Human LS174T Research Articles

沉默 c2orf68 基因对结直肠癌细胞增殖的影响

Objective: To explore the c2orf68 gene expression of human colorectal adenocarcinoma SW480, SW620 and LS174T cell lines. Further more,to observe the change of their biological characteristics in three cell lines by applying RNA interference technique to silence c2orf68 and to clarify its in the pathogenesis of colorectal cancer. Methods: Using siRNA online design software designed and synthesised of siRNA interference fragments, and transported them to the three cell lines. The mRNA expression of the c2orf68 were detected by RT-PCR,the cell proliferation was checked by MTT,and the cell apoptosis was measured by flow-cytometry. Results: The mRNA expression of the c2orf68 was inhibited by the two pairs siRNA in SW480,SW620 and LS174T cell lines. It showed that the inhibition rates were as follows: 50. 05%,50. 05%; 49. 69%,49. 69%; 50. 15%,45. 79%. Then,siRNA1 of high interference efficiency was selected. The cell proliferation inhibitory rate was 21. 2% in LS174T cell line( P 0. 05). The cell apoptosis rate was 18. 13% in LS174T cell line( P 0. 05). Conclusions: The successfully obtained siRNA fragement which specifically degraded its c2orf68 mRNA,inhibited the growth of the test cells,promoted cell apoptosis. It indicated that c2orf68 might be associated with the proliferation on colorectal cancer cells,its pathogenesis might be through gene expression,further regulated the change of the corresponding biological characteristics,and caused the occurrence of colorectal cancer.

Involvement of SET in the Wnt signaling pathway and the development of human colorectal cancer.

The SET oncoprotein is involved in cancer progression by modulating multiple cellular processes, including the inhibition of the tumor suppressor, protein phosphatase 2 (PP2A). Based upon these multiple activities, we hypothesized that targeted inhibition of SET is likely to have multiple discrete and measurable effects on cancer cells. In the present study, the mRNA expression levels of SET, PP2A and β-catenin were examined in 31 pairs of human colorectal adenocarcinoma tissues and corresponding adjacent normal colorectal tissues by quantitative real-time polymerase chain reaction (qPCR). A small interfering RNA targeting SET was transfected into the human colon carcinoma cell lines, LS174T and SW480. The mRNA levels of SET, PP2A, β-catenin, c-Myc, E-cadherin and p53 were determined by qPCR analysis and the protein levels of SET, c-Myc, PP2A and β-catenin were examined by western blot analysis. mRNA expression levels of SET and β-catenin were found to be elevated in 22 (70.9%) samples, while PP2A expression levels were upregulated in eight (25.8%) samples. In addition, the knockdown of SET mRNA expression caused the upregulation of PP2A and c-Myc in the two cell lines, whereas β-catenin, E-cadherin and p53 mRNA expression was downregulated. Consistent with these results, the protein expression of β-catenin and c-Myc was found to be downregulated, whereas PP2A was upregulated at the protein level. Based on these results, we proposed that SET is essential in the carcinogenesis of human colorectal adenocarcinoma. In addition, it is suggested that SET promotes carcinogenesis through regulation of the Wnt signaling pathway.

Numerical Comparison of Iodine-Based and Indium-Based Antibody Biodistributions

Single-step iodination is often performed in preference to use of a radiometal label for initial animal biodistributions. Yet loss of iodine occurs in vivo so that it is important to measure uptake (%ID/g) differences between radiolabels. Murine biodistributions of four radioiodinated and (111)In-labeled cognate anti-carcinoembryonic antigen antibodies were compared. Uptakes were obtained in athymic mice out to 96 hours for diabody, minibody, scFv-Fc, and intact humanized (M5A) versions of the T84.66 antibody. Tissues included liver, spleen, kidneys, lungs, and human LS174T colorectal xenografts. Ratios (R) of iodine uptake to indium uptake were calculated. For all cognates, no significant differences were found in the blood uptakes between the two labels. In normal solid organs, iodine was generally cleared more rapidly with decreasing molecular weight (MW). In liver and spleen by 24 hours, the R value was 5% for diabody and minibody, whereas a value of 50% was seen for the intact mAb. Renal differences were even more marked for the two lower MW species. Tumor losses, however, were found to be essentially independent of MW and were modest; 50% by 48 hours. To test the generality of the results, comparisons were then made for normal organs and tumors of the R values found above for M5A and MN-14 intact antibody literature results. Good agreement, within estimated errors in R, was seen between these two cases, although (111)In and (88)Y were the respective radiometals. Loss of iodine from labeled antibodies can be marked in normal organs and that correction (by 1/R) of the iodine biodistribution to estimate the associated radiometal result may be possible. Explicit differences between the two types of biodistribution also imply that separate uptake results need to be considered when evaluating the impact on imaging and therapy using various possible radiolabels.

Bromelain and N-acetylcysteine inhibit proliferation and survival of gastrointestinal cancer cells in vitro : significance of combination therapy

BackgroundBromelain and N-acetylcysteine are two natural, sulfhydryl-containing compounds with good safety profiles which have been investigated for their benefits and application in health and disease for more than fifty years. As such, the potential values of these agents in cancer therapy have been variably reported in the literature. In the present study, the efficacy of bromelain and N-acetylcysteine in single agent and combination treatment of human gastrointestinal carcinoma cells was evaluated in vitro and the underlying mechanisms of effect were explored.MethodsThe growth-inhibitory effects of bromelain and N-acetylcysteine, on their own and in combination, on a panel of human gastrointestinal carcinoma cell lines, including MKN45, KATO-III, HT29-5F12, HT29-5M21 and LS174T, were assessed by sulforhodamine B assay. Moreover, the influence of the treatment on the expression of a range of proteins involved in the regulation of cell cycle and survival was investigated by Western blot. The presence of apoptosis was also examined by TUNEL assay.ResultsBromelain and N-acetylcysteine significantly inhibited cell proliferation, more potently in combination therapy. Drug-drug interaction in combination therapy was found to be predominantly synergistic or additive. Mechanistically, apoptotic bodies were detected in treated cells by TUNEL assay. Furthermore, Western blot analysis revealed diminution of cyclins A, B and D, the emergence of immunoreactive subunits of caspase-3, caspase-7, caspase-8 and cleaved PARP, withering or cleavage of procaspase-9, overexpression of cytochrome c, reduced expression of anti-apoptotic Bcl-2 and pro-survival phospho-Akt, the emergence of the autophagosomal marker LC3-II and deregulation of other autophagy-related proteins, including Atg3, Atg5, Atg7, Atg12 and Beclin 1. These results were more prominent in combination therapy.ConclusionWe report for the first time to our knowledge the growth-inhibitory and cytotoxic effects of bromelain and N-acetylcysteine, in particular in combination, on a panel of gastrointestinal cancer cell lines with different phenotypes and characteristics. These effects apparently resulted from cell cycle arrest, apoptosis and autophagy. Towards the development of novel strategies for the enhancement of microscopic cytoreduction, our results lay the basis for further evaluation of this formulation in locoregional approaches to peritoneal surface malignancies and carcinomatosis.

SREBP-2 negatively regulates FXR-dependent transcription of FGF19 in human intestinal cells

Sterol regulatory element-binding protein-2 (SREBP-2) is a basic helix-loop-helix-leucine zipper transcription factor that positively regulates transcription of target genes involved in cholesterol metabolism. In the present study, we have investigated a possible involvement of SREBP-2 in human intestinal expression of fibroblast growth factor (FGF)19, which is an endocrine hormone involved in the regulation of lipid and glucose metabolism. Overexpression of constitutively active SREBP-2 decreased FGF19 mRNA levels in human colon-derived LS174T cells. In reporter assays, active SREBP-2 overexpression suppressed GW4064/FXR-mediated increase in reporter activities in regions containing the IR-1 motif (+848 to +5200) in the FGF19 gene. The suppressive effect disappeared in reporter activities in the region containing the IR-1 motif when the mutation was introduced into the IR-1 motif. In electrophoretic mobility shift assays, binding of the FXR/retinoid X receptor α heterodimer to the IR-1 motif was attenuated by adding active SREBP-2, but SREBP-2 binding to the IR-1 motif was not observed. In chromatin immunoprecipitation assays, specific binding of FXR to the IR-1-containing region of the FGF19 gene (+3214 to +3404) was increased in LS174T cells by treatment with cholesterol and 25-hydroxycholesterol. Specific binding of SREBP-2 to FXR was observed in glutathione-S-transferase (GST) pull-down assays. These results suggest that SREBP-2 negatively regulates the FXR-mediated transcriptional activation of the FGF19 gene in human intestinal cells.

Comparison between two labeled agents in mice using a coinjection-ratio approach in contrast to a conventional group approach

The differences between two agents often need to be accurately defined in vivo. Usually they are injected respectively into two groups of subjects. However, if the two agents do not interact with each other in vivo, a coinjection would serve the same purpose. We believe some individual differences in biodistribution may be circumvented through this approach by calculating organ level ratios. A model system of MORF/cMORF pretargeting (MORF/cMORF is a complementary pair of DNA analogues) was employed in connection with an on-going tumor therapeutic project. Human LS174T cells were implanted into the flank of severely immuno-compromised NOD-scid IL2rg(null) mice. The tumor was confirmed to express TAG-72 antigens. At 16 days post tumor inoculation, mice received IV 60 μg of MORF-conjugated CC49 (an antiTAG-72 antibody), followed 2 days later by a low-mass-dose IV coinjection containing 2.5 μg of (90)Y-cMORF and 2.5 μg of (99m)Tc-cMORF. At 3 h post radioactivity injection, the distribution of (99m)Tc was imaged on a SPECT/CT camera and then organs were excised and counted for (90)Y and (99m)Tc. Because the two labeled cMORFs do not react or interact with each other in vivo, the two groups of (90)Y and (99m)Tc data enabled a conventional group comparison. In a new effort, (90)Y/(99m)Tc ratios were calculated. Student's t-test and retrospective power analysis were performed for both approaches. In the new approach, the ratios were set at 1 as the null hypothesis. The Student's t-test in the conventional group approach indicated that the two labeled cMORFs distributed similarly, but significant differences were observed in salivary gland and large intestines. The coinjection-ratio approach certainly did not subvert the results of the conventional approach but revealed subtler differences. The P values were reduced, the powers were increased in most organs, and more significant differences were observed. The increased sensitivity was due to the reduced CV%s (SD/average*100%) of the (90)Y/(99m)Tc ratios. Therefore, some individual differences were circumvented and notably the ratio approach differentiated individual differences into ratio-correctable and ratio-uncorrectable. Although the conventional approach is reliable, the coinjection-ratio approach using organ level ratios is more sensitive and therefore is recommended whenever possible. In addition, it differentiates individual differences into "coinjection correctable" and "coinjection uncorrectable".

Effects of anthocyanins on the AhR–CYP1A1 signaling pathway in human hepatocytes and human cancer cell lines

Anthocyanins are plant pigments occurring in flowers and berry fruits. Since a phenomenon of food–drug interactions is increasingly emerging, we examined the effects of 21 major anthocyanins and the extracts from 3 food supplements containing anthocyanins on the aryl hydrocarbon receptor (AhR)–cytochrome P450 CYP1A1 signaling pathway in human hepatocytes and human hepatic HepG2 and intestinal LS174T cancer cells. Pelargonidin-3-O-rutinoside (PEL-2) and cyanidin-3,5-O-diglucoside (CYA-3) dose-dependently activated AhR, as revealed by gene reporter assay. PEL-2 and CYA-3 induced CYP1A1 mRNA but not protein in HepG2 and LS174T cells. Neither compounds induced CYP1A1 mRNA and protein in four different primary human hepatocytes cultures. The effects of PEL-2 and CYA-3 on AhR occurred by ligand-dependent and ligand-independent mechanisms, respectively, as demonstrated by ligand binding assay. In a direct enzyme inhibition assay, none of the antocyanins tested inhibited the CYP1A1 marker activity to less than 50% even at 100μM concentration. PEL-2 and CYA-3 at 100μM inhibited CYP1A1 to 79% and 65%, respectively. In conclusion, with exception of PEL-2 and CYA-3, there were no effects of 19 major anthocyanins and 3 food supplements containing anthocyanins on AhR–CYP1A1 signaling, implying zero potential of these compounds for food–drug interactions with respect to AhR–CYP1A1 pathway.

Abstract 3743: Effect of folic acid supplementation on epigenetics and cancer stem cell populations in established colorectal cancer cell lines.

Abstract Mandated fortification of folic acid, a B vitamin involved in nucleotide synthesis and DNA CpG methylation, has led to a subsequent increase in blood folate concentration as well as a simultaneous increase in colorectal cancer incidence in North America. Recent findings suggest that a proportion of tumor cells, termed cancer stem cells (CSC), share properties with native stem cells and are vital in the development and perpetuation of tumor metastasis. Both epigenetic DNA modifications as well as the activation of established stem cell signaling pathways are responsible for the activation and maintenance of (CSC). CSC share properties with native stem cells and are vital in the development of primary lesions and the perpetuation of tumor metastasis. We hypothesize that folate, a CpG methylation altering agent, and extracellular microenvironment factors can influence the development and maintenance of CSCs. By isolating and characterizing the CSC fraction in established human colorectal cancer cell lines HCT116, Caco2, and SW480, LS174T and PC7, a subline of DLD-1, we have assessed the effect of folate on CSC signaling pathways and global DNA methylation. Cell lines were grown in suspension and supplemented with specialized stem cell media to promote the growth and expansion of the CSC fraction. Western blot analysis and immunofluorescence were used to characterize the protein expression and nuclear localization of proteins associated with established CSC maintenance pathways, respectively. Global DNA methylation status was assessed using an ELISA-based assay. HCT116 and SW480 show an increased ability to form colonospheres in culture compared to Caco2 and IEC18 (normal rat intestinal cell line). Cells grown in suspension show variable activation of stem cell renewal pathways compared to monolayer. Low folic acid levels led to reduced methylation status and colonosphere yield while high folic acid levels led to increased global methylation and an increased in colonosphere yield. Thus, altered folic acid exposure in vitro can influence the methylation status and CSC self-renewal ability of human colorectal cancer cells. The relationship between folic acid and cancer stem cell maintenance may be an important factor in the apparent duel modulatory effect of folic acid on colorectal cancer and warrants further investigation. Citation Format: Nathan Farias, Brenda Coomber. Effect of folic acid supplementation on epigenetics and cancer stem cell populations in established colorectal cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3743. doi:10.1158/1538-7445.AM2013-3743

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.

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.

Systemic TNFα Gene Therapy Synergizes With Liposomal Doxorubicine in the Treatment of Metastatic Cancer

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

Troy, a Tumor Necrosis Factor Receptor Family Member, Interacts With Lgr5 to Inhibit Wnt Signaling in Intestinal Stem Cells

The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice. We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc(+/Min) and Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization. We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer-derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist. TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.

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.

Parthenolide suppresses tumor growth in a xenograft model of colorectal cancer cells by inducing mitochondrial dysfunction and apoptosis

Parthenolide (PT), a principal active component in medicinal plants, has been used conventionally to treat migraine and inflammation. This component has recently been reported to induce apoptosis in cancer cells, through mitochondrial dysfunction. In the present study, we investigated PT-mediated cell death signaling pathway by focusing on the involvement of Bcl-2 family members in human colorectal cancer cells. We also investigated the inhibitory effect of PT on tumor growth in xenografts. Using the human colorectal cancer cell lines HT-29, SW620 and LS174T, we demonstrated that treatment of these cancer cells with PT induces apoptosis using MTT, Annexin V assay and Hoechst 33258 staining. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, cytochrome c release and caspase activation. Moreover, intraperitoneal injection of PT showed significant inhibition of tumor growth, angiogenesis in the xenograft model. These results demonstrate that PT exhibits anti-cancer activity in human colorectal cancer in vitro and in vivo. These findings may also provide a novel approach for the treatment of colorectal cancer.

Study of the upregulation of the activity of cytochrome P450 3A isoforms by Astragalus injection and Astragalus granules in rats and in cells

Astragalus injection (AI) and Astragalus granules (AG) are the two representative clinical preparations from Astragali Radix. In order to investigate the regulation of metabolism, AI and AG were tested for their ability to affect the major enzyme cytochrome P450 3A isoforms in vivo and in vitro. In the study of CYP3A1 enzyme activity, male rats were pretreated with AI and AG. The "cocktail" approach-based LC-MS/MS results showed that AI pretreatment at 0.16, 0.8 and 4gkg(-1)day(-1) significantly increased the rat liver microsome CYP3A1 activity by 1.62-, 1.68- and 2.00-fold, and AG pretreatment at 32, 160 and 800mgkg(-1)day(-1) significantly increased the rat CYP3A1 activity by 1.86-, 2.16- and 1.76-fold. The effects of AI and AG on liver microsome CYP3A1 mRNA expression in rats were analyzed using real-time PCR technique. The results showed that AI and AG pretreatments significantly increased the CYP3A1 mRNA expression. The induction of CYP3A4 enzyme activity by AI and AG in vitro was measured using a CYP3A4 luciferase reporter gene assay in transiently transfected human intestinal LS174T cells. Compared to the control group, AI at 62.5-1,000mg/ml could significantly induce CYP3A4 reporter gene luciferase activity of 1.36- to 1.88-fold for 48-h incubated PXR-transfected LS174T cells, and AG at 62.5-1,000μg/ml significantly transactivated CYP3A4 reporter gene luciferase activity of 1.36- to 2.05-fold. However, the CYP3A4 reporter gene construct was not significantly transactivated by the AI and AG in CAR-transfected LS174T cells. These CYP3A isoforms upregulation results can help us to use AI and AG rationally in the clinic.

ABCB1 (P-glycoprotein) reduces bacterial attachment to human gastrointestinal LS174T epithelial cells

The aim of this project was to show elevated P-glycoprotein (P-gp) expression decreasing bacterial association with LS174T human gastrointestinal cells, and that this effect could be reversed upon blocking functional P-gp efflux. Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Lactobacillus acidophilus and numerous strains of Escherichia coli, from commensal to enteropathogenic and enterohaemorrhagic strains (O157:H7) were fluorescently labelled and incubated on LS174T cultures either with or without P-gp amplification using rifampicin. PSC-833 was used as a potent functional P-gp blocking agent. Staphylococcus and Pseudomonas displayed the greatest association with the LS174T cells. Surprisingly, lactobacilli retained more fluorescence than enteropathogenic -E. coli in this system. Irrespective of attachment differences between the bacterial species, the increase in P-gp protein expression decreased bacterial fluorescence by 25–30%. This included the GFP-labelled E. coli, and enterohaemorrhagic E. coli (O157:H7). Blocking P-gp function through the co-administration of PSC-833 increased the amount of bacteria associated with P-gp expressing LS174T cells back to control levels. As most bacteria were affected to the same degree, irrespective of pathogenicity, it is unlikely that P-gp has a direct influence on adhesion of bacteria, and instead P-gp may be playing an indirect role by secreting a bank of endogenous factors or changing the local environment to one less suited to bacterial growth in general.

Adenovirus-mediated tissue-targeted expression of the CDglyTk gene for the treatment of breast cancer

The aim of this study was to evaluate the selective killing efficacy of adenovirus (Ad)-mediated double suicide genes driven by the kinase domain-containing receptor (KDR) promoter in human breast cancer cells and vascular endothelial cells. Two Ad-mediated double suicide gene systems [with the two suicide genes, thymidine kinase (TK) and cytosine deaminase (CD)] with the KDR promoter (Ad-KDRP-CDglyTK) and the cytomegalovirus (CMV) promoter (Ad-CMV-CDglyTK) were established and transfected into the KDR-expressing MCF7 human breast cancer, EC304 human vascular endothelial and LS174T human colon carcinoma, which does not express KDR, cell lines. The selective killing efficiency and specificity of the double suicide gene system were measured in vitro by the analysis of cellular proliferation and assayed in vivo by subcutaneous injection of MCF7 cells into nude mice. The microvessel density (MVD) in the transplanted tumor was determined by immunohistochemical staining of CD34 cells. Our results showed that the transgenic CDglyTK genes were expressed in three cell lines (MCF7, ECV304 and LS174T) infected with Ad-CMV-CDglyTK. However, of the cells infected with Ad-KDRP-CDglyTK, the transgenic CDglyTK gene was only expressed in the KDR-expressing MCF7 and ECV304 cells, but not in the KDR-deficient LS174T cells. Cell proliferation was significantly reduced in a dose-dependent manner by pre-treatment with ganciclovir (GCV) and 5-fluorocytosine (5-FC) in MCF7 and ECV304 cells with transfected KDRP-CDglyTK genes and the three cell lines transfected with the CMV-CDglyTK genes. Similar results were not observed in the LS174T cells with transfected KDRP-CDglyTK genes. The results of this study show that the tumor-targeted expression of CDglyTK driven by the KDR promoter has a high specificity and performance. The killing effect of the CD/TK fusion gene in the target cells was significantly increased compared with the single suicide gene. The cell cycle of MCF7 and ECV304 cells transfected with KDRP-CDglyTK genes was arrested at the S phase following treatment with the prodrugs. The tumors formed by the MCF7 cells with the double suicide gene system were much smaller and the MVD of the tumor tissue was significantly decreased compared with the control. This study demonstrates that tumor‑targeted expression of the CDglyTK gene driven by the KDR promotor may be a novel strategy for the gene therapy of human breast cancer.

Abstract 3225: Growth inhibition of glycolytic tumors by targeting basigin/lactate-H+ symporters (MCTs): Metformin sensitizes MCT inhibition

Abstract Intense conversion of glucose to lactic acid via glycolysis is a feature of rapidly growing cells often encountered in hypoxic tumor microenvironments. To survive and expand, tumor cells must efficiently export lactic acid to maintain intracellular pH. Cells possess several systems for lactic acid extrusion. A family of H+-linked MonoCarboxylate Transporters (MCTs) is represented by the ubiquitously expressed MCT1, a H+/lactate symporter that operates in both directions. MCT4, a close relative of MCT1, is up-regulated by HIF-1 and is highly expressed in aggressive malignant tumors. In addition, the functional expression of MCT1/MCT4 requires the interaction with the glycoprotein CD147/Basigin also known as EMMPRIN, a protumoral protein involved in invasion. Objectives: 1) Demonstrate that tumor growth is dependent on lactic acid export and that both transporters MCT1/MCT4 represent key anticancer targets. 2) Demonstrate whether the protumoral function of Basigin/CD147 is primary linked to lactic acid export or to other invasive functions. 3) Demonstrate whether Metformin, an inhibitor of mitochondrial complex I, sensitizes glycolytic tumor cells to MCTs inhibitors. Methods: We exploited two tumoral models. i) Ras transformed fibroblasts expressing only MCT1/MCT2 and ii) the human colon adenocarcinoma cell line LS174T expressing MCT1 and MCT4. We inhibited MCT1/MCT2 with the specific astraZeneca compound AR-C155858 and knocked-down MCT1, MCT4 and CD147 with inducible shRNAs. In addition we knocked-out mct4 or basigin with Zinc Finger Nucleases in LS174T cells. Results: First we demonstrated that silencing or pharmacological blockage of MCTs, reduced pHi, the rate of glycolysis and tumor growth in mice xenografts. This tumor growth inhibition was recapitulated by a single silencing or knock out of basigin/CD147 gene that also reduced the plasma membrane expression of MCT1 and MCT4 and lactate transport up to 10-fold. Second, to gain insight into CD147/Basigin function, we uncoupled MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin-high expressors, suppressed tumor growth. Conversely in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Thirdly, we showed that tumor cells lacking Basigin or MCT4 become highly sensitive to MCT1 inhibition when treated with Metformin. This synthetic lethality demonstrated in vitro and currently being tested in vivo will be discussed. Conclusions: These findings highlight that a major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer approach. Furthermore, we reveal that Metformin, by sensitizing normoxic cells to inhibitors of lactic export (MCTs), could offer an interesting novel anticancer strategy for rapidly growing tumors. 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 3225. doi:1538-7445.AM2012-3225

Effects of flavored mineral waters on AhR–CYP1A1 signaling pathway in primary human hepatocytes and in human hepatic and intestinal cancer cells

Food–drug interaction is an emerging phenomenon, comprising pharmacokinetic or toxicokinetic interactions between food constituents and drugs. The mechanisms include inhibition of enzymes and transporters, and induction of drug metabolizing enzymes. A prominent regulator of drug-metabolizing enzymes is an aryl hydrocarbon receptor (AhR) that transcriptionally regulates CYP1 enzymes, phase II enzymes and many other genes. In the current paper, we have examined the effects of 28 different flavored mineral waters on AhR–CYP1A1 signaling pathway in primary cultures of human hepatocytes and in human cancer cell lines HepG2 (hepatic) and LS174T (intestinal). The techniques of Western blot, RT-PCR and gene reporter assays were employed to determine the expression of CYP1A1 mRNA, protein and activation of AhR, respectively. We have identified four flavored mineral waters which activated AhR and/or induced CYP1A1. These data imply a potential of some flavored mineral waters to cause food–drug interactions. In addition, activation of AhR–CYP1A1 signaling may result in chemically-induced carcinogenesis and alteration of intermediary metabolism.

Inflammatory cues modulate the expression of secretory product genes, Golgi sulfotransferases and sulfomucin production in LS174T cells

The signals that mediate goblet cell expression of specific mucin chemotypes are poorly defined. Animal and in vitro studies show that acidomucin chemotypes may be altered by inflammation and changes in intestinal microbiota. To examine factors that may elicit this response, human adenocarcinoma-derived LS174T cells, which have a goblet cell-like phenotype and produce both sulfo- and sialomucins, were used to examine the effects of selected microbial and host factors on expression of goblet cell secretory product genes, sulfotransferases and sulfomucin production. Expression of genes encoding mucin 2 (MUC2), resistin-like molecule β (RETNLB), and trefoil factor 3 (TFF3) and Golgi sulfotransferases, carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 5 (CHST5) and galactose-3-O-sulfotransferase 2 (GAL3ST2), was measured by quantitative reverse transcriptase-polymerase chain reaction following treatment with bacterial flagellin, tumor necrosis factor α (TNF-α) or the mucogenic cytokine interleukin-13 (IL-13). Expression of the toll-like receptor 5 (TLR5) gene was also analysed. Sulfomucin expression was examined via high-iron diamide/alcian blue (HID/AB) histochemistry and immunofluorescent staining for the Sulfo Le(a) antigen, which is synthesized in part by GAL3ST2. Flagellin, IL-13 and TNF-α all significantly increased GAL3ST2, MUC2, TFF3 and TLR5 expression, while only IL-13 increased RETNLB and CHST5 expression. Based on HID/AB histochemistry, mucin sulfation was significantly increased in response to both flagellin and IL-13 but not TNF-α. Only treatment with flagellin increased the expression of the Sulfo Le(a) antigen. Collectively, these results indicate that bacterial flagellin, IL-13 and TNF-α differentially modulate the expression of goblet cell secretory product genes, sulfotransferases and sulfomucin production.