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ABSTRACT: Apoptosis plays a critical role in the development and homeostasis of multicellular organisms, and endoplasmic reticulum stress (ERS) is one of the intrinsic apoptosis pathways. Previous studies have shown that adenosine induces apoptosis in several cancer cell lines. However, the molecular mechanism remains poorly understood. In this study, we explored whether adenosine triggers apoptosis of EC109 esophageal carcinoma (EC) cells by ERS. The MTT assay was used to determine cell proliferation; cell cycle detection (FCM) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay were performed to determine cell apoptosis. The subcellular distribution and expression of the ERS-related proteins GRP78, cleaved caspase-3, cleaved caspase-4, CHOP and NF-κB p65 were detected by western blot techniques. NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). The MTT assay demonstrated that adenosine inhibited EC109 cell proliferation in a dose- and time-dependent manner. FCM and TUNEL assay verified that adenosine caused an apoptotic peak in cell cycle arrest and a higher percentage of apoptotic cells. Western blot analysis confirmed that the expression of GRP78, cleaved caspase-4, CHOP, NF-κB p65 and cleaved caspase-3 were upregulated in a dose-dependent manner after adenosine treatment. EMSA revealed that adenosine activated NF-κB p65. This is the first demonstration that adenosine inhibits cell proliferation, increases GRP78 and NF-κB p65 expression and induces apoptosis by CHOP and caspase-4 pathways. The ERS pathway is involved in adenosine-induced apoptosis in EC109 cells.
International Journal of Molecular Medicine 08/2012; · 1.98 Impact Factor
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ABSTRACT: Endoplasmic reticulum stress (ERS)-mediated cell apoptosis has been implicated in the development of multiple diseases such as cancer, neurodegenerative diseases and ischemic reperfusion damage. Previous studies have demonstrated the adenosine-induced apoptosis in several tumor cell lines. However, the role of ERS in adenosine-induced human hepatoma HepG2 cell apoptosis remains unclear. The present study was designed to determine whether ERS is involved in adenosine-induced HepG2 cell apoptosis. The MTT assay was used to determine proliferation, and DAPI staining of cell nuclei was performed to determine cell apoptosis. The translocation of CHOP and caspase-3 was observed by immunofluorescence analysis, and the protein expression of CHOP, caspase-4 and caspase-3 was detected by Western blotting. The MTT assay demonstrated that adenosine inhibited HepG2 cell proliferation in a dose-dependent manner. DAPI staining of cell nuclei and cell cycle analysis verified cell apoptosis. The immunofluorescence assay demonstrated that adenosine induced the translocation of CHOP and of caspase-3 from the cytoplasm to the nucleus. Western blotting confirmed that CHOP, caspase-4 and caspase-3 were up-regulated in HepG2 cells after treatment with adenosine. However, JNK protein expression was not altered. These results show that ERS is involved in the adenosine-induced HepG2 cell apoptosis.
Oncology Reports 07/2011; 26(1):73-9. · 1.84 Impact Factor
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ABSTRACT: Adenosine can exhibit cytotoxic activity in vivo and in vitro, though its mechanisms are still uncertain. In this study, we investigated the adenosine-mediated apoptotic signaling pathway and the role of NF-kappaB in human hepatocellular carcinoma HepG2 cells. HepG2 cells were treated with different concentrations of adenosine for 12-48 h, and the effect of adenosine on cell proliferation was evaluated by MTT assay. The cytotoxicity of adenosine alone or in combination with an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was also evaluated by MTT assay and the mode of cell death was detected by Hoechst 33342 staining. Cell cycle progress was performed by flow cytometry with PI staining. The protein expressions of Bcl-2, p53, NF-kappaB subunit p65, and caspase-3 were assayed by Western blot. Caspase-3 activity was measured by spectrophotomteric assay. The results showed that adenosine significantly reduced the viability of HepG2 cells in a dose- and time-dependent manner, with IC 50 (24 and 48 h) of 2.52 and 1.89 mmol x L(-1), respectively. The apoptotic index (percentage of sub-G1 phase) of HepG2 cells in adenosine treatment alone for 12 and 24 h or in combination with PDTC were 8.30%, 22.32% and 20.18%, 30.89%, respectively. All of them were higher than that in the control group (0.81%, p < 0.01). The characteristic changes of cell apoptosis (chromatin condensation and sub-G1 peak) were observed under fluorescent microscopy and flow cytometry. We also found that the apoptotic process triggered by adenosine was involved in G0-G1 cell-cycle arrest, enhanced the activity of caspase-3, upregulated p53 and NF-kappaB p65 expression, and downregulated Bcl-2 expression. Inhibition of NF-kappaB by PDTC decreased NF-kappaB p65 expression, enhanced cell apoptosis ratio, and increased caspase-3 activity. NF-kappaB may play an anti-apoptosis role in adenosine-induced HepG2 cytotoxicity.
Biochemistry and Cell Biology 08/2010; 88(4):705-14. · 2.67 Impact Factor
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ABSTRACT: To investigate effects of adenosine on cell proliferation and apoptosis in human HepG2 cells.
HepG2 cells were incubated in the presence of adenosine (0.1-5 mmol/L) for 12-48 h, and the effect of adenosine on cell proliferation was evaluated by using 3-(4,5-dimethyl-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Hoechst 33342 fluorescent staining, dUTP-fluorescein isothiocyanate (FITC) fluorescence and flow cytometric analysis techniques were used to observe cell apoptosis. The effects of adenosine receptor (A1, A2a, A3 and nonspecific receptor) antagonists (8-cpt, DMPX, MRS1191, and theophylline) and an adenosine transporter protein inhibitor (dipyridamole) on adenosine-induced cell apoptosis were observed. Mitochondrial membrane potential was analyzed using DePsipher fluorescent staining, and caspase activity was detected using a Fluorometric assay kit and a fluorescence microplate reader.
Adenosine significantly reduced cell viability in a dose- and time-dependent manner. The cytotoxicity of adenosine was related to the induction of cell apoptosis. Four adenosine receptor antagonists had no effect on cell apoptosis. However, dipyridamole significantly reduced the percentage of adenosine-induced apoptotic cells from 27.3% to 7.1% (P<0.05). At 48 h after treatment, 3 mmol/L adenosine increased caspase-3 activity 3.5-fold; dipyridamole markedly decreased caspase-3 activity 1.6-fold, and decreased apoptotic cell numbers. When HepG2 cells were treated with 3 mmol/L adenosine, mitochondrial membrane potential and the activity of caspase-8 or -9 remained unchanged.
Our results suggest that adenosine-induced apoptosis in HepG2 cells is related to intracellular events rather than cell surface receptors, and that a caspase-3 cascade activation is required, which is not mediated via a mitochondrial pathway.
Acta Pharmacologica Sinica 04/2006; 27(4):477-84. · 1.95 Impact Factor
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ABSTRACT: Aim: To investigate effects of adenosine on cell proliferation and apoptosis in human HepG2 cells. Methods: HepG2 cells were incubated in the presence of adenosine (0.1–5 mmol/L) for 12–48 h, and the effect of adenosine on cell proliferation was evaluated by using 3-(4,5 -dimethyl-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Hoechst 33342 fluorescent staining, dUTP-fluorescein isothiocyanate (FITC) fluorescence and flow cytometric analysis techniques were used to observe cell apoptosis. The effects of adenosine receptor (A1, A2a, A3 and nonspecific receptor) antagonists (8-cpt, DMPX, MRS 1191, and theophylline) and an adenosine transporter protein inhibitor (dipyridamole) on adenosine-induced cell apoptosis were observed. Mitochondrial membrane potential was analyzed using DePsipher fluorescent staining, and caspase activity was detected using a Fluorometric assay kit and a fluorescence microplate reader. Results: Adenosine significantly reduced cell viability in a dose- and time-dependent manner. The cytotoxicity of adenosine was related to the induction of cell apoptosis. Four adenosine receptor antagonists had no effect on cell apoptosis. However, dipyridamole significantly reduced the percentage of adenosine-induced apoptotic cells from 27.3% to 7.1% (P<0.05). At 48 h after treatment, 3 mmol/L adenosine increased caspase-3 activity 3.5-fold; dipyridamole markedly decreased caspase-3 activity 1.6-fold, and decreased apoptotic cell numbers. When HepG2 cells were treated with 3 mmol/L adenosine, mitochondrial membrane potential and the activity of caspase-8 or -9 remained unchanged. Conclusion: Our results suggest that adenosine-induced apoptosis in HepG2 cells is related to intracellular events rather than cell surface receptors, and that a caspase-3 cascade activation is required, which is not mediated via a mitochondrial pathway.
Acta Pharmacologica Sinica 03/2006; 27(4):477 - 484. · 1.95 Impact Factor
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ABSTRACT: To determine the expression of c-fos in gastric myenteric plexus and spinal cord of rats with cervical spondylosis and its clinical significance.
A cervical spondylosis model was established in rats by destroying the stability of cervical posterior column, and the cord segments C(4-6) and gastric antrum were collected 3, 4 and 5 mo after the operation. Rats with sham operation were used as controls. c-fos neuronal counter-staining was performed with an immunohistochemistry method. Every third sections from C(4-6) segments were drawn. The 10 most labeled c-fos-immunoreactive (Fos-IR) neurons were counted, and the average number was used for statistical analysis. The mean of Fos-IR neurons in myenteric plexus was calculated after counting Fos-IR neurons in 25 ganglia from each antral preparation, and expressed as a mean count per myenteric ganglion.
There were a few c-fos-positive neurons in the cervical cord and antrum in the control group. There was an increased c-fos expression in model group 3, 4 and 5 mo after operation, whereas there was no significant increase in c-fos expression in the control group at 3, 4 and 5 mo. More importantly, there was a significant difference in c-fos expression between rats followed up for 3 mo and those for 5 mo in the model group (11.20+/-2.26 vs 27.68+/-4.36, P<0.05, for the cervical cord; and 11.3+/-2.3 vs 29.3+/-4.6, P<0.05, for the gastric antrum). There was no significant difference between rats followed up for 3 mo and those for 4 mo and between rats followed up for 4 mo and those for 5 mo in the model group.
c-fos expression in gastric myenteric plexus was dramatically associated with that in the spinal cord in rats with cervical spondylosis, suggesting that the gastrointestinal function may be affected by cervical spondylosis. If this hypothesis is confirmed by further studies, functional gastrointestinal diseases such as functional dyspepsia and irritable bowel syndrome could be explained by neurogastroenterology.
World Journal of Gastroenterology 01/2005; 11(4):529-33. · 2.47 Impact Factor
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ABSTRACT: To evaluate the value of miniprobe sonography (MPS), spiral CT and MR imaging (MRI) in the tumor and regional lymph node staging of esophageal cancer.
Eight-six patients (56 men and 30 women; age range of 39-73 years, mean 62 years) with esophageal carcinoma were staged preoperatively with imaging modalities. Of them, 81 (94 %) had squamous cell carcinoma, 4(5 %) adenocarcinoma, and 1(1 %) adenoacanthoma. Eleven patients (12 %) had malignancy of the upper one third, 41 (48 %) of the mid-esophagus and 34 (40 %) of the distal one third. Forty-one were examined by spiral CT in whom 13 were co-examined by MPS, and forty-five by MRI in whom 18 were also co-examined by MPS. These imaging results were compared with the findings of the histopathologic examination for resected specimens.
In staging the depth of tumor growth, MPS was significantly more accurate (84 %) than spiral CT and MRI (68 % and 60 %, respectively, P<0.05). The specificity and sensitivity were 82 % and 85 % for MPS; 60 % and 69 % for spiral CT; and 40 % and 63 % for MRI, respectively. In staging regional lymph nodes, spiral CT was more accurate (78 %) than MPS and MRI (71 % and 64 %, respectively), but the difference was not statistically significant. The specificity and sensitivity were 79 % and 77 % for spiral CT; 75 % and 68 % for MPS; and 68 % and 62 % for MRI, respectively.
MPS is superior to spiral CT or MRI for T staging, especially in early esophageal cancer. However, the three modalities have the similar accuracy in N staging. Spiral CT or MRI is helpful for the detection of far-distance metastasis in esophageal cancer.
World Journal of Gastroenterology 02/2003; 9(2):219-24. · 2.47 Impact Factor
