Aberrant expression of miR-133a in endothelial cells inhibits angiogenesis by reducing pro-angiogenic but increasing anti-angiogenic gene expression.

Angiogenesis is a multi-factorial physiological process deregulated in human diseases characterised by excessive or insufficient blood vessel formation. Emerging evidence highlights a novel role for microRNAs as regulators of angiogenesis. Previous studies addressing the effect of miR-133a expression in endothelial cells during blood vessel formation have reported conflicting results. Here, we have assessed the specific effect of mature miR-133a strands in angiogenesis and the expression of endothelial angiogenic genes. Transfection of miR-133a-3p or -5p mimics in primary human endothelial cells significantly inhibited proliferation, migration, and tubular morphogenesis of transfected cells. Screening of gene arrays related to angiogenic processes, and further validation by TaqMan qPCR, revealed that aberrant expression of miR-133a-3p led to a decrease in the expression of genes encoding pro-angiogenic molecules, whilst increasing those with anti-angiogenic functions. Ingenuity Pathway Analysis of a collection of genes differentially expressed in cells harbouring miR-133a-3p, predicted decreased cellular functions related to vasculature branching and cell cycle progression, underlining the inhibitory role of miR-133a-3p in angiogenic cellular processes. Our results suggest that controlled delivery of miR-133a-3p mimics, or antagomirs in diseased endothelial cells, might open new therapeutic interventions to treat patients suffering from cardiovascular pathologies that occur with excessive or insufficient angiogenesis.

PLGA-Nano-Encapsulated Disulfiram Inhibits Hypoxia-Induced NF-κB, Cancer Stem Cells, and Targets Glioblastoma In Vitro and In Vivo.

Glioblastoma stem cell (GSC) is the major cause of glioblastoma multiforme (GBM) chemotherapy failure. Hypoxia is one of the determinants of GSC. NF-κB plays a pivotal link between hypoxia and cancer stem cells (CSCs). Disulfiram, an antialcoholism drug, has very strong NF-κB-inhibiting and anti-CSC activity. In this study, the in vitro anti-GSC activity of disulfiram and in vivo anti-GBM efficacy of poly lactic-co-glycolic acid nanoparticle-encapsulated disulfiram (DS-PLGA) were examined. We attempt to elucidate the molecular network between hypoxia and GSCs and also examined the anti-GSC activity of disulfiram in vitro and in vivo. The influence of GSCs and hypoxia on GBM chemoresistance and invasiveness was studied in hypoxic and spheroid cultures. The molecular regulatory roles of NF-κB, hypoxia-inducible factor-1α (HIF1α), and HIF2α were investigated using stably transfected U373MG cell lines. The hypoxia in neurospheres determines the cancer stem cell characteristics of the sphere-cultured GBM cell lines (U87MG, U251MG, U373MG). NF-κB is located at a higher hierarchical position than HIF1α/HIF2α in hypoxic regulatory network and plays a key role in hypoxia-induced GSC characters. DS inhibits NF-κB activity and targets hypoxia-induced GSCs. It showed selective toxicity to GBM cells, eradicates GSCs, and blocks migration and invasion at very low concentrations. DS-PLGA efficaciously inhibits orthotopic and subcutaneous U87MG xenograft in mouse models with no toxicity to vital organs.

Cangrelor versus Ticagrelor in Patients Treated with Primary Percutaneous Coronary Intervention: Impact on Platelet Activity, Myocardial Microvascular Function and Infarct Size: A Randomized Controlled Trial.

BACKGROUND: Oral P2Y12 inhibitors take more than 2 hours to achieve full effect in healthy subjects and this action is further delayed in patients with acute myocardial infarction. Intravenous P2Y12 inhibition might lead to more timely and potent anti-platelet effect in the context of emergency primary angioplasty, improving myocardial recovery. OBJECTIVES: This article compares the efficacy of intravenous cangrelor versus ticagrelor in a ST-elevation myocardial infarction (STEMI) population treated with primary percutaneous coronary intervention (PPCI). MATERIALS AND METHODS: In an open-label, prospective, randomized controlled trial, 100 subjects with STEMI were assigned 1:1 to intravenous cangrelor or oral ticagrelor. The co-primary endpoints were platelet P2Y12 inhibition at infarct vessel balloon inflation time, 4 and 24 hours. Secondary endpoints included indices of coronary microcirculatory function: index of microvascular resistance (IMR), initial infarct size (troponin at 24 hours) and final infarct size at 12 weeks (cardiac magnetic resonance). Secondary endpoints included indices of coronary microcirculatory function (index of microvascular resistance [IMR]), initial infarct size (troponin at 24 hours), final infarct size at 12 weeks (cardiac magnetic resonance), corrected thrombolysis in myocardial infarction (TIMI) frame count, TIMI flow grade, myocardial perfusion grade, and ST-segment resolution (ClinicalTrials.gov NCT02733341). RESULTS: P2Y12 inhibition at first balloon inflation time was significantly greater in cangrelor-treated patients (cangrelor P2Y12 reaction unit [PRU] 145.2 ± 50.6 vs. ticagrelor 248.3 ± 55.1). There was no difference in mean PRU at 4 and 24 to 36 hours post-dosing. IMR, final infarct size, angiographic and electrocardiographic measures of reperfusion were all similar between groups. CONCLUSION: Cangrelor produces more potent P2Y12 inhibition at the time of first coronary balloon inflation time compared with ticagrelor. Despite this enhanced P2Y12 inhibition, coronary microvascular function and final infarct size did not differ between groups.

tmem33 is essential for VEGF-mediated endothelial calcium oscillations and angiogenesis.

Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33-mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development.

Poly-Gamma-Glutamic Acid (γ-PGA)-Based Encapsulation of Adenovirus to Evade Neutralizing Antibodies.

In recent years, there has been an increasing interest in oncolytic adenoviral vectors as an alternative anticancer therapy. The induction of an immune response can be considered as a major limitation of this kind of application. Significant research efforts have been focused on the development of biodegradable polymer poly-gamma-glutamic acid (γ-PGA)-based nanoparticles used as a vector for effective and safe anticancer therapy, owing to their controlled and sustained-release properties, low toxicity, as well as biocompatibility with tissue and cells. This study aimed to introduce a specific destructive and antibody blind polymer-coated viral vector into cancer cells using γ-PGA and chitosan (CH). Adenovirus was successfully encapsulated into the biopolymer particles with an encapsulation efficiency of 92% and particle size of 485 nm using the ionic gelation method. Therapeutic agents or nanoparticles (NPs) that carry therapeutics can be directed specifically to cancerous cells by decorating their surfaces using targeting ligands. Moreover, in vitro neutralizing antibody response against viral capsid proteins can be somewhat reduced by encapsulating adenovirus into γ-PGA-CH NPs, as only 3.1% of the encapsulated adenovirus was detected by anti-adenovirus antibodies in the presented work compared to naked adenoviruses. The results obtained and the unique characteristics of the polymer established in this research could provide a reference for the coating and controlled release of viral vectors used in anticancer therapy.

Downregulation of aquaporin 3 inhibits cellular proliferation, migration and invasion in the MDA-MB-231 breast cancer cell line.

Aquaporins are membrane proteins that regulate cellular water flow. Recently, aquaporins have been proposed as mediators of cancer cell biology. A subset of aquaporins, referred to as aquaglyceroporins are known to facilitate the transport of glycerol. The present study describes the effect of gene knockdown of the aquaglyceroporin AQP3 on MDA-MB-231 breast cancer cell proliferation, migration, invasion, adherence and response to the chemotherapeutic agent 5-fluorouracil. shRNA mediated AQP3 gene knockdown induced a 28% reduction in cellular proliferation (P<0.01), a 39% decrease in migration (P<0.0001), a 24% reduction in invasion (P<0.05) and a 25% increase in cell death at 100 µM 5-FU (P<0.01). Analysis of cell permeability to water and glycerol revealed that MDA-MB-231 cells with knocked down AQP3 demonstrated a modest decrease in water permeability (17%; P<0.05) but a more marked decrease in glycerol permeability (77%; P<0.001). These results suggest that AQP3 has a role in multiple aspects of breast cancer cell pathophysiology and therefore represents a novel target for therapeutic intervention.

Investigation of the key chemical structures involved in the anticancer activity of disulfiram in A549 non-small cell lung cancer cell line.

BACKGROUND: Disulfiram (DS), an antialcoholism medicine, demonstrated strong anticancer activity in the laboratory but did not show promising results in clinical trials. The anticancer activity of DS is copper dependent. The reaction of DS and copper generates reactive oxygen species (ROS). After oral administration in the clinic, DS is enriched and quickly metabolised in the liver. The associated change of chemical structure may make the metabolites of DS lose its copper-chelating ability and disable their anticancer activity. The anticancer chemical structure of DS is still largely unknown. Elucidation of the relationship between the key chemical structure of DS and its anticancer activity will enable us to modify DS and speed its translation into cancer therapeutics. METHODS: The cytotoxicity, extracellular ROS activity, apoptotic effect of DS, DDC and their analogues on cancer cells and cancer stem cells were examined in vitro by MTT assay, western blot, extracellular ROS assay and sphere-reforming assay. RESULTS: Intact thiol groups are essential for the in vitro cytotoxicity of DS. S-methylated diethyldithiocarbamate (S-Me-DDC), one of the major metabolites of DS in liver, completely lost its in vitro anticancer activity. In vitro cytotoxicity of DS was also abolished when its thiuram structure was destroyed. In contrast, modification of the ethyl groups in DS had no significant influence on its anticancer activity. CONCLUSIONS: The thiol groups and thiuram structure are indispensable for the anticancer activity of DS. The liver enrichment and metabolism may be the major obstruction for application of DS in cancer treatment. A delivery system to protect the thiol groups and development of novel soluble copper-DDC compound may pave the path for translation of DS into cancer therapeutics.

Reduced expression of PMCA1 is associated with increased blood pressure with age which is preceded by remodelling of resistance arteries.

Hypertension is a well-established risk factor for adverse cardiovascular events, and older age is a risk factor for the development of hypertension. Genomewide association studies have linked ATP2B1, the gene for the plasma membrane calcium ATPase 1 (PMCA1), to blood pressure (BP) and hypertension. Here, we present the effects of reduction in the expression of PMCA1 on BP and small artery structure and function when combined with advancing age. Heterozygous PMCA1 null mice (PMCA1Ht ) were generated and conscious BP was measured at 6 to 18 months of age. Passive and active properties of isolated small mesenteric arteries were examined by pressure myography. PMCA1Ht mice exhibited normal BP at 6 and 9 months of age but developed significantly elevated BP when compared to age-matched wild-type controls at ≥12 months of age. Decreased lumen diameter, increased wall thickness and increased wall:lumen ratio were observed in small mesenteric arteries from animals 9 months of age and older, indicative of eutrophic remodelling. Increases in mesenteric artery intrinsic tone and global intracellular calcium were evident in animals at both 6 and 18 months of age. Thus, decreased expression of PMCA1 is associated with increased BP when combined with advancing age. Changes in arterial structure precede the elevation of BP. Pathways involving PMCA1 may be a novel target for BP regulation in the elderly.

Poly lactic-co-glycolic acid controlled delivery of disulfiram to target liver cancer stem-like cells.

Disulfiram (DS), an anti-alcoholism drug, shows very strong cytotoxicity in many cancer types. However its clinical application in cancer treatment is limited by the very short half-life in the bloodstream. In this study, we developed a poly lactic-co-glycolic acid (PLGA)-encapsulated DS protecting DS from the degradation in the bloodstream. The newly developed DS-PLGA was characterized. The DS-PLGA has very satisfactory encapsulation efficiency, drug-loading content and controlled release rate in vitro. PLGA encapsulation extended the half-life of DS from shorter than 2minutes to 7hours in serum. In combination with copper, DS-PLGA significantly inhibited the liver cancer stem cell population. CI-isobologram showed a remarkable synergistic cytotoxicity between DS-PLGA and 5-FU or sorafenib. It also demonstrated very promising anticancer efficacy and antimetastatic effect in liver cancer mouse model. Both DS and PLGA are FDA approved products for clinical application. Our study may lead to repositioning of DS into liver cancer treatment.

Vascular endothelial growth factor and hypoxia-inducible factor-1α gene polymorphisms and coronary collateral formation in patients with coronary chronic total occlusions.

INTRODUCTION: We evaluated the association between two single nucleotide polymorphisms of the vascular endothelial growth factor gene and one of the hypoxia-inducible factor-1α gene and the degree of coronary collateral formation in patients with a coronary chronic total occlusion. METHODS: Totally, 98 patients with symptomatic coronary artery disease and a chronic total occlusion observed during coronary angiography were recruited. Genotyping of two vascular endothelial growth factor promoter single nucleotide polymorphisms (-152G>A and -165C>T) and the C1772T single nucleotide polymorphism of hypoxia-inducible factor-1α were performed using polymerase chain reaction and restriction fragment length polymorphism analysis. The presence and extent of collateral vessel filling was scored by blinded observers using the Rentrop grade. RESULTS: We found no association between the vascular endothelial growth factor -152G>A, -165C>T and hypoxia-inducible factor-1α -1772C>T with the presence and filling of coronary collateral vessels. A history of percutaneous coronary intervention and transient ischaemic attack/cerebrovascular accident were associated with the presence of enhanced collateral vessel formation following binary logistic regression analysis. CONCLUSION: The study findings suggest that coronary collateral formation is not associated with the tested polymorphic variants of vascular endothelial growth factor and hypoxia-inducible factor-1α in patients with symptomatic coronary artery disease and the presence of a chronic total occlusion.

The cytotoxic mechanisms of disulfiram and copper(ii) in cancer cells.

The anticancer activity of disulfiram (DS) is copper(ii) (Cu)-dependent. This study investigated the anticancer mechanisms of DS/Cu using in vitro cytotoxicity and metabolic kinetic analysis. Our study indicates that DS/Cu targets cancer cells by the combination of two types of actions: (1) instant killing executed by DS/Cu reaction generated reactive oxygen species; (2) delayed cytotoxicity introduced by the end product, DDC-Cu. Nanoencapsulation of DS might shed light on repositioning of DS into cancer treatment.

Liposome encapsulated Disulfiram inhibits NFκB pathway and targets breast cancer stem cells in vitro and in vivo.

Breast cancer stem cells (BCSCs) are pan-resistant to different anticancer agents and responsible for cancer relapse. Disulfiram (DS), an antialcoholism drug, targets CSCs and reverses pan-chemoresistance. The anticancer application of DS is limited by its very short half-life in the bloodstream. This prompted us to develop a liposome-encapsulated DS (Lipo-DS) and examine its anticancer effect and mechanisms in vitro and in vivo. The relationship between hypoxia and CSCs was examined by in vitro comparison of BC cells cultured in spheroid and hypoxic conditions. To determine the importance of NFκB activation in bridging hypoxia and CSC-related pan-resistance, the CSC characters and drug sensitivity in BC cell lines were observed in NFκB p65 transfected cell lines. The effect of Lipo-DS on the NFκB pathway, CSCs and chemosensitivity was investigated in vitro and in vivo. The spheroid cultured BC cells manifested CSC characteristics and pan-resistance to anticancer drugs. This was related to the hypoxic condition in the spheres. Hypoxia induced activation of NFκB and chemoresistance. Transfection of BC cells with NFκB p65 also induced CSC characters and pan-resistance. Lipo-DS blocked NFκB activation and specifically targeted CSCs in vitro. Lipo-DS also targeted the CSC population in vivo and showed very strong anticancer efficacy. Mice tolerated the treatment very well and no significant in vivo nonspecific toxicity was observed. Hypoxia induced NFκB activation is responsible for stemness and chemoresistance in BCSCs. Lipo-DS targets NFκB pathway and CSCs. Further study may translate DS into cancer therapeutics.

Near patient anti-platelet response testing over time and gene analysis in patients admitted with acute coronary syndromes.

We sought to assess the relationships between platelet reactivity at different time points, CYP2C19*2 and ABCB1 status and clinical outcomes in patients with acute coronary syndromes (ACS). Anti-platelet response to clopidogrel was studied prospectively using the VerifyNow (VN) P2Y12 assay at the time of angiography and at 30 days post procedure in 151 patients admitted with ACS who underwent percutaneous coronary intervention (PCI). Troponin T levels were measured at angiography and 16-24 hour following PCI. DNA was extracted and the presence of CYP2C19*2 allele and ABCB1 polymorphisms were determined. Adverse cardiovascular and cerebral events (ACCE) were assessed at 12 months. High VN P2Y12 response at angiography was associated with a greater peri-procedural rise in troponin T, but not ACCE. However, VN P2Y12 response measured at 30 days was associated with ACCE (p = 0.017). CYP2C19*2 status was associated with higher VN P2Y12 response at angiography (p < 0.0001) and 30 days (p = 0.006) but not ACCE. Near-patient testing for clopidogrel response was associated with subsequent ACCE when performed 30 days following PCI, but not at angiography.

Disruption of the interaction between PMCA2 and calcineurin triggers apoptosis and enhances paclitaxel-induced cytotoxicity in breast cancer cells.

Cancer is caused by defects in the signalling mechanisms that govern cell proliferation and apoptosis. It is well known that calcium-dependent signalling pathways play a critical role in cell regulation. A tight control of calcium homeostasis by transporters and channel proteins is required to assure a proper functioning of the calcium-sensitive signal transduction pathways that regulate cell growth and apoptosis. The plasma membrane calcium ATPase 2 (PMCA2) has been recently identified as a negative regulator of apoptosis that can play a significant role in cancer progression by conferring cells resistance to apoptosis. We have previously reported an inhibitory interaction between PMCA2 and the calcium-activated signalling molecule calcineurin in breast cancer cells. Here, we demonstrate that disruption of the PMCA2/calcineurin interaction in a variety of human breast cancer cells results in activation of the calcineurin/NFAT pathway, upregulation in the expression of the pro-apoptotic protein Fas Ligand and in a concomitant loss of cell viability. Reduction in cell viability is the consequence of an increase in cell apoptosis. Impairment of the PMCA2/calcineurin interaction enhances paclitaxel-mediated cytotoxicity of breast tumoral cells. Our results suggest that therapeutic modulation of the PMCA2/calcineurin interaction might have important clinical applications to improve current treatments for breast cancer patients.

Endothelial nitric oxide synthase activity is inhibited by the plasma membrane calcium ATPase in human endothelial cells.

AIMS: Nitric oxide (NO) plays a pivotal role in the regulation of cardiovascular physiology. Endothelial NO is mainly produced by the endothelial nitric oxide synthase (eNOS) enzyme. eNOS enzymatic activity is regulated at several levels, including Ca(2+)/calmodulin binding and the interaction of eNOS with associated proteins. There is emerging evidence indicating a role for the plasma membrane calcium ATPase (PMCA) as a negative regulator of Ca(2+)/calmodulin-dependent signal transduction pathways via its interaction with partner proteins. The aim of our study was to investigate the possibility that the activity of eNOS is regulated through its association with endothelial PMCA. METHODS AND RESULTS: We show here a novel interaction between endogenous eNOS and PMCA in human primary endothelial cells. The interaction domains were located to the region 735-934 of eNOS and the catalytic domain of PMCA. Ectopic expression of PMCA in endothelial cells resulted in an increase in phosphorylation of the residue Thr-495 of endogenous eNOS. However, disruption of the PMCA-eNOS interaction by expression of the PMCA interaction domain significantly reversed the PMCA-mediated effect on eNOS phosphorylation. These results suggest that eNOS activity is negatively regulated via interaction with PMCA. Moreover, NO production by endothelial cells was significantly reduced by ectopic expression of PMCA. CONCLUSION: Our results show strong evidence for a novel functional interaction between endogenous PMCA and eNOS in endothelial cells, suggesting a role for endothelial PMCA as a negative modulator of eNOS activity, and, therefore, NO-dependent signal transduction pathways.

Triptolide simultaneously induces reactive oxygen species, inhibits NF-kappaB activity and sensitizes 5-fluorouracil in colorectal cancer cell lines.

The in vitro chemosensitizing effect of triptolide (PG490) on the cytotoxicity of 5-fluorouracil (5-FU) was determined in three colorectal cancer (CRC) cell lines. PG490 alone was toxic to CRC cell lines (IC(50)s: 1.39-5.51nM). The cytotoxicity of 5-FU to CRC cell lines was 4-15-fold sensitized by combining use of low concentration of PG490. CI-isobologram indicated that the effect of PG490 plus 5-FU was synergistic. PG490 and 5-FU treatment induced activated caspase 3 and Bax expression and inhibited Bcl-2 expression. PG490 simultaneously induced generation of reactive oxygen species (ROS) and inhibited NF-kappaB transcriptional activity.

Disulfiram/copper complex inhibiting NFkappaB activity and potentiating cytotoxic effect of gemcitabine on colon and breast cancer cell lines.

Most of the gemcitabine (dFdC) resistant cell lines manifested high NFkappaB activity. The NFkappaB activity can be induced by dFdC and 5-FU exposure. The chemosensitizing effect of disulfiram (DS), an anti-alcoholism drug and NFkappaB inhibitor, and copper (Cu) on the chemoresistant cell lines was examined. The DS/Cu complex significantly enhanced the cytotoxicity of dFdC (resistant cells: 12.2-1085-fold) and completely reversed the dFdC resistance in the resitant cell lines. The dFdC-induced NFkappaB activity was markedly inhibited by DS/Cu complex. The data from this study indicated that DS may be used in clinic to improve the therapeutic effect of dFdC in breast and colon cancer patients.

Plasma membrane calcium ATPase proteins as novel regulators of signal transduction pathways.

Emerging evidence suggests that plasma membrane calcium ATPases (PMCAs) play a key role as regulators of calcium-triggered signal transduction pathways via interaction with partner proteins. PMCAs regulate these pathways by targeting specific proteins to cellular sub-domains where the levels of intracellular free calcium are kept low by the calcium ejection properties of PMCAs. According to this model, PMCAs have been shown to interact functionally with the calcium-sensitive proteins neuronal nitric oxide synthase, calmodulin-dependent serine protein kinase, calcineurin and endothelial nitric oxidase synthase. Transgenic animals with altered expression of PMCAs are being used to evaluate the physiological significance of these interactions. To date, PMCA interactions with calcium-dependent partner proteins have been demonstrated to play a crucial role in the pathophysiology of the cardiovascular system via regulation of the nitric oxide and calcineurin/nuclear factor of activated T cells pathways. This new evidence suggests that PMCAs play a more sophisticated role than the mere ejection of calcium from the cells, by acting as modulators of signaling transduction pathways.

Tumor suppressor Ras-association domain family 1 isoform A is a novel regulator of cardiac hypertrophy.

BACKGROUND: Ras signaling regulates a number of important processes in the heart, including cell growth and hypertrophy. Although it is known that defective Ras signaling is associated with Noonan, Costello, and other syndromes that are characterized by tumor formation and cardiac hypertrophy, little is known about factors that may control it. Here we investigate the role of Ras effector Ras-association domain family 1 isoform A (RASSF1A) in regulating myocardial hypertrophy. METHODS AND RESULTS: A significant downregulation of RASSF1A expression was observed in hypertrophic mouse hearts, as well as in failing human hearts. To further investigate the role of RASSF1A in cardiac (patho)physiology, we used RASSF1A knock-out (RASSF1A(-)(/)(-)) mice and neonatal rat cardiomyocytes with adenoviral overexpression of RASSF1A. Ablation of RASSF1A in mice significantly enhanced the hypertrophic response to transverse aortic constriction (64.2% increase in heart weight/body weight ratio in RASSF1A(-)(/)(-) mice compared with 32.4% in wild type). Consistent with the in vivo data, overexpression of RASSF1A in cardiomyocytes markedly reduced the cellular hypertrophic response to phenylephrine stimulation. Analysis of molecular signaling events in isolated cardiomyocytes indicated that RASSF1A inhibited extracellular regulated kinase 1/2 activation, likely by blocking the binding of Raf1 to active Ras. CONCLUSIONS: Our data establish RASSF1A as a novel inhibitor of cardiac hypertrophy by modulating the extracellular regulated kinase 1/2 pathway.