Essential oil from Fructus Alpinia zerumbet ameliorates atherosclerosis by activating PPARγ-LXRα-ABCA1/G1 signaling pathway.

BACKGROUND: Atherosclerosis (AS) is a progressive chronic disease. Currently, cardiovascular diseases (CVDs) caused by AS is responsible for the global increased mortality. Yanshanjiang as miao herb in Guizhou of China is the dried and ripe fruit of Fructus Alpinia zerumbet. Accumulated evidences have confirmed that Yanshanjiang could ameliorate CVDs, including AS. Nevertheless, its effect and mechanism on AS are still largely unknown. PURPOSE: To investigate the role of essential oil from Fructus Alpinia zerumbet (EOFAZ) on AS, and the potential mechanism. METHODS: A high-fat diet (HFD) ApoE-/- mice model of AS and a oxLDL-induced model of macrophage-derived foam cells (MFCs) were reproduced to investigate the pharmacological properties of EOFAZ on AS in vivo and foam cell formation in vitro, respectively. The underlying mechanisms of EOFAZ were investigated using Network pharmacology and molecular docking. EOFAZ effect on PPARγ protein stability was measured using a cellular thermal shift assay (CETSA). Pharmacological agonists and inhibitors and gene interventions were employed for clarifying EOFAZ's potential mechanism. RESULTS: EOFAZ attenuated AS progression in HFD ApoE-/- mice. This attenuation was manifested by the reduced aortic intima plaque development, increased collagen content in aortic plaques, notable improvement in lipid profiles, and decreased levels of inflammatory factors. Moreover, EOFAZ inhibited the formation of MFCs by enhancing cholesterol efflux through activiting the PPARγ-LXRα-ABCA1/G1 pathway. Interestingly, the pharmacological knockdown of PPARγ impaired the beneficial effects of EOFAZ on MFCs. Additionally, our results indicated that EOFAZ reduced the ubiquitination degradation of PPARγ, and the chemical composition of EOFAZ directly bound to the PPARγ protein, thereby increasing its stability. Finally, PPARγ knockdown mitigated the protective effects of EOFAZ on AS in HFD ApoE-/- mice. CONCLUSION: These findings represent the first confirmation of EOFAZ's in vivo anti-atherosclerotic effects in ApoE-/- mice. Mechanistically, its chemical constituents can directly bind to PPARγ protein, enhancing its stability, while reducing PPARγ ubiquitination degradation, thereby inhibiting foam cell formation via activation of the PPARγ-LXRα-ABCA1/G1 pathway. Simultaneously, EOFAZ could ameliorates blood lipid metabolism and inflammatory microenvironment, thus synergistically exerting its anti-atherosclerotic effects.

Therapeutic targeting miR130b counteracts diffuse large B-cell lymphoma progression via OX40/OX40L-mediated interaction with Th17 cells.

MicroRNAs (miRNAs) are involved in lymphoma progression by regulating the tumor microenvironment. Serum miR130b is overexpressed in diffuse large B-cell lymphoma (DLBCL), inducing Th17 cell alterations. To further illustrate its biological significance and therapeutic rationale, miR130b was detected by quantitative real-time PCR in the serum samples of 532 newly diagnosed DLBCL patients. The mechanism of miR130b on lymphoma progression and the tumor microenvironment was investigated both in vitro and in vivo. Therapeutic targeting miR130b was also evaluated, including OX40 agonistic antibody and lipid nanoparticles (LNPs)-miR130b antagomir. The results showed that serum miR130b significantly correlated with tumor miR130b and serum interleukin-17, indicating lymphoma relapse and inferior survival of DLBCL patients. MiR130b overexpression altered tumor microenvironment signaling pathways and increased Th17 cell activity. As mechanism of action, miR130b downregulated tumor OX40L expression by directly targeting IFNAR1/p-STAT1 axis, recruiting Th17 cells via OX40/OX40L interaction, thereby promoting immunosuppressive function of Th17 cells. In co-culture systems of B-lymphoma cells with immune cells, miR130b inhibited lymphoma cell autophagy, which could be counteracted by OX40 agonistic antibody and LNPs-miR130b antagomir. In murine xenograft model established with subcutaneous injection of A20 cells, both OX40 agonistic antibody and LNPs-miR130b antagomir remarkably inhibited Th17 cells and retarded miR130b-overexpressing tumor growth. In conclusion, as an oncogenic biomarker of DLBCL, miR130b was related to lymphoma progression through modulating OX40/OX40L-mediated lymphoma cell interaction with Th17 cells, attributing to B-cell lymphoma sensitivity towards OX40 agonistic antibody. Targeting miR130b using LNPs-miR130b antagomir could also be a potential immunotherapeutic strategy in treating OX40-altered lymphoid malignancies.

A Small Ligand That Selectively Binds to the G-quadruplex at the Human Vascular Endothelial Growth Factor Internal Ribosomal Entry Site and Represses the Translation.

G-quadruplexes are believed to have important biological functions, so many small molecules have been screened or developed for targeting G-quadruplexes. However, it is still a major challenge to find molecules that recognize specific G-quadruplexes. Here, by using a combination of surface plasmon resonance, electrospray ionization mass spectrometry, circular dichroism, Western blot, luciferase assay, and reverse transcriptase stop assay, we observed a small molecule, namely, oxymatrine (OMT) that could selectively bind to the RNA G-quadruplex in 5'-untranslated regions (UTRs) of human vascular endothelial growth factor (hVEGF), but could not bind to other G-quadruplexes. OMT could selectively repress the translation of VEGF in cervical cancer cells. Furthermore, it could recognize VEGF RNA G-quadruplexes in special conformations. The results indicate that OMT may serve as a potentially special tool for studying the VEGF RNA G-quadruplex in cells and as a valuable scaffold for the design of ligands that recognize different G-quadruplexes.

YgfY Contributes to Stress Tolerance in Shewanella oneidensis Neither as an Antitoxin Nor as a Flavinylation Factor of Succinate Dehydrogenase.

YgfY(SdhE/CptB) is highly conserved while has controversial functions in bacteria. It works as an antitoxin and composes a type IV toxin-antitoxin system with YgfX(CptA) typically in Escherichia coli, while functions as an flavinylation factor of succinate dehydrogenase and fumarate reductase typically in Serratia sp. In this study, we report the contribution of YgfY in Shewanella oneidensis MR-1 to tolerance of low temperature and nitrite. YgfY deficiency causes several growth defects of S. oneidensis MR-1 at low temperature, while YgfX do not cause a growth defect or morphological change of S. oneidensis MR1-1 and E. coli. YgfY do not interact with FtsZ and MreB nor with YgfX examined by bacterial two-hybrid assay. YgfY effect on growth under low temperature is not attributed to succinate dehydrogenase (SDH) because a mutant without SDH grows comparably with the wild-type strain in the presence of succinate. The ygfY mutant shows impaired tolerance to nitrite. Transcription of nitrite reductase and most ribosome proteins is significantly decreased in the ygfY mutant, which is consistent with the phenotypes detected above. Effects of YgfY on growth and nitrite tolerance are closely related to the RGXXE motif in YgfY. In summary, this study demonstrates pleiotropic impacts of YgfY in S. oneidensis MR-1, and sheds a light on the physiological versatility of YgfY in bacteria.

Intraarterial Lidocaine Administration for Pain Control by Water-in-Oil Technique in Transarterial Chemoembolization: in vivo and Randomized Clinical Trial.

OBJECTIVE: To investigate the sustained release of lidocaine from a lidocaine-epirubicin-lipiodol emulsion created by water-in-oil (W/O) technique in vivo and evaluate the efficacy and safety of intraarterial lidocaine administration for intra- and postoperative pain control in transarterial chemoembolization (TACE) for hepatocellular carcinoma (HCC). METHODS: The in vivo concentrations of lidocaine were determined in tumor tissues after VX2 rabbit models for hepatic tumor were administered with intra-arterial lidocaine-epirubicin-lipiodol emulsion. A prospective randomized controlled clinical trial was performed, enrolling 70 consecutive patients who underwent TACE. Patients were randomized into two groups: Group A received an immediate bolus intraarterial lidocaine injection before TACE, and Group B received a lidocaine-epirubicin-lipiodol emulsion during TACE. Pain intensity was compared between the two groups using a visual analog scale (VAS) score before (Tbefore) and at 0 h (T0), 4 h (T4), 8 h (T8), 24 h (T24), 48 h (T48), and 72 h (T72) after the procedure. Adverse events and intake of analgesics were evaluated and compared between the two groups. RESULTS: The concentrations of lidocaine in tumor tissues were higher in experimental group than in control group at T0.5 (P=0.004), T1 (P=0.038), T4 (P=0.036), and T8 (P=0.029). In the clinical trial, VAS scores in Group B were significantly lower than in Group A at T0 (P=0.006), T4 (P=0.001), T8 (P=0.002), and T24 (P=0.005). The tramadol intake in Group B was significantly lower than in Group A (P=0.021). No significant difference was observed regarding the incidence of adverse events between the two groups. CONCLUSION: This study demonstrated the effectiveness and safety of intraarterial lidocaine administration using the W/O technique in controlling intra- and post-TACE pain.

Gastrodin ameliorates learning and memory impairment in rats with vascular dementia by promoting autophagy flux via inhibition of the Ca2+/CaMKII signal pathway.

Vascular dementia (VD) is a common disease that occurs during human aging. Gastrodin (GAS) has potential benefits for the prevention and treatment of VD. In the present study, we investigated the effects of GAS on cognitive dysfunction in rats with VD induced by permanent middle cerebral artery occlusion (pMCAO) and explored the underlying mechanism. Immunohistochemical and western blot analyses revealed that GAS attenuated hippocampal levels of LC3 (microtubule-associated protein 1 light chain 3), p62, and phosphorylated CaMKII (Ca2+-calmodulin stimulated protein kinase II) in VD rats. Additionally, our results revealed that cobalt chloride blocked autophagic flux in HT22 cells, which was confirmed by increased levels of LC3 and p62 when combined with chloroquine. Notably, GAS ameliorated the impaired autophagic flux. Furthermore, we confirmed that GAS combined with KN93 (a CaMKII inhibitor) or CaMKII knockdown did not impact the reduced p62 levels when compared with GAS treatment alone. Furthermore, a co-immunoprecipitation assay demonstrated that endogenous p62 bound to CaMKII, as confirmed by mass spectrometric analysis after the immunoprecipitation of p62 from HT22 cells. These findings revealed that GAS attenuated autophagic flux dysfunction by inhibiting the Ca2+/CaMKII signaling pathway to ameliorate cognitive impairment in VD.

Inhibitory effect of resveratrol on the pharmacokinetics of ticagrelor in vivo and in vitro.

This study was to evaluate the effect of resveratrol on the pharmacokinetics of ticagrelor in rats and the metabolism of ticagrelor in human cytochrome P450 (CYP) 3A4 (CYP3A4) and liver microsomes. Eighteen Sprague-Dawley rats were randomly divided into three groups: group A (control group), group B (50 mg/kg resveratrol), and group C (150 mg/kg resveratrol). After 30 min administration of resveratrol, a single dose of ticagrelor (18 mg/kg) was administered orally. The in vitro experiment was performed to examine the influence of resveratrol on ticagrelor metabolism in CYP3A4*1, human, and rat liver microsomes. Serial biological samples were assayed by validated ultra high-performance liquid chromatography - tandem mass spectrometer methods. For the in vivo study, the area under the concentration-time curve and mean peak plasma concentrations of ticagrelor in group B and C appeared to be significantly higher than the control group, while volume of distribution in terminal phase and apparent clearance of ticagrelor in group B and C were significantly decreased. For the in vitro study, resveratrol exhibited an inhibitory effect on CYP3A4*1, human and rat liver microsomes. The half-maximal inhibitory concentration values of resveratrol were 56.75 µM, 69.07 µM, and 14.22 µM, respectively. Our results indicated that resveratrol had an inhibitory effect on the metabolism of ticagrelor in vitro and in vivo. Further research should focus on the clinical combination of resveratrol with ticagrelor, and ticagrelor plasma concentration should be monitored to avoid the occurrence of adverse reaction.

Functional Measurement of CYP2C9 and CYP3A4 Allelic Polymorphism on Sildenafil Metabolism.

AIM: We aimed to systematically examine the effects of enzymatic activity of 38 human CYP2C9 alleles and 21 human CYP3A4 alleles, including wild-type CYP2C9.1 and CYP3A4.1, which contain the 24 CYP2C9 novel alleles (*36-*60) and 6 CYP3A4 novel alleles (*28-*34) newly found in the Chinese population, on sildenafil metabolism through in vitro experiment. METHODS: The recombinant cytochrome P450 alleles protein of CYP2C9 and CYP3A4 expressed in insect baculovirus expression system were reacted with 10-500 µM sildenafil for 30 minutes at 37°C, and the reaction was terminated by cooling to -80°C immediately. Next, we used ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) detection system to detect sildenafil and its active metabolite N-desmethyl sildenafil. RESULTS: The intrinsic clearance (Vmax/Km) values of most CYP2C9 variants were significantly altered when compared with the wild-type CYP2C9*1, with most of these variants exhibiting either reduced Vmax and/or increased Km values. Four alleles (CYP2C9*11, *14, *31, *49) exhibited no markedly decreased relative clearance (1-fold). The relative clearance of the remaining thirty-three variants exhibited decrease in different levels, ranging from 1.81% to 88.42%. For the CYP3A4 metabolic pathway, when compared with the wild-type CYP3A4*1, the relative clearance values of four variants (CYP3A4*3, *10, *14 and *I335T) showed significantly higher relative clearance (130.7-134.9%), while five variants (CYP3A4*2, *5, *24, *L22V and *F113I) exhibited sharply reduced relative clearance values (1.80-74.25%), and the remaining nine allelic variants showed no statistical difference. In addition, the kinetic parameters of two CYP3A4 variants (CYP3A4*17 and CYP3A4*30) could not be detected, due to the defect of the CYP3A4 gene. CONCLUSION: These findings were the first evaluation of all these infrequent CYP2C9 and CYP3A4 alleles for sildenafil metabolism; when treating people who carry these CYP2C9 and CYP3A4 variants, there should be more focus on the relation of dose intensity, side effects and therapeutic efficacy when administering sildenafil. The study will provide fundamental data on effect of CYP2C9 and CYP3A4 allelic variation on sildenafil metabolism for further clinical research.

The effect of apatinib on pharmacokinetic profile of buspirone both in vivo and in vitro.

OBJECTIVE: In this study, we aimed to investigate the potential interaction of apatinib and buspirone and underlying mechanism. METHODS: UPLC-MS/MS assay was applied to determine the concentrations of buspirone and its main metabolites (1-PP and 6-OH buspirone) after incubated with liver microsomes. Moreover, the connection of in vitro and in vivo was further determined. Sprague Dawley rats were randomly divided into two groups: group A (20 mg/kg buspirone) and group B (buspirone vs 40 mg/kg apatinib). Tail vein blood was collected and subjected to the UPLC-MS/MS detection. KEY FINDINGS: Apatinib inhibited the generations of 1-PP and 6-OH buspirone dose-dependently with IC50 of 1.76 and 2.23 µm in RLMs, and 1.51 and 1.48 µm in HLMs, respectively. There was a mixed mechanism underlying such an inhibition effect. In rat, AUC(0- t ) , AUC(0-∞) , Tmax and Cmax of buspirone and 6-OH buspirone increased significantly while co-administering with apatinib, but Vz/F and CLz/F decreased obviously while comparing group A with group B . CONCLUSIONS: Apatinib suppresses the CYP450 based metabolism of buspirone in a mixed mechanism and boosted the blood exposure of prototype drug and 6-OH buspirone dramatically. Therefore, extra caution should be taken when combining apatinib with buspirone in clinic.

Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes.

BACKGROUND: Calpain-2 is a Ca2+-dependent cysteine protease, and high calpain-2 activity can enhance apoptosis mediated by multiple triggers. AIM: To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum (ER) stress-related apoptosis in rat hepatocyte BRL-3A cells. METHODS: BRL-3A cells were treated with varying doses of dithiothreitol (DTT), and their viability and apoptosis were quantified by 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2-H-tetrazolium bromide and flow cytometry. The expression of ER stress- and apoptosis-related proteins was detected by Western blot analysis. The protease activity of calpain-2 was determined using a fluorescent substrate, N-succinyl-Leu-Leu-Val-Tyr-AMC. Intracellular Ca2+ content, and ER and calpain-2 co-localization were characterized by fluorescent microscopy. The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified. RESULTS: DTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis. DTT treatment significantly upregulated 78 kDa glucose-regulated protein, activating transcription factor 4, C/EBP-homologous protein expression by >2-fold, and enhanced PRKR-like ER kinase phosphorylation, caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence. DTT treatment also significantly increased intracellular Ca2+ content, calpain-2 expression, and activity by >2-fold in BRL-3A cells. Furthermore, immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2. Moreover, calpain-2 silencing to decrease calpain-2 expression by 85% significantly mitigated DTT-enhanced calpain-2 expression, caspase-12 cleavage, and apoptosis in BRL-3A cells. CONCLUSION: The data indicated that Ca2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.

A novel prognostic scoring model for newly diagnosed FLT3-ITD-positive acute myeloid leukemia.

FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML). However, the molecular structure characteristics and widely accepted prognostic factors for FLT3-ITD are still not well described. This study aimed to retrospectively examine 81 patients with FLT3-ITD-positive AML diagnosed and treated at the First Affiliated Hospital of Zhejiang University from December 2013 to March 2018 using the next-generation sequencing 185-gene platform. High variant allele frequency (VAF) [> 0.48, P = 0.0089 for overall survival (OS), P = 0.13 for relapse-free survival (RFS)], multiple ITDs (> 1 ITDs, P = 0.011 for OS, P = 0.033 for RFS) and longer insertion length (> 69 bp, P = 0.14 for OS, P = 0.0078 for RFS) predicted poor survival. The study further proposed an easily applicable scoring model for OS using the Least Absolute Shrinkage and Selector Operation (LASSO) Cox regression model. Also, an independent cohort of 30 patients was used for external model validation. The mode was expressed as follows: 0.659 × FLT3-ITD VAF + 0.375 × FLT3-ITD number + 0.807 × Age + 0.688 × DNMT3A + 1.939 × U2AF1 (FLT3-ITD VAF > 0.48 scored 1; FLT3-ITD number scored 1 if carried 1 ITD, 2 if carried ≥ 2 ITDs; age > 44 years scored 1, the presence of DNMT3A or U2AF1 scored 1; 0 for other conditions). It categorized patients into low-risk (L-R, score < 1, n = 20) and high-risk (H-R, score ≥ 1, n = 61) groups based on the risk score with a significant difference in survival (3-year OS, P < 0.0001; 3-year RFS, P = 0.0005). A prognostic nomogram that integrated these five factors was developed with a concordance index calculation [OS: 0.68, 95% CI (0.64-0.72)].

Effects of 24 CYP2D6 variants found in Chinese population on the metabolism of clonidine in vitro.

BACKGROUND AND OBJECTIVES: Clonidine has been clinically used to treat Tourette's syndrome for decades. There was research finding that clonidine possessed the best risk-benefit ratio, especially for patients associated with attention deficit hyperactivity disorder. CYP2D6 is a significant member of Cytochrome P450 enzymes. The genetic polymorphisms of CYP2D6 greatly affect the clinical effects of drugs even lead to side effects and medical malpractice. Our goal is to research the effect of CYP2D6 genetic polymorphism on the metabolism of clonidine and evaluate the functions of 22 CYP2D6 allelic variants in vitro, which were discovered in Chinese Han population recently. METHODS: This study was carried out through a mature incubation system. The wild-type CYP2D6*1 and 24 variants (CYP2D6*2, CYP2D6*10 and 22 novel CYP2D6 variants) were expressed in insect cells, and the catalytic activity of all the variants were assessed by substrate clonidine. Metabolite 4-OH clonidine was accurately detected via ultra-performance liquid-chromatography tandem mass spectrometry to evaluate the effect of CYP2D6 genetic polymorphism on the clonidine. RESULT: Among the 22 novel CYP2D6 variants, the intrinsic clearance (Vmax/Km) of 21 variants were significantly decreased (from 1.53% to 83.25%) compared to the wild-type. In particular, the following seven variants (CYP2D6* 2, CYP2D6* 10, CYP2D6* 93, CYP2D6* 95, E215K, V327 M and R497C) attract more attention, of which the intrinsic clearance decreased more than 70% compared to the wild-type. Because the variants with significantly reduced intrinsic clearance are more likely to cause adverse reactions than the variants with increased or little changed intrinsic clearance. In addition, the related pharmacokinetic parameters of CYP2D6*92 and CYP2D6*96 could not be acquired for the defect of CYP2D6 nucleotide. CONCLUSION: We comprehensively evaluated the effect of 22 novel CYP2D6 variants on the metabolism of clonidine for the first time and hoped corresponding data provide a reference for metabolism of clonidine for further studies in vivo, and extend our understanding of the clinical drug toxicity or ineffectiveness by CYP2D6 genetic polymorphism.

Enzymatic Activities of CYP3A4 Allelic Variants on Quinine 3-Hydroxylation In Vitro.

Cytochrome P450 3A4 (CYP3A4) enzyme activity is known to show considerable ethnic heterogeneity and inter-individual differences, affecting the outcome of drug treatment. CYP3A4 genetic polymorphisms are believed to be one of the important causes, leading to inter-individual variability in drug metabolism. Quinine is an antipyretic drug with antimalarial properties that is metabolized primarily by CYP3A4. Quinine 3-hydroxylation has been proven as a biomarker reaction for evaluating CYP3A4 ability. Quinine has frequent adverse effects and there are distinct inter-individual differences in quinine sensitivity. The open reading frame for 30 CYP3A4 allelic variants were constructed from wild-type CYP3A4*1A by an overlap extension polymerase chain reaction. Recombinant CYP3A4 variants were expressed using baculovirus-insect cell expression system, and their catalytic activities towards quinine hydroxylation were determined and evaluated. Of the 30 CYP3A4 allelic variants, 23 variants exhibited significantly reduced intrinsic clearance towards quinine, 2 variants showed increased intrinsic clearance for quinine, 2 variants possessed no significant differences towards quinine, compared with CYP3A4*1A, and 3 variants had no detected expression and enzyme activity. Our assessment on the enzymatic activities of CYP3A4 variants towards quinine may contribute to laying an experimental foundation for further clinical studies so as to accelerate the process of determining the associations between genetic variations and clinical phenotypes.

Shikonin inhibits triple-negative breast cancer-cell metastasis by reversing the epithelial-to-mesenchymal transition via glycogen synthase kinase 3ß-regulated suppression of ß-catenin signaling.

Triple-negative breast cancer (TNBC) is characterized by elevated metastasis, low survival, and poor response to therapy. Although many specific and effective agents for treating TNBC have been investigated, promising therapeutic options remain elusive. Here, we screened the inhibitory activities of three main components of Lithospermum erythrorhizon Sieb. et Zucc (shikonin, acetylshikonin, and ß,ß-dimethylacrylshikonin) on TNBC cells. The results revealed that shikonin potently decreased the viabilities of TNBC MDA-MB-231 and 4T1 cells but showed less cytotoxicity to normal mammary epithelial MCF-12A cells. Additionally, shikonin reversed the epithelial-to-mesenchymal transition (EMT) in MDA-MB-231 and 4T1 cells. Shikonin depressed cell migration and invasion, upregulated E-cadherin levels, downregulated N-cadherin, vimentin, and Snail levels, and reorganized the cytoskeletal proteins F-actin and vimentin. Shikonin reversed EMT by inhibiting activation of ß-catenin signaling through attenuating ß-catenin expression, nuclear accumulation, binding to T-cell factor consensus oligos, and transcription of its targeted EMT-related genes. Moreover, shikonin upregulated glycogen synthase kinase 3ß (GSK-3ß) levels, leading to enhanced phosphorylation and decreased levels of ß-catenin. Furthermore, shikonin administration significantly inhibited lung metastasis of MDA-MB-231 cells in NOD/SCID mice accompanied by low systemic toxicity. Histological analysis confirmed that shikonin elevated levels of E-cadherin, phosphorylated ß-catenin, and GSK-3ß, and decreased levels of vimentin and ß-catenin in pulmonary metastatic foci. These results indicated that shikonin potently inhibits TNBC metastasis by targeting the EMT via GSK-3ß-regulated suppression of ß-catenin signaling, which highlights the importance of shikonin as a potential candidate for novel anticancer therapeutics against TNBC.

Hepatitis B virus X protein decreases nephrin expression and induces podocyte apoptosis via activating STAT3.

The gene for hepatitis B virus X protein (HBx) comprises the smallest open reading frame in the HBV genome, and the protein product can activate various cell signaling pathways and regulate apoptosis, among other effects. However, in different cell types and under different external conditions, its mechanism of action differs. In the present study, the effect of HBx on the viability and apoptosis of mouse podocyte clone 5 (MPC5) cells was investigated. The cells were transfected with the HBx gene using pEX plasmid, and real-time quantitative PCR and western blot analysis were used to test the transfection efficiency and assess related protein expression. The highest expression of HBx occurred at 48 h after MPC5 cells were transfected with HBx. The expression of nephrin protein in the HBx transfection group was lower than that in blank and negative control groups. Following transfection of the HBx gene, podocyte viability was suppressed, while the rate of cell apoptosis was increased; moreover, the expression of signal transducer and activator of transcription 3 (STAT3) and phospho-STAT3 was increased compared with in the control groups. The present study suggests that STAT3 activation may be involved in the pathogenic mechanism of renal injuries caused by HBV injection. Thus STAT3 is a potential molecular target in the treatment of HBV-GN.

Proteomic analysis of the effects of cell culture density on the metastasis of breast cancer cells.

Cancer cell progression and proliferation increase cell density, resulting in changes to the tumour site, including the microenvironment. What is not known is if increased cell density influences the aggressiveness of cancer cells, especially their proliferation, migration, and invasion capabilities. In this study, we found that dense cell culture enhances the aggressiveness of the metastatic cancer cell lines, 4T1 and ZR-75-30, by increasing their proliferation, migration, and invasion capabilities. However, a less metastatic cell line, MCF-7, did not show an increase in aggressiveness, following dense cell culture conditions. We conducted a differential proteomic analysis on 4T1 cells cultured under dense or sparse conditions and identified an increase in expression for proteins involved in migration, including focal adhesion, cytoskeletal reorganization, and transendothelial migration. In contrast, 4T1 cells grown under sparse conditions had higher expression levels for proteins involved in metabolism, including lipid and phospholipid binding, lipid and cholesterol transporter activity, and protein binding. These results suggest that the high-density tumour microenvironment can cause a change in cellular behaviour, leading towards more aggressive cancers. SIGNIFICANCE OF THE STUDY: Metastasis of cancer cells is an obstacle to the clinical treatment of cancer. We found that dense cultures made metastatic cancer cells more potent in terms of proliferation, migration, and invasion. The proteomic and bioinformatic analyses provided some valuable clues for further intensive studies about the effects of cell density on cancer cell aggressiveness, which were associated with events such as pre-mRNA splicing and RNA transport, focal adhesion and cytoskeleton reorganization, ribosome biogenesis, and transendothelial migration, or associated with proteins, such as JAM-1 and S100A11. This investigation gives us new perspectives to investigate the metastasis mechanisms related to the microenvironment of tumour sites.

Preconditioning with fludarabine, busulfan and cytarabine versus standard BuCy2 for patients with acute myeloid leukemia: a prospective, randomized phase II study.

To compare the efficacy and toxicity of a novel regimen called FBA, consisting of fludarabine, busulfan, and cytarabine, with the standard BuCy2 regimen for younger adult patients with acute myeloid leukemia, we conducted a prospective randomized phase II study. Patients in complete remission were randomly assigned to receive either the FBA (n = 56) or the BuCy2 regimen (n = 55). The difference in 100-day transplant-related mortality (TRM) was not statistically significant between the two arms (1.79% for FBA versus 5.45% for BuCy2, P = 0.260), as were the cumulative incidences of relapse, TRM, overall survival (OS) and event-free survival (EFS) at 3 years. However, the 100-day cumulative incidences of grades II-IV and III-IV acute graft-versus-host disease (aGVHD) were lower in the FBA group [(8.93% versus 21.86%, P = 0.032) (1.79% versus 9.09%, P = 0.025)]. The 3-year GVHD and relapse-free survival (GRFS) was 31.20% for the FBA group and 14.96% for the BuCy2 group (P = 0.004). The incidences of diarrhea and severe oral mucositis within the first 30 days post-transplantation were lower in the FBA group [(28.57% versus 65.45%; P < 0.001) (51.79% versus 70.91%; P = 0.039)]. In conclusion, allogenic transplantation with the FBA regimen achieved similar TRM, relapse rate, OS and EFS, as that with the BuCy2 regimen but with less frequent and less severe complications in early stage after transplantation and a trend toward higher GRFS.

Functional characterization of CYP2C19 variants in nebivolol 4-hydroxlation in vitro.

Cytochrome P450 2C19 (CYP2C19) allelic variants are thought to play an important part in inter-individual variability in drug metabolism. We evaluated the in vitro hydroxylation of nebivolol by 31 CYP2C19 alleles identified in a Chinese Han population recently. Wild-type CYP2C19*1B and 30 isoforms were highly expressed in insect cells, and the enzymatic activities of CYP2C19 variants towards nebivolol hydroxylation were characterized. Among the 30 CYP2C19 alleles, most of the recombinant CYP2C19 variants exhibited no or significantly low activity compared with CYP2C19*1B. Three variants, CYP2C19*29 (K28I), L16F, and CYP2C19*23 (G91R), showed increased intrinsic clearance of >140% CYP2C19*1B. Combined with a previous study on the effects of CYP2D6 variants on nebivolol metabolism, our comprehensive analyses on the enzymatic activities of CYP2C19 variants towards nebivolol in the present study may contribute to determination of the optimal doses of nebivolol for the treatment of hypertension and understanding of "individualized" medication.

Time Window Is Important for Adenosine Preventing Cold-induced Injury to the Endothelium.

Cold cardioplegia is used to induce heart arrest during cardiac surgery. However, endothelial function may be compromised after this procedure. Accordingly, interventions such as adenosine, that mimic the effects of preconditioning, may minimize endothelial injury. Herein, we investigated whether adenosine prevents cold-induced injury to the endothelium. Cultured human cardiac microvascular endothelial cells were treated with adenosine for different durations. Phosphorylation and expression of endothelial nitric oxide synthase (eNOS), p38MAPK, ERK1/2, and p70S6K6 were measured along with nitric oxide (NO) production using diaminofluorescein-2 diacetate (DAF-2DA) probe. Cold-induced injury by hypothermia to 4°C for 45 minutes to mimic conditions of cold cardioplegia during open heart surgery was induced in human cardiac microvascular endothelial cells. Under basal conditions, adenosine stimulated NO production, eNOS phosphorylation at serine 1177 from 5 minutes to 4 hours and inhibited eNOS phosphorylation at threonine 495 from 5 minutes to 6 hours, but increased phosphorylation of ERK1/2, p38MAPK, and p70S6K only after exposure for 5 minutes. Cold-induced injury inhibited NO production and the phosphorylation of the different enzymes. Importantly, adenosine prevented these effects of hypothermic injury. Our data demonstrated that adenosine prevents hypothermic injury to the endothelium by activating ERK1/2, eNOS, p70S6K, and p38MAPK signaling pathways at early time points. These findings also indicated that 5 minutes after administration of adenosine or release of adenosine is an important time window for cardioprotection during cardiac surgery.

Establishment of a novel human lymphoblastic cell strain with the long arm of chromosome 11 aberration without MLL rearrangement.

At present, all cell strains derived from acute lymphoblastic leukemia (ALL) patients with the long arm of chromosome 11 aberration are accompanied with mixed lineage leukemia (MLL) gene rearrangement. In this study, we established a permanent ALL cell strain CHH-1 with the long arm of chromosome 11 aberration and without MLL rearrangement, hoping that it could be used for the research of ALL with such genetic abnormality. CHH-1 cell strain was certified through morphology, immunophenotype, genetics and immunoglobulin (Ig) gene rearrangement analysis. Cell characteristics including tumorigenic ability, semisolid colony forming ability, telomerase activity, autocrine and invasion were further detected. Cells were with an add(11)(q23) structural abnormality without MLL rearrangement, and were consistent with the genetic abnormality of the patient. In addition, these cells had features of tumor-forming ability, high colony forming capacity, unique cytokine autocrine mode, high telomerase activity, and high invasion ability. CHH-1 may prove to be a useful cell model for the research of human leukemia with genetic aberration in chromosome 11, and help explore the role of such genetic abnormality in the pathogenesis, progression and prognosis of ALL, and in developing new target drugs.