Der-An Tsao

Enhancing detection of circulating tumor cells with activating KRAS oncogene in patients with colorectal cancer by weighted chemiluminescent membrane array method.

BackgroundIn 2008, the National Comprehensive Cancer Network suggested conducting a KRAS mutations test in metastatic colorectal cancer (mCRC) patients prior to administering therapy that uses anti-epidermal growth factor receptor (EGFR) monoclonal antibody. However, tests of KRAS mutations have been limited when traditional molecular techniques, such as polymerase chain reaction (PCR) combined direct sequencing, are used to obtain and analyze patients’ cancer tissues. If the primary tumor or metastatic tissues of patients with mCRC is unavailable, then such analysis will not be feasible. Our laboratory has successfully established a colorimetric membrane array analysis platform that could detect activating KRAS mutations from the peripheral blood of patients with various malignancies.MethodsThe current research aims to improve the above-mentioned technique not only by using chemiluminescence detection to replace color development, but also to add scores weighted according to the relevance of each gene to activating KRAS mutations.ResultsOur results show that the described weighted chemiluminescent membrane array (WCHMA) can detect circulating tumor cells (CTCs) harboring activating KRAS mutations in the peripheral blood in CRC. The sensitivity, specificity, and accuracy were 90.2, 94.9, and 93.5%, respectively, and the detection limitation was three colon tumor cells per millimeter of blood. The current study would significantly improve the detection sensitivity and accuracy over that of our previously designed membrane array method.ConclusionsThese findings also highlight the need to prompt further prospective studies on more cases of CRC to further establish the clinical relevance of activating KRAS mutation detection from peripheral blood in anti-EGFR-based chemotherapy that uses activating KRAS detection chips and the WCHMA analysis method.

Expression of nitric oxide synthases in keratinocytes after UVB irradiation

The importance of nitric oxide (NO) in mediating vasodilation, neurotransmission, and immune and inflammatory responses has been demonstrated. Human keratinocyte express inducible nitric oxide synthase (iNOS) and the neuronal constitutive isoform of NOS (ncNOS). We established an in vitro model in keratinocytes to investigate changes in NO, iNOS and ncNOS expression after UVB exposure. We demonstrated a large induction of NO after UVB exposure and that the source of NO produced in UVB-exposed keratinocytes was increased expression of iNOS and ncNOS. The increased NO production with increased expression of iNOS and ncNOS may contribute to the pathological and physiological features of UVB-induced erythema and skin inflammation.

A fast and convenient new technique to detect the therapeutic target, K-ras mutant, from peripheral blood in non-small cell lung cancer patients.

Activating mutation of the K-ras gene was one of the earliest discoveries of genetic alterations in lung cancer. Moreover, K-ras somatic mutations might be suggested for predicting resistance to molecular antibodies targeting the epidermal growth factor receptor (EGFR). However, activated K-ras mutant detection methods are limited to traditional techniques. The techniques are complicated and are used only in tissue samples, which are limited for clinical applications. In a previous study, we established a low-cost, convenient, and easy technique for detecting activated K-ras in a small number of circulating tumor cells by the colorimetric membrane array method (CLMA). However, the sensitivity still needs further improvement. The aim of this study is to develop a new platform with chemiluminescence as reporter and weighted values of target genes on the chip in order to achieve a more sensitive, easier to read, and more accurate platform-weighted chemiluminecent membrane array (WCHMA). In advance, we collected 209 peripheral blood samples of non-small cell lung cancer (NSCLC) from patients to evaluate clinical K-ras activation detection using Activating KRAS Detection Chip both conducted by CLMA and WCHMA. Results show 71 specimens with K-ras mutation, of which 59 were identified as positive through CLMA and 66 were positive through WCHMA. After statistical analysis, the sensitivity of CLMA was found to be 83% and the specificity was 96%. On the other hand, the sensitivity of WCHMA increased to 93% and the specificity remained at 94%. Results of the detection limitation of peripheral blood on two platforms are: 3cancer cells/cm(3) blood using WCHMA, which is better than 5cancer cells/cm(3) blood using CLMA. Further analysis on the correlation between the test results and clinical pathological features shows that the mean score obtained using WCHMA is significantly correlated to TNM stage, tumor size, and metastasis.

Nitric oxide enhances expression of raf kinase inhibitor protein in keratinocytes

Abstract:  Several reports have focused on the potential of nitric oxide (NO) to influence the proliferation and differentiation cascade in a number of mammalian cells. The purpose of this study was to determine the relationship between expression of raf kinase inhibitor protein (RKIP) and proliferation in keratinocyte with NO treatment. Normal human keratinocytes were treated with SNAP (NO donor) doses of 10−7, 10−6, 10−5, 10−4 and 0 m (control group) separately. Expression of protein and mRNA of RKIP, cell proliferation and apoptosis have been measured. These results showed that elevated expression of RKIP in keratinocyte with NO treatment may contribute to the pathological and physiological features of NO‐inhibited proliferation.

Hexavalent chromium inhibited the expression of RKIP of heart in vivo and in vitro

BACKGROUND Chromium (Cr) is considered to be a risk factor to the cardiovascular effects of fine particulate matter components to PM2.5 from traffic in highway patrol officers. RKIP (raf kinase inhibitor protein) is a physiological inhibitor of GRK-2 (G-protein-coupled receptor kinase 2) and affects β-adrenergic signaling and contractile activity in cardiomyocytes. OBJECTIVES In this study, we explored the change of RKIP in heart of chromium (VI)-exposed rats and cultured myocardial cells with chromium (VI) treatment. METHOD Wistar rats were divided into six groups which were chronically fed with 250, 500, 750, 1000, and 1250 ppm Na(2)Cr(2)O(7) and water for 60 days, respectively. Na(2)Cr(2)O(7) dose of 0.25, 0.5, 1.5, 3, 4.5, and 0 ppm (control group) was applied in cultured myocardial cells. The level of heart Cr (VI) was determined by electrothermal atomic absorption spectrometry. The expression of RKIP was measured by Western blot method. The MTT assay was used to measure the toxicity of myocardial cells with Cr (VI) treatment. The apoptosis test of myocardial cells was determined by caspase-3 colorimetric assay kit. RESULT The result showed that the expression of RKIP in heart (in vivo) and myocardial cells (in vitro) was decreased following Cr (VI) dose-dependent treatment. CONCLUSION We suggested that the decrement of RKIP of heart and myocardial cells with Cr (VI) treatment resulted in the function of cardiovascular system decreased.

The expression of RKIP, RhoGDI, galectin, c‐Myc and p53 in gastrointestinal system of Cr(VI)‐exposed rats

Hexavalent chromium (CrVI) is considered to be a risk factor in the formation of human cancer. Raf kinase inhibitor protein (RKIP), Rho‐GDIα, galectin, c‐Myc and p53 play important roles in cancer formation. The purpose of this study was to determine if Cr(VI) induces the formation of gastrointestinal cancer. We explored the expression of RKIP, Rho‐GDIα, galectin, c‐Myc and p53 in the colon and stomach in rats exposed to chromium (CrVI). Thirty Wistar rats were divided into six groups which were chronically fed with 250, 500, 750, 1000 and 1250 ppm Na2Cr2O7 and water for 60 days. The level of Cr(VI) was determined by electrothermal atomic absorption spectrometry. The expression of RKIP, Rho‐GDIα, galectin, c‐Myc and p53 of stomach and colon was measured by western blot. The gene expression of RKIP, Rho‐GDIα, galectin, c‐Myc and p53 of the stomach and colon was determined by RT‐PCR. The results showed that the expression of p53 and Rho‐GDIα was decreased in the stomach and colon of rats with Cr(VI) treatment. The expression of RKIP was decreased in the stomach and colon of rats treated with high‐dose Cr(VI). The expression of c‐Myc and gelectin‐1 was increased in the stomach and colon of rats with Cr(VI) treatment. We concluded that the anomalous expression of RKIP, Rho‐GDIα, galectin, c‐Myc and p53 might be a dangerous index of cancer formation in the stomach and colon of rats with Cr(VI) exposure. Copyright

RKIP expression of liver and kidney after arsenic exposure

Arsenic is associated with cancers of kidney and liver. Raf kinase inhibitor protein (RKIP) has been identified as a member of a novel class of molecules that suppress the metastatic spread of tumors. In order to investigate the effect of arsenic to RKIP of liver and kidney, the expression of RKIP of liver and kidney with As (III) was explored in this study. Thirty male mice were chronically fed with 42.5 ppm, 85 ppm NaAsO2 and water for 180 days. The kidney and liver accumulation levels of As (III) in mice were determined by electro‐thermal atomic absorption spectrometry. The method of RT‐PCR, Western blotting analysis and immunohistochemistry were used to determine gene expression and protein expression of RKIP. The results showed that arsenic level was significantly increased in kidney and liver of As (III)‐exposed mice as compared with control group. The gene expression and protein expression of RKIP was significantly decreased in kidney and liver of As (III)‐exposed mice in comparison with these of control mice. These data suggested that RKIP decrease of liver and kidney with As (III) may be dangerous index in formation of cancer.

The KRAS Mutation is Highly Correlated With EGFR Alterations in Patients With Non-small Cell Lung Cancer

To understand epidermal growth factor receptor ( EGFR ) and KRAS alterations among tissues of lung cancer patients in Taiwan, and to further understand how they are related, we used tumor tissues from the non-small cell lung cancer patients and performed a complete analysis. In the experimental results, coexisting EGFR and KRAS mutations were found in 1% (2/180) of the samples. This shows that these mutations tend not to coexist. Furthermore, nearly no coexistence was found between KRAS mutations and EGFR overexpression. These outcomes greatly assist the efficacy prediction of current anti- EGFR drugs for cancer patients.

Identification and Authentication of Burdock (Arctium lappa Linn) Using PCR Sequencing

The traditional visual identification of Chinese herbs is not objective. Molecular biological techniques can be used to accurately identify the origin of each herbal species. This can enable the further purification and control of important herbal species. Molecular techniques involving polymerase chain reaction and sequencing were used to provide a relatively simple and objective means of identifying burdock at the species level. This study proved that the length of the ITS1–5.8S rRNA-ITS2 sequence was 358 base-pairs (bp) for six types of Arctium lappa Linn (the following breeds: Pingtung Gueilai, General Snow, Japanese, Fengshan, Wholesaler, and Tainan). Automatic sequencing analysis found that ITS DNA sequences for Pingtung Gueilai and Japanese breeds were the same, and the General Snow breed differed from them at its 277 bp. This study used DNA sequencing to analyze the high specificity regions of A. lappa Linn ITS, originated in different parts of Taiwan, and discovered the breed identification single-nucleotide polymorphism at the 277 bp position for local differentiation.

Single Nucleotide Polymorphism of TCF7L2 and Adiponectin Genes for Type 2 Diabetes Mellitus in Taiwan

In Taiwan, type 2 diabetes mellitus (T2DM) is increasing and accounts for a high proportion of medical costs. Recent genome-wide scans have mapped three diabetes susceptibility loci on chromosomes 10q25.3 and 3q27, where the transcription factor 7-like 2 gene ( TCF7L2 ) and adiponectin gene ( APM1 ) are located, respectively. This study aimed to explore the TCF7L2 and adiponectin gene polymorphisms in Taiwanese T2DM patients. In order to determine whether genetic polymorphisms of TCF7L2 and adiponectin are associated with T2DM, PCR-restriction fragment length polymorphism and PCR product sequencing experiments were performed to identify genetic polymorphisms in TCF7L2 rs12255372, rs7903146, and adiponectin single-nucleotide polymorphism (SNP)-45/SNP-276. We collected blood from unrelated T2DM patients and unrelated healthy controls. Our data suggest that TCF7L2 polymorphisms are rare in Taiwanese T2DM patients. Regarding the adiponectin gene SNP-45, T2DM patients with genotype TT had lower high-density lipoproteincholesterol levels than those with genotypes TG and GG ( p = 0.011). Females with genotype GG had lower levels than males with genotype GG ( p = 0.035). Our data show that cholesterol level might be correlated with the adiponectin SNP gene for T2DM. The adiponectin SNP might be associated with increased cardiovascular risk in T2DM patients.