PSMB8 regulates glioma cell migration, proliferation, and apoptosis through modulating ERK1/2 and PI3K/AKT signaling pathways.

Glioma has been considered as one of the most aggressive and popular brain tumors of patients. It is essential to explore the mechanism of glioma. In this study, we established PSMB8 as a therapeutic target for glioma treatment. Expression of PSMB8 as well as Ki-67 was higher in glioma tissues demonstrated by western blot and immunohistochemistry. Then, the role of PSMB8 in migration and proliferation of glioma cells was investigated by conducting wound-healing, trans-well assay, cell counting kit (CCK)-8, flow cytometry assay and colony formation analysis. The data showed that interfering PSMB8 may inhibit the migration and proliferation of glioma cells by reducing expression of cyclin A, cyclin B1, cyclin D1, Vimentin, and N-cadherin, and by increasing expression of E-cadherin. Additionally, interfering PSMB8 may induce apoptosis of glioma cells by upregulating caspase-3 expression. Furthermore, these in vitro findings were validated in vivo and the ERK1/2 and PI3k/AKT signaling pathways were involved in PSMB8-triggered migration and proliferation of glioma cells. In an in vivo model, downregulation of PSMB8 suppressed tumor growth. In conclusion, PSMB8 is closely associated with migration, proliferation, and apoptosis of glioma cells, and might be considered as a novel prognostic indicator in patients with gliomas.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR). METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Mouse Feces.

Mouse models are widely used for studying gastrointestinal (GI) tract-related diseases. It is necessary and important to develop a new set of primers to monitor the mouse gut microbiota. In this study, 16S rRNA gene-targeted group-specific primers for Firmicutes, Actinobacteria, Bacteroidetes, Deferribacteres, "Candidatus Saccharibacteria," Verrucomicrobia, Tenericutes, and Proteobacteria were designed and validated for quantification of the predominant bacterial species in mouse feces by real-time PCR. After confirmation of their accuracy and specificity by high-throughput sequencing technologies, these primers were applied to quantify the changes in the fecal samples from a trinitrobenzene sulfonic acid-induced colitis mouse model. Our results showed that this approach efficiently predicted the occurrence of colitis, such as spontaneous chronic inflammatory bowel disease in transgenic mice. The set of primers developed in this study provides a simple and affordable method to monitor changes in the intestinal microbiota at the phylum level.

Protective Effect of Procyanidin B2 against CCl4-Induced Acute Liver Injury in Mice.

Procyanidin B2 has demonstrated several health benefits and medical properties. However, its protective effects against CCl4-induced hepatotoxicity have not been clarified. The present study aimed to investigate the hepatoprotective effects of procyanidin B2 in CCl4-treated mice. Our data showed that procyanidin B2 significantly decreased the CCl4-induced elevation of serum alanine aminotransferase activities, as well as improved hepatic histopathological abnormalities. Procyanidin B2 also significantly decreased the content of MDA but enhanced the activities of antioxidant enzymes SOD, CAT and GSH-Px. Further research demonstrated that procyanidin B2 decreased the expression of TNF-α, IL-1ß, cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as inhibited the translocation of nuclear factor-kappa B (NF-κB) p65 from the cytosol to the nuclear fraction in mouse liver. Moreover, CCl4-induced apoptosis in mouse liver was measured by (terminal-deoxynucleotidyl transferase mediated nick end labeling) TUNEL assay and the cleaved caspase-3. Meanwhile, the expression of apoptosis-related proteins Bax and Bcl-xL was analyzed by Western blot. Results showed that procyanidin B2 significantly inhibited CCl4-induced hepatocyte apoptosis, markedly suppressed the upregulation of Bax expression and restored the downregulation of Bcl-xL expression. Overall, the findings indicated that procyanidin B2 exhibited a protective effect on CCl4-induced hepatic injury by elevating the antioxidative defense potential and consequently suppressing the inflammatory response and apoptosis of liver tissues.

FADD is essential for glucose uptake and survival of thymocytes.

Fas-associated protein with death domain (FADD) has been implicated in T lymphocytes, but the nature of FADD-dependent mechanism in early T cell development has not been completely elucidated. In this study, using T-cell specific deletion mice, we observed that FADD deficiency in thymocytes led to increased apoptosis and reduced cell numbers, which may be attributed to the reduction of Glut1 expression and correspondingly decreased glucose uptake. Furthermore, an abnormal transduction of Akt signaling was discovered in FADD(-/-) thymocytes, which may be responsible for the declined Glut1 expression. Collectively, our results demonstrate the new function of FADD in glucose metabolism and survival of early T cells.

[Expression characteristics of microRNA in mice with schistosomiasis and praziquantel treatment].

OBJECTIVE: To investigate the expression characteristics of miR-155 and miR-146a in mice with schistosomiasis and praziquantel (PZQ) treatment. METHODS: Totally 40 BABL/c mice were divided into 4 groups: a normal group, a 6W infected group that were infected cutaneously with 10 Schistosoma japonicum cercariae for 6 weeks, a 12W infected group that were infected cutaneously with 10 Schistosoma japonicum cercariae for 12 weeks, and a praziquantel treated group that were infected cutaneously with 10 Schistosomajaponicum cercariae and intragastrically administered with PZQ (300 mg/kg/day) for 1 day in 6 weeks post-infection and continuing surviving 6 weeks. The animals were sacrificed in 6 weeks and 12 weeks post-infection respectively. The left front lobe of each liver was stained with hematoxylin-eosin (HE) to detect pathological lesions. The levels of mRNA expressions of miR-155, miR-146a and pro-inflammatory cytokines (TNF-alpha, IL-1beta and IL-6) in the liver tissue were determined by using quantitative real-time PCR. RESULTS: The levels of mRNA expressions of miR-155, miR-146a and pro-inflammatory cytokines (TNF-alpha, IL-1beta and IL-6) in the 6W infected mice were significantly higher than those of the normal mice and of the 12W infected mice. Compared with the 12W infected mice, the inflammation response of liver egg granuloma in the PZQ-treated mice was ameliorated. Furthermore, there was a marked increase in the levels of mRNA expressions of miR-155, miR-146a and three pro-inflammatory cytokines in the PZQ-treated mice compared to the 12W infected mice. CONCLUSION: miR-155 and miR-146a may play a role in schistosomiasis liver inflammation response and the inflammation regulation of praziquantel treatment.

Tumor-targeting Salmonella typhimurium, a natural tool for activation of prodrug 6MePdR and their combination therapy in murine melanoma model.

The PNP/6-methylpurine 2'-deoxyriboside (6MePdR) system is an efficient gene-directed enzyme prodrug therapy system with significant antitumor activities. In this system, Escherichia coli purine nucleoside phosphorylase (ePNP) activates nontoxic 6MePdR into potent antitumor drug 6-methylpurine (6MeP). The Salmonella typhimurium PNP (sPNP) gene has a 96-% sequence homology in comparison with ePNP and also has the ability to convert 6MePdR to 6MeP. In this study, we used tumor-targeting S. typhimurium VNP20009 expressing endogenous PNP gene constitutively to activate 6MePdR and a combination treatment of bacteria and prodrug in B16F10 melanoma model. The conversion of 6MePdR to 6MeP by S. typhimurium was analyzed by HPLC and the enzyme activity of sPNP was confirmed by in vitro (tetrazolium-based colorimetric assay) MTT cytotoxicity assay. After systemic administration of VNP20009 to mice, the bacteria largely accumulated and specifically delivered endogenous sPNP in the tumor. In comparison with VNP20009 or 6MePdR treatment alone, combined administration of VNP20009 followed by 6MePdR treatment significantly delayed the growth of B16F10 tumor and increased the CD8(+) T-cell infiltration. In summary, our results demonstrated that the combination therapy of S. typhimurium and prodrug 6MePdR is a promising strategy for cancer therapy.

Synergistic antitumor activity of oridonin and arsenic trioxide on hepatocellular carcinoma cells.

Although arsenic trioxide (As2O3) has been successfully employed in treatment of patients with APL (acute promyelocytic leukemia), the sensitivity of solid tumor cells to this treatment was much lower than APL cells. The single agent of As2O3 was inefficient for treatment of hepatocellular carcinoma (HCC) in phase II trial demonstrating that new modalities of treatment with enhanced therapeutic effect are needed. In this study, we showed that oridonin, a diterpenoid isolated from traditional Chinese medicine Rabdosia rubescences, greatly potentiated apoptosis induced by As2O3 in hepatocellular carcinoma cells. The synergistic pro-apoptosis effect of combination of these two drugs led to increase in intracellular reactive oxygen species (ROS) level and N-acetyl-L-cysteine (NAC), a thiol-containing anti-oxidant, was able to completely block the effect. The combination treatment induced ROS-dependent decrease in mitochondrial membrane potential (MMP) decrease, and relocation of Bax and cytochrome C. Besides, oridonin dramatically increased the intracellular Ca2+ overload triggered by As2O3. Furthermore, the co-treatment of oridonin and As2O3 induced ROS-mediated down-regulation of Akt and XIAP, and inhibition of NF-κB activation. The two drug combination enhanced tumor suppression activity in murine HCC model compared with single agent treatment in vivo. These findings demonstrate that oridonin can sensitize hepatocellular carcinoma cells to As2O3 treatment and will facilitate the optimization of As2O3 therapy for HCC patients.