LS174T Cells Research Articles

Interaction of silencing mediator for retinoid and thyroid receptors with steroid and xenobiotic receptor on multidrug resistance 1 promoter

AimsThe steroid and xenobiotic receptor (SXR) regulates the transcription of its target genes by interacting with various nuclear receptor cofactors. We have previously shown that silencing mediator for retinoid and thyroid receptors (SMRT) interacts with SXR even in the presence of rifampicin on cytochrome P450 monooxygenase 3A4 (CYP3A4) promoter in HepG2 cells. To examine the specificity of such interaction, the involvement of SMRT on SXR-mediated transcription through multidrug resistance (MDR) 1 gene promoter was examined using LS174T intestine-derived clonal cells. Main methodsTransient transfection-based reporter gene assay was carried out to examine the effect of SMRT or nuclear receptor corepressor (NCoR) on SXR-mediated transcription in LS174T cells. Semi-quantitative RT-PCR was performed to confirm the expression of MDR1 mRNA in LS174T cells. To examine the interaction of SMRT with SXR, we carried out mammalian one-hybrid assay in CV-1 cells and immunoprecipitation study in HEK-293 cells. Key findingsSMRT, but not NCoR suppressed rifampicin-induced SXR-mediated transcription. The SXR-mediated MDR1 mRNA expression was augmented in the presence of rifampicin, whereas it suppressed the expression following the overexpression of SMRT. In mammalian one-hybrid assay, only SMRT but not NCoR interacted with SXR on MDR1 promoter in the presence of rifampicin. In immunoprecipitation study, SMRT bound to SXR regardless of the presence or absence of rifampicin. SignificanceSMRT may be recruited in the SXR-cofactor complex even in the presence of ligand. SMRT may be involved not only in SXR-mediated suppression without ligand, but also in ligand-activated transcription to suppress the overactivation of transcription.

Colonic MUC2 mucin regulates the expression and antimicrobial activity of β‐defensin 2

The MUC2 mucus layer is the first line of host defense in the gut. How MUC2 interacts with antimicrobial peptides in host defense is not well understood. In this study we investigated the interactions between MUC2 and β‐defensin. Of the colonic cells tested, β‐ defensin 2 expressions was the highest in MUC2 producing human LS 174T and the lowest in Caco‐2 cells. Similarly, expression of murine β‐defensin 4 (orthologous to human β‐defensin 2) was low in the colon of Muc2−/−mice as compared to Wt. Purified MUC2 attenuated the stimulation of β‐defensin as IL‐1β and sodium butyrate induced the expression of β‐defensin in Caco‐2, but not LS 174T cells. The antimicrobial activity of β‐defensin 2 differed among enteric bacteria; Escherichia coli were more susceptible than Bacteroides vulgatus and Clostridium difficile in killing assays. Our studies revealed that MUC2 served as a food source for enteric bacteria, which explains why Muc2−/− harbored less commensal Bacteroides and Firmicutes spp. Mechanistically, MUC2 N‐ and O‐linked oligosaccharides bound bacteria and protected them against β‐defensin 2 antimicrobial activities. MUC2 protected the microbiota by serving as a food source and by inhibiting the antimicrobial activity of β‐defensin. When the mucus barrier is compromised, β‐defensin kills susceptible bacteria whereas β‐defensin resistant bacteria may colonize and cause disease.

Direct modulation of goblet cell function by galacto‐oligosaccharides

Prebiotic galacto‐oligosaccharides (GOS) are non‐digestible food ingredients that are thought to benefit the host indirectly by stimulating the growth and/or activity of beneficial bacteria in the colon. Herein, human adenocarcinoma derived LS174T cells, which exhibit a goblet cell like phenotype, were used to examine possible direct effects of prebiotic GOS on goblet cell function. LS174T cells were treated with GOS and the expression of goblet cell secretory product genes mucin 2 (MUC2), trefoil factor 3 (TFF3), resistin like molecule beta (RETNLB) and Golgi sulfotransferase genes, carbohydrate (N‐acetylglucosamine 6‐O) sulfotransferase 5 (CHST5) and galactose 3‐O‐sulfotransferase 2 (GAL3ST2), was determined by qRT‐PCR and Western blot analysis. QRT‐PCR analysis demonstrated that MUC2 and CHST5 expression was upregulated 2–4 fold while RETNLB and TFF3 expression were upregulated 6–8 fold at 72h. Immunofluorescence microscopy and Western blot data revealed that GOS treatment increased the level of RETNLB, TFF3 and CHST5 at 96h treatment. In addition, IL‐13 with GOS resulted in synergistic induction of RETNLB and CHST5. GOS treatment did not alter IL‐8 secretion. Collectively, the data indicate that GOS may directly enhance mucosal barrier function by modulating the expression of goblet cell products independent of an inflammatory response.

Mucus and adiponectin deficiency: role in chronic inflammation-induced colon cancer.

This study aims to define the role of adiponectin (APN) in preventing goblet cell apoptosis and in differentiation of epithelial cells to goblet cell lineage resulting in greater mucus production and hence greater protection from chronic inflammation-induced colon cancer (CICC). Six- to eight-week-old male APNKO and C57BL/6 (WT) mice were randomly distributed to three treatment groups: DSS, DMH, DSS + DMH and control. Chronic inflammation was induced in DSS and DSS + DMH group by administrating 2% DSS in drinking water for 5days followed by 5days of normal drinking water and this constitutes one DSS cycle. Three cycles of DSS were administered to induce chronic inflammation. Cancer was induced in both APNKO and WT mice in DMH and DSS + DMH groups by intraperitoneal injections of DMH (20mg/kg body weight) once for DSS + DMH group and once per week for 12weeks for DMH group. On day 129, the colon tissue was dissected for mucus thickness measurements and for genomic studies. HT29-C1.16E and Ls174T cells were used for several genomic and siRNA studies. APNKO mice have more tumors and tumor area in DSS + DMH group than WT mice. APN deficiency downregulated goblet to epithelial cell ratio and enhanced the colonic mucosal erosion with reduced mucus thickness. APN increases Muc2 production with no affect on Muc1 production. APN abated goblet cell apoptosis, while APN deficiency reduced epithelial to goblet cell differentiation. APN may be involved in reducing the severity of CICC by preventing goblet cell apoptosis and increasing epithelial to goblet cell differentiation.

Pelargonidin activates the AhR and induces CYP1A1 in primary human hepatocytes and human cancer cell lines HepG2 and LS174T

We examined the effects of anthocyanidins (cyanidin, delphinidin, malvidin, peonidin, petunidin, pelargonidin) on the aryl hydrocarbon receptor (AhR)-CYP1A1 signaling pathway in human hepatocytes, hepatic HepG2 and intestinal LS174T cancer cells. AhR-dependent reporter gene expression in transfected HepG2 cells was increased by pelargonidin in a concentration-dependent manner at 24h. Similarly, pelargonidin induced the expression of CYP1A1 mRNA up to 5-fold in HepG2 and LS174T cells relative to the induction by 5nM 2,3,7,8-tetrachlorodibenzodioxin (TCDD), the most potent activator of AhR. CYP1A1 and CYP1A2 mRNAs were also increased by pelargonidin in three primary human hepatocytes cultures (approximately 5% of TCDD potency) and the increase in CYP1A1 protein in HepG2 and LS174T cells was comparable to the increase in catalytic activity of CYP1A1 enzyme. Ligand binding analysis demonstrated that pelargonidin was a weak ligand of AhR.Enzyme kinetic analyses using human liver microsomes revealed inhibition of CYP1A1 activity by delphinidin (IC50 78μM) and pelargonidin (IC50 33μM).Overall, although most anthocyanidins had no effects on AhR-CYP1A1 signaling, pelargonidin can bind to and activate the AhR and AhR-dependent gene expression, and pelargonidin and delphinidin inhibit the CYP1A1 catalytic activity.

Bacteria Regulate Intestinal Epithelial Cell Differentiation Factors Both In Vitro and In Vivo

BackgroundThe human colon harbours a plethora of bacteria known to broadly impact on mucosal metabolism and function and thought to be involved in inflammatory bowel disease pathogenesis and colon cancer development. In this report, we investigated the effect of colonic bacteria on epithelial cell differentiation factors in vitro and in vivo. As key transcription factors we focused on Hes1, known to direct towards an absorptive cell fate, Hath1 and KLF4, which govern goblet cell.MethodsExpression of the transcription factors Hes1, Hath1 and KLF4, the mucins Muc1 and Muc2 and the defensin HBD2 were measured by real-time PCR in LS174T cells following incubation with several heat-inactivated E. coli strains, including the probiotic E. coli Nissle 1917+/− flagellin, Lactobacilli and Bifidobacteria. For protein detection Western blot experiments and chamber-slide immunostaining were performed. Finally, mRNA and protein expression of these factors was evaluated in the colon of germfree vs. specific pathogen free vs. conventionalized mice and colonic goblet cells were counted.ResultsExpression of Hes1 and Hath1, and to a minor degree also of KLF4, was reduced by E. coli K-12 and E. coli Nissle 1917. In contrast, Muc1 and HBD2 expression were significantly enhanced, independent of the Notch signalling pathway. Probiotic E. coli Nissle 1917 regulated Hes1, Hath1, Muc1 and HBD2 through flagellin. In vivo experiments confirmed the observed in vitro effects of bacteria by a diminished colonic expression of Hath1 and KLF4 in specific pathogen free and conventionalized mice as compared to germ free mice whereas the number of goblet cells was unchanged in these mice.ConclusionsIntestinal bacteria influence the intestinal epithelial differentiation factors Hes1, Hath1 and KLF4, as well as Muc1 and HBD2, in vitro and in vivo. The induction of Muc1 and HBD2 seems to be triggered directly by bacteria and not by Notch.

Microenvironment and cytokines in pseudomyxoma peritonei.

433 Background: Pseudomyxoma Peritonei (PMP), a peritoneal mucinous neoplasm of appendix origin, is associated with inflammation and fibrosis which is central to the disease biology. Here, we examine possible biomarkers or regulators of PMP biology in peritoneal tumor microenvironment and in serum. Methods: Ascites, peritoneal washings and serum were collected from PMP patients. Cytokines and C-reactive protein (CRP) levels were determined using Milliplex immunoassays. Immunohistochemistry (IHC) was performed on formalin-fixed tissue sections. Statistical analysis was performed by the Mann Whitney U test and Bivariate analysis. Results: Serum CRP was significantly elevated in PMP patients (n=29) versus controls (n=4) (0.124 vs 0.004 mg/dl, p = 0.003). CRP levels were lower in ascites (0.028 vs 0.124 mg/dl, p = 0.014), but correlated with serum levels (R = 0.65, p < 0.001). Cytokines typically elevated after infection or injury—TNF-α, IL-1α, IL-1β and γ INF—were not significantly elevated in PMP ascites; however, IL-6, IL-8, IP-10 and MCP-1 were consistently higher (p < 0.001) in PMP with ascites (n=29) when compared to control peritoneal washings (n=9). Both serum and ascites from 10 patients were evaluated for these four cytokines. Ascites levels were significantly elevated compared to their respective serum for IL-6 (1107 vs 3.18 pg/ml, p < 0.001), IL-8 (167 vs 1.59 pg/ml, p < 0.001), IP-10 (5454 vs 504 pg/ml, p < 0.001) and MCP-1 (2420 vs 234 pg/ml, p = 0.002) but the serum levels were not significantly elevated compared to controls (n=6) (p = 0.31, 0.2, 0.63, 0.37 respectively). IHC revealed staining for IL-6 and IL-8 in stromal cells and focal staining of tumor for IP-10 and MCP-1. Recombinant IL-6-treated LS174T cells (mucinous colon cancer cell line) showed increased proliferation (p<0.009) which was inhibited by anti-IL-6 antibodies (p<0.004). Conclusions: CRP, a liver-synthesized marker of systemic inflammation, is elevated in PMP, and may have clinical utility. The results support local synthesis for cytokines IL-6, IL-8, IP-10 and MCP-1 in the peritoneum. Interestingly, the pattern of cytokines appears to be distinct from those following injury or infection. Further studies may yield novel targets for future treatment.

Anti-proliferative and apoptosis-inducing activities of juglone in LS-174T cells

Anti-proliferative and apoptosis-inducing effects of juglone in LS-174T cells were investigated. In this study, we showed that juglone inhibited the proliferation of LS-174T cells in a time and dose-dependent manner, treatment of juglone resulted in the activation of caspase-9 and caspase-3, decrease of Bcl-2. N-acetylcysteine significantly attenuate LS-174T cell death induced by juglone (p<0.001). In addition, NAC could reverse caspase-3 and caspase-9 activation, increase expression of Bcl-2 protein. Taken together, these findings indicated that juglone-mediated oxidative injury may act as upstream change, trigger ROS release, Bcl-2 modulation, caspase activation, and consequently leading cell apoptosis in LS-174T cells. In conclusion, these findings suggest that juglone may be an effective way for treating human cancers.

Effects of iodonium-class flavin dehydrogenase inhibitors on growth, reactive oxygen production, cell cycle progression, NADPH oxidase 1 levels, and gene expression in human colon cancer cells and xenografts

Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10–250nM for DPI, 0.5–2.5μM for DTI, and 155nM to 10μM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.

Epigenetic regulation of CXCR4 expression by the ocular microenvironment.

Expression of the chemokine receptor CXCR4 by tumors is associated with metastatic migration and invasion of tumor cells. The importance of CXCR4 expression by uveal melanomas in metastasis to the liver was recently demonstrated when injection of CXCR4-negative uveal melanoma cells into mice resulted in reduced liver metastasis compared with CXCR4-positive uveal melanoma cells. Factors in the eye can induce downregulation of genes by epigenetic mechanisms. This study examined whether epigenetic regulation by the ocular environment induced downregulation of CXCR4 expression. LS174T colon cancer cells were injected in the anterior chamber (AC), subcutaneously (SC), or in the spleen capsule to induce liver metastasis in immune-deficient mice. CXCR4 gene transcription was analyzed by RT-PCR, and protein expression was determined by flow cytometry. Methyltransferase and histone deacetylase activities were determined by ELISA. Treatment with either 5-Aza-2-deoxycytidine (5-Aza) or trichostatin A (TSA) was used to induce demethylation or inhibit histone deacetylases, respectively. AC-derived LS174T cells showed lower CXCR4 gene expression compared with SC-, liver-derived, or wild-type tumor cells. AC-derived LS174T tumor cells expressed methyltransferase activity compared with SC-, liver-derived, and wild-type tumor cells. Deacetylase activity was elevated in AC-derived LS174T tumor cells compared with SC-derived, liver-derived, and wild-type tumor cells. Treatment of AC-derived LS174T tumor cells with 5-Aza upregulated CXCR4 expression. TSA treatment did not restore CXCR4 expression. These studies demonstrate that ocular microenvironment factors induce methylation and downregulation of tumor CXCR4 expression.

PXR-Mediated Upregulation of CYP3A Expression by Herb Compound Praeruptorin C fromPeucedanum praeruptorumDunn

We recently reported that Praeruptorin C effectively transactivated the mRNA, protein expression, and catalytic activity of CYP3A4 via the CAR-mediated pathway, but whether and how PC could affect the expression and catalytic activity of CYP3A4 via PXR pathway remains unknown. Therefore, in this study, the effect of PC on the CYP3A gene expression was investigated in mice primary hepatocytes after knockdown of PXR by transient transfection of PXR siRNA, and the gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells with PXR overexpression were determined by real-time PCR, western blot analysis, and LC-MS/MS-based CYP3A4 substrate assay, respectively. We found that the level of CYP3a11 gene expression in mouse primary hepatocytes was significantly increased by praeruptorin C, but such an induction was suppressed after knockdown of pregnane X receptor by its siRNA. In PXR-overexpressed LS174T cells, PC significantly enhanced CYP3A4 mRNA, protein expression, and functional activity through PXR-mediated pathway; conversely, no such increase was found in the untransfected cells. These findings suggest that PC can significantly upregulate CYP3A level via the PXR-mediated pathway, and this should be taken into consideration to predict any potential herb-drug interactions between PC, Qianhu, and the other coadministered drugs.

Clinacanthus nutansExtracts Are Antioxidant with Antiproliferative Effect on Cultured Human Cancer Cell Lines

Clinacanthus nutans Lindau leaves (CN) have been used in traditional medicine but the therapeutic potential has not been explored for cancer prevention and treatment. Current study aimed to evaluate the antioxidant and antiproliferative effects of CN, extracted in chloroform, methanol, and water, on cancer cell lines. Antioxidant properties of CN were evaluated using DPPH, galvinoxyl, nitric oxide, and hydrogen peroxide based radical scavenging assays, whereas the tumoricidal effect was tested on HepG2, IMR32, NCL-H23, SNU-1, Hela, LS-174T, K562, Raji, and IMR32 cancer cells using MTT assay. Our data showed that CN in chloroform extract was a good antioxidant against DPPH and galvinoxyl radicals, but less effective in negating nitric oxide and hydrogen peroxide radicals. Chloroform extract exerted the highest antiproliferative effect on K-562 (91.28 ± 0.03%) and Raji cell lines (88.97 ± 1.07%) at 100 μg/ml and the other five cancer cell lines in a concentration-dependent manner, but not on IMR-32 cells. Fourteen known compounds were identified in chloroform extract, which was analysed by gas chromatography—mass spectra analysis. In conclusion, CN extracts possess antioxidant and antiproliferative properties against cultured cancer cell lines, suggesting an alternate adjunctive regimen for cancer prevention or treatment.

Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7

The largest EHEC outbreak up to now in Germany occurred in 2011. It was caused by the non-O157:H7 Shiga-toxinogenic enterohemorrhagic E. coli strain O104:H4. This strain encodes in addition to the Shiga toxin 2 (Stx2), responsible for the hemolytic uremic syndrome (HUS), several adhesins such as aggregative adherence fimbriae. Currently, there is no effective prophylaxis and treatment available for EHEC infections in humans. Especially antibiotics are not indicated for treatment as they may induce Stx production, thus worsening the symptoms. Alternative therapeutics are therefore desperately needed. We tested the probiotic Escherichia coli strain Nissle 1917 (EcN) for antagonistic effects on two O104:H4 EHEC isolates from the 2011 outbreak and on the classical O157:H7 EHEC strain EDL933. These tests included effects on adherence, growth, and Stx production in monoculture and co-culture together with EcN. The inoculum of each co-culture contained EcN and the respective EHEC strain either at a ratio of 1:1 or 10:1 (EcN:EHEC). Adhesion of EHEC strains to Caco-2 cells and to the mucin-producing LS-174T cells was reduced significantly in co-culture with EcN at the 1:1 ratio and very dramatically at the 10:1 ratio. This inhibitory effect of EcN on EHEC adherence was most likely not due to occupation of adhesion sites on the epithelial cells, because in monocultures EcN adhered with much lower bacterial numbers than the EHEC strains. Both EHEC strains of serotype O104:H4 showed reduced growth in the presence of EcN (10:1 ratio). EHEC strain EDL933 grew in co-culture with EcN only during the first 2h of incubation. Thereafter, EHEC counts declined. At 24h, the numbers of viable EDL933 was at or slightly below the numbers at the time of inoculation. The amount of Stx2 after 24h co-incubation with EcN (EcN:EHEC ratio 10:1) was for all 3 EHEC strains tested significantly reduced in comparison to EHEC monocultures.Obviously, EcN shows very efficient antagonistic activity on the EHEC strains of serotype O104:H4 and O157:H7 tested here regarding adherence to human gut epithelial cells, bacterial growth, and Stx2 production in vitro.

Clinicopathological and biological significance ofcriptooverexpression in human colon cancer

To assess the clinicopathological and biological significance of cripto in human colorectal cancer. Real-time reverse-transcription polymerase chain reaction (PCR) was used to examine cripto mRNA levels in primary colon cancer and normal colon tissues as well as normal and metastatic lymph nodes from colon cancers. Human colon cancer LS-174T cells were transfected with cripto small interfering RNA (siRNA), and mRNA and protein levels were evaluated using real-time PCR and western blot analysis, respectively. The growth of cancer cells was evaluated using the MTT assay and colony formation in soft agar. Invasion was examined using a Transwell assay, and the expressions of matrix metalloproteinase (MMP)-7 and MMP-9 were determined using western blot assay. Cripto was significantly overexpressed in primary colon cancer and metastatic lymph nodes. Silencing cripto gene expression with cripto siRNA resulted in a significant decrease in colony formation in soft agar in the colon cancer cell line LS-174T. Cripto siRNA treatment decreased the migration and invasion capabilities of the colon cancer cell line LS-174T in vitro. Furthermore, cripto siRNA treatment inhibited the expression of matrix MMP-7 and MMP-9. The results provide evidence that cripto siRNA could be an effective approach for the inhibition of cancer cell invasion and migration and thus has potential for use in devising novel preventive and therapeutic strategies for colon cancer metastasis.

IL-10 Promotes Production of Intestinal Mucus by Suppressing Protein Misfolding and Endoplasmic Reticulum Stress in Goblet Cells

Protein misfolding and endoplasmic reticulum (ER) stress have been observed in intestinal secretory cells from patients with inflammatory bowel diseases and induce intestinal inflammation in mice. However, it is not clear how immune factors affect ER stress and therefore disease symptoms. We analyzed the effects of interleukin (IL)-10 on ER stress in intestinal tissues in wild-type C57BL/6, Winnie, IL-10(-/-), and Winnie × IL-10(+/-) mice. In Winnie mice, misfolding of the intestinal mucin Muc2 initiates ER stress and inflammation. We also analyzed the effects of different inhibitors of IL-10 signaling and the N-glycosylation inhibitor tunicamycin in cultured human LS174T goblet cells. Administration of neutralizing antibodies against IL-10 or its receptor (IL-10R1) to Winnie mice rapidly exacerbated ER stress and intestinal inflammation compared with mice given vehicle (controls). Antibodies against IL-10 also increased accumulation of misfolded Muc2 in the ER of goblet cells of Winnie mice and increased T-cell production of inflammatory cytokines. Winnie × IL-10(+/-) mice and IL-10(-/-) mice with a single Winnie allele each developed more severe inflammation than Winnie mice or IL-10(-/-) mice. Administration of tunicamycin to wild-type mice caused intestinal ER stress, which increased when IL-10R1 was blocked. In LS174T cells, induction of ER stress with tunicamycin and misfolding of MUC2 were reduced by administration of IL-10; this reduction required STAT1 and STAT3. In LS174T cells incubated with tunicamycin, IL-10 up-regulated genes involved in MUC2 folding and in ER-associated degradation and maintained correct folding of MUC2, its transport from the ER, and its O-glycosylation and secretion. IL-10 prevents protein misfolding and ER stress by maintaining mucin production in goblet cells and helps the intestine preserve the mucus barrier.

Induction of apoptosis by D-limonene is mediated by inactivation of Akt in LS174T human colon cancer cells

D-limonene is recognized as a potential chemotherapeutic agent, however, the details of this mechanism remain unclear. In this study, we investigated the effects of d-limonene on colon cancer cell viability and its potential mechanism of action invitro. After 48h of treatment, d-limonene suppressed the viability of LS174T cells in a dose-dependent manner and caused a dose-dependent apoptotic cell death. D-limonene activated caspase-3 and -9 and PARP cleavage in a dose-dependent manner. Moreover, an increase in Bax protein and cytosol cytochromec from mitochondria and a decrease in bcl-2 protein were observed following treatment with d-limonene. In addition, d-limonene decreased the levels of p-Akt (Ser473), p-Akt (Thr308) and p-GSK-3β (Ser9), suggesting that d-limonene induced apoptosis via the mitochondrial death pathway and the suppression of the PI3K/Akt pathway.

Sodium butyrate restores ASC expression and induces apoptosis in LS174T cells

Sodium butyrate (NaBu) is a short-chain fatty acid (SCFA), which has been proposed as a potential anticancer agent. Apoptosis-associated speck-like protein (ASC) is a pro-apoptotic signaling factor that is subjected to epigenetic silencing in human cancers. Modulation by the aberrant methylation of CpG islands of ASC is a well-characterized epigenetic mechanism, and the methylation-induced silencing of ASC has been observed in several types of tumors. NaBu induces cell cycle arrest, markers of cell differentiation and apoptosis in colon cancer. NaBu promotes transcriptional activation by relaxing the DNA conformation and displays anti-proliferative and differentiating activity in a wide variety of cancers. Thus, we used NaBu to investigate the relationship between the status of cell proliferation and the re-expression of ASC in colon carcinoma LS174T cells. Our experiments determined ASC re-expression at the protein level using western blotting. In addition, we used reverse transcription-polymerase chain reaction to detect the expression levels of ASC mRNA and an MTT assay to detect the inhibitory rate of cell growth. The apoptosis rate was also detected for further validation of the re-expression of ASC. The results showed that ASC re-expression was significantly increased in the LS174 cells following NaBu treatment in a time- and dose-dependent manner. The expression of ASC also induced the apoptosis of LS174T cells. These results suggest that NaBu plays a role in the reactivation of ASC expression and that the latter promotes the apoptosis of LS174T cells.

The relative roles of charge and a recognition peptide in luminal targeting of colorectal cancer by fluorescent polyacrylamide

Real time detection of biomarkers at the mucosal level is imperative for the prevention and efficient treatment of colorectal cancer.Cationized polyacrylamide (CPAA) with increasing charge densities was prepared by radical polymerization of acrylamide and different mol% ratios of N-acryloyl, N′-(tert-butyl-carbonyl) diaminoethane. The NIR fluorophore derivative of IR-783, IR-783-S-Ph-COOH, was attached to the CPAA to give CPAA-783. After selecting the optimal IR-783-S-Ph-COOH ratio that avoids quenching, the preferential binding of the polymer was tested in SW-620, SW-480, HT-29, and LS-174T cancer cells. The optimal polymeric product was tested in situ in gut sac preparations of the dimethylhydrazine induced rat model.To increase the detection capabilities of CPAA-783, the FITC-labeled peptide EPPT1, that targets the cell transmembrane underglycosylated MUC-1 (uMUC-1), was conjugated to the polymer to obtain CPAA-783-EPPT1. The dually labeled modified polymer was tested in HT-29 and LS-174T cells (over expressing uMUC-1), followed by an examination in an orthotopic mouse model.CPAA-783 preferentially bound to the cancer cells, depending on CRC staging. The best binding occurred when the fraction of the cationic monomer was 100mol%, labeled with 0.75mol% of IR-783-S-Ph-COOH. An increase in the recognition of the dually labeled polymeric product, CPAA-783-EPPT1, towards HT-29 and LS-174T cells occurred in the lowest EPPT1molar ratio (0.63mol%) only, probably due to quenching phenomena and steric hindrance. Similar observation was obtained in the orthotopic mice.It is concluded that fluorescently tagged CPAA can be used for the detection of malignant tissues in colorectal cancer after luminal instillation. Dually targeted CPAA with EPPT1is feasible, but requires further optimization.

Colon cancer cell chemosensitisation by fish oil emulsion involves apoptotic mitochondria pathway

Adjuvant use of safe compounds with anti-tumour properties has been proposed to improve cancer chemotherapy outcome. We aimed to investigate the effects of fish oil emulsion (FOE) rich in n-3 PUFA with the standard chemotherapeutic agents 5-fluorouracil (5-FU), oxaliplatin (OX) or irinotecan (IRI) on two human colorectal adenocarcinoma cells with different genetic backgrounds. The HT-29 (Bax+/+) and LS174T (Bax-/-) cells were co-treated for 24-72 h with 1 μm-5-FU, 1 μm-OX or 10 μm-IRI and/or FOE dilution corresponding to 24 μm-EPA and 20·5 μm-DHA. Soyabean oil emulsion (SOE) was used as isoenergetic and isolipid control. Cell viability, apoptosis and nuclear morphological changes were evaluated by cytotoxic colorimetric assay, flow cytometry analysis with annexin V and 4',6'-diamidino-2-phenylindole staining, respectively. A cationic fluorescent probe was used to evaluate mitochondrial dysfunction, and protein expression involved in mitochondrial apoptosis was determined by Western blot. In contrast to SOE, co-treatment with FOE enhanced significantly the pro-apoptotic and cytotoxic effects of 5-FU, OX or IRI in HT-29 but not in LS174T cells (two-way ANOVA, P <0.01). These results were confirmed by the formation of apoptotic bodies in HT-29 cells. A significant increase in mitochondrial membrane depolarisation was observed after the combination of 5-FU or IRI with FOE in HT-29 but not in LS174T cells (P <0.05). Co-administration of FOE with the standard agents, 5-FU, OX and IRI, could be a good alternative to increase the efficacy of chemotherapeutic protocols through a Bax-dependent mitochondrial pathway.

Up-regulation of FXR isoforms is not required for stimulation of the expression of genes involved in the lack of response of colon cancer to chemotherapy

Several mechanisms are involved in the poor response of colorectal adenocarcinoma (CRAC) to pharmacological treatment. Since preliminary evidences have suggested that the enhanced expression of farnesoid X receptor (FXR) results in the stimulation of chemoresistance, we investigated whether FXR up-regulation is required for the expression of genes that characterize the multidrug resistance (MDR) phenotype of CRAC. Samples of tumours and adjacent healthy tissues were collected from naive patients. Using Taqman Low-Density Arrays, the abundance of mRNA of 87 genes involved in MDR was determined. Relevant changes were re-evaluated by conventional RT-QPCR. In healthy tissue the major FXR isoforms were FXRα2(+/−) (80%). In tumours this predominance persisted (91%) but was accompanied by a consistent reduction (3-fold) in total FXR mRNA. A lower FXR expression was confirmed by immunostaining, in spite of which there was a significant change in the expression of MDR genes. Pharmacological challenge was simulated “in vitro” using human CRAC cells (LS174T cells). Short-term (72h) treatment with cisplatin slightly increased the almost negligible expression of FXR in wild-type LS174T cells, whereas long-term (months) treatment induced a cisplatin-resistant phenotype (LS174T/R cells), which was accompanied by a 350-fold up-regulation of FXR, mainly FXRα1(+/−). However, the changed expression of MDR genes in LS174T/R cells was not markedly affected by incubation with the FXR antagonist Z-guggulsterone. In conclusion, although the enhanced expression of FXR may be involved in the stimulation of chemoresistance that occurs during pharmacological treatment, FXR up-regulation is not required for the presence of the MDR phenotype characteristic of CRAC.