Porphyromonas gingivalis infection exacerbates oesophageal cancer and promotes resistance to neoadjuvant chemotherapy.

BACKGROUND: The effect of Porphyromonas gingivalis (Pg) infection on oesophageal squamous cell carcinoma (ESCC) prognosis, chemotherapeutic efficacy, and oesophageal cancer cell apoptosis resistance and proliferation remain poorly understood. METHODS: Clinicopathological data from 312 ESCC oesophagectomy patients, along with the computed tomography imaging results and longitudinal cancerous tissue samples from a patient subset (n = 85) who received neoadjuvant chemotherapy (NACT), were analysed. Comparison of overall survival and response rate to NACT between Pg-infected and Pg-uninfected patients was made by multivariate Cox analysis and Response Evaluation Criteria in Solid Tumours v.1.1 criteria. The influence of Pg on cell proliferation and drug-induced apoptosis was examined in ESCC patients and validated in vitro and in vivo. RESULTS: The 5-year overall survival was lower in Pg-positive patients, and infection was associated with multiple clinicopathological factors and pathologic tumour, node, metastasis stage. Of the 85 patients who received NACT, Pg infection was associated with a lower response rate and 5-year overall survival. Infection with Pg resulted in apoptosis resistance in ESCC and promoted ESCC cell viability, which was confirmed in longitudinal cancerous tissue samples. Pg-induced apoptosis resistance was dependent on fimbriae and STAT3. CONCLUSIONS: Pg infection is associated with a worse ESCC prognosis, reduced chemotherapy efficacy, and can potentiate the aggressive behaviour of ESCC cells.

Oxysophocarpine reduces oxidative stress and inflammation in tuberculosis-infected neutrophils and mouse lungs.

Tuberculosis (TB) is a chronic inflammatory infectious disease caused by Mycobacterium tuberculosis (Mtb), which induces irreversible pulmonary damage. Oxysophocarpine (OSC) is a natural alkaloid that exhibits multiple pharmacological activities, including anti-inflammation; however, the protective effects of OSC against TB and the mechanisms involved are unknown. Here, we established murine and cellular models of TB with C3HeB/FeJ mice and neutrophils infected with H37Rv to investigate the biological functions of OSC in TB. We found that OSC reduced the mortality, inhibited the pulmonary H37Rv growth, and alleviated the lung pathology injury in the Mtb-infected mice. OSC also repressed neutrophil recruitment to the lesions of the Mtb-infected mice as evidenced by a decrease in the number and percentage of neutrophils in the lungs. OSC hampered the production of proinflammatory cytokines and chemokines, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, macrophage inflammatory protein-2 (MIP-2), granulocyte colony stimulating factor (G-CSF), and keratinocyte chemoattractant (KC) in the lungs of Mtb-infected mice. The results of the in vitro experiments showed that OSC repressed the adhesion and F-actin polymerization of the Mtb-infected neutrophils by inhibiting the toll-like receptor 2/myeloid differentiation primary response gene 88/Src/extracellular signal-regulated kinase 1/2 signaling. Moreover, OSC abolished the Mtb-induced expression and release of TNF-α, IL-1ß, IL-6, MIP-2, G-CSF, and KC in neutrophils. Overall, these findings indicate that OSC can treat TB partly by lessening the neutrophilic recruitment and inflammation.

The novel targets of DL-3-n-butylphthalide predicted by similarity ensemble approach in combination with molecular docking study.

BACKGROUND: DL-3-n-butylphthalide (NBP) is a drug for treating acute ischemic stroke, and may play a neuroprotective role by acting on multiple active targets. The aim of this study was to predict the target proteins of NBP in mammalian cells. METHODS: The similarity ensemble approach search tool (SEArch), one of the commonly used public bioinformatics tools for target prediction, was employed in the experiment. The molecular docking of NBP to target proteins was performed by using the three-dimensional (3-D) crystal structure, substrate free. The software AutoDock Vina was used for all dockings. The binding targets of NBP were illustrated as 3-D and 2-D diagrams. RESULTS: Firstly, the results showed that NBP bounded to the same binding site on NAD(P)H quinone oxidoreductases (NQO1) as the substrate FAD, leading to competitive inhibition for the catalytic site with -7.2 kcal/mol. This might break the 3-D structure of NQO1 and bring about P53 degradation, resulting in a decrease of p53-mediated apoptosis in ischemic brain cells. Secondly, NBP might exert its therapeutic effect on acute ischemic stroke via modulating indoleamine 2,3-dioxygenase (IDO) bioactivity after associating with it. NBP could alleviate the depression following ischemic stroke by inhibiting IDO. Thirdly, NBP might modulate the function of NADH-ubiquinone oxidoreductase by competitively embedding itself into this complex, further affecting mitochondrial respiration in cerebrovascular diseases as an anti-oxidant agent. CONCLUSIONS: Three potential target proteins of NBP were identified, which may provide a novel aspect for better understanding the protective effects of NBP on the nervous system at the molecular level.

Niche astrocytes promote the survival, proliferation and neuronal differentiation of co-transplanted neural stem cells following ischemic stroke in rats.

Niche astrocytes have been reported to promote neuronal differentiation through juxtacrine signaling. However, the effects of astrocytes on neuronal differentiation following ischemic stroke are not fully understood. In the present study, transplanted astrocytes and neural stem cells (NSCs) were transplanted into the ischemic striatum of transient middle cerebral artery occlusion (MCAO) model rats 48 h following surgery. It was observed that the co-transplantation of astrocytes and NSCs resulted in a higher ratio of survival and proliferation of the transplanted NSCs, and neuronal differentiation, in MCAO rats compared with NSC transplantation alone. These results demonstrate that the co-administration of astrocytes promotes the survival and neuronal differentiation of NSCs in the ischemic brain. These results suggest that the co-transplantation of astrocytes and NSCs is more effective than NSCs alone in the production of neurons following ischemic stroke in rats.

Overexpression of ß-Catenin Induces Cisplatin Resistance in Oral Squamous Cell Carcinoma.

Abnormal expression of ß-catenin contributes to tumor development, progression, and metastasis in various cancers. However, little is known about the relationship between abnormal expression of ß-catenin and cisplatin chemotherapy in oral squamous cell carcinoma (OSCC). The present study aimed to investigate the effect of ß-catenin on OSCC cisplatin resistance and evaluated the drug susceptibility of stable cell lines with ß-catenin knockin and knockdown. In this study, we found that higher expression level of ß-catenin can be observed in CDDP-treated cell lines as compared with the control group. Furthermore, the expression levels of ß-catenin increased in both a concentration- and time-dependent manner with the cisplatin treatment. More importantly, the nuclear translocation of ß-catenin could also be observed by confocal microscope analysis. Stable cell lines with CTNNB1 knockin and knockdown were established to further investigate the potential role and mechanism of ß-catenin in the chemoresistance of OSCC in vitro and in vivo. Our findings indicated that overexpression of ß-catenin promoted cisplatin resistance in OSCC in vitro and in vivo. We confirmed that GSK-3ß, C-myc, Bcl-2, P-gp, and MRP-1 were involved in ß-catenin-mediated drug resistance. Our findings indicate that the Wnt/ß-catenin signaling pathway may play important roles in cisplatin resistance in OSCC.

Dissociation of E-cadherin/ß-catenin complex by MG132 and bortezomib enhances CDDP induced cell death in oral cancer SCC-25 cells.

E-cadherin/ß-catenin complex plays an important role in maintaining the homeostasis of tissues and regulating cell proliferation, survival and apoptosis. To address the relationships between the change of E-cadherin/ß-catenin complex and cell apoptosis, human oral squamous carcinoma SCC-25 cells were used to investigate whether the dissociation of the E-cadherin/ß-catenin complex was the main reason of MG132- or bortezomib-induced apoptosis. We found that MG132 or bortezomib alone induced remarkable loss of cell integrity and contact, inhibited cell growth, survival, migration and caused cell cycle arrest, intracellular ROS production. Further experiments showed that colony formations were significantly decreased by MG132 and bortezomib alone or plus cis-diaminedichloroplatinum (CDDP). Immunofluorescence staining showed that SCC-25 cells exhibited remarkable accumulations of ß-catenin in cytoplasm and few E-cadherin in cell membranes after MG132 or bortezomib treatment. Western blot results showed that MG132 or bortezomib induced high accumulation of ubiquitinated proteins and activation of apoptosis related protein caspase-3. Meanwhile, the combinational use of MG132 or bortezomib with CDDP led to synergistic effects on SCC-25 cells. However, knockdown of ß-catenin could decrease MG132 or bortezomib induced cell death. Taken together, our data suggest that the regulation of E-cadherin/ß-catenin complex could be a promising therapeutic target to overcome the multidrug resistance of oral cancer.

Progranulin expression in neural stem cells and their differentiated cell lineages: an immunocytochemical study.

Progranulin (PGRN) is a neurotrophic factor that regulates neurite outgrowth and enhances neuronal survival. The association between PGRN and neural stem cell (NSC) differentiation may aid in elucidating the underlying pathogenesis and potential treatments for neurodegenerative diseases. To investigate the association between PGRN and NSCs and their lineages, primary NSCs were prepared from the subventricular zone of neonatal Sprague Dawley pups (age, 1 day) and cultured in the neural stem cell medium. After 7 days in culture, NSCs aggregated into neurospheres and were maintained in the differential medium for 7 days following three passages. In addition, PGRN expression in neurospheres and differentiated cells (neurons, astrocytes and oligodendrocytes) were analyzed by immunocytochemistry. The PGRN expression and localization was also investigated in the brain tissues of neonatal rats (age, 1 and 7 days) by double immunofluorescence staining. The data indicated that PGRN was highly expressed in NSCs and their differentiated cell lineages in vitro. The results also demonstrated that PGRN was predominantly expressed in neurons and microglia in vivo, and marginally in NSCs, astrocytes and oligodendrocytes. The present study suggested that PGRN may be involved in the regulation of NSC differentiation.

Transient focal cerebral ischemia upregulates immunoproteasomal subunits.

This study was designed to determine whether focal cerebral ischemia alters the expression of the immunoproteasomal (i-proteasomal) subunits. Transient cerebral ischemia significantly increased the expression of the i-proteasomal subunits, 20S ß1i (LMP2) and ß5i (LMP7) in the parietal cortex and hippocampus. This alteration was associated with a remarkable increase in ubiquitinated proteins. It is likely that the postischemic induction of the i-proteasome plays an important role in coping with the damaged proteins and thus may have an important effect on neuronal survival and death.

Exocytosis of MTT formazan could exacerbate cell injury.

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method is one of the most widely used methods to analyze cell proliferation and viability. It is taken up through endocytosis and is reduced by mitochondrial enzymes as well as endosomal/lysosomal compartments, then is transported to cell surfaces to form needle-like MTT formazans; however the effect of MTT itself still remains elusive. Our objective was to investigate the direct effects of MTT on in vitro SH-SY5Y cells. Results showed that the endocytosis of MTT did not cause obvious lesion and induce cell death, but the metabolism and exocytosis of MTT could dramatically damage cells. Our results also indicated that MTT could activate apoptosis related factors such as caspase-8, caspase-3 or accelerate the leakage of cell contents after the appearance of MTT formazan crystals. The present data suggest MTT method should be carefully chosen; otherwise the cell viability would be underestimated and incomparable.

Effects of DL-3-n-butylphthalide on vascular dementia and angiogenesis.

3-n-Butylphthalide (NBP) is a compound extracted from Chinese celery and is used as an anti-hypertensive herbal medicine for treating stroke patients. The aim of this study is to demonstrate the effects and mechanisms of this compound through in vitro and in vivo experiments. Culture experiments were performed by adding hydrogen peroxide (H(2)O(2)) to SH-SY5Y cells. From the MTT assay result, enhanced cell survival was observed with DL-NBP treatment, regardless of whether they are added before, simultaneously with or after the addition of H(2)O(2). For the in vivo experiment, Spontaneously Hypertensive rats and Wistar Kyoto control rats with chronic cerebral ischemia, which were induced by bilateral transection of the common carotid arteries, were given DL-NBP. Their performances in the place navigation test and spatial probe test in the Morris Water Maze have significantly improved compared with the DL-NBP untreated animals, indicating an improvement in spatial learning and memory in the ischemic-animals. In addition, in the chick embryonic chorioallantoic membrane assay, angiogenesis was more vigorous under the effects of DL-NBP, together with increased expression of growth factors, VEGF, VEGF-receptor and bFGF. All these suggested that one of the mechanisms of DL-NBP might be ameliorating vascular dementia and promoting angiogenesis.

The toxic effect of ketamine on SH-SY5Y neuroblastoma cell line and human neuron.

Ketamine used as an injectable anesthetic in human and animal medicine is also a recreational drug used primarily by young adults often at all night dance parties in nightclubs. The percentage of ketamine users has grown very fast in the last 5 years worldwide. However, this leads to the serious question of the long-term adverse effects of ketamine on our nervous system, particularly the brain, because ketamine as an NMDA antagonist could cause neurons to commit apoptosis. Our study therefore aimed to find out the chronic effect of ketamine on neuron using prolonged incubation (48 h) of neuronal cells with ketamine in culture. Our results showed that differentiated neuronal cells were prone to the toxicity of ketamine but probably less susceptible than undifferentiated neuronal cells and fibroblasts. This suggested that the ketamine abuse would be harmful to many other organs as well as the brain. Our results also confirmed that the toxicity of ketamine is related to apoptosis via the Bax/Bcl-2 ratio pathway and caspase-3 in the differentiated neuronal cells. Therefore, long-term ketamine treated cell or animal models should be sought to study this multiorgan effects of ketamine.

Pien Tze Huang, a composite Chinese traditional herbal extract, affects survival of neuroblastoma cells.

Pien Tze Huang is a popular Chinese medicine for liver diseases. In the investigations of possible effects of Pien Tze Huang on the central nervous system, we first studied the in vitro anti-cancer activity of Pien Tze Huang on neuroblastoma cells (SH-SY5Y) as compared with normal fibroblasts (NIH-3T3). Results showed that Pien Tze Huang significantly decreased (p < .05) cell survival of SH-SY5Y as compared to NIH-3T3. Furthermore, the decreases in cell survival of SH-SY5Y were significantly and linearly dose-dependent (p < .05) from 400 to 1,000 microg/ml. This supports further in vivo and animal studies for anti-cancer effect, neuroprotection, and their mechanisms.

Oxidative stress on the astrocytes in culture derived from a senescence accelerated mouse strain.

Astrocytes are one of the predominant glial cell types in the adult central nervous system functioning as both supportive and metabolic cells for the brain. Our objective in this experiment is to study the direct effects of hydrogen peroxide induced oxidative stress on astrocytes in culture. These astrocytes were derived from both an aged mouse strain (P8) and a matched control strain (R1). The astrocytes for both the P8 and R1 strains were treated with increasing concentrations of hydrogen peroxide. Our results showed that the oxidative stress had a similar effect in both strains of astrocytes; decreases in 3-(4,5-dimethylthiazol-2-yl)-2,2-diphenyltetrazolium bromide (MTT) and glial fibrillary acidic protein (GFAP) levels, and increases in terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling (TUNEL) staining, lactate dehydrogenase (LDH) staining, and superoxide dismutase (SOD), caspase-3 and B-cell lymphoma 2-associated protein X (bax) levels. At a hydrogen peroxide concentration of 400 microM , the differences of the above parameters between P8 cultures and R1 cultures were statistically significant (p<0.05). This strongly suggested that astrocytes derived from P8 and R1 strains reacted to oxidative stress with similar mechanisms and consequences. However, the mechanisms were not able to compensate for the oxidative stress in the P8 strain at a hydrogen peroxide concentration of 400 microM. The inability of the P8 astrocytes to counteract the oxidative stress might lead to inadequate protection from neuronal loss possibly resulting in significantly more astrocytic death. Our results suggested that the changes of astrocytes in peroxide detoxification may play a role in aging of the central nervous system, and further aging studies should examine the oxidative status of the samples.