In vitro and in silico study on the effect of carvedilol and sorafenib alone and in combination on the growth and inflammatory response of melanoma cells.

Melanoma is an aggressive skin cancer. Increasing evidence has shown the role of ß-adrenergic receptors in the pathogenesis of melanoma. Carvedilol is a widely used non-selective ß-AR antagonist with potential anticancer activity. The purpose of the study was to estimate the influence of carvedilol and sorafenib alone and in combination on the growth and inflammatory response of C32 and A2058 melanoma cells. Furthermore, this study also aimed to predict the probable interaction of carvedilol and sorafenib when administered together. Predictive study of the interaction of carvedilol and sorafenib was performed using the ChemDIS-Mixture system. Carvedilol and sorafenib alone and in combination showed a growth inhibitory effect on cells. The greatest synergistic antiproliferative effect on both cell lines was observed at Car 5 µM combined with Sor 5 µM. Analysis in silico identified diseases, proteins, and metabolic pathways that can be affected by the interaction of carvedilol and sorafenib. The results obtained demonstrated that carvedilol and sorafenib modulated the secretion of IL-8 by IL-1ß-stimulated by melanoma cell lines but the use of a combination of both drugs did not intensify the effect. In summary, the results presented indicate that the combination of carvedilol and sorafenib may have a promising anticancer effect on melanoma cells.

Pterostilbene-Mediated Inhibition of Cell Proliferation and Cell Death Induction in Amelanotic and Melanotic Melanoma.

Melanoma is one of the fastest-growing cancers worldwide. Treatment of advanced melanoma is very difficult; therefore, there is growing interest in the identification of new therapeutic agents. Pterostilbene is a natural stilbene that has been found to have several pharmacological activities. The aim of this study was to evaluate the influence of pterostilbene on the proliferation and apoptosis of human melanoma cells. Proliferation of pterostilbene-treated amelanotic (C32) and melanotic (A2058) melanoma cells was determined by BRDU assay. Flow cytometric analyses were used to determine cell cycle progression, and further molecular investigations were performed using real-time RT-qPCR. The expression of the p21 protein and the DNA fragmentation assay were determined by the ELISA method. The results revealed that pterostilbene reduced the proliferation of both amelanotic and melanotic melanoma cells. Pterostilbene induced apoptosis in amelanotic C32 melanoma cells, and this effect was mediated by an increase in the expression of the BAX, CASP9, and CASP9 genes; induction of caspase 3 activity; and DNA degradation. Pterostilbene did not affect the activation of apoptosis in the A2058 cell line. It may be concluded that pterostilbene has anticancer potential against human melanoma cells; however, more studies are still needed to fully elucidate the effects of pterostilbene on amelanotic and melanotic melanoma cells.

Mechanism of Pterostilbene-Induced Cell Death in HT-29 Colon Cancer Cells.

Pterostilbene is a dietary phytochemical that has been found to possess several biological activities, such as antioxidant and anti-inflammatory. Recent studies have shown that it exhibits the hallmark characteristics of an anticancer agent. The aim of the study was to investigate the anticancer activity of pterostilbene against HT-29 human colon cancer cells, focusing on its influence on cell growth, differentiation, and the ability of this stilbene to induce cell death. To clarify the mechanism of pterostilbene activity against colon cancer cells, changes in the expression of several genes and proteins that are directly related to cell proliferation, signal transduction pathways, apoptosis, and autophagy were also evaluated. Cell growth and proliferation of cells exposed to pterostilbene (5-100 µM) were determined by SRB and BRDU assays. Flow cytometric analyses were used for cell cycle progression. Further molecular investigations were performed using quantitative real-time RT-PCR. The expression of the signaling proteins studied was determined by the ELISA method. The results revealed that pterostilbene inhibited proliferation and induced the death of HT-29 colon cancer cells. Pterostilbene, depending on concentration, caused inhibition of proliferation, G1 cell arrest, and/or triggered apoptosis in HT-29 cells. These effects were mediated by the down-regulation of the STAT3 and AKT kinase pathways. It may be concluded that pterostilbene could be considered as a potential therapeutic option in the treatment of colon cancer in the future.

Regulation of MicroRNA-155 and Its Related Genes Expression by Inositol Hexaphosphate in Colon Cancer Cells.

Inositol hexaphosphate (IP6), a natural dietary component, has been found as an antitumor agent by stimulating apoptosis and inhibiting cancer cell proliferation, their migration, and metastasis in diverse cancers including colon cancer. However, molecular mechanisms of its action have not been well understood. In recent years, microRNAs (miRNAs) have been reported to play important roles in a broad range of biologic processes, such as cell growth, proliferation, apoptosis, or autophagy. These small noncoding molecules regulate post-transcriptional expression of targets genes via degradation of transcript or inhibition of protein synthesis. Aberrant expression and/or dysregulation of miRNAs have been characterized during tumor development and progression, thus, they are potential molecular targets for cancer prevention. The aim of this study was to investigate the effect of IP6 on the miRNAs expression profile in Caco-2 colon cancer cells. 84 miRNAs were analyzed in Caco-2 cells treated with 2.5 mM and 5 mM IP6 by the use of PCR (Polymerase Chain Reaction) array. The effect of 5 mM IP6 on selected potential miR-155 targets was determined by real-time (RT)-qPCR and ELISA (quantitative Polymerase Chain Reaction and Enzyme-Linked Immunosorbent Assay )method. The results indicated alteration in the specific 10 miRNA expression in human colon cancer cells following their treatment with 5 mM IP6. It down-regulated 8 miRNAs (miR-155, miR-210, miR-144, miR-194, miR-26b, miR-126, miR-302c, and miR-29a) and up-regulated 2 miRNAs (miR-223 and miR-196b). In silico analysis revealed that FOXO3a, HIF-1α, and ELK3 mRNAs are those of predicted targets of miR-155. IP6 at the concentration of 5 mM markedly induced FOXO3a and HIF-1a genes' expression at both mRNA and protein level and decreased the amount of ELK3 mRNA as well as protein concentration in comparison to the control. In conclusion, the present study indicates that one of the mechanisms of antitumor potential of IP6 is down-regulation of the miR-155 expression in human colon cancer cells. Moreover, the expression of genes that are targeted by miRNA are also modulated by IP6.

Inositol Hexaphosphate Inhibits Proliferation and Induces Apoptosis of Colon Cancer Cells by Suppressing the AKT/mTOR Signaling Pathway.

Abstract: 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.

Modulating effect of inositol hexaphosphate on arachidonic acid-dependent pathways in colon cancer cells.

Cyclooxygenase (COX) and lipoxygenase (LOX) are key enzymes of arachidonic acid metabolism. Their products, prostaglandins and leukotrienes, are involved in the pathogenesis of inflammatory bowel diseases and colorectal cancer. The aim of the study was to examine the influence of inositol hexaphosphate (IP6), a naturally occurring phytochemical, on the expression of genes encoding COX and LOX isoforms and synthesis of their products (PGE2 and LTB4) in colon cancer cell line Caco-2 stimulated with pro-inflammatory agents (IL-1ß/TNFα). Real-time RT-qPCR was used to validate mRNAs level of examined genes. The concentrations of COX-2 and 5-LOX proteins as well as PGE2 and LTB4 were determined by the ELISA method. Based on these studies it may be concluded that IP6 may limit inflammatory events in the colonic epithelium and prevent colon carcinomas by modulating the expression of genes encoding COX and LOX isoforms at both mRNA and protein levels as well as by affecting the synthesis and secretion of prostaglandins and leukotrienes.

ANTIPROLIFERATIVE EFFECT OF INOSITOL HEXAPHOSPHATE ON HUMAN SKIN MELANOMA CELLS IN VITRO.

Human malignant melanoma is a highly metastatic tumor with poor prognosis. The majority of metastatic melanomas are resistant to diverse chemotherapeutic agents. Consequently, the search for novel antimelanoma agents continues. In recent years, the interest in plants and their biologically active constituents as a source of novel potential drugs significantly increased. Inositol hexaphosphate (IP6) is a naturally occurring compound that has been shown to inhibit the growth of a wide variety of tumor cells in multiple experimental model systems. The aim of this study was to evaluate the antiproliferative and cytotoxic influence of IP6 on melanotic melanoma cells in vitro. The A2058 cells used as a model of human skin melanoma malignum were exposed to different concentrations of IP6 (0.1-5 mM) for a various period of time and their growth was determined by sulforhodamine B assay after 24, 48 and 72 h. The cytotoxicity of IP6 was measured at 24 and 72 h by XTT assay. IP6 has been found to cause dose-dependent growth suppression of A2058 melanoma cells. At low concentrations (0.1 and 0.5 mM) it did not exert any influence on the cell proliferation as compared to control cultures. Higher concentrations of IP6 (from 1 to 5 mM) had a statistically significant, suppressive effect on cell proliferation after 24 h incubation. When the experimental time period was increased up to 72 h, statistically significant inhibition of cell proliferation was monitored at all IP6 concentrations used. Data obtained from XTT assay indicated that IP6 had dose- and time-dependent cytotoxic effect on melanoma cells. The results demonstrate the antiproliferative and cytotoxic properties of IP6 in a wide range of concentrations on human A2058 melanoma cells. Hence, it can be suggested that IP6 could have a promising therapeutic significance in treating cancer.

THE EFFECT OF SULFASALAZINE AND 5-AMINOSALICYLIC ACID ON THE SECRETION OF INTERLEUKIN 8 BY HUMAN COLON MYOFIBROBLASTS.

Sulfasalazine (SAS) and its therapeutically active derivative--5-aminosalicylic acid (5-ASA) are used in the treatment of inflammatory bowel disease. 5-ASA mechanism of action on the one hand, involves the inhibition of the cyclooxygenase and lipoxygenase activity, and thus decrease of synthesis of prostaglandins, leukotrienes and free radicals, on the other hand, the suppression of the immune response in the intestinal mucosa. Myofibroblasts, which are located just below the basement membrane, are important element of the mucosa. Due to its secretory activity they may interact with other cells, including epithelial cells. Examining SAS and 5-ASA cytotoxic properties on human normal, colon subepithelial myofibroblasts (CSEMF) it was found that the first of these compounds in a concentration of 1 mM significantly reduced the number of these cells as compared to the control, while the latter exhibited an action at the 5-fold higher concentration (5 mM). Moreover, SAS concentration greater than 0.25 mM reduced IL-8 secretion by CSEMF, and 5-ASA had no effect in the tested range of concentrations, i.e., up to 7.5 mM.

DOWN-REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE EXPRESSION BY INOSITOL HEXAPHOSPHATE IN HUMAN COLON CANCER CELLS.

Inflammatory bowel disease (IBD) is chronic inflammatory condition associated with increased risk of developing colorectal cancer. A number of mediators of inflammation, such as pro-inflammatory cytokines, prostaglandins and nitric oxide have been involved in carcinogenesis, especially in the promotion and progression stages. NO is synthesized from L-arginine by constitutively expressed endothelial and neuronal nitric oxide synthases (eNOS and nNOS, respectively) and an inducible NOS (iNOS) isoform expressed under inflammatory conditions. A selective inhibitors of iNOS could be, therefore, considered to be good candidates as chemopreventive agents against colon cancer. In this study, the effect of inositol hexaphosphate (IP6), dietary phytochemical, on the mRNA expression of iNOS stimulated with bacterial lipopolysaccharides (Escherichia coli and Salmonella typhimurium) and IL-1ß in intestinal cells Caco-2 for 6 and 12 h was investigated. A transcription level of iNOS with the use real time QRT-PCR technique was determined in cells treated with 1 and 2.5 mM IP6. Stimulation of Caco-2 with pro-inflammatory factors (LPS and IL-1ß) resulted in an up-expression of iNOS mRNA at 6 and 12 h. Cells exposed to IP6 only revealed significant reduction in iNOS gene transcription after 12 h. A decrease in iNOS transcription by IP6 following the gene induction by proinflammatory agents in 6 and 12 h lasting cultures was also determined. The findings of this study suggest that one of the anti-cancer and anti-inflammatory abilities of IP6 can be realized by suppressing the expression of gene encoding inducible nitric oxide synthase isoform at the transcriptional level.

Influence of inositol hexaphosphate on the expression of selected proliferation markers in IL-1ß-stimulated intestinal epithelial cells.

The aim of the present study was to examine the influence of IP6, a naturally occurring phytochem- ical, on the expression of genes coding for proliferation markers, i.e., cyclin D1 (CCND1) and histone H3 in IL-1ß-stimulated intestinal cancer cell line Caco-2. Quantification of genes expression was carried out using real time RT-QPCR technique in Caco-2 cells after treatment with IL-1ß, 1 and 2.5 mM of IP6 for 3, 6 and 12 h. In separate cultures, cells were incubated with IL-1ß for the indicated times. The untreated Caco-2 cells were used as the control. In a time course experiment, stimulation of cells with IL-1ß only resulted in an overex- pression of both CCND1 and histone H3 mRNAs as compared with control. IP6 had no influence on IL-1ß-stimulated CCND1 expression for 3 and 6 h. After 12 h, statistically significant decrease in CCND1 mRNA was observed in cells exposed to IL-1ß and IP6 (1 and 2.5 mM) in relation to cells treated with IL-1ß only. The levels of H3 mRNA in IL-1ß-stimulated cells and cells treated with IL-1ß and IP6 revealed no statistically significant differences after 3 h. IP6 at 1 and 2.5 mM enhanced IL1ß-stimulated transcription of H3 gene after 6 h. Subsequently (12 h), the combination of IP6 and IL-1ß decreased H3 mRNA level compared to IL1ß-treated cells. In conclusion, pro-inflammatory cytokine IL-1ß up-regulates CCND1 and histone H3 mRNAs expression in Caco-2 cells. These results suggest that the ability of IP6 to inhibit colon cancer cells proliferation may be mediated through downregulation of genes encoding cyclin D1 and histone H3 at the mRNA level.

In vitro evaluation of antiproliferative and cytotoxic properties of pterostilbene against human colon cancer cells.

Colon cancer has been remaining the second leading cause of cancer mortality in Poland in the last years. Epidemiological, preclinical and clinical studies reveal that dietary phytochemicals may exert chemopreventive and therapeutic effect against colorectal cancer. There is a growing interest in identifying new biologically active agents from dietary sources in this respect. Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) is a naturally occurring stilbene, that has been found to have antioxidative, anti-inflammatory and antipro- liferative properties. Compared to other stilbenes, pterostilbene has greater bioavailability, and so, a greater potential for clinical applications. Recent studies showed that pterostilbene exhibits the hallmark characteristics of an anticancer agent. The aim of this study was to analyze antiproliferative and cytotoxic effects of pterostilbene on human colon cancer Caco-2 cells. They were cultured using standard techniques and exposed to increasing doses of pterostilbene (5-100 µM) for 48 and 72 h. Cell proliferation was determined by sulforhodamine B assay. The growth of treated cells was expressed as a percentage of that of untreated control cells. Pterostilbene decreased proliferation rate of Caco-2 cells in a dose- and time-dependent manner. Its concentrations = 25 µM did not affect cell growth after 48 h treatment period. Significant growth inhibition was observed in cultures incubated with higher concentrations of pterostilbene (40-100 µM). Pterostilbene at all concentrations used (5-100 µM) caused significant inhibition of cell proliferation when the experimental time period was elongated to 72 h. The maximum growth reduction was observed at 100 mM pterostilbene. The cytotoxicity of pterostilbene was evaluated in 48 h cultures based on lactate dehydrogenase (LDH) leakage into the culture medium and showed dose-related pattern. The findings of this study showed significant dose-dependent antiproliferative and cytotoxic effects of pterostilbene against human colon cancer cells in vitro.

Induction of the expression of genes encoding TGF-beta isoforms and their receptors by inositol hexaphosphate in human colon cancer cells.

Transforming growth factors-beta (TGF-beta) are multifunctional cytokines involved in the regulation of cell development, differentiation, survival and apoptosis. They are also potent anticancer agents that inhibit uncontrolled proliferation of cells. Incorrect TGF-beta regulation has been implicated in the pathogenesis of many diseases including inflammation and cancer. In humans, the TGF-beta family consists of three members (TGF-beta1, 2, 3) that show high similarity and homology. TGF-betas exert biological activities on various cell types including neoplastic cells via their specific receptors. Inositol hexaphosphate (phytic acid, IP6), a phytochemical has been reported to possess various health benefits. The aim of this study was to examine the effect of IP6 on the expression of genes encoding TGF-beta1, TGF-beta2, TGF-beta3 isoforms and their receptors TbetaRI, TbetaRII, TbetaRIII in human colorectal cancer cell line Caco-2. The cells were treated with 0.5, 1 and 2.5 mM IP6 for 3, 6 and 12 h. The untreated Caco-2 cells were used as the control. Quantification of genes expression was performed by real time QRT-PCR technique with a SYBR Green I chemistry. The experimental data revealed that the TGF-beta1 mRNA was the predominant isoform in Caco-2 cells and that IP6 enhanced transcriptional activity of genes of all three TGF-beta isoforms and their receptors TbetaRI, TbetaRII TbetaRIII in these cells. At concentrations up to 1 mM, IP6 over-expressed the genes in 6 h lasting cultures, and its higher dose (2.5 mM) caused successively increasing transcript level of TGF-beta isoforms and receptors with the duration of experiment up to 12 h. The findings of this study indicate that one of anti-cancer abilities of IP6 can be realized by enhancing the gene expression of TGF-beta isoforms and their receptors at the transcriptional level.

Role of protein tyrosine kinase in the effect of IP6 on IL-8 secretion in intestinal epithelial cells.

Phytic acid (IP6) is a major fiber-associated component of a diet physiologically present in human intestines. Studies showed that this phytochemical can modulate immune functions of intestinal epithelium through regulation of proinflammatory cytokines secretion but mechanisms underlying these cellular response to IP6 have weakly been examined, as yet. The aim of this study was to determine the role of protein tyrosine kinase (PTK) in secretion of IL-8, a central proinflammatory cytokine, by unstimulated and IL-1beta-stimulated intestinal epithelial cells Caco-2 treated with IP6 (1 and 2.5 mM). To study the involvement of PTK signal pathway in IL-8 secretion, inhibitors of phosphotyrosine phosphatase (sodium orthovanadate, OV) and tyrosine kinase (genistein, GEN) were incubated with Caco-2 cells prior to IP6 treatment. IP6 had suppressive effect on basal and IL-1beta-stimulated IL-8 secretion by cells. The effect of OV on IL-8 release by cells treated with IP6 was different under constitutive and stimulated conditions. Secretion of IL-8 was significantly down-regulated in cells with GEN and GEN plus IP6 treatment. In addition, total PTK activity in both unstimulated and IL-1beta stimulated cells was determined in the presence of IP6. The results suggest that physiological intestinal concentrations of IP6 may have an inhibitory effect on IL-8 secretion by Caco-2 cells and one of the mechanisms of its action is the inhibition of PTK signaling cascade. The study revealed for the first time that PTKs could be one of the molecular targets for IP6 effects in the intestinal epithelial cells.

Differential influence of inositol hexaphosphate on the expression of genes encoding TGF-ß isoforms and their receptors in intestinal epithelial cells stimulated with proinflammatory agents.

Transforming growth factor ß (TGF-ß) is a multifunctional cytokine recognized as an important regulator of inflammatory responses. The effect of inositol hexaphosphate (IP6), a naturally occurring phytochemical, on the mRNA expression of TGF- ß1, TGF-ß2, TGF-ß3 and TßRI, TßRII, and TßRIII receptors stimulated with bacterial lipopolysaccharides (Escherichia coli and Salmonella typhimurium) and IL-1ß in intestinal cells Caco-2 for 3 and 12 h was investigated. Real-time qRT-PCR was used to validate mRNAs level of examined genes. Bacterial endotoxin promoted differential expression of TGF-ßs and their receptors in a time-dependent manner. IL-1ß upregulated mRNA levels of all TGF-ßs and receptors at both 3 h and 12 h. IP6 elicited the opposed to LPS effect by increasing downregulated transcription of the examined genes and suppressing the expression of TGF- ß1 at 12 h. IP6 counteracted the stimulatory effect of IL-1ß on TGF-ß1 and receptors expression by decreasing their mRNA levels. IP6 enhanced LPS- and IL-1ß-stimulated mRNA expression of TGF-ß2 and -ß3. Based on these studies it may be concluded that IP6 present in the intestinal milieu may exert immunoregulatory effects and chemopreventive activity on colonic epithelium under inflammatory conditions or during microbe-induced infection/inflammation by modulating the expression of genes encoding TGF-ßs and their receptors at transcriptional level.

The effect of inositol hexaphosphate on the expression of selected metalloproteinases and their tissue inhibitors in IL-1ß-stimulated colon cancer cells.

INTRODUCTION: Matrix metalloproteinases (MMPs) have repeatedly been shown to play a very active role in extracellular matrix degradation associated with tumor invasion and metastasis. Tissue inhibitors of MMPs (TIMPs) are well-known for their ability to inhibit MMP activity thereby inhibiting malignant progression. Inositol hexaphosphate (IP6 phytic acid) has been recognized to have both preventive and therapeutic effects against various cancers including that of colon. In in vitro studies, IP6 has been demonstrated to inhibit cancer cell adhesion and migration. In the present study, the effect of IP6 on the expression of MMP and TIMP genes was evaluated in unstimulated and IL-1ß-stimulated colon cancer cell line Caco-2. MATERIALS AND METHODS: Real-time QRT-PCR was used to validate the transcription level of selected MMP and TIMP genes in Caco-2 cells after treatment with 1 ng/ml of IL-1ß, 2.5 mM of IP6, and both for 6, 12, and 24 h. RESULTS: Stimulation of cells with IL-1ß only resulted in an overexpression of MMP and their TIMP mRNAs. A significant decrease in MMP-13, MMP-3, MMP-2, and TIMP-1 basal expression was achieved by IP6. IP6 was also an efficient downregulator of MMP-1, MMP-9, and TIMP-2 genes transcription stimulated by IL-1ß in 6 h lasting culture. After 12 h, IL-1ß-induced MMP-2 mRNA expression was significantly reduced by IP6. CONCLUSION: Proinflammatory cytokine IL-1ß upregulates MMP and TIMP mRNAs expression in colon cancer epithelial cells Caco-2. IP6 (2.5 mM) influences constitutive expression of both MMP and TIMP genes and downregulates IL-1ß stimulated transcription of some of these genes. IP6 exerts its anti-metastatic activity through modulation of MMP and TIMP genes expression to prevent cancer cell migration and invasion.

Phytic acid down-regulates IL-8 secretion from colonic epithelial cells by influencing mitogen-activated protein kinase signaling pathway.

Phytic acid (IP6) is an essential component of high fiber diet physiologically present in human large gut. It has been recognized to possess various significant health benefits effects including chemopreventive and have antineoplastic activity against various types of cancer. Moreover, its role in immune response through modulation of the secretion of proinflammatory cytokines and chemokines has been postulated. One of the signal transduction pathways involved in a variety of inflammatory responses is p38 mitogen-activated protein kinase (MAPK) pathway. The aim of this study was to examine effect of IP6 on human p38alpha MAP kinase activity and the expression of gene encoding p38 MAP kinase in unstimulated and IL-1beta-stimulated Caco-2 cells. Furthermore, the role of signaling pathways involving p38 MAP kinase in IP6-induced down-regulation of IL-8 secretion by unstimulated and IL-1beta-stimulated cells in the presence of p38 MAP kinase activator (anisomycin) and inhibitor (SB 203580) was evaluated. IP6 inhibited activity of recombinant p38 MAPK activity in dose-dependent manner. Treatment of cells with IP6 for 3 h resulted in decreased p38 MAP kinase expression in both unstimulated and stimulated with IL-1beta cells. The similar level of p38alpha mRNA was found in untreated and treated with IP6 cells after 6 and 12 h. Incubation of Caco-2 cells with anisomycin resulted in upregulation of IL-8 secretion and their pretreatment with anisomycin prior to IP6 addition showed down-regulation of IL-8 secretion compared to cells treated with anisomycin alone. The findings of this study show that p38 MAPK could be one of the molecular targets for IP6 in the intestinal epithelial cells and that IP6 inhibitory effect on IL-8 secretion by Caco-2 cells could be mediated by its inhibition of p38 activity.

The effect of phytic acid on the expression of NF-kappaB, IL-6 and IL-8 in IL-1beta-stimulated human colonic epithelial cells.

Intestinal epithelial cells play an important role in the mucosal immune and inflammatory reactions via the expression and secretion of proinflammatory cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8). The expression of both interleukins is regulated by nuclear factor KB (NF-kappaB). Phytic acid (IP6) is an essential component of high fiber diet. It is physiologically present in the human large gut at concentrations reaching 4 mM. It exhibits pleiotropic health beneficial effects including anti-oxidant and anti-tumor activities. Recent studies showed that IP6 can modulate immune functions of intestinal epithelium through regulation of proinflammatory cytokines secretion. The aim of this study was to analyze the effect of IP6 on the expression of IL-6 and IL-8 as well as p50 and p65 subunits of NF-kappaB and its inhibitor IkappaBalpha in Caco-2 cells stimulated with IL-1beta. A kinetic study of mRNAs expression in cells was performed after their treatment with 1 and 2.5 mM IP6 for 3, 6 and 12 h. Quantification of the genes expression was carried out using real time QRT-PCR technique. IP6 at all used concentrations had no influence on transcription of p65 gene and modulated expression of p50 and IkappaBalpha genes in Caco-2 cells. Treatment of cells with IP6 resulted in a marked decrease in both IL-6 (at 3 and 6 h) and IL-8 expression (3 h). The results of these studies suggest that IP6 may exert immunoregulatory effects on intestinal epithelium by influencing transcriptional activity of genes encoding p50 subunit of NF-kappaB, its inhibitor IkappaBalpha and proinflammatory cytokines IL-6 and IL-8.

Inhibitory effect of inositol hexaphosphate on metalloproteinases transcription in colon cancer cells stimulated with phorbol-12-myristate 13-acetate.

Inositol hexaphosphate (IP6) is a naturally occurring phytochemical, found in abundance in cereals, legumes and other high-fiber-content diets. IP6 has shown promising efficacy against a wide range of cancers. Its anti-cancer activity involves anti-proliferative, pro-apoptotic and anti-metastatic effects. Both matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), are implicated in tumor growth, metastasis, and angiogenesis. Phorbol-12-myristate 13-acetate (PMA) is a well-known inflammatory stimulator and tumor promoter that activates PKC and increases the invasiveness of various types of cancer cells by activating MMPs. The aim of the present study was to examine the influence of IP6 on the expression of selected MMPs, i.e., MMP-1, -2, -3, -9, 10, -13 and their TIMP-1 and -2 in unstimulated and PMA-stimulated colon cancer cell line Caco-2. Quantification of genes expression in Caco-2 cells treated with 100 ng/mL of PMA, 2.5 mM of IP6 and both for 6 and 12 h was carried out using real time QRT-PCR technique. Stimulation of cells with PMA resulted in an up-expression of MMP-2, MMP-3, MMP-9, MMP-10, MMP-13 and TIMP-1 mRNAs and decrease in MMP-1 gene expression. The quantity of TIMP-2 transcript was reduced by PMA. A significant decrease in MMP-2, MMP-3, MMP-10, MMP-13, and TIMP-1 expression in response to IP6 was observed. IP6 down-regulated MMP-9 transcription induced by PMA and decreased the level of both MMP-2 and MMP-3 mRNAs in PMA-stimulated cells. Caco-2 treated with both PMA and IP6 showed a significant decrease in MMP-1 expression in comparison to PMA-stimulated cells. The results of this study show that PMA can modulate MMP and TIMP genes transcription in colon cancer cells Caco-2. IP6 exerts an influence of basal mRNA expression of some MMPs and their tissue inhibitors and down-regulates MMP-1, MMP-2, MMP-3 and MMP-9 in cells treated with PMA. IP6 could be an effective anti-metastatic agent that suppresses expression of MMP genes at transcription level.

Evaluation of the expression of metalloproteinases 2 and 9 and their tissue inhibitors in colon cancer cells treated with phytic acid.

Matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) belong to a zinc dependent family of enzymes that degrade components of extracellular matrix. One postulated mechanism by which inositol hexaphosphate (phytic acid, IP6), an ubiquitous plant component, prevents the activation of MMPs may be due to its ability to chelate minerals. The aim of the study was to evaluate the expression profile of MMP-2, MMP-9 and their tissue inhibitors TIMP-1 and TIMP-2 at the mRNA level in human colorectal cancer cell line Caco-2 treated with IP6. A kinetic study of MMP-2, MMP-9 and TIMP-1, TIMP-2 mRNAs was performed after cells treatment with 1; 2.5; 5 mM IP6 for 1, 6, 12 and 24 h. Quantification of genes expression was carried out using real time QRT-PCR technique. The gene encoding MMP-9 was neither constitutively expressed nor induced by IP6 in Caco-2 cells. IP6 at the concentration of 1 mM evoked increase in MMP-2 transcript level, however, its higher doses (2.5; 5 mM) caused a decrease in this gene expression at 1 h incubation. In 24 h lasting culture along with increasing IP6 concentration, the cells expressed lower and lower MMP-2 mRNA level. In response to 1 and 2.5 mM at 6 h, the cells demonstrated an increased transcriptional activity of the TIMP-2 gene which was accompanied by a decrease in TIMP-1 gene transcription. Treatment of cells with 2.5 mM IP6 at 12 h resulted in a strong increase in both TIMP-1 and TIMP-2 expression. The results of this study show that IP6 modulates MMP-2, TIMP-1 and TIMP-2 genes expression in colon cancer cells at the transcriptional level in a way dependent on its concentration and time of interaction.