An Updated Review of the Cross-talk Between MicroRNAs and Epigenetic Factors in Cancers.

BACKGROUND: Growing evidence indicates that miRs have critical activities in adjusting cellular processes, e.g., cell death, proliferation, and cell-cycle. INTRODUCTION: This study aimed to provide a concise review of the recent findings regarding tumoral miRs and the cross-talk between miRs and epigenetic factors. RESULTS: Like the protein-coding genes, the expression levels of miRs are mediated by various transcriptional networks. Indeed, the expression of miRs could be epigenetically modulated by DNA methylation factors and histone modifiers. Furthermore, miRs can suppress critical factors, which mediate epigenetic modifications. Besides, miRs have been implicated in cancer development, metastasis, and chemo-resistance. The aberrant expression of miRs and dysregulated modulatory circuits between miRs and epigenetic factors participate in tumor progression. CONCLUSION: Identifying tumoral miRs can provide ample opportunity to overcome chemo- resistance and bring a forefront treatment for affected patients.

The Safe Soluble Compound Dehydroascorbic Acid Inhibits Various Upstream and Downstream Effectors of PI3K and KRAS Signaling Pathways in Undruggable PIK3CA/KRAS-Mutant Colorectal Cancer Stem-Like Cells.

Efforts to develop effective drugs targeting PI3K and KRAS signaling pathways in PIK3CA/KRAS-mutant colorectal cancer stem cells (CRCSCs) remain challenging. Finding safe compounds that can easily enter CRCSCs with the ability to target metastasis-driver gene CXCR4 and pluripotency network genes as key upstream and downstream effectors of both PI3K and KRAS signaling pathways may provide promising results. PIK3CA/KRAS-mutant CRCSCs display high expression of glucose transporters (GLUTs) on their cell membrane and a glycolytic phenotype providing an opportunity to deliver antiglycolytic compounds into these cells via the GLUTs. CRC patients with low levels of vitamin C in their plasma show a shorter survival suggesting the ability of this vitamin at the physiologic levels for caspase-3 activation and apoptosis in CRCSCs. Vitamin C in an oxidized form (L-dehydroascorbic acid; L-DHA) with antiglycolytic activity can be taken up into CRC cells via the GLUTs. This may provide selective toxicity on CRCSCs and affect CXCR4 and stemness markers genes expression in these cells. To this end, we treated PIK3CA/KRAS-mutant LS174T cells with high glycolytic activity as an attractive model for CRCSCs with L-DHA equal to the pharmacological levels of vitamin C in human plasma, after which cell numbers, metabolic activity, proliferation-rate, CXCR4 and pluripotency network genes expression, caspase-3 activity with apoptosis were evaluated. 48 h post-treatment with 100- to 1000 µM L-DHA, cell numbers were decreased and measured to be 70-47% control. L-DHA with selective toxicity on LS174T cells diminished metabolic activity and cell proliferation-rate to 1.4-0.8 (Control OD = 1.5) and 92-54.5% respectively with no toxicity on PBMCs. L-DHA decreased CXCR4, Bmi-1, Sox-2 and Oct-4 expression to 45%, 85%, 45% and 48% control respectively followed by caspase-3 reactivation by 2.5 to 4.9-fold increases and induction of apoptosis ranging from 0.5% to 58.3% for 100- to 1000 µM L-DHA. According to our data, CRC stem-like cells were highly sensitive to L-DHA in in-vitro. L-DHA selectively targeted LS174T cells and successfully reactivated caspase-3 and apoptosis in these cells. CXCR4, stemness marker genes and metabolic activity appear to be promising targets of L-DHA. Our results may provide a new therapeutic approach to target selectively GLUT-overexpressing PIK3CA/KRAS-mutant CRCSCs using L-DHA with no toxicity on normal cells.

The response of PIK3CA/KRAS-mutant colorectal cancer stem-like cells to RGD-peptide FraC produced by the strawberry anemone: A promising water-soluble peptide-based inhibitor of metastasis-driver gene CXCR4, stem cell regulatory genes and self-renewal.

Colorectal cancer (CRC) is a stem cell-based disease. PIK3CA/KRAS-mutant CRC stem cells (CRCSCs) display high self-renewal, metastatic properties, high activity of PI3K and KRAS signaling pathways with chemoresistant phenotypes. Recently, RGD peptide (containing Arg-Gly-Asp motif)-based therapy of solid tumor cells has attracted much attention. However, little is known whether this method can target self-renewal capacity, key effectors of PI3K and KRAS signaling pathways such as metastasis-driver gene CXCR4 and stem cell regulatory genes with caspase-3 reactivation in CRCSCs overexpressing RGD-dependent integrins. The sea anemone Actinia fragacea produces a water-soluble RGD-peptide fragacea toxin C (FraC) suggesting the possible activity of FraC against PIK3CA/KRAS-mutant CRCSCs. Recombinant FraC was expressed via pET-28a(+)-FraC in E. coli and purified through affinity chromatography followed by performing SDS-PAGE and hemolytic activity assay. Next, PIK3CA/KRAS-mutant HCT-116 cells that serve as an attractive model for CRCSCs were treated with FraC. Thereafter, cell numbers, viability, proliferation, LDH activity, cytotoxicity index, CXCR4 and pluripotency network genes expression, self-renewal capacity, caspase-3 activity with apoptosis were evaluated. Caspase-1, -2, -3,…, -9 sequences were analyzed for RGD-binding motifs. FraC sequence and structure were also evaluated by bioinformatics software. FraC altered cellular morphology to round shapes and disrupted cell connections. 48 h post-treatment with 0.056- to 7.2 µM FraC resulted in 12 %-99 % and 8 %-97.6 % decreases in cell numbers and viabilities respectively and increased LDH activity by 0.2 %-66.7 % in a dose-dependent manner. The results of the cytotoxicity index showed that FraC induces significant toxicity on HCT-116 cells compared to PBMCs and Huvec cells. FraC dramatically decreased the expression of CXCR4 and pluripotency network genes Bmi-1, Sox-2, Oct-4 and Nanog followed by remarkable decreases in self-renewal capacity ranged from 91- to 0 colonies per well for 0.056- to 3.6 µM FraC after 2 weeks. Caspase-3 was found to contain an RGD-binding motif and its activity increased with increasing FraC concentrations followed by apoptosis induction. Potential RGD-binding motifs for FraC were also found in caspase-1, -7, -8 and -9. Unique advantages of FraC peptide, such as low molecular weight, water solubility, high sensitivity of CRC stem-like cells with more selective toxicity to this compound, targeting tumor cell membrane and self-renewal capacity along with the modulation of CXCR4 and stem cell regulatory genes as upstream and downstream effectors of undruggable PI3K and KRAS signaling pathways may open up avenues for FraC peptide-based therapy of PIK3CA/KRAS-mutant CRCSCs with lower toxicity on healthy cells.

Modulating Pluripotency Network Genes with Omega-3 DHA is followed by Caspase- 3 Activation and Apoptosis in DNA Mismatch Repair-Deficient/KRAS-Mutant Colorectal Cancer Stem-Like Cells.

BACKGROUND: Targeting DNA mismatch repair-deficient/KRAS-mutant Colorectal Cancer Stem Cells (CRCSCs) with chemical compounds remains challenging. Modulating stemness factors Bmi-1, Sox-2, Oct-4 and Nanog in CRCSCs which are direct downstream targets of carcinogenesis pathways may lead to the reactivation of caspase-3 and apoptosis in these cells. Omega-3 DHA modulates different signaling pathways involved in carcinogenesis. However, little is known, whether in vitro concentrations of DHA equal to human plasma levels are able to modulate pluripotency genes expression, caspase-3 reactivation and apoptosis in DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells. METHODS: DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells (LS174T cells) were treated with DHA, after which, cell number and proliferation-rate, Bmi-1, Sox-2, Nanog and Oct-4 expression, caspase-3 activation and apoptosis were evaluated with different cellular and molecular techniques. RESULTS: DHA changed the morphology of cells to apoptotic forms and disrupted cell connections. After 48h treatment with 50- to 200µM DHA, cell numbers and proliferation-rates were measured to be 86%-35% and 93.6%-45.7% respectively. Treatment with 200 µM DHA dramatically decreased the expression of Bmi-1, Sox- 2, Oct-4 and Nanog by 69%, 70%, 97.5% and 53% respectively. Concurrently, DHA induced caspase-3 activation by 1.8-4.7-fold increases compared to untreated cells. An increase in the number of apoptotic cells ranging from 9.3%-38.4% was also observed with increasing DHA concentrations. CONCLUSIONS: DHA decreases the high expression level of pluripotency network genes suggesting Bmi-1, Sox-2, Oct-4 and Nanog as promising molecular targets of DHA. DHA reactivates caspase-3 and apoptosis in DNA mismatch repair-deficient/KRAS-mutant CRC stem-like cells, representing the high potential of this safe compound for therapeutic application in CRC.

Modulating Survivin as a Radioresistant Factor, Caspase-3, and Apoptosis by Omega-3 Docosahexaenoic Acid Sensitizes Mutant-p53 Colorectal Cancer Cells to γ-Irradiation.

Mutant-p53 colorectal cancer (CRC) cells are often resistant to radiotherapy. Loss of suppressor activity of wt-p53 on survivin is responsible for the enhanced expression of survivin as a radioresistant factor in tumor cells. Yet, no survivin-modulating drug has been approved for clinical application in CRC. Thus, the search for safe compounds that modulate survivin expression and induce apoptosis irrespective of p53 status may potentiate the efficacy of radiotherapy in mutant-p53 CRC cells. Omega-3 docosahexaenoic acid (DHA) induces apoptosis in malignant cells without cytotoxicity in normal cells. However, little is known whether in vitro concentrations of DHA equal to the human plasma levels are able to modulate expression of survivin and sensitize mutant-p53 CRC cells to γ-irradiation. Radioresistant mutant-p53 HT-29 cells were pretreated with 50- and 100-µM DHA for 48-h before 2-, 4-, 6-, 8-, and 10-Gy of γ-irradiation. Thereafter, proliferation rates were measured after 6 d. HT-29 cells were also pretreated with 50- and 100-µM DHA for 4-h before 2- and 10-Gy of γ-irradiation after which, cell number, survivin expression, caspase-3 activation, apoptosis, and ED50 (γ-irradiation dose causing 50% growth inhibition) were evaluated. Pretreatment of HT-29 cells with 50- and 100-µM DHA for 48-h followed by 2- to 10-Gy of γ-irradiation induced a dose-dependent additive decrease in cell proliferation rate and ED50 values were decreased by 88%, 44%, 41%, and 27% for 500-, 1500-, 2500-, and 5000 cells per well pretreated with 100-µM DHA respectively. Pretreatment of 5 × 105 HT-29 cells per well with 100-µM DHA for 4-h followed by 2- or 10-Gy of irradiation resulted in 53% and 86% decreases in cell numbers, 2- and 5.1-fold activation in caspase-3 followed by 66% and 60% decreases in survivin mRNA levels respectively. DHA in combination with radiation increased total apoptotic rate 48-h post-treatment. DHA decreases survivin expression and induces caspase-3 activation irrespective of p53 status. Significant decreases in ED50 values at concentrations of DHA equal to human plasma levels, suggesting that DHA could be used as an attractive radiosensitizer agent in CRC patients with mutant-p53.

Omega-3 fatty acid DHA modulates p53, survivin, and microRNA-16-1 expression in KRAS-mutant colorectal cancer stem-like cells.

BACKGROUND: The presence of chemotherapy-resistant colorectal cancer stem cells (CCSCs) with KRAS mutation is thought to be one of the primary causes for treatment failure in colorectal cancer (CRC). P53, survivin, and microRNA-16-1 are challenging targets for anticancer drugs which are associated with chemoresistance in CRC. Yet, no p53-, survivin-, and microRNA-16-1-modulating drug with low toxicity but high efficacy against KRAS-mutant CCSCs have been approved for clinical application in CRC. Here, we investigated whether in vitro concentrations of DHA equal to human plasma levels, are able to modulate, Wt-p53, survivin, and microRNA-16-1 in CRC cells with stem cell-like properties. METHODS: Wt-p53/KRAS-mutant CRC cells (HCT-116) with stem cell-like properties were treated with 100-, 150- and 200-µM/L DHA, after which cell number, viability, growth inhibition, Wt-p53, survivin and microRNA-16-1 expression, caspase-3 activation and apoptotic-rate were evaluated by different cellular and molecular techniques. RESULTS: After 24-, 48-, and 72-h treatments with 100- to 200-µM/L DHA, growth inhibition- rates were measured to be 54.7% to 59.7%, 73.% to 75.8%, and 63.3% to 97.7%, respectively. Treatment for 48 h with indicated DHA concentrations decreased cell number and viability. In addition, we observed a decrease in both the transcript and protein levels of survivin followed by 1.3- to 1.7- and 1.1- to 4.7-fold increases in the Wt-p53 accumulation and caspase-3 activation levels respectively. Treatment with 100 and 150 µM/L DHA increased microRNA-16-1 expression levels by 1.3- to 1.7-fold and enhanced the microRNA-16-1/survivin mRNA, p53/survivin, and caspase-3/survivin protein ratios by 1.7- to 1.8-, 1.3- to 2.6-, and 1.3- to 2-fold increases respectively. A decrease in the number of live cells and an increase in the number of apoptotic cells were also observed with increasing DHA concentrations. CONCLUSION: Wt-p53, survivin, and microRNA-16-1 appear to be promising molecular targets of DHA. Thus, DHA might represent an attractive anti-tumor agent directed against KRAS-mutant CCSCs.

Functions of the Heterologous Intron-Derived Fragments Intra and Extra Factor IX-cDNA Coding Region on the Human Factor IX Expression in HepG2 and Hek-293T Cells.

BACKGROUND: Human 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. OBJECTIVES: We 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 METHODS: HepG2 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. RESULTS: Our 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. CONCLUSIONS: The 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.

Fish-Oil-Derived DHA-mediated Enhancement of Apoptosis in Acute Lymphoblastic Leukemia Cells is Associated with Accumulation of p53, Downregulation of Survivin, and Caspase-3 Activation.

In acute lymphoblastic leukemia (ALL), resistance to chemotherapy is associated with inactivation of p53 and upregulation of survivin. Thus, targeting the p53 and survivin expression may provide an attractive strategy for ALL treatment. It has been shown that fish-oil-derived docosahexaenoic acid (DHA) activates several antitumorigenic mechanisms in tumor cells, but little is known regarding the role of DHA on modulating p53 and survivin expression in ALL cells. In this study, we investigated the alterations of the p53 and survivin expression and induction of apoptosis in DHA-treated Molt-4 cells that serve as a model for ALL cells. Molt-4 cells were treated with 50, 100, 150, and 200 µM DHA after which cell proliferation, survivin mRNA and protein levels, p53 protein level, caspase-3 activation, and apoptotic rates were evaluated by different cellular and molecular techniques. After 48- and 72-h treatments with DHA at concentrations ranging from 50 to 200 µM, cell proliferation rates were measured to be 80.5-44.4%, and 73.4-14.4%, respectively, compared to untreated cells. We also found that treatment for 48 h with 200 µM DHA resulted in 10.8- and 3.6-fold increase in p53 protein level and caspase-3 activation followed by 4.7-and 1.6-fold decrease in survivin mRNA and protein levels, respectively, compared to untreated cells. Treatment of cells with different concentrations of DHA dramatically increased the p53/survivin and caspase-3/survivin ratios by 2.8- to 16.9-fold and 3.3 to 5.6-fold increases, respectively, compared to untreated cells. A decrease in the number of cells ranging from 16% to 70% and an increase in the number of apoptotic cells ranging from 9.3% to 93% was also observed with increasing DHA concentrations. In conclusion, p53 and survivin may provide promising targets of DHA in ALL cells and this compound with high proapoptotic capacity represents the possibility of its therapeutic application for ALL treatment.

Treatment of undifferentiated colorectal cancer cells with fish-oil derived docosahexaenoic acid triggers caspase-3 activation and apoptosis.

BACKGROUND: The effectiveness of chemotherapy is often limited by the side effects on normal tissues. Consequently, the search for new therapeutic agents with minimal toxicity is of particular interest in cancer management. Many studies have shown that docosahexaenoic acid (DHA) have cytotoxic effects against different kinds of cancer cells. However, little attention has been paid to explore the effect of DHA on undifferentiated colorectal cancer cells. In this study, the effects of DHA on LS174T cells as an early stage of tumor initiation were investigated. MATERIALS AND METHODS: Tumor cells were treated to various concentrations of DHA and proliferation, survivin expression, caspase-3 activation, and apoptosis were evaluated by different cellular and molecular techniques. RESULTS: Following 48 h treatment, proliferation was measured to be 73 ± 4.5% (P = 0.000), 53 ± 5.7% (P = 0.000) and 26.3 ± 3.5% (P = 0.000) for 50, 100, and 150 µM DHA, respectively compared to untreated cells. This molecule induced 63% (P = 0.001) and 46% (P = 0.000) decrease in survivin messenger ribonucleic acid (mRNA) level as well as 1.8 (P = 0.001) and three-fold (P = 0.000) increase in caspase-3 activation for 50 and 100 µM DHA, respectively compared to untreated cells. Our evidence showed that survivin mRNA is expressed at the early stage of colorectal cancer cells and DHA-treated cells expressed markedly a lower survivin mRNA compared to untreated cells. CONCLUSIONS: DHA is an attractive repressor of survivin expression, increases caspase-3 and apoptosis in colorectal cancer cells and may provide a novel approach to the treatment of colorectal cancer at the early stage of tumor initiation.

Bioengineering of differentiated hepatocytes with human factor IX-expressing plasmids in vitro.

For somatic gene therapy of hemophilia B, hepatocytes as the main cellular host for expression of hFIX are attractive targets. In gene therapy protocols, an efficient expression vector equipped with cis-regulatory elements such as introns is required. With this in mind, hFIX-expressing plasmids equipped with different combinations of 2 human ß-globin (hBG) introns inside the hFIX-cDNA and Kozak element were used for bioengineering of HepG2 cells as a model for differentiated hepatocytes and CHO cells a cell line generally used to produce recombinant hFIX (rhFIX). In HepG2 cells, the highest hFIX secretion level occurred for the intron-less plasmid with 8.5 to 53.8- fold increases, while in CHO cells, the hBG intron-I containing plasmid induced highest hFIX secretion level with 2.3 to 14.3-fold increases as compared to other plasmids. The first hBG intron appears to be more effective than the second one in both cell lines. The expression level was further increased upon the inclusion of the Kozak element. The highest hFIX activity was obtained from the cells that carrying the intron-less plasmids with 470 mU/ml and 25 mU/ml for HepG2 and CHO cells respectively. Secretion of active hFIX by all constructs was documented except for hBG intron-II containing construct in both cell lines. HepG2 cells were able to secret higher hFIX levels by 0.6 to 112.2-fold increases with activity by 5.3 to 16.4-fold increases compared to CHO cells transfected with the same constructs. Presence of both hBG intron-I and II inside the hFIX-cDNA provides properly spliced hFIX transcripts in both cell lines. In conclusion, the advantages of hBG introns as attractive cis-regulatory elements to obtain higher expression level of hFIX particularly in CHO cells were demonstrated. Hepatocytes could be effectively bioengineered with the use of plasmid vectors and this strategy may provide a potential in-vitro source of functional hepatocytes for ex-vivo gene therapy of hemophilias and production of rhFIX in vitro.

Effective Targeting Survivin, Caspase-3 and MicroRNA-16-1 Expression by Methyl-3-pentyl-6-methoxyprodigiosene Triggers Apoptosis in Colorectal Cancer Stem-Like Cells.

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.

Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids.

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 ß-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.

Treatment of LS174T colorectal cancer stem-like cells with n-3 PUFAs induces growth suppression through inhibition of survivin expression and induction of caspase-3 activation.

PURPOSE: Colorectal cancer stem cells (CCSCs) are thought to contribute to tumor initiation, progression, metastasis, chemo-resistance and therapy failure. Therefore, assessment of the effectiveness of agents with anti-proliferative activities against CCSCs is warranted. Several studies have shown that different tumorigenic steps, ranging from initiation to metastasis, can be affected by n-3 polyunsaturated fatty acids (PUFAs). Here, we evaluated the effects of the PUFA components docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), alone or in combination, on LS174T cells that serve as a model for colorectal cancer initiating cells with stem cell-like properties. METHODS: LS174T cells were treated with 50, 100 and 150 µM DHA and EPA, or equal mixtures of DHA/EPA (i.e., 25/25, 50/50 and 75/75 µM), after which cell number, viability, growth inhibition, survivin expression, caspase-3 activation and apoptotic rate were evaluated. RESULTS: We found that treatment of LS174T cells with increasing PUFA concentrations significantly increased growth inhibition in a dose- and time-dependent manner. After a 72 h treatment with 150 µM DHA and EPA, or their combination (75/75 µM), growth rates were inhibited by 80.3 ± 5.5%, 79.3 ± 5% and 71.1 ± 1%, respectively, compared to untreated cells. We also found that treatment for 48 h with 100 µM DHA and EPA, or their combination (50/50 µM), resulted in 2.9-, 3- and 2.6-fold increases in caspase-3 activation, as well as 54, 62.4 and 100% decreases in survivin mRNA expression levels, respectively, compared to untreated cells. Low survivin mRNA levels combined with high caspase-3 activity levels were found to correlate with a higher growth inhibition in PUFA-treated cells. DHA appears to be a more potent growth inhibitor than EPA and the DHA/EPA combination. An increase in the number of apoptotic cells (early + late), ranging from 12.9 to 44.7%, was observed with increasing DHA doses. CONCLUSION: From our data we conclude that PUFAs induce growth inhibition via targeting survivin expression in LS174T cells, which serve as a model for CCSCs.

Prodigiosin isolated from cell wall of Serratia marcescens alters expression of apoptosis-related genes and increases apoptosis in colorectal cancer cells.

Colorectal cancer remains often refractory to classic therapies. In consequence, the search for new anti-tumor agents with minimal toxicity is of particular interest in colon cancer treatment. Prodigiosin as a secondary metabolite of Serratia marcescens induces apoptosis in various kinds of cancer cells with low toxicity on normal cells. In the present study, we evaluated the effect of prodigiosin on proliferation and expression of apoptotic-related genes in HT-29 cells. Malignant cells were treated to various concentrations of prodigiosin and proliferation rate, survivin, Bcl-2, Bax and Bad mRNA levels, caspase 3 activation and apoptosis were evaluated by different cellular and molecular techniques. Treatment of cells with increasing concentration of prodigiosin decreased significantly cell proliferation in a dose- and time-dependent manner. Following 48-h treatment, growth rate was measured to be 77 ± 6.8, 41.3 ± 3.1 and 46 ± 6.3 % for 100, 400 and 600 nM prodigiosin, respectively, compared to untreated cells. This molecule induced 61.7, 90 and 89 % decrease in survivin mRNA level as well as 1.9-, 2.8- and 2.2-fold increase in caspase 3 activation for indicated concentrations of prodigiosin, respectively. The level of Bcl-2 mRNA was inversely proportional to Bax and Bad mRNA levels. Low mRNA levels of Bcl-2 combined with high levels of Bax and Bad mRNAs were correlated to higher apoptosis rate in treated cells. Our data suggest that prodigiosin-induced apoptosis may ascribe to Bcl-2 and survivin inhibition in HT-29 cells and these genes may provide promising molecular targets of prodigiosin. Collectively, prodigiosin may have a great potential for colorectal cancer-directed therapy.

Enhancement of the human factor IX expression, mediated by an intron derived fragment from the rat aldolase B gene in cultured hepatoma cells.

Combinations of a liver-specific rat aldolase B intronic enhancer (rABE) with either of the hepatocyte-specific human α1-antitrypsin promoter (hAATp) and cytomegalovirus enhancer/promoter (CMVp) were used to construct a number of plasmids expressing non-viral human factor IX (hFIX). The efficacies of the plasmids were evaluated in a hepatocyte cell line (HepG2). Potential of the rABE was evidenced, by 300%--and 800% increase of the hFIX expression levels when it was combined with the CMVp and hAATp, respectively. The highest hFIX expression level was obtained when the rABE was combined with the CMVp for which the maximum intracellular accumulation of hFIX was also evidenced. Therefore, the rABE is suggested as a suitable cis-acting element for protein expression in hepatocytes. Considering the potential of introns during post-transcriptional processes, the function of the human ß-globin (hBG) intron-II, within the hFIX coding region, in the second generations of the hFIX expressing plasmids was also examined, which leaded to reduction of the hFIX expression level, probably due to improper splicing of the hBG intron-II.

A systematic study of the function of the human beta-globin introns on the expression of the human coagulation factor IX in cultured Chinese hamster ovary cells.

BACKGROUND: Intronic sequences have the potential to improve gene expression in eukaryotes by a variety of mechanisms. In this context, human beta-globin (hBG) introns were inserted into the human factor IX (hFIX) cDNA in cytomegalovirus (CMV)-regulated plasmids. The resulting construct was then used for further expression analysis in vitro. METHODS: Seven hFIX-expressing plasmids with different combinations of the two hBG introns and the Kozak element were constructed and used for a systematic expression analysis in cultured Chinese hamster ovary (CHO) cells. In parallel, the hBG intronic sequences were analysed for the presence of possible regulatory elements. RESULTS: All the constructed plasmids resulted in transient expression of the hFIX. However, the coagulation activities varied according to the particular constructs used. Based on the hFIX antigenic assay, a wide range of variation was observed during persistent expression. The second hBG intron appears to be more effective than the first one. The expression level was further increased upon the inclusion of the Kozak element. Sequence analysis has detected several transcription factor binding (TFB) motifs in both of the introns, but with a higher frequency in the second one. CONCLUSIONS: Potentials of hBG introns as enhancer-like elements for the expression of the hFIX in cultured CHO cells and a higher activity with respect to the second hBG intron compared to the first one were demonstrated. The larger number of TFBs in the second hBG intron reflects its stronger effect. The results obtained suggest possible synergistic functions of the hBG introns and Kozak on the expression level of hFIX in vitro.

Longitudinal determination of hepatitis B surface antigen-specific B lymphocyte frequency in healthy high responder adults after booster vaccination.

OBJECTIVES: The influence of booster vaccination on hepatitis B surface antigen (HBsAg)-specific B lymphocytes in humans has not been well characterized. Considering the low frequency of circulating B cells specific for HBsAg in vaccine high responder subjects, determination of this frequency at different time intervals after booster dose injection may provide invaluable information for evaluation of immune response to rHBsAg and identification of the most appropriate timing for isolation of specific B cells and generation of human monoclonal antibodies. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from 7 healthy high responder adults at 1, 2, 4, 8 and 16 weeks following administration of booster vaccination with an rHBsAg. The cells were transformed with Epstein-Barr virus and cultured at different cell densities over a feeder of human fetal foreskin fibroblasts. Following transformation, total hIg and HBsAg-specific antibody were screened in culture supernatant using ELISA, and primary frequency of specific B cells was calculated by limiting dilution assay based on Poisson analysis. Actual frequency was determined taking into consideration the percent of B cells in each PBMC population and efficiency of EBV transformation. RESULTS: The mean frequencies of specific B cells after booster vaccination were found to be 1/13,462, 1/3,318, 1/5,224, 1/8,861 and 1/10,714 for the specified time intervals, respectively. Significant differences were observed between the frequencies of samples collected at all time intervals with the exception of week 1 versus weeks 8 and 16, week 2 versus week 4, and week 8 versus week 16. CONCLUSIONS: Our results may provide an indirect measure for immunological memory and may help optimize immunization strategies for novel vaccines and generate human monoclonal antibodies.