ABSTRACT
Low levels of nitric oxide (NO) produced by constitutively expressed inducible NO synthase (NOS2) in tumor cells may be an important factor in their development. NOS2 expression is associated with high mortality rates for various cancers. Alternative splicing of NOS2 down-regulates its enzymatic activity, resulting in decreased intracellular NO concentrations. Specific probes to detect alternative splicing of NOS2 were used in two isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620). Splicing variant of NOS2 S3, lacking exons 9, 10, and 11, was overexpressed in SW480 cells. NOS2 S3 was silenced in SW480 cells. Flow-cytometry analysis was used to estimate the intracellular NO levels and to analyze the cell cycle of the studied cell lines. Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine apoptosis and autophagy markers. SW480 and SW620 cells expressed NOS2 S3. Overexpression of the NOS2 S3 in SW480 cells downregulated intracellular NO levels. SW480 cells with knocked down NOS2 S3 (referred to as S3C9 cells) had higher intracellular levels of NO compared to the wild-type SW480 cells under serum restriction. Higher NO levels resulted in the loss of viability of S3C9 cells, which was associated with autophagy. Induction of autophagy by elevated intracellular NO levels in S3C9 cells under serum restriction, suggests that autophagy operates as a cytotoxic response to nitrosative stress. The expression of NOS2 S3 plays an important role in regulating intracellular NO production and maintaining viability in SW480 cells under serum restriction. These findings may prove significant in the design of NOS2/NO-based therapies for colon cancer.
Subject(s)
Adenocarcinoma/enzymology , Autophagy , Colonic Neoplasms/enzymology , Nitric Oxide Synthase Type II/metabolism , Nitric Oxide/metabolism , Nitrosative Stress , Adenocarcinoma/genetics , Adenocarcinoma/secondary , Cell Line, Tumor , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Humans , Nitric Oxide Synthase Type II/genetics , Protein Isoforms , Signal TransductionABSTRACT
Antiproliferative effect of Amaranthus mantegazzianus proteins and peptides released after simulated gastrointestinal digestion (DH% 37.8 ± 3.8) was investigated on human colon cancer cell line HT-29. Inhibition of proliferation of HT-29 cells was exhibited after a 24 h treatment with different concentrations of amaranth protein isolate (API) and the peptides released after digestion (DGS), presenting IC50 values of 1.35 ± 0.12 and 0.30 ± 0.07 mg soluble protein/mL, respectively. Lactate dehydrogenase assay indicated that both samples caused the loss of membrane integrity and cell lysis over HT-29 cells, and DAPI fluorescence microscopies evidenced typical apoptotic features. Moreover, Annexin V-FITC flow cytometry showed a significant increase of early apoptotic and late apoptotic/necrotic HT-29 cells compared to untreated ones, and caspase-3 assay confirmed the apoptosis induction with a 43.0 ± 10.3 and 65.8 ± 12.7% increase of caspase-3 activity produced by a 2 mg/mL treatment of API and DGS, respectively. In conclusion, amaranth peptides successfully released after simulated gastrointestinal digestion would exert a potential antiproliferative activity over HT-29 tumor cells. This effect was linked to the induction of cell necrosis and apoptosis, supporting the idea of using amaranth proteins as a potential food alternative ingredient for functional foods.
Subject(s)
Amaranthus/chemistry , Cell Proliferation/drug effects , Functional Food , Peptides/pharmacology , Plant Proteins/pharmacology , Apoptosis/drug effects , Digestion , HT29 Cells , HumansABSTRACT
Magnetic iron oxide nanoparticles (MNPs) have been prepared and stabilized with three organic acids (tartaric, malic and ascorbic) in order to obtain biocompatible and water dispersible MNPs with potential to bind specifically to tumoral cancer cells. An in deep characterization was performed aiming to verify the presence and effect of the coating and stabilizer on MNPs surface. Besides the mechanisms followed by the different acids to bind MNPs were elucidated and used to justify the differences in the physicochemical properties of each formulation. Data related to characterization revealed that MNPs coated with ascorbic acid (MNPs-AA) resulted the most suitable in terms of their size, surface charge and stability along the time. Besides, ascorbic acid may be recognized by GLUTs receptors that are overexpressed in several kinds of tumoral cells. Therefore, MNPs-AA was selected to explore its performance in both MRI and in vitro assays using human colon cancer cells HCT 116. MRI experiments were performed in clinical equipment using a series of aqueous dispersions of MNPs-AA that were evaluated as T2 contrast agent. The T2- weighted images obtained as well as the calculated r2, indicated that MNPs-AA could act as efficient T2 contrast agent for MRI. Regarding in vitro assays, MNPs-AA did not alter the cellular function neither exert cytotoxicity using the three explored doses. The internalization of the nanoparticles on the cellular structure was confirmed quanti and qualitatively using atomic absorption spectroscopy and Prussian blue techniques respectively. From these results, it emerges that ascorbic acid coated-magnetite nanoparticles may be used as alternative contrast agent to avoid or minimize some toxicological issues related to the widely used gadolinium.
Subject(s)
Contrast Media/chemistry , Ferric Compounds/chemistry , Magnetic Resonance Imaging , Magnetite Nanoparticles/chemistry , Neoplasms/diagnostic imaging , Ascorbic Acid/chemistry , Humans , Particle Size , Surface PropertiesABSTRACT
Parathyroid hormone-related peptide (PTHrP) is associated with several human cancers such as colon carcinoma. This disease is a complex multistep process that involves enhanced cell cycle progression and migration. Recently we obtained evidence that in the human colorectal adenocarcinoma Caco2 cells, exogenous PTHrP increases the proliferation and positively modulates cell cycle progression via ERK1/2, p38 MAPK and PI3K. The purpose of this study was to explore if the serine/threonine kinase RSK, which is involved in the progress of many cancers and it is emerging as a potential therapeutic target, mediates PTHrP effects on cancer colon cells. Western blot analysis revealed that PTHrP increases RSK phosphorylation via ERK1/2 signaling pathway but not through p38 MAPK. By performing subcellular fractionation, we found that the peptide also induces the nuclear localization of activated RSK, where many of its substrates are located. RSK participates in cell proliferation, in the upregulation of cyclin D1 and CDK6 and in the downregulation of p53 induced by PTHrP. Wound healing and transwell filter assays revealed that cell migration increased after PTHrP treatment. In addition, the hormone increases the protein expression of the focal adhesion kinase FAK, a regulator of cell motility. We observed that PTHrP induces cell migration and modulates FAK protein expression through ERK/RSK signaling pathway but not via p38 MAPK pathway. Finally, in vivo studies revealed that the hormone activates RSK in xenografts tumor. Taken together, our findings provide new insights into the deregulated cell cycle and migration that is characteristic of tumor intestinal cells.
Subject(s)
Colonic Neoplasms/genetics , Gene Expression Regulation, Neoplastic , MAP Kinase Signaling System/genetics , Parathyroid Hormone-Related Protein/pharmacology , Protein Serine-Threonine Kinases/genetics , Animals , Caco-2 Cells , Cell Movement/drug effects , Cell Survival/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclin D1/genetics , Cyclin D1/metabolism , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , HCT116 Cells , Humans , Injections, Intralesional , Male , Mice , Mice, Nude , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Neoplasm Transplantation , Parathyroid Hormone-Related Protein/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Alternative pre-mRNA splicing plays key roles in determining tissue- and species-specific cell differentiation as well as in the onset of hereditary disease and cancer, being controlled by multiple post- and co-transcriptional regulatory mechanisms. We report here that airborne particulate matter, resulting from industrial pollution, inhibits expression and specifically affects alternative splicing at the 5' untranslated region of the mRNA encoding the bone morphogenetic protein BMP4 in human colon cells in culture. These effects are consistent with a previously reported role for BMP4 in preventing colon cancer development, suggesting that ingestion of particulate matter could contribute to the onset of colon cell proliferation. We also show that the underlying mechanism might involve changes in transcriptional elongation. This is the first study to demonstrate that particulate matter causes non-pleiotropic changes in alternative splicing.
Subject(s)
Alternative Splicing/drug effects , Colonic Neoplasms/pathology , Particulate Matter/pharmacology , RNA Precursors/genetics , RNA, Messenger/genetics , Base Sequence , Bone Morphogenetic Protein 4/genetics , Cell Line, Tumor , Colonic Neoplasms/genetics , DNA Primers , HEK293 Cells , Humans , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND AND AIMS: Interferon-α (IFN-α) treatment is associated with up-regulation of epidermal growth factor receptor (EGFR) expression and marked growth inhibition of colon cancer cell lines. We aimed to determine the effect of combining IFN-α and gefitinib in the growth of human colon cancer cell lines. METHODS: Two human colon cancer cell lines SW480 and LOVO were treated with IFN-α alone or gefitinib alone or IFN-α plus gefitinib. Proliferation of colon cancer cells was measured by methyl thiazolyl tetrazolium (MTT) assay; the apoptosis rate was analysed by flow cytometry (FCM). The expression of XIAP, XAF1 mRNA was detected by RT-PCR and the expression of XIAP, XAF1 protein was detected by western blotting. RESULTS: Methyl thiazolyl tetrazolium showed that IFN-α, gefitinib and IFN-α plus gefitinib significantly inhibited SW480 and LOVO cells in a dose-dependent manner (p < 0.05). The FCM revealed that IFN α, gefitinib and IFN-α plus gefitinib could markedly upgrade the apoptosis rate (p < 0.05). The expression of XIAP mRNA down-regulated markedly (p < 0.05) while the expression of XAF1 mRNA up-regulated significantly (p < 0.05). The expression of XIAP protein was down-regulated markedly (p < 0.05) while the expression of XAF1 protein was up-regulated significantly (p < 0.05). CONCLUSION: IFN-α promotes the antiproliferaative effect of gefitinib on human colon cancer cell lines and the mechanism may be related to up-regulation expression of EGFR by IFN-α.
ANTECEDENTES Y OBJETIVOS: El tratamiento con interferón α (IFN-α) se halla asociado con la regulación por incremento de la expresión del receptor del factor de crecimiento epidérmico y la acentuada inhibición del crecimiento de las líneas celulares del cáncer colorrectal. El presente trabajo tuvo por objetivo determinar el efecto que se produce al combinar el IFN-α y el gefitinib en el crecimiento de las líneas celulares del cáncer de colon. MÉTODOS: Dos líneas celulares de cáncer del colon en humanos - SW480 y LOVO - fueron tratadas con IFN-α solamente, gefitinib solamente, o IFN-α más gefitinib. La proliferación de las células cancerosas del colon se midió mediante ensayo de metil tiazolil tetrazolio (MTT); la tasa de apoptosis se analizó mediante citometría de flujo (CMF); la expresión de XIAP/XAF1 mRNA fue detectada mediante RT-PCR y la expresión de la proteína XIAP/XAF1 fue detectada mediante inmunoblot (western blot). RESULTADOS: El MTT mostró que el IFN-α, el gefitinib, y el IFN-α más gefitinib inhibían de forma significativa las células SW480 y LOVO en dependencia de la dosis (p < 0.05). La CMF reveló que el IFN-α, el gefitinib, y el IFN-α más gefitinib podían aumentar notablemente la tasa de apoptosis (p < 0.05). La expresión de XIAP mRNA tuvo una marcada regulación por decremento (p < 0.05) mientras que la expresión de XAF1 mRNA tuvo una significativa regulación por incremento (p < 0.05); la expresión de la proteína XIAP fue notablemente regulada por decremento (p < 0.05) mientras que la expresión de la proteína XAF1 fue regulada por incremento de manera significativa (p < 0.05). CONCLUSIÓN: El IFN-α promueve el efecto antiproliferativo del gefitinib sobre las líneas celulares del cáncer colorrectal, y el mecanismo puede hallarse relacionado con la expresión de la regulación por incremento del EGFR mediante el IFN-α.