7,8-Dihydroxy Flavone Induces Apoptosis via Upregulation of Caspase-3 in Human Hepatocarcinoma Cell.

OBJECTIVE: 7,8-Dihydroxyflavone, a tyrosine kinase receptor agonist, is a flavonoid that has recently gained the attention of researchers due to its anticancer properties. Nevertheless, molecular pathways of 7,8-dihydroxyflavone for hepatocarcinoma are uncertain. Our aim was to identify the impact of 7,8-dihydroxyflavone on human hepatocarcinoma. MATERIAL AND METHODS: Human hepatocarcinoma cell line-7 cells were used as human hepatocarcinoma cells, and 7,8-dihydroxyflavone was applied to the cells at various doses. The cytotoxic and apoptotic effects of 7,8-dihydroxyflavone were determined with Alamar Blue and flow cytometry. The properties of 7,8-dihydroxyflavone on the mRNA expressions related with Bcl-2, Bax, cleaved-caspase-3 genes, and protein expressions were determined via quantitative real-time polymerase chain reaction and western blot analysis, respectively. RESULTS: 7,8-Dihydroxyflavone-enhanced cell death in human hepatocarcinoma cell line-7 via the overexpression of cleaved-caspase-3 (P=.003) and decreased Bcl-2 (P=.038) protein levels. Furthermore, cleavedcaspase-3 mRNA overexpression (P=.001) markedly led to 7,8-dihydroxyflavone-induced apoptosis. CONCLUSION: 7,8-Dihydroxyflavone could promote apoptotic cell death by modulating caspase pathways and suppressing antiapoptotic protein. These characteristics may mediate to clinical practice of 7,8-dihydroxyflavone for prevention and therapy of hepatocarcinoma.

Measurement of kynurenine pathway metabolites by tandem mass spectrometry.

Objectives: Recent studies have shown that derangements in kynurenine pathway metabolite levels are associated with various pathologies such as neurodegenerative diseases, schizophrenia, depression, bipolar disorder, rheumatoid arthritis, and cancer. Therefore, reliable, accurate, fast, and multiplex measurement methods for kynurenines have become increasingly important. This study aimed to validate a new mass spectrometric method for analyzing tryptophan metabolites. Methods: A tandem mass spectrometric method, including protein precipitation and evaporation steps, was developed to measure serum levels of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid. Samples were separated using a Phenomenex Luna C18 reversed-phase column. The kynurenine pathway metabolites were detected by tandem mass spectrometry. The developed method was validated according to Clinical & Laboratory Standards Institute (CLSI) guidelines and applied to hemodialysis samples. Results: The developed method was linear at the concentrations of 48.8 - 25,000, 0.98 - 500, 1.2-5000, 1.2-5000, and 0.98-250 ng/mL for tryptophan, kynurenic acid, kynurenine, 3-hydroxyanthranilic acid, and 3-hydroxykynurenine, respectively. The imprecisions were less than 12 %. The median serum concentrations of tryptophan, kynurenine, kynurenic acid, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid were 10530, 1100, 218, 17.6, and 25.4 ng/mL in pre-dialysis blood samples, respectively. They were 4560, 664, 135, 7.4, and 12.8 ng/mL in post-dialysis blood samples, respectively. Conclusions: A fast, simple, cost-effective, accurate, robust, and validated tandem mass spectrometric method was developed, and the method was successfully used for the quantitation of kynurenine pathway metabolite concentrations in hemodialysis patients.

Altered kynurenine pathway metabolism in patients with ankylosing spondylitis.

BACKGROUND: Various studies reported that increased proinflammatory cytokines in patients with ankylosing spondylitis (AS). Proinflammatory cytokines can affect the expression of various kynurenine pathway enzymes and therefore lead to metabolic changes that can affect the inflammatory response and immunity. Our aim was to measure serum levels of kynurenine pathway metabolites in patients with AS. METHODS: The study included 85 patients with AS and 50 healthy volunteers. Serum tryptophan, kynurenine, kynurenic acid, 3-hydroxyanthranilic acid, 3-hydroxykynurenine, quinolinic acid concentrations were measured with tandem mass spectrometry. In addition, participants were divided into four groups according to the treatment regimen: TNF-α inhibitor group, conventional therapy group, control group and newly diagnosed AS group. These groups were compared in terms of kynurenine pathways metabolites, interleukin 6 (IL-6), erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels. RESULTS: Serum tryptophan, kynurenic acid, 3-hydroxykynurenine levels were significantly decreased (p < 0.05) in both AS groups compared to the control group, while the levels of kynurenine, quinolinic acid, CRP, ESR, and IL-6 were higher (p < 0.05). The Kynurenine/Tryptophan ratio and CRP levels of the conventional therapy and anti-TNF therapy group were significantly lower than the newly diagnosed AS patients (p < 0.05). CONCLUSION: As a result of our study, we found that altered kynurenine pathway metabolism in patients with AS. Conventional therapy and anti-TNF-α therapy are effective in reducing the Kynurenine/Tryptophan ratio and CRP levels, although the effect of both treatments on other metabolites appears to be limited.

Comparison of anticarcinogenic properties of Viburnum opulus and its active compound p-coumaric acid on human colorectal carcinoma.

Resistance to therapeutic agents and the highly toxic side effects of synthetic drugs has spurred new research in the treatment of colon cancer, which has high morbidity and mortality ratios. This study aims to clarify the molecular mechanisms of the anticarcinogenic properties of methanol extract of Viburnum opulus L. (EVO)and its main active compound, trans-p -coumaric acid ( p -CA), on human colon cancer cells (DLD-1, HT-29, SW-620, Caco-2) and healthy colon epithelial cells (CCD-18Co). The effects of EVO on controlled cell death (apoptosis) and the cell division cycle were determined by flow cytometry. Alteration in mRNA and protein expressions of switch genes in colorectal carcinoma (APC, MLH1, TP53, SMAD4, KRAS, and BRAF) were determined by qRT-PCR and Western blot, respectively. Our results show that EVO possesses a strong reducing capacity and free-radical scavenging activity. HPLC analyses prove that p -CAis the main compound of EVO. EVO and p -CA inhibit the proliferation of human colon cancer cells DLD-1 and HT-29 in a dose-dependent manner. EVO increases apoptosis of DLD-1 cells and halts the cell cycle in the G2 stage in HT-29 cells. mRNA and protein expressions of p53 and SMAD-4 are upregulated, while BRAFs are downregulated. The results were directly proportional to p -CA. EVO and p -CA up- and downregulate switch genes and protein expressions of DLD-1 cells, which alter the expression of 186 other genes. This is the first study of pharmacological exploration of V.opulus in human colon cancer. Its antiproliferative effects may be due to the presence of p -CA.

Determination of serum imatinib and its' metabolite in patients chronic myeloid leukemia.

INTRODUCTION: Imatinib has favorable pharmacokinetic properties, but primary and secondary resistance mechanisms may cause a decrease in clinical response over time. There is a positive correlation between serum imatinib concentrations and treatment response. Our aim was to develop a method for the measurement of imatinib and its' active metabolite N-desmethyl imatinib. METHODS: Serum imatinib and N-desmethyl imatinib levels were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and validation studies were carried out according to CLSI (The Clinical & Laboratory Standards Institute) protocols. Serum samples were collected from 40 patients with chronic myeloid leukemia (CML) and analyzed with LC-MS/MS and ultra high-performance liquid chromatography (UHPLC) methods. RESULTS: The linearity range and correlation coefficient were 12.2-12,500 ng/mL and 0.9987 for LC-MS/MS method, respectively. Limit of quantitation was determined as 24.4 ng/mL. The retention times of imatinib and N-desmethyl imatinib were 1.66 and 1.60 min, respectively. There was no statistically significant difference between the results of both methods. DISCUSSION: This LC-MS/MS method is cost-effective and has adavantages such as using low serum volumes, requiring simple pretreatment steps (only protein precipitation) and reduced turnaround times for analysis.