There is an increasing need to understand cell-cell interactions for cell and tissue engineering purposes, such as optimizing cell sheet constructs, as well as for examining adhesion defect diseases. For cell-sheet engineering, one major obstacle to sheet function is that cell sheets in suspension are fragile and, over time, will contract. While the role of the cytoskeleton in maintaining the structure and adhesion of cells cultured on a rigid substrate is well-characterized, a systematic examination of the role played by different components of the cytoskeleton in regulating cell sheet contraction and cohesion in the absence of a substrate has been lacking.
In this study, keratinocytes were cultured until confluent and cell sheets were generated using dispase to remove the influence of the substrate. The effects of disrupting actin, microtubules or intermediate filaments on cell-cell interactions were assessed by measuring cell sheet cohesion and contraction. Keratin intermediate filament disruption caused comparable effects on cell sheet cohesion and contraction, when compared to actin or microtubule disruption. Interfering with actomyosin contraction demonstrated that interfering with cell contraction can also diminish cell cohesion.
All components of the cytoskeleton are involved in maintaining cell sheet cohesion and contraction, although not to the same extent. These findings demonstrate that substrate-free cell sheet biomechanical properties are dependent on the integrity of the cytoskeleton network.
Cell sheet; Cytoskeleton; Adhesion; Contraction
AIM: To examine the clinical features and analyze prognostic factors in a prospective study of primary biliary cirrhosis (PBC) patients.
METHODS: From 1995 to 2010, PBC patients without hepatic decompensation seen at the Peking Union Medical College Hospital were enrolled. Clinical signs and manifestations (pruritus, persistent fatigue, jaundice and pain in the right hypochondrium), laboratory parameters (auto-antibodies for autoimmune hepatic disease, biliary and hepatic enzymes, immunoglobulin, bilirubin, and albumin) and imaging findings were recorded at entry and at specific time points during follow-up. Cox regression and Kaplan-Meier analyses, respectively, assessed the risk factors for hepatic decompensation and survival.
RESULTS: Two hundred and sixty-two PBC patients were enrolled with a median follow-up of 75.2 mo (range, 21-201 mo). The 240 patients were aged 51.5 ± 10.2 years at diagnosis and 91.6% were female. Two hundred and forty-five (93.5%) were seropositive for anti-mitochondrial antibodies. At presentation, 170 patients (64.9%) were symptomatic, while 96 patients (36.6%) had extra-hepatic autoimmune disease. During the follow-up period, 62 (23.7%) patients developed hepatic decompensation of whom four underwent liver transplantation and 17 died. The cumulative survival rate and median survival time were 83.9% and 181.7 mo, respectively. Cox regression analysis revealed that an incomplete ursodeoxycholic acid (UDCA) response or inconsistent treatment [P < 0.001; hazard risk (HR) 95%CI = 2.423-7.541], anti-centromere antibodies (ACA) positivity (P < 0.001; HR 95%CI = 2.516-7.137), alanine aminotransferase ratio (AAR) elevations (P < 0.001; HR 95%CI = 1.357-2.678), and histological advanced liver disease (P = 0.006; HR 95%CI = 1.481-10.847) were predictors of hepatic decompensation. The clinical features and survival of PBC in China are consistent with those described in Western countries.
CONCLUSION: Incomplete UDCA response or inconsistent treatment, ACA positivity, AAR elevations, and advanced histological stage are predictors of decompensation.
Primary biliary cirrhosis; Risk factor; Hepatic decompensation; Survival; Ursodeoxycholic acid response; Anti-centromere antibodies; Histological stage
Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z’-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538), was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.
Extracorporeal Shock Wave Lithotriptors are very popular for the treatment of urinary stones all over the world. They depend basically upon either X-ray fluoroscopy or ultrasound scans to detect the stones before therapy begins. To increase the effectiveness of treatment this study took advantage of both X-ray and ultrasound to develop a dual stone locating system with image processing modules. Its functions include the initial stone locating mode with stone detection by fluorescent images and the follow-up automatic stone tracking mode made by constant ultrasound scanning. The authors have integrated both apparatus and present the operating principles for both modes. The system used two in vitro experiments to justify its abilities of stone location in all procedures.
X-ray; ultrasound; image processing; Extracorporeal Shock Wave Lithotriptor; stone location; stone tracking
This study was designed to investigate whether Resveratrol (Res) could be a prophylactic factor in the prevention of I/R injury and to shed light on its underlying mechanism. Primary culture of neonatal rat cardiomyocytes were randomly distributed into three groups: the normal group (cultured cardiomyocytes were in normal conditions), the I/R group (cultured cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion), and the Res+I/R group (100 µmol/L Res was administered before cardiomyocytes were subjected to 2 h simulated ischemia followed by 4 h reperfusion). To test the extent of cardiomyocyte injury, several indices were detected including cell viability, LDH activity, Na+-K+-ATPase and Ca2+-ATPase activity. To test apoptotic cell death, caspase-3 activity and the expression of Bcl-2/Bax were detected. To explore the underlying mechanism, several inhibitors, intracellular calcium, SOD activity and MDA content were used to identify some key molecules involved. Res increased cell viability, Na+-K+-ATPase and Ca2+-ATPase activity, Bcl-2 expression, and SOD level. While LDH activity, capase-3 activity, Bax expression, intracellular calcium and MDA content were decreased by Res. And the effect of Res was blocked completely by either L-NAME (an eNOS inhibitor) or MB (a cGMP inhibitor), and partly by either DS (a PKC inhibitor) or Glybenclamide (a KATP inhibitor). Our results suggest that Res attenuates I/R injury in cardiomyocytes by preventing cell apoptosis, decreasing LDH release and increasing ATPase activity. NO, cGMP, PKC and KATP may play an important role in the protective role of Res. Moreover, Res enhances the capacity of anti-oxygen free radical and alleviates intracellular calcium overload in cardiomyocytes.
Telomere length plays an important role in chromosomal stability and tumorigenesis, and its measurement in peripheral white blood cell DNA may be a predictor of the development of lung cancer.
Using a new method - monochrome multiplex quantitative PCR -which reduces measurement variability, we compared telomere length relative to standard DNA in white blood cell DNA in 229 incident male lung cancer cases and 229 matched controls within the prospective Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of male smokers.
Median (10th, 90th percentile) telomere length was 1.13 (0.86, 1.45) in cases and 1.08 (0.85, 1.38) in controls (P = 0.038). Telomere length was inversely associated with pack-years of smoking (Spearman correlation r = −0.16, P = 0.02) among controls. Compared to subjects with shorter telomere length (≤ median), subjects with greater telomere length (> median) had a 1.6-fold (95% CI, 1.06–2.36) increased risk of lung cancer. There was a significant linear relationship between quartiles of telomere length and risk of lung cancer (odds ratios (95% confidence intervals) by quartile: 1.00, 0.98 (0.55–1.73), 1.62 (0.95–2.77), and 1.50 (0.84–2.68); Ptrend = 0.05). In addition, subgroup analysis showed that greater telomere length was associated with increased risk of lung cancer among heavy smokers (> 38 years) (OR, 1.90; 95% CI, 1.00–3.59) but not among light smokers (≤ 38 years) (OR, 1.08; 95% CI, 0.56–2.11) (Pinteraction = 0.01).
Our results suggest that greater telomere length may be associated with higher risk of lung cancer among male smokers.
Telomere length; lung cancer; cohort study
The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography.
Pyruvate kinase (PYK) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit Leishmania mexicana PYK (LmPYK) activity in a time- (and dose-) dependent manner; a characteristic of irreversible inhibition. The crystal structure of 4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid (DBS) complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.
Leishmania mexicana; lysine covalent modification; nucleotide binding; pyruvate kinase; saccharin analogues; covalent inhibitor
The development of tumor drug resistance is one of the biggest obstacles on the way to achieve a favorable outcome of chemotherapy. Among various strategies that have been explored to overcome drug resistance, the combination of current chemotherapy with plant polyphenols as a chemosensitizer has emerged as a promising one. Plant polyphenols are a group of phytochemicals characterized by the presence of more than one phenolic group. Mechanistic studies suggest that polyphenols have multiple intracellular targets, one of which is the proteasome complex. The proteasome is a proteolytic enzyme complex responsible for intracellular protein degradation and has been shown to play an important role in tumor growth and the development of drug resistance. Therefore, proteasome inhibition by plant polyphenols could be one of the mechanisms contributing to their chemosensitizing effect. Plant polyphenols that have been identified to possess proteasome-inhibitory activity include (−)-epigallocatechins-3-gallate (EGCG), genistein, luteolin, apigenin, chrysin, quercetin, curcumin and tannic acid. These polyphenols have exhibited an appreciable effect on overcoming resistance to various chemotherapeutic drugs as well as multidrug resistance in a broad spectrum of tumors ranging from carcinoma and sarcoma to hematological malignances. The in vitro and in vivo studies on polyphenols with proteasome-inhibitory activity have built a solid foundation to support the idea that they could serve as a chemosensitizer for the treatment of cancer. In-depth mechanistic studies and identification of optimal regimen are needed in order to eventually translate this laboratory concept into clinical trials to actually benefit current chemotherapy.
Polyphenols; flavonoids; EGCG; genistein; curcumin; structure-activity relationship; ubiquitin-proteasome pathway; proteasome inhibitors; bortezomib; chemotherapy; drug resistance; NF-κB; Bcl-2; chemosensitization; clinical trials
Control of intracellular reactive oxygen species (ROS) concentrations is critical for cancer cell survival. We show that, in human lung cancer cells, acute increases in intracellular concentrations of ROS caused inhibition of the glycolytic enzyme pyruvate kinase M2 (PKM2) through oxidation of Cys358. This inhibition of PKM2 is required to divert glucose flux into the pentose phosphate pathway and thereby generate sufficient reducing potential for detoxification of ROS. Lung cancer cells in which endogenous PKM2 was replaced with the Cys358 to Ser358 oxidation-resistant mutant exhibited increased sensitivity to oxidative stress and impaired tumor formation in a xenograft model. Besides promoting metabolic changes required for proliferation, the regulatory properties of PKM2 may confer an additional advantage to cancer cells by allowing them to withstand oxidative stress.
Drug repurposing for cancer treatment is an emerging approach to discover clinically approved drugs that demonstrate antineoplastic effect. The effective therapeutics for patients with advanced adrenocortical carcinoma(ACC) are greatly needed. The objective of this study was to identify and validate drugs with antineoplastic effect in ACC cells using a novel quantitative high-throughput drug screening (qHTS) technique.
A quantitative high-throughput proliferation assay of 2,816 clinically approved drugs was performed in the NCI-H295R ACC cell line. We validated the antiproliferative effect of candidate compounds in NCI-H295R cells. Further validation was performed in 3-dimensional multicellular aggregates (MCA) of NCI-H295R and SW-13 cell lines.
We identified 79 active compounds against ACC cells; 21 had an efficacy ≥60% and IC50 <1 μM. The top drug categories enriched were cardiotonic, antiseptic, and antineoplastic. We selected Bortezomib, ouabain, Methotrexate, pyrimethamine for validation. All had an antiproliferative effect in monolayer culture of NCI-H295R cells at clinical achievable serum level. Bortezomib and ouabain inhibited growth of MCA in both cell lines at a low concentration (10 fold below IC50). Methotrexate inhibited growth and caused disintegration of MCA in both cell lines at concentrations well below the maximum serum level (10 to 100 fold of IC50). Pyrimethamine caused growth inhibition in both cell lines at 10 fold of IC50 concentration.
qHTS of previously approved compounds is an effective and efficient method to identify anticancer drugs for a rare cancer such as ACC. We have validated the antineoplastic effect of Bortezomib, ouabain, Methotrexate and pyrimethamine, which could be translated into clinical trials in patients with locally advanced and/or metastatic ACC.
Adrenocortical cancer; High throughput drug screening; Chemotherapy; Drug repurposing; Indication switching
An integrin found on platelets, αIIbβ3 mediates platelet aggregation, and αIIbβ3 antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the β subunit metal ion–dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the αIIb subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the β3 subunits. They induce conformational changes in the β3 subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of αIIbβ3 (RUC-1) that binds exclusively to the αIIb subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ~100-fold higher affinity. RUC-2 does not induce major conformational changes in β3 as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2–IIbβ3 headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb the way RUC-1 does, but in addition, it binds to the β3 MIDAS residue glutamic acid 220, thus displacing Mg2+ from the MIDAS. When the Mg2+ concentration was increased to 20 mM, however, Mg2+ was identified in the MIDAS and RUC-2 was absent. RUC-2′s ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg2+ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other αIIbβ3 antagonists and may offer advantages as a therapeutic agent.
Polyethylene glycol-grafted polyethylenimine (PEG-g-PEI) which was functionalized with a neuroblastoma cell-specific ligand, the GD2 single chain antibody (scAbGD2), was synthesized in order to effectively deliver Bcl-2 siRNA into neuroblastoma cells. This polymer was complexed first with superparamagnetic iron oxide nanoparticle (SPION) to get a MRI-visible targeted non-viral vector (scAbGD2-PEG-g-PEI-SPION) and then with Bcl-2 siRNA to form nanoparticles showing low cytotoxicity. The targeting capacity of scAbGD2-PEG-g-PEI-SPION was successfully verified in vivo and in vitro by magnetic resonance imaging. The single chain antibody encoded targeted polyplex was more effective in transferring Bcl-2 siRNA than the nontargeting one in SK-N-SH cells, a human neuroblastoma cell line, resulting in a 46.34% inhibition in the expression of Bcl-2 mRNA. Consequently, a high level of cell apoptosis up to 50.76% and a significant suppression of tumor growth were achieved, which indicates that scAbGD2-PEG-g-PEI-SPION is a promising magnetic resonance imaging-visible non-viral vector for targeted neuroblastoma siRNA therapy and diagnosis.
tumor targeting; GD2; non-viral vector; Bcl-2 small interfering RNA; magnetic resonance imaging
Immune deficiency is one of the best characterized and strongest known risk factors for non-Hodgkin lymphoma (NHL). We studied the association between single nucleotide polymorphisms (SNPs) in integrin genes that are important components in human innate immunity and the risk of NHL in a population-based case–control study of women in Connecticut, USA. A total of 373 tag SNPs in 33 gene regions were included in the analysis of 448 cases and 525 controls. The ADAM19 rs11466782 SNP was associated with an increased risk of lymphoma (OR, 1.73; 95 % CI, 1.28–2.35; P additive = 0.0004), and the ICAM3 rs2304240 (OR, 0.67; 95 % CI, 0.52–0.86; P additive = 0.002) and the PTGDR rs708486 SNPs (OR, 0.75; 95 % CI, 0.63–0.90; P additive = 0.002) were associated with reduced risk of lymphoma. Two gene regions (ADAM19 (P=0.009) and ICAM3 (P=0.009)) displayed global associations with lymphoma risk at the P<0.01 level. While our results suggest that genetic polymorphisms in integrin genes may play a role in the genesis of lymphoma in women, they should be viewed as exploratory until they are replicated in additional populations.
lymphoma; integrin; innate immunity; single nucleotide polymorphism
Continued examination of substituted 6-arylquinazolin-4-amines as Clk4 inhibitors resulted in selective inhibitors of Clk1, Clk4, Dyrk1A and Dyrk1B. Several of the most potent inhibitors were validated as being highly selective within a comprehensive kinome scan.
Clk1; Clk2; Clk3; Clk4; Dyrk1A; Dyrk1B; Pre-mRNA splicing; kinase inhibition; quinazoline
Evidence suggests that de novo, therapy-related and benzene-induced acute myeloid leukemias (AML) occur via similar cytogenetic and genetic pathways, several of which involve aneuploidy, the loss or gain of chromosomes. Aneuploidy of specific chromosomes has been detected in benzene-related leukemia patients as well as in healthy benzene-exposed workers, suggesting that aneuploidy precedes and may be a potential mechanism underlying benzene-induced leukemia. Here, we analyzed the peripheral blood lymphocytes of 47 exposed workers and 27 unexposed controls using a novel OctoChrome fluorescence in situ hybridization (FISH) technique that simultaneously detects aneuploidy in all 24 chromosomes. Through this chromosome-wide aneuploidy study (CWAS) approach, we found heterogeneity in the monosomy and trisomy rates of the 22 autosomes when plotted against continuous benzene exposure. In addition, statistically significant, chromosome-specific increases in the rates of monosomy [5, 6, 7, 10, 16 and 19] and trisomy [5, 6, 7, 8, 10, 14, 16, 21 and 22] were found to be dose dependently associated with benzene exposure. Furthermore, significantly higher rates of monosomy and trisomy were observed in a priori defined ‘susceptible’ chromosome sets compared with all other chromosomes. Together, these findings confirm that benzene exposure is associated with specific chromosomal aneuploidies in hematopoietic cells, which suggests that such aneuploidies may play roles in benzene-induced leukemogenesis.
Chemotherapy remains the staple of treatment for many types of leukemia. Despite the positive impact on extending overall survival in patients with hematological malignancies, new treatment strategies are needed to reduce the nonspecific toxicity and improve the efficacy of treatment. Celastrol, derived from the ‘Thunder God Vine’ and Pro-EGCG, a pre-drug version of green tea polyphenol EGCG have shown potent biological activity in vitro and in vivo. Whether these natural products augment the efficacy of conventional chemotherapy in the treatment of leukemia cells has yet to be demonstrated. Here we demonstrate that these natural products could sensitize the effect of chemotherapy in both K-562 and Jurkat T human leukemia cells. Accordingly, this potent biological activity was associated with increased levels of leukemia cell killing, caspase 3 activation, and poly(ADP-ribose) polymerase cleavage. Furthermore, the higher levels of apoptotic indices were associated with decreased levels of Bcr-Abl oncoprotein in K-562 cells. Taken together, our findings present a compelling rationale for the development of combination strategies using natural products in the treatment of hematological malignancies.
natural products; pro-drug; chemosensitizing; chemotherapy; apoptosis; leukemia
Aim. To investigate the combination effects and mechanisms of valsartan (angiotensin II type 1 receptor blocker) and LAF237 (DPP-IV inhibitor) on prevention against oxidative stress and inflammation injury in db/db mice aorta. Methods. Db/db mice (n = 40) were randomized to receive valsartan, LAF237, valsartan plus LAF237, or saline. Oxidative stress and inflammatory reaction in diabetic mice aorta were examined. Results. Valsartan or LAF237 pretreatment significantly increased plasma GLP-1 expression, reduced apoptosis of endothelial cells isolated from diabetic mice aorta. The expression of NAD(P)H oxidase subunits also significantly decreased resulting in decreased superoxide production and ICAM-1 (fold change: valsartan : 7.5 ± 0.7, P < 0.05; LAF237: 10.2 ± 1.7, P < 0.05), VCAM-1 (fold change: valsartan : 5.2 ± 1.2, P < 0.05; LAF237: 4.8 ± 0.6, P < 0.05), and MCP-1 (fold change: valsartan: 3.2 ± 0.6, LAF237: 4.7 ± 0.8; P < 0.05) expression. Moreover, the combination treatment with valsartan and LAF237 resulted in a more significant increase of GLP-1 expression. The decrease of the vascular oxidative stress and inflammation reaction was also higher than monotherapy with valsartan or LAF237. Conclusion. These data indicated that combination treatment with LAF237 and valsartan acts in a synergistic manner on vascular oxidative stress and inflammation in type 2 diabetic mice.
The present study was to investigate the effects and mechanism of Luteolin on myocardial infarct size, cardiac function and cardiomyocyte apoptosis in diabetic rats with myocardial ischemia/reperfusion (I/R) injury.
Diabetic rats underwent 30 minutes of ischemia followed by 3 h of reperfusion. Animals were pretreated with or without Luteolin before coronary artery ligation. The severity of myocardial I/R induced LDH release, arrhythmia, infarct size, cardiac function impairment, cardiomyocyte apoptosis were compared. Western blot analysis was performed to elucidate the target proteins of Luteolin. The inflammatory cytokine production were also examined in ischemic myocardium underwent I/R injury. Our results revealed that Luteolin administration significantly reduced LDH release, decreased the incidence of arrhythmia, attenuated myocardial infarct size, enhanced left ventricular ejection fraction and decreased myocardial apoptotic death compared with I/R group. Western blot analysis showed that Luteolin treatment up-regulated anti-apoptotic proteins FGFR2 and LIF expression, increased BAD phosphorylation while decreased the ratio of Bax to Bcl-2. Luteolin treatment also inhibited MPO expression and inflammatory cytokine production including IL-6, IL-1a and TNF-a. Moreover, co-administration of wortmannin and Luteolin abolished the beneficial effects of Luteolin.
This study indicates that Luteolin preserves cardiac function, reduces infarct size and cardiomyocyte apoptotic rate after I/R injury in diabetic rats. Luteolin exerts its action by up-regulating of anti-apoptotic proteins FGFR2 and LIF expression, activating PI3K/Akt pathway while increasing BAD phosphorylation and decreasing ratio of Bax to Bcl-2.
SLE is an autoimmune inflammatory disease in which various pro- and anti-inflammatory cytokines, including TGF-β, IL-10, BAFF, IL-6, IFN-α, IFN-γ, IL-17, and IL-23, play crucial pathogenic roles. Virtually, all these cytokines can be generated by both innate and adaptive immune cells and exert different effects depending on specific local microenvironment. They can also interact with each other, forming a complex network to maintain delicate immune homeostasis. In this paper, we elaborate on the abnormal secretion and functions of these cytokines in SLE, analyze their potential pathogenic roles, and probe into the possibility of them being utilized as targets for therapy.
Pulmonary hypertension (PH) contributes to the mortality of
patients with lung and heart diseases. However, the underlying
mechanism has not been completely elucidated. Accumulating
evidence suggests that inflammatory response may be involved in
the pathogenesis of PH. Macrophage migration inhibitory factor
(MIF) is a critical upstream inflammatory mediator which promotes
a broad range of pathophysiological processes. The aim of the
study was to investigate the role of MIF in the pulmonary vascular
remodeling of hypoxia-induced PH. We found that MIF mRNA and
protein expression was increased in the lung tissues from hypoxic
pulmonary hypertensive rats. Intensive immunoreactivity for MIF
was observed in smooth muscle cells of large pulmonary arteries
(PAs), endothelial cells of small PAs, and inflammatory cells of
hypoxic lungs. MIF participated in the hypoxia-induced PASMCs
proliferation, and it could directly stimulate proliferation of
these cells. MIF-induced enhanced growth of PASMCs was attenuated
by MEK and JNK inhibitor. Besides, MIF antagonist ISO-1 suppressed
the ERK1/2 and JNK phosphorylation induced by MIF. In conclusion,
the current finding suggested that MIF may act on the
proliferation of PASMCs through the activation of the ERK1/2 and
JNK pathways, which contributes to hypoxic pulmonary hypertension.
The genetic basis of chronic lymphocytic leukemia (CLL) has not been elucidated to date. Although it is the most common hematological malignancy in Caucasians, it is uncommon among Asians. A recent genome-wide scan of CLL in Caucasians identified 6 variants showing strong association. We attempted to replicate these findings in 71 cases of CLL and 1273 controls in Hong Kong Chinese. Three of the 6 variants were significantly associated with CLL. The rs872071 variant (Odds Ratio (95% Confidence Interval) = 1.78 (1.25–2.53), p = 0.0013) in the IRF4 gene region showed the strongest association, similar to that reported in the United Kingdom study. Polymorphisms in SP140 and ACOXL and were also associated with risk of CLL. Further, the mean allele frequencies of the 6 variants were moderately (59%) to extremely (0.5%) lower in the Chinese population compared with Caucasians. These results suggest that variants in three loci may contribute to risk of CLL among Chinese.
genetic polymorphism; chronic lymphocytic leukemia; Chinese
Compared to normal differentiated cells, cancer cells have altered metabolic regulation to support biosynthesis and the expression of the M2 isozyme of pyruvate kinase (PKM2) plays an important role in this anabolic metabolism. While the M1 isoform is a highly active enzyme, the alternatively spliced M2 variant is considerably less active and expressed in tumors. While the exact mechanism by which decreased pyruvate kinase activity contributes to anabolic metabolism remains unclear, it is hypothesized that activation of PKM2 to levels seen with PKM1 may promote a metabolic program that is not conducive to cell proliferation. Here we report the third chemotype in a series of PKM2 activators based on the 2-oxo-N-aryl-1,2,3,4-tetrahydroquinoline-6-sulfonamide scaffold. The synthesis, structure activity relationships, selectivity and notable physiochemical properties are described.
PKM2; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
Small molecule kinase inhibitors are important tools for studying cellular signaling pathways, phenotypes and are, occasionally, useful clinical agents. With stereochemistry pervasive throughout the molecules of life it is no surprise that a single stereocenter can bestow a ligand with distinct binding affinities to various protein targets. While the majority of small molecule kinase inhibitors reported to date are achiral, a number of asymmetric compounds show great utility as tools for probing kinase-associated biomolecular events as well as promising therapeutic leads. The mechanism by which chirality is introduced varies but includes screening of chiral libraries, incorporation of chiral centers during optimization efforts and the rational installation of a chiral moiety as guided by structural and modeling efforts. Here we discuss several advanced chiral small molecule kinase inhibitors where stereochemistry plays an important role in terms of potency and selectivity.
Lucanthone and hycanthone are thioxanthenone DNA intercalators used in the 1980s as antitumor agents. Lucanthone is in Phase I clinical trial, whereas hycanthone was pulled out of Phase II trials. Their potential mechanism of action includes DNA intercalation, inhibition of nucleic acid biosyntheses, and inhibition of enzymes like topoisomerases and the dual function base excision repair enzyme apurinic endonuclease 1 (APE1). Lucanthone inhibits the endonuclease activity of APE1, without affecting its redox activity. Our goal was to decipher the precise mechanism of APE1 inhibition as a prerequisite towards development of improved therapeutics that can counteract higher APE1 activity often seen in tumors. The IC50 values for inhibition of APE1 incision of depurinated plasmid DNA by lucanthone and hycanthone were 5 µM and 80 nM, respectively. The KD values (affinity constants) for APE1, as determined by BIACORE binding studies, were 89 nM for lucanthone/10 nM for hycanthone. APE1 structures reveal a hydrophobic pocket where hydrophobic small molecules like thioxanthenones can bind, and our modeling studies confirmed such docking. Circular dichroism spectra uncovered change in the helical structure of APE1 in the presence of lucanthone/hycanthone, and notably, this effect was decreased (Phe266Ala or Phe266Cys or Trp280Leu) or abolished (Phe266Ala/Trp280Ala) when hydrophobic site mutants were employed. Reduced inhibition by lucanthone of the diminished endonuclease activity of hydrophobic mutant proteins (as compared to wild type APE1) supports that binding of lucanthone to the hydrophobic pocket dictates APE1 inhibition. The DNA binding capacity of APE1 was marginally inhibited by lucanthone, and not at all by hycanthone, supporting our hypothesis that thioxanthenones inhibit APE1, predominantly, by direct interaction. Finally, lucanthone-induced degradation was drastically reduced in the presence of short and long lived free radical scavengers, e.g., TRIS and DMSO, suggesting that the mechanism of APE1 breakdown may involve free radical-induced peptide bond cleavage.