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.

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.

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.

BackgroundThe 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.MethodsWt-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.ResultsAfter 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.ConclusionWt-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.

The determination of the frequency of antigen‐specific lymphocytes may provide invaluable information for the evaluation of the immune response to different antigens and pathogens. Different methods are employed to determine the frequency of specific B lymphocytes in peripheral blood. In this study, the frequency of hepatitis B surface antigen (HBsAg)‐specific B lymphocytes was determined by a limiting dilution assay (LDA) and an enzyme‐linked immunospot assay (ELISPOT) in seven healthy adult high responders to recombinant HBsAg. Peripheral blood mononuclear cells isolated at different time intervals (1, 2, 4, 8 and 16 weeks) following administration of a booster dose were either transformed with Epstein–Barr virus (LDA) or stimulated with Pokeweed mitogen (ELISPOT). In an LDA, anti‐HBs positive wells were screened by a sandwich ELISA and the frequency of specific B lymphocytes was estimated based on the Poisson statistical analysis. In an ELISPOT, coloured spots representing specific B lymphocytes were finally enumerated by stereomicroscope. Our results showed a significant increase in the number of specific B lymphocytes in the first week by an ELISPOT compared with an LDA (1:190 versus 1:13,462) (P < 0.001). No significant differences were observed at other time intervals. A significant correlation was observed between the serum titer of anti‐HBs antibody and frequency of HBsAg‐specific B cells obtained by LDA and ELISPOT methods at different time intervals. The highest correlation was found at fourth week in LDA (r = 0.83, P < 0.01) and ELISPOT (r = 0.85, P < 0.01) assays. Furthermore, a significant correlation was observed between an LDA and ELISPOT at different time intervals (highest correlation in second week, r = 0.88, P < 0.008). These findings suggest that in addition to technical advantages, such as speed and simplicity, an ELISPOT is a more sensitive assay, compared with an LDA.