Histone Deacetylase Inhibitors as Anticancer Drugs.

Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC) and histone acetyltransferases (HAT). HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.

Sarcosine Up-Regulates Expression of Genes Involved in Cell Cycle Progression of Metastatic Models of Prostate Cancer.

The effects of sarcosine on the processes driving prostate cancer (PCa) development remain still unclear. Herein, we show that a supplementation of metastatic PCa cells (androgen independent PC-3 and androgen dependent LNCaP) with sarcosine stimulates cells proliferation in vitro. Similar stimulatory effects were observed also in PCa murine xenografts, in which sarcosine treatment induced a tumor growth and significantly reduced weight of treated mice (p < 0.05). Determination of sarcosine metabolism-related amino acids and enzymes within tumor mass revealed significantly increased glycine, serine and sarcosine concentrations after treatment accompanied with the increased amount of sarcosine dehydrogenase. In both tumor types, dimethylglycine and glycine-N-methyltransferase were affected slightly, only. To identify the effects of sarcosine treatment on the expression of genes involved in any aspect of cancer development, we further investigated expression profiles of excised tumors using cDNA electrochemical microarray followed by validation using the semi-quantitative PCR. We found 25 differentially expressed genes in PC-3, 32 in LNCaP tumors and 18 overlapping genes. Bioinformatical processing revealed strong sarcosine-related induction of genes involved particularly in a cell cycle progression. Our exploratory study demonstrates that sarcosine stimulates PCa metastatic cells irrespectively of androgen dependence. Overall, the obtained data provides valuable information towards understanding the role of sarcosine in PCa progression and adds another piece of puzzle into a picture of sarcosine oncometabolic potential.

Relation of exposure to amino acids involved in sarcosine metabolic pathway on behavior of non-tumor and malignant prostatic cell lines.

BACKGROUND: Sarcosine (N-methylglycine) was previously delineated as a substantial oncometabolite of prostate cancer (PCa) and its metabolism seems to be significantly involved in PCa development and behavior. METHODS: We focused on investigation whether the exposure of prostate cells (PNT1A, 22Rv1, and PC-3) to sarcosine-related amino acids (glycine, dimethylglycine, and sarcosine) affects their aggressiveness (cell mobility and division rates, using real-time cell based assay). The effect of supplementation on expression of glycine-N-methyltransferase (GNMT) mRNA was examined using qRT-PCR. Finally, post-treatment amino acids patterns were determined with consequent statistical processing using the Ward's method, factorial ANOVA and principal component analysis (P < 0.05). RESULTS: The highest migration induced sarcosine and glycine in metastatic PC-3 cells (a decrease in relative free area about 53% and 73%). The highest cell division was achieved after treatment of 22Rv1 and PC-3 cells with sarcosine (time required for division decreased by 65% or 45%, when compared to untreated cells). qRT-PCR revealed also significant effects on expression of GNMT. Finally, amino acid profiling shown specific amino acid patterns for each cell line. In both, treated and untreated PC-3 cells significantly higher levels of serine, glutamic acid, and aspartate, linked with prostate cancer progression were found. CONCLUSIONS: Sarcosine-related amino acids can exceptionally affect the behavior of benign and malignant prostate cells.

Use of brightness wavelet transformation for automated analysis of serum metallothioneins- and zinc-containing proteins by Western blots to subclassify childhood solid tumours.

In this study, we determined serum levels of metallothioneins (MTs) and zinc in children with solid tumours (neuroblastoma, Hodgkin lymphoma, medulloblastoma, osteosarcoma, Ewing sarcoma and nephroblastoma) by differential pulse voltammetry Brdicka reaction and ELISA. Zn(II) level in patients sera was 40% compared to controls, contrariwise, MT level was 4.2 × higher in patients. No significant differences among single diagnoses were found both for Zn(II) and MT. When determined Zn(II)/MT ratio, in controls its value was 24.6, but it was 2.6 in patients. After Western-blotting with anti-MT and anti-Zn chicken antibodies, variable intensities of the bands within the samples were observed. The brightness curve obtained for each sample both for MT- and Zn blots was further analysed to produce a list of band positions together with some complementary information related to the intensity of the observed bands by the optimised algorithm. We constructed from those profiles decision trees that enable to distinguish different groups of tumours. The blood samples were heat-treated, in which we supposed mainly MT, but samples contained other thermostable Zn-containing proteins that were helpful for identification of embryonal tumours with 88% accuracy and for identification of sarcomas with 78% accuracy. In MT blots the accuracies were 53 and 45%, respectively. Simultaneous analysis of MT and Zn blots did not increased accuracy of identification neither in embryonal tumours (80%) nor in sarcomas. Those results are promising not only from diagnostic point of view but particularly in the area of studying of individual MT isoforms and their aggregates in malignant tumours.

Anticancer agent ellipticine combined with histone deacetylase inhibitors, valproic acid and trichostatin A, is an effective DNA damage strategy in human neuroblastoma.

OBJECTIVES: Valproic acid (VPA) and trichostatin A (TSA) exert antitumor activity as histone deacetylase inhibitors, whereas ellipticine action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of cytochrome P450 (CYP)- and peroxidase-mediated covalent DNA adducts. This is the first report on the molecular mechanism of combined treatment of human neuroblastoma UKF-NB-3 and UKF-NB-4 cells with these compounds. METHODS: HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites formed by microsomes and peroxidases. Covalent DNA modifications by ellipticine in neuroblastoma cells and in incubations with microsomes and peroxidases were detected by 32P-postlabeling. Expression of CYP enzymes, peroxidases and cytochrome b5 was examined by Western blot. RESULTS: The cytotoxicity of ellipticine to neuroblastomas was increased by pre-treating these cells with VPA or TSA. A higher sensitivity of cells to ellipticine correlated with an increase in formation of covalent ellipticine-derived DNA adducts in these cells. To evaluate the mechanisms of this finding, we investigated the modulation by VPA and TSA of CYP- and peroxidase-mediated ellipticine-derived DNA adduct formation in vitro. The effects of ellipticine in the presence of VPA and TSA on expression of CYPs and peroxidases relevant for ellipticine activation and levels of cytochrome b5 and P-glycoprotein in neuroblastoma cells were also investigated. Based on these studies, we attribute most of the enhancing effects of VPA and TSA on ellipticine cytotoxicity to enhanced ellipticine-DNA adduct formation caused by an increase in levels of cytochrome b5, CYP3A4 and CYP1A1 in neuroblastoma cells. A lower sensitivity of UKF-NB-4 cells to combined effects of ellipticine with VPA and TSA than of UKF-NB-3 cells is also attributable to high levels of P-glycoprotein expressed in this cell line. CONCLUSION: The results found here warrant further studies and may help in the design of new protocols geared to the treatment of high risk neuroblastomas.

Enhancement of monoclonal antibody production by lysine-containing peptides.

In the search for peptides that could effectively enhance the monoclonal antibody production of a model hybridoma, the performance of five lysine-containing peptides was compared. The capacity of the peptides to enhance the monoclonal antibody yield correlated with their growth-suppressing activity. No correlation of the production-enhancing activity with the character of the distribution of cell-cycle phases could be found. All of the tested peptides, including the negative control peptide Gly-Phe-Gly, altered the cell-cycle phases distribution in favor of the proportion of the S phase. The peptides added to the hybridoma culture were found to be gradually decomposed into dipeptides and free amino acids. Among the set of tested lysine-containing di- to pentapeptides, the best results were obtained with the tripeptide Gly-Lys-Gly. The growth-suppressing and production-enhancing capacity of this peptide supplement was obviously associated with the temporary presence of the intact peptide molecule in the culture media, because the addition of a mixture of free amino acids constituting this peptide, i.e., glycine and lysine, displayed a different effect-a slight promotion of cell growth.