differentiation of macrophages into OCLs. Fisetin inhibited the formation of OCLs in a dose-dependent manner and suppressed the bone-resorbing activity of OCLs. Moreover, fisetin-treated OCLs showed markedly decreased phosphorylation of extracellular signal-regulated kinase, Akt, and Jun N-terminal kinase, but fisetin did not inhibit p38 phosphorylation. Fisetin up-regulated mRNA expression of phase II antioxidant enzymes including HO-1 and interfered with RANKL-mediated reactive oxygen species (ROS) production. Studies with RNA interference showed that suppression of NF-E2–related factor 2 (Nrf2), a key transcription factor for phase II antioxidant enzymes, rescued fisetin-mediated inhibition of OCL differentiation. Furthermore, fisetin significantly decreased RANKL-induced nuclear translocation of cFos and nuclear factor of activated T cells cytoplasmic-1 (NFATc1), which is a transcription factor critical for osteoclastogenic gene regulation. Therefore, fisetin inhibits OCL differentiation through blocking RANKL-mediated ROS production by Nrf2-mediated up-regulation of phase II antioxidant enzymes.
This study aimed to clarify the correlation between the estrous cycle and prevalence rate of endometriosis by sequential laparoscopy in Wistar-Imamichi female rats. The peritoneal implantation of endometrial tissue was performed in four estrous cycle rats (proestrus, estrus, metestrus, and diestrus). One week after implantation, the volume of the ectopic endometriosis was measured, and sequential laparoscopy was performed for 4 weeks to observe the prevalence rate. Five weeks after implantation, the volume of the ectopic endometriosis was measured again after laparoscopy. One week after implantation, the volume of endometriosis was significantly larger in proestrus and estrus rats than metestrus and diestrus rats. Prevalence rate was decreased with time. Five weeks after implantation, the prevalence rate and volume were higher and larger in the metestrus, diestrus, and estrus rats than in the proestrus rats. These results show that the estrous cycle affects the change of ectopic endometriosis. The decrease of prevalence rate was slow in metestrus, diestrus, and estrus rats as compared to that in proestrus rats. The volume of ectopic endometriosis showed little decrease with time when the endometrial tissue was implanted during the metestrus and diestrus portion of the cycle. Moreover, sequential laparoscopy made it possible to observe the prevalence rate of endometriosis.
Atrophic gastritis caused by infection with Helicobacter pylori is characterized by parietal cell loss, which is a main risk factor for gastric cancer. Parietal cells play a crucial role in the regulation of cell lineage maturation and proliferation in the gastric units. Among the classical cadherins, E-cadherin plays an important role not only in epithelial cell–cell connections, but also in the maintenance of epithelial polarity and gastric glandular architecture and regulation of cell proliferation. The aim of this study is to elucidate how parietal cells and E-cadherin are altered in gastritis with Helicobacter pylori infection. We studied the effects of Helicobacter pylori on gastric mucosal E-cadherin 2 weeks after inoculation and investigated the relationship between parietal cell loss and the amount of E-cadherin on parietal cells in Mongolian gerbils. The number of parietal cells and amount of staining of E-cadherin below the isthmus were investigated by immunohistochemistry. It was shown that a reduction in intercellular E-cadherin preceded the disappearance of parietal cells. The gastric glands where parietal cells were lost were replaced by mucus secreting cells without E-cadherin. These results suggest that Helicobacter pylori damaged E-cadherin on parietal cells and caused massive parietal cell loss, leading to the deregulation of gastric morphology.
Lung fibroblasts are responsible for collagen secretion during normal tissue repair and the development of fibrosis. Many other prostaglandins have been reported to regulate collagen synthesis in lung fibroblasts, but the role of prostaglandin D2 (PGD2) is unknown. In this study, we investigated the effect of PGD2 on type I collagen secretion in human lung fibroblasts. Pretreatment with PGD2 (0.1 - 10 μM, 1 h) significantly attenuated type I collagen secretion to the cell supernatant induced by transforming growth factor-β (TGF-β). Although an agonist on chemoattractant receptorhomologous molecule expressed on Th2 cells (CRTH2) did not have any effect, the prostanoid DP-receptor agonist BW245C (0.01 - 1 μM) suppressed TGF-β-induced collagen secretion. PGD2 and BW245C significantly increased intracellular cAMP level. One-hour pretreatment with forskolin (0.1 - 10 μM), dibutyryl-cAMP (0.01 - 1 mM), and the protein kinase A (PKA)-activator N6-phenyl-cyclic AMP (100 μM) significantly reduced TGF-β-induced collagen secretion, while exchange protein activated by cAMP (Epac) activator 8-bromo-2′-O-methyladenosine-3′,5′-cyclic AMP (10 μM) did not affect collagen deposition. These results suggest that PGD2 inhibits TGF-β-induced collagen secretion via intracellular cAMP accumulation through activating DP receptor.
We investigated the effects of endothelium-derived hyperpolarizing factor (EDHF) and the role of hydrogen sulphide (H2S) in the cerebral vasorelaxation induced by acetylcholine (ACh) in global cerebral ischemia–reperfusion (CIR) rats. CIR was induced by occlusion of bilateral carotid and vertebral arteries. Isolated arterial segments from the cerebral basilar (CBA) and middle artery (MCA) of CIR rats were studied in a pressurized chamber. Transmembrane potential was recorded using glass microelectrodes to evaluate hyperpolarization. In the CIR CBAs and MCAs preconstricted by 30 mM KCl, ACh induced concentration-dependent vasorelaxation and hyperpolarization that were partially attenuated by NG-nitro-l-arginine methyl ester (l-NAME, 30 μM) and l-NAME plus indomethacin (10 μM). The residual responses were abolished by the H2S inhibitor dl-propargylglycine (PPG, 100 μM). The H2S donor NaHS and l-Cys, the substrate of endogenous H2S synthase, elicited similar responses to ACh and was inhibited by tetraethylamonine (1 mM) or PPG. ACh induces EDHF-mediated vasorelaxation and hyperpolarization in rat cerebral arteries. These responses are up-regulated by ischemia–reperfusion while NO-mediated responses are down-regulated. Further, the ACh-induced, EDHF-mediated relaxation, and hyperpolarization and the inhibition of these responses are similar to the H2S-induced responses, suggesting that H2S is a possible candidate for EDHF in rat cerebral vessels.
There is so far no generally accepted animal model of chronic cystitis by which potential therapies can be evaluated. In this study, we aimed to establish a new mouse model of cystitis based on the proinflammatory effects of reactive oxygen species. A single intravesical injection of 1.5% hydrogen peroxide (H2O2) significantly increased the numbers of voids by 1 day after injection in female mice, which lasted up to 7 days. The H2O2 injection rapidly increased the bladder weight by 3 h in parallel with the histological damage and hyperpermeability of urothelial barrier. Although the urothelial dysfunction was recovered to normal by 7 days, increase in bladder weight, edematous thickening of the submucosa, and vascular hyperpermeability were apparent even 7 days after injection. During the time course, massive infiltration of neutrophils and increased expression of inflammatory cytokines were observed in the bladder. An intraperitoneal administration of oxybutynin, amitriptyline, indomethacin, or morphine attenuated the H2O2-induced frequent urination. These findings suggest that an intravesical injection of H2O2 induces relatively long-lasting inflammatory and overactive bladder, compared with existing cystitis models. The intravesical H2O2 injection model may be a simple and useful tool in the pathological study and drug discovery for chronic cystitis.
Canine hemangiopericytoma (CHP) is characterized by frequent local recurrence and increased invasiveness. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in tumors. The aim of the present study was to investigate the effect of a single dose of bevacizumab on a xenograft model of CHP. VEGF protein was secreted from cultured CHP cells and interacted with bevacizumab. Bevacizumab treatment suppressed tumor growth by inhibiting tumor angiogenesis, whereas no significant differences were observed in the proliferation index and apoptosis rates of treated and untreated mice. Thus, bevacizumab had antitumor effects in a xenograft model of CHP.
We explored the neuroprotection by atorvastatin in the ischemia/reperfusion model of rat and its microRNA-related mechanisms. At first, we uncovered a previously unknown alteration in temporal expression of a large set of microRNAs following spinal cord ischemia–reperfusion injury (IRI). The target genes for the differentially expressed microRNAs include genes encoding components that are involved in the inflammation, apoptosis, and neural damage that are known to play important roles in IRI. Atorvastatin pretreatment restored part of the up or down regulations. These findings suggest that altered expression of microRNAs may contribute to the mechanism of neuroprotection of statins in spinal cord IRI.
Prostaglandin E2 (PGE2) was shown to induce neuronal death in the CNS. To characterize the neurotoxicity of PGE2 and E-prostanoid receptors (EP) in motor neurons, we investigated PGE2-induced cell death and the type(s) of EP responsible for mediating it in NSC-34, a motor neuron-like cell line. Immunoblotting studies showed that EP2 and EP3 were dominantly expressed in NSC-34 cells and motor neurons in mice. Exposure to PGE2 and butaprost, an EP2 agonist, but not sulprostone, an EP1/3 agonist, resulted in decreased viability of these cells. These results suggest that PGE2 induces cell death by activation of EP2 in NSC-34 cells.
Nitric oxide (NO) induces cytotoxicity in neuronal and glial cells via activation of the Na+/Ca2+ exchanger (NCX). This study examined the role of the predominant brain-specific NCX splice variant NCX1.5 in NO-induced cytotoxicity in the HEK293 cell expression system. Cells were transfected with the plasmid construct pcDNA3.1/V5-His containing full-length rat NCX1.5 cDNA. There was no difference in the cytotoxic effects of the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine between control and transfected cells. These results suggest that NO cytotoxicity is not dependent on NCX1.5.