Metastatic Colon Carcinoma Cells Research Articles

Sulforaphane has an additive anticancer effect to FOLFOX in highly metastatic human colon carcinoma cells.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Patients with CRC may need chemotherapy (CTx) in a neoadjuvant, adjuvant or palliative setting through the course of the disease. Unfortunately, its effect is limited by chemoresistance and chemotoxicity. Novel more effective and non‑toxic CTx regimens are needed to further improve CRC treatment outcomes. Thus, the present study was designed to test the hypothesis that non‑toxic sulforaphane (SF) is effective against CRC and has additive effects in combination with conventional 5‑fluorouracil, oxaliplatin and folinic acid (FOLFOX) CTx invitro. Highly metastatic human colon cancer cells, CX‑1, and fibroblasts were treated with FOLFOX ± SF. Cell viability was assessed using an MTT assay. The level of apoptosis and the expression of apoptotic proteins were measured by TUNEL assay and quantitative PCR analysis. Aldehyde dehydrogenase isoform 1 (ALDH1) and multidrug resistance protein 2 (MRP2) levels were evaluated. The ability of cells to form spheroids was measured in three‑dimensional cell culture. SF alone and in combination with FOLFOX effectively decreased the viability of the CX‑1 cells, promoted apoptosis within the CX‑1 cells, prevented cellular spheroid formation and decreased ALDH1 activity. However, SF promoted MRP2 expression and protein levels. In conclusion, SF together with conventional FOLFOX has additive anticancer effects against highly metastatic human CRC invitro.

Concise synthesis and biological evaluation of 2-Aryl-3-Anilinobenzo[b]thiophene derivatives as potent apoptosis-inducing agents

Many clinically used agents active in cancer chemotherapy exert their activity through the induction of cell death (apoptosis) by targeting microtubules, altering protein function or inhibiting DNA synthesis. The benzo[b]thiophene scaffold holds a pivotal place as a pharmacophore for the development of anticancer agents, and, in addition, this scaffold has many pharmacological activities. We have developed a flexible method for the construction of a new series of 2-aryl-3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophenes as potent antiproliferative agents, giving access to a wide range of substitution patterns at the 2-position of the 6-methoxybenzo[b]thiophene common intermediate. In the present study, all the synthesized compounds retained the 3-(3,4,5-trimethoxyanilino)-6-methoxybenzo[b]thiophene moiety, and the structure–activity relationship was examined by modification of the aryl group at its 2-position with electron-withdrawing (F) or electron-releasing (alkyl and alkoxy) groups. We found that small substituents, such as fluorine or methyl, could be placed in the para-position of the 2-phenyl ring, and these modifications only slightly reduced antiproliferative activity relative to the unsubstituted 2-phenyl analogue. Compounds 3a and 3b, bearing the phenyl and para-fluorophenyl at the 2-position of the 6-methoxybenzo[b]thiophene nucleus, respectively, exhibited the greatest antiproliferative activity among the tested compounds. The treatment of both Caco2 (not metastatic) and HCT-116 (metastatic) colon carcinoma cells with 3a or 3b triggered a significant induction of apoptosis as demonstrated by the increased expression of cleaved-poly(ADP-ribose) polymerase (PARP), receptor-interacting protein (RIP) and caspase-3 proteins. The same effect was not observed with non-transformed colon 841 CoN cells. A potential additional effect during mitosis for 3a in metastatic cells and for 3b in non-metastatic cells was also observed.

Lymph Node Subcapsular Sinus Microenvironment-On-A-ChipModeling Shear Flow Relevant to Lymphatic Metastasis and Immune Cell Homing.

SummaryA lymph node sinus-on-a-chip adhesion microfluidic platform that recapitulates the hydrodynamic microenvironment of the lymph node subcapsular sinus was engineered. This device was used to interrogate the effects of lymph node remodeling on cellular adhesion in fluid flow relevant to lymphatic metastasis. Wall shear stress levels analytically estimated and modeled after quiescent and diseased/inflamed lymph nodes were experimentally recapitulated using a flow-based microfluidic perfusion system to assess the effects of physiological flow fields on human metastatic cancer cell adhesion. Results suggest that both altered fluid flow profiles and presentation of adhesive ligands, which are predicted to manifest within the lymph node subcapsular sinus as a result of inflammation-induced remodeling, and the presence of lymph-borne monocytic cells may synergistically contribute to the dynamic extent of cell adhesion in flow relevant to lymph node invasion by cancer and monocytic immune cells during lymphatic metastasis.

SUV39H1 regulates human colon carcinoma apoptosis and cell cycle to promote tumor growth.

Trimethylation of histone 3 lysine 9 (H3K9me3) at gene promoters is a major epigenetic mechanism that silences gene expression. We have developed a small molecule inhibitor for the H3K9me3-specific histone methyltransferase SUV39H1. We report here that FAS expression is significantly down-regulated and SUV39H1 expression is significantly up-regulated in human colorectal carcinoma (CRC) as compared to normal colon. SUV39H1-selective inhibitor F5446 decreased H3K9me3 deposition at the FAS promoter, increased Fas expression, and increased CRC cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, inhibition of SUV39H1 altered the expression of genes with known functions in DNA replication and cell cycle in the metastatic colon carcinoma cells, which is associated with cell cycle arrest at S phase in the metastatic human colon carcinoma cells, resulting in tumor cell apoptosis and growth inhibition in a concentration-dependent manner in vitro. Moreover, F5446 increased 5-FU-resistant human CRC sensitivity to both 5-FU- and FasL-induced apoptosis and inhibited tumor cell growth in vitro. More importantly, F5446 suppressed human colon tumor xenograft growth in vivo. Our data indicate that pharmacological inhibition of SUV39H1 is an effective approach to suppress human CRC.

Effects of α-lactalbumin and sulindac on primary and metastatic human colon cancer cell lines

Abstract Background HAMLET (human α-lactalbumin made lethal to tumor cells) and sulindac are active ingredients which are used in the treatment of cancers. Under both in vivo and in vitro conditions, HAMLET binds to the surface of tumor cells and enters the cells. Sulindac is one of nonsteroidal anti-inflammatory drugs. It inhibits the growth of tumor cells by inducing the apoptosis. In our study, we aimed to investigate effects of α-lactalbumin and sulindac on COLO-320 primary and COLO-741 metastatic human colon carcinoma cell lines via mitochondrial apoptotic pathway by indirect immunocytochemistry and TUNEL assays. Materials and methods The effects of α-lactalbumin and sulindac were assessed by using MTT assay at 24, 48 and 72 h and apoptosis markers caspase-3, caspase-9 and cytochrome-c were detected using immunocytochemistry and TUNEL methods. Results It was appeared that α-lactalbumin and sulindac may triggger mechanisms of apoptosis in both primary and metastatic colon carcinoma cell lines and the primary colon carcinoma cell line was affected more than the metastatic cells. Conclusion It is proposed that α-lactalbumin and sulindac can be used in cancer treatments and future in vivo experiments.

Increased glucocorticoid receptor activity and proliferation in metastatic colon cancer

Metastasis is regarded as the fatal hallmark for colon cancer, but molecular mechanisms responsible for it have remained poorly defined. Glucocorticoid receptor (GR) within the tumor microenvironment mediates the effects of stress hormones which are used in clinics for their inflammation-modulatory and immunosuppressive properties. Further, epigenetic activation of GR promotes tumor heterogeneity and metastasis. Here, we sought to investigate the correlation between GR activation and proliferation and invasion in metastatic colon cancer microenvironment. We used proliferation/invasion assays, western blot, RT-qPCR, immunofluorescence staining and quantitative methylation to study glucocorticoid-GR signaling, including the involvement of CDK1, in human colon adenocarcinoma cell lines HT29 and T84 (a representative metastatic cell line). Nuclear expression levels of GR were significantly upregulated in metastatic T84 cells, and glucocorticoid derivative, dexamethasone (DEX) treatment caused increased proliferation and invasion in T84 cell, compared to HT29 cell. DEX treatment induced CDK1 expression which was accompanied by reduced CDK1 methylation, indicating epigenetic regulation. Depletion of GR suppressed proliferation of metastatic colon carcinoma cells and depletion of CDK1 had similar suppressing effects on proliferation as well as invasion of metastatic cells. Our study suggests that glucocorticoid-GR-CDK1 signaling induces proliferation and invasion of colon cancer cells and therapies involving the use of glucocorticoids need to exercise caution and re-evaluation.

An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers.

Metastasis is a major cause of mortality in cancer patients. However, the mechanisms governing the metastatic process remain elusive, and few accurate biomarkers exist for predicting whether metastasis will occur, something that would be invaluable for guiding therapy. We report here that the carboxypeptidase E gene (CPE) is alternatively spliced in human tumors to yield an N-terminal truncated protein (CPE-ΔN) that drives metastasis. mRNA encoding CPE-ΔN was found to be elevated in human metastatic colon, breast, and hepatocellular carcinoma (HCC) cell lines. In HCC cells, cytosolic CPE-ΔN was translocated to the nucleus and interacted with histone deacetylase 1/2 to upregulate expression of the gene encoding neural precursor cell expressed, developmentally downregulated gene 9 (Nedd9)--which has been shown to promote melanoma metastasis. Nedd9 upregulation resulted in enhanced in vitro proliferation and invasion. Quantification of mRNA encoding CPE-ΔN in HCC patient samples predicted intrahepatic metastasis with high sensitivity and specificity, independent of cancer stage. Similarly, high CPE-ΔN mRNA copy numbers in resected pheochromocytomas/paragangliomas (PHEOs/PGLs), rare neuroendocrine tumors, accurately predicted future metastasis or recurrence. Thus, CPE-ΔN induces tumor metastasis and should be investigated as a potentially powerful biomarker for predicting future metastasis and recurrence in HCC and PHEO/PGL patients.

Analyzing Mechanisms of Metastatic Cancer Cell Adhesive Phenotype Leveraging Preparative Adhesion Chromatography Microfluidic.

An integrated, parallel-plate microfluidic device is engineered to interrogate and fractionate cells based on their adhesivity to a substrate surface functionalized with adhesive ligand in a tightly controlled flow environment to elucidate associated cell-intrinsic pathways. Wall shear stress levels and endothelial presentation of E-selectin are modeled after the inflamed vasculature microenvironment in order to simulate in vitro conditions under which in vivo hematogenous metastasis occurs. Based on elution time from the flow channel, the collection of separate fractions of cells-noninteracting and interacting-at high yields and viabilities enables multiple postperfusion analyses, including flow cytometry, in vivo metastasis modeling, and transcriptomic analysis. This platform enables the interrogation of flow-regulated cell molecular profiles, such as (co)expression levels of natively expressed selectin ligands sLex , CD44, and carcinoembryonic antigen, and cancer stem cell marker CD24. This additionally reveals E-selectin adhesivity exhibited by metastatic human colon carcinoma cells to be a transient phenotype. Facile and rapid, this methodology for unbiased, label free sorting of large populations of cells based on their adhesion in flow represents a method of studying flow-regulated adhesion in vitro for the identification of molecular drug targets for development as antimetastatic cancer therapeutics.

Insulin-like growth factor-1 activates different catalytic subunits p110 of PI3K in a cell-type-dependent manner to induce lipogenesis-dependent epithelial-mesenchymal transition through the regulation of ADAM10 and ADAM17.

The activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) is critical for the induction of epithelial-mesenchymal transition (EMT) by growth factors, including insulin-like growth factor 1 (IGF-1). The activation of intracellular lipogenesis provides proliferative and survival signals for cancer cells. In this study, we investigated the connection between lipogenesis-related EMT processes and IGF-1-mediated PI3K p110 isoform activation in primary (SW480 cells) and metastatic (SW620) colon carcinoma cells. We also examined the underlying signaling pathway that promotes fatty acid synthesis in IGF-1-activated colon cancer cells. IGF-1 stimulation upregulated the expression of lipogenic enzymes as well as the activation of Nardilysin (N-arginine dibasic convertase, NRD1) and its downstream targets, a disintegrin and metalloproteases 10 (ADAM10) and ADAM17. The upregulation of the Lyn/Syk-mediated PI3K p110δ isoform in SW480 cells and the Lyn-dependent PI3K p110α isoform in SW620 cells triggered fatty acid production and cell motility in IGF-1-activated colon cancer cells. Pharmacological inhibition with A66 (PI3K p110α specific inhibitor) and CAL-101 (PI3K p110δ specific inhibitor) efficiently inhibited EMT in colon cancer cells by blocking the NRD1/ADAM family protein signaling pathway. Gene silencing of NRD1 and ADAM family proteins attenuated the generation of intracellular fatty acid and the migratory activity of colon cancer cells. Our results suggest that the different isoforms of the PI3K p110 subunit could be therapeutic targets for primary and metastatic colon cancer and that regulation of the NRD1/ADAM signaling pathway controls lipogenesis-mediated EMT in IGF-1-stimulated colon cancer cells.

The type I insulin-like growth factor regulates the liver stromal response to metastatic colon carcinoma cells.

Hepatic stellate cells (HSC) play a major role in initiating the liver fibrogenic (wounding) response of the liver and can also orchestrate a pro-metastatic microenvironment in the liver in response to invading cancer cells. Here we explored the role of the hepatic stellate cells in colon carcinoma liver metastasis with emphasis on the contribution of the insulin-like growth factor (IGF) axis to their activation and function. To this end, we used mice with a Tamoxifen inducible liver IGF-I deficiency. We found that in mice with a sustained IGF-I deficiency, recruitment and activation of HSC into tumor-infiltrated areas of the liver were markedly diminished, resulting in decreased collagen deposition and reduced tumor expansion. In addition, IGF-I could rescue HSC from apoptosis induced by pro-inflammatory factors such as TNF-α known to be upregulated in the early stages of liver metastasis. Moreover, in surgical specimens, activated IGF-IR was observed on HSC-like stromal cells surrounding colorectal carcinoma liver metastases. Finally, IGF-targeting in vivo using an IGF-Trap caused a significant reduction in HSC activation in response to metastatic colon cancer cells. Therefore, our data identify IGF as a survival factor for HSC and thereby, a promoter of the pro-metastatic microenvironment in the liver. IGF-targeting could therefore provide a strategy for curtailing the pro-metastatic host response of the liver during the early stages of liver metastasis.

Interaction Study of Lipopeptide Biosurfactant viscosin with DPPC and Cholesterol by Langmuir Monolayer Technique

Langmuir monolayers at the air/water interface are frequently used to establish a pseudo-physiological environment needed to study peptides and proteins properties to mimic their interaction with the cell membrane. The self-assembled monolayers formed at the air/water interface that respond to external stimuli and re-structure upon interaction with viscosin peptide in controlled environment have been studied in details. The mechanical and thermodynamic properties of the resulting monolayer are also influenced by temperature and pressure of the resulting mixture. Herein, two distinct pathways were found to contribute to equilibrium formation of DPPC and Cholesterol monolayers. Our data provided an estimate for Gibbs free energy of mixing, the elastic modulus and the classification of the final mixing behavior. Fluorescence microscopy (FM) imaging was also used to assess the morphological and structural changes within the fabricated monolayers. A molecular biological view has been established between our in vitro results and the clinical inhibition of viscosin toward the metastatic cancer and colon carcinoma cells.

H3K9 Trimethylation Silences Fas Expression To Confer Colon Carcinoma Immune Escape and 5-Fluorouracil Chemoresistance.

The Fas-FasL effector mechanism plays a key role in cancer immune surveillance by host T cells, but metastatic human colon carcinoma often uses silencing Fas expression as a mechanism of immune evasion. The molecular mechanism under FAS transcriptional silencing in human colon carcinoma is unknown. We performed genome-wide chromatin immunoprecipitation sequencing analysis and identified that the FAS promoter is enriched with H3K9me3 in metastatic human colon carcinoma cells. The H3K9me3 level in the FAS promoter region is significantly higher in metastatic than in primary cancer cells, and it is inversely correlated with Fas expression level. We discovered that verticillin A is a selective inhibitor of histone methyltransferases SUV39H1, SUV39H2, and G9a/GLP that exhibit redundant functions in H3K9 trimethylation and FAS transcriptional silencing. Genome-wide gene expression analysis identified FAS as one of the verticillin A target genes. Verticillin A treatment decreased H3K9me3 levels in the FAS promoter and restored Fas expression. Furthermore, verticillin A exhibited greater efficacy than decitabine and vorinostat in overcoming colon carcinoma resistance to FasL-induced apoptosis. Verticillin A also increased DR5 expression and overcame colon carcinoma resistance to DR5 agonist drozitumab-induced apoptosis. Interestingly, verticillin A overcame metastatic colon carcinoma resistance to 5-fluorouracil in vitro and in vivo. Using an orthotopic colon cancer mouse model, we demonstrated that tumor-infiltrating cytotoxic T lymphocytes are FasL(+) and that FasL-mediated cancer immune surveillance is essential for colon carcinoma growth control in vivo. Our findings determine that H3K9me3 of the FAS promoter is a dominant mechanism underlying FAS silencing and resultant colon carcinoma immune evasion and progression.

Abstract 2756: Ceramide analog targets xIAP and cIAP1 to sensitize metastatic colon carcinoma cells to apoptosis induction to suppress tumor progression

Abstract Sphingolipids are bioeffectors that mediate various cellular processes. We aimed to identify molecular targets of ceramide in the apoptosis pathway and to determine the efficacy of a ceramide analog in overcoming cancer cell resistance to apoptosis induction. We observed that exposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to FasL-induced apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to FasL-induced apoptosis. LCL85 resembles Smac mimetic in sensitization of colon carcinoma cells to FasL-induced apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. Consistent with its apoptosis sensitization activity, LCL85 suppressed colon carcinoma cell metastatic potential in an experimental colon carcinoma lung metastasis mouse model. Taken together, we determined that a sublethal dose of LCL85 is effective as a sensitizer in overcoming apoptosis resistance of metastatic human colon carcinoma cells, and LCL85 is effective in suppressing tumor metastasis in vivo. Citation Format: Amy Paschall, Mary Zimmerman, Christina Torres, Dafeng Yang, May Chen, Xia Li, Erhard Bieberich, Aiping Bai, Jacek Bielawski, Alicja Bielawska, Kebin Liu. Ceramide analog targets xIAP and cIAP1 to sensitize metastatic colon carcinoma cells to apoptosis induction to suppress tumor progression. [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 2756. doi:10.1158/1538-7445.AM2014-2756

Ceramide targets xIAP and cIAP1 to sensitize metastatic colon and breast cancer cells to apoptosis induction to suppress tumor progression

BackgroundCeramide is a bioeffector that mediates various cellular processes, including apoptosis. However, the mechanism underlying ceramide function in apoptosis is apparently cell type-dependent and is not well-understood. We aimed at identifying molecular targets of ceramide in metastatic human colon and breast cancer cells, and determining the efficacy of ceramide analog in suppression of colon and breast cancer metastasis.MethodsThe activity of and mechanism underlying ceramide as a cytotoxic agent, and as a sensitizer for Fas-mediated apoptosis was analyzed in human cell lines established from primary or metastatic colon and breast cancers. The efficacy of ceramide analog LCL85 in suppression of metastasis was examined in preclinical mouse tumor models.ResultsExposure of human colon carcinoma cells to ceramide analog LCL85 results in apoptosis in a dose-dependent manner. Interestingly, a sublethal dose of LCL85 increased C16 ceramide content and overcame tumor cell resistance to Fas-mediated apoptosis. Subsequently, treatment of tumor cells with exogenous C16 ceramide resulted in increased tumor cell sensitivity to Fas-mediated apoptosis. LCL85 resembles Smac mimetic BV6 in sensitization of colon carcinoma cells to Fas-mediated apoptosis by inducing proteasomal degradation of cIAP1 and xIAP proteins. LCL85 also decreased xIAP1 and cIAP1 protein levels and sensitized metastatic human breast cancer cells to Fas-mediated apoptosis. Silencing xIAP and cIAP1 with specific siRNAs significantly increased the metastatic human colon carcinoma cell sensitivity to Fas-mediated apoptosis, suggesting that IAP proteins mediate apoptosis resistance in metastatic human colon carcinoma cells and ceramide induces IAP protein degradation to sensitize the tumor cells to apoptosis induction. Consistent with its apoptosis sensitization activity, subtoxic doses of LCL85 suppressed colon carcinoma cell metastatic potential in an experimental lung metastasis mouse model, as well as breast cancer growth and spontaneous lung metastasis in an orthotopic breast cancer mouse model.ConclusionWe have identified xIAP and cIAP1 as molecular targets of ceramide and determined that ceramide analog LCL85 is an effective sensitizer in overcoming resistance of human cell lines established from metastatic colon and breast cancers to apoptosis induction to suppress metastasis in vivo.

PDGF Suppresses the Sulfation of CD44v and Potentiates CD44v-Mediated Binding of Colon Carcinoma Cells to Fibrin under Flow

Fibrin(ogen) mediates sustained tumor cell adhesion and survival in the pulmonary vasculature, thereby facilitating the metastatic dissemination of tumor cells. CD44 is the major functional fibrin receptor on colon carcinoma cells. Growth factors, such as platelet-derived growth factor (PDGF), induce post-translational protein modifications, which modulate ligand binding activity. In view of the roles of PDGF, fibrin(ogen) and CD44 in cancer metastasis, we aimed to delineate the effect of PDGF on CD44-fibrin recognition. By immunoprecipitating CD44 from PDGF-treated and untreated LS174T colon carcinoma cells, which express primarily CD44v, we demonstrate that PDGF enhances the adhesion of CD44v-coated beads to immobilized fibrin. Enzymatic inhibition studies coupled with flow-based adhesion assays and autoradiography reveal that PDGF augments the binding of CD44v to fibrin by significantly attenuating the extent of CD44 sulfation primarily on chondroitin and dermatan sulfate chains. Surface plasmon resonance assays confirm that PDGF enhances the affinity of CD44v-fibrin binding by markedly reducing its dissociation rate while modestly increasing the association rate. PDGF mildly reduces the affinity of CD44v-hyaluronan binding without affecting selectin-CD44v recognition. The latter is attributed to the fact that CD44v binds to selectins via sialofucosylated O-linked residues independent of heparan, dermatan and chondroitin sulfates. Interestingly, PDGF moderately reduces the sulfation of CD44s and CD44s-fibrin recognition. Collectively, these data offer a novel perspective into the mechanism by which PGDF regulates CD44-dependent binding of metastatic colon carcinoma cells to fibrin(ogen).

339 Wnt/Beta Catenin Pathway Differences After Treatment of Primary and Metastatic Colon Carcinoma Cell Lines

339 Wnt/Beta Catenin Pathway Differences After Treatment of Primary and Metastatic Colon Carcinoma Cell Lines

Comparative label‐free LC‐MS/MS analysis of colorectal adenocarcinoma and metastatic cells treated with 5‐fluorouracil

A label-free mass spectrometric strategy was used to examine the effect of 5-fluorouracil (5-FU) on the primary and metastatic colon carcinoma cell lines, SW480 and SW620, with and without treatment. 5-FU is the most common chemotherapeutic treatment for colon cancer. Pooled biological replicates were analyzed by nanoLC-MS/MS and protein quantification was determined via spectral counting. Phenotypic and proteomic changes were evident and often similar in both cell lines. The SW620 cells were more resistant to 5-FU treatment, with an IC(50) 2.7-fold higher than that for SW480. In addition, both cell lines showed pronounced abundance changes in pathways relating to antioxidative stress response and cell adhesion remodeling due to 5-FU treatment. For example, the detoxification enzyme NQO1 was increased with treatment in both cell lines, while disparate members of the peroxiredoxin family, PRDX2 or PRDX5 and PRDX6, were elevated with 5-FU exposure in either SW480 or SW620, respectively. Cell adhesion-associated proteins CTNNB1 and RhoA showed decreased expression with 5-FU treatment in both cell lines. The differential quantitative response in the proteomes of these patient-matched cell lines to drug treatment underscores the subtle molecular differences separating primary and metastatic cancer cells.

Decitabine and Vorinostat Cooperate To Sensitize Colon Carcinoma Cells to Fas Ligand-Induced Apoptosis In Vitro and Tumor Suppression In Vivo

The death receptor Fas and its physiological ligand (FasL) regulate apoptosis of cancerous cells, thereby functioning as a critical component of the host cancer immunosurveillance system. To evade Fas-mediated apoptosis, cancer cells often downregulate Fas to acquire an apoptosis-resistant phenotype, which is a hallmark of metastatic human colorectal cancer. Therefore, targeting Fas resistance is of critical importance in Fas-based cancer therapy and immunotherapy. In this study, we demonstrated that epigenetic inhibitors decitabine and vorinostat cooperate to upregulate Fas expression in metastatic human colon carcinoma cells. Decitabine also upregulates BNIP3 and Bik expression, whereas vorinostat decreased Bcl-x(L) expression. Altered expression of Fas, BNIP3, Bik, and Bcl-x(L) resulted in effective sensitization of the metastatic human colon carcinoma cells to FasL-induced apoptosis. Using an experimental metastasis mouse model, we further demonstrated that decitabine and vorinostat cooperate to suppress colon carcinoma metastasis. Analysis of tumor-bearing lung tissues revealed that a large portion of tumor-infiltrating CD8(+) T cells are FasL(+), and decitabine and vorinostat-mediated tumor-suppression efficacy was significantly decreased in Fas(gld) mice compared with wild-type mice, suggesting a critical role for FasL in decitabine and vorinostat-mediated tumor suppression in vivo. Consistent with their function in apoptosis sensitization, decitabine and vorinostat significantly increased the efficacy of CTL adoptive transfer immunotherapy in an experimental metastasis mouse model. Thus, our data suggest that combined modalities of chemotherapy to sensitize the tumor cell to Fas-mediated apoptosis and CTL immunotherapy is an effective approach for the suppression of colon cancer metastasis.

Abstract 4888: Decitabine and vorinostat cooperate to sensitize metastatic human colon carcinoma cells to Fas-mediated apoptosis

Abstract The death receptor Fas and its physiological ligand, FasL, which is expressed on the surface of cytotoxic T-cells (CTLs), regulate apoptosis of cancerous cells. While normal colon epithelial cells express high levels of Fas, Fas expression is diminished in colon carcinomas. Metastatic colon cancers acquire an apopotosis-resistant phenotype often due to the loss of Fas expression and function. Previous studies have implicated Fas-mediated apoptosis as an important contributor of tumor regression. Therefore, targeting Fas resistance is of critical importance in treating colon cancer through Fas-based cancer therapy and immunotherapy. In this study, we explore whether epigenetic pharmacological agents can restore Fas expression and re-establish sensitivity to Fas-mediated apoptosis in metastatic human colon carcinoma cells. First we determined that the epigenetic drugs Decitabine, a DNA methyltransferase inhibitor, and Vorinostat, a HDAC inhibitor, each are capable of up-regulating Fas expression. When combined, these two inhibitors cooperate to increase Fas expression to an even greater extent than each agent alone. Pre-treatment with decitabine, vorinostat, or both drugs followed by incubation with FasL, was able to overcome resistance to FasL-induced apoptosis. Analysis of bisulfite-modified genomic DNA, isolated from human metastatic colon carcinoma cells revealed that the Fas promoter is sporadically methylated; therefore sensitization of Fas receptor expression on the cell surface by decitabine was unlikely through direct inhibition of Fas promoter meythlation. Although the Fas promoter was not heavily methylated, the promoters for BNIP3 and Bik, which are downstream of Fas signaling, were methylated. After treatment with these pharmacological epigenetic inhibitors, alone or in combination, protein levels of both BNIP3 and Bik were increased. Our data suggest that combined modalities of chemotherapy and Fas-based immunotherapy may be an effective approach for the suppression of colon cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4888. doi:1538-7445.AM2012-4888

Effect of AA861 combined with celecoxib on metastatic colon carcinoma cell

Objective To investigate the effects of cyclooxygenase-2 (COX-2) selective inhibitor combined with 5-lipoxygenase (5-LOX) inhibitor docebenone (AA861) on cell proliferation and migration of human colon cancer cell line HT-29. Methods Cultured in vitro of HT-29 cells in high metastatic colon cancer, A A861 and celecoxib on colon cancer cell drugs for 48 h, the cell proliferation was assayed by MTT; the migration of cell was detected by Transwell; immunofluorescence staining of intracellular changes in ICAM-1 protein, RT-PCR detect the gene expression of ICAM-1 and VEGF. Results MTT and Transwell experiments showed that the inhibition on celecoxib and AA861 on colon cancer was dose-dependent and time-dependent. And the inhibition of AA861 and celecoxib 100 μmol/L alone on colon cancer cells proliferation were 43.2 % and 42.8%, and when the two drugs 100 μmol/L combined on colon cancer cells the inhibition was 53.8%, and the difference between alone group and combined group was statistical (P ＜0.001). The inhibition of AA861 and celecoxib 100 μmol/L alone on colon cancer cells migration were 32.0 % and 29.3%, and when the two drugs 100 μ,mol/L combined on colon cancer cells the inhibition is 57.8 %, and the difference of between alone group and combined group was statistical (P ＜0.001). Immunefluorescence staining and RT-PCR results suggested that the two drugs can inhibit ICAM-1 and VEGF protein and gene expression, and when the two drugs combined, a stronger inhibition effect appeared than used alone (P＜0.001). Conclusion Low-dose celecoxib and AA861 combined has a synergistic inhibited effect on colon cancer cells invasion and metastasis, and the mechanism relates with the VEGF and ICAM-1 expression. Key words: Neoplasms, experimental; Colonic neoplasms; Drug therapy, combination; Neoplasms metastasis