Putative role of the mTOR/4E-BP1 signaling pathway in the carcinogenesis and progression of gastric cardiac adenocarcinoma.

The mammalian target of rapamycin/eukaryotic translation inititiation factor 4E binding protein 1 (mTOR/4E-BP1) transduction pathway is activated in a range of malignant cancers, but its role in human gastric cardiac adenocarcinoma (GCA) has not been well defined. The present study used western blotting and reverse transcription polymerase chain reaction (RT-PCR) to assess the expression of mTOR, 4E-BP1 and eukaryotic translation initiation factor 4E (eIF4E) at the protein and mRNA levels in 33 cases of GCA and paired adjacent normal gastric mucosal tissues. The expression of mTOR at the protein level in GCA was significantly lower than that in the corresponding normal gastric mucosa (0.296 ± 0.27 vs. 1.348 ± 0.80, P<0.05), but the ratio of p-mTOR to mTOR was significantly increased in tumor tissues (1.425 ± 1.07 vs. 0.450 ± 0.24, P<0.05). The expression of 4E-BP1 was significantly decreased in GCA compared with normal tissues (p<0.05), while the levels of phosphorylated 4E-BP1 (p-4E-BP1) were markedly increased in tumor tissues (p<0.05). The levels of phosphorylated eIF4E (p­eIF4E) were significantly higher in the tumors in comparison to the corresponding normal tissues (1.822 ± 0.63 vs. 0.997 ± 0.38, P<0.05), and the levels of p-eIF4E were closely correlated with lymph node metastasis (p<0.05). The mTOR/4E-BP1 signaling pathway is activated in GCA, with mTOR activated mainly through increased mTOR phosphorylation rather than protein overexpression.

[Inhibitory effects of rapamycin on proliferation of chronic myelogenous leukemia cells and its mechanism].

OBJECTIVE: To explore the inhibitory effects of rapamycin on proliferation of chronic myelogenous leukemia (CML) cells and its possible mechanism. METHODS: The effects of rapamycin at various concentrations on cell proliferation of CML cell line K562 cells were analyzed by MTT. The expressions of mTOR, 4E-BP1 and p70S6K at protein and mRNA level in K562 cells with rapamycin treatment were detected by Western blot and RT-PCR. The protein expressions and phosphorylation of mTOR, 4E-BP1 and p70S6K in primary bone marrow cells from CML patients at chronic phase (CP) were also investigated by Western blot, bone marrow cells from healthy people were used as control. Data were analyzed by the χ(2) test, Fisher's exact test and one-way analysis of variance (ANOVA). RESULTS: The phosphorylation of mTOR, 4E-BP1 and p70S6K were significantly increased in CML bone marrow cells compared with that of normal control (70.6% vs 30.0%, 76.5% vs 40.0%, 73.5% vs 20.0%, respectively, P < 0.05). The proliferation of K562 cells was significantly inhibited with 20 nmol/L and more rapamycin treatment. The phosphorylation of mTOR was decreased after rapamycin treatment, as well as the expressions of 4E-BP1 and p70S6K at protein and mRNA level (P < 0.05). CONCLUSION: mTOR signaling played an important role in CML pathogenesis, and rapamycin could decrease CML cells proliferation by inhibiting the activity of mTOR signaling in vitro.

[Construction of human TAP1 expression vector and the effects of it on HLA-I expression in GES-1 cells in vitro].

AIM: To construct the human TAP1 expression vector and to evaluate its effects on HLA-I expression in GES-1 cells in vitro. METHODS: The human TAP1 expression vector (pcDNA3.1/V5-His-TAP1) was constructed by gene recombination technology. The expression of HLA-I on human gastric epithelial cell line (GES-1 cells) after transfection was detected by RT-PCR, Western blot and flow cytometry (FCM). RESULTS: Human full-length TAP1 gene was obtained from human peripheral blood mononuclear cells by RT-PCR reaction, then TAP1 gene was inserted into pcDNA3.1/V5-HisB vector to get the TAP1 expression vector (pcDNA3.1/V5-His-TAP1) by recombination technology including digestion with restriction enzymes, ligation and transformation. The vector was sequenced to ensure the sequence fidelity.To further evaluate the function of the TAP1 plasmid we constructed, GES-1 cells were selected as the target cell to be transfected. Firstly RT-PCR and Western blot results showed that the expression of TAP in GES-1 cells was increased after pcDNA3.1/V5-His-TAP1 transfection. Based on the high efficiency of transfection in GES-1 cell, we then detected the expression of HLA-I. The results showed that the expressions of HLA-A, HLA-B and HLA-C at mRNA level were all increased by TAP1 transfection, but no change was found in beta2m mRNA. HLA-I protein level was increased correspondingly with the TAP expression in cells by FCM and Western blot assay. CONCLUSION: The TAP1 expression vector was successfully constructed, and it can induce the expression of HLA-I on the GES-1 cells after TAP1 transfection. The results confirm that the TAP1 plays a cruical role in the HLA-I antigen expression and antigen presentation pathway.

[Analysis of the changing trends of frequency and localization of gastric cancers arising from different sites of the stomach in population of the high incidence area of esophageal and gastric cancers in Hebei province].

OBJECTIVE: To analyze the changing trends of frequency and localization of gastric cancers arising from the gastric cardia, corpus and antrum during the past 14 years in population of the high incidence area of esophageal and gastric carcinoma in Hebei province, China. METHODS: The clinicopathological data of 4334 cases of gastric carcinomas among the local residents of Cixian and Zanhuang counties, initially diagnosed in our department from 1993 to 2006, were retrospectively analyzed. The proportion of gastric carcinomas arising from the gastric cardia, corpus and antrum in different years and in patients with different sex and ages were analyzed and compared, and the changing trends of the frequency of gastric carcinoma arising from different sites of the stomach were statistically analyzed. RESULTS: Among all the 4334 gastric carcinomas, gastric cardia carcinoma accounted for 68.0%, significantly higher than that of corpus (24.2%) and antrum (7.9%; chi(2) = 124.396, P < 0.0001). An increasing tendency in the proportion of gastric cardia carcinoma from 1993 to 2006 was seen. The percentage of cardiac carcinoma in the high incidence area of esophageal carcinoma (Cixian county) was higher than that in the high incidence area of gastric cancer (Zanhuang county) (71.2% vs. 51.2%; chi(2) = 109.648, P < 0.0001). The increase in the incidence of cardiac carcinoma in Cixian county was mainly due to the increase of cardiac carcinoma from 1993 to 2006, while the contributing factor for the increase in the proportion of cardiac carcinomas was resulted from the decrease of incidence of antrum carcinoma in Zanhuang county during the same period. The occurring site of gastric carcinoma was related with age of patients (chi(2) = 58.380, P < 0.0001). The percentage of carcinoma of the gastric body was highest in < 50 year age group, while that in the gastric cardia was highest in 61 - 70 year age group (71.6%). CONCLUSION: The major occurring site of gastric carcinoma is the gastric cardia among the local residents in population of the high incidence areas of esophageal and gastric carcinomas during the past 14 years in Hebei province, China. The increasing trend of cardiac carcinoma and decreasing trend of corpus carcinoma in Cixian county and antrum carcinoma in Zanhuang county will be maintained in the coming years if the epidemiological conditions will not be changed.

Protective effects of endomorphins, endogenous opioid peptides in the brain, on human low density lipoprotein oxidation.

Neurodegenerative disorders are associated with oxidative stress. Low density lipoprotein (LDL) exists in the brain and is especially sensitive to oxidative damage. Oxidative modification of LDL has been implicated in the pathogenesis of neurodegenerative diseases. Therefore, protecting LDL from oxidation may be essential in the brain. The antioxidative effects of endomorphin 1 (EM1) and endomorphin 2 (EM2), endogenous opioid peptides in the brain, on LDL oxidation has been investigated in vitro. The peroxidation was initiated by either copper ions or a water-soluble initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes, thiobarbituric acid reactive substances, and the relative electrophoretic mobility. Low density lipoprotein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. Endomorphins markedly and in a concentration-dependent manner inhibited Cu2+ and AAPH induced the oxidation of LDL, due to the free radical scavenging effects of endomorphins. In all assay systems, EM1 was more potent than EM2 and l-glutathione, a major intracellular water-soluble antioxidant. We propose that endomorphins provide protection against free radical-induced neurodegenerative disorders.

Endomorphins, endogenous opioid peptides, induce apoptosis in human leukemia HL-60 cells.

Opioids play a role in the apoptosis machinery. We studied the induction of apoptosis in endomorphin 1 (EM1) and endomorphin 2 (EM2), 2 newly isolated endogenous mu-opioid receptor agonists. These endomorphins were able to reduce the viability of cultured HL-60 cells. The antiproliferative properties of endomorphins appeared to be attributable to their induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 indicated down-regulation, but the Bax, Fas, and FasL expression showed up-regulation as compared with the untreated control cells. These data support the idea that endomorphins induce apoptosis in HL-60 cells through the activation of the Bcl-2-Bax and the Fas-FasL pathway. We suggest that endomorphins may play an important role in the regulation of tumor cell death.