Inhibition of UV-induced matrix metabolism by a myristoyl tetrapeptide.

Regulation of extracellular matrix (ECM) composition is important in tissue homeostasis and function. We screened small peptides for their ability to inhibit ultraviolet (UV)-induced cell metabolism in epidermal fibroblasts. We found that UV irradiation increased matrix metalloproteinase (MMP) expression and inflammatory gene expression in human Hs68 fibroblast cells. We also demonstrated that a myristoyl tetrapeptide with the amino acid sequence Gly-Leu-Phe-Trp (mGLFW) suppressed the UV-induced expression of MMPs and inflammatory genes. Moreover, mGLFW stimulated the expression of ECM proteins in Hs68 fibroblasts. In order to provide the mechanism of action for mGLFW, we investigated UV-induced signaling changes in the presence of mGLFW using a cDNA microarray. UV exposure increased the expression of MMP genes, such as MMP1, MMP3, and MMP14, and inflammation-related genes, including interleukin 1 receptor and peroxisome proliferator-activated receptor gamma (PPARγ). Treatment with mGLFW abrogated the UV-induced expression of MMP-related genes and inflammatory genes. In addition, mGLFW increased the expression of collagen genes, including COL1A1, COL1A2, and COL5A1. We examined whether the activation of AP-1, a UV-activated transcription factor, is suppressed by mGLFW. The results demonstrated that AP-1 expression increased upon UV exposure and that this expression was inhibited by mGLFW. In conclusion, our results demonstrate that mGLFW reversed the effects of UV exposure by enhancing the expression of collagen proteins and suppressing the expression of MMPs, which degrade the ECM.

Smad-induced alterations of matrix metabolism by a myristoyl tetra peptide.

Regulation of extracellular matrix (ECM) components is essential for tissue homeostasis and function. We screened a small peptide that induces ECM protein synthesis for its usefulness in protecting keratinocytes. In this report, we demonstrate that myristoyl tetrapeptide Ala-Ala-Pro-Val (mAAPV) stimulates the expression of ECM proteins and inhibits the expression of metalloproteinases (MMPs) that degrade ECM proteins in Hs68 human fibroblast cells. In order to elucidate the underlying molecular mechanisms for the effects of mAAVP, we investigated the changes in gene expression in the presence of mAAPV using a cDNA microarray. Treatment with mAAPV resulted in decreased expression of MMP-related genes such as MMP1, MMP3, TIMP1 and TIMP3 and increased expression of collagen genes, including COL1A1, COL1A2, COL3A1, COL5A1 and COL6A3. The pattern of gene expression regulated by mAAPV was very similar to that of gene expression induced by transforming growth factor (TGF)-ß, indicating that the TGF-ß signaling pathway is crucial for simultaneous activation of several ECM-related genes by mAAPV. We examined whether the activation of SMAD, a downstream protein of TGF-ß receptor, is involved in the signal transduction pathway induced by mAAPV. The results demonstrate that mAAVP directly activates SMAD2 and induces SMAD3 to bind to DNA. In conclusion, our results demonstrate that mAAPV both enhances the expression of collagen and inhibits its degradation via production of protease inhibitors that prevent enzymatic breakdown of the ECM. The results suggest that mAAPV would be a useful ECM-protecting agent.

Association of single nucleotide polymorphisms on Interleukin 17 receptor A (IL17RA) gene with aspirin hypersensitivity in asthmatics.

AIM: To investigate the association of single nucleotide polymorphisms (SNP) on IL17RA gene with Aspirin Exacerbated Respiratory Disease (AERD) and the functional effect of these variants on expression of IL17RA gene products. MATERIAL & METHODS: 15 SNPs of IL17RA gene were analyzed in 825 normal controls and 143 subjects with AERD and 411 with aspirin-tolerant asthma (ATA) and functionally characterized using measurement of protein and m-RNA expression. RESULT: Minor alleles frequencies of the three SNPs (-1075 A>G, -947 A>G, -50 C>T) and one haplotype (BL1_ht1) were significantly lower in AERD compared to those in ATA (p(corr)=0.002-0.03). IL17RA protein expression and mRNA amount in CD14(+) peripheral blood monocytes and mononuclear cells were significantly increased in subjects carrying the common alleles homozygote compared with those carrying the minor alleles. CONCLUSIONS: The minor alleles of the three SNPs may decrease the risk of AERD via attenuation of IL17RA gene expression.

Association analysis of N-acetyl transferase-2 polymorphisms with aspirin intolerance among asthmatics.

AIMS: Cysteinyl leukotrienes are inactivated by acetyl coenzyme A-dependent N-acetyltransferase (NAT). Thus, functional alterations of the NAT gene may contribute to the risk of aspirin-intolerant asthma. MATERIALS & METHODS: Asthmatics (n = 438) were categorized into aspirin-intolerant asthma (15% or greater decrease in the forced expiratory volume in 1 s or cutaneous reactions, n = 170) or aspirin-tolerant asthma (n = 268) groups. In total, 14 polymorphisms of the NAT2 gene were genotyped by a single-base extension method. RESULTS: The distributions of all loci of the 14 SNPs were in Hardy-Weinberg equilibrium (p > 0.05). Among the 14 SNPs, six common SNPs (minor allele frequency >5%) in a Korean population were used for haplotype construction and further statistical analysis. The logistic regression analysis demonstrated that NAT2 -9246G>C and haplotype 2 (TCACGG) were significantly associated with the risk of aspirin-intolerant asthma. The rare allele frequencies of the SNP and Ht2 were significantly higher in the aspirin-intolerant asthma group than in the aspirin-tolerant asthma group (p(corr) = 0.03 and p(corr) = 0.02 in codominant model). CONCLUSION: In a large genetic epidemiology study of aspirin-intolerant asthma in a Korean population, genetic polymorphisms of NAT2 were found to be related to a risk of aspirin hypersensitivity among asthmatics.

Quinoline derivative KB3-1 potentiates paclitaxel induced cytotoxicity and cycle arrest via multidrug resistance reversal in MES-SA/DX5 cancer cells.

AIMS: The resistance to chemotherapeutic drugs is a major problem for successful cancer treatment. Multidrug resistance (MDR) phenotype is characterized by over-expression of P-glycoprotein (P-gp) on the cancer cell plasma membrane that extrudes drugs out of the cells. Therefore, novel MDR reversal agents are desirable for combination therapy to reduce MDR and enhance anti-tumor activity. Thus, the present study was aimed to evaluate the potent efficacy of novel quinoline derivative KB3-1 as a potent MDR-reversing agent for combined therapy with TAX. MAIN METHODS: MDR reversing effect and TAX combined therapy were examined by Rhodamine accumulation and efflux assay and Confocal immunofluorescence microscopy, Western blotting, TUNEL assay, and cell cycle analysis. KEY FINDINGS: The discovery of quinoline-3-carboxylic acid [4-(2-[benzyl-3[-(3,4-dimethoxy-phenyl)-propionyl]-amino]-ethyl)-phenyl]-amide (KB3-1) as a novel MDR-reversal agent. KB3-1 significantly enhanced the accumulation and retention of a P-gp substrate, rhodamine-123 in the P-gp-expressing MES-SA/DX5 uterine sarcoma cells but not in the P-gp-negative MES-SA cells at non-toxic concentrations of 1 microM and 3 microM. Similarly, fluorescence microscopy observation revealed that KB3-1 reduced the effluxed rhodamine-123 expression on the membrane of MES-SA/DX5 cells. Consistent with decreased P-gp pumping activity, confocal microscopic observation revealed that KB3-1 effectively diminished the expression of P-gp in paclitaxel (TAX)-treated MES-SA/DX-5 cells. Furthermore, Western blotting confirmed that KB3-1 reduced P-gp expression and enhanced cytochrome C release and Bax expression in TAX treated MES-SA/DX-5 cells. In addition, KB3-1 enhanced TAX-induced apoptotic bodies in MES-SA/DX5 cells by TdT-mediated-dUTP nick-end labeling (TUNEL) staining assay aswell as potentiated TAX- induced cytotoxicity, G2/M phase arrest and sub-G1 apoptosis in MES-SA/DX5 cells but not in MES-SA cells. Interestingly, KB3-1 at 3 microM had comparable MDR-reversal activity to 10 microM verapamil, a well-known MDR- reversal agent. SIGNIFICANCE: KB3-1 can be a MDR-reversal drug candidate for combination chemotherapy with TAX.

DMNQ-S17 inhibits constitutive NF-kappaB activation leading to induction of apoptosis through the activation of caspase-3 in human myeloid leukemia U937 cells.

Through cytotoxicity screening with naphthoquinone derivatives, a novel compound 6-(1-oxoallkyl)-5,8-dimethoxy-1,4-naphthoquinone-S17 (DMNQ-S17) showed its potency against human myeloid leukemia U937 cells. Thus, to elucidate the apoptotic mechanism of DMNQ-S17, this study was performed in myeloid leukemia U937 cells by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) assay, eletrophoretic mobility shift assay (EMSA), terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay, 4',6-diamidino-2-phenylindole (DAPI) staining, and Western blotting. In the present study, DMNQ-S17 inhibited constitutive NF kappaB activation and its transcriptional activity in U937 cells. In addition, DMNQ-S17 induced apoptotic features such as apoptotic bodies, cell shrinkage and chromatin condensation in U937 cells. Consistently, flow cytometric analysis showed that DMNQ-S17 increased sub-G1 portion and TUNEL positive cells in a concentration-dependent manner. Furthermore, DMNQ-S17 effectively attenuated mitochondrial membrane potential, released cytochrome C, activated caspase-3 expression, and cleaved poly (ADP-ribose) polymerase (PARP). Reversely, caspase-3 and -9 inhibitors also blocked the DMNQ-S17 induced caspase-3 activation and PARP cleavage in U937 cells. Taken together, these findings suggest that DMNQ-S17 can be a potent anticancer candidate for myeloid leukemias by the suppression of NF-kappaB activation leading to the activation of caspase-3 in human myeloid leukemia U937 cells.

Complete spinal cord injury treatment using autologous bone marrow cell transplantation and bone marrow stimulation with granulocyte macrophage-colony stimulating factor: Phase I/II clinical trial.

To assess the safety and therapeutic efficacy of autologous human bone marrow cell (BMC) transplantation and the administration of granulocyte macrophage-colony stimulating factor (GM-CSF), a phase I/II open-label and nonrandomized study was conducted on 35 complete spinal cord injury patients. The BMCs were transplanted by injection into the surrounding area of the spinal cord injury site within 14 injury days (n = 17), between 14 days and 8 weeks (n = 6), and at more than 8 weeks (n = 12) after injury. In the control group, all patients (n = 13) were treated only with conventional decompression and fusion surgery without BMC transplantation. The patients underwent preoperative and follow-up neurological assessment using the American Spinal Injury Association Impairment Scale (AIS), electrophysiological monitoring, and magnetic resonance imaging (MRI). The mean follow-up period was 10.4 months after injury. At 4 months, the MRI analysis showed the enlargement of spinal cords and the small enhancement of the cell implantation sites, which were not any adverse lesions such as malignant transformation, hemorrhage, new cysts, or infections. Furthermore, the BMC transplantation and GM-CSF administration were not associated with any serious adverse clinical events increasing morbidities. The AIS grade increased in 30.4% of the acute and subacute treated patients (AIS A to B or C), whereas no significant improvement was observed in the chronic treatment group. Increasing neuropathic pain during the treatment and tumor formation at the site of transplantation are still remaining to be investigated. Long-term and large scale multicenter clinical study is required to determine its precise therapeutic effect. Disclosure of potential conflicts of interest is found at the end of this article.