BRAF gene mutations have been observed in 30–50 % of malignant melanoma patients. Recent development of therapeutic intervention using BRAF inhibitors requires an accurate and rapid detection system ...for BRAF mutations. In addition, the clinical characteristics of the melanoma associated with BRAF mutations in Japanese patients have not been investigated on a large scale evaluation. We recently established quenching probe system (QP) for detection of an activating BRAF mutation, V600E and evaluated 113 melanoma samples diagnosed in Saga University Hospital from 1982 to 2011. The QP system includes fully automated genotyping, based on analysis of the probe DNA melting curve, which binds the target mutated site using a fluorescent guanine quenched probe. BRAF mutations were detected in 54 of 115 (47 %) including 51 of V600E and 3 of V600 K in Japanese melanoma cases. Among clinical subtypes of melanoma, nodular melanoma showed high frequency (12 of 15; 80 %) of mutation followed by superficial spreading melanoma (13 of 26; 50 %). The QP system is a simple and sensitive method to determine BRAF V600E mutation, and will be useful tool for patient-oriented therapy with BRAF inhibitors. Introduction
Abstract Mina53, a novel target gene product of c-Myc, is overexpressed in various malignancies. We previously demonstrated that Mina53 is overexpressed in lung cancer patients from the early ...clinical stages. In this paper, the association between disease prognosis and Mina53 expression in lung cancer patients is analyzed; we found that overexpression of Mina53 in lung cancer patients is associated with favorable prognosis. Statistical analysis using the Kaplan–Meier method showed that patients with negative staining for Mina53 had significantly shorter survival than patients with positive staining for Mina53, especially in stage I or with squamous cell carcinoma. Because the major cause of death in lung cancer patients after surgery is distant metastasis, the effect on cancer cell invasiveness was analyzed for the mechanisms involved in the association with favorable outcome. Overexpression of Mina53 in H226B, a lung squamous cell carcinoma cell line, inhibited cancer cell invasion. Transfection with mina53 shRNA increased the number of invading cells. These results suggest that Mina53 immunostaining is a useful prognostic marker – especially in the early stage of lung cancer – and that Mina53 negative patients should be managed particularly carefully after surgery.
Re-challenge with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) has been suggested to potentially improve survival in certain populations of patients with advanced lung ...cancer, but predictive markers for the success of EGFR-TKI re-challenge have not been identified. The present study analyzed 16 re-challenges with EGFR-TKI undertaken in 12 patients with lung adenocarcinoma by investigating T790M and hepatocyte growth factor (HGF) in plasma coupled with clinical characteristics.
mutations in plasma DNA were detected using the wild inhibiting PCR and quenched probe system for exon 19 deletions, and T790M and L858R were detected using the mutation-biased PCR and quenched probe system. HGF levels in the plasma were measured by enzyme-linked immunosorbent assay, and the ratio of HGF levels prior to re-challenge to those prior to the previous EGFR-TKI treatment was calculated. Two re-challenges demonstrated partial response, six remained as stable disease and eight had progressive disease (PD). A total of 4 of the 5 patients with a history of T790M positivity based on plasma DNA levels had PD. A total of 7 of the 8 patients who had ≥1.5-fold elevation of HGF prior to re-challenge with EGFR-TKI suffered PD. Elevation of the HGF ratio to ≥1.5 was significantly associated with poor response to EGFR-TKI re-challenge. Having no history of T790M and an HGF ratio <1.5 was significantly associated with a positive response to EGFR-TKI re-challenge. A combination of T790M detection and HGF quantification using plasma is a potentially useful assay system for predicting the effect of EGFR-TKI re-challenge. Future prospective studies are required to confirm the predictive validity of these markers.
Bernard–Soulier syndrome (BSS) is an inherited bleeding disorder caused by a defect in the platelet glycoprotein (GP) Ib/IX complex. The GPIX W127X mutation is the most common genetic defect in ...Japanese patients with BSS, which is often misdiagnosed as immune thrombocytopenic purpura, presumably due to residual expression of GPIbα. Neither the mechanism by which this mutation leads to a mild bleeding diathesis, nor whether functional GPIbα is expressed on platelet surfaces is known. We investigated GPIbα expression and function in platelets with a GPIX W127X mutation (GPIXW127X). GPIbα complexed with GPIbβ by disulfide bonding was expressed on GPIXW127X platelets and stable CHO-K1 cells lacking GPIX but expressing GPIbα and GPIbβ. Expression of GPIbα/β on GPIXW127X platelets was sufficient to support adhesion to immobilized von Willebrand factor and type III collagen and ristocetin-induced platelet agglutination. A residual amount of functional GPIbα/β heteromer expressed on GPIXW127X platelets partially compensates for the absence of the GPIb/IX complex. This may account for the mild bleeding phenotype of the BSS variant characterized by a non-sense mutation in GPIX.
Circulating cell-free nucleic acids are noninvasive diagnostic tools for cancer detection. Heterogeneous nuclear ribonucleoprotein (hnRNP) B1, an RNA binding protein, has been found overexpressed in ...the early stage of lung cancer, including bronchial dysplasia, a premalignant lesion of lung squamous cell carcinoma. To determine the utility of plasma hnRNP B1 RNA and as cancer detection markers for lung cancer, we analyzed plasma
hnRNP B1 mRNA of lung cancer patients by real-time RT-PCR. Plasma RNA was extracted from plasma of 44 lung cancer patients, 7 lung neoplasm patients, 24 benign lung diseases and 25 healthy volunteers. Mean concentration of plasma
hnRNP B1 mRNA in lung cancer patients was 0.99
pg/μg RNA, whereas that in healthy volunteers and in benign lung diseases was 0.23
pg/μg RNA and 0.30
pg/μg RNA, respectively (
p
<
0.05). Twenty of 44 (45.5%) lung cancer patients showed more than 0.70
pg/μg RNA of plasma
hnRNP B1 mRNA, compared with only 3 of 25 (12.0%) healthy volunteers. Looking at histological subtype, squamous cell carcinoma patients showed higher
hnRNP B1 mRNA in the plasma than did adenocarcinoma patients, which is consistent with our previous immunohistochemistry results. These results indicate that plasma
hnRNP B1 mRNA is a useful non-invasive markers for detection of lung cancer.
Purpose
In 2008, we reported that 10 Japanese-size cups of green tea daily, supplemented with tablets of green tea extract (GTE), reduced the recurrence of colorectal adenoma by 51.6 % in patients ...after polypectomy. Based on these results, we paid special attention to Japanese cancer patients, who consume green tea every day and are administered anticancer drugs. This encouraged us to study whether the combination of green tea catechins and anticancer drugs has the potential to enhance the efficacy of the drugs.
Results and discussion
The combination of GTE and NSAIDs synergistically inhibited tumor development in rodents through the activation of the GADD153–DR5–TRAIL apoptotic pathway. Since then, this study was further extended by various investigators to the combinations of EGCG and other green tea catechins with anticancer compounds, the latter of which include NSAIDs, phytochemicals, and anticancer drugs. In order to demonstrate whether diversity of the combinations would generally induce synergistic anticancer effects on numerous human cancer cell lines, we studied the results of 42 in vitro combination experiments and the synergistic inhibition of tumor volume of 13 combination experiments using xenograft mouse models, which were previously reported by other investigators. The various combinations of EGCG and anticancer compounds induced similar synergistic anticancer effects for both in vitro and in vivo experiments, and showed an average reduction in tumor volume by 70.3 %. Considering the evidence showing that treatment with EGCG inhibited self-renewal of cancer stem cells, the combination shows a great advantage.
Conclusion
Green tea is a cancer preventive for humans, showing a new trend of green tea catechins as synergists with anticancer compounds.
(–)‐Epigallocatechin gallate (EGCG), the main constituent of green tea, and green tea extract show growth inhibition of various cancer cell lines, such as lung, mammary, and stomach. We studied how ...tea polyphenols induce growth inhibition of cancer cells. Since green tea extract contains various tea polyphenols, such as EGCG, (–)‐epigallocatechin (EGC), (–)‐epicatechin gallate (ECG), and (–)‐epicatechin (EC), the inhibitory potential of each tea polyphenol on the growth of a human lung cancer cell line, PC‐9 cells, was first examined. EGC and ECG inhibited the growth of PC‐9 cells as potently as did EGCG, but EC did not show significant growth inhibition. The mechanism of growth inhibition by EGCG was studied in relation to cell cycle regulation. Flow cytometric analysis revealed that treatment with 50 μM and 100 μM EGCG increased the percentages of cells in the G2‐M phase from 13.8% to 15.6% and 24.1%, respectively. The DNA histogram after treatment with 100 μM EGCG was similar to that after treatment with genistein, suggesting that EGCG induces G2‐M arrest in PC‐9 cells. Moreover, we found by microautoradiography that 3HEGCG was incorporated into the cytosol, as well as the nuclei. These results provide new insights into the mechanisms of action of EGCG and green tea extract as cancer‐preventive agents in humans.
Heterogeneous nuclear ribonucleoprotein B1, an RNA binding protein, is overexpressed from the early stage of lung cancers; it is evident even in bronchial dysplasia, a premalignant lesion. We ...evaluated the proteins bound with hnRNP B1 and found that hnRNP B1 interacted with DNA-dependent protein kinase (DNA-PK) complex, and recombinant hnRNP B1 protein dose-dependently inhibited DNA-PK activity in vitro. To test the effect of hnRNP B1 on DNA repair, we performed comet assay after irradiation, using normal human bronchial epithelial (HBE) cells treated with siRNA for hnRNP A2/B1: reduction of hnRNP B1 treated with siRNA for hnRNP A2/B1 induced faster DNA repair in normal HBE cells. Considering these results, we assume that overexpression of hnRNP B1 occurring in the early stage of carcinogenesis inhibits DNA-PK activity, resulting in subsequent accumulation of erroneous rejoining of DNA double-strand breaks, causing tumor progression.
Green tea is a daily beverage, a non-oxidized non-fermented product containing at least four green tea catechins. Considering our first results when repeated applications of (-)-epigallocatechin ...gallate (EGCG) prevented tumor promotion in mouse skin, we have continued to look at green tea as a possible cancer preventive agent. 1) The 10-year prospective cohort study by Drs. K. Nakachi and K. Imai revealed that drinking 10 Japanese-size cups (120 mL/cup) of green tea per day delayed cancer onset in humans by 7.3 years among females and by 3.2 years among males. The delay of cancer onset is of course significant evidence of primary cancer prevention in humans. 2) In collaboration with Dr. H. Moriwaki's group we successfully presented a prototype of tertiary cancer prevention showing that 10 Japanese-size cups of green tea daily, supplemented with tablets of green tea extract (G.T.E), reduced recurrence of colorectal adenomas in polypectomy patients by 51.6% (from 31% to 15%). 3) In 1999, we first reported that the combination of green tea catechins and non-steroidal anti-inflammatory drugs showed synergistic anticancer effects in both in vitro and in vivo experiments, along with elucidation of the mechanism. 4) Further studies by other investigators have revealed that various combinations of EGCG or green tea extract and anticancer compounds inhibit tumor volume in xenograft mouse models implanted with various human cancer cell lines. Green tea is a cancer preventive, and green tea catechins act as synergists with anticancer compounds.
Heterogeneous nuclear ribonucleoproteins (hnRNPs) play an important role as the autoantigens in certain autoimmune disorders including neurological diseases such as HTLV‐1–associated ...myelopathy/tropical spastic paraparesis and paraneoplastic neurological syndromes. To clarify their implication in multiple sclerosis (MS), we assayed antibodies (Abs) against hnRNP A and B proteins in sera and cerebrospinal fluid (CSF) of MS patients and compared the results with 25 patients with other neurological diseases (ONDs). Using recombinant hnRNP A1, A2, and B1 proteins and Western blotting for the assay, we found Abs against hnRNP B1 in CSF from 32 of 35 MS patients (91.4%) but not in any sera or CSF of the 25 OND patients. Most notably, no Abs against hnRNP B1 were found in sera of all 22 MS patients examined. Although Abs against hnRNP A1 and A2 were concomitantly found in CSF reacting with B1, their incidence and immunoreactivity were lower or weaker than those of anti–hnRNP B1 Abs. There was no correlation between the reactivity of CSF with hnRNP B1 and CSF parameters—such as the number of the cells and the IgG level—or clinical parameters—such as duration of illness and disease activity. The selective generation of Abs against hnRNP B1 in CSF was shown to be highly specific for MS, which makes them a disease marker. Ann Neurol 2004
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