Impaired nitrergic relaxation in pyloric sphincter of the 6-OHDA Parkinson's disease rat.

Patients with Parkinson's disease (PD) often suffer from delayed gastric emptying, but the underlying mechanism remains unclear. We have shown previously that a PD rat model comprising bilateral substantia nigra destruction by 6-hydroxydopamine (6-OHDA rats) exhibits gastroparesis with alteration of neural nitric oxide synthase (nNOS) and acetylcholine in gastric corpus. However, changes in pyloric motility in the 6-OHDA rats have not been characterized. Solid gastric emptying test, immunofluorescence, Western blot, and in vitro pyloric motility recordings were used to assess pyloric motor function in the 6-OHDA rats. The 6-OHDA-treated rats displayed delayed solid gastric emptying and a lower basal pyloric motility index. In the 6-OHDA rats, high K+-induced transient contractions were weaker in pyloric sphincters. Electric field stimulation (EFS)-induced pyloric sphincter relaxation was lower in the 6-OHDA rats. NG-nitro-l-arginine methyl ester (l-NAME), a nonselective inhibitor of NOS, markedly inhibited the EFS-induced relaxation in both control and 6-OHDA rats. Pretreatment of tetrodotoxin abolished the effect of EFS on the pyloric motility. In addition, nNOS-positive neurons were extensively distributed in the pyloric myenteric plexus, whereas the number of nNOS-immunoreactive neurons and the protein expression of nNOS were significantly decreased in the pyloric muscularis of 6-OHDA rats. However, sodium nitroprusside-induced pyloric relaxations were similar between the control and 6-OHDA rats. These results indicate that the pyloric sphincters of 6-OHDA rats exhibit both weakened contraction and relaxation. The latter may be due to reduced nNOS in the pyloric myenteric plexus. The dysfunction of the pyloric sphincter might be involved in the delayed gastric emptying.NEW & NOTEWORTHY Reduced nitrergic neurons in pyloric myenteric plexus potently contributed to the attenuated relaxation in 6-hydroxydopamine (6-OHDA) rats, subsequently affecting gastric emptying. SNP could well improve the relaxation of pylori in 6-OHDA rats. The present study provides new insight into the diagnosis and treatment of delayed gastric emptying in patients with PD.

MicroRNAs of bone marrow mesenchymal stem cell-derived exosomes regulate acute myeloid leukemia cell proliferation and apoptosis.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant hematological disease, originating from hematopoiesis stem cell differentiation obstruction and clonal proliferation. New reagents or biologicals for the treatment of AML are urgently needed, and exosomes have been identified as candidate biomarkers for disease diagnosis and prognosis. This study aimed to investigate the effects of exosomes from bone marrow mesenchymal stem cells (BMSCs) on AML cells as well as the underlying microRNA (miRNA)-mediated mechanisms. METHODS: Exosomes were isolated using a precipitation method, followed by validation using marker protein expression and nanoparticle tracking analysis. Differentially expressed miRNAs were identified by deep RNA sequencing and confirmed by quantitative real-time polymerase chain reaction (qPCR). Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt method, and cell cycle progression and apoptosis were detected by flow cytometry. Functional gene expression was analyzed by qPCR and Western blotting (WB). Significant differences were determined using Student's t test or analysis of variance. RESULTS: BMSCs-derived exosomes effectively suppressed cell proliferation (both P < 0.0001 at 10 and 20 µg/mL) and cell cycle progression (P < 0.01 at G0-G1 stage), and also significantly enhanced cell apoptosis (P < 0.001) in KG-1a cells. There were 1167 differentially expressed miRNAs obtained from BMSCs-derived exosomes compared with KG-1a cell-derived exosomes (P < 0.05). Knockdown of hsa-miR-124-5p in BMSCs abrogated the effects of BMSCs-derived exosomes in regulating KG-1a such as the change in cell proliferation (both P < 0.0001 vs. normal KG-1a cell [NC] at 48 and 72 h). KG-1a cells treated with BMSCs-derived exosomes suppressed expression of structural maintenance of chromosomes 4 (P < 0.001 vs. NC by qPCR and P < 0.0001 vs. NC by WB), which is associated with the progression of various cancers. This BMSCs-derived exosomes effect was significantly reversed with knockdown of hsa-miR-124-5p (P < 0.0001 vs. NC by WB). CONCLUSIONS: BMSCs-derived exosomes suppress cell proliferation and cycle progression and promote cell apoptosis in KG-1a cells, likely acting through hsa-miR-124-5p. Our study establishes a basis for a BMSCs-derived exosomes-based AML treatment.

Actual long-term survival in HCC patients with portal vein tumor thrombus after liver resection: a nationwide study.

BACKGROUND: Liver resection for hepatocellular carcinoma (HCC) patients with portal vein tumor thrombus (PVTT) offers a chance of cure, although survival is often limited. The actual 3-year survival and its associated prognostic factors have not been reported. METHODS: A nationwide database of HCC patients with PVTT who underwent liver resection with 'curative' intent was analyzed. The clinicopathologic characteristics, the perioperative, and survival outcomes for the actual long-term survivors were compared with the non-long-term survivors (patients who died within 3 years of surgery). Univariable and multivariable regression analyses were performed to identify predictive factors associated with long-term survival outcomes. RESULTS: The study included 1590 patients with an actuarial 3-year survival of 16.6%, while the actual 3-year survival rate was 11.7%. There were 171 patients who survived for at least 3 years after surgery and 1290 who died within 3 years of surgery. Multivariable regression analysis revealed that total bilirubin > 17.1 µmol/l, AFP > 400 ng/ml, types of hepatectomy, extent of PVTT, intraoperative blood loss > 400 ml, tumor diameter > 5 cm, tumor encapsulation, R0 resection, liver cirrhosis, adjuvant TACE, postoperative early recurrence (< 1 year), and recurrence treatments were independent prognostic factors associated with actual long-term survival. CONCLUSION: One in nine HCC patients with PVTT reached the long-term survival milestone of 3 years after resection. Major hepatectomy, controlling intraoperative blood loss, R0 resection, adjuvant TACE, and 'curative' treatment for initial recurrence should be considered for patients to achieve better long-term survival outcomes.

Source of dopamine in gastric juice and luminal dopamine-induced duodenal bicarbonate secretion via apical dopamine D2 receptors.

BACKGROUND AND PURPOSE: Dopamine protects the duodenal mucosa. Here we have investigated the source of dopamine in gastric juice and the mechanism underlying the effects of luminal dopamine on duodenal bicarbonate secretion (DBS) in rodents. EXPERIMENTAL APPROACH: Immunofluorescence, UPLC-MS/MS, gastric incubation and perfusion were used to detect gastric-derived dopamine. Immunofluorescence and RT-PCR were used to examine the expression of dopamine receptors in the duodenal mucosa. Real-time pH titration and pHi measurement were performed to investigate DBS. KEY RESULTS: H+ -K+ -ATPase was co-localized with tyrosine hydroxylase and dopamine transporters in gastric parietal cells. Dopamine was increased in in vivo gastric perfusate after intravenous infusion of histamine and in gastric mucosa incubated, in vitro, with bethanechol chloride or tyrosine. D2 receptors were the most abundant dopamine receptors in rat duodenum, mainly distributed on the apical membrane of epithelial cells. Luminal dopamine increased DBS in a concentration-dependent manner, an effect mimicked by a D2 receptor agonist quinpirole and inhibited by the D2 receptor antagonist L741,626, in vivo D2 receptor siRNA and in D2 receptor -/- mice. Dopamine and quinpirole raised the duodenal enterocyte pHi . Quinpirole-evoked DBS and PI3K/Akt activity were inhibited by calcium chelator BAPTA-AM or in D2 receptor-/- mice. CONCLUSION AND IMPLICATIONS: Dopamine in the gastric juice is derived from parietal cells and is secreted along with gastric acid. On arrival in the duodenal lumen, dopamine increased DBS via an apical D2 receptor- and calcium-dependent pathway. Our data provide novel insights into the protective effects of dopamine on the duodenal mucosa.

Radiofrequency ablation for hepatic hemangiomas: A consensus from a Chinese panel of experts.

Recent studies have shown that radiofrequency (RF) ablation therapy is a safe, feasible, and effective procedure for hepatic hemangiomas, even huge hepatic hemangiomas. RF ablation has the following advantages in the treatment of hepatic hemangiomas: minimal invasiveness, definite efficacy, high safety, fast recovery, relatively simple operation, and wide applicability. It is necessary to formulate a widely accepted consensus among the experts in China who have extensive expertise and experience in the treatment of hepatic hemangiomas using RF ablation, which is important to standardize the application of RF ablation for the management of hepatic hemangiomas, regarding the selection of patients with suitable indications to receive RF ablation treatment, the technical details of the techniques, therapeutic effect evaluations, management of complications, etc. A final consensus by a Chinese panel of experts who have the expertise of using RF ablation to treat hepatic hemangiomas was reached by means of literature review, comprehensive discussion, and draft approval.

PIM-1 kinase inhibitor SMI-4a exerts antitumor effects in chronic myeloid leukemia cells by enhancing the activity of glycogen synthase kinase 3ß.

The development of targeted tyrosine kinase inhibitors (TKIs) has succeeded in altering the course of chronic myeloid leukemia (CML). However, a number of patients have failed to respond or experienced disease relapse following TKI treatment. Proviral integration site for moloney murine leukemia virus­1 (PIM­1) is a serine/threonine kinase that participates in regulating apoptosis, cell cycle, signal transduction and transcriptional pathways, which are associated with tumor progression, and poor prognosis. SMI­4a is a selective PIM­1 kinase inhibitor that inhibits PIM­1 kinase activity in vivo and in vitro. The present study aimed to explore the mechanism underlying the antitumor effect of SMI­4a in K562 and imatinib­resistant K562 (K562/G) cell lines. It was demonstrated that SMI­4a inhibited the proliferation of K562 and K562/G cells using a WST­8 assay. The Annexin V­propidium iodide assay demonstrated that SMI­4a induced apoptosis of K562 and K562/G cells in a dose­, and time­dependent manner. Furthermore, Hoechst 33342 staining was used to verify the apoptosis rate. The clone formation assay revealed that SMI­4a significantly inhibited the colony formation capacity of K562 and K562/G cells. Western blot analysis demonstrated that SMI­4a decreased phosphorylated (p)­Ser9­glycogen synthase kinase (GSK) 3ß/pGSK3ß and inhibited the translocation of ß­catenin. In addition, the downstream gene expression of apoptosis regulator Bax and poly(ADP­ribose) polymerase­1 was upregulated, and apoptosis regulator Bcl­2 and Myc proto­oncogene protein expression levels were downregulated. Immunofluorescence results demonstrated changes in the expression level of ß­catenin in the plasma and nucleus. The results of the present study suggest that SMI­4a is an effective drug to use in combination with current chemotherapeutics for the treatment of imatinib-resistant CML.

Celecoxib exerts antitumor effects in HL-60 acute leukemia cells and inhibits autophagy by affecting lysosome function.

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been demonstrated to exert antitumor activity in a variety of cancer cells. The underlying mechanism involves inhibition of cell cycle progression and induction of apoptosis. Besides, celecoxib has also been found to induce autophagy in some solid tumor cells. The aim of this study was to investigate the effect of celecoxib on cell proliferation in HL-60 human acute leukemia cells and to explore the potential mechanism. HL-60 cells were exposed to various concentrations of celecoxib and cell viability was evaluated by the MTT assay. Apoptosis was analyzed with flow cytometry and the amount of autophagosome was evaluated by LysoTracker probe labelling. The expression of apoptosis- and autophagy-related proteins was assayed by Western blot and LysoSensor probe labelling was used to detect the effect of celecoxib on the lysosomal functions. The results of this study indicated that celecoxib inhibited cell proliferation in a time- and dose-dependent fashion. The flow cytometry analysis showed that celecoxib induced apoptosis at low concentrations and mainly cell necrosis at high concentrations. The Western blot test confirmed the induction of apoptosis by the upregulation of apoptosis-related proteins cleaved caspase-3 and cleaved PARP. Furthermore, this study demonstrated that celecoxib prevented the autophagic flux by inhibiting lysosome function; the fluorescence intensity of the LysoTracker probe and the level of autophagy-related proteins LC3-II and p62 were increased, but the fluorescence intensity of the LysoSensor probe was weakened. These findings show that celecoxib is an autophagy suppresser and has antitumor effects in HL-60 cells by inducing cell apoptosis and necrosis.

Zoledronic acid overcomes adriamycin resistance in acute myeloid leukemia cells by promoting apoptosis.

Zoledronic acid (ZOL), a nitrogen­containing bisphosphonate, is widely used in metastatic bone disease. Previous studies indicate that ZOL has marked anti­leukemia activity, however, the underlying mechanism of action remains to be elucidated. The present study aimed to explore the mechanism of the anti­leukemia effect of ZOL in leukemia cells. It was observed that ZOL inhibited the proliferation of HL­60 and adriamycin­resistant HL­60 (HL­60/A) cells using a WST­8 assay. An Annexin V­propidium iodide indicated that ZOL induced apoptosis of the two cell types in a dose­ and time­dependent manner. Hoechst 33342 staining was also used to verify the levels of apoptosis. The colony formation assay demonstrated that ZOL significantly inhibited colony formation capacity in acute myeloid leukemia (AML) cells. This was achieved by the induction of S­phase cell cycle arrest, downregulation of B­cell lymphoma 2 (Bcl­2) and upregulation of Bcl­2 associated X protein and cleaved poly (ADP­ribose) polymerase. The results indicate that ZOL inhibited cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and this anti­leukemic activity appeared notably enhanced in HL­60/A cells. As ZOL is already available for clinical use, these results indicate that it may be an effective addition to the chemotherapeutic strategies for AML.

RUNX3 plays an important role in As2O3­induced apoptosis and allows cells to overcome MSC­mediated drug resistance.

The interaction between bone marrow stromal cells and leukemia cells is critical for the persistence and progression of leukemia, and this interaction may account for residual disease. However, the link between leukemia cells and their environment is still poorly understood. In our study, runt­related transcription factor 3 (RUNX3) was identified as a novel target gene affected by As2O3 and involved in mesenchymal stem cell (MSC)­mediated protection of leukemia cells from As2O3­induced apoptosis. We observed induction of RUNX3 expression and the translocation of RUNX3 into the nucleus after As2O3 treatment in leukemia cells. In K562 chronic myeloid leukemia cells, downregulation of endogenous RUNX3 compromised As2O3­induced growth inhibition, cell cycle arrest, and apoptosis. In the presence of MSC, As2O3­induced expression of RUNX3 was reduced significantly and this reduction was modulated by CXCL12/CXCR4 signaling. Furthermore, overexpression of RUNX3 restored, at least in part, the sensitivity of leukemic cells to As2O3. We conclude that RUNX3 plays an important role in As2O3­induced cellular responses and allows cells to overcome MSC­mediated drug resistance. Therefore, RUNX3 is a promising target for therapeutic approaches to overcome MSC­mediated drug resistance.

Altered expression of dopamine receptors in cholinergic motoneurons of the hypoglossal nucleus in a 6-OHDA-induced Parkinson's disease rat model.

Parkinson's disease (PD) is a common neurodegenerative disorder that is often associated with weak tongue motility. However, the link between the degenerated dopaminergic neurons in the substantia nigra (SN) and lingual dysfunction remains unclear. In the present study, we investigated the localization of dopamine receptor 1 (D1) and dopamine receptor 2 (D2) and alternations in their expression in cholinergic motoneurons of the hypoglossal nucleus (HN) using double-label immunofluorescence, Western blotting and semi-quantitative reverse transcription and polymerase chain reaction (SqRT-PCR) in rats that received microinjections of 6-hydroxydopamine bilaterally into the SN (6-OHDA rats). The results revealed that a large population of choline acetyltransferase immunoreactive (ChAT-IR) neurons was distributed throughout HN and that almost all of the ChAT-IR motoneurons were also D1-IR and D2-IR. Several tyrosine hydroxylase (TH)-IR profiles were observed in a nonuniform pattern near the ChAT-IR, D1-IR or D2-IR somas, suggesting potent dopaminergic innervation. In the 6-OHDA rats, TH immunoreactivity in the SN was significantly decreased, but food residue was increased and treadmill occupancy time was shortened. In the HN, protein expression of TH and D2 was increased, whereas that of ChAT and D1 was decreased. A similar pattern was observed in mRNA levels. The present study suggests that dopamine may modulate the activity of cholinergic neurons via binding with D1 and D2 in the HN. Changes in the expression of ChAT, TH, D1 and D2 in the HN of 6-OHDA rats might be associated with the impaired tongue motility in PD. These findings should be further investigated.

Distinctive expression and cellular distribution of dopamine receptors in the pancreatic islets of rats.

Activation of the dopamine (DA) D2 receptor inhibits glucose-stimulated insulin secretion in isolated rodent islets in vitro; however, no information is available regarding the cellular localization of DA receptors (DRs, including D1-D5 receptors) in pancreatic islets in situ. We investigate the protein expression and cellular localization of five types of DRs in pancreatic islets by means of Western blotting and double-labeling immunofluorescence in both normal control and alloxan-induced type 1 diabetes model (T1DM) rats. In control rats, D1 immunoreactivity (-IR) was distributed in the core of the islet and co-localized with insulin-IR, D2-IR was peripherally distributed and found only in somatostatin-immunoreactive cells and D5-IR was co-localized with glucagon-IR and pancreatic polypeptide-IR. No IR for either the D3 or D4 receptor was observed in rat islets. The protein level of the D1 receptor was reduced in T1DM rats (D1/D-glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 0.63 ± 0.05 in control rats compared with 0.16 ± 0.03 in T1DM rats, n = 8, P < 0.05) but no significant alteration was detected in the protein expression of either the D2 receptor (D2/GAPDH, 0.48 ± 0.04 compared with 0.43 ± 0.04, n = 8, P = 0.42) or the D5 receptor (D5/GAPDH, 0.50 ± 0.04 compared with 0.47 ± 0.04, n = 8, P = 0.58). The present study is the first clear demonstration of the protein expression and cellular localization of the D1, D2 and D5 receptors in rat pancreatic islets and provides crucial morphological evidence for further investigations of the underlying mechanism regarding the DA regulation of pancreatic endocrine function.

Diagnosis of Felty's syndrome, distinguished from hematological neoplasm: A case report.

Felty's syndrome (FS) is characterized by the three conditions of rheumatoid arthritis (RA), neutropenia and splenomegaly, and occurs in few cases of longstanding erosive RA. Discriminating between rare occurrences of autoimmune diseases and malignancies is crucial. The present study describes the case of a 17-year-old female with a two-year history of RA, presenting with an irregular fever, hepatosplenomegaly and enlarged lymph nodes. The antinuclear antibody titer was 1:320, while antibody results for anti-dsDNA, anti-Sm and rheumatoid factor were negative. The clinical presentation was similar to that of lymphoma. However, the fluorodeoxyglucose-positron emission tomography and biopsy examinations of the liver and cervical lymph node did not support the diagnosis of lymphoma. According to the laboratory results and clinical symptoms, the differential diagnosis indicated FS, and immunosuppressive agents were administered. Two weeks later, the patient no longer had a fever, and the transaminase levels were normal, associated with shrinkage of the liver and spleen.

New perspectives of vesicular monoamine transporter 2 chemical characteristics in mammals and its constant expression in type 1 diabetes rat models.

Vesicular monoamine transporter 2 (VMAT2) has been exploited as a biomarker of ß-cell mass in human islets. However, a current report suggested no immunoreactivity of VMAT2 in the ß cells of rat islets. To investigate the cellular localization of VMAT2 in islets further, the pancreatic tissues from monkeys and humans were compared with those of rats and mice. The study was performed using among-species comparisons and a type 1 diabetes model (T1DM) for rats by Western blotting, double-label immunofluorescence, and confocal laser scanning microscopy. We found that VMAT2-immunoreactivity (IR) was distributed peripherally in the islets of rodents, but was widely scattered throughout the islets of primates. Consistent with rodent islets, VMAT2-IR did not exist in insulin (INS)-IR cells but was abundantly present in glucagon (GLU)-IR and pancreatic polypeptide (PP)-IR cells in monkey and human islets. VMAT2-IR had no colocalization with INS-IR in any part of the rat pancreas (head, body, and tail). INS-IR cells were reduced dramatically in T1DM rat islets, but no significant alteration in the proportion of VMAT2-IR cells and GLU-IR cells was observed. Furthermore, a strong colocalization of VMAT2-IR with GLU-IR was distributed in the peripheral regions of diabetic islets. For the first time, the current study demonstrates the presence of VMAT2 in α cells and PP cells but not in ß cells in the islets of monkeys and humans. This study provides convinced morphologic evidence that VMAT2 is not present in ß cells. There needs to be studies for new markers for ß cell mass.

Alterations in TH- and ChAT-immunoreactive neurons in the DMV and gastric dysmotility in an LPS-induced PD rat model.

To study movement disorder in Parkinson's disease (PD), an animal model of PD can be created by injecting lipopolysaccharide (LPS) into the substantia nigra of rats. In addition to body movement disorders, patients with PD often experience gastrointestinal (GI) dysfunction, such as gastroparesis. However, the underlying mechanism of these disorders remains unclear. The dorsal motor nucleus of vagus (DMV) is a well-known visceral nucleus that regulates GI function. The present study investigated alterations in DMV neurons and gastric motility in rats with LPS-induced PD (LPS-PD rats). Gastric motility was recorded using a strain gauge force transducer in vivo. The distributions of tyrosine hydroxylase (TH)- and choline acetyltransferase (ChAT)-positive neurons in the DMV were determined using immunofluorescence and confocal laser microscopy. Our results indicated that in LPS-PD rats, the number of neurons in the substantia nigra, including neurons with TH immunoreactivity, was markedly reduced, although glial cell proliferation was clearly observed. However, enhanced TH immunoreactivity and decreased ChAT immunoreactivity were found in the DMV. Furthermore, weakened gastric motility was recorded in anesthetized LPS-PD rats. In conclusion, rats with LPS-induced PD exhibited gastric dysmotility with an alteration in DMV neurons. This PD model may be used to study autonomic nervous system disorders that are often observed in patients with early-stage PD.

Distribution of dopamine receptors D1- and D2-immunoreactive neurons in the dorsal motor nucleus of vagus in rats.

The dorsal motor nucleus of vagus (DMV) plays an important role in the regulation of gastrointestinal function. Dopamine (DA) exerts potent neuromodulatory effects on the motoneurons in the DMV via dopamine receptors (DRs). However, the distribution of DRs and their neurochemical phenotypes in the DMV are unclear. In the present study, the distribution of DRs D1- and D2-immunoreactive (IR) neurons and their neurochemical phenotypes in the DMV of rats were investigated using a double-labeling immunofluorescence technique combined with confocal microscopy. The results indicated that a considerable quantity of D1 and D2 was expressed throughout the DMV. A large amount of choline acetyltransferase (ChAT)-IR and a few tyrosine hydroxylase (TH)-IR neurons were observed in the DMV. Nearly all of the neurons were also D1-IR and D2-IR. In conclusion, the present study demonstrates the wide distribution of D1 and D2 in the cholinergic and catecholaminergic neurons in the DMV of rats. The DRs might play an important role in the regulation of DA on the activity of cholinergic and catecholaminergic neurons in the DMV.

Dopamine D1 receptors mediate dopamine-induced duodenal epithelial ion transport in rats.

Dopamine (DA) is synthesized in gastrointestinal epithelial cells and performs important regulatory effects on the duodenal mucosa. However, the underlying mechanism remains largely unknown. The present study investigated the effect of DA on the duodenal epithelial ion transport in rats by means of short-circuit current (ISC), real-time pH titration, enzyme-linked immunosorbent assay, and immunohistochemistry. The results indicate that basolateral, but not apical, application of DA induced a concentration-dependent ISC downward deflection with an apparent IC50 of 5.34 µmol/L. Basolateral application of dopaminergic receptor D1 (D1) antagonist, SCH-23390, inhibited DA-induced change in ISC (△ISC) in a dose-dependent manner. D1 agonist, SKF38393, mimicked the effect of DA on the ISC. The clear immunoreactivity of D1 subtype D5 (D1b) was at the both apical and basolatoral sides of Brunner's glands and intestinal crypts. Basolateral pretreatment with adenylate cyclase inhibitor, MDL12330A, significantly inhibited DA- and forskolin-induced △ISC. DA and SKF38393 increased the level of intracellular cyclic adenosine monophosphate (cAMP) from 1.55 ± 0.11 to 2.07 ± 0.11 and 5.91 ± 0.25 pmol/L·mg(-1), respectively. Furthermore, the serosal DA-induced △ISC was remarkably inhibited by apical administration of K(+) channel blockers, Ba(2+) and tetraethylammonium, but not by Cl(-) channel blockers. Serosal DA and D1 agonist did not affect duodenal HCO3(-) secretion. In conclusion, the present results demonstrate that serosal DA is able to promote rat duodenal epithelial K(+) secretion, not HCO3(-) secretion through D1-mediated and cAMP-dependent pathway. The study provides a new insight in the modulation of DA on the ion transport of duodenal epithelia in rats.

RUNX3 is involved in caspase-3-dependent apoptosis induced by a combination of 5-aza-CdR and TSA in leukaemia cell lines.

PURPOSE: Epigenetic therapy has had a significant impact on the management of haematologic malignancies. The aim of this study was to assess whether 5-aza-CdR and TSA inhibit the growth of leukaemia cells and induce caspase-3-dependent apoptosis by upregulating RUNX3 expression. METHODS: K562 and Reh cells were treated with 5-aza-CdR, TSA or both compounds. RT-PCR and Western blot analyses were used to examine the expression of RUNX3 at the mRNA and protein levels, respectively. Immunofluorescence microscopy was used to detect the cellular location of RUNX3. Additionally, after K562 cells were transfected with RUNX3, apoptosis and proliferation were studied using Annexin V staining and MTT assays. RESULTS: The expression of RUNX3 in leukaemia cell lines was markedly less than that in the controls. Demethylating drug 5-aza-CdR could induce RUNX3 expression, but the combination of TSA and 5-aza-CdR had a greater effect than did treatment with a single compound. The combination of 5-aza-CdR and TSA induced the translocation of RUNX3 from the cytoplasm into the nucleus. TSA enhanced apoptosis induced by 5-aza-CdR, and Annexin V and Hoechst 33258 staining showed that the combination induced apoptosis but not necrosis. Furthermore, apoptosis was dependent on the caspase-3 pathway. RUNX3 overexpression in K562 cells led to growth inhibition and apoptosis and potentiated the effects of 5-aza-CdR induction. CONCLUSION: RUNX3 plays an important role in leukaemia cellular functions, and the induction of RUNX3-mediated effects may contribute to the therapeutic value of combination TSA and 5-aza-CdR treatment.

Down-regulation of telomerase activity and activation of caspase-3 are responsible for Tanshinone I-induced apoptosis in monocyte leukemia cells in vitro.

Tanshinone I (Tan-I) is a diterpene quinone extracted from the traditional herbal medicine Salvia miltiorrhiza Bunge. Recently, Tan-I has been reported to have anti-tumor effects. In this study, we investigated the growth inhibition and apoptosis inducing effects of Tan-I on three kinds of monocytic leukemia cells (U937, THP-1 and SHI 1). Cell viability was measured by MTT assay. Cell apoptosis was assessed by flow cytometry (FCM) and AnnexinV/PI staining. Reverse transcriptase polymerase chain reaction (RT-PCR) and PCR-enzyme-linked immunosorbent assay (ELISA) were used to detect human telomerase reverse transcriptase (hTERT) expression and telomerase activity before and after apoptosis. The activity of caspase-3 was determined by Caspase colorimetric assay kit and Western blot analysis. Expression of the anti-apoptotic gene Survivin was assayed by Western blot and Real-time RT-PCR using the ABI PRISM 7500 Sequence Detection System. The results revealed that Tan-I could inhibit the growth of these three kinds of leukemia cells and cause apoptosis in a time- and dose-dependent manner. After treatment by Tan-I for 48 h, Western blotting showed cleavage of the caspase-3 zymogen protein with the appearance of its 17-kD subunit, and a 89-kD cleavage product of poly (ADP-ribose) polymerase (PARP), a known substrate of caspase-3, was also found clearly. The expression of hTERT mRNA as well as activity of telomerase were decreased concurrently in a dose-dependent manner. Moreover, Real-time RT-PCR and Western blot revealed a significant down-regulation of Survivin. We therefore conclude that the induction of apoptosis by Tan-I in monocytic leukemia U937 THP-1 and SHI 1 cells is highly correlated with activation of caspase-3 and decreasing of hTERT mRNA expression and telomerase activity as well as down-regulation of Survivin expression. To our knowledge, this is the first report about the effects of Tan-I on monocytic leukemia cells.

[Significance of DNA methylation status of runx3 gene promoter region in acute leukemia].

This study was aimed to investigate the effects of methylation of runx3 gene promoter on pathogenesis of acute leukemia (AL) and its clinical significance. The methylation of runx3 gene promoter in cells of bone marrow or peripheral blood from 40 cases of AL and 10 healthy persons as well as in CHRF, U937 and K562 cell lines were assuaged by methylation specific polymerase chain reaction (MS-PCR), the expression of runx3 gene were detected with reverse transcription polymerase chain reaction (RT-PCR). The results indicated that no methylation was detected in all of cell lines and healthy persons while expression of runx3 gene could be deteted, methylation of runx3 gene promoter was found in 35% (14/40) AL patients and its percentage was significant higher than that healthy persons (0%), the difference in methylation for runx3 between two kinds of samples was statistically significant (p<0.05), while methylation rate in AML was 30.43% (7/23), ALL was 41.18% (7/17), there was no significant difference between them (p>0.25). All of methylated samples had no expression of runx3 gene. Patients without methylation of runx3 gene had a lower percentage of blasts in bone marrow and a higher complete remission rate of first chemotherapy than those with methylation of runx3 gene. It is concluded that methylation of runx3 gene promoter probably plays a role in the pathogenesis of AL and may have clinical significance in predicting prognosis of AL.