Over-expression of the proto-oncogene survivin in colorectal cancer stem cells (CCSCs) is thought to be one the primary causes for therapy failure. It has also been reported that tumor suppressor miR-16-1 is down-regulated in colorectal cancer (CRC) cells. Therefore, the search for new anti-proliferative agents which target survivin or miR-16-1 in CCSCs is warranted. Several studies have shown that prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in different kinds of cancer cells. Here, we investigated the effects of prodigiosin on HCT-116 cells that serve as a model for CRC initiating cells with stem-like cells properties. HCT-116 cells were treated with 100, 200 and 400 nM prodigiosin after which cell number, viability, growth-rate, survivin and miRNA-16-1 expression, caspase-3 activation and apoptotic rate were evaluated. Prodigiosin decreased significantly growth-rate in a dose-and time-dependent manner. After a 48 h treatment with 100, 200 and 400 nM prodigiosin, growth-rates were measured to be 84.4 ± 9.2 %, 58 ± 6.5 % and 46.3 ± 5.2 %, respectively, compared to untreated cells. We also found that treatment for 48 h with indicated concentrations of prodigiosin resulted in 41 %, 54.5 % and 63 % decrease in survivin mRNA levels and induced 32 %, 48 % and 61 % decrease in survivin protein levels as well as resulted in 128.3 ± 10 %, 178.7 ± 6.1 % and 205 ± 7.6 % increase in caspase-3 activation respectively compared to untreated cells. Prodigiosin caused a significant increase in miRNA-16-1 expression at a concentration of 100 nM and treatment with different concentrations of prodigiosin resulted in 2.2- to 3-fold increase in miRNA-16-1/survivin ratios compared to untreated cells. An increase in number of apoptotic cells ranging from 28.2 % to 86.8 % was also observed with increasing prodigiosin concentrations. Our results provide the first evidence that survivin and miRNA-16-1 as potential biomarkers could be targeted in CRC initiating cells with stem-like cells properties by prodigiosin and this compound with high pro-apoptotic capacity represents the possibility of its therapeutic application directed against CCSCs.

Salvia macrosiphon Boiss. is an aromatic perennial herb belonging to the family Lamiaceae. Phytochemical studies and biological activities of this plant have been rarely documented in the literature. The current study aimed to investigate antibacterial and cytotoxic activity of different fractions of aerial parts of S. macrosiphon. Also, we tried to isolate and identify cytotoxic compounds from the plant. In this respect, the hydroalcoholic extract of the corresponding parts of the plant was fractionated into four fractions. Then, antibacterial and cytotoxic activity of each fraction were examined. It was found that the chloroform fraction had a good antibacterial activity against gram-positive and gram-negative bacteria. The most potent cytotoxicity was also obtained by the n-hexane fraction comparing with etoposide as the reference drug which was selected for the study and characterization of secondary metabolites. Accordingly, 13-epi manoyl oxide (1), 6α-hydroxy-13-epimanoyl oxide (2), 5-hydroxy-7,4'-dimethoxyflavone (3), and β-sitosterol (4) were isolated and evaluated for their cytotoxic activity. Among them, compound 1 revealed significant cytotoxicity against A549, MCF-7, and MDA-MB-231. It merits mentioning that it showed high selectivity index ratio regarding the low cytotoxic effects on Human Dermal Fibroblast which can be considered as a promising anticancer candidate.

Ex-vivo gene therapy of hemophilias requires suitable bioreactors for secretion of hFIX into the circulation and stem cells hold great potentials in this regard. Viral vectors are widely manipulated and used to transfer hFIX gene into stem cells. However, little attention has been paid to the manipulation of hFIX transgene itself. Concurrently, the efficacy of such a therapeutic approach depends on determination of which vectors give maximal transgene expression. With this in mind, TF-1 (primary hematopoietic lineage) and rat-bone marrow mesenchymal stem cells (BMSCs) were transfected with five hFIX-expressing plasmids containing different combinations of two human β-globin (hBG) introns inside the hFIX-cDNA and Kozak element and hFIX expression was evaluated by different methods. In BMSCs and TF-1 cells, the highest hFIX level was obtained from the intron-less and hBG intron-I,II containing plasmids respectively. The highest hFIX activity was obtained from the cells that carrying the hBG intron-I,II containing plasmids. BMSCs were able to produce higher hFIX by 1.4 to 4.7-fold increase with activity by 2.4 to 4.4-fold increase compared to TF-1 cells transfected with the same constructs. BMSCs and TF-1 cells could be effectively bioengineered without the use of viral vectors and hFIX minigene containing hBG introns could represent a particular interest in stem cell-based gene therapy of hemophilias.

EPA induces strongly wt-p53 with a remarkable decrease in survivin expression, representing an attractive compound to modulate wt-p53 and survivin in ALL cells.

BackgroundHuman FIX (hFIX) gene transfer into hepatocytes has provided a novel approach for treatment of hemophilia B. To obtain an improved expression of hFIX, the functional hFIX-expressing plasmids with appropriate intron-derived fragments which facilitate transcription and promote an efficient 3′-end formation of mRNAs are required.ObjectivesWe aim to evaluate the functions of the heterologous intron-derived fragments intra and extra hFIX-cDNA coding region with respect to the hFIX expression in the hepatocytes and kidney cells.Materials and MethodsHepG2 cells as differentiated hepatocytes and Hek-293T cells as embryonic kidney cells were transfected with the different hFIX-expressing plasmids containing various combinations of the two human beta-globin (hBG) introns within the hFIX-cDNA and Kozak sequence. In the next stage, as a hepatocyte-specific sequence, the rat aldolase B intronic enhancer sequence (rABE), was isolated from the first intron of the rat aldoase B gene and inserted within the upstream CMV promoter (CMVp) and efficacies of the engineered vectors were investigated in the stably-transfected HepG2 cells.ResultsOur data indicate that the intron-less construct and hBG intron-I containing construct are more effective with regard to hFIX expression compared to other constructs in Hek-293 cells. In HepG2 cells, the rABE in combination with CMVp in context of intron-less plasmid induced an increase in total expression of hFIX protein dramatically; ranging from 2.3 to 40 folds increase compared to other constructs. The rABE in combination with CMVp in the hBG intron-I, hBG intron-II, and hBG intron-I,II containing plasmids induced 3.7, 2, and 1.6-fold increase in the total expression of hFIX protein, respectively. The presence of both hBG intronic sequences within the hFIX-cDNA induced a higher secretion level of hFIX than either intron-I or II alone and provided correctly spliced hFIX transcripts in HepG2 and kidney cell lines. The intron-less construct with or without rABE induced the highest hFIX mRNA levels in HepG2 and Hek-293T cells respectively compared to other constructs.ConclusionsThe embryonic kidney cells in addition to the differentiated hepatic cell lines could be successfully targeted by plasmid vectors. The intron-less and hBG intron-I containing plasmids represent a particular interest in producing recombinant hFIX in Hek-293T cells. The synergistic function on the hFIX expression that was achieved by combining the CMVp with the liver-specific rABE would be a useful approach for future designing of the expression cassettes for hepatocyte-mediated gene expression in hemophilia B.

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