Hesperetin Inhibits TGF-ß1-Induced Migration and Invasion of Triple Negative Breast Cancer MDA-MB-231 Cells via Suppressing Fyn/Paxillin/RhoA Pathway.

Triple-negative breast cancer is an aggressive subtype of breast cancer with poor clinical outcomes and poor prognosis. Hesperetin is an active component extracted from Citrus fruits and Traditional Chinese Medicine has a wide range of pharmacological effects. Here, we assessed the anti-migration and anti-invasive effects and explored inhibitory mechanisms of hesperetin on metastasis of human triple negative breast cancer MDA-MB-231 cells. Cell viability experiments revealed that 200 µM hesperetin has a clear inhibitory effect on MDA-MB-231 cells. TGF-ß1 treatment induces apparent tumor progression in MDA-MB-231 cells including aberrant wound-healing and invasion ability, which is effectively suppressed by hesperetin co-treatment. Additionally, hesperetin inhibited the TGF-ß1-mediated actin stress fiber formation. Western blot results showed that hesperetin suppressed the TGF-ß1-mediated (i) activation of Fyn, (ii) phosphorylation of paxillin at Y31, Y88, and Y118 sites, (iii) the increased expression of RhoA, and (iv) activation of Rho-kinase. We demonstrated the increased interaction of Fyn with paxillin and RhoA protein in the TGF-ß1-induced metastasis of MDA-MB-231 cells. Small interfering RNA Fyn inhibited phosphorylation of paxillin (Y31) and activation of Rho-kinase induced by TGF-ß1. In conclusion, hesperetin has a significant inhibitory effect on migration and invasion of MDA-MB-231 cells induced by TGF-ß1, which might be attributed to inhibiting the Fyn/paxillin/RhoA pathway.

Keratin 18 induces proliferation, migration, and invasion in gastric cancer via the MAPK signalling pathway.

PURPOSE: Keratin 18 (KRT18) is a cytoskeleton protein that plays a key role in multiple cancers. The present study aims to further investigate the roles of KRT18 in gastric cancer (GC) tissues and cells. METHODS: The KRT18 protein expression levels of GC tissues and cells were detected using immunohistochemistry and western blot. The relationship between KRT18 expression levels and the prognosis of GC patients was further analyzed. To explore this relationship, small interfering RNA (siRNA) was used to inhibit the endogenous expression of KRT18 in GC cells. Furthermore, the effects of KRT18 on the proliferation, invasion, migration, and apoptosis of GC cells were analyzed in vitro. In addition, the role of KRT18 in GC-specific processes was investigated. RESULTS: Keratin 18 expression was shown to be up-regulated in GC tissues and associated with poor prognosis. Following KRT18 silencing with siRNA, the proliferation, invasion, and migration ability of GC cells were significantly inhibited, while the apoptotic process was promoted. Furthermore, the activation of the MAPK signalling pathway was identified as the potential mechanism through which KRT18 influenced GC processes. CONCLUSIONS: Keratin 18 plays a cancer-promoting role and might be a potential therapeutic target in the treatment of GC.

Treatment of solitary hepatocellular carcinoma up to 2 cm: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: In recent years, there has been considerable uncertainty about the optimal treatment option for very early hepatocellular carcinoma (HCC) with tumor size less than 2 cm. Therefore, we performed a systematic review and meta-analysis to evaluate the outcomes of the different treatments. METHODS: This study was designed in accordance with the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA). PubMed, EMBASE, and Cochrane library were searched for calculating the survival rates, and the "time to event" method was used to compare the outcomes of liver resection (LR) and radiofrequency ablation (RFA). All studies focusing on the treatment of solitary HCC up to 2 cm by different techniques were included in our analysis. The Hazard ratios (HR) and 95% confidence intervals (CI) derived from multivariate and univariate analysis were utilized to assess the treatment risks. RESULTS: We included 32 studies in our systematic review. The median 5-year overall survival (OS) and recurrence-free survival rate (RFS) for LR were 73% and 47%, respectively, and those for RFA were 73% and 43%, respectively. RFA was found to be associated with increased risk of mortality and recurrence compared to LR (HR = 1.61, 95% CI: 1.35-1.92, P < .0001 for OS and HR = 1.75, 95% CI: 1.56-1.96, P < .0001 for RFS). CONCLUSION: Our meta-analysis demonstrated that LR is superior to RFA in the treatment of solitary HCC up to 2 cm, with reduction in mortality and recurrence risk and improved long-term outcome.

[Effects of curcumin on protein expression of glucose regulated protein 78 and caspase-12 of myocardial endoplasmic reticulum stress related factors in type 2 diabetes rats].

OBJECTIVE: To study the effect of curcumin on the expression of glucose regulated protein 78 kD(GRP78) and cysteinyl aspartate specific proteinase-12(caspase-12) of myocardial endoplasmic reticulum stress related factors in type 2 diabetes rats. METHODS: Type 2 diabetes rats model was established by high-fat drink feeding and one-time intraperitoneal injecting streptozotocin(35 mg/kg). After model rats were built, rats was randomly divided into diabetic model group and low dose of curcumin group(200 mg/kg), high dose of curcumin group(400 mg/kg) and captopril group(60 mg/kg) with 10 rats in each group. The rats in each group were ig administered with corresponding drugs once a day. Continuous administration for 12 w. The levels of fasting blood glucose(FBG) and lactate dehydrogenase(LDH), electrocardiogram and heart weight index(HWI) were measured respectively. The myocardial pathological changes were observed by HE staining. The levels of collagen fiber expression in myocardial tissue were performed by Masson staining. The protein expression levels of GRP78 and caspase-12 in myocardium of rats were observed by immunohistochemistry. RESULTS: The result showed that compared with control group, the levels of FBG and LDH of serum were increased obviously, HWI was increased, myocardial cells were hypertrophy, the collagen fibers of intercellular space of cell were increased, the protein expressions of GRP78 and caspase-12 of myocardium were increased in rats, myocardial cell apoptosis was increased in the model group(P<0. 05). Compared with model group, FBG and LDH levels and HWI were reduced, the collagen fiber of intercellular space were decreased, the protein expression levels of GRP78 and caspase-12 were lowered in high dose of curcumin group(P<0. 05). CONCLUSION: It indicates that Cur defends myocardium tissue in type 2 diabetes rats, which may be related to decreasing the level of blood glucose and the protein expressions of GRP78 and caspase-12, and blocking the ERS-initiated apoptotic.

Endothelial progenitor cells in peripheral blood may serve as a biological marker to predict severe acute pancreatitis.

AIM: To investigate the significance of endothelial progenitor cells (EPCs) in predicting severe acute pancreatitis (SAP). METHODS: We recruited 71 patients with acute pancreatitis (AP) and excluded 11 of them; finally, cases of mild acute pancreatitis (MAP) (n = 30) and SAP (n = 30), and healthy volunteers (n = 20) were internalized to investigate levels of EPCs, C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), fibrinogen (FIB) and white blood cells (WBC) in peripheral blood. RESULTS: The levels of TNF-α, WBC, FIB and CRP were higher both in SAP and MAP cases than in healthy volunteers (P < 0.05, all). Interestingly, the level of EPCs was higher in SAP than MAP (1.63% ± 1.47% vs 6.61% ± 4.28%, P < 0.01), but there was no significant difference between the MAP cases and healthy volunteers (1.63% ± 1.47% vs 0.55% ± 0.54%, P > 0.05). Receiver operating characteristics curve (ROC) showed that EPCs, TNF-α, CRP and FIB were significantly associated with SAP, especially EPCs and CRP were optimal predictive markers of SAP. When the cut-off point for EPCs and CRP were 2.26% and 5.94 mg/dL, the sensitivities were 90.0% and 73.3%, and the specificities were 83.3% and 96.7%. Although, CRP had the highest specificity, and EPCs had the highest sensitivity and highest area under the curve value (0.93). CONCLUSION: Data suggest that EPCs may be a new biological marker in predicting SAP.

Enhancement of Gastric Ulcer Healing and Angiogenesis by Hepatocyte Growth Factor Gene Mediated by Attenuated Salmonella in Rats.

The present study developed an oral hepatocyte growth factor (HGF) gene therapy strategy for gastric ulcers treatment. An attenuated Salmonella typhimurium that stably expressed high HGF (named as TPH) was constructed, and the antiulcerogenic effect of TPH was evaluated in a rat model of gastric ulcers that created by acetic acid subserosal injection. From day 5 after injection, TPH (1 × 109 cfu), vehicle (TP, 1 × 109 cfu), or sodium bicarbonate (model control) was administered orally every alternate day for three times. Then ulcer size was measured at day 21 after ulcer induction. The ulcer area in TPH-treated group was 10.56 ± 3.30 mm², which was smaller when compared with those in the TP-treated and model control groups (43.47 ± 4.18 and 56.25 ± 6.38 mm², respectively). A higher level of reepithelialization was found in TPH-treated group and the crawling length of gastric epithelial cells was significantly longer than in the other two groups (P < 0.05). The microvessel density in the ulcer granulation tissues of the TPH-treated rats was 39.9 vessels/mm², which was greater than in the TP-treated and model control rats, with a significant statistical difference. These results suggest that TPH treatment significantly accelerates the healing of gastric ulcers via stimulating proliferation of gastric epithelial cells and enhancing angiogenesis on gastric ulcer site.

Ajuba Preferentially Binds LXRα/RXRγ Heterodimer to Enhance LXR Target Gene Expression in Liver Cells.

The liver X receptors (LXRs) are important regulators of lipid, cholesterol, and glucose homeostasis by transcriptional regulation of many key genes in these processes, and the transcriptional activities of LXRs are finely controlled by cooperating with retinoid X receptors and many other coregulators. Here, we report that the LIM protein Ajuba binds to the hinge and the ligand binding domains of LXRα via its C-terminal tandem LIM motifs and enhances LXR target gene expression in liver cells. Depletion of Ajuba in HepG2 cells and in mouse primary hepatocytes decreases LXR target gene expression, whereas stable expression of Ajuba in HepG2 cells results in increased expression of these genes. Mechanistic investigations found that Ajuba selectively interacts with LXRα/retinoid X receptor-γ heterodimer to form a ternary complex, which displays a higher transactivation activity to LXR target genes. Moreover, Ajuba and LXR mutually affect their DNA binding activity at endogenous target chromatins and the cooperation between Ajuba and LXRα is dependent on the functional LXR response elements located in the target promoters. Together, our studies demonstrate that Ajuba is a novel coactivator for LXRs and may play important role in lipid and glucose metabolism.

Activation of the Liver X Receptor by Agonist TO901317 Improves Hepatic Insulin Resistance via Suppressing Reactive Oxygen Species and JNK Pathway.

Activation of Liver X receptors (LXRs), key transcriptional regulators of glucose metabolism, normalizes glycemia and improves insulin sensitivity in rodent models with insulin resistance. However, the molecular mechanism is unclear. This study is aimed to elucidate the mechanism of LXRs-mediated liver glucose metabolic regulation in vitro and in vivo. Db/db mice were used as an in vivo model of diabetes; palmitate (PA)-stimulated HepG2 cells were used as an in vitro cell model with impairment of insulin signaling. TO901317 (TO) was chosen as the LXRs agonist. We demonstrated that TO treatment for 14 days potently improved the hepatic glucose metabolism in db/db mice, including fasting blood glucose, fasting insulin level, and HOMA-IR. TO had no effect on the glucose metabolism in normal WT mice. TO-mediated activation of hepatic LXRs led to strong inhibition of ROS production accompanied by inactivation of JNK pathway and re-activation of Akt pathway. TO also suppressed the expression of gluconeogenic genes such as PEPCK and G-6-pase in db/db mice, but not in WT mice. In HepG2 cells, TO almost completely restored PA-induced Akt inactivation, and suppressed PA-stimulated ROS production and JNK activation. Interestingly, basal level of ROS was also inhibited by TO in HepG2 cells. TO significantly inhibited PA-stimulated expressions of gluconeogenic genes. Finally, we found that anti-oxidative genes, such as Nrf2, were up-regulated after LXRs activation by TO. These results strongly support the notion that activation of LXRs is critical in suppression of liver gluconeogenesis and improvement of insulin sensitivity in diabetic individuals. At molecular levels, the mode of action appears to be as fellows: under diabetic condition, ROS production is increased, JNK is activated, and Akt activity is inhibited; TO-mediated LXR activation potently inhibits ROS production, increases anti-oxidative gene expressions, suppresses JNK activation, and restores Akt activity. Our data provide new evidence to support LXRs as promising therapeutic targets for anti-diabetic drug development.

[Effect of flavonoids from Sophora flavescens in aging mice induced by D-galactos].

To investigate the effect of flavonoids from Sophora flavescens in aging mice induced by D-galactose and its mechanism. Totally 60 mice were randomly divided into six groups: the control group, the model group, the piracetam group (positive control group) and flavonoids from S. flavescens low, medium and high doses groups. Except for the control group, all of the rest groups were subcutaneously injected with D-galactose (160 mg x kg(-1)) for successively 30 days to establish the sub-acute senescent model. Meanwhile, flavonoids from S. flavescens low, medium and high doses groups were respectively administered with 150, 300 and 600 mg xkg-('1)of flavonoids from S. flavescens for 30 days. The learning and memory abilities of mice were determined by avoiding darkness ex-eriment and jumping stair experiment. The contents of malondialdehyde (MDA) tumor necrosis factor-aα NF-aα the activities of superoxide dismutase (SOD) monoamine oxidase-B (MAO-B) Na'(+)K'(+)-ATPase and Ca2(+ )-ATPase in the brain of mice were deter-ined respectively after the behavioral experiments. The activity of lactic dehydrogenase ( DH) in blood serum was also determined and analyzed by microscope after HE staining to observe the changes in hippocampal organizational structure. Compared with the model group, flavonoids from S. favescens medium and high doses groups showed significantly increases in the latency of avoiding darkness and jumping stair experiments; flavonoids from S. fllvescens low, medium and high doses groups and the piracetam group showed de-reases in the numbers of errors in avoiding darkness experiment; the flavonoids from S. flavescens high dose group and the piracetam group showed reduction- n the number of errors in jumping stair experiment (P <0 . 5 or P <0 . 1). Flavonoids from S. flavescens me-ium and high doses groups and the piracetam group showed improvements in the activities of SOD, Na'(+)K'(+)ATPase in the brain of mice and declines in the contents of MDA and TNF-aα the activity of MAO-B in the brain of mice, the activity of LDH in blood serum (P <0 . 5 or P <0 . 1). Flavonoids from S. flavescens low, medium and high doses groups and the piracetam group also showed im-rovement in the activity of Ca2(+ )ATPase, with statistical difference from the model group (P <0 . 5 or P <0 . 1). The pathological result showed decreases in the number of cells of hippocampal dentate gyrus area, sparse cell arrangement, incomplete cellular mor-hology, scarce cytoplasm, blurred boundary between nucleus and cytoplasm, nuclei anachromasis, irregular pyknosis and unconspicu-us nucleoli in the model group. Compared with the model group, flavonoids from S. flavescens low, medium and high doses groups and the piracetam group showed improvements in hippocampal organization tissues. Flavonoids from S. favescens can improve the learning and memory ability of senescent mice induced by D-galactose. Its mechanism may be correlated with the enhancement of anti-oxidative actions by lowering TNF-aαcontent, which results in the stability of cell membrane and the reduction in MAO-B activity.

[Effects of schizandrins on learning-memory disorder in mice].

OBJECTIVE: To observe the effects of schizandrins on the learning and memory disorder in mice, and explore its mechanism. METHOD: The memory impairment model was established by using the pentobarbital sodium (20 mg x kg(-1)) intraperitoneally injected in mice. Schizandrins (0.5, 1.0, 2.0 g x kg(-1)) were administered through intragavage for consecutive 14 days. Morris Water Maze test was used to evaluate the impairment of learning and memory. The energy of superoxide dismutase (SOD), nitric oxide (NO) and catalase (CAT) of brain tissue were measured. And the positive expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65), caspase-3 in the hippocampus CA1 region were determined by immunohistochemical analysis. At the cellular level, 24 h after schizandrins (0.062 5, 0.125, 0.25 g x L(-1)) were pre-administered, the apoptosis model of PC12 cell was induced by H2O2, and activity of PC12 cell was detected by MTT colorimetric assay, the energy of NO in cell serum were measured. The expression of Bcl-2 was determined by the combination of immunocytochemical staining and image analysis software. RESULT: Morris Water Maze test showed that the model group mice took shorter searching time and distance on the previous flat area than those in the control group (P < 0.05), which could be prolonged after schizandrins treatment (P < 0.05, P < 0.01). Compared with the control group, the level of NO increased while the activity of SOD, CAT decreased in the model group (both P < 0.01). After treated with schizandrins, the level of NO significantly decreased (P < 0.01), while the activity of SOD increased (P < 0.01). Immunohistochemistry analysis showed that the protein expression of NF-kappaB p65, Caspase-3 in the hippocampal CA1 region significantly increased after modeling, while schizandrins (1.0 g x kg(-1)) can significantly inhibit the protein expression of NF-kappaB p65, Caspase-3 (P < 0.05, P < 0.01). Compared with the H2O2, model group, schizandrins (0.125, 0.25 g x L(-1)) can significantly increased PC12 cell activity and decreased the NO level (P < 0.05, P < 0.01), the expression of Bcl-2 in the schizandrins group (0.125, 0.25 g x L(-1)) was up-regulated. CONCLUSION: Schizandrins could improve the learning-memory dysfunction induced by the sodium pentobarbital in mice, and its protective mechanism is related to the lowering oxidative damage and inhibiting the cell apoptosis through up-regulating the expression of Bcl-2.

Tumor selectivity of stealth multi-functionalized superparamagnetic iron oxide nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPIO-NPs) have traditionally been used as MRI contrast agent for disease imaging via passive targeting. However, there has been an increasing interest in the development of SPIO-NPs to cellular-specific targeting for imaging and drug delivery currently. The objective of our study was to develop a novel active tumor-targeting SPIO-NPs system by surface-modifying superparamagnetic iron oxide nanoparticles (SPIO-NPs) with o-carboxymethyl chitosans (OCMCS) and folic acid (FA) to improve their biocompatibility and ability to target specific tumor cells as well as to evade reticuloendothelial system (RES). The results in vitro indicated the covalent surface-modification of SPIO-NPs with OCMCS significantly reduced not only the nano-cytotoxicity but also the capture of SPIO-NPs by macrophage cells. On the other hand, the folic acid modification promoted the uptake of nanoparticles by FR-positive tumor cell lines, but had little impact on other cells without folate receptor (FR). MRI image and tumor histological analysis demonstrated the FA-OCMCS-SPIO-NPs had the ability to target tumor cells with FR in vivo. OCMCS and folic acid modification of SPIO-NPs could significantly improve both the SPIO-NPs biocompatibility and the FR target for MRI imaging, potential carrier for drug targeting and hyperthermia.

Prepubertal octylphenol exposure up-regulate BRCA1 expression, down-regulate ERalpha expression and reduce rat mammary tumorigenesis.

BACKGROUND: Endogenous estrogens play an important role in the development of breast cancer. Octylphenol (OP) and genistein (GEN) are estrogen-like chemicals. Prepubertal estradiol and genistein exposure can up-regulate BRCA1 mRNA in mammary gland and reduce futuer breast cancer risk. In the present study, the effects of prepubertal exposure to high-dose OP and GEN on mammary carcinogenesis and the association with the expression of BRCA1 and ERalpha were investigated. METHODS: Prepubertal female Sprague-Dawley rats were exposed to 20, 40, 80mg/kg OP daily from postnatal day (PND) 22-28, subsequently, the rats were given a single dose of 100mg/kg 7,12-dimethylbenz [a] anthracene (DMBA) on PND42 to induce mammary tumor. RESULTS: The incidence of DMBA-induced mammary tumors significantly decreased when rats were treated with 40mg/kg OP. BRCA1 mRNA and protein expression were found up-regulated and ERalpha expression was down-regulated in the mammary tumor when rats were exposed to 40mg/kg octylphenol. CONCLUSION: Exposure 40mg/kg octylphenol can reduce later breast cancer risk in prepubertal Sprague-Dawley rats, the protective effect of OP is associated with persistent up-regulation of BRCA1 and down-regulation of ERalpha in the mammary tumor.