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1.
Cancer Res ; 77(21): 5741-5754, 2017 11 01.
Article in English | MEDLINE | ID: mdl-28923859

ABSTRACT

The trans-sulfuration enzyme cystathionine-ß-synthase (CBS) and its product hydrogen sulfide (H2S) are aberrantly upregulated in colorectal cancers, where they contribute to tumor growth and progression by both autocrine and paracrine mechanisms. However, it is unknown whether the CBS/H2S axis plays a role in colorectal carcinogenesis. Here, we report upregulation of CBS in human biopsies of precancerous adenomatous polyps and show that forced upregulation of CBS in an adenoma-like colonic epithelial cell line is sufficient to induce metabolic and gene expression profiles characteristic of colorectal cancer cells. Differentially expressed metabolites (65 increased and 20 decreased) clustered into the glycolytic pathway, nucleotide sugars, intermediates of the pentose phosphate pathway, and lipogenesis, including primarily phospholipids, sphingolipids, and bile acids. CBS upregulation induced broad changes in the NCM356 cell transcriptome with over 350 differentially expressed genes. These genes overlapped significantly with gene sets related to glycolysis, hypoxia, and a colon cancer cell phenotype, including genes regulated by NF-κB, KRAS, p53, and Wnt signaling, genes downregulated after E-cadherin knockdown, and genes related to increased extracellular matrix, cell adhesion, and epithelial-to-mesenchymal transition. The CBS-induced switch to an anabolic metabolism was associated with increased NCM356 cell bioenergetics, proliferation, invasion through Matrigel, resistance to anoikis, and CBS-dependent tumorigenesis in immunocompromised mice. Genetic ablation of CBS in CBS heterozygous mice (CBS+/- ) reduced the number of mutagen-induced aberrant colonic crypt foci. Taken together, these results establish that activation of the CBS/H2S axis promotes colon carcinogenesis. Cancer Res; 77(21); 5741-54. ©2017 AACR.


Subject(s)
Adenomatous Polyps/genetics , Colon/metabolism , Cystathionine beta-Synthase/genetics , Intestinal Mucosa/metabolism , Up-Regulation , Adenomatous Polyps/metabolism , Animals , Carcinogenesis/genetics , Carcinogenesis/metabolism , Cell Line , Cell Movement/genetics , Colon/pathology , Cystathionine beta-Synthase/metabolism , Female , Gene Expression Profiling/methods , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Hydrogen Sulfide/metabolism , Intestinal Mucosa/pathology , Male , Metabolomics/methods , Mice, Knockout , Mice, Nude , Transplantation, Heterologous
2.
Oncotarget ; 8(33): 55332-55352, 2017 Aug 15.
Article in English | MEDLINE | ID: mdl-28903423

ABSTRACT

Tumor cells undergo a critical remodeling of intracellular Ca2+ homeostasis that contribute to important cancer hallmarks. Store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway modulated by mitochondria, is dramatically enhanced in colon cancer cells. In addition, most cancer cells display the Warburg effect, a metabolic switch from mitochondrial metabolism to glycolysis that provides survival advantages. Accordingly, we investigated mitochondria control of store-operated currents (SOCs) in two cell lines previously selected for representing human normal colonic cells and colon cancer cells. We found that, in normal cells, mitochondria are important for SOCs activity but they are unable to prevent current inactivation. In contrast, in colon cancer cells, mitochondria are dispensable for SOCs activation but are able to prevent the slow, Ca2+-dependent inactivation of SOCs. This effect is associated to increased ability of tumor cell mitochondria to take up Ca2+ due to increased mitochondrial potential (ΔΨ) linked to the Warburg effect. Consistently with this view, selected non-steroidal anti-inflammatory drugs (NSAIDs) depolarize mitochondria, inhibit mitochondrial Ca2+ uptake and promote SOC inactivation, leading to inhibition of both SOCE and cancer cell proliferation. Thus, mitochondria sustain store-operated currents in colon cancer cells but not in normal colonic cells and this effect is counteracted by selected NSAIDs providing a mechanism for cancer chemoprevention.

3.
Int J Mol Sci ; 18(5)2017 Apr 27.
Article in English | MEDLINE | ID: mdl-28448473

ABSTRACT

Colorectal cancer (CRC) cells undergo the remodeling of intracellular Ca2+ homeostasis, which contributes to cancer hallmarks such as enhanced proliferation, invasion and survival. Ca2+ remodeling includes critical changes in store-operated Ca2+ entry (SOCE) and Ca2+ store content. Some changes have been investigated at the molecular level. However, since nearly 100 genes are involved in intracellular Ca2+ transport, a comprehensive view of Ca2+ remodeling in CRC is lacking. We have used Next Generation Sequencing (NGS) to investigate differences in expression of 77 selected gene transcripts involved in intracellular Ca2+ transport in CRC. To this end, mRNA from normal human colonic NCM460 cells and human colon cancer HT29 cells was isolated and used as a template for transcriptomic sequencing and expression analysis using Ion Torrent technology. After data transformation and filtering, exploratory analysis revealed that both cell types were well segregated. In addition, differential gene expression using R and bioconductor packages show significant differences in expression of selected voltage-operated Ca2+ channels and store-operated Ca2+ entry players, transient receptor potential (TRP) channels, Ca2+ release channels, Ca2+ pumps, Na⁺/Ca2+ exchanger isoforms and genes involved in mitochondrial Ca2+ transport. These data provide the first comprehensive transcriptomic analysis of Ca2+ remodeling in CRC.


Subject(s)
Calcium Channels/genetics , Calcium/metabolism , Gene Expression Profiling , Calcium Channels/metabolism , Cell Line, Tumor , Cluster Analysis , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Gene Expression Regulation , HT29 Cells , High-Throughput Nucleotide Sequencing , Humans , Principal Component Analysis , Sequence Analysis, RNA , Sodium-Calcium Exchanger/genetics , Sodium-Calcium Exchanger/metabolism , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolism
4.
Oncotarget ; 6(29): 27403-15, 2015 Sep 29.
Article in English | MEDLINE | ID: mdl-26299804

ABSTRACT

Previous studies suggest the anti-inflammatory drug, sulindac inhibits tumorigenesis by a COX independent mechanism involving cGMP PDE inhibition. Here we report that the cGMP PDE isozymes, PDE5 and 10, are elevated in colon tumor cells compared with normal colonocytes, and that inhibitors and siRNAs can selectively suppress colon tumor cell growth. Combined treatment with inhibitors or dual knockdown suppresses tumor cell growth to a greater extent than inhibition from either isozyme alone. A novel sulindac derivative, ADT-094 was designed to lack COX-1/-2 inhibitory activity but have improved potency to inhibit PDE5 and 10. ADT-094 displayed >500 fold higher potency to inhibit colon tumor cell growth compared with sulindac by activating cGMP/PKG signaling to suppress proliferation and induce apoptosis. Combined inhibition of PDE5 and 10 by treatment with ADT-094, PDE isozyme-selective inhibitors, or by siRNA knockdown also suppresses ß-catenin, TCF transcriptional activity, and the levels of downstream targets, cyclin D1 and survivin. These results suggest that dual inhibition of PDE5 and 10 represents novel strategy for developing potent and selective anticancer drugs.


Subject(s)
Acetamides/chemistry , Colonic Neoplasms/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Indenes/chemistry , Phosphodiesterase Inhibitors/chemistry , Phosphoric Diester Hydrolases/metabolism , beta Catenin/metabolism , Apoptosis , Caco-2 Cells , Cell Line, Tumor , Cell Proliferation , Computer Simulation , Cyclin D1/metabolism , Gene Expression Regulation, Neoplastic , HCT116 Cells , HT29 Cells , Humans , Inhibitor of Apoptosis Proteins/metabolism , Inhibitory Concentration 50 , RNA, Small Interfering/metabolism , Signal Transduction , Sulindac/chemistry , Survivin , Transcription, Genetic , beta Catenin/antagonists & inhibitors
5.
Cancer Lett ; 365(1): 68-78, 2015 Aug 28.
Article in English | MEDLINE | ID: mdl-26021766

ABSTRACT

Cancer cells rely mostly on glycolysis to meet their energetic demands, producing large amounts of lactate that are extruded to the tumour microenvironment by monocarboxylate transporters (MCTs). The role of MCTs in the survival of colorectal cancer (CRC) cells is scarce and poorly understood. In this study, we aimed to better understand this issue and exploit these transporters as novel therapeutic targets alone or in combination with the CRC classical chemotherapeutic drug 5-Fluorouracil. For that purpose, we characterized the effects of MCT activity inhibition in normal and CRC derived cell lines and assessed the effect of MCT inhibition in combination with 5-FU. Here, we demonstrated that MCT inhibition using CHC (α-cyano-4-hydroxycinnamic acid), DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and quercetin decreased cell viability, disrupted the glycolytic phenotype, inhibited proliferation and enhanced cell death in CRC cells. These results were confirmed by specific inhibition of MCT1/4 by RNA interference. Notably, we showed that 5-FU cytotoxicity was potentiated by lactate transport inhibition in CRC cells, either by activity inhibition or expression silencing. These findings provide novel evidence for the pivotal role of MCTs in CRC maintenance and survival, as well as for the use of these transporters as potential new therapeutic targets in combination with CRC conventional therapy.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Colorectal Neoplasms/metabolism , Lactic Acid/metabolism , Monocarboxylic Acid Transporters/antagonists & inhibitors , Antimetabolites, Antineoplastic/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Dose-Response Relationship, Drug , Fluorouracil/pharmacology , Glycolysis/drug effects , Humans , Membrane Transport Modulators/pharmacology , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Muscle Proteins/antagonists & inhibitors , Muscle Proteins/genetics , Muscle Proteins/metabolism , RNA Interference , Symporters/antagonists & inhibitors , Symporters/genetics , Symporters/metabolism , Time Factors , Transfection
6.
Am J Pathol ; 185(4): 1135-44, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25794709

ABSTRACT

Methionine adenosyltransferase 2B (MAT2B) encodes for variant proteins V1 and V2 that interact with GIT1 to increase ERK activity and growth in human liver and colon cancer cells. MAT2B or GIT1 overexpression activates MEK. This study explores the mechanism for MEK activation. We examined protein-protein interactions by co-immunoprecipitation and verified by confocal microscopy and pull-down assay using recombinant or in vitro translated proteins. Results were confirmed in an orthotopic liver cancer model. We found that MAT2B and GIT1-mediated MEK1/2 activation was not mediated by PAK1 or Src in HepG2 or RKO cells. Instead, MAT2B and GIT1 interact with B-Raf and c-Raf and enhance recruitment of Raf proteins to MEK1/2. MAT2B-GIT1 activates c-Raf, which is the key mediator for MEK/12 activation, because this still occurred in RKO cells that express constitutively active B-Raf mutant. The mechanism lies with the ability of MAT2B-GIT1 to activate Ras and promote B-Raf/c-Raf heterodimerization. Interestingly, MAT2B but not GIT1 can directly interact with Ras, which increases protein stability. Finally, increased Ras-Raf-MEK signaling occurred in phenotypically more aggressive liver cancers overexpressing MAT2B variants and GIT1. In conclusion, interaction between MAT2B and GIT1 serves as a scaffold and facilitates signaling in multiple steps of the Ras/Raf/MEK/ERK pathway, further emphasizing the importance of MAT2B/GIT1 interaction in cancer growth.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Cycle Proteins/metabolism , Colonic Neoplasms/metabolism , Liver Neoplasms/metabolism , Methionine Adenosyltransferase/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Proto-Oncogene Proteins B-raf/metabolism , ras Proteins/metabolism , Cell Line, Tumor , Colonic Neoplasms/enzymology , Colonic Neoplasms/pathology , Enzyme Activation , Humans , Liver Neoplasms/enzymology , Liver Neoplasms/pathology , Protein Binding , Protein Multimerization , Proto-Oncogene Proteins c-raf/metabolism , Up-Regulation , p21-Activated Kinases/metabolism , src-Family Kinases/metabolism
7.
Proc Natl Acad Sci U S A ; 111(46): 16520-5, 2014 Nov 18.
Article in English | MEDLINE | ID: mdl-25368155

ABSTRACT

Colorectal tumorigenesis is driven by genetic alterations in the adenomatous polyposis coli (APC) tumor suppressor pathway and effectively inhibited by nonsteroidal antiinflammatory drugs (NSAIDs). However, how NSAIDs prevent colorectal tumorigenesis has remained obscure. We found that the extrinsic apoptotic pathway and the BH3 interacting-domain death agonist (BID) are activated in adenomas from NSAID-treated patients. Loss of BID abolishes NSAID-mediated tumor suppression, survival benefit, and apoptosis in tumor-initiating stem cells in APC(Min/+) mice. BID-mediated cross-talk between the extrinsic and intrinsic apoptotic pathways is responsible for selective killing of neoplastic cells by NSAIDs. We further demonstrate that NSAIDs induce death receptor signaling in both cancer and normal cells, but only activate BID in cells with APC deficiency and ensuing c-Myc activation. Our results suggest that NSAIDs suppress intestinal tumorigenesis through BID-mediated synthetic lethality triggered by death receptor signaling and gatekeeper mutations, and provide a rationale for developing more effective cancer prevention strategies and agents.


Subject(s)
Adenomatous Polyposis Coli/prevention & control , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Apoptosis , BH3 Interacting Domain Death Agonist Protein/physiology , Genes, APC , Adenomatous Polyposis Coli/pathology , Animals , Apoptosis Regulatory Proteins/physiology , BH3 Interacting Domain Death Agonist Protein/antagonists & inhibitors , BH3 Interacting Domain Death Agonist Protein/deficiency , BH3 Interacting Domain Death Agonist Protein/genetics , Caspases/physiology , Cell Line, Tumor , Colon/pathology , Gene Expression Regulation, Neoplastic , Genes, myc , Humans , Indomethacin/pharmacology , Intestine, Small/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mitochondria/metabolism , Organ Specificity , Pyrazoles/pharmacology , RNA, Small Interfering/pharmacology , Receptors, Death Domain/physiology , Stem Cells/metabolism , Stem Cells/pathology , Sulfonamides/pharmacology , Sulindac/pharmacology
8.
J Biol Chem ; 289(42): 28765-82, 2014 Oct 17.
Article in English | MEDLINE | ID: mdl-25143380

ABSTRACT

We have investigated the molecular basis of intracellular Ca(2+) handling in human colon carcinoma cells (HT29) versus normal human mucosa cells (NCM460) and its contribution to cancer features. We found that Ca(2+) stores in colon carcinoma cells are partially depleted relative to normal cells. However, resting Ca(2+) levels, agonist-induced Ca(2+) increases, store-operated Ca(2+) entry (SOCE), and store-operated currents (ISOC) are largely enhanced in tumor cells. Enhanced SOCE and depleted Ca(2+) stores correlate with increased cell proliferation, invasion, and survival characteristic of tumor cells. Normal mucosa cells displayed small, inward Ca(2+) release-activated Ca(2+) currents (ICRAC) mediated by ORAI1. In contrast, colon carcinoma cells showed mixed currents composed of enhanced ICRAC plus a nonselective ISOC mediated by TRPC1. Tumor cells display increased expression of TRPC1, ORAI1, ORAI2, ORAI3, and STIM1. In contrast, STIM2 protein was nearly depleted in tumor cells. Silencing data suggest that enhanced ORAI1 and TRPC1 contribute to enhanced SOCE and differential store-operated currents in tumor cells, whereas ORAI2 and -3 are seemingly less important. In addition, STIM2 knockdown decreases SOCE and Ca(2+) store content in normal cells while promoting apoptosis resistance. These data suggest that loss of STIM2 may underlie Ca(2+) store depletion and apoptosis resistance in tumor cells. We conclude that a reciprocal shift in TRPC1 and STIM2 contributes to Ca(2+) remodeling and tumor features in colon cancer.


Subject(s)
Calcium/metabolism , Cell Adhesion Molecules/metabolism , Colorectal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , TRPC Cation Channels/metabolism , Apoptosis , Carcinogenesis , Cell Line, Tumor , Cell Proliferation , Cell Survival , Colon/metabolism , Electrophysiological Phenomena , Gene Expression Profiling , Gene Silencing , Humans , Inositol 1,4,5-Trisphosphate/chemistry , Intestinal Mucosa/pathology , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Stromal Interaction Molecule 1 , Stromal Interaction Molecule 2
9.
Environ Sci Technol ; 48(12): 6743-53, 2014 Jun 17.
Article in English | MEDLINE | ID: mdl-24840005

ABSTRACT

As the use of alternative drinking water treatment increases, it is important to understand potential public health implications associated with these processes. The objective of this study was to evaluate the formation of disinfection byproducts (DBPs) and cytotoxicity of natural organic matter (NOM) concentrates treated with chlorine, chloramine, and medium pressure ultraviolet (UV) irradiation followed by chlorine or chloramine, with and without nitrate or iodide spiking. The use of concentrated NOM conserved volatile DBPs and allowed for direct analysis of the treated water. Treatment with UV prior to chlorine in ambient (unspiked) samples did not affect cytotoxicity as measured using an in vitro normal human colon cell (NCM460) assay, compared to chlorination alone when toxicity is expressed on the basis of dissolved organic carbon (DOC). Nitrate-spiked UV+chlorine treatment produced greater cytotoxicity than nitrate-spiked chlorine alone or ambient UV+chlorine samples, on both a DOC and total organic halogen basis. Samples treated with UV+chloramine were more cytotoxic than those treated with only chloramine using either dose metric. This study demonstrated the combination of cytotoxicity and DBP measurements for process evaluation in drinking water treatment. The results highlight the importance of dose metric when considering the relative toxicity of complex DBP mixtures formed under different disinfection scenarios.


Subject(s)
Chloramines/toxicity , Chlorine/toxicity , Drinking Water/chemistry , Toxicity Tests , Ultraviolet Rays , Water Purification/methods , Cell Death/drug effects , Cell Death/radiation effects , Cell Line , Disinfection , Halogenation/drug effects , Halogenation/radiation effects , Humans , Inhibitory Concentration 50 , Iodine/analysis , Water Pollutants, Chemical/analysis
10.
Cancer Cell ; 25(4): 469-83, 2014 Apr 14.
Article in English | MEDLINE | ID: mdl-24735923

ABSTRACT

MicroRNA deregulation is frequent in human colorectal cancers (CRCs), but little is known as to whether it represents a bystander event or actually drives tumor progression in vivo. We show that miR-135b overexpression is triggered in mice and humans by APC loss, PTEN/PI3K pathway deregulation, and SRC overexpression and promotes tumor transformation and progression. We show that miR-135b upregulation is common in sporadic and inflammatory bowel disease-associated human CRCs and correlates with tumor stage and poor clinical outcome. Inhibition of miR-135b in CRC mouse models reduces tumor growth by controlling genes involved in proliferation, invasion, and apoptosis. We identify miR-135b as a key downsteam effector of oncogenic pathways and a potential target for CRC treatment.


Subject(s)
Colonic Neoplasms/genetics , MicroRNAs/genetics , Animals , Cell Growth Processes/genetics , Cell Line, Tumor , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Disease Models, Animal , Disease Progression , Heterografts , Humans , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Nude , MicroRNAs/metabolism , Transfection
11.
Mol Cancer Ther ; 12(9): 1848-59, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23804703

ABSTRACT

Nonsteroidal anti-inflammatory drugs (NSAID) display promising antineoplastic activity for colorectal and other cancers, but toxicity from COX inhibition limits their long-term use for chemoprevention. Previous studies have concluded that the basis for their tumor cell growth inhibitory activity does not require COX inhibition, although the underlying mechanism is poorly understood. Here, we report that the NSAID sulindac sulfide inhibits cyclic guanosine 3',5'-monophosphate phosphodiesterase (cGMP PDE) activity to increase intracellular cGMP levels and activate cGMP-dependent protein kinase (PKG) at concentrations that inhibit proliferation and induce apoptosis of colon tumor cells. Sulindac sulfide did not activate the cGMP/PKG pathway, nor affect proliferation or apoptosis in normal colonocytes. Knockdown of the cGMP-specific PDE5 isozyme by siRNA and PDE5-specific inhibitors tadalafil and sildenafil also selectively inhibited the growth of colon tumor cells that expressed high levels of PDE5 compared with colonocytes. The mechanism by which sulindac sulfide and the cGMP/PKG pathway inhibits colon tumor cell growth involves the transcriptional suppression of ß-catenin to inhibit Wnt/ß-catenin T-cell factor transcriptional activity, leading to downregulation of cyclin D1 and survivin. These observations suggest that safer and more efficacious sulindac derivatives can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP-degrading isozymes.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Colonic Neoplasms/pathology , Cyclic GMP-Dependent Protein Kinases/metabolism , Cyclic GMP/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5/metabolism , Sulindac/analogs & derivatives , Wnt Signaling Pathway/drug effects , Antineoplastic Agents/analysis , Apoptosis/drug effects , Caco-2 Cells , Carbolines/pharmacology , Cell Line , Colonic Neoplasms/metabolism , Cyclic GMP/genetics , Cyclin D1/metabolism , HCT116 Cells , HT29 Cells , Humans , Inhibitor of Apoptosis Proteins/metabolism , Phosphodiesterase 5 Inhibitors/pharmacology , Piperazines/pharmacology , Purines/pharmacology , Sildenafil Citrate , Sulfones/pharmacology , Sulindac/analysis , Sulindac/pharmacology , Survivin , Tadalafil
12.
Am J Physiol Gastrointest Liver Physiol ; 303(11): G1270-8, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-22982339

ABSTRACT

Subepithelial myofibroblasts are involved in the initiation and coordination of intestinal epithelial repair, but the molecular signaling pathways are largely unknown. The cellular adaptations that occur during repair range from dedifferentiation and migration to proliferation and redifferentiation, in a way that is strongly reminiscent of normal crypt-to-villus epithelial maturation. We therefore hypothesized that Wnt/ß-catenin signaling may have a pivotal role in intestinal epithelial wound repair. We used the established scratch wound method in Caco-2 cells and in nontransformed NCM460 cells to monitor the effects of IL-1ß-stimulated colonic myofibroblasts (CCD-18co) on intestinal epithelial repair, with immunoblotting and immunodepletion to examine the conditioned media. Conditioned media from IL-1ß-stimulated, but not -untreated, myofibroblasts increased Caco-2 wound closure twofold over 24 h. IL-1ß-stimulated myofibroblasts downregulated the differentiation marker sucrase-isomaltase in the Caco-2 cells, whereas the proliferation marker c-myc was upregulated. Array expression profiling identified Wnt-5a as the Wnt-related gene that was most upregulated (28-fold) by IL-1ß stimulation of CCDs. Recombinant Wnt-5a enhanced proliferation of Caco-2 and NCM460 cells. In scratch assays, it increased migration of the leading edge in both cell lines. Wnt-5a immunodepletion of the IL-1ß-CCD conditioned media abrogated the ability to enhance the repair. Wnt-5a often acts through a noncanonical signal transduction pathway. Further experiments supported this pathway in epithelial wound healing: IL-1ß-CCD-mediated repair was not affected by the addition of the canonical Wnt antagonist Dickkopf-1. Furthermore, media from stimulated myofibroblasts (but not Wnt-5a-depleted media) increased c-jun in Caco-2 cell nuclear extracts. Myofibroblast-mediated noncanonical Wnt-5a signaling is therefore important in the dedifferentiation and migration stages of epithelial wound repair.


Subject(s)
Interleukin-1beta/pharmacology , Myofibroblasts/drug effects , Proto-Oncogene Proteins/physiology , Wnt Proteins/physiology , Wound Healing/drug effects , Caco-2 Cells , Cell Dedifferentiation , Cell Line , Cell Movement , Culture Media, Conditioned/pharmacology , Down-Regulation , Humans , Myofibroblasts/physiology , Proto-Oncogene Proteins/biosynthesis , Signal Transduction/drug effects , Up-Regulation , Wnt Proteins/biosynthesis , Wnt-5a Protein , Wound Healing/physiology , beta Catenin/metabolism
13.
Cancer Lett ; 324(1): 98-108, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-22579651

ABSTRACT

We recently demonstrated that p38α is required to maintain colorectal cancer (CRC) metabolism, as its inhibition leads to FoxO3A activation, autophagy, cell death, and tumor growth reduction both in vitro and in vivo. Here we show that inhibition of p38α is followed by TRAIL-mediated activation of caspase-8 and FoxO3A-dependent HER3 upregulation with consequent overactivation of the MEK-ERK1/2 survival pathway. p38α and MEK combined inhibition specifically induces apoptosis by enabling TRAIL signaling propagation through t-Bid and caspase-3, and fosters cell death in CRC cells and preclinical mouse models. Current MEK1-directed pharmacological strategies could thus be exploited, in combination with p38α inhibition, to develop new approaches for CRC treatment.


Subject(s)
Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Aged , Aged, 80 and over , Animals , Apoptosis/drug effects , Apoptosis/genetics , Benzamides/pharmacology , Caspase 8/metabolism , Cell Death/drug effects , Cell Death/genetics , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Diphenylamine/analogs & derivatives , Diphenylamine/pharmacology , Enzyme Inhibitors/pharmacology , Female , HT29 Cells , Humans , Imidazoles/pharmacology , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , Male , Mice , Mice, Nude , Middle Aged , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Mitogen-Activated Protein Kinase 14/metabolism , Phosphorylation , Pyridines/pharmacology , Xenograft Model Antitumor Assays , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors
14.
J Nat Prod ; 75(1): 26-33, 2012 Jan 27.
Article in English | MEDLINE | ID: mdl-22216935

ABSTRACT

Hamamelis virginiana (witch hazel) bark is a rich source of condensed and hydrolyzable tannins reported to exert a protective action against colon cancer. The present study characterizes different witch hazel tannins as selective cytotoxic agents against colon cancer. To cover the structural diversity of the tannins that occur in H. virginiana bark, the hydrolyzable tannins, hamamelitannin and pentagalloylglucose, together with a proanthocyanidin-rich fraction (F800H4) were selected for the study. Treatment with these compounds reduced tumor viability and induced apoptosis, necrosis, and S-phase arrest in the cell cycle of HT29 cells, with hamamelitannin being the most efficient. Owing to polyphenol-mediated H(2)O(2) formation in the incubation media, the antiproliferative effect was determined in the presence and absence of catalase to rule out any such interference. The presence of catalase significantly changed the IC(50) only for F800H4. Furthermore, at concentrations that inhibit the growth of HT29 cells by 50%, hamamelitannin had no harmful effects on NCM460 normal colonocytes, whereas pentagalloylglucose inhibited both cancerous and normal cell growth. Using the TNPTM assay, we identified a highly reactive phenolic position in hamamelitannin, which may explain its efficacy at inhibiting colon cancer growth.


Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Gallic Acid/analogs & derivatives , Hamamelis/chemistry , Hexoses/isolation & purification , Hexoses/pharmacology , Hydrolyzable Tannins/isolation & purification , Hydrolyzable Tannins/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Apoptosis/drug effects , Colonic Neoplasms , Drug Screening Assays, Antitumor , Gallic Acid/chemistry , Gallic Acid/isolation & purification , Gallic Acid/pharmacology , Hexoses/chemistry , Humans , Hydrogen Peroxide/analysis , Hydrolyzable Tannins/chemistry , Molecular Structure , Plant Bark/chemistry
15.
Nutr Cancer ; 64(1): 128-35, 2012.
Article in English | MEDLINE | ID: mdl-22171558

ABSTRACT

Methylselenol is hypothesized to be a critical selenium metabolite for anticancer action, and differential chemopreventive effects of methylselenol on cancerous and noncancerous cells may play an important role. In this study, the submicromolar concentrations of methylselenol were generated by incubating methionase with seleno-L methionine, and colon-cancer-derived HCT-116 cells and noncancerous colon NCM460 cells were exposed to methylselenol. Methylselenol exposure inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase and an induction of apoptosis in HCT116, but to a much lesser extent in NCM460 colon cells. Similarly, the examination of mitogen-activated protein kinase (MAPK) and cellular myelocytomatosis oncogene (c-Myc) signaling status revealed that methylselenol inhibited the phosphorylation of extracellular-regulated kinase1/2 and p38 mitogen-activated protein kinase and the expression of c-Myc in HCT116 cells, but also to a lesser extent in NCM460 cells. The other finding is that methylselenol inhibits sarcoma kinase phosphorylation in HCT116 cells. In contrast, methylselenol upregulated the phosphorylation of sarcoma and focal adhesion kinase survival signals in the noncancerous NCM460 cells. Collectively, methylselenol's stronger potential of inhibiting cell proliferation/survival signals in the cancerous HCT116 cells when compared with that in noncancerous NCM460 cells may partly explain the potential of methylselenol's anticancer action.


Subject(s)
Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Methanol/analogs & derivatives , Organoselenium Compounds/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Line , Cell Proliferation/drug effects , Colon/cytology , Colon/drug effects , Colonic Neoplasms/drug therapy , Dose-Response Relationship, Drug , Enzyme Activation , Focal Adhesion Kinase 1/metabolism , G1 Phase/drug effects , Humans , Methanol/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins c-myc/metabolism , S Phase/drug effects , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism
16.
Nutr Cancer ; 63(2): 248-55, 2011.
Article in English | MEDLINE | ID: mdl-21271458

ABSTRACT

Sulforaphane (SFN) is a naturally occurring chemopreventive agent; the induction of cell cycle arrest and apoptosis is a key mechanism by which SFN exerts its colon cancer prevention. However, little is known about the differential effects of SFN on colon cancer and normal cells. In this study, we demonstrated that SFN (15 µmol/L) exposure (72 h) inhibited cell proliferation by up to 95% in colon cancer cells (HCT116) and by 52% in normal colon mucosa-derived (NCM460) cells. Our data also showed that SFN exposure (5 and 10 µmol/L) led to the reduction of G1 phase cell distribution and an induction of apoptosis in HCT116 cells, but to a much lesser extent in NCM460 cells. Furthermore, the examination of mitogen-activated protein kinase (MAPK) signaling status revealed that SFN upregulated the phosphorylation of extracellular-regulated kinase 1/2 (ERK1/2) in NCM460 cells but not in HCT116 cells. In contrast, SFN enhanced the phosphorylation of stress-activated protein kinase (SAPK) and decreased cellular myelocytomatosis oncogene (c-Myc) expression in HCT116 cells but not NCM460 cells. Taken together, the activation of survival signaling in NCM460 cells and apoptotic signaling in HCT116 cells may play a critical role in SFN's stronger potential of inhibiting cell proliferation in colon cancer cells than in normal colon cells.


Subject(s)
Anticarcinogenic Agents/pharmacology , Apoptosis , Signal Transduction , Thiocyanates/pharmacology , Cell Cycle , Cell Line , Cell Proliferation , Colon/cytology , Colon/metabolism , Colonic Neoplasms/metabolism , G1 Phase , Gene Expression Regulation, Neoplastic , HCT116 Cells , Humans , Isothiocyanates , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Sulfoxides
17.
Anticancer Res ; 30(6): 1881-5, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20651330

ABSTRACT

BACKGROUND/AIM: Methionine inhibits proliferation of breast and prostate cancer cells. This study aimed to determine cell cycle effects of methionine and selectivity for cancer cells. MATERIALS AND METHODS: MCF-7 (breast), LNCaP (prostate), and LS-174 (colon) cancer cells (wild-type p53), DU-145 (prostate) and SW480 (colon) cancer cells (mutated p53), and immortalized, non-tumorigenic MCF-10A (breast), BPH-1 (prostate), and NCM-460 (colon) epithelial cells were used. Cell cycle effects were assessed by flow cytometry and cell cycle-related gene expression by microarray analysis and QRT-PCR. RESULTS: L-Methionine at 5 mg/ml for 72 hours (non-apoptotic) arrested cell cycle in LNCaP, DU145, and MCF-7 cells, but not in untransformed cells, nor in LS-174 cells. LNCaP and MCF-7 cells were arrested at G(1), but DU-145 at S. Methionine up-regulated CDKIs and down-regulated CDKs. CONCLUSION: L-Methionine selectively inhibits proliferation of breast and prostate cancer cells, but not non-tumorigenic cells, and may thus have therapeutic benefits. p53 status appeared to determine the cell cycle stage at which methionine acts.


Subject(s)
Breast Neoplasms/drug therapy , Methionine/pharmacology , Pancreatic Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Cycle/drug effects , Cell Line, Tumor , Female , Gene Expression Profiling , Humans , Male , Pancreatic Neoplasms/pathology , Reverse Transcriptase Polymerase Chain Reaction , Tumor Suppressor Protein p53/analysis
18.
J Cell Physiol ; 225(1): 73-83, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20648625

ABSTRACT

The extracellular Ca(2+)-sensing receptor (CaR) is increasingly implicated in the regulation of multiple cellular functions in the gastrointestinal tract, including secretion, proliferation and differentiation of intestinal epithelial cells. However, the signaling mechanisms involved remain poorly defined. Here we examined signaling pathways activated by the CaR, including Ca(2+) oscillations, in individual human colon epithelial cells. Single cell imaging of colon-derived cells expressing the CaR, including SW-480, HT-29, and NCM-460 cells, shows that stimulation of this receptor by addition of aromatic amino acids or by an elevation of the extracellular Ca(2+) concentration promoted striking intracellular Ca(2+) oscillations. The intracellular calcium oscillations in response to extracellular Ca(2+) were of sinusoidal pattern and mediated by the phospholipase C/diacylglycerol/inositol 1,4,5-trisphosphate pathway as revealed by a biosensor that detects the accumulation of diacylglycerol in the plasma membrane. The intracellular calcium oscillations in response to aromatic amino acids were of transient type, that is, Ca(2+) spikes that returned to baseline levels, and required an intact actin cytoskeleton, a functional Rho, Filamin A and the ion channel TRPC1. Further analysis showed that re-expression and stimulation of the CaR in human epithelial cells derived from normal colon and from colorectal adenocarcinoma inhibits their proliferation. This inhibition was associated with the activation of the signaling pathway that mediates the generation of sinusoidal, but not transient, intracellular Ca(2+) oscillations. Thus, these results indicate that the CaR can function in two signaling modes in human colonic epithelial cells offering a potential link between gastrointestinal responses and food/nutrients uptake and metabolism.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Cell Proliferation , Colon/cytology , Epithelial Cells/physiology , Receptors, Calcium-Sensing/metabolism , Actins/metabolism , Cell Line , Contractile Proteins/metabolism , Cytoskeleton/metabolism , Epithelial Cells/cytology , Filamins , Humans , Microfilament Proteins/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Calcium-Sensing/genetics , TRPC Cation Channels/metabolism , rho GTP-Binding Proteins/metabolism
19.
Nature ; 464(7291): 1058-61, 2010 Apr 15.
Article in English | MEDLINE | ID: mdl-20348907

ABSTRACT

Cancer chemoprevention uses natural, synthetic, or biological substances to reverse, suppress, or prevent either the initial phase of carcinogenesis or the progression of neoplastic cells to cancer. It holds promise for overcoming problems associated with the treatment of late-stage cancers. However, the broad application of chemoprevention is compromised at present by limited effectiveness and potential toxicity. To overcome these challenges, here we developed a new chemoprevention approach that specifically targets premalignant tumour cells for apoptosis. We show that a deficiency in the adenomatous polyposis coli (APC) gene and subsequent activation of beta-catenin lead to the repression of cellular caspase-8 inhibitor c-FLIP (also known as CFLAR) expression through activation of c-Myc, and that all-trans-retinyl acetate (RAc) independently upregulates tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors and suppresses decoy receptors. Thus, the combination of TRAIL and RAc induces apoptosis in APC-deficient premalignant cells without affecting normal cells in vitro. In addition, we show that short-term and non-continuous TRAIL and RAc treatment induce apoptosis specifically in intestinal polyps, strongly inhibit tumour growth, and prolong survival in multiple intestinal neoplasms C57BL/6J-Apc(Min)/J (Apc(Min)) mice. With our approach, we further demonstrate that TRAIL and RAc induce significant cell death in human colon polyps, providing a potentially selective approach for colorectal cancer chemoprevention by targeting APC-deficient cells for apoptosis.


Subject(s)
Adenomatous Polyposis Coli Protein/deficiency , Apoptosis/drug effects , Colorectal Neoplasms/pathology , Colorectal Neoplasms/prevention & control , Vitamin A/analogs & derivatives , Adenomatous Polyposis Coli Protein/genetics , Animals , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Cell Proliferation/drug effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Diterpenes , Gene Expression Regulation/drug effects , Genes, APC , Humans , Intestinal Polyps/drug therapy , Intestinal Polyps/pathology , Mice , Mice, Inbred C57BL , Precancerous Conditions/drug therapy , Precancerous Conditions/genetics , Precancerous Conditions/metabolism , Precancerous Conditions/pathology , Proto-Oncogene Proteins c-myc/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Retinyl Esters , Signal Transduction/drug effects , Survival Rate , TNF-Related Apoptosis-Inducing Ligand/administration & dosage , TNF-Related Apoptosis-Inducing Ligand/pharmacology , TNF-Related Apoptosis-Inducing Ligand/therapeutic use , Time Factors , Vitamin A/administration & dosage , Vitamin A/pharmacology , Vitamin A/therapeutic use , beta Catenin/metabolism
20.
Int J Cancer ; 126(4): 864-75, 2010 Feb 15.
Article in English | MEDLINE | ID: mdl-19697327

ABSTRACT

Expression of gastrin and cholecystokinin 2 (CCK(2)) receptor splice variants (CCK(2)R and CCK(2i4sv)R) are upregulated in human colonic adenomas where they are thought to contribute to tumor growth and progression. To determine the effects of ectopic CCK(2) receptor variant expression on colonic epithelial cell growth in vitro and in vivo, we employed the non-tumorigenic colonic epithelial cell line, NCM356. Receptor expression was induced using a retroviral expression vector containing cDNAs for either CCK(2i4sv)R or CCK(2)R. RT-PCR and intracellular Ca(2+) ([Ca(2+)](i)) imaging of RIE/CCK(2)R cells treated with conditioned media (CM) from NCM356 revealed that NCM356 cells express gastrin mRNA and secrete endogenous, biologically active peptide. NCM356 cells expressing either CCK(2)R or CCK(2i4sv)R (71 and 81 fmol/mg, respectively) grew faster in vitro, and exhibited an increase in basal levels of phosphorylated ERK (pERK), compared with vector. CCK(2) receptor selective antagonist, YM022, partially inhibited the growth of both receptor-expressing NCM356 cells, but not the control cells. Inhibitors of mitogen activated protein kinase pathway (MEK/ERK) or protein kinase C (PKC) isozymes partially inhibited the elevated levels of basal pERK and in vitro growth of receptor-expressing cells. Vector-NCM356 cells did not form tumors in nude mice, whereas, either CCK(2) receptor-expressing cells formed large tumors. Autocrine activation CCK(2) receptor variants are sufficient to increase in vitro growth and tumorigenicity of non-transformed NCM356 colon epithelial cells through a pathway involving PKC and the MEK/ERK axis. These findings support the hypothesis that expression of gastrin and its receptors in human colonic adenomas contributes to tumor growth and progression.


Subject(s)
Colon/physiology , Colorectal Neoplasms/pathology , Intestinal Mucosa/physiology , Receptor, Cholecystokinin B/genetics , Adenoma/pathology , Animals , Calcium/metabolism , Carcinoma/genetics , Cell Culture Techniques/methods , Cell Division/genetics , Colon/metabolism , Colon/pathology , Colorectal Neoplasms/genetics , DNA Primers , Disease Progression , Gastrins/genetics , Gastrins/metabolism , Genetic Variation , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Mice , Mutation , Neoplasm Staging , Reverse Transcriptase Polymerase Chain Reaction
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