lithium-chloride and Colonic-Neoplasms

Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide. It is also the second most common cause of cancer‑associated mortality; it accounted for about 9.2% of all cancer deaths in 2018, most of which were due to resistance to therapy. The main treatment for CRC is surgery, generally associated with chemotherapy, radiation therapy and combination therapy. However, while chemo‑radiotherapy kills differentiated cancer cells, mesenchymal stem‑like cells are resistant to this treatment, and this can give rise to therapy‑resistant tumors. Our previous study isolated T88 primary colon cancer cells from a patient with sporadic colon cancer. These cells exhibited mesenchymal and epithelial features, high levels of epithelial‑to‑mesenchymal transition transcription factors, and stemness markers. In addition, it was revealed that lithium chloride (LiCl), a specific glycogen synthase kinase (GSK)‑3β inhibitor, induced both the mesenchymal‑to‑epithelial transition and differentiation, and also reduced cell migration, stemness features and cell plasticity in these primary colon cancer cells. The aim of the present study was to investigate the effect of LiCl treatment on the viability of primary colon cancer cells exposed to 7 Gy delivered by high‑energy photon beams, which corresponds to 6 megavolts of energy. To achieve this aim, the viability of irradiated T88 cells was compared with that of irradiated T88 cells pre‑treated with LiCl. As expected, it was observed that LiCl sensitized primary colon cancer cells to high‑energy photon irradiation treatment. Notably, the decrease in cell viability was greater with combined therapy than with irradiation alone. To explore the molecular basis of this response, the effect of LiCl on the expression of Bax, p53 and Survivin, which are proteins involved in the apoptotic mechanism and in death escape, was analyzed. The present study revealed that LiCl upregulated the expression of pro‑apoptotic proteins and downregulated the expression of proteins involved in survival. These effects were enhanced by high‑energy photon irradiation, suggesting that LiCl could be used to sensitize colon cancer cells to radiation therapy.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Humans; Lithium Chloride; Photons; Radiation Tolerance; Radiation-Sensitizing Agents; Radiotherapy, High-Energy; Signal Transduction

Metastasis-associated in colon cancer 1 (MACC1) has been reported to be overexpressed in multiple cancers and promote proliferation, metastasis, cancer stem cell-like properties, and drug resistance of cancer cells. Despite its significance and the considerable knowledge accumulated on the function of MACC1 in various types of human malignancies, regulatory mechanisms underlying MACC1 expression remain unclear. Here we report that MACC1 is a direct target of Wnt/β-catenin signaling pathway in colon cancer cells and that DBC1 functions as a coactivator for Wnt-mediated MACC1 expression by promoting the activity of a LEF1/β-catenin-dependent enhancer located in intron 1 of MACC1 gene. DBC1 is required for LEF1/β-catenin complex formation on the MACC1 enhancer and for long-distance enhancer-promoter interaction of the MACC1 locus. MACC1 expression was increased in colonosphere cells compared to adherent colon cancer cells, and DBC1 overexpression further increased MACC1 expression in colonospheres and promoted sphere-forming abilities of colon cancer cells and drug resistance of colonospheres. Importantly, expressions of MACC1 and DBC1 are positively correlated with each other, upregulated in high-risk groups of colorectal cancer patients, and associated with poor survival. Our results establish MACC1 as a transcriptional target of Wnt/β-catenin signaling and suggest that DBC1 plays a key role in colorectal cancer progression through Wnt/β-catenin-MACC1 signaling axis.

Topics: Adaptor Proteins, Signal Transducing; beta Catenin; Cell Line, Tumor; Chromatin; Colonic Neoplasms; Enhancer Elements, Genetic; Gene Expression Regulation, Neoplastic; Humans; Kaplan-Meier Estimate; Lithium Chloride; Neoplastic Stem Cells; Prognosis; Promoter Regions, Genetic; Protein Binding; RNA Interference; RNA, Small Interfering; Trans-Activators; Transcription Factors; Wnt Signaling Pathway

Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of β-catenin on the plasma membrane and inhibition of its transcriptional activity. To better characterize the causal relationship between β-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting (FH535 treatment, transcription factor 7-like 2 siRNA inactivation, Wnt withdrawal) or stimulating (LiCl treatment) β-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the β-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of β-catenin in CRC. The ability of MUCDHL to sequester and inhibit β-catenin appears to provide a positive feedback enforcing the effect of β-catenin inhibitors rather than serving as the primary mechanism responsible for β-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with β-catenin inhibitory activity.

Topics: beta Catenin; Caco-2 Cells; Cadherin Related Proteins; Cadherins; Colonic Neoplasms; Enterocytes; Feedback, Physiological; Gene Expression Regulation, Neoplastic; HCT116 Cells; Humans; Lithium Chloride; Primary Cell Culture; RNA, Small Interfering; Sulfonamides; Tissue Culture Techniques; Transcription Factor 7-Like 2 Protein; Wnt Signaling Pathway

Epithelial‑to‑mesenchymal transition (EMT) confers stem cell‑like phenotype and more motile properties to carcinoma cells. During EMT, the expression of E‑cadherin decreases, resulting in loss of cell‑cell adhesion and increased migration. Expression of Twist1 and other pleiotropic transcription factors, such as Snail, is known to activate EMT. We established primary colon cancer cell cultures from samples of operated patients and validated cultures by cytogenetic and molecular biology approaches. Western blot assay, quantitative real‑time PCR and immunofluorescence were performed to investigate the expression of E‑cadherin, vimentin, β‑catenin, cytokeratin‑20 and ‑18, Twist1, Snail, CD44, cyclooxygenase‑2 (COX2), Sox2, Oct4 and Nanog. Moreover, cell differentiation was induced by incubation with LiCl‑containing medium for 10 days. We observed that these primary colorectal cancer (CRC) cells lost expression of the E‑cadherin epithelial marker, which was instead expressed in cancer and normal colon mucosa of the same patient, while overexpressed vimentin (mesenchymal marker), Twist1, Snail (EMT markers) and COX2. Cytokeratin‑18 was expressed both in tissues and cell cultures. Expression of stem cell markers, such as CD44, Oct4 and Nanog, were also observed. Following differentiation with the glycogen synthase kinase 3β (GSK3β) inhibitor LiCl, the cells began to express E‑cadherin and, at once, Twist1 and Snail expression was strongly downregulated, suggesting a MET‑reverting process. In conclusion, we established primary colon mesenchymal cancer cell cultures expressing mesenchymal and epithelial biomarkers together with high level of EMT transcription factors. We propose that they could represent a good model for studying EMT and its reverting mechanism, the mesenchymal‑to‑epithelial transition (MET). Our observation indicates that LiCl, a GSK3β inhibitor, induces MET in vitro, suggesting that LiCl and GSK3β could represent, respectively, interesting drug, and target for CRC therapy.

Topics: Adjuvants, Immunologic; Biomarkers, Tumor; Blotting, Western; Cell Differentiation; Colonic Neoplasms; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; Humans; Lithium Chloride; Neoplasm Proteins; Real-Time Polymerase Chain Reaction; Tumor Cells, Cultured

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/β-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/β-catenin, where the flavonoid acts downstream of β-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/β-catenin and should be further investigated as a potential novel anti-tumoral agent.

Topics: Active Transport, Cell Nucleus; Animals; Antineoplastic Agents; beta Catenin; Blotting, Western; Body Patterning; Cell Line; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Colonic Neoplasms; Early Growth Response Protein 2; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; HCT116 Cells; Humans; Immunohistochemistry; In Situ Hybridization; Lithium Chloride; Quercetin; Reverse Transcriptase Polymerase Chain Reaction; Wnt Signaling Pathway; Xenopus; Xenopus Proteins

Evasion from chemotherapy-induced apoptosis due to p53 loss strongly contributes to drug resistance. Identification of specific targets for the treatment of drug-resistant p53-null tumors would therefore increase the effectiveness of cancer therapy.. By using a kinase-directed short hairpin RNA library and HCT116p53KO drug-resistant colon carcinoma cells, glycogen synthase kinase 3 beta (GSK3B) was identified as a target whose silencing bypasses drug resistance due to loss of p53. p53-null colon cancer cell lines with different sets of mutations were used to validate the role of GSK3B in sustaining resistance and to characterize cell death mechanisms triggered by chemotherapy when GSK3B is silenced. In vivo xenograft studies were conducted to confirm resensitization of drug-resistant cells to chemotherapy upon GSK3 inhibition. Colon cancer samples from a cohort of 50 chemotherapy-treated stage II patients were analyzed for active GSK3B expression.. Downregulation of GSK3B in various drug-resistant p53-null colon cancer cell lines abolished cell viability and colony growth after drug addition without affecting cell proliferation or cell cycle in untreated cells. Cell death of 5-fluorouracil (5FU)-treated p53-null GSK3B-silenced colon carcinoma cells occurred via PARP1-dependent and AIF-mediated but RIP1-independent necroptosis. In vivo studies showed that drug-resistant xenograft tumor mass was significantly reduced only when 5FU was given after GSK3B inhibition. Tissue microarray analysis of colon carcinoma samples from 5FU-treated patients revealed that GSK3B is significantly more activated in drug-resistant versus responsive patients.. Targeting GSK3B, in combination with chemotherapy, may represent a novel strategy for the treatment of chemotherapy-resistant tumors.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Colonic Neoplasms; DNA Damage; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Activation; Female; Fluorouracil; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HCT116 Cells; Humans; Kaplan-Meier Estimate; Lithium Chloride; Mice; Mice, Nude; Necrosis; RNA, Small Interfering; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Wnt/β-catenin signaling is highly regulated and critical for intestinal epithelial development and repair; aberrant β-catenin signaling is strongly associated with colon cancer. The small GTPase Rac1 regulates β-catenin nuclear translocation and signaling. Here we tested the hypothesis that β1Pix, a Cdc42/Rac guanine nucleotide exchange factor (GEF), regulates β-catenin-dependent transcriptional activity and cell function. We report the novel observations that β1Pix binds directly to β-catenin, an action requiring both the β1Pix DH and dimerization domains but not β1Pix GEF activity. In human colon cancer cells, activation of β-catenin signaling with LiCl decreased β1Pix/β-catenin association in the cytosol and increased nuclear binding of β-catenin to β1Pix. Nuclear association of β1Pix and β-catenin was independent of Rac1 expression and activation; down- and up-regulating Rac1 expression levels did not alter nuclear β1Pix/β-catenin association. Ectopic β1Pix expression enhanced LiCl-induced β-catenin transcriptional activity. Conversely, siRNA knockdown of β1Pix attenuated both LiCl-induced β-catenin transcriptional activity and colon cancer cell proliferation. Ectopic expression of β1Pix stimulated β-catenin transcriptional activity, whereas β1PixΔ(602-611), which is unable to bind β-catenin, had no effect. Altogether, these findings suggest that β1Pix functions as a transcriptional regulator of β-catenin signaling through direct interaction with β-catenin, an action that may be functionally relevant to colon cancer biology.

Topics: Adjuvants, Immunologic; beta Catenin; Caco-2 Cells; Cell Nucleus; Cell Proliferation; Colonic Neoplasms; Cytosol; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Lithium Chloride; Neoplasm Proteins; Protein Multimerization; Protein Structure, Tertiary; Rho Guanine Nucleotide Exchange Factors; Transcription, Genetic

Lithium is a common mood stabilizer to treat bipolar disorder. It has a narrow window of therapeutic action and its mechanism of action and possible side effects are still not fully understood. Lithium is a potent inhibitor of glycogen synthase kinase 3β (GSK-3β). Previous studies indicated that lithium can induce cell cycle arrest by stabilization of p53. In order to further elucidate the signaling mechanism of lithium-induced cell cycle arrest and its potential pharmacological effect on p53 transformed cell lines, we studied the effect of lithium on the rat fibroblast cell line R6 and a p53(Val135) transformed cell line R6T2 (hereafter referred to as T2).. We monitored the effects of lithium on cell cycle progression by FACS analysis and the activation of MAPK signaling pathways by Western blot using anti-phospho-MAPK antibodies in R6 and T2.. We report here lithium can induce G2/M arrest in T2 independent of β-catenin signals. Lithium increases phosphorylation of extracellular signal-regulated kinases (ERKs) leading to the up-regulation of p53 levels and subsequent G2/M arrest. Lithium also induced phosphorylation of p38 MAPK, consequently downregulated p53 and alleviated G2/M cell cycle arrest. We further showed the gate-keeping role of p53 in the lithium-induced G2/M arrest in the T2 cell line.. Our results reveal a novel mechanism underlying the differential response of the transformed and normal R6 to lithium-induced G2/M cell cycle arrest and delineate the multiplicity of signaling pathways dictating the cell fate in responding to cell stress signals.

Topics: Adenocarcinoma; Animals; Antimanic Agents; beta Catenin; Blotting, Western; Cell Line; Cell Line, Tumor; Colonic Neoplasms; Fibroblasts; Flow Cytometry; G2 Phase Cell Cycle Checkpoints; Humans; Lithium Chloride; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Rats; Signal Transduction; Tumor Suppressor Protein p53

PRL-3 is a key gene associated with progression and metastasis of colorectal cancer. Recently PRL-3 was suggested to promote epithelial mesenchymal transition (EMT) by downregulating E-cadherin expression. But the mechanisms of EMT induced by PRL-3 remain largely unknown. Here we found that PRL-3 could also promote EMT in a colorectal cancer cell model SW480 with deficient E-cadherin expression in vivo and in vitro. PRL-3 stable overexpression or knockdown SW480 cells were injected subcutaneously into nude mice. Immunohistochemical analyses of tumor samples from nude mice showed that PRL-3 promoted upregulation of mesenchymal marker vimentin and downregulation of epithelial markers E-cadherin and cytokeratin. Glycogen synthase kinase-3beta inactivated by PRL-3 as assessed by phosphospecific antibodies was a key event in EMT induced by PRL-3. Inhibition of glycogen synthase kinase-3beta by lithium chloride, a highly selective inhibitor, leading to phosphorylation of glycogen synthase kinase-3beta increased Snail expression. In order to identify the direct effects of PRL-3, we isolated CDH22, one member of cadherin family, as a new candidate of interacting proteins of PRL-3 in yeast two-hybrid systems, and the interaction was confirmed in vitro by GST pull-down assay or in exogenous cell systems and endogenous colorectal cancer cells by co-immunoprecipitation assay and co-localization analysis. We observed that PRL-3 promoted downregulation of CDH22 expression. Interestingly, expression of E-cadherin was recovered in SW480 cells after PRL-3 was knocked-down. Our results first linked PRL-3 to cadherin directly. It provided new insights into the regulatory mechanisms of EMT induced by PRL-3.

Topics: Adenocarcinoma; Animals; Cadherins; Cell Adhesion; Cell Line, Tumor; Cell Transdifferentiation; Colonic Neoplasms; Epithelial Cells; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Mesoderm; Mice; Mice, Nude; Neoplasm Proteins; Protein Interaction Mapping; Protein Tyrosine Phosphatases; Snail Family Transcription Factors; Transcription Factors

Atoh1 plays a crucial role in intestinal cell differentiation. We have demonstrated that its human homolog Hath1 protein is targeted by the Wnt-GSK3 axis, resulting in the proteasomal degradation in human colon cancer. However, the contribution of Hath1 degradation to the undifferentiated state of colon cancer remains unknown. In this study, we demonstrated that both constitutive expression of mutant Hath1 and stabilization of Hath1 protein by a GSK3 inhibitor in colon cancer cells increased the expression of MUC2 known as a representative function of differentiated goblet cells. This means that Hath1 protein degradation may be required for maintaining the undifferentiated state of colon cancers, and that GSK3 inhibitors have potential for use in cancer therapy.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell Line, Tumor; Colonic Neoplasms; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lithium Chloride; Mucin-2; Mucins; Proteasome Endopeptidase Complex; RNA, Messenger; Transcription, Genetic; Wnt Proteins

Inappropriate activation of the Wnt/APC/beta-catenin signaling pathways plays a critical role at early stages in a variety of human cancers. However, their respective implication in tumor cell invasion is still hypothetical. Here, we show that two activators of the canonical Wnt/beta-catenin transcription pathway, namely Dvl-2, the Axin 501-560 fragment binding glycogen synthase kinase -3beta (GSK-3beta), and the negative Wnt regulator wt-Axin did not alter cell invasion into type I collagen. In addition, both Dvl-2 and Axin 501-560 exerted a permissive action on the proinvasive activity of HGF and intestinal trefoil factor. Upstream activation of Wnt signaling by the Wnt-2 and Wnt-3a ligands, stable overexpression of Wnt-2, as well as GSK-3beta inhibition by lithium, SB216763, and GSK-3beta dominant negative forms (K85R and R96E) conferred the invasive phenotype through several proinvasive pathways. Induction of the matrix metalloprotease MMP-7 (matrilysin) gene and protein by Wnt-2 was abolished by inactivation of the AP-1 binding site in the promoter. Accordingly, invasion induced by Wnt-2 was prevented by soluble FRP-3 and FRP-1, sequestration of Gbetagamma subunits, depletion of the GSK-3beta protein by RNA interference, the c-Jun dominant negative mutant TAM67 and was not reversed by wt-Axin. Thus, the proinvasive activity of Wnt-2 is mediated by a noncanonical Wnt pathway using GSK-3beta and the AP-1 oncogene. Our data provide a potential clue for our understanding of the action and crosstalk between Wnt activators and other proinvasive pathways, in relation with matrix substrates and proteases in human cancers.

Topics: Animals; Antibodies, Monoclonal; Axin Protein; Cell Line; Cell Line, Transformed; Cell Line, Tumor; Colonic Neoplasms; Colorectal Neoplasms; Dogs; Epithelial Cells; Genetic Vectors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Glycoproteins; Heterotrimeric GTP-Binding Proteins; HT29 Cells; Humans; Indoles; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Kidney; Kidney Neoplasms; Ligands; Lithium Chloride; Maleimides; Matrix Metalloproteinase 7; Neoplasm Invasiveness; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Repressor Proteins; Retroviridae; Signal Transduction; Transcription Factor AP-1; Transcription, Genetic; Wnt Proteins; Wnt2 Protein

Colorectal cancer (CRC) may be particularly amenable to gene therapy because CRCs exhibit constitutive upregulation of Wnt signaling. We have previously demonstrated that butyrate, found in the colonic lumen, modulates Wnt signaling and nonspecifically upregulates transcription from minimal promoters. Because both of these actions may influence the efficiency and specificity of Wnt-targeted expression, the effects of butyrate on Wnt-targeted gene therapy were determined. Lithium is another agent known to upregulate Wnt activity in HCT-116 CRC cells and therefore may induce Wnt-targeted CRC cell kill. CRC cells were cotransfected with an expression vector for green fluorescent protein (GFP) and different versions of vectors coupling Wnt-sensitive promoters to FADD or diphtheria toxin A-chain (DT) effector genes. Cells were treated with butyrate and/or lithium chloride and flow cytometry was used to determine the percentage of remaining transfected (GFP-positive) cells. We demonstrate that promoter and cell type-specific differences occur in Wnt-specific cell kill induced by FADD and DT. A Wnt-sensitive version of the CMV promoter (TcfCMV) exhibited the optimum combination of efficient SW620 CRC cell kill and Wnt specificity; in addition, treatment with a physiologically relevant concentration of butyrate enhanced cell kill induced by TcfCMV-FADD while maintaining specificity. In HCT-116 CRC cells, optimum results were achieved utilizing TcfFos-DT constructs and cotreatment with both butyrate and lithium. The findings suggest that effective CRC cell kill can be achieved by gene therapy through modulation of Wnt signaling by butyrate and/or lithium.

Topics: Apoptosis; Butyrates; Cell Line, Tumor; Colonic Neoplasms; Humans; Intercellular Signaling Peptides and Proteins; Lithium Chloride; Wnt Proteins

Fibroblast growth factor-binding protein (FGF-BP) releases immobilized FGFs from the extracellular matrix and can function as an angiogenic switch molecule in cancer. Here we show that FGF-BP is up-regulated in early dysplastic lesions of the human colon that are typically associated with a loss of adenomatous polyposis coli and up-regulation of beta-catenin. In addition, FGF-BP expression is induced in dysplastic lesions in ApcMin/+ mice in parallel with the up-regulation of beta-catenin. Also, in cell culture studies FGF-BP is induced by beta-catenin through direct activation of the FGF-BP gene promoter. We conclude that FGF-BP is a target gene of beta-catenin.

Topics: Adenoma; Animals; beta Catenin; Carrier Proteins; Colonic Neoplasms; Cytoskeletal Proteins; Gene Expression Regulation, Neoplastic; Humans; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Lithium Chloride; Mice; Mice, Inbred C57BL; Promoter Regions, Genetic; RNA, Messenger; Trans-Activators; Transfection; Up-Regulation