Developing and Evaluating the HRM Technique for Identifying Cytochrome P450 2D6 Polymorphisms.

BACKGROUND: Cytochrome P450 2D6 is one of the important enzymes involved in the metabolism of many widely used drugs. Genetic polymorphisms of CYP2D6 can affect its activity. Therefore, an efficient method for identifying CYP2D6 polymorphisms is clinically important. METHODS: We developed a high-resolution melting (HRM) analysis to investigate CYP2D6 polymorphisms. Genomic DNA was extracted from peripheral blood samples from 71 healthy individuals. All nine exons of the CYP2D6 gene were sequenced before screening by HRM analysis. This method can detect the most genotypes (*1, *2, *4, *10, *14, *21 *39, and *41) of CYP2D6 in Chinese. RESULTS: All samples were successfully genotyped. The four most common mutant CYP2D6 alleles (*1, *2, *10, and *41) can be genotyped. The single nucleotides polymorphism (SNP) frequencies of 100C > T (rs1065852), 1039C > T (rs1081003), 1661G > C (rs1058164), 2663G > A (rs28371722), 2850C > T (rs16947), 2988G > A (rs28371725), 3181A > G, and 4180G > C (rs1135840) were 58%, 61%, 73%, 1%, 13%, 3%, 1%, 73%, respectively. We identified 100% of all heterozygotes without any errors. The two homozygous genotypes (1661G > C and 4180G > C) can be distinguished by mixing with a known genotype sample to generate an artificial heterozygote for HRM analysis. Therefore, all samples could be identified using our HRM method, and the results of HRM analysis are identical to those obtained by sequencing. Our method achieved 100% sensitivity, specificity, positive prediction value and negative prediction value. CONCLUSION: HRM analysis is a nongel resolution method that is faster and less expensive than direct sequencing. Our study shows that it is an efficient tool for typing CYP2D6 polymorphisms.

Direct assessment of cytochrome P450 2D6 genotypes by high-resolution melting analysis and DNA sequencing.

We developed a CYP2D6 genotyping method that required only one polymerase chain reaction (PCR) followed by a high-resolution melting curve analysis (HRM) and DNA sequencing. DNA was extracted from peripheral blood samples obtained from 100 normal individuals. From the HRM analysis using three fragments of amplicons (exons 1, 6, and 9), we successfully identified four common CYP2D6 gene polymorphisms (100C>T, 2850C>T, 2988G>A, and 4180G>C). Exons 3 and 7 were also screened by HRM analysis. The heteroduplexes, wild-type homoduplexes, and homoduplexes of compound mutations showed distinct melting plots. The other four exons (exons 2, 4, 5, and 8) were directly analyzed by DNA sequencing. In conclusion, we developed an HRM and DNA sequencing based method to assess the CYP2D6 gene directly without the need for nested PCR. This method is quick and cost-effective; it reduces the chance of PCR contamination and is suitable for clinical application.

Short and long-term impact of lipectomy on expression profile of hepatic anabolic genes in rats: a high fat and high cholesterol diet-induced obese model.

OBJECTIVE: To understand the molecular basis of the short and long-term effects of an immediate shortage of energy storage caused by lipectomy on expression profile of genes involved in lipid and carbohydrate metabolism in high fat and high cholesterol diet-induced obese rats. METHODS: The hepatic mRNA levels of enzymes, regulator and transcription factors involved in glucose and lipid metabolism were analyzed by quantitative real time polymerase chain reaction (RT-qPCR) ten days and eight weeks after lipectomy in obese rats. Body and liver weights and serum biochemical parameters, adiponectin, leptin and insulin were determined. RESULTS: No significant difference was observed on the food intake between the lipectomized and sham-operated groups during the experimental period. Ten days after the operation, the lipectomized animals showed significant higher triacylglycerol, glucose and insulin levels, a lower adiponectin concentration than the sham-operated rats, along with significant higher hepatic mRNA levels of hepatocyte nuclear factor 4α (HNF4α) and the enzymes involved in lipogenesis, sterol biosynthesis and gluconeogenesis. The results of immunohistochemical (IHC) analysis also confirmed increased levels of lipogenic enzymes in the liver of lipectomized versus sham-operated animals. The lipectomized group had a significantly lower adiponectin/leptin ratio that was positively correlated to the level of LDL (r = 0.823, P<0.05) and negatively to glucose and insulin (r = -0.821 and -0.892 respectively, P<0.05). Eight weeks after the operation, the lipectomized animals revealed significant higher body and liver weights, weight gain, liver to body weight ratio, hepatic triacylglycerol and serum insulin level. CONCLUSIONS: In response to lipectomy a short term enhancement of the expression of hepatic anabolic genes involved in lipid and carbohydrate metabolism was triggered that might eventually lead to the final extra weight gain. These metabolic changes could be the results of reduced circulating adiponectin that further influences the functions of insulin and hepatic HNF4α.

Detection of KRAS codon 12 and 13 mutations by mutant-enriched PCR assay.

BACKGROUND: The identification of KRAS mutations before the administration of anti-epidermal growth factor receptor (EGFR) therapy of metastatic colorectal cancer (mCRC) has become important. The aim of the present study was to develop a novel technology that can increase detection sensitivity for KRAS mutations. METHODS: DNAs were extracted from colorectal cancer tissues and formalin-fixed, paraffin-embedded (FFPE) colorectal cancer samples. Mutant-enriched PCR assay utilizes the exceptionally thermostable endonucleases, PspGI for codon 12 and PhoI for codon 13, for specific amplifying KRAS mutations from mixed samples. The amplified PCR products were subjected to single-base primer extension or sequencing. Digital PCR was used to evaluate some of the results. RESULTS: We compared the results with that from direct sequencing. In the FFPE samples, thirteen discordant samples were found. We showed that the mutant-enriched PCR assay can identify the codons 12 and 13 mutation in a mixed population of mutant and wild type DNA sequences at 1:1000 and 1:400, respectively. The sensitivity of this method is lower than the digital PCR. CONCLUSIONS: We developed a rapid and highly sensitive method to detect codons 12 and 13 mutations of the KRAS gene. This method is a powerful tool for finding low-abundance variations in genomic DNA.

Inhibitory effect of Piper betel leaf extracts on copper-mediated LDL oxidation and oxLDL-induced lipid accumulation via inducing reverse cholesterol transport in macrophages.

Piper betel leaf (PBL) has the biological capabilities of detoxification and can work as an anti-inflammatory agent and an anti-oxidant. In this study, we evaluated the anti-oxidative activity of the extract of Piper betel leaves (PBLs) on the basis of Cu(2+)-mediated oxidation, and its ability to prevent foam cell formation in a model for oxidised low density lipoprotein (oxLDL)-induced lipid accumulation in macrophages. Our data demonstrated that PBLs were able to inhibit LDL oxidation in vitro and are able to reduce the lipid accumulation in macrophages. We showed the underlying mechanisms to be the following: PBLs up-regulated the protein levels of the class A and class B scavenger receptors, the membrane lipid transporter ABCA1, and its upstream regulator Liver X receptor (LXR) in the macrophages exposed to oxLDL. The results suggested that PBLs activated the reverse cholesterol transport mechanism to enhance the metabolism of the oxLDL that could prevent both lipid accumulation and foam cell formation and further minimise the possible damage of vessels caused by the oxLDL.

The preparation and characterization of gold-conjugated polyphenol nanoparticles as a novel delivery system.

Nanogold particles are commonly used in nanomedicine. We generated physical nanogold (pNG) conjugated with different ratios of epigallocatechin-3-gallate (EGCG) and evaluated its physicochemical properties, antioxidant activity, and cytotoxicity in vitro as well as anticancer activity in vivo. Results showed that the EGCG-pNG conjugates were successfully prepared at ratios between 23:1 and 23:5, with the percentage of EGCG content increasing with the EGCG:pNG ratio from 23:1 (2.0% ± 0.02%) to 23:5 (28% ± 0.3%). EGCG-pNG particles at ratios of 23:1 and 23:5 demonstrated significantly decreased size from 500 to 20 nm and decreasing zeta potentials of 21 mV to -22 mV, respectively. At a ratio of 23:2.5, the EGCG-pNG particles (27% EGCG, 50 nm in size, zeta potential of -8 mV) showed longer EGCG activity half-life (110 days vs 5 hours), controlled release (2 hours vs 30 minutes), and higher antioxidant activity (four times), as well as inhibition of tumor cell growth, than controls. The present study indicated that EGCG-pNG possesses promising therapeutic potential, based on its strong free-radical scavenging and anticancer activities.

Rapid identification of the copy number of α-globin genes by capillary electrophoresis analysis.

OBJECTIVES: The current study aimed at the rapid identification of the copy number of α-globin genes for the diagnosis of α-thalassemia. DESIGN AND METHODS: To identify the copy number of α-globin genes in α-thalassemia, we developed a novel method using a multiplex polymerase chain reaction (PCR) in combination with the CE analysis. RESULTS: The proposed method provides a rapid detection of the common α-globin gene deletions. Sixty-six patients with α-thalassemia and 46 normal controls were included in the present study. The obtained results showed good correlation with those obtained by gap PCR. Moreover, a low amount of maternal cell contamination in the fetus specimen for the prenatal diagnosis of hemoglobin Barts hydrops fetalis as well as the rare multiplicated α-globin genes can be identified using this method. CONCLUSION: This method provides a convenient and efficient tool for the rapid identification of the copy number of α-globin genes in α-thalassemia and the individuals with α-globin gene multiplication.

Expression of protein kinase C family in human hepatocellular carcinoma.

Protein kinase Cs (PKCs) play important roles in signal transduction, cell regulation, and tumor formation. In the present study, we analyzed the expression of PKCs in human hepatocellular carcinoma (HCC) tissues and explored their roles in the development of HCC. Real-time quantitative PCR and immunohistochemistry showed that PKCbeta and PKCtheta were down-regulated in HCC tissues. Reduced expression of PKCtheta is well correlated with the grade of cancer cells (p = 0.009), and the down-regulated expression of PKCbetaII is associated with HBV infection (p = 0.035). Our findings suggest particular roles of the two PKC isoenzymes in the hepatocarcinogenesis of human HCC.

Analysing the expression of protein kinase C eta in human hepatocellular carcinoma.

AIMS: Protein kinase Cs (PKCs) play important roles in cell proliferation, differentiation, apoptosis, migration and tumorigenesis. In this report, we investigated the expression of PKCeta in human hepatocellular carcinoma (HCC) tissues and explored its role in the development of HCC. METHODS: We used real-time quantitative RT-PCR, mutation analysis, and immunohistochemical staining to analyse the expression of PKCeta in 50 pairs of human hepatocellular carcinoma (HCC) tissues. RESULTS: Expression of PKCeta was down-regulated in 82% of HCC tissues and the reduction of PKCeta was associated with poorer long-term survival of HCC patients. CONCLUSION: Reduced expression of PKCeta may represent a molecular lesion in the development of more aggressive disease of HCC.

Fast simultaneous detection of K-RAS mutations in colorectal cancer.

BACKGROUND: RAS genes acquire the most common somatic gain-of-function mutations in human cancer, and almost all of these mutations are located at codons 12, 13, 61, and 146. METHODS: We present a method for detecting these K-RAS hotspot mutations in 228 cases of colorectal cancer. The protocol is based on the multiplex amplification of exons 2, 3 and 4 in a single tube, followed by primer extension of the PCR products using various sizes of primers to detect base changes at codons 12, 13, 61 and 146. We compared the clinicopathological data of colorectal cancer patients with the K-RAS mutation status. RESULTS: K-RAS mutation occurred in 36% (83/228) of our colorectal cancer cases. Univariate analysis revealed a significant association between K-RAS mutation at codon 12 of exon 2 and poor 5-year survival (p = 0.023) and lymph node involvement (p = 0.048). Also, K-RAS mutation at codon 13 of exon 2 correlates with the size of the tumor (p = 0.03). Multivariate analysis adjusted for tumor size, histologic grade, and lymph node metastasis also indicated K-RAS mutations at codon 12 and 13 of exon 2 correlate significantly with overall survival (p = 0.002 and 0.025). No association was observed between codon 61 and 146 and clinicopathological features. CONCLUSION: We demonstrated a simple and fast way to identify K-RAS mutation.