CCY-1a-E2 induces G2/M phase arrest and apoptotic cell death in HL-60 leukemia cells through cyclin-dependent kinase 1 signaling and the mitochondria-dependent caspase pathway.

Our previous study demonstrated that 2-[(3-methoxybenzyl)oxy]benzaldehyde (CCY-1a-E2) is a potent compound that acts against multiple human leukemia cell lines. CCY-1a-E2 was also shown to have efficacious anti­leukemic activity in vivo. However, the molecular mechanism of action of CCY­1a­E2 attributed to its anticancer effect remains poorly understood. In the present study, CCY­1a­E2 suppressed cell viability in multiple leukemia cell lines (HL­60, K562, KG­1 and KG­1a) via inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. CCY­1a­E2 exhibited a marked toxic effect on HL­60 cells and displayed low cytotoxicity in normal human peripheral blood mononuclear cells (PBMCs). Results from flow cytometric analysis indicated that CCY­1a­E2 promoted G2/M phase arrest and promoted apoptosis in the HL­60 cells. CCY­1a­E2 treatment upregulated cyclin B, cyclin­dependent kinase 1 (CDK1), cell division cycle 25C (cdc25C) and p21 protein expression. CCY­1a­E2 caused apoptotic cell death and DNA fragmentation as determined by 4',6­diamidino­2­phenylindole (DAPI) staining and DNA gel electrophoresis. Elevated activities of caspase­8, ­9 and ­3 were observed during CCY­1a­E2­induced cell apoptosis; their specific inhibitors were found to block CCY­1a­E2­induced apoptosis, respectively. Moreover, CCY­1a­E2 time­dependently disrupted the mitochondrial membrane potential (ΔΨm), and it enhanced the protein levels of Fas/CD95, cytochrome c, Bax, cleaved PARP, as well as attenuated Bcl­2 expression in the HL­60 cells. Our results provide direct evidence that supports the future potential therapeutic application of CCY-1a-E2 in leukemia.

In vivo evaluation of the synthesized novel 2-benzyloxybenzaldehyde analog CCY-1a-E2 for the treatment of leukemia in the BALB/c mouse WEHI-3 allograft model.

Our previous study demonstrated that the 2-benzyloxybenzaldehyde analog CCY-1a-E2 is a potent compound against HL-60 human leukemia cell lines. To investigate the potential therapeutic application of CCY-1a-E2 for leukemia, we analyzed the antileukemic effects and safety of CCY-1a-E2 in the BALB/c mouse WEHI-3 allograft model. Our results showed that CCY-1a-E2 decreased the percentage of viable cells in a concentration-dependent manner. The IC(50) of CCY-1a-E2 was 5 µM for the 24-h treatment of WEHI-3 cells. We examined the antileukemic activity of CCY-1a-E2 in the BALB/c mouse WEHI-3 allograft model. The CCY-1a-E2 (100 mg/kg) group was not found to have significantly decreased body weight compared with the control group, while the leukemia group was found to have significantly decreased body weight compared with the control mice. The CCY-1a-E2 (100 mg/kg) group showed no difference in spleen and liver weight, but significantly decreased levels of CD11b and CD45 compared with the leukemia group. In safety evaluation analysis, CCY-1a-E2 had no adverse effects on renal, hepatic and hematological parameters. Based on these observations, CCY-1a-E2 has efficacious antileukemic activity in the BALB/c mouse WEHI-3 allograft model.

Proteomics-based identification of haptoglobin as a novel plasma biomarker in oral squamous cell carcinoma.

BACKGROUND: Identification of tumor biomarkers to assist early diagnosis and monitoring of disease progression may potentially decrease the mortality and morbidity associated with oral cancer. METHODS: A mouse model with oral squamous cell carcinoma (OSCC) induced by 4-nitroquinoline 1-oxide (4-NQO)/arecoline in drinking water was established to discover stage-associated biomarkers. A proteomics approach, immunoblot and immunohistochemical analysis were used to validate the expressed biomarkers in mice with OSCC. Human plasma samples were also collected and candidate biomarkers were evaluated using enzyme-linked immunosorbent assay. RESULTS: Proteomic profiling of mouse plasma samples indicated that haptoglobin and apolipoprotein A1 precursor were up-regulated in the mice with OSCC. Immunoblotting of plasma samples and immunohistochemical analysis of oral tissues showed a significantly higher level of haptoglobin in the OSCC mice than in the control mice. The expression of haptoglobin in human plasma samples from 52 patients with OSCC indicated a strong correlation between the increasing levels of haptoglobin and the clinical stages of OSCC (P<0.01). CONCLUSIONS: These results suggest that haptoglobin has a great potential as a sensitive plasma biomarker for early detection of patients with OSCC.

Co-treating with arecoline and 4-nitroquinoline 1-oxide to establish a mouse model mimicking oral tumorigenesis.

The aim of this study was to establish an effective mouse model of oral cancer and to use this model to identify potential markers of oral tumor progression. C57BL/6JNarl mice were treated with arecoline, 4-nitroquinoline 1-oxide (4-NQO), or both arecoline and 4-NQO in high and low doses for 8 weeks to induce oral tumor. The induced oral lesions were observed for 20 weeks to assess the efficiency of cancer induction and survival rate of the mice. In addition, two target proteins that are frequently overexpressed during tongue cancer tumorigenesis, alphaB-crystallin and Hsp27, were examined by immunohistochemical analysis. In mice exposed to 4-NQO (200 microg/mL) and arecoline (500 microg/mL), the tongue lesions showed evidence of hyperplasia, papilloma, dysplasia, and carcinoma, and the lesions were pathologically similar to those lesions in human oral cancer. The tongue tumor incidence rate was 100% in mice exposed to concomitant 4-NQO (200 microg/mL) and arecoline (500 microg/mL) treatment, 57% in mice exposed to 4-NQO only, and 0% in mice exposed to arecoline only. Immunohistochemical analysis demonstrated that, consistent with human studies, alphaB-crystallin and Hsp27 were upregulated in murine oral tumors. In conclusion, we have established a powerful animal model that enables the study of the promoting effects of arecoline on tongue tumorigenesis. Data subsequently attained from this mouse model support a role for alphaB-crystallin and Hsp27 as clinical markers for tumor progression.

Houttuynia cordata Thunb extract induces apoptosis through mitochondrial-dependent pathway in HT-29 human colon adenocarcinoma cells.

The Houttuynia cordata Thunb (HCT) extract has been used as a traditional Chinese herb medicine and as well as an effective drug for treating allergic inflammation for thousands of years. In this study, we investigated the anti-cancer activity of HCT and its molecular mechanisms in the human colon adenocarcinoma cell line HT-29. HCT inhibited HT-29 cell viability in a dose- and time-dependent manner by MTT assay. Treatment with 450 microg/ml of HCT for 48 and 72 h led to DNA damage and apoptosis by DAPI staining and comet assay. HCT increased reactive oxygen species production and decreased the levels of mitochondria membrane potential (MMP) in HT-29 cells by flow cytometry analysis. HCT caused the release of cytochrome c, Apaf-1, pro-caspase-9 and AIF from mitochondria via a decrease of the MMP. The decrease of MMP was then associated with a decrease in the ratio of Bax/Bcl-2 and activation of caspase-9 and -3 by Western blotting and caspase activity assay. Caspase-9 and -3 inhibitors almost completely suppressed HCT-induced caspase-9 and -3 activities. Our results demonstrated that the HCT-induced apoptosis in human colon adenocarcinoma cell line HT-29 might be related to a mitochondrial-dependent pathway.

Functional genomic analysis identified epidermal growth factor receptor activation as the most common genetic event in oral squamous cell carcinoma.

A 250K single-nucleotide polymorphism array was used to study subchromosomal alterations in oral squamous cell carcinoma (OSCC). The most frequent amplification was found at 7p11.2 in 9 of 29 (31%) oral cancer patients. Minimal genomic mapping verified a unique amplicon spanning from 54.6 to 55.3 Mb on chromosome 7, which contains SEC61G and epidermal growth factor receptor (EGFR). Results from fluorescence in situ hybridization, transcriptome, and immunohistochemistry analyses indicated that the expression level of EGFR, but not of SEC61G, was up-regulated and tightly correlated with DNA copy number in 7p11.2 amplified tumors. Among the members of the erbB family, EGFR (HER1) was found to be the most frequently amplified and highly expressed gene in both human and mouse oral tumors (P < 0.01). Genes for downstream effectors of EGFR, including KRAS, mitogen-activated protein kinase 1, and CCND1, were also found amplified or mutated, which resulted in activation of EGFR signaling in 55% of OSCC patients. Head and neck squamous cancer cells with different EGFR expression levels showed differential sensitivity to antitumor effects of AG1478, a potent EGFR inhibitor. AG1478-induced EGFR inactivation significantly suppressed tumor development and progression in a mouse oral cancer model. Our data suggest that EGFR signaling is important in oral cancer development and that anti-EGFR therapy would benefit patients who carry the 7p11.2 amplicon in their tumors.

Apolipoprotein E4 allele influences the response of plasma triglyceride levels to tamoxifen in breast cancer patients.

BACKGROUND: Apolipoprotein E4 (APOE4) allele is an important risk factor for breast cancer and affects clearance of chylomicron remnants. Tamoxifen therapy increases serum triglyceride levels and sometimes inducing severe hypertriglyceridemia in breast cancer patients. METHODS: Thirty-three women with breast cancer were recruited to examine the APOE polymorphism and fasting plasma lipid profiles before and after tamoxifen treatment for 6 months. RESULTS: We found that plasma lipid profiles changed in accordance with the APOE4 allele after tamoxifen treatment for 6 months. Especially plasma triglyceride levels significantly decreased in the APOE4-positive patients (p=0.025), while there was no change in APOE4-negative patients (p=0.189). The total plasma cholesterol levels were reduced in APOE-4 positive patients after 6-month tamoxifen treatment (p=0.014). The levels of plasma low density lipoprotein cholesterol and high density lipoprotein cholesterol significantly decreased in both APOE4-negative and APOE4-positive patients. CONCLUSIONS: These findings indicate that the effects of tamoxifen on plasma triglyceride levels are modified by APOE polymorphism. Breast cancer patients with APOE4 allele have low plasma triglyceride levels when receiving tamoxifen therapy. Therefore, we suggest that APOE gene polymorphism is a critical validation before tamoxifen treatment in breast cancer patients.

Cloning, sequencing, expression, and single-step purification of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) from Salmonella typhimurium ATCC 19585.

The bi-functional enzyme, adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU), is involved in the biosynthesis of cobalamin in Salmonella typhimurium, and, therefore, can be used for the in vitro synthesis of analogs of B(12). Previously, five different steps were required to purify the recombinant enzyme from Escherichia coli. Here, we describe the cloning, sequencing, and expression of the cobU gene from S. typhimurium ATCC 19585 and, without introducing a purification tag sequence to the N- or C-terminus of the recombinant enzyme, a new single-step purification method based on hydrophobic interaction chromatography.

Synthesis and anticancer activity of benzyloxybenzaldehyde derivatives against HL-60 cells.

A series of benzyloxybenzaldehyde derivatives were prepared and tested against the HL-60 cell line for anticancer activity. Preliminary structure-activity relationships were established. It was discovered that 2-(benzyloxy)benzaldehyde (17), 2-(benzyloxy)-4-methoxybenzaldehyde (26), 2-(benzyloxy)-5-methoxybenzaldehyde (27), 2-(benzyloxy)-5-chlorobenzaldehyde (28), 2-[(3-methoxybenzyl)oxy]benzaldehyde (29), 2-[(2-chlorobenzyl)oxy]benzaldehyde (30), and 2-[(4-chlorobenzyl)oxy]benzaldehyde (31) exhibited significant activity at 1-10 microM. Among them, compound 29 was the most potent one. The morphological assessment and DNA fragmentation analysis indicated that these compounds arrested cell cycle progression at G2/M phase and induced cell apoptosis. They resulted in the loss of mitochondrial membrane potential after 12h of treatment.