We evaluated whether the inhibitory effects of vascular endothelial growth factor (VEGF)-targeted drugs on the proliferation of cancer cells differed according to VEGF receptor (VEGFR) genes, Flt1 and KDR, promoter methylation status. Five hyper-VEGFR-methylation and six no-VEGFR-methylation cancer cells were used for the present study, together with human umbilical endothelial cells (HUVECs) as a control. No-VEGFR-methylation cancer cells showed higher expression of Flt1 and KDR than hyper-VEGFR-methylation cancer cells. Hyper-VEGFR-methylation cancer cells only showed increased expression and protein levels of Flt1 and KDR after treatment with the demethylase 5-aza-2'-deoxycytidine. Two drugs (a VEGF-specific-antibody, bevacizumab, and a KDR-specific-antibody) targeting extracellular VEGF-VEGFR signaling and two VEGF-specific-tyrosine kinase inhibitors (PTK/ZK and sunitinib) targeting intracellular VEGFR signaling were used in the cell proliferation assay. HUVECs showed dose- and time-dependent proliferation decrease with all tested drugs over a 72 h incubation period. No- or hyper-VEGFR-methylation cancer cells showed no significant proliferation differences after treatment with VEGF-specific-antibody or VEGFR2-specific-antibody. After PTK/ZK or sunitinib treatment, no-VEGFR-methylation cancer cells showed dose- or time-dependent decreases in proliferation. Hyper-VEGFR-methylation cancer cells also showed proliferation inhibition by VEGF-specific-tyrosine kinase inhibitors after demethylation of Flt1 and KDR. Proliferation inhibition synergistically increased after combination of demethylation with PTK/ZK in hyper-VEGF-methylation cancer cells. We observed that intracellular targeting of VEGF-VEGFR signaling could be more effective than extracellular targeting of the pathway in the suppression of proliferation of some cancer cells. In particular, the efficacy of intracellular targeting of VEGF-specific-tyrosine kinase inhibitors might be influenced by the epigenetic alteration of VEGFRs.

BackgroundThe vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) signaling pathway is involved in cancer-related biological functions and is a therapeutic target in cancer. However, the influence of epigenetic regulation of VEGF-VEGFR signaling-related genes remains unclear. Here, we evaluated the effects of FLT1 and KDR promoter hypermethylation combined with drugs targeting VEGF-VEGFR signaling on cancer-related phenotypes in renal cancer cells (RCCs) and examined changes in FLT1 and KDR promoter hypermethylation in tissues from patients with renal cancer.ResultsIn vitro experiments were performed to evaluate the effects of beavacizumab (an anti-VEGF antibody), an anti-FLT1 peptide, an anti-KDR antibody, and the VEGFR tyrosine kinase inhibitors (TKIs) sunitinib and axitinib in 13 RCC lines with different levels of FLT1 and/or KDR promoter methylation and in 2 FLT1 or KDR in vitro knockdown models. The synergistic effects of sunitinib and axitinib treatment were also evaluated in four RCC lines having different levels of FLT1 and/or KDR methylation. In our in vitro experiments, bevacizumab and an anti-KDR antibody did not affect the proliferation of RCCs having FLT1 and/or KDR hypermethylation. In contrast, in RCCs with FLT1 hypermethylation, proliferation inhibition was counteracted by treatment with an anti-FLT1 peptide and both VEGF-TKIs (sunitinib and axitinib). Demethylation with sunitinib or axitinib synergistically increased proliferation inhibition in the RCCs exhibiting FLT1 hypermethylation. Using in vitro FLT1 or KDR knockdown models, decreased proliferation inhibition following anti-FLT1 peptide, sunitinib, and axitinib treatment was observed only in FLT1-knockdown cells. In patients with renal cancer who received sunitinib, FLT1 promoter methylation was higher in renal cancer tissues from eight nonresponders (stable or progressive disease assessed by the Response Evaluation Criteria in Solid Tumors) than in cancer tissues from five responders (complete response or partial response).ConclusionsThe present data showed that hypermethylated FLT1 was important for the efficacy of anti-VEGF/VEGFR drugs targeting FLT1 or intracellular VEGFR signaling. FLT1 hypermethylation causing alterations of FLT1 function could serve as a useful biomarker for predicting changes in FLT1 status in RCCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0134-9) contains supplementary material, which is available to authorized users.

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Alcohol is one of the main causes of liver diseases such as fatty liver, alcoholic hepatitis, and chronic hepatitis with liver fibrosis or cirrhosis. To reproduce the conditions of alcohol-induced liver diseases and to identify the disease-causing mechanisms at the cellular level, several methods have been used to expose the cells to ethanol. As ethanol evaporates easily, it is difficult to mimic chronic alcohol exposure conditions at the cellular level. In this study, we developed a glass capillary system containing ethanol, which could steadily release ethanol from the polyethylene tubing and hydrogel portion at both sides of the capillary. The ethanol-containing capillary could release ethanol in the cell culture medium for up to 144 h, and the concentration of ethanol in the cell culture medium could be adjusted by controlling the number of capillaries. A long-term exposure to ethanol by the capillary system led to an increased toxicity of cells and altered the cellular physiologies, such as increasing the lipid accumulation and hepatic transaminase release in cells, as compared to the traditional direct ethanol addition method. Ethanol capillaries showed different gene expression patterns of lipid accumulation- or chronic alcoholism-related genes. Our results suggest that our ethanol-containing capillary system can be used as a valuable tool for studying the mechanism of chronic alcohol-mediated hepatic diseases at the cellular level.

The SAS macro faces program by Friendly (1992) is for Chernoff face analysis, which is one of methods for the visualization representation of multivariate data. In this paper, we examined 18 face features used in the program and presented the modified program depending on the definition of a good face in days present. In addition, a good bank evaluation for 15 domestic banks was performed through Chernoff face analysis based on 11 bank economic indicators representing stability, the consumer satisfaction, soundness, and banks profitability.