The effects of folic acid on global DNA methylation and colonosphere formation in colon cancer cell lines.

Folate and its synthetic form, folic acid (FA), are essential vitamins for the regeneration of S-adenosyl methionine molecules, thereby maintaining adequate cellular methylation. The deregulation of DNA methylation is a contributing factor to carcinogenesis, as alterations in genetic methylation may contribute to stem cell reprogramming and dedifferentiation processes that lead to a cancer stem cell (CSC) phenotype. Here, we investigate the potential effects of FA exposure on DNA methylation and colonosphere formation in cultured human colorectal cancer (CRC) cell lines. We show for the first time that HCT116, LS174T, and SW480 cells grown without adequate FA demonstrate significantly impaired colonosphere forming ability with limited changes in CD133, CD166, and EpCAM surface expression. These differences were accompanied by concomitant changes to DNA methyltransferase (DNMT) enzyme expression and DNA methylation levels, which varied depending on cell line. Taken together, these results demonstrate an interaction between FA metabolism and CSC phenotype in vitro and help elucidate a connection between supplemental FA intake and CRC development.

Lack of efficacy of combined antiangiogenic therapies in xenografted human melanoma.

Antiangiogenic therapy is theoretically a promising anticancer approach but does not always produce significant tumor control. Combinations of antiangiogenic therapies are therefore being investigated as strategies to enhance clinical benefit. Common targets for angiogenic blockade include endothelial specific receptors, such as Tie2/Tek, which signal blood vessel stabilization via recruitment and maturation of pericytes. Here, we report on the effects of targeted Tie2 antiangiogenic therapy (TekdeltaFc) in combination with nontargeted metronomic cyclophosphamide (LDM CTX) (reported to also act in antiangiogenic fashion) in xenografted human melanoma. Individually, these therapies showed transient antitumor activity, but, in combination, there was no significant reduction in tumor growth. In addition, while TekdeltaFc caused the expected increased pericyte coverage in treated blood vessels, LDM CTX alone or in combination with TekdeltaFc resulted in increased levels of VEGF production. Collectively, our data highlight the complexity of molecular interactions that may take place when antiangiogenic regimens are combined.

Sodium dichloroacetate (DCA) reduces apoptosis in colorectal tumor hypoxia.

We examined the effect of hypoxia on apoptosis of human colorectal cancer (CRC) cells in vitro and in vivo. All cell lines tested were susceptible to hypoxia-induced apoptosis. DCA treatment caused significant apoptosis under normoxia in SW480 and Caco-2 cells, but these cells displayed decreased apoptosis when treated with DCA combined with hypoxia, possibly through HIF-1alpha dependent pathways. DCA treatment also induced significantly increased growth of SW480 tumor xenografts, and a decrease in TUNEL positive nuclei in hypoxic but not normoxic regions of treated tumors. Thus DCA is cytoprotective to some CRC cells under hypoxic conditions, highlighting the need for further investigation before DCA can be used as a reliable apoptosis-inducing agent in cancer therapy.

Modulation of the tumor suppressor protein alpha-catenin by ischemic microenvironment.

Dysregulation or mislocalization of cell adhesion molecules and their regulators, such as E-cadherin, beta-catenin, and alpha-catenin, usually correlates with loss of polarity, dedifferentiation, invasive tumor growth, and metastasis. A subpopulation of alpha-catenin-negative cells within the DLD-1 colorectal carcinoma cell line causes it to display a heterogeneous morphological makeup, thus providing an excellent model system in which to investigate the role of alpha-catenin in tumorigenesis. We re-established expression of alpha-catenin protein in an alpha-catenin-deficient subpopulation of the DLD-1 cell line and used it to demonstrate that loss of alpha-catenin resulted in increased in vitro tumorigenic characteristics (increased soft agarose colony formation, clonogenic survival after suspension, and survival in suspension). When the cells were used to form tumor xenografts, those lacking alpha-catenin showed faster growth rates because of increased cellular cycling but not increased tumor microvascular recruitment. alpha-Catenin-expressing cells were preferentially located in well perfused areas of xenografts when tumors were formed from mixed alpha-catenin-positive and -negative cells. We therefore evaluated the role of the ischemic tumor microenvironment on alpha-catenin expression and demonstrated that cells lose expression of alpha-catenin after prolonged exposure in vitro to hypoglycemic conditions. Our findings illustrate that the tumor microenvironment is a potent modulator of tumor suppressor expression, which has implications for localized nutrient deficiency and ischemia-induced cancer progression.

Induction of DNA hypomethylation by tumor hypoxia.

In cancer, the extensive methylation found in the bulk of chromatin is reduced, while the normally unmethylated CpG islands become hypermethylated. Regions of solid tumors are transiently and/or chronically exposed to ischemia (hypoxia) and reperfusion, conditions known to contribute to cancer progression. We hypothesized that hypoxic microenvironment may influence local epigenetic alterations, leading to inappropriate silencing and re-awakening of genes involved in cancer. We cultured human colorectal and melanoma cancer cell lines under severe hypoxic conditions, and examined their levels of global methylation using HPLC to quantify 5-methylcytosine (5-mC), and found that hypoxia induced losses of global methylation. This was more extensive in normal human fibroblasts than cancer cell lines. Cell lines from metastatic colorectal carcinoma or malignant melanoma were found to be markedly more hypomethylated than cell lines from their respective primary lesions, but they did not show further reduction of 5-mC levels under hypoxic conditions. To explore these epigenetic changes in vivo, we established xenografts of the same cancer cells in immune deficient mice. We used Hypoxyprobe to assess the magnitude of tissue hypoxia, and immunostaining for 5-mC to evaluate DNA methylation status in cells from different regions of tumors. We found an inverse relationship between the presence of extensive tumor hypoxia and the incidence of methylation, and a reduction of 5-mC in xenografts compared to the levels seen in the same cancer cell lines in vitro, verifying that methylation patterns are also modulated by hypoxia in vivo. This suggests that epigenetic events in solid tumors may be modulated by microenvironmental conditions such as hypoxia.

Ischemia-induced K-ras mutations in human colorectal cancer cells: role of microenvironmental regulation of MSH2 expression.

Mutation of the K-ras gene is one of the most common genetic alterations in solid tumors, including colorectal cancer. The relatively late emergence of K-ras mutations in colorectal cancer is particularly striking in the class of mismatch repair-deficient tumors associated with early-onset microsatellite instability. We, therefore, tested the hypothesis that the microsatellite instability phenotype itself does not efficiently trigger K-ras mutations in colorectal cancer cells, but rather that tumor-associated microenvironmental conditions (e.g., hypoxia and hypoglycemia) contribute to this event by modulating genetic instability. We examined K-ras(G13D) mutation using PCR-RFLP analysis in two different microsatellite instability colorectal cancer cell lines (HCT116 and DLD-1) and their variants in which the mutant (but not the wild-type) K-ras allele has been genetically disrupted (Hkh-2 and Dks-8). We found K-ras(G13D) mutation to occur at far greater incidence in cells derived from xenografted tumors or exposed to conditions of combined hypoxia and hypoglycemia in vitro. Interestingly, this mutagenesis was neither enhanced by induced oxidative damage nor prevented by the antioxidant vitamin E. Moreover, the accumulation of K-ras mutations was paralleled by down-regulation of the key mismatch repair protein MSH2 in xenografted tumors, particularly in hypoperfused areas and under hypoglycemic conditions (in vitro). In contrast, the microsatellite stable colorectal cancer cell line Caco-2 neither accumulated K-ras mutations nor showed down-regulation of MSH2 under these conditions. Thus, our study suggests that ischemia may not simply select for, but can actually trigger, increased mutation rate in crucial colorectal cancer oncoproteins. This finding establishes a novel linkage between genetic instability, tumor ischemia, and genetic tumor progression and carries important implications for applying anticancer therapies involving tumor hypoxia (e.g., antiangiogenesis) in microsatellite instability cancers.

Oncogenic events regulate tissue factor expression in colorectal cancer cells: implications for tumor progression and angiogenesis.

Tissue factor (TF) is the primary cellular initiator of blood coagulation and a modulator of angiogenesis and metastasis in cancer. Indeed, systemic hypercoagulability in patients with cancer and TF overexpression by cancer cells are both closely associated with tumor progression, but their causes have been elusive. We now report that in human colorectal cancer cells, TF expression is under control of 2 major transforming events driving disease progression (activation of K-ras oncogene and inactivation of the p53 tumor suppressor), in a manner dependent on MEK/mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI3K). Furthermore, the levels of cell-associated as well as circulating (microvesicle-associated) TF activity are linked to the genetic status of cancer cells. Finally, RNA interference experiments suggest that TF expression is an important effector of the K-ras-dependent tumorigenic and angiogenic phenotype in vivo. Thus, this study establishes a causal link between cancer coagulopathy, angiogenesis, and genetic tumor progression.

Inhibition by heparin of protein kinase C activation and hydroxyl radical generation in puromycin aminonucleoside treated isolated rat hepatocytes.

Heparin has been reported to have many actions similar to calcium-dependent protein kinase (PKC) inhibitors. We have found that puromycin aminonucleoside (PAN) increases hydroxyl radical generation and this was prevented by H-7, a PKC inhibitor in isolated rat hepatocytes. In this study, we investigate the effect of heparin on the increased hydroxyl radical generation as well as PKC activation by PAN in isolated rat hepatocytes. To estimate the amount of hydroxyl radical generation, we measured methylguanidine (MG) and creatol which are the products from the reaction of creatinine and hydroxyl radical. Synthetic rate of MG plus creatol in isolated rat hepatocytes incubated in Krebs-Henseleit bicarbonate buffer containing creatinine and tested reagents were recorded. This rate with or without PAN was 231 +/- 11 or 112 +/- 5.6 nmol/g wet cells/4 h (mean +/- S.E., n = 5), respectively. Heparin concentrations of 3.3, 6.6 and 10 U/ml inhibited MG plus creatol synthesis in the presence of PAN by 30, 38 and 39%, and without PAN by 8.4, 27 and 34%, respectively. Statistical significance was observed except for 3.3 U/ml without PAN. The ratio of PKC in membrane/cytoplasmic fraction, an indicator of PKC activation, increased 2.8- and 3-fold that of the 0 time after 60 and 120 min incubation with PAN while heparin at 10 U/ml almost completely suppressed this increase in the ratio of PKC. The PKC ratio of the membrane/cytoplasmic fraction obtained from hepatocytes with heparin alone or without PAN and heparin was almost unchanged during the tested period. Variation of PKC levels in membrane fraction is similar to that of PKC ratio of the membrane/cytoplasmic fraction. Increased creatol synthesis by PAN and its inhibition by heparin were observed in the same samples as those used for the PKC study. These results indicate that heparin inhibits the increase in hydroxyl radical generation induced by PAN through inhibition of PKC activation in isolated rat hepatocytes.

Impact of water-dispersible beadlets as a vehicle for the delivery of carotenoids to cultured cells.

Water-dispersible beadlets of carotenoids were used as supplements for human umbilical vein endothelial cells (HUVECs), human peripheral blood mononuclear cells (PBMCs) and human monocytes. Stability, cellular association and cytotoxicity of the carotenoid beadlets were compared with carotenoids delivered with tetrahydrofuran (THF). Incubations with lycopene, beta-carotene, lutein and astaxanthin dissolved in THF resulted in a lower stability in the medium, lower cellular association, and a higher standard deviation. Beadlets provided 60, 4, 6, and 2 times greater accumulation of lycopene, beta-carotene, lutein and astaxanthin, respectively, by PBMCs than THF. The cellular association of carotenoids delivered by THF seems to be more carotenoid-specific than when carotenoids are delivered by beadlets. After 48 h of incubation under cell culture conditions all of the four carotenoids (1 microM) delivered by beadlets to the medium showed a reduction less than 30%. In addition, no cytotoxic effect of the carotenoid beadlets or the vehicle alone was detected in a concentration range of 0.5-5 microM. The results show that beadlets are a non-toxic vehicle for supplementing and stabilizing carotenoids in culture media offering a reasonable compromise in term of cell accumulation efficiency.