[Role of microRNA-155 in Brain Metastasis of Hypoxic Lung Cancer].

OBJECTIVE: To clarify the relationship between hypoxia stress and the microRNA-155 released from lung cancer cells, to reveal the possible mechanism of brain metastases of lung cancer. METHODS: The hypoxia model of A549 lung cancer cells was established. Lung cancer cells were cultured under the hypoxia condition or normal oxygen condition as control for 0.5, 2, 4, 8, 12 and 24 h immunofluorescence and Western blot methods were used to determine the expression level of heat shock protein 70 (hsp70) in lung cancer cells. Hsp70 overexpressed lung cancer cell line was established, the levels of microRNA-155 in A549 and hsp70 overexpressed A549 cell culture medium were determined by qRT-PCR.An in vitro blood-brain barrier model was established, and was treated with A549 cell culture medium collected at different hypoxia time points. Horseradish peroxidase (HRP) was used to detect the changes of permeability of the in vitro blood-brain barrier model, automatic cell technical instrument was used to count A549 lung cancer cells in the culture medium in under Transwell room. Culture mediums of A549 lung cancer cells collected at different hypoxia time points were injected into rats via tail vein, Western blot was used to analyze the expression of occludin in brain tissue, Evans blue was used to detect the change of blood-brain barrier permeability in animals. RESULTS: When lung cancer cells were hypoxic cultured for 8 h, both the expression level of hsp70 in lung cancer cells and microRNA-155 in culture medium reached the highest level (P < 0.05). Compared with A549 cells, the enhancement of microRNA-155 level in culture medium of hsp70 overexpressed cell was more notably under hypoxia condition. At the same time, the permeability of blood-brain barrier was the highest, and the number of lung cancer cells crossed the blood-brain barrier model was the most. In animal experiment, after injection the lung cancer cell culture fluid with hypoxia 8 h, the tight junction protein occludin expression in blood-brain barrier was lowest, and the permeability of blood-brain barrier was the largest. CONCLUSION: Hypoxia can cause the increase of hsp70 production in lung cancer cells. Increased hsp70 promotes the synthesis and release of microRNA-155, which in turn leads to reduced expression of occludin protein in the blood-brain barrier, resulting in increased permeability of the blood-brain barrier and eventually causing lung cancer cells to metastasize into the brain.

[Effect of Hypoxia Inducible Factor-1 Alpha on Brain Metastasis from Lung Cancer and Its Mechanism].

OBJECTIVE: To study the relationship between hypoxia and the hypoxia inducible factor-1α (HIF-1α) from lung cancer cells, to reveal the possible mechanism of brain metastases of lung cancer. METHODS: The hypoxia model of A549 lung cancer cells was established. After hypoxia culture of A549 cells for 0.5, 2, 4, 8, 12 and 24 h (normal oxygen culture at the same time point was set as the control group), the mass concentration of HIF-1α in A549 lung cancer cell culture medium were determined by ELISA. Transwell chamber was used to construct an in vitro blood brain barrier model, was treated with A549 lung cancer cell culture medium after different time points of hypoxia, Tran endothelial resistance (TER) change of blood-brain barrier model in instrument, to reflect the changes of blood-brain barrier permeability in vitro; A549 lung cancer cells in the culture medium were counted under Transwell room. A549 lung cancer cells with hypoxia at different time points injected into Wistar rats via tail vein, Western blot method was used to menstruate expression of tight junction associated protein Claudin-5 in the brain tissues, Evans blue to detect the change of blood brain barrier permeability in rats. RESULTS: Compared with the control group, the HIF-1α mass concentration in the cell culture solution of A549 increased, the in vitro blood-brain barrier model TER decreased, and the cell number of A549 that passed through transwell into the lower chamber increased (all P<0.05) after hypoxia 2 h, the above effect was most obvious when hypoxia 8 h (all P<0.01). After hypoxia 24 h, it was restored to the control group level. In the in vivo experiment of rats, compared with the control group, the mass percent of Evans blue in rat brain tissues increased after A549 cell culture solution with hypoxia 2 h was injected via caudal vein, meaning increased the permeability of rat blood brain barrier, while the expression of Claudin-5 protein in rat brain tissues decreased (all P<0.05). The effect was most obvious when A549 cell culture solution with hypoxia 8 h was injected into rat tail vein (P<0.01 ). Ejectionof hypoxia 24 h A549 cell culture solution yielded the same effects as those in the control group. CONCLUSION: Hypoxia can induce the increase of HIF-1α in lung cancer cells. The increase of HIF-1α results in the decrease of Claudin-5 expression and increase of blood-brain barrier permeability, leading to lung cancer cells metastasis into the brain.

[Effect of Aspirin on Brain Metastasis of Lung Cancer and Its Possible Mechanism].

OBJECTIVE: To determine the effect of prostaglandin E2 (PGE2) on brain metastasis of lung cancer,and to explore the possible mechanism of aspirin (PGE2 inhibitor) reducing brain metastasis of lung cancer. METHODS: Radioimmunoassay was performed to measure the expression level of PGE2 in cell supernatant collected from cells treated with or without aspirin (8 mmol/L) at different time points. After establishing in vitro blood-brain barrier (BBB) model using Transwell, lung cancer cells was added to upper chamber of transwell and was then treated with aspirin (8 mmol/L). Western blot was used to examine the expression of occludin protein in brain microvascular endothelial cells. The permeability changes of BBB model in vitro were determined using horseradish peroxides. The number of lung cancer cells passing through BBB model in vitro was counted with Hemocytometer. Effect of aspirin on brain metastasis of lung cancer was observed in nude mice in the animal level. RESULTS: PGE2 level decreased and reached minimum level 120 min after aspirin treatment in lung cancer cells culture fluid. Occludin expression increased and reached maximum level 120 min after aspirin treatment in brain microvascular endothelial cells. At the same time,permeability of BBB and number of lung cancer cells passing through BBB also reached the lowest value 120 min after aspirin treatment. Aspirin significantly reduced the incidence of brain metastasis of lung cancer in animal model. CONCLUSION: Aspirin reduced occurrences of the brain metastasis of lung cancer in animal model,which may be caused by inhibition of PGE2 released by lung cancer cells and upregulation of occludin expression therefore leading to decrease in BBB permeability.

[Effect of Brucea Javanica Oil Emulsion on the Invasiveness of Glioma Cells and Its Possible Mechanism].

OBJECTIVE: To investigate the effect of brucea javanica oil emulsion on the invasiveness of glioma cells and hosphatidylinositol 3 kinase/protein kinase B (PI3K/AKT) signaling pathway. METHODS: C6 glioma cells were treated by brucea javanica oil emulsion. The inhibition rate of glioma cells was detected by MTT, Western blot was used to detect protein expression levels of PI3K, AKT, and nuclear factor (NF)-κB in glioma cells. The number of the glioma cells migrated through polycarbonate membrane was detected by crystal violet staining method. RESULTS: Brucea javanica oil emulsion inhibited PI3K, AKT, and NF-κB protein expression which reached the highest inhibition at 30 min, 60 min, and 120 min after brucea javanica oil emulsion, respectively. Maximum suppression on the proliferation of C6 glioma cells reached at 180 min after brucea javanica oil emulsion, while the number of glioma cells migrated through polycarbonate membrane was the least. CONCLUSION: Brucea javanica oil emulsion inhibit the proliferation and invasiveness of glioma cells, which may be related to the inhibition of PI3K/AKT signal pathway.

[The Methylation of p16 Gene Promoter in Carcinogenesis and Development of Breast Cancer].

OBJECTIVE: To investigate the protein expression of the p16 gene and the methylation of its promoter in breast cancer, and to analyze the correlation between the p16 DNA methylation and the clinicopathological features. METHODS: Immuno-histochemistry technique (SP method) and methylation-specific-PCR (MSP) were used to detect p16 protein expression and the methylation of the p16 promoter in 47 breast cancer samples as well as in 20 hyperplasia samples of mammary glands. Results The p16 protein expression in breast cancer samples significantly lower when compared with those of hyperplasia samples (48. 9% vs. 70. 0%) and p16 methylation was more frequent in breast-tumor tissues when compared with those of hyperplasia samples (38. 3% vs. 20. 0%), but the statistical significance wasn't found (P> 0. 05). Down-regulation of p16 protein was negatively correlation with p16 gene hypermethylation (r= -0. 33, P =0. 02). Meanwhile, p16 methylation in breast cancer tissues correlated with histological type, lymph node metastasis, but not correlated with the age, tumor diameter, TNM stage, expression of estrogen receptor (ER) and progesterone receptor (PR) gene status. CONCLUSION: The downregulation of p16 protein induced by promoter methylation of p16 gene may not contribute to early cancinogenesis, but may contribute to progression of breast cancer.

The molecular mechanism of dexamethasone-mediated effect on the blood-brain tumor barrier permeability in a rat brain tumor model.

This study was performed to determine whether dexamethasone (DEX) had an effect on calcium-activated potassium channels (K(Ca) channels) and Occludin protein in blood-brain tumor barrier (BTB). Using a rat brain glioma model, we found that the expression of K(Ca) channels protein and Occludin protein was significantly increased in brain tumor tissue after DEX treatment for 3 days. Compared with non-DEX-treated animals, Evans Blue levels were greatly attenuated in DEX-treated animals. These effects were significantly reversed by the glucocorticoid receptor antagonist RU38486. In addition, DEX treatment enhanced the density of I(KCa) in the rat brain microvascular endothelial cells (RBMECs) in vitro BTB. All of these results strongly suggest that DEX could be involved in the regulation of both transcellular and paracellular pathway.

Bradykinin-induced blood-tumor barrier opening is mediated by tumor necrosis factor-alpha.

Bradykinin has been shown to increase the permeability of blood-tumor barrier (BTB) selectively. This study was performed to determine whether tumor necrosis factor-alpha (TNF-alpha) was involved in the regulation of this biological process. We found that the levels of TNF-alpha mRNA and heat shock factor-1 (HSF1) protein in C6 cells were markedly up-regulated by bradykinin via real-time RT-PCR and Western blot methods. And the most obvious increase of HSF1 protein and TNF-alpha mRNA in C6 cells were observed at 5 min and 10 min of bradykinin perfusion, respectively. In addition, the radioactivity of TNF-alpha in C6 cells' culture fluid also mostly increased at 15 min of bradykinin perfusion. And the Evans blue content of brain tumor tissues in rats and the concentration of TNF-alpha reached the maximum at 15 min of bradykinin perfusion. Our results suggested that the bradykinin-mediated BTB permeability increase is due to accelerated release of TNF-alpha, which could cause the increase of BTB permeability by promoting to the release HSF1 from neurospongioma cells.

Interleukin-1ß induces the upregulation of caveolin-1 expression in a rat brain tumor model.

The aim of the present study was to investigate the expression of caveolin-1 in rat brain glioma tissue, and to determine whether interleukin-1ß (IL-1ß) has a role in this process. Using glioma cells, a tumor-burdened rat model was established, and the expression of caveolin-1 protein in the tumor sites was significantly increased following intracarotid infusion of IL-1ß (3.7 ng/kg/min), as indicated by western blot analysis. The maximum value of the caveolin-1 expression was observed in tumor-burdened rats after 60 min of IL-1ß perfusion, and which was significantly enhanced by vascular endothelial growth factor (VEGF). In addition, VEGF also significantly increased IL-1ß-induced blood tumor barrier (BTB) permeability. The results suggest that the IL-1ß-induced BTB permeability increase may be associated with the expression of caveolin-1 protein, and VEGF may be involved in this process.

Cyclooxygenase inhibitor induces the upregulation of connexin-43 expression in C6 glioma cells.

The present study was performed to determine whether aspirin, a cyclooxygenase (COX) inhibitor, has an effect on the expression of connexin 43 (Cx43) in C6 glioma cells. Using an in vitro glioma invasion model, the expression of Cx43 protein in C6 cells was significantly increased following aspirin treatment at a dose of 8 mmol/l for 30, 60 and 120 min via western blot analysis. The peak value of the Cx43 expression was observed in C6 cells after 120 min of aspirin treatment, which was significantly reduced by prostaglandin E2 (PGE2). In addition, aspirin also significantly increased the gap junction intercellular communication (GJIC) activity and reduced glioma invasion, which was induced by PGE2. This led to the conclusion that the aspirin-induced glioma invasion decrease may be associated with the increased expression of Cx43 protein and formation of GJIC.

Antitumor Activity of Tenacissoside H on Esophageal Cancer through Arresting Cell Cycle and Regulating PI3K/Akt-NF-κB Transduction Cascade.

Objective. The purpose of the study was to elucidate the molecular mechanism of tenacissoside H (TDH) inhibiting esophageal carcinoma infiltration and proliferation. Methods. In vitro, EC9706 cells were treated with TDH. Cells proliferation and cell cycle were assayed. PI3K and NF-κB mRNAs expression were determined by real time PCR. In vivo, model of nude mice with tumor was established. Mice were treated with TDH. Inhibition ratio of tumor volume was calculated. PCNA expression was examined. Protein expression in PI3K/Akt-NF-κB signaling pathway was determined. Results. In vitro, TDH significantly inhibited cells proliferation in a time-and-dose-dependent manner. TDH arrested the cell cycle in S phase and significantly inhibited PI3K and NF-κB mRNA expression, compared with blank controlled group (P < 0.05). In vivo, TDH strongly inhibits tumor growth and volume. PCNA expression was significantly decreased after treatment of TDH. TDH downregulated proteins expression in PI3K/Akt-NF-κB transduction cascade (P < 0.05). Conclusion. TDH inhibited esophageal carcinoma infiltration and proliferation both in vitro and in vivo. The anticancer activity has relation to arresting the cell cycle at the S phase, inhibited the PCNA expression of transplanted tumors in nude mice, and regulated the protein expression in the PI3K/Akt-NF-κB transduction cascade.

Histone acetylation and expression of mono-aminergic transmitters synthetases involved in CUS-induced depressive rats.

Histone acetylation has been linked to depression, the etiology of which involves many factors such as genetics, environments, and epigenetics. The aim of the present study was to investigate whether it was associated with epigenetic histone modification and gene expression of enzymes responsible for the biosynthesis of norepinephrine and serotonin in rat depression model induced by chronic unpredictable stress (CUS). Eight-week-old male Sprague-Dawley rats were exposed to CUS over 28 days. It was shown that the CUS-induced rats displayed remarked anxiety- and depression-like behavior with weakened locomotor activity in open field test and prolonged immobility in forced swimming test. Western blot revealed that CUS led to significant decrease in acetylation of H3 at Lysine 9 (K9) and H4 at Lysine 12 (K12) with obviously increasing histone deacetylases 5 (HDAC5) expression in hippocampus of CUS-induced rats. Meanwhile, there was an obviously decreased expression of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) both at protein and mRNA levels. Administration of sodium valproate (VPA), a histone deacetylase 5 (HDAC5) inhibitor, not only significantly relieved the anxiety- and depression-like behaviors of CUS-induced rats but also clearly blunted decrease of H3(K9) and H4(K12) acetylation and expression of TH and TPH, and prevented increase of HDAC5 expression. The results indicate that there exists possible interrelation between TH and TPH gene expression and epigenetic histone acetylation in CUS-induced depressive rats, which at least partly contributes to the etiology of depression.

Antidepressive effect of paroxetine in a rat model: upregulating expression of serotonin and norepinephrine transporter.

Paroxetine is a selective serotonin reuptake inhibitor used for the treatment of depression; this study investigated its other mechanisms by studying the expression and therefore involvement of norepinephrine transporter (NET) and serotonin transporter (5-HTT). Male Sprague-Dawley rats were divided into a vehicle-treated control group (VC), a paroxetine-treated control group (PC), a vehicle-treated model group (VM), and a paroxetine-treated model group (PM). The depression model was established by chronic unpredicted stress. Paroxetine (1.8 mg/kg once daily) was administered to rats (PM and PC groups) by an intragastric gavage, and the same dosage of vehicle was administered to rats in the VM and VC groups. Rat behaviors, superoxide dismutase and catalase activities, malondialdehyde level in the serum, and expression of 5-HTT in the hippocampus and NET in the pons were determined, respectively. Compared with VM rats, the PM rats showed significant relief of depression-like behaviors, decrease in the malondialdehyde level, increase in superoxide dismutase and catalase activities, and increase in 5-HTT and NET expression. The results may suggest that the antidepressive effect of paroxetine is at least partly related to reversing oxidative stress imbalance and elevating the expression of 5-HTT and NET.

Role of ROS/RhoA/PI3K/PKB signaling in NS1619-mediated blood-tumor barrier permeability increase.

The calcium-activated potassium channel (K (Ca) channel) activator, NS1619, has been shown to selectively and time-dependently increase the permeability of the blood-tumor barrier (BTB) by downregulating the expression of tight junction (TJ) protein. However, the role of signaling cascades in this process has not been precisely elucidated. This study was performed to determine the role of signaling cascades involving reactive oxygen species (ROS)/RhoA/PI3K/PKB in increasing the permeability of the BTB induced by NS1619. Using an in vitro BTB model and selective inhibitors of signaling pathways, we investigated whether ROS/RhoA/PI3K/PKB pathway plays a key role in the process of the increase in BTB permeability induced by NS1619. The results revealed that the BTB permeability was increased and the expression of TJ proteins were significantly decreased by NS1619, and selective inhibitors of identified signaling pathways reversed the observed alterations. Moreover, the significant increases in ROS, RhoA activity, and PKB phosphorylation after NS1619 administration were observed, which were partly inhibited by N-2-mercaptopropionyl glycine or C3 exoenzyme or LY294002 pretreatment. The present study demonstrates that the activation of signaling cascades involving ROS/RhoA/PI3K/PKB in rat brain microvascular endothelial cells was required for the increase in BTB permeability induced by NS1619.

Hyperthermia promotes apoptosis and suppresses invasion in C6 rat glioma cells.

Gliomas are a group of heterogeneous primary central nervous system tumors. Hyperthermia has proven to be a potential therapeutic tool for cancers in the clinic. However, the molecular mechanisms of hyperthermia remain unclear. The objective of this study was to investigate the effects of hyperthermia on the invasiveness in C6 glioma cells and related molecular pathways. Here our data show hyperthermia stimulated the release of tumor necrosis factor-alpha (TNF-α) and decreased C6 glioma cell migration and invasive capability at 30, 60, 120 and 180 min; with increased spontaneous apoptosis in C6 glioma cells at 120 min. We also found mitogen-activated protein kinase (P38 MAPK) protein expression to be increased and nuclear factor-kappa B (NF-κB) protein expression decreased. Based on the results, we conclude that hyperthermia alone reduced invasion of C6 glioma cells through stimulating TNF-α signaling to activate apoptosis, enhancing P38 MAPK expression and inhibiting the NF-κB pathway, a first report in C6 rat glioma cells.

Dexamethasone enhances calcium-activated potassium channel expression in blood-brain tumor barrier in a rat brain tumor model.

This study was performed to determine whether dexamethasone (DEX) had an effect on calcium-activated potassium channels (KCa channels) in blood-brain tumor barrier (BTB).Using a rat brain glioma model, we found that the expression of KCa channels protein was significantly increased in brain tumor tissue. And bradykinin-induced increase of KCa channels protein was further enhanced after DEX pretreatment for 3 days. In addition, DEX pretreatment enhanced bradykinin-mediated up-regulation of the density of IKCa in the rat brain C6 cells in vitro BTB. Bradykinin markedly increased BTB permeability independent of DEX pretreatment. All of these results strongly suggest that DEX could regulate the target in the transcellular pathway of BTB-KCa channels.

[Protective effects of local heat preconditioning on hepatic ischemia/reperfusion injury].

AIM: To study the protective mechanism of HSP70 induced by the heat stress pretreatment on the hepatic ischemia/ reperfusion (I/R) injury. METHODS: To establish the models of the hepatic ischemia/ reperfusion injury using pringle's maneuver with or without heat stress pretreatment. The rats were randomly divided into pretreatment group (HP + I/R) and non-pretreatment group (I/R), in which the expression of HSP70, the MDA contents and SOD activity in liver, the activities of serum AST and ALT, and the pathological changes in liver were detected at 0, 4, 8, 12 and 24 h after I/R. RESULTS: At any time point set after I/R, the SOD activity in the liver in HP + I/R group were higher than those in IR group, HSP70 expression maximum was attained at 12 h after heat pre-treatment. While in HP+ I/R group the levels of liver enzymes and the production of MDA significantly reduced and the pathological changes improved compared with those in I/R group. CONCLUSION: HSP70 induced by heat pretreated protecting liver against I/R injury may be through increasing the SOD content and then reducing the insults of oxygen free radicals in the liver.