Applications of homemade kit in mutation detection of genes

With the accomplishment of Human Genome Project (HGP), single nucleotide polymorphism (SNP) and mutation detection in human genome are becom-ing a new researching focus. These researches can help us to understand the phenotype diversity of indi-vidual, disease susceptibility and drug resistance of different colonies. Traditional method used for muta-tion detection is slab gel electrophoresis, which re-mains labor-intensive and time-consuming because of the requirement of radioactivity or te…     

Network pharmacology-based approach of novel traditional Chinese medicine formula for treatment of acute skin inflammation in silico

Matrix metalloproteinase-9 (MMP-9) appears to play an important role in acute skin inflammation. Subantimicrobial dose of tetracycline has been demonstrated to inhibit the activity of MMP-9 protein. However, long-term use tetracycline will induce side effect. The catalytic site of MMP-9 is located at zinc-binding amino acids, His401, His405 and His411. We attempted to search novel medicine formula as MMP-9 inhibitors from traditional Chinese medicine (TCM) database by using in silico studies. We utilized high-throughput virtual screening to find which natural compounds could bind to the zinc-binding site. The quantitative structure-activity relationship (QSAR) models, which constructed by scaffold of MMP-9 inhibitors and its activities, were employed to predict the bio-activity of the natural compounds for MMP-9. The results showed that Celacinnine, Lobelanidine and Celallocinnine were qualified to interact with zinc-binding site and displayed well predictive activity. We found that celallocinnine was the best TCM compound for zinc binging sites of MMP-9 because the stable interactions were observed under dynamic condition. In addition, Celacinnine and Lobelanidine could interact with MMP-9 related protein that identified by drug-target interaction network analysis. Thus, we suggested the herbs Hypericum patulum, Sedum acre, and Tripterygium wilfordii that containing Celallocinnine, Celacinnine and Lobelanidine might be a novel medicine formula to avoid the side effect of tetracycline and increase the efficacy of treatment.     

A functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin- β1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral- MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.     

Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry

Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.     

Cholesterol secosterol aldehydes induce amyloidogenesis and dysfunction of wild-type tumor protein p53

Epidemiologic and clinical evidence points to an increased risk for cancer when coupled with chronic inflammation. However, the molecular mechanisms that underpin this interrelationship remain largely unresolved. Herein we show that the inflammation-derived cholesterol 5,6-secosterol aldehydes, atheronal-A (KA) and -B (ALD), but not the polyunsaturated fatty acid (PUFA)-derived aldehydes 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), induce misfolding of wild-type p53 into an amyloidogenic form that binds thioflavin T and Congo red dyes but cannot bind to a consensus DNA sequence. Treatment of lung carcinoma cells with KA and ALD leads to a loss of function of extracted p53, as determined by the analysis of extracted nuclear protein and in activation of p21. Our results uncover a plausible chemical link between inflammation and cancer and expand the already pivotal role of p53 dysfunction and cancer risk.     

Seven nonsynonymous SNPs in the gene encoding human deoxyribonuclease II may serve as a functional SNP potentially implicated in autoimmune dysfunction

Many nonsynonymous SNPs in the human DNase II gene (DNASE2), potentially relevant to autoimmunity in conditions such as rheumatoid arthritis, have been identified, but only limited population data are available and no studies have evaluated whether such SNPs are functional. Genotyping of all the 15 nonsynonymous human DNase II SNPs was performed in three ethnic groups including 16 different populations using the PCR‐restriction fragment length polymorphism technique. A series of constructs corresponding to each SNP was examined. Fifteen nonsynonymous SNPs in the gene, except for p.Val206Ile in a Korean population, exhibited a mono‐allelic distribution in all of the populations. On the basis of alterations in the activity levels resulting from the corresponding amino acid substitutions, four activity‐abolishing and five activity‐reducing SNPs were confirmed to be functional. The amino acid residues in activity‐abolishing SNPs were conserved in animal DNase II. All the nonsynonymous SNPs that affected the catalytic activity of human DNase II showed extremely low genetic heterogeneity. However, a minor allele of seven SNPs producing a loss‐of‐function or extremely low activity‐harboring variant could serve as a genetic risk factor for autoimmune dysfunction. These functional SNPs in DNASE2 may have clinical implications in relation to the prevalence of autoimmune diseases.     

Electrochemical analysis of single nucleotide polymorphisms of p53 gene

Single nucleotide polymorphisms (SNPs) of cancer repression gene p53 were analyzed electrochemically with ferrocenyl naphthalene diimide (1) as a hybridization indicator. The SNPs studied were the transition to A from G in the codon for amino acid at positions 175, 248 or 273 and the transversion to C from G in the codon for the amino acid at position 72. Thus, 20-meric oligonucleotides carrying the SNP site were used both as a sample and a probe with the latter immobilized on an electrode. Even one base difference on the p53 gene resulted in a significant difference in the current response of 1 and the magnitude of the response correlated with the amount of the DNA hybrid on the electrode. Moreover, when PCR products of exon 4, on which the P72/R72 SNP resides, of the p53 gene were analyzed by this method, the heterozygote and homozygotes were discriminated with modest precision.     

Sequence-based analysis of 5′UTR and coding regions of CASP3 in terms of miRSNPs and SNPs in targetting miRNAs

Apoptosis is described as a mechanism of cell death occurring after adequate cellular harm. Deregulation of apoptosis occurs in many human conditions such as autoimmune disorders, ischemic damage, neurodegenerative diseases and different cancer types. Information relating miRNAs to cancer is increasing. miRNAs can affect development of cancer via many different pathways, including apoptosis. Polymorphisms in miRNA genes or miRNA target sites (miRSNPs) can change miRNA activity. Although polymorphisms in miRNA genes are very uncommon, SNPs in miRNA-binding sites of target genes are quite common. Many researches have revealed that SNPs in miRNA target sites improve or decrease the efficacy of the interaction between miRNAs and their target genes. Our aim was to specify miRSNPs on CASP3 gene (caspase-3) and SNPs in miRNA genes targeting 5′UTR and coding exons of CASP3, and evaluate the effect of these miRSNPs and SNPs of miRNA genes with respect to apoptosis. We detected 141 different miRNA binding sites (126 different miRNAs) and 7 different SNPs in binding sites of miRNA in 5′UTR and CDS of CASP3 gene. Intriguingly, miR-339-3p’s binding site on CASP3 has a SNP (rs35372903, G/A) on CASP3 5′UTR and its genomic sequence has a SNP (rs565188493, G/A) at the same nucleotide with rs35372903. Also, miR-339-3p has two other SNPs (rs373011663, C/T rs72631820, A/G) of which the first is positioned at the binding site. Here, miRSNP (rs35372903) at CASP3 5′UTR and SNP (rs565188493) at miR-339-3p genomic sequence cross-matches at the same site of binding region. Besides, miR-339-3p targets many apoptosis related genes (ZNF346, TAOK2, PIM2, HIP1, BBC3, TNFRSF25, CLCF1, IHPK2, NOL3) although it had no apoptosis related interaction proven before. This means that miR-339-3p may also have a critical effect on apoptosis via different pathways other than caspase-3. Hence, we can deduce that this is the first study demonstrating a powerful association between miR-339-3p and apoptosis upon computational analysis.     

Simvastatin inhibits induction of matrix metalloproteinase-9 in rat alveolar macrophages exposed to cigarette smoke extract

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IκB, and nuclear AP-1 or NF-κB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IκB-NF-κB are involved.     

High mutation frequency at Ha-ras exons 1-4 in squamous cell carcinomas from PUVA-treated psoriasis patients

Clinical follow-up studies have revealed that PUVA-treated patients are at increased risk of skin cancer, particularly squamous cell carcinoma (SCC). However, since psoralen and UVA (PUVA) is not only a potent mutagen and carcinogen but also an immunosuppressor, and since other (co)carcinogenic factors often exist in psoriasis patients, the exact causes and mechanisms of PUVA-associated SCC are still not completely understood. In order to fill this gap the tools of molecular epidemiology are being used to study the SCC mutational spectra of p53 and Ha-ras, two of the most commonly mutated genes in human cancers. A previous mutation analysis revealed that SCC in PUVA-treated patients often carried mutated p53 genes and that many of the mutations had the UV fingerprint (i.e. C-->T or CC-->TT transitions at dipyrimidine sites). In the present study DNA-sequencing analysis revealed a total of 18 Ha-ras missense or nonsense mutations at exons 1-4 in 13 of 17 SCC (76%) from 8 of 11 (73%) PUVA-treated psoriasis patients. Six of the 18 mutations (33%) were of UV-fingerprint type (C-->T transitions), five (28%) were at 5'-TpG sites (i.e. potential psoralen-binding sites and thus potentially caused by PUVA) and seven were of other type (39%), including six G:C-->T:A transversions at hotspot codon 12. In addition, in the case of 6 of the 11 subjects (55%) both tumor and normal skin samples contained a T:A-->C:G base change at codon 27 (a 5'-ATT site), a change previously hypothesized to be a possible silent Ha-ras polymorphism at one allele. When we compared the present Ha-ras mutation spectrum with the p53 mutation spectrum from a previous study of the samples, we found that approximately half of the tumors harbored mutations in both Ha-ras and p53. Together, our results indicate that Ha-ras mutations are present in a large proportion of PUVA-associated SCC and that UVB, PUVA and other agents may induce Ha-ras mutations and act together with p53 in the formation of SCC in psoriasis patients.     

Enhanced induction of Bax gene expression in H460 and H1299 cells with the combined treatment of cisplatin and adenovirus mediated wt-p53 gene transfer

Cytotoxic effect of either cisplatin or p53 gene transfection of lung cancer cells may be different depending on the p53 status of cells. We investigated cytotoxic effects on the combined treatment of cisplatin and adenovirus mediated p53 gene transfer (Avp53) in both H460 and H1299 cells in vitro. The results showed the highest numbers of apoptotic cells in both H460 and H1299 cells following the combined treatment regardless of p53 status in comparison with either cisplatin or Avp53 alone. The expression levels of p53, p21, Bax and ICE were examined to understand a possible cellular signal path of the combined treatment. In western analyses, the patterns of phosphorylated p53 protein were different between Avp53 and combined treatment. The expressions of p21 and Bax were increased in combined treatment, whereas the cleaved form of ICE (20 kD) was not detected. These results suggest that cisplatin induced p53 protein phosphorylation and may activate the downstream of p53 gene expression such as p21 and Bax. The enhanced apoptosis of lung cancer cells by the combined treatment may be useful in the development of clinical therapeutic modality of lung tumors.     

Regulation of expression of matrix metalloproteinase-9 by JNK in Raw 264.7 cells: presence of inhibitory factor(s) suppressing MMP-9 induction in serum and conditioned media

Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/chemokines. Whereas the MEK-ERK and PI3K-Akt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-α, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125-mediated MMP-9 induction, similar to IFN-γ. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-γ. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.     

Triptolide sensitizes lung cancer cells to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by inhibition of NF-kappaB activation

TNF-related apoptosis-inducing ligand (TRAIL/Apo- 2L), a newly identified member of the TNF family promotes apoptosis by binding to the transmembrane receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). TRAIL known to activate NF-kappaB in number of tumor cells including A549 (wt p53) and NCI-H1299 (null p53) lung cancer cells exerts relatively selective cytotoxic affects to the human tumor cell lines without much effect on the normal cells. We set out to identify an agent that would sensitize lung cancer cells to TRAIL-induced apoptosis through inhibition of NF-kappaB activation. We found that triptolide, an oxygenated diterpene extracted and purified from the Chinese herb Tripterygium wilfordii sensitized A549 and NCI-H1299 cells to TRAIL-induced apoptosis through inhibition of NF-kappaB activation. Pretreatment with MG132 which is a well-known NF-kappaB inhibitor by blocking degradation of IkappaBalpha also greatly sensitized lung cancer cells to TRAIL-induced apoptosis. Triptolide did not block DNA binding of NF-kappaB activated by TRAIL as in the case of TNF-alpha. It has been already proven that triptolide blocks transactivation of p65 which plays a key role in NF-kappaB activation. These observations suggest that triptolide may be a potentially useful drug to enhance TRAIL-induced tumor killing in lung cancer.     

Inhibition of cell proliferation,invasion and migration by the cardenolides digitoxigenin monodigitoxoside and convallatoxin in human lung cancer cell line

Cardiac glycosides consist of a large family of naturally derived compounds that are clinically used to treat congestive heart failure, and also present anticancer properties. In this study, the cytotoxic effects of two cardenolides, digitoxigenin monodigitoxoside (DGX) and convallatoxin (CON) were screened in four human tumour cell lines. Both compounds showed anti-proliferative effects in all tumour cells, at nanomolar concentrations. Since the human lung cancer cell line A549 was the most sensitive, we investigated the anti-proliferative, anti-migratory and anti-invasive effects of these cardenolides. DGX and CON reduced A549 cell migration, being able to reduce more than 90% of cell invasion. Their effects on the expression of key regulators of metastatic mechanism showed decreased levels of MMP-2, MMP-9 and p-FAK. Both compounds also presented low toxicity for healthy cells. Finally, this work provides the first insights into the effects of these cardenolides on key steps of lung cancer metastasis.     

Genetic and expression analysis of all non‐synonymous single nucleotide polymorphisms in the human deoxyribonuclease I‐like 1 and 2 genes

Members of the human DNase I family, DNase I‐like 1 and 2 (DNases 1L1 and 1L2), with physiological role(s) other than those of DNase I, possess three and one non‐synonymous SNPs in the genes, respectively. However, only limited population data are available, and the effect of these SNPs on the catalytic activity of the enzyme remains unknown. Genotyping of all the non‐synonymous SNPs was performed in three ethnic groups including six different populations using the PCR‐RFLP method newly developed. Asian and African groups including Japanese, Koreans, Ghanaians and Ovambos were typed as a single genotype at each SNP, but polymorphism at only SNP V122I in DNase 1L1 was found in Caucasian groups including Germans and Turks; thus a Caucasian‐specific allele was identified. The DNase 1L1 and 1L2 genes show relatively low genetic diversity with regard to these non‐synonymous SNPs. The level of activity derived from the V122I, Q170H and D227A substituted DNase 1L1 corresponding to SNPs was similar to that of the wild‐type, whereas replacement of the Asp residue at position 197 in the DNase 1L2 protein with Ala, corresponding to SNP D197A, reduced its activity greatly. Thus, SNP V122I in DNase 1L1 exhibiting polymorphism exerts no effect on the catalytic activity, and furthermore SNP D197A in DNase 1L2, affecting its catalytic activity, shows no polymorphism. These findings permit us to postulate that the non‐synonymous SNPs identified in the DNase 1L1 and 1L2 genes may exert no influence on the activity levels of DNases 1L1 and 1L2 in human populations.     

Constituents of Aquilaria sinensis Leaves Upregulate the Expression of Matrix Metalloproteases 2 and 9

In this novel study, we isolated 28 compounds from the leaves of Aquilaria sinensis (Lour.) Gilg based on a bioassay-guided procedure and also discovered the possible matrix metalloprotease 2 (MMP-2) and 9 (MMP-9) modulatory effect of pheophorbide A (PA). To evaluate the regulatory activity on MMP-2 and MMP-9, the HT-1080 human fibrosarcoma cells were treated with various concentrations of extracted materials and isolated compounds. PA was extracted by methanol from the leaves of A. sinensis and separated from the fraction of the partitioned ethyl acetate layer. PA is believed to be an active component for MMP expression since it exhibited significant stimulation on MMP-2 and proMMP-9 activity. When treating with 50 μM of PA, the expression of MMP-2 and MMP-9 were increased 1.9-fold and 2.3-fold, respectively. PA also exhibited no cytotoxicity against HT-1080 cells when the cell viability was monitored. Furthermore, no significant MMP activity was observed when five PA analogues were evaluated. This study is the first to demonstrate that C-17 of PA is the deciding factor in determining the bioactivity of the compound. The MMP-2 and proMMP-9 modulatory activity of PA indicate its potential applications for reducing scar formation and comparative medical purposes.