Prodigiosin produced by Serratia marcescens inhibits expression of MMP-9 and survivin and promotes caspase-3 activation with induction of apoptosis in acute lymphoblastic leukaemia cells.

AIMS: Matrix metalloproteinase-9 (MMP-9) and survivin are involved in several steps of carcinogenesis in acute lymphoblastic leukaemia (ALL). Yet, no MMP-9 and survivin-modulating drugs with low toxicity on normal cells but high efficacy against high MMP-9- and survivin-expressing leukaemia cells have been approved for clinical application in ALL. Prodigiosin, a secondary metabolite of Serratia marcescens, induces apoptosis in different kinds of cancer cells with low toxicity on normal cells. However, little is known about the effects of this compound on the high MMP-9- and survivin-expressing leukaemia cells. METHODS AND RESULTS: CCRF-CEM cells as a model for high MMP-9- and survivin-expressing ALL cells were treated with 100, 200 and 400 nmol l-1 prodigiosin after which cell number, proliferation rate, MMP-9 and survivin expression, caspase-3 activation and apoptosis were evaluated. After 24-, 48-, and 72-h treatments with 100, 200 and 400 nmol l-1 prodigiosin, proliferation rates were measured to be 92·3-76·7%, 82-63% and 63·7-46·6% respectively. Treatment with prodigiosin for 48 h decreased MMP-9 mRNA levels followed by decreases in secreted (S) and intracellular (I) MMP-9 protein levels by 20-22% and 69-72% for 100-400 nmol l-1 prodigiosin respectively. Prodigiosin decreased survivin protein levels from 40 to 26% followed by 3·7-5·6-fold increases in caspase-3 activation for the aforementioned prodigiosin concentration ranges. Treatment with 100-400 nmol l-1 prodigiosin increased the caspase-3/survivin, caspase-3/I-MMP-9 and caspase-3/S-MMP-9 ratios by 6-7·3-, 11·5-19·1- and 4·9-6·8-fold increases respectively. A dramatic increase in the number of apoptotic cells was also observed with increasing prodigiosin concentrations. CONCLUSION: The inhibitory effects of prodigiosin on MMP-9 and survivin expression, as well as its pro-apoptotic capacity, represent a novel therapeutic avenue against ALL cells. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings provide an important and interesting basis to develop a new therapeutic compound with high potential against ALL cells.

Regulation of p53 and survivin by prodigiosin compound derived from Serratia marcescens contribute to caspase-3-dependent apoptosis in acute lymphoblastic leukemia cells.

Tumor suppressor p53 and proto-oncogene survivin are challenging targets for anticancer drugs in acute lymphoblastic leukemia (ALL) which are associated with chemoresistance. Yet, no p53 and survivin-modulating drug with low toxicity and high efficacy has been approved for clinical application in ALL. Consequently, the search for novel compounds which target p53 or survivin is needed to further advance ALL treatment. Prodigiosin, a secondary metabolite of Serratia marcescens induces apoptosis in cancer cells with no toxicity on normal cells. However, the possible potential of prodigiosin as p53- and survivin-modulating agent in ALL cells has not been investigated. Wt-p53 Molt-4 cells were treated with 100 to 600 nM prodigiosin, after which, viability, cell proliferation rates, survivin and p53 protein levels, caspase-3 activation, and apoptosis were evaluated. After 24-, 48-, and 72-h treatments with 100 to 600 nM prodigiosin, cell proliferation rates were measured to be 93.7-77.3%, 75.5-58.3%, and 55-23.3%, respectively. Treatment for 48 hours with 100 to 600 nM prodigiosin resulted in 41-19% decrease in survivin protein levels followed by 450-950% increases in caspase-3 activation levels. Prodigiosin induced remarkably p53 accumulation and increased p53/survivin and caspase-3/survivin ratios by 6.1 to 11.3 and 10.3 to 47.5-fold at 100 to 600 nM, respectively. Survivin protein levels were inversely proportional to p53 accumulation levels. Low survivin protein levels combined with high levels of p53 accumulation were correlated to higher apoptotic rates. P53 and survivin as molecular targets of prodigiosin contribute to caspase-3-dependent apoptosis in ALL cells and this compound represents an attractive p53- and survivin-modulating agent in ALL.

Fish-oil-derived eicosapentaenoic acid decreases survivin expression and induces wt-p53 accumulation with caspase-3 activation in acute lymphoblastic leukemia cells.

BACKGROUND: Defects in modulating wild-type (wt) p53 and survivin are associated with a resistant disease in acute lymphoblastic leukemia (ALL). Yet, no wt-p53 and survivin modulating drugs have been approved for clinical application in ALL. Here, we investigated if in vitro eicosapentaenoic acid (EPA) concentrations equal to human plasma levels are able to target wt-p53 and survivin. METHODS: Wt-p53 Molt-4 cells (ALL cell line) were treated with 50, 100, 150, and 200 µM of EPA after which cell number, viability, proliferation rate, survivin expression, wt-p53 accumulation, caspase-3 activation, and apoptosis were evaluated. RESULTS: After 48- and 72-h treatments with EPA at concentrations ranging from 50 to 200 µM, cell proliferation rates were measured to be 71.5-32.6% and 68.2-13.7% and metabolic activities were measured to be 77-44% and 71-26%, respectively. Treatment with 50-200 µM of EPA for 48 h resulted in 14.1-74.6% and 69.5-45.5% decreases in survivin mRNA and protein levels, respectively. EPA induced 1.3-6 and 1.9-20-fold increases in caspase-3 activation and wt-p53 accumulation, respectively. Increase in wt-p53/survivin and caspase-3/survivin ratios from 1 in untreated cells to 20.3 and 5.8 was measured for 150 µM of EPA. Low necrotic rates ranging from 0.3% to 2.8% and an increase in the number of total apoptotic cells (early + late) ranging from 9.8% to 81% were also observed with increasing EPA concentrations. CONCLUSION: 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.

Targeting survivin with prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in hepatocellular carcinoma cells.

BACKGROUND: Abnormal activation of the Wnt/ß-catenin signaling pathway increases survivin expression that is involved in hepatocarcinogenesis. Therefore, downregulation of survivin may provide an attractive strategy for treatment of hepatocellular carcinoma. In this regard, little is known about the anticancer effects of prodigiosin isolated from cell wall of Serratia marcescens on the survivin expression and induction of apoptosis in hepatocellular carcinoma cells. METHODS: Human hepatocellular carcinoma (HepG2) cells were treated with 100-, 200-, 400-, and 600-nM prodigiosin after which morphology of cells, cell number, growth inhibition, survivin expression, caspase-3 activation, and apoptotic rate were evaluated by inverted microscope, hemocytometer, MTT assay, RT-PCR, fluorometric immunosorbent enzyme assay, and flow cytometric analysis, respectively. RESULTS: Prodigiosin changed morphology of cells to apoptotic forms and disrupted cell connections. This compound significantly increased growth inhibition rate and decreased metabolic activity of HepG2 cells in a dose- and time-dependent manner. After 24-, 48-, and 72-h treatments with prodigiosin at concentrations ranging from 100 nM to 600 nM, growth inhibition rates were measured to be 1.5-10%, 24-47.5%, and 55.5-72.5%, respectively, compared to untreated cells. At the same conditions, metabolic activities were measured to be 91-83%, 74-53%, and 47-31% for indicated concentrations of prodigiosin, respectively, compared to untreated cells. We also found that treatment of HepG2 cells for 48 h decreased significantly cell number and survivin expression and increased caspase-3 activation in a dose-dependent manner. Specifically, treatment with 600-nM prodigiosin resulted in 77% decrease in cell number, 88.5% decrease in survivin messenger RNA level, and 330% increase in caspase-3 activation level compared to untreated cells. An increase in the number of apoptotic cells (late apoptosis) ranging from 36.9% to 97.4% was observed with increasing prodigiosin concentrations. CONCLUSION: From our data, prodigiosin is an attractive compound that turns the profile of high-level survivin expression in hepatocellular carcinoma cells into that of normal cells and may provide a novel approach to the hepatocellular carcinoma-targeted therapy.

Frequency analysis of HBsAg-specific B lymphocytes in high-responder individuals to recombinant hepatitis B vaccine: comparison of LDA and ELISPOT assays.

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.