Human Colon Cancer Caco-2 Research Articles

Abstract 28: The influence of PDGF and VEGF on tumor proliferation in colon cancer

Abstract Background: The platelet derived growth factor (PDGF) and Vascular Endothelial Growth Factor (VEGF) are known to play a crucial role in different tumor entities. Involved in cell migration and proliferation of stromal cells and tumor cells they represent key targets in cancer therapy. The aim of the study was to analyse the specific role of PDGF and VEGF stimulation on tumor cell proliferation and metabolism in colorectal cancer (CRC). Methods: The human colon cancer cell line HT-29 and Caco-2 were cultured and stimulated with PDGF and VEGF in a time-dependent manner. Whole cell extracts and RNA extracts were analyzed by Western Blot and RT-q-PCR for PDGF- and VEGF receptors and for components of cellular metabolism. To discover effects of PDGF and VEGF on proliferation MTS proliferation assays were performed. Results: Western Blot analysis and RT-qPCR showed no relevant PDGF-receptor α and β expression in HT-29 cells but varying expression in Caco-2 cells. Stimulation with PDGF or VEGF resulted in increased proliferation compared to untreated controls while simultaneous stimulation with both growth factors did not result in intensified proliferation. Additionally, under stimulation altered expression of various metabolic components was detected. Conclusion: Our results demonstrate a promoting effect on tumor cell proliferation of both growth factors VEGF and PDGF. Simultaneous stimulation did not show synergistic effects. PDGF in HT-29 cells resulted in proliferation in the absence of PDGF receptors with changes in tumor cell metabolism after stimulation. Moreover in HT-29 cells glucose metabolism changed during stimulation. In conclusion, our data give evidence for direct receptor/ligand signalling of PDGF not only on stromal cells but also on colon cancer cells even in the absence of the PDGF receptor. Citation Format: Romana Mönch, Tanja Grimmig, Vinicius Kannen, Christoph T. Germer, Ana Maria Waaga-Gasser, Martin Gasser. The influence of PDGF and VEGF on tumor proliferation in colon cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 28. doi:10.1158/1538-7445.AM2014-28

Abstract 5358: IL-4/IL-13 induce Duox2/DuoxA2 expression and reactive oxygen production in human pancreatic and colon cancer cells

Abstract NADPH oxidase (NOX)-derived reactive oxygen species (ROS) contribute significantly to inflammation-associated carcinogenesis. Expression of dual oxidase 2 (Duox2), one of seven members of the NOX gene family, is up-regulated in inflammatory bowel disease, chronic pancreatitis, and in many human malignancies including carcinomas of the prostate, lung, and breast. Previously, we demonstrated that Stat1 and/or NF-κB play a critical role in modulating the enhanced expression of Duox2, and its cognate maturation factor DuoxA2, by IFN-γ and lipopolysaccharide in human pancreatic cancer cells. This cytokine-mediated increase in Duox2 expression is responsible for a consequent significant increase in tumor cell H2O2 production and DNA damage. Using Caco2 and T84 human colon cancer lines, and BxPC-3 pancreatic cancer cells, we now report that two other pro-inflammatory cytokines, IL-4 and IL-13, also strongly induce Duox2/DuoxA2 RNA and protein expression (>5-10-fold after 24h treatment with 50 ng/ml cytokine). In the BxPC-3 line, IL-4 exposure activates the Stat6 signaling pathway, enhances expression of Duox2/DuoxA2 in a time- and concentration-dependent manner, and leads to a significant increase in the generation of extracellular H2O2. The transcription and translation inhibitors actinomycin D and cyclohexamide both individually suppress IL-4-induced Duox2 expression, suggesting that IL-4-mediated Duox2 up-regulation is transcriptionally regulated, but that new protein synthesis is also necessary. We found that Stat6 is essential for IL-4-induced Duox2 expression because silencing Stat6 with Stat6-specific siRNA significantly attenuates IL-4-induced Duox2 RNA and protein levels in BxPC-3 cells. To evaluate the potential role of Duox2 in inflammation-associated human cancers further, immunohistochemical studies were performed to semi-quantitatively examine Duox expression using human pancreatic and colon cancer tissue arrays. We found that a majority of samples of pancreatic intra-epithelial neoplasia and frank pancreatic cancer demonstrated high-level expression of Duox , often in association with inflammatory cell infiltrates; high-level Duox expression was also found in most colorectal cancer samples. These studies, in concert with our previous experiments, strongly suggest that ROS, produced as a consequence of cytokine-induced Duox up-regulation, may be associated with the development of inflammation-related gastrointestinal malignancies. Note: This abstract was not presented at the meeting. Citation Format: Yongzhong Wu, James H. Doroshow. IL-4/IL-13 induce Duox2/DuoxA2 expression and reactive oxygen production in human pancreatic and colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5358. doi:10.1158/1538-7445.AM2014-5358

Oxidative stress, cell cycle arrest and differentiation contribute toward the antiproliferative action of BSO and calcitriol on Caco-2 cells

The prognosis and incidence of colon cancer are linked to vitamin D3 serum levels. To evaluate the effects of D,L-buthionine-S,R-sulfoximine (BSO), 1,25(OH)2D3 and their combination on intestinal Caco-2 cell growth, to elucidate the possible cellular mechanisms involved in their antiproliferative action, and to determine whether BSO acts as a sensitizer to 1,25(OH)2D3 treatment, enabling minimization of the toxic effects caused by high doses of the steroid. Human colon cancer Caco-2 cells were treated with 1,25(OH)2D3, BSO, both, or vehicle. Cell proliferation was evaluated by crystal violet staining. Cell cycle and mitochondrial membrane potential were measured by flow cytometry. Total glutathione, catalase, superoxide dismutase, superoxide anion levels, and alkaline phosphatase activities were analyzed by spectrophotometry. DNA fragmentation was evaluated using the terminal dUTP nick end labeling assay. BSO and 1,25(OH)2D3 inhibited Caco-2 cell growth, an effect that was higher with the combined treatment. The antiproliferative effect produced by the combination could be protected by ascorbic acid. BSO plus 1,25(OH)2D3 induced cell cycle arrest and suppressed cell division. Total glutathione decreased and superoxide anion increased with BSO and BSO plus 1,25(OH)2D3. Catalase activity increased with the combined treatment. Mitochondrial membrane potential and alkaline phosphatase activity were altered by 1,25(OH)2D3 alone or plus BSO. The percentage of terminal dUTP nick end labeling-positive cells was increased. BSO increases the antiproliferative effect of 1,25(OH)2D3 on Caco-2 cells through induction of oxidative stress, which occurs simultaneously with DNA breakage. The antioxidant system can partially compensate the damage induced by BSO plus 1,25(OH)2D3. Cell differentiation induction is also involved in the response to the combined treatment.

Comments on the business of genetic testing

Evidence has shown that extracellular adenosine induces apoptosis in variety of cancer cells, mainly through two pathways: an intrinsic pathway relative to adenosine uptake into the cells, and an extrinsic pathway involving the adenosine receptors. We elucidated the mechanisms underlying the adenosine-induced anticancer effects.Extrinsic pathway analysis showed that extracellular adenosine induces apoptosis in CW2 human colon cancer and RCR-1 rat astrocytoma cells through the A1 adenosine receptor; in Caco-2 human colon cancer and HepG2 human hepatoma cells through the A2a adenosine receptor; and through the A3 adenosine receptor in A549, Lu-65, and SBC-3 human lung cancer cells, RCC4-VHL human renal cancer cells, 5637 human bladder cancer cells, and human malignant pleural mesothelioma cells. In the intrinsic pathways, intracellularly transported adenosine induces apoptosis in GT3-TKB human lung cancer cells, human malignant pleural mesothelioma cells, HuH-7 and HepG2 human hepatoma cells, and MCF-7 human breast cancer cells by a) activating AMPK, b) upregulating p53, c) downregulating c-FLIP expression, d) neutralizing caspase-3 inhibition due to inhibition of apoptosis protein (IAP) in cooperation with DIABLO, e) accumulating AMID in the nucleus, f) regulating apoptosis-related gene transcription, or g) promoting GATA-2-regulated p53 gene transcription.Adenosine exerts its anticancer action on a wide variety of cancer cell types through diverse signaling pathways. Therefore, adenosine and its signaling cascades can be useful as possible targets in the development of promising anticancer therapies.

224: Effect of HuIFN-α and Royal jelly on the proliferation of human colon cancer (CaCo-2) cells in vitro

224: Effect of HuIFN-α and Royal jelly on the proliferation of human colon cancer (CaCo-2) cells in vitro

Evaluation of the Cytotoxicity of Cholesterol Oxides in Human Colon Cancer Caco-2 Cells

The content of cholesterol oxides (COPs) in foods varies between 0.1 and 294.3 mg/g. These oxides are formed by auto-oxidative enzymatic processes promoted by the heat treatment of food and/or to exposition of them to the presence of oxygen and sunlight during storage. Their importance is that they are associated with pathological processes like apoptosis, dyslipidemia and pro-oxidative states, among others. The objective was to evaluate the cytotoxicity, by means MTT assay, of 7-keto cholesterol (7KC), cholestane-triol (Triol), α-epoxy cholesterol (α-epoxy C), β-epoxy cholesterol (β-epoxy C) in differentiated Caco-2 cells at 120μM for 24, 48 and 72h.Triol was the most cytotoxic COP, with a percentage of cell viability among 3.2 to 3.4 at all times studied. The β-epoxy C and 7KC showed similar percentages from 65.4 to 6.9 and 67.4 to 6.5, respectively. The α-epoxy C was the least cytotoxic (83.3 to 48.7% of cell viability).COPs in the study conditions, showed a cytotoxic effect on Caco-2 cells due to disruption of the integrity of mitochondrial function, being the most toxic compound the triol.

Anti-proliferative and pro-apoptotic activity of whole extract and isolated indicaxanthin from Opuntia ficus-indica associated with re-activation of the onco-suppressor p16INK4a gene in human colorectal carcinoma (Caco-2) cells

Phytochemicals may exert chemo-preventive effects on cells of the gastro-intestinal tract by modulating epigenome-regulated gene expression. The effect of the aqueous extract from the edible fruit of Opuntia ficus-indica (OFI extract), and of its betalain pigment indicaxanthin (Ind), on proliferation of human colon cancer Caco-2 cells has been investigated. Whole extract and Ind caused a dose-dependent apoptosis of proliferating cells at nutritionally relevant amounts, with IC50 400±25mg fresh pulp equivalents/mL, and 115±15μM (n=9), respectively, without toxicity for post-confluent differentiated cells. Ind accounted for ∼80% of the effect of the whole extract. Ind did not cause oxidative stress in proliferating Caco-2 cells. Epigenomic activity of Ind was evident as de-methylation of the tumor suppressor p16INK4a gene promoter, reactivation of the silenced mRNA expression and accumulation of p16INK4a, a major controller of cell cycle. As a consequence, decrease of hyper-phosphorylated, in favor of the hypo-phosphorylated retinoblastoma was observed, with unaltered level of the cycline-dependent kinase CDK4. Cell cycle showed arrest in the G2/M-phase. Dietary cactus pear fruit and Ind may have chemo-preventive potential in intestinal cells.

GC-MS Lipidomic Profiling of the Echinoderm Marthasterias glacialis and Screening for Activity Against Human Cancer and Non-Cancer Cell Lines

Marine organisms have been increasingly regarded as an excellent source of bioactive molecules for human health. As part of an on-going study to elucidate the chemical composition and biological activity of Marthastherias glacialis, we report here the lipidomic profile of this organism and screening for anticancer activity. Two classes of biologically active metabolites were identified and quantified by a GC-MS optimized method for screening several classes of metabolites in a single run. Free fatty acids and sterols were found, including ergosterol, β-sitosterol and cholesterol derivatives, some of which are reported in this species for the first time. Distinct assays were performed to screen extract's effect on viability, membrane integrity and density of three human cancer cell lines: human oestrogen receptor-positive (ER(+)) breast cancer (MCF-7), human neuroblastoma (SH-SY5Y) and human colon cancer (Caco-2). Differential activity towards the three cancer cell lines was found, SH-SY5Y cell line being the most susceptible. No activity was found for Caco-2 cell line. Non-cancer cell lines (human dermal fibroblasts and human foreskin fibroblasts) were also tested and revealed to be less susceptible. This work establishes M. glacialis as a potential source of bioactive molecules for further studies.

Adenosine exerts potent anticancer effects through diverse signaling pathways

Abstract Purpose Evidence has shown that extracellular adenosine induces apoptosis in variety of cancer cells, mainly through two pathways: an intrinsic pathway relative to adenosine uptake into the cells, and an extrinsic pathway involving the adenosine receptors. We elucidated the mechanisms underlying the adenosine-induced anticancer effects. Study section and results Extrinsic pathway analysis showed that extracellular adenosine induces apoptosis in CW2 human colon cancer and RCR-1 rat astrocytoma cells through the A 1 adenosine receptor; in Caco-2 human colon cancer and HepG2 human hepatoma cells through the A 2a adenosine receptor; and through the A 3 adenosine receptor in A549, Lu-65, and SBC-3 human lung cancer cells, RCC4-VHL human renal cancer cells, 5637 human bladder cancer cells, and human malignant pleural mesothelioma cells. In the intrinsic pathways, intracellularly transported adenosine induces apoptosis in GT3-TKB human lung cancer cells, human malignant pleural mesothelioma cells, HuH-7 and HepG2 human hepatoma cells, and MCF-7 human breast cancer cells by a) activating AMPK, b) upregulating p53, c) downregulating c-FLIP expression, d) neutralizing caspase-3 inhibition due to inhibition of apoptosis protein (IAP) in cooperation with DIABLO, e) accumulating AMID in the nucleus, f) regulating apoptosis-related gene transcription, or g) promoting GATA-2-regulated p53 gene transcription. Conclusions Adenosine exerts its anticancer action on a wide variety of cancer cell types through diverse signaling pathways. Therefore, adenosine and its signaling cascades can be useful as possible targets in the development of promising anticancer therapies.

A novel and essential role for FcγRIIa in cancer cell–induced platelet activation

AbstractPlatelets play a role in cancer by acting as a dynamic reservoir of effectors that facilitate tumor vascularization, growth, and metastasis. However, little information is available about the mechanism of tumor cell–induced platelet secretion (TCIPS) or the molecular machinery by which effector molecules are released from platelets. Here we demonstrate that tumor cells directly induce platelet secretion. Preincubation of platelets with human colon cancer (Caco-2), prostate cancer (PC3M-luc), or breast cancer cells (MDA-MB-231;MCF-7) resulted in a marked dose-dependent secretion of dense granules. Importantly, TCIPS preceded aggregation which always displayed a characteristic lag time. We investigated the role of platelet receptors and downstream molecules in TCIPS. The most potent modulators of TCIPS were the pharmacologic antagonists of Syk kinase, phospholipase C and protein kinase C, all downstream mediators of the immunoreceptor tyrosine-based activation motif (ITAM) cascade in platelets. Supporting this, we demonstrated a central role for the immune Fcγ receptor IIa (FcγRIIa) in mediating platelet-tumor cell cross-talk. In conclusion, we demonstrate that cancer cells can promote platelet dense-granule secretion, which is required to augment platelet aggregation. In addition, we show a novel essential role for FcγRIIa in prostate cancer cell–induced platelet activation opening the opportunity to develop novel antimetastatic therapies.

MK886 inhibits cell proliferation and promotes apoptosis in human colon cancer cell lines SW480 and Caco-2

MK886 inhibits cell proliferation and promotes apoptosis in human colon cancer cell lines SW480 and Caco-2

Growth inhibition of colon cancer cells by compounds affecting AMPK activity.

To determine if other molecules reported to modulate AMP-dependent protein kinase (AMPK) activity would have effects resembling those of metformin and phenformin on colon cancer cell proliferation and metabolism. Studies were performed with four human colon cancer cell lines, Caco-2, HCT116, HT29 and SW1116. The compounds that were studied included A-769662, 5-aminoimidazole-4-carboxamide-1-ribofuranoside, butyrate, (-)-epigallocatechin gallate (EGCG), KU-55933, quercetin, resveratrol and salicylates. The parameters that were measured were cell proliferation and viability, glucose uptake, lactate production and acidification of the incubation medium. Investigations with several molecules that have been reported to be associated with AMPK activation (A-769662, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside, EGCG, KU-55933, quercetin, resveratrol and salicylates) or AMPK inhibition (compound C) failed to reveal increased medium acidification and increased glucose uptake in colon cancer cells as previously established with metformin and phenformin. The only exception was 5-aminosalicylic acid with which there were apparently lower glucose levels in the medium after incubation for 72 h. Further study in the absence of cells revealed that the effect was an artifact due to inhibition of the enzyme-linked glucose assay. The compounds were studied at concentrations that inhibited cell proliferation. It was concluded that treatment with several agents that can affect AMPK activity resulted in the inhibition of the proliferation of colon cancer cells under conditions in which glucose metabolism is not enhanced, in contrast to the effect of biguanides.

Individual profiles of free ceramide species and the constituent ceramide species of sphingomyelin and neutral glycosphingolipid and their alteration according to the sequential changes of environmental oxygen content in human colorectal cancer Caco-2 cells

We previously performed a systematic analysis of free ceramide (Cers) species, the constituent ceramide species of sphingomyelins and neutral glycosphingolipids (NGSLs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with high-energy collision-induced dissociation. As a result, distinct species differences were found among Cers, sphingomyelins and NGSLs in the kidneys. Using this method, we investigated various sphingolipid species from human colon cancer Caco-2 cells as well as the influence of environmental oxygen on these species in detail. Unexpectedly, even in normoxia, all Cers species were composed of dihydrosphingosine (d18:0) and non-hydroxy fatty acid (NFA), and 34% of sphingomyelins were composed of dihydrosphingomyelins with NFA. In contrast, major constituent ceramide species of NGSLs were composed of the usual long-chain base of sphingosine (d18:1) and hydroxy fatty acid (HFA). When the cells were cultured under hypoxic condition for 3 days, all the Cers and nearly 80% of the sphingomyelins were dihydrosphingolipids composed of d18:0-NFAs, but a significant proportion of d18:1-HFAs still remained in the NGSLs. When the cells were transferred from conditions of hypoxia to normoxia again (reoxygenation), Cer species composed of d18:1-NFAs, which were not found in Cers under the original normoxic conditions, appeared. Such Cers were probably synthesized as precursors for the constituent ceramides of sphingomyelins and NGSLs.

Genistein-induced G2/M cell cycle arrest of human intestinal colon cancer Caco-2 cells is associated with Cyclin B1 and Chk2 down-regulation

Genistein is an isoflavonic phyto-oestrogen contained in soya beans. It is thought to display anti-cancer effects. This study was designed to investigate its effect on human intestinal colon cancer Caco-2 cells. MTT assay, flow cytometric analysis and western blotting were used to investigate the effect of genistein on cell proliferation, cell cycle progression and protein alterations of selected cell cycle-related proteins in Caco-2 cells. Our results showed that genistein and daidzein significantly suppressed cell proliferation. Genistein treatment was demonstrated to modulate cell cycle distribution through accumulation of cells at G2/M phase, with a significant decreasing effect of Cyclin B1 and Serine/threonine-protein kinase 2 (Chk2) proteins expression. However, daidzein did not alter the cell cycle progression in Caco-2 cells. All these observation strongly indicate that genistein has anti-proliferative effect in human intestinal colon cancer Caco-2 cells through the down-regulation of cell cycle check point proteins, Cyclin B1 and Chk2.

Effects of autophagy modulator on autophagy and uridine 5'-diphospho-glucuronosyltransferase 1A1 induced by sulforaphane

To explore the effects of 3-methyladenine (3-MA) and rapamycin (Rapa) on autophagy and uridine 5'-diphospho-glucuronosyltransferase 1A1 (UGT1A1) induced by sulforaphane (SFN) in human colon cancer Caco-2 cells. Western blot was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) and UGT1A1 proteins. And immunocytochemistry was employed to observe the intracellular distribution of LC3 and nuclear localization of NF-E2-related factor 2 (Nrf2). Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed to examine the mRNA expression of UGT1A1 and human pregnane X receptor (hPXR). After the treatment of SFN, the LC3-II protein was induced in a dose and time-dependent manner. SFN-induced LC3-II protein could be attenuated and enhanced by 3-MA and Rapa respectively. In comparison with the control group, UGT1A1 mRNA levels increased significantly after the treatment of Rapa, SFN or their combination (2.4, 4.12 and 2.41 folds respectively, all P < 0.01). And the combination of SFN and Rapa possessed the highest level. UGT1A1 protein band intensity was also enhanced in three groups. There was no obvious nuclear staining of Nrf2 in control group while intense nuclear fluorescent labeling of Nrf2 could be observed in the SFN-treated groups, especially the combination group of SFN and Rapa. The hPXR mRNA levels increased significantly in the Rapa and combination groups (1.82 and 1.4 folds respectively, both P < 0.01). The treatment of 3-MA or Rapa may attenuate or enhance SFN-induced autophagy respectively. And Rapa also potentiates SFN-induced UGT1A1 expression. The mechanism for the synergic effect of Rapa and SFN on UGT1A1 induction may be a simultaneous activation of Nrf2 and hPXR signaling pathway.

MiR‐214 and hypoxia down‐regulate Necl‐2/CADM1 and enhance ErbB2/ErbB3 signaling

Necl-2/CADM1 is down-regulated by the promoter hypermethylation and/or the loss of heterozygosity at chromosome 11q23.2 in many types of cancers and serves as a tumor suppressor by interacting in cis with ErbB3 and suppressing the ligand-induced ErbB2/ErbB3 signaling for cell movement and death. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers. We investigated here other mechanisms that down-regulate Necl-2. miR-214, that is frequently up-regulated in a variety of cancers, targeted the 3'UTR of the Necl-2 mRNA directly, suppressed the translation of Necl-2 and enhanced the ligand-induced ErbB2/ErbB3 signaling in human colon cancer Caco-2 cells. Hypoxia reduced the Necl-2 protein level in a manner independent of miR-214 or hypoxia-inducible factor-1α in Caco-2 cells. These results indicate that miR-214 and hypoxia are novel regulators that down-regulate Necl-2 and enhance ErbB2/ErbB3 signaling.

Enhancing the antitumor cell proliferation and Cu2+-chelating effects of black soybeans through fermentation with Aspergillus awamori

In the present study, black soybeans were fermented with Aspergillus awamori at 30°C for 3 days. The effect of fermentation on the antiproliferative effect against human colon cancer cells, Caco-2 and HT-29 as well as Cu(2+)-chelating effect of black soybeans was investigated. It was found that the water, 80% methanol or 80% ethanol extract of fermented black soybeans showed a significantly higher (P<0.05) antiproliferative and Cu(2+)-chelating effect than did the respective extract of non-fermented black soybeans. Generally, the methanol extract and the ethanol extract of fermented black soybeans exerted higher antiproliferative effect on both Caco-2 and HT-29 cells. While water extract of fermented black soybeans showed the highest Cu(2+)-chelating effect among the various extracts examined. Taking into account of extraction yields further revealed that bioactive principles that exhibit Cu(2+)-chelating effect could be extracted to the largest extent with water as the extraction solvent. With same amount of sample, water extract obtained from fermented black soybeans possesses the highest Cu(2+)-chelating abilities.

Aldose Reductase Inhibition Prevents Colon Cancer Growth by Restoring Phosphatase and Tensin Homolog Through Modulation of miR-21 and FOXO3a

We have shown earlier that inhibition of aldose reductase (AR), an oxidative stress-response protein, prevents colon cancer cell growth in vitro and in vivo. Changes in microribonucleic acid (miR) expression can contribute to cancer by modulating the functional expression of critical genes involved in cancer growth and metastasis. However, the molecular mechanisms by which AR regulates miR expression and their dependent mitogenic effects in cancer cells are not known. Therefore, we investigated how AR regulates growth factor-induced expression of miRs and growth of colon cancer cells. Inhibition of AR significantly downregulated growth factor-induced miR-21 expression in human colon cancer cells, HT29, SW480, and Caco-2. Further, AR inhibition also increased phosphatase and tensin homolog (PTEN) (a direct target of miR-21) and forkhead box O3A (FOXO3a) in colon cancer cells. Our results obtained with HT29 cells ablated with FOXO3a siRNA showed increased activator protein-1 (AP-1) activation and miR-21 expression, indicating that FOXO3a represses miR-21 via AP-1 inactivation. Inhibition of AR also prevented the epidermal growth factor-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (AKT), c-Jun, c-Fos, PTEN, and FOXO3a, and deoxyribonucleic acid (DNA)-binding activity of AP-1. More importantly, in human colon adenocarcinoma xenograft tissues, miR-21 expression was lower, and PTEN and FOXO3a levels were significantly higher in AR inhibitor-treated mice compared to controls. These findings demonstrate a novel role of AR in the regulation of miR-21 and its target PTEN in growth factor-induced colon cancer cell growth. Collectively, these results show a novel role of AR in mediation of growth factor-induced colon cancer growth by modulating miR-21, PTEN, and FOXO3a expression through reactive oxygen species (ROS)/PI3K/AKT/AP-1.

Evidence of cell damage induced by major components of a diet-compatible mixture of oxysterols in human colon cancer CaCo-2 cell line

Cholesterol oxidation products, termed oxysterols, have been shown to be more reactive than unoxidized cholesterol, possessing marked pro-inflammatory and cytotoxic effects in a number of cells and tissues. Oxysterols, absorbed with the diet as products of cholesterol auto-oxidation, have recently been suggested to potentially interfere with homeostasis of the mucosal intestinal epithelium, by promoting and sustaining irreversible damage. However, the treatment of colon cancer cells with a diet-compatible mixture of oxysterols does not elicit the same responses than individual components added to the cells at the same concentrations at which they are present in the mixture. SixtyμM oxysterol mixture showed a slight pro-apoptotic effect on human colon cancer CaCo-2 cell line, evaluated in terms of caspase-3 and caspase-7 activation; conversely, 7α-hydroxycholesterol, 7β-hydroxycholesterol and 5α,6α-epoxycholesterol were identified to be able to induce a significant pro-apoptotic effect if added to cell culture singly; 7β-hydroxycholesterol had stronger action than other compounds. The enhanced production of reactive oxygen species through up-regulation of the colonic NADPH-oxidase isoform NOX1 appeared to be the key event in oxysterol-induced apoptosis in these colon cancer cells. As regards pro-inflammatory effects of oxysterols, IL-8 and MCP-1 were evaluated for their chemotactic activity. Only MCP-1 production was significantly induced by 7β-hydroxycholesterol, as well as by cholesterol and oxysterol mixture. However, oxysterol-induced inflammation appeared to be NOX1-independent, suggesting a secondary role of this enzyme in inducing inflammation in colon cancer cells. A selective cell death induced by specific oxysterols against colon cancer cells, mainly exploiting their ability to activate NOX1 in generating oxidative reactions, might represent a promising field of investigation in colorectal cancer, and might bring new insights on strategies in anticancer therapy.

Cellular location and expression of Na+, K+-ATPase α subunits affect the anti-proliferative activity of oleandrin

The purpose of this study was to investigate whether intracellular distribution of Na(+), K(+) -ATPase α3 subunit, a receptor for cardiac glycosides including oleandrin, is differentially altered in cancer versus normal cells and whether this altered distribution can be therapeutically targeted to inhibit cancer cell survival. The cellular distribution of Na(+), K(+) -ATPase α3 isoform was investigated in paired normal and cancerous mucosa biopsy samples from patients with lung and colorectal cancers by immunohistochemical staining. The effects of oleandrin on α3 subunit intracellular distribution, cell death, proliferation, and EKR phosphorylation were examined in differentiated and undifferentiated human colon cancer CaCO-2 cells. While Na(+), K(+) -ATPase α3 isoform was predominantly located near the cytoplasmic membrane in normal human colon and lung epithelia, the expression of this subunit in their paired cancer epithelia was shifted to a peri-nuclear position in both a qualitative and quantitative manner. Similarly, distribution of α3 isoform was also shifted from a cytoplasmic membrane location in differentiated human colon cancer CaCO-2 cells to a peri-nuclear position in undifferentiated CaCO-2 cells. Intriguingly, oleandrin exerted threefold stronger anti-proliferative activity in undifferentiated CaCO-2 cells (IC50, 8.25 nM) than in differentiated CaCO-2 cells (IC50, >25 nM). Oleandrin (10 to 20 nM) caused an autophagic cell death and altered ERK phosphorylation in undifferentiated but not in differentiated CaCO-2 cells. These data demonstrate that the intracellular location of Na(+), K(+) -ATPase α3 isoform is altered in human cancer versus normal cells. These changes in α3 cellular location and abundance may indicate a potential target of opportunity for cancer therapy.