Oncolytic Bovine Herpesvirus 1 Inhibits Human Lung Adenocarcinoma A549 Cell Proliferation and Tumor Growth by Inducing DNA Damage.

Bovine herpesvirus 1 (BoHV-1) is a promising oncolytic virus with broad antitumor spectrum; however, its oncolytic effects on human lung adenocarcinoma in vivo have not been reported. In this study, we report that BoHV-1 can be used as an oncolytic virus for human lung adenocarcinoma, and elucidate the underlying mechanism of how BoHV-1 suppresses tumor cell proliferation and growth. First, we examined the oncolytic activities of BoHV-1 in human lung adenocarcinoma A549 cells. BoHV-1 infection reduced the protein levels of histone deacetylases (HDACs), including HDAC1-4 that are promising anti-tumor drug targets. Furthermore, the HDAC inhibitor Trichostatin A (TSA) promoted BoHV-1 infection and exacerbated DNA damage and cytopathology, suggesting a synergy between BoHV-1 and TSA. In the A549 tumor xenograft mouse model, we, for the first time, showed that BoHV-1 can infect tumor and suppressed tumor growth with a similar high efficacy as the treatment of TSA, and HDACs have potential effects on the virus replication. Taken together, our study demonstrates that BoHV-1 has oncolytic effects against human lung adenocarcinoma in vivo.

The Role of Epidermal Growth Factor Receptor Signaling Pathway during Bovine Herpesvirus 1 Productive Infection in Cell Culture.

Accumulating studies have shown that the epidermal growth factor receptor (EGFR) signaling pathway plays an essential role in mediating cellular entry of numerous viruses. In this study, we report that bovine herpesvirus 1 (BoHV-1) productive infection in both the human lung carcinoma cell line A549 and bovine kidney (MDBK) cells leads to activation of EGFR, as demonstrated by the increased phosphorylation of EGFR at Tyr1068 (Y1068), which in turn plays important roles in virus infection. A time-of-addition assay supported that virus replication at post-entry stages was affected by the EGFR specific inhibitor Gefitinib. Interestingly, both phospholipase C-γ1 (PLC-γ1) and Akt, canonical downstream effectors of EGFR, were activated following virus infection in A549 cells, while Gefitinib could inhibit the activation of PLC-γ1 but not Akt. In addition, virus titers in A549 cells was inhibited by chemical inhibition of PLC-γ1, but not by the inhibition of Akt. However, the Akt specific inhibitor Ly294002 could significantly reduce the virus titer in MDBK cells. Taken together, our data suggest that PLC-γ1 is stimulated in part through EGFR for efficient replication in A549 cells, whereas Akt can be stimulated by virus infection independent of EGFR, and is not essential for virus productive infection, indicating that Akt modulates BoHV-1 replication in a cell type-dependent manner. This study provides novel insights on how BoHV-1 infection activates EGFR signaling transduction to facilitate virus replication.

CCR7 mediates human breast cancer cell invasion, migration by inducing epithelial-mesenchymal transition and suppressing apoptosis through AKT pathway.

Chemokine and the chemokine receptor have a key role in the tumor progress. Here, we supposed that CCR7 might induce the invasion, migration, and epithelial-mesenchymal transition (EMT) process of breast cancer. In this research, human breast cancer MCF-7 and MDA-MB-231cells were treated with CCL19 and small-interfering RNA (CCR7 siRNA) for activation and inhibition of CCR7, respectively. Cell invasion and transwell assays were used to detect the effect of CCR7 on invasion and migration. The results demonstrated that CCL19 mediated cell invasion and migration by inducing the EMT, with downregulation of E-cadherin and up-regulation of N-cadherin and vimentin levels. On the other hand, knockdown of CCR7 revealed the changes compared with CCL19 group and the control group. Knockdown of CCR7 inhibits CCL19-induced breast cancer cell proliferation, the cell cycle, migration, invasion and EMT. Moreover, we demonstrated that CCL19-induced AKT phosphorylation; however, CCR7 siRNA suppressed CCL19-induced AKT phosphorylation, a key regulator of tumor metastasis. In conclusion, all findings demonstrated that CCL19/CCR7 axis regulated EMT progress in breast cancer cells and mediated the tumor cell invasion and migration process via activation of AKT signal pathway. Our results suggested that CCR7 may regard as a therapeutic target for the breast cancer treatment.

Function of ghrelin and ghrelin receptors in the network regulation of gastric motility.

Numerous previous studies have demonstrated that ghrelin promotes gastric motility when administered peripherally. This effect appears to be regulatory but not directly stimulatory, and therefore may involve a number of complex mechanisms. In the periphery, ghrelin may affect gastric motility through intercellular networks among interstitial cells of Cajal, myenteric nerve cells and smooth muscle cells. The aim of the present study was to investigate the effects and possible mechanisms underlying this hypothesis. The effects of ghrelin on the contraction force of gastric antrum smooth muscle strips of rats were studied in the presence or absence of carbachol (CCh), [D­Lys3]­GHRP­6, atropine, tetrodotoxin (TTX) and nimodipine in vitro. The expression of ghrelin receptors (GHS­Rs) on different cell types in gastric muscle layers was observed by means of immunofluorescence. Ghrelin enhanced smooth muscle strip contraction induced by CCh, but when CCh was absent, this effect was eliminated. Atropine and nimodipine eradicated the muscle strip contraction enhanced by ghrelin, while [D­Lys3]­GHRP­6 was only able to partly block this effect and TTX had no effect on muscle strip contraction. It was identified that ghrelin had no effect on the contractive rhythm of the strips. GHS­R1s were located differentially depending on the cell type, including myenteric nerve cells, interstitial cells of Cajal and smooth muscle cells. In conclusion the present study demonstrated that ghrelin may act as an adjuvant to regulate gastric smooth muscle contraction induced by CCh through GHS­R1s, which are expressed on myenteric nerve cells, Cajal cells and smooth muscle cells. Ghrelin may exert its effects by influencing the functional status of different cell types in the gastric muscle layer to subsequently enhance the contractive effect of cholinergic neurotransmitters and enhance gastric motility.

Comparison of the effects of Mg-6Zn and Ti-3Al-2.5V alloys on TGF-ß/TNF-α/VEGF/b-FGF in the healing of the intestinal tract in vivo.

To evaluate the different effects of Mg-6Zn alloy and Ti-3Al-2.5V alloy implants in intestinal tract healing, we compared these two different alloys with respect to their effect on a rat's intestinal tract, using serum magnesium, radiology, pathology and immunohistochemistry in vivo. It was found using the scanning electron microscope that the Mg-6Zn alloy began to degrade during the first week and that the Ti-3Al-2.5V alloy was non-degradable throughout the process. The Mg-6Zn alloy did not have an impact on serum magnesium. Superior to the Ti-3Al-2.5V alloy, the Mg-6Zn alloy enhanced the expression of transforming growth factor-ß1 in healing tissue, and promoted the expression of both the vascular endothelial growth factor and the basic fibroblast growth factor, which helped angiogenesis and healing. The Mg-6Zn alloy reduced the expression of the tumor necrosis factor (TNF-α) at different stages and decreased inflammatory response, which may have been related to the zinc inhibiting TNF-α. In general, the Mg-6Zn alloy performed better than Ti-3Al-2.5V at promoting healing and reducing inflammation. The Mg-6Zn alloy may be a promising candidate for use in the pins of circular staplers for gastrointestinal reconstruction in medicine.

Comparison of the effects of Mg-6Zn and titanium on intestinal tract in vivo.

To evaluate the ability of Mg-6Zn to replace titanium nails in the reconstruction of the intestinal tract in general surgery, we compared the Mg-6Zn and titanium implants with respect to their effects on rat's intestinal tract by biochemical, radiological, pathological and immunohistochemical methods. The results indicated that Mg-6Zn implants started to degrade at the third week and disintegrate at the fourth week. No bubbles appeared, which may be associated with intestinal absorption of the Mg-6Zn implants. Pathological analyses (containing liver, kidney and cecum tissues) and biochemical measurements, including serum magnesium, creatinine, blood urea nitrogen, glutamic-pyruvic-transaminase and glutamic-oxaloacetic-transaminase proved that degradation of Mg-6Zn did not harm the important organs, which is an improvement over titanium implants. Immunohistochemical results showed that Mg-6Zn could enhance the expression of transforming growth factor-ß1. Mg-6Zn reduced the expression of tumor necrosis factor at different stages. In general, our study demonstrates that the Mg-6Zn alloy had good biocompatibility in vivo and performed better than titanium at promoting healing and reducing inflammation. It may be a promising candidate for stapler pins in intestinal reconstruction.

[Growth hormone secretagogue participates in two-way regulation of the motility of small intestinal smooth muscle in rats].

OBJECTIVE: To investigate the effect of growth hormone secretagogue(ghrelin) on the contraction and relaxation of small intestinal smooth muscle in rats and its mechanism. METHODS: Twenty-four vagotomized rats were injected intraperitoneally with different concentrations of ghrelin (0, 20, 40, 80 µg/kg). The small intestinal transit were observed. The effect of ghrelin(0.01, 0.1, 0.5, 1.0 µmol/L) on the contraction and relaxation of rat small intestinal smooth muscle strips was observed in vitro in the presence of carbachol(50 nmol/L), the locations of ghrelin receptors(GHS-R1a) on different cells in small intestinal muscle layers were detected by immunofluorescence. RESULTS: With the increase of concentrations, ghrelin elevated the percentage of small intestinal transit[(25.4±1.0)%, (33.7±1.9)%, (39.3±2.4)%, (44.7±2.1)%] in a dose-dependent manner, and the differences were statistically significant among groups(P<0.05). Ghrelin could also enhance the contraction [(67.0±2.4)%,(149.5±3.3)%, (187.1±4.7)%, (213.5±3.4)%] and relaxation[(35.3±1.1)%, (62.9±3.8)%, (79.6±2.7)%, (94.6±2.2)%] of smooth muscle strips mediated by Cch in a dose-dependent manner, and the differences were statistically significant among groups(P<0.05). Immunofluorescence revealed that ghrelin receptors mainly located on membrane of the nerve cells in the muscle layers, while no receptors were observed on membrane of the smooth muscle cells. CONCLUSION: Ghrelin may enhance the effect of the contraction and relaxation of the rat small intestinal smooth muscle mediated by cholinergic neurotransmitters by activating the nerve cells in the enteric plexus.

Down-regulation of ghrelin receptors in the small intestine delays small intestinal transit in vagotomized rats.

Vagal nerve injury may occur in esophageal and gastric surgeries. The aim of this study was to observe the effects of ghrelin on small intestinal motility upon vagal nerve injury and the possible co-relationship between changes in ghrelin receptor expression in the small intestine and delayed small intestinal transit after vagotomy. The effects of intraperitoneal administration of ghrelin (20, 40 and 80 µg/kg) and the ghrelin receptor antagonist [D-Lys3]-GHRP-6 (1.5 µmol/kg) on small intestinal transit were studied in control and vagotomized rats in vivo. The effects of ghrelin (0.01, 0.1, 0.5, 1.0 and 2.0 µmol/l) on the contraction force of smooth muscle strips from the jejunum were studied in the presence or absence of carbachol (50 nmol/l) and [D-Lys3]-GHRP-6 (10 µmol/l) in vitro. Ghrelin receptor expression was assessed in intestinal muscle layers by means of Western blotting. The results indicated that ghrelin dose-dependently increased small intestinal transit in the control and model rats. In addition, ghrelin enhanced smooth muscle strip contraction induced by carbachol. Ghrelin receptor antagonist [D-Lys3]-GHRP-6 blocked the effect of ghrelin. Ghrelin receptor expression in the small intestinal muscle layers was down-regulated in the vagotomized rats. Down-regulation of growth hormone secretagogue receptor 1a in small intestinal muscle layers, which affected the function of ghrelin, may be one of the mechanisms behind delayed small intestinal transit after vagotomy.

Therapeutic effects of ghrelin and growth hormone releasing peptide 6 on gastroparesis in streptozotocin-induced diabetic guinea pigs in vivo and in vitro.

BACKGROUND: Diabetic gastroparesis is a disabling condition with no consistently effective treatment. In normal animals, both ghrelin and its synthetic peptide, growth hormone releasing peptide 6 (GHRP-6), increase gastric emptying. Thus, we investigated the potential therapeutic significance of ghrelin and GHRP-6 in diabetic guinea pigs with gastric motility disorders. METHODS: A diabetic guinea pig model was produced by intraperitoneal (i.p.) injection of streptozotocin (STZ, 280 mg/kg). Diabetic guinea pigs were injected i.p. with ghrelin or GHRP-6 (10 - 100 microg/kg), and the effects on gastric emptying were measured after intragastric application of phenol red. The effect of atropine or a growth hormone secretagogue receptor (GHS-R) antagonist, D-Lys(3)-GHRP-6, on the gastroprokinetic effects of ghrelin or GHRP-6 (100 microg/kg) was also investigated. Further, the in vitro effects of ghrelin or GHRP-6 (0.01 - 10 micromol/L) on spontaneous or carbachol-induced contractile amplitude in gastric fundic circular strips taken from diabetic guinea pigs were examined. Growth hormone secretagogue receptor transcripts in the fundic strips of diabetic guinea pigs were detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: We established a guinea pig model of delayed gastric emptying. Ghrelin (20, 50, or 100 microg/kg) and GHRP-6 (20, 50, or 100 microg/kg) accelerated gastric emptying in diabetic guinea pigs with gastroparesis (n = 6, P < 0.05). In the presence of atropine, which delayed gastric emptying, ghrelin and GHRP-6 (100 microg/kg) failed to accelerate gastric emptying (n = 6, P < 0.05). D-Lys(3)-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist (n = 6, P < 0.05). Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundic strips taken from diabetic guinea pigs (n = 6, P < 0.05). RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations. CONCLUSIONS: Ghrelin and GHRP-6 increased gastric emptying in diabetic guinea pigs with gastroparesis, potentially, by activating the peripheral cholinergic pathways in the enteric nervous system.

Prokinetic effects of a ghrelin receptor agonist GHRP-6 in diabetic mice.

AIM: To investigate the effects of a ghrelin receptor agonist GHRP-6 on delayed gastrointestinal transit in alloxan-induced diabetic mice. METHODS: A diabetic mouse model was established by intraperitoneal injection with alloxan. Mice were randomized into two main groups: normal mice and diabetic mice treated with GHRP-6 at doses of 0, 20, 50, 100 and 200 microg/kg ip. Gastric emptying (GE), intestinal transit (IT), and colonic transit (CT) were studied in mice after they had a phenol red meal following injection of GHRP-6. Based on the most effective GHRP-6 dosage, atropine was given at 1 mg/kg for 15 min before the GHRP-6 injection for each measurement. The mice in each group were sacrificed 20 min later and the percentages of GE, IT, and CT were calculated. RESULTS: Percentages of GE, IT, and CT were significantly decreased in diabetic mice as compared to control mice. In the diabetic mice, GHRP-6 improved both GE and IT, but not CT. The most effective dose of GHRP-6 was 200 microg/kg and atropine blocked the prokinetic effects of GHRP-6 on GE and IT. CONCLUSION: GHRP-6 accelerates delayed GE and IT, but has no effect on CT in diabetic mice. GHRP-6 may exert its prokinetic effects via the cholinergic pathway in the enteric nervous system, and therefore, has therapeutic potential for diabetic patients with delayed upper gastrointestinal transit.

Ghrelin improves delayed gastrointestinal transit in alloxan-induced diabetic mice.

AIM: To investigate the effects of ghrelin on delayed gastrointestinal transit in alloxan-induced diabetic mice. METHODS: A diabetic mouse model was established by intraperitoneal injection with alloxan. Mice were randomized into two main groups: normal mice group and diabetic mice group treated with ghrelin at doses of 0, 20, 50, 100 and 200 mug/kg ip. Gastric emptying (GE), intestinal transit (IT), and colonic transit (CT) were studied in mice after they had a phenol red meal following injection of ghrelin. Based on the most effective ghrelin dosage, atropine was given at 1 mg/kg 15 min before the ghrelin injection for each measurement. The mice in each group were sacrificed 20 min later and their stomachs, intestines, and colons were harvested immediately. The amount of phenol red was measured. Percentages of GE, IT, and CT were calculated. RESULTS: Percentages of GE, IT, and CT were significantly decreased in diabetic mice as compared to control mice (22.9 +/- 1.4 vs 28.1 +/- 1.3, 33.5 +/- 1.2 vs 43.2 +/- 1.9, 29.5 +/- 1.9 vs 36.3 +/- 1.6, P < 0.05). In the diabetic mice, ghrelin improved both GE and IT, but not CT. The most effective dose of ghrelin was 100 mug/kg and atropine blocked the prokinetic effects of ghrelin on GE and IT. CONCLUSION: Ghrelin accelerates delayed GE and IT but has no effect on CT in diabetic mice. Ghrelin may exert its prokinetic effects via the cholinergic pathway in the enteric nervous system, and therefore has therapeutic potential for diabetic patients with delayed upper gastrointestinal transit.

Gastric motor effects of ghrelin and growth hormone releasing peptide 6 in diabetic mice with gastroparesis.

AIM: To investigate the potential therapeutic significance of ghrelin and growth hormone releasing peptide 6 (GHRP-6) in diabetic mice with gastric motility disorders. METHODS: A diabetic mouse model was established by intraperitoneal (ip) injection of alloxan. Diabetic mice were injected ip with ghrelin or GHRP-6 (20-200 microg/kg), and the effects on gastric emptying were measured after intragastric application of phenol red. The effect of atropine, NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) or D-Lys3-GHRP-6 (a growth hormone secretagogue receptor (GHS-R) antagonist) on the gastroprokinetic effect of ghrelin or GHRP-6 (100 microg/kg) was also investigated. The effects of ghrelin or GHRP-6 (0.01-10 micromol/L) on spontaneous or carbachol-induced contractile amplitude were also investigated in vitro, in gastric fundic circular strips taken from diabetic mice. The presence of growth hormone secretagogue receptor 1a transcripts in the fundic strips of diabetic mice was detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: We established a diabetic mouse model with delayed gastric emptying. Ghrelin and GHRP-6 accelerated gastric emptying in diabetic mice with gastroparesis. In the presence of atropine or L-NAME, which delayed gastric emptying, ghrelin and GHRP-6 (100 microg/kg) failed to accelerate gastric emptying. D-Lys3-GHRP-6 also delayed gastric emptying induced by the GHS-R agonist. Ghrelin and GHRP-6 increased the carbachol-induced contractile amplitude in gastric fundic strips taken from diabetic mice. RT-PCR confirmed the presence of GHS-R mRNA in the strip preparations. CONCLUSION: Ghrelin and GHRP-6 increase gastric emptying in diabetic mice with gastroparesis, perhaps by activating peripheral cholinergic pathways in the enteric nervous system.

[Effect and mechanism of ghrelin and its synthetic peptide growth hormone releasing peptide 6 on gastric motor in mice].

OBJECTIVE: To investigate the effect and mechanism of ghrelin and its synthetic peptide GHRP-6 on gastric motor in mice. METHODS: In vivo, the dose-dependent effects of ghrelin (20,50,100,200 mug/kg) and GHRP-6 (20,50,100,200 mug/kg) on gastric emptying were measured by intragastric application of phenol red test which was adapted for use in mice. The effects of atropine, NG-nitro-L-arginine methyl ester (L-NAME), and D-Lys(3)-GHRP-6 (GHS-R antagonist) on the gastric motor induced by ghrelin and GHRP-6 (100 mug/kg) were also investigated. In vitro, the effects of ghrelin (0.01,0.1,1.0,10.0 mumol/L) and GHRP-6 (0.01,0.1,1.0,10.0 mumol/L) on spontaneous contraction of mice fundic muscle strips were studied as well. RESULTS: Both ghrelin (50,100,200 mug/kg) and GHRP-6 (50,100,200 mug/kg) significantly accelerated gastric emptying (P<0.05), but they failed to accelerate gastric emptying in the presence of atropine, L-NAME and D-Lys(3)-GHRP-6 (P<0.05). Ghrelin (0.1, 1.0, 10.0 mumol/L) and GHRP-6 (0.1, 1.0, 10.0 mumol/L) induced significant contraction of fundic muscle strips in concentration-dependent manner (P<0.05), which could be blocked by tetrodotoxin. CONCLUSION: Ghrelin and its synthetic peptide GHRP-6 accelerate gastric emptying perhaps by activating GHS-R of cholinergic excitatory pathways and nitrergic nervous pathways in the enteric nervous system.

Involvement of ghrelin-growth hormone secretagogue receptor system in pathoclinical profiles of digestive system cancer.

Ghrelin receptor has been shown to be expressed along the human gastrointestinal tract. Recent studies showed that ghrelin and a synthetic ghrelin receptor agonist improved weight gain and lean body mass retention in a rat model of cancer cachexia by acting on ghrelin receptor, that is, growth hormone secretagogue receptor (GHS-R). This study aims to explore the expression and the distribution of ghrelin receptor in human gastrointestinal tract cancers and to investigate the possible involvement of the ghrelin-GHS-R system in human digestive cancers. Surgical human digestive cancer specimens were obtained from various portions of the gastrointestinal tract from different patients. The expression of ghrelin receptor in these tissues was detected by tissue microarray technique. Our results showed that ghrelin receptor was expressed in cancers throughout the gastrointestinal tract, mainly in the cytoplasm of mucosal layer cells. Its expression level possibly correlated with organ type, histological grade, tumor-nodes-metastases stage, and nutrition status (weight loss) of the patients. For the first time, we identified the distribution of ghrelin receptor in digestive system cancers. Our results implied that the ghrelin-GHS-R system might be involved in the pathoclinical profiles of digestive cancers.