Mammalian Metallothionein-2A and Oxidative Stress.

Mammalian metallothionein-2A (MT2A) has received considerable attention in recent years due to its crucial pathophysiological role in anti-oxidant, anti-apoptosis, detoxification and anti-inflammation. For many years, most studies evaluating the effects of MT2A have focused on reactive oxygen species (ROS), as second messengers that lead to oxidative stress injury of cells and tissues. Recent studies have highlighted that oxidative stress could activate mitogen-activated protein kinases (MAPKs), and MT2A, as a mediator of MAPKs, to regulate the pathogenesis of various diseases. However, the molecule mechanism of MT2A remains elusive. A deeper understanding of the functional, biochemical and molecular characteristics of MT2A would be identified, in order to bring new opportunities for oxidative stress therapy.

[Corrigendum] SCF increases cardiac stem cell migration through PI3K/AKT and MMP­2/­9 signaling.

Following the publication of this article, the authors have realized that they mistakenly used the total AKT blot featured in Fig. 4A for the GAPDH blot in Fig. 3B on p. 116. The corrected version of Fig. 3, featured the correct data for the GAPDH experiment, is shown opposite. The authors regret that this error was not picked up upon before the paper was sent to press, and thank the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish a corrigendum. The error did not affect either the results or the conclusions reported in the study, and all the authors agree to this corrigendum. Furthermore, they regret any inconvenience caused to the readership. [the original article was published in International Journal of Molecular Medicine 34: 112­118, 2014; DOI: 10.3892/ijmm.2014.1773].

High glucose promotes vascular smooth muscle cell proliferation by upregulating proto-oncogene serine/threonine-protein kinase Pim-1 expression.

Serine/threonine kinase proviral integration site for Moloney murine leukemia virus 1 (Pim-1) plays an essential role in arterial wall cell proliferation and associated vascular diseases, including pulmonary arterial hypertension and aortic wall neointima formation. Here we tested a role of Pim-1 in high-glucose (HG)-mediated vascular smooth muscle cell (VSMC) proliferation. Pim-1 and proliferating cell nuclear antigen (PCNA) expression levels in arterial samples from streptozotocin-induced hyperglycemia rats were increased, compared with their weak expression in normoglycemic groups. In cultured rat VSMCs, HG led to transient Pim-1 expression decline, followed by sustained expression increase at both transcriptional and translational levels. Immunoblot analysis demonstrated that HG increased the expression of the 33-kDa isoform of Pim-1, but at much less extent to its 44-kDa plasma membrane isoform. D-glucose at a concentration of 25 mmol/L showed highest activity in stimulating Pim-1 expression. Both Pim-1 inhibitor quercetagetin and STAT3 inhibitor stattic significantly attenuated HG-induced VSMC proliferation and arrested cell cycle progression at the G1 phase. Quercetagetin showed no effect on Pim-1 expression but decreased the phosphorylated-Bad (T112)/Bad ratio in HG-treated VSMCs. However, stattic decreased phosphorylated-STAT3 (Y705) levels and caused transcriptional and translational down-regulation of Pim-1 in HG-treated VSMCs. Our findings suggest HG-mediated Pim-1 expression contributes to VSMC proliferation, which may be partly due to the activation of STAT3/Pim-1 signaling.

Effects of high glucose on expression of OPG and RANKL in rat aortic vascular smooth muscle cells.

OBJECTIVE: To explore effect of high glucose on expression of osteoprotegerin (OPG) and receptor activator of NF- κ B ligand (RANKE) in rat aortic vascular smooth muscle cells. METHODS: SD rats were intraperitoneally injected with streptozotocin, OPG and RANKL expression in rat thoracic aortas were detected by immunohistochemical staining. In cultured vascular smooth muscle cells (VSMCs) (A7r5), qRT-PCR and Western blot analysis were used to examine the mRNA and protein levels of OPG and RANKL. RESULTS: Our results demonstrated that OPG expression was increased in hyperglycemic rat aortic VSMCs, while RANKL expression was decreased. Besides, in vitro experiments high glucose induced OPG expression, but depressed RANKL expression by dose- and time-dependent manner in cultured A7r5. CONCLUSIONS: Our findings suggested that high glucose could promote the expression of OPG, and inhibit the expression of RANKL in VSMCs, which may be partly be the molecular mechanism of diabetic vascular calcification.

SCF increases cardiac stem cell migration through PI3K/AKT and MMP­2/­9 signaling.

The transplantation of cardiac stem cells (CSCs) is thought to be responsible for improving the performance of injured heart induced by myocardial infarction (MI). However, the mechanisms involved in the migration of activated CSCs post­MI remain to be clarified. In this study, CSCs were isolated from rat hearts and a cellular migration assay was performed using a 24­well Transwell system. Stem cell factor (SCF) induced CSC migration in a concentration­dependent manner, which could be blocked with an SCF antibody as well as a PI3K/AKT inhibitor, LY294002. Moreover, SCF induced the expression and activity of matrix metalloproteinase (MMP)­2 and MMP­9 in a concentration­ and time­dependent manner, as measured by quantitative RT­PCR, western blot analysis and gelatin zymography. Results of western blot analysis revealed phosphorylated AKT was markedly increased in SCF­treated CSCs and that inhibition of SCF/c­Kit signaling or phospho­AKT activity significantly attenuated the SCF­induced expression of MMP­2 and MMP­9. Thus, our results showed that SCF partially mediated CSC migration via the activation of PI3K/AKT/MMP­2/­9 signaling.

p42/p44 mitogen-activated protein kinases inhibit atrial natriuretic peptide mRNA transcription in gp130-mediated hypertrophic ventricular myocytes.

OBJECTIVE: To understand the role of ANP mRNA transcription regulation in gp130-mediated cardiomyocyte hypertrophy, and the involved mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK, also called p42/p44 MAPK) signaling pathway. METHODS: Isolated neonatal ventricular myocytes were treated with different concentrations of CT-1 (10(-9), 10(-8)and 10(-7)mol/L). MTT was used to analyze the viability and RT-PCR was used to detect ANP mRNA levels in cardiomyocyte. To inhibit p42/p44 MAPK activity in hypertrophic cardiomyocytes, the cells were pretreated with a specific MEK1 inhibitor. RESULTS: CT-1 significantly induced ANP mRNA expression and the viability of cardiomyocytes in a dose- and time-dependent manner. Furthermore, blocking p42/p44 MAPK activity by the special MEK1 inhibitor upregulated the ANP mRNA. CONCLUSIONS: p42/p44 MAPK have an important role in suppressing ANP mRNA transcription and cell activity in gp130-mediated hypertrophic ventricular myocytes.

Construction and characterization of a full-lengh cDNA library from non-fresh Giardia lamblia.

OBJECTIVE: To construct rapidly a full-length cDNA library from nanogram amounts total RNA of Giardia lamblia (G. lamblia) trophozoites stocked in RNA stabilization reagent. METHODS: Total RNA of Giardia was extracted using Trizol reagent. A full-length cDNA library of G. lamblia trophozoites was constructed by a long-distance PCR (LD-PCR) method. The recombinant rate and the coverage rate of full-length clones of the library were evaluated. The inserted fragments were identified and sequenced by PCR amplification. RESULTS: The titer of cDNA library was 3.85 × 10(7) pfu/mL. The length of inserted fragments ranged from 0.4 to 2.5 kb, and the recombination efficiency accounted for 100% (20/20). The coverage rate of full-length clones is high (17/20). CONCLUSIONS: The RNA stabilization reagent may be used to fix the cells and prevent the RNA in cells even though delivered under normal atmospheric temperature. The long-distance PCR can be used to construct a full-length cDNA library rapidly and it needs less RNA than the traditional method from mRNA.

Inhibition of Pim-1 attenuates the proliferation and migration in nasopharyngeal carcinoma cells.

OBJECTIVE: To explore the role of proto-oncogene Pim-1 in the proliferation and migration of nasopharyngeal carcinoma (NPC) cells. METHODS: Pim-1 expressions in NPC cell lines CNE1, CNE1-GL, CNE-2Z and C666-1 were examined by RT-PCR, western blotting and immunoflucesence, respectively. After CNE1, CNE1-GL and C666-1 cells were treated with different concentrations of Pim-1 special inhibitor, quercetagetin, the cell viability, colony formation rate and migration ability were analyzed. RESULTS: Pim-1 expression was negative in well-differentiated CNE1 cells, whereas expressed weakly positive in poor-differentiated CNE-2Z cells and strongly positive in undifferentiated C666-1 cells. Interestingly, CNE1-GL cells that derived from CNE1 transfected with an Epstein Barr virus latent membrane protein-1 over-expression plasmid displayed stronger expression of Pim-1. Treatment of CNE1-GL and C666-1 cells with quercetagetin significantly decreased the cell viability, colony formation rate and migration ability but not the CNE1 cells. CONCLUSIONS: These findings suggest that Pim-1 overexpression contributes to NPC proliferation and migration, and targeting Pim-1 may be a potential treatment for anti-Pim-1-expressed NPCs.

Toxicarioside A inhibits SGC-7901 proliferation, migration and invasion via NF-κB/bFGF signaling.

AIM: To investigate the inhibitory role of toxicarioside A on the gastric cancer cell line human gastric cancer cell line (SGC-7901) and determine the underlying molecular mechanism. METHODS: After SGC-7901 cells were treated with toxicarioside A at various concentrations (0.5, 1.5, 4.5, 9.0 µg/mL) for 24 h or 48 h, cell viability was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, and the motility and invasion of tumor cells were assessed by the Transwell chamber assay. Immunofluorescence staining, reverse transcription polymerase chain reaction and Western blotting were performed to detect the expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR1), and nuclear factor-kappa B (NF-κB) activation was examined by electrophoretic mobility shift assay. RESULTS: The results showed that toxicarioside A was capable of reducing cell viability, inhibiting cell growth, and suppressing cell migration and invasion activities in a time- and dose-dependent manner in SGC-7901 cells. Further analysis revealed that not only the expression of bFGF and its high-affinity receptor FGFR1 but also the NF-κB-DNA binding activity were effectively blocked by toxicarioside A in a dose-dependent manner compared with the control group (P < 0.05 or P < 0.01). Interestingly, application of the NF-κB specific inhibitor, pyrrolidinedithiocarbamate (PDTC), to SGC-7901 cells significantly potentized the toxicarioside A-induced down-regulation of bFGF compared with the control group (P < 0.05). CONCLUSION: These findings suggest that toxicarioside A has an anti-gastric cancer activity and this effect may be achieved partly through down-regulation of NF-κB and bFGF/FGFR1 signaling.