No abstract

3,6-Diazaphenothiazines were obtained in cyclization of 3-amino-3'-nitro-2,4'-dipyridinyl sulfide and the reaction of sodium 3-amino-2-pyridinethiolate with 4-chloro-3-nitropyridine followed by alkylation and heteroarylation. The thiazine ring formation ran via the Smiles rearrangement. The structure elucidation was based on 2D NMR and X-ray analysis of N-methylated product. 3,6-Diazaphenothiazines were investigated for antitumor activity using glioblastoma SNB-19, melanoma C-32 and breast cancer MCF-7 cells. 10H-3,6-diazaphenothiazine was 10 times more active (IC50 < 0.72 μg/mL) than cisplatin. Two diazaphenothiazines with the 2-pyrimidinyl and dimethylaminopropyl substituents were selectively active against MCF-7 and C-32 cells. The expressions of H3 (proliferation marker), TP53, CDKN1A (cell cycle regulators), BAX and BCL-2 (proapoptopic and antiapoptopic genes) were detected by RT-QPCR method. The expression analysis suggests the cell cycle arrest and the mitochondrial apoptosis pathway activation in MCF-7 and SNB-19 cells.

AKT, a serine/threonine protein kinase and mammalian target of rapamycin (mTOR) plays a critical role in the proliferation and resistance to apoptosis that are essential to the development and progression of colon cancer. Therefore, AKT/mTOR signaling pathway has been recognized as an attractive target for anticancer therapy. Inositol hexaphosphate (InsP6), a natural occurring phytochemical, has been shown to have both preventive and therapeutic effects against various cancers, however, its exact molecular mechanisms of action are not fully understood. The aim of the in vitro study was to investigate the anticancer activity of InsP6 on colon cancer with the focus on inhibiting the AKT1 kinase and p70S6K1 as mTOR effector, in relation to proliferation and apoptosis of cells. The colon cancer Caco-2 cells were cultured using standard techniques and exposed to InsP6 at different concentrations (1 mM, 2.5 mM and 5 mM). Cellular proliferative activity was monitored by 5-bromo-2′-deoxyuridine (BrdU) incorporation into cellular DNA. Flow cytometric analysis was performed for cell cycle progression and apoptosis studies. Real-time RT-qPCR was used to validate mRNA levels of CDNK1A, CDNK1B, CASP3, CASP9, AKT1 and S6K1 genes. The concentration of p21 protein as well as the activities of caspase 3, AKT1 and p70S6K1 were determined by the ELISA method. The results revealed that IP6 inhibited proliferation and stimulated apoptosis of colon cancer cells. This effect was mediated by an increase in the expression of genes encoding p21, p27, caspase 3, caspase 9 as well a decrease in transcription of AKT1 and S6K1. InsP6 suppressed phosphorylation of AKT1 and p70S6K1, downstream effector of mTOR. Based on these studies it may be concluded that InsP6 can reduce proliferation and induce apoptosis through inhibition of the AKT/mTOR pathway and mTOR effector followed by modulation of the expression and activity of several key components of these pathways in colon cancer cells.

The objective of this study was to determine the frequencies of human cytomegalovirus (HCMV) infection and HCMV genome copy number in blood of consecutive (treated from several months to several years) systemic lupus erythematosus (SLE) patients (22 women). The obtained results were compared to the healthy controls (15 women). All patients fulfilled at least four of the 1982 revised American rheumatism association (ARA) classification criteria for SLE. Our patients demonstrated three or four of the nine possible organ systems involved and most of them had mild SLE with SLE disease activity index (SLEDAI) score < 10 at time when blood samples were collected to detect HCMV. Quantitative analysis of HCMV genome was performed with aid of sequence analyzer ABI PRISM 7,700 Perkin Elmer. Primers and probe were constructed on the basis of IE4 region of HCMV genome. The viral load was expressed as log(10) of calculated HCMV genome copy number. Qualitative analysis revealed that 100% of our SLE patients were infected with HCMV, whereas in the control group only 73% of persons were HCMV positive. Statistically significant difference was demonstrated when the strength of the association between SLE or controls and infection of HCMV was calculated (estimated by Fisher's exact test, P value=0.02). Higher viral DNA copy number was observed in whole blood of SLE patients than in the control group (338.45+/- 221.76 and 229.00+/- 405.61 copies/ml respectively) but did not reach statistical significance level (95% confidence interval from 170.41 to 249.32, P=0.71). Furthermore percentage of patients with HCMV-DNA copy number >2.0 x 10(2) copies/ml was statistically significantly higher than this one in controls. The data show association between HCMV infection and SLE, which should be taken into account during the course of SLE.

New derivatives of two isomeric types of azaphenothiazines, 1,8-and 2,7-diazaphenothiazine, containing the triple bond substituents and additionally tertiary cyclic and acyclic amine groups, were synthesized and tested for their anticancer activity. The compounds exhibited differential inhibitory activities. Better results were obtained when the acetylenic group was transformed via the Mannich reaction to the dialkylaminobutynyl groups. The most active was 2,7-diazaphenothiazine with the N-methylpiperazine-2-butynyl substituent against the human ductal breast epithelial tumor cell line T47D, more potent than cisplatin. The 2,7-diazaphenothiazine system turned out to be more active than isomeric 1,8-diaza one. For the most active compound, the expression of TP53, CDKN1A, BCL-2 and BAX genes was detected by the RT-QPCR method. The gene expression ratio BACL-2/BAX suggests the mitochondrial apoptosis in T47D cells. The synthesis makes possible to obtain many new bioactive phenothiazines with the dialkylaminoalkynyl substituents inserting various tertiary cyclic and acyclic amine moieties to the substituents.