LS180 Cells Research Articles

Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells.

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter-like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high-affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium-3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na(+)-independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H(+) gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K(+) concentration. In addition, the Na(+)-independent uptake of choline was saturable, and the Km value was estimated to be 108 µm. These findings suggest that the uptake of choline into LS180 cells is membrane potential-dependent, but not outward H(+) gradient-dependent.

Abstract 2602: KRAS G12D and G12V specific alkylating agent (KR12) inhibits growth of colon cancer with those KRAS mutations in vitro as well as in vivo

Abstract Despite extensive efforts to develop chemotherapeutic drug(s) targeting mutated RAS, the successful drug discovery, especially targeting KRAS codon 12 mutation, has never been made. We have found that Pyrrole-Imidazole polyamide seco-CBI conjugate (KR12) selectively recognized mutated KRAS sequence (ACGCCT-A/T-CA) at codon 12 and significantly suppressed tumor growth specifically in human colon cancer cells with G12D or G12V mutation. KRAS expression suppressed preferentially at mutated allele and active KRAS were markedly reduced. G2/M arrest, senescence and subsequent apoptosis by activating the p53 pathway were observed in KR12-exposed LS180 cells with G12D heterozygous mutation. In LS180 and SW480 (G12V homozygous mutation) xenograft colon cancer models, KR12 treatment induced massive tumor growth suppression with low host toxicity. Collectively, our current results strongly suggest that KR12 is a specific alkylating agent against KRAS codon 12 mutations, and could become a powerful candidate compound for the unmet need of KRAS-mutant tumor treatment. Citation Format: Hiroki Nagase, Kiriko Hiraoka, Takahiro Inoue, Takayoshi Watanabe, Ken-Ichi Shinohara, Nobuko Koshikawa, Ozaki Toshinori. KRAS G12D and G12V specific alkylating agent (KR12) inhibits growth of colon cancer with those KRAS mutations in vitro as well as in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2602. doi:10.1158/1538-7445.AM2014-2602

Role of Bcl-xL/Beclin-1 in synergistic apoptotic effects of secretory TRAIL-armed adenovirus in combination with mitomycin C and hyperthermia on colon cancer cells.

In this study, we attempted to develop a multimodality approach using chemotherapeutic agent mitomycin C, biologic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo-2L), and mild hyperthermia to treat colon cancer. For this study, human colon cancer LS174T, LS180, HCT116 and CX-1 cells were infected with secretory TRAIL-armed adenovirus (Ad.TRAIL) and treated with chemotherapeutic agent mitomycin C and hyperthermia. The combinatorial treatment caused a synergistic induction of apoptosis which was mediated through an increase in caspase activation. The combinational treatment promoted the JNK-Bcl-xL-Bak pathway which transmitted the synergistic effect through the mitochondria-dependent apoptotic pathway. JNK signaling led to Bcl-xL phosphorylation at serine 62, dissociation of Bak from Bcl-xL, oligomerization of Bak, alteration of mitochondrial membrane potential, and subsequent cytochrome c release. Overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed the synergistic death effect. Interestingly, Beclin-1 was dissociated from Bcl-xL and overexpression of dominant-negative mutant of Bcl-xL (S62A), but not dominant-positive mutant of Bcl-xL (S62D), suppressed dissociation of Beclin-1 from Bcl-xL. A combinatorial treatment of mitomycin C, Ad.TRAIL and hyperthermia induced Beclin-1 cleavage, but the Beclin-1 cleavage was abolished in Beclin-1 double mutant (D133A/D146A) knock-in HCT116 cells, suppressing the apoptosis induced by the combination therapy. We believe that this study supports the application of the multimodality approach to colon cancer therapy.

Rapid production of novel pre-microRNA agent hsa-mir-27b in Escherichia coli using recombinant RNA technology for functional studies in mammalian cells.

Noncoding microRNAs (miRNAs or miRs) have been revealed as critical epigenetic factors in the regulation of various cellular processes, including drug metabolism and disposition. However, research on miRNA functions is limited to the use of synthetic RNA and recombinant DNA agents. Herein, we show that novel pre-miRNA-27b (miR-27b) agents can be biosynthesized in Escherichia coli using recombinant RNA technology, and recombinant transfer RNA (tRNA)/mir-27b chimera was readily purified to a high degree of homogeneity (>95%) using anion-exchange fast protein liquid chromatography. The tRNA-fusion miR-27b was revealed to be processed to mature miRNA miR-27b in human carcinoma LS-180 cells in a dose- and time-dependent manner. Moreover, recombinant tRNA/miR-27b agents were biologically active in reducing the mRNA and protein expression levels of cytochrome P450 3A4 (CYP3A4), which consequently led to lower midazolam 1'-hydroxylase activity. These findings demonstrate that pre-miRNA agents can be produced by recombinant RNA technology for functional studies.

Binary and ternary combinations of anti-HIV protease inhibitors: effect on gene expression and functional activity of CYP3A4 and efflux transporters

The purpose of this study is to identify the effect of binary and ternary combinations of anti-HIV protease inhibitors (PIs) on the expression of metabolizing enzyme (CYP3A4) and efflux transporters [multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP)] in a model intestinal cell line (LS-180). LS-180 cells were treated with various combinations of PIs (amprenavir, indinavir, saquinavir and lopinavir), and the mRNA expression levels of metabolizing enzyme and efflux transporters were measured using quantitative reverse transcription polymerase chain reaction. The alteration of gene expression was further correlated to the expression of nuclear hormone receptor PXR. Uptake of fluorescent and radioactive substrates was carried out to study the functional activity of these proteins. Cytotoxicity and adenosine triphosphate (ATP) assays were carried out to measure stress responses. Binary and ternary combinations of PIs appeared to modulate the expression of CYP3A4, MRP2, P-gp and BCRP in a considerable manner. Unlike the individual PIs, their binary combinations showed much greater induction of metabolizing enzyme and efflux proteins. However, such pronounced induction was not observed in the presence of ternary combinations. The observed trend of altered mRNA expression was found to correlate well with the change in expression levels of PXR. The gene expression was found to correlate with activity assays. Lack of cytotoxicity and ATP activity was observed in the treatment samples, suggesting that these alterations in expression levels were probably not stress responses. In the present study, we demonstrated that combinations of drugs can have serious consequences toward the treatment of HIV infection by altering their bioavailability and disposition.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induced cell-specific drug transporters with acquired cisplatin resistance in cisplatin sensitive cancer cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can induce drug transporter genes such as the ATP-binding cassette G member 2 (ABCG2), which contributes to multidrug resistance. We investigated the effect of TCDD pretreatment on drug transporters induction from cancer cells of various origins. Cell viabilities after treatment of cisplatin were measured to evaluate acquiring cisplatin resistance by TCDD. Acquring cisplatin resistance was found only in cisplatin senstivie cancer cells including gastric SNU601, colon LS180, brain CRT-MG and lymphoma Jurkat cells which showed a significant increase in cell viability after combined treatment with TCDD and cisplatin. High increase of ABCG2 gene expression was found in SNU601 and LS180 cells with a mild increase in the expression of the ABCC3, ABCC5,and SLC29A2 genes in SNU601 cells, and of major vault protein (MVP) in LS180 cells. The AhR inhibitor kaempferol suppressed the upregulation of ABCG2 expression and reversed the TCDD-induced increase in cell viability in LS180 cells. However, in CRT-MG cells, other transporter genes including ABCC1, ABCC5, ABCA3, ABCA2, ABCB4, ABCG1, and SLC29A1 were up-regulated. These findings suggested the acquiring cisplatin resistance by TCDD associated with cancer cell-type-specific induction of drug transporters.Graphical

Variability of Bioavailability and Intestinal Absorption Mechanisms of Metoprolol

We previously reported that aging and/or cytochrome P450 2D6 polymorphism are responsible for the interindividual variability in the systemic clearance (CL) and bioavailability (F) of metoprolol. The aim of the present study was to evaluate the residual variability of F of metoprolol in routinely treated Japanese patients and to investigate the intestinal absorption mechanism of the drug using human intestinal epithelial LS180 cells. We first re-analyzed the blood concentration data for metoprolol in 34 Japanese patients using a nonlinear mixed effects model. The oral clearance (CL/F) of metoprolol was positively correlated with the apparent volume of distribution (V/F), suggesting the residual variability of F. The uptake of metoprolol into LS180 cells was significantly decreased by the acidification of extracellular medium pH, and was dependent on temperature and intracellular pH. Furthermore, the cellular uptake of metoprolol was saturable, and was significantly decreased in the presence of hydrophobic cationic drugs such as diphenhydramine, procainamide, bisoprolol, and quinidine. These findings indicate that residual variability of F is one of the causes of the interindividual pharmacokinetic variability of metoprolol, and that the interindividual variability of not only presystemic first-pass metabolism, but also intestinal absorption, may be responsible for the variable F of the drug.

Analysis of flavonoids regulating the expression of UGT1A1 via xenobiotic receptors in intestinal epithelial cells

UDP-glucuronosyltransferase (UGT) 1A1 is one of the major metabolic enzymes for the detoxification of harmful xenobiotics in intestines, and its expression is regulated by transcription factors like the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). A screening assay using real-time PCR showed that baicalein and 3-hydroxyflavone induced human UGT1A1 mRNA expression in LS180 cells. Experimental results confirmed that these flavonoids increased UGT1A protein expression as well as its enzymatic activity. The results indicated that baicalein and 3-hydroxyflavone increased the transcriptional activity of UGT1A1 via AhR and PXR, respectively. Observation via immunofluorescence microscopy suggested that baicalein and 3-hydroxyflavone further induced nuclear translocation of AhR and PXR, respectively. In addition, direct interaction between baicalein and AhR or 3-hydroxyflavone and PXR were observed using the quartz crystal microbalance method. These results elucidate the molecular mechanism of flavonoid-induced UGT1A1 gene expression via xenobiotic receptors in the intestines.

Expression of the Orphan Cytosolic Sulfotransferase SULT1C3 in Human Intestine: Characterization of the Transcript Variant and Implications for Function

The cystolic sulfotransferse 1C3 (SULT1C3) gene was identified by computational analysis of the human genome and suggested to contain duplications of its last two exons (7a/b and 8a/b). Although the SULT1C3 isoform containing the more downstream exons 7b and 8b (SULT1C3d) has been expressed in Escherichia coli, crystallized, and characterized for activity, there is currently no evidence that SULT1C3 is expressed in any human tissue. Using reverse-transcription polymerase chain reaction, we detected SULT1C3 mRNA in the colorectal adenocarcinoma cell line (LS180), colon, and small intestine, but the amplified fragment contained the more upstream exons 7a and 8a. 3'-Rapid amplification of cDNA ends (RACE) confirmed that the SULT1C3 transcript expressed in LS180 cells contained exons 7a/8a, whereas 5'-RACE identified a noncoding exon 1. Full-length SULT1C3 transcript containing exons 7a/8a was amplified from LS180 and intestinal RNA, and in vitro transcription-translation of the cloned cDNA indicated that translation primarily began at the first of three in-frame ATG codons. Since SULT1C3 containing exons 7a/8a (SULT1C3a) would differ by 30 amino acids from SULT1C3d containing exons 7b/8b, we considered the functional implications of expressing one or the other isoform by generating structural models based on the reported crystal structure for SULT1C3d. Comparison of the structures indicated that five of the residues forming the substrate-binding pocket differed between the two isoforms, resulting in a change in both electron density and charge distribution along the inner wall of the substrate-binding pocket. These data indicate that SULT1C3 is expressed in human intestine but suggest that the expressed isoform is likely to differ functionally from the isoform that has been previously characterized.

Regulation of Human Cytosolic Sulfotransferases 1C2 and 1C3 by Nuclear Signaling Pathways in LS180 Colorectal Adenocarcinoma Cells

Cytosolic sulfotransferases (SULTs) catalyze the sulfate conjugation of a myriad of endogenous and xenobiotic substrates. Among the 13 human SULTs, little is known regarding regulation of the SULT1C subfamily. We evaluated the effects of a panel of transcription factor activators on levels of SULT1C mRNA (1C2 and 1C3) and protein (1C2) in LS180 colorectal adenocarcinoma cells. Treatment with 3-[3-[N-(2-chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl)amino]propyloxy]phenylacetic acid hydrochloride [GW3965, liver X receptor (LXR) activator], 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole [GW4064, farnesoid X receptor (FXR)], or rifampicin [pregnane X receptor (PXR)] moderately (≤2-fold) increased both SULT1C2 and SULT1C3 mRNA levels. 1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3, vitamin D receptor (VDR)] selectively upregulated SULT1C2, whereas ciprofibrate [peroxisome proliferator-activated receptor α (PPARα)], rosiglitazone (PPARγ), and 2,3,7,8-tetrachlorodibenzo-p-dioxin [aryl hydrocarbon receptor (AhR)] selectively increased SULT1C3 mRNA levels. SULT1C2 protein content was strongly increased by 1,25(OH)2D3 treatment and moderately increased by GW3965, GW4064, and rifampicin. To evaluate SULT1C2 transcriptional regulation, treatment effects were determined on reporter activity from transfected constructs containing ∼10 kb of the SULT1C2 gene. Treatment with GW3965, GW4064, or 1,25(OH)2D3 increased reporter activity ∼2-, 5-, and 5.5-fold, respectively, from a construct containing mostly intron 1 of the SULT1C2 gene. Expression of AhR, LXRα, LXRβ, PPARα, PPARγ, PXR, and VDR was confirmed in LS180 cells using quantitative reverse-transcription polymerase chain reaction; however, FXR expression was negligible, suggesting that GW4064 increased SULT1C expression through an FXR-independent mechanism. Collectively, our findings are the first to characterize the regulation of human SULT1C2 and SULT1C3 expression by several transcription factor activators. Further, we determined that responsive regions for LXR and VDR are likely contained within intron 1 of the SULT1C2 gene.

Pharmacokinetic variability of flecainide in younger Japanese patients and mechanisms for renal excretion and intestinal absorption

The aims of the present study were to evaluate the variability of pharmacokinetics of flecainide in young Japanese patients and to investigate the mechanisms of renal excretion and intestinal absorption of the drug using cultured epithelial cells. First the plasma concentration data of flecainide was analysed in 16 Japanese patients aged between 0.07 and 18.30 years using a one-compartment model. Considerable interindividual variability was observed in the oral clearance (CL/F) and the apparent volume of distribution (V/F) of flecainide in the young patients. Flecainide was transported selectively in the basolateral-to-apical direction in P-glycoprotein-expressing renal epithelial LLC-GA5-COL150 cell monolayers. The uptake of flecainide into intestinal epithelial LS180 cells was decreased significantly by acidification of the extracellular medium, and was inhibited by tertiary amines, such as diphenhydramine and quinidine. These findings in the present study suggest that flecainide is excreted by P-glycoprotein in the renal tubule and is taken up by the postulated H(+)/tertiary amine antiporter in the intestine, and that functional variability of not only the hepatic drug-metabolizing enzymes, but also the transporters in the kidney and intestine, may be responsible for the interindividual variability of systemic clearance (CL) and/or the bioavailability (F) of flecainide.

Epigenetic regulation of the tissue-specific expression of human UDP-glucuronosyltransferase (UGT) 1A10

Human UDP-glucuronosyltransferase (UGT) 1A10 is not expressed in the liver; however, UGT1A10 is highly expressed in the intestine, contributing to presystemic first-pass metabolism. Earlier studies revealed that hepatocyte nuclear factor (HNF) 1α and Sp1, as well as an intestine-specific transcription factor, caudal type homeobox (Cdx) 2, are involved in the constitutive expression of UGT1A10. However, why UGT1A10 is not expressed in the liver, where HNF1α and Sp1 are abundantly expressed, is unknown. In this study, we sought to elucidate the mechanism, focusing on epigenetic regulation. Bisulfite sequence analysis revealed that the CpG-rich region (−264 to +117) around the UGT1A10 promoter was hypermethylated (89%) in hepatocytes, whereas the UGT1A10 promoter was hypomethylated (11%) in the epithelium of the small intestine. A luciferase assay revealed that the methylation of the UGT1A10 promoter by SssI methylase abrogated transactivity even with overexpressed Cdx2 and HNF1α. The UGT1A10 promoter was highly methylated (86%) in liver-derived HuH-7 cells, where UGT1A10 is not expressed. In contrast, the UGT1A10 promoter was hardly methylated (19%) in colon-derived LS180 cells, where UGT1A10 is expressed. Treatment with 5-aza-2′-deoxycitidine (5-Aza-dC), an inhibitor of DNA methylation, resulted in an increase in UGT1A10 expression only in HuH-7 cells. Moreover, overexpression of HNF1α and Cdx2 further increased UGT1A10 expression only in the presence of 5-Aza-dC. Collectively, we found that DNA hypermethylation would interfere with the binding of HNF1α and Cdx2, resulting in the defective expression of UGT1A10 in human liver. Thus, epigenetic regulation is one of the mechanisms that determine the tissue-specific expression of UGT1A10.

Telaprevir is a substrate and moderate inhibitor of P-glycoprotein, a strong inductor of ABCG2, but not an activator of PXR in vitro

Triple therapy combining the protease inhibitor telaprevir with interferon-α and ribavirin is a promising new option for long-term treatment of hepatitis C. The interaction potential of telaprevir has not yet been fully elucidated. The in vitro potency of telaprevir to inhibit P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) was assessed and its substrate characteristics for P-gp, BCRP and the multidrug resistance-associated proteins (MRPs, ABCCs) 1–3 were evaluated. The inducing properties of telaprevir on important drug-metabolising enzymes and transporters were also assessed and its ability to activate the pregnane X receptor (PXR) was investigated. Using growth inhibition assays, it was confirmed that telaprevir is a substrate of P-gp and it was demonstrated for the first time that it is not transported by BCRP and MRPs. Telaprevir only moderately inhibited P-gp in the calcein assay and did not inhibit BCRP in the pheophorbide A assay. In LS180 cells, telaprevir strongly induced mRNA expression of ABCG2 (4.3-fold at 30μmol/L) and weakly induced ABCB11, CYP2C19 and UGT1A3. In contrast, telaprevir had no significant influence on mRNA expression of CYP3A4, UGT1A9, ABCB1, ABCC2 and SLCO1B1. In a reporter gene assay, telaprevir did not activate PXR. Thus, it appears unlikely that telaprevir induces CYP3A4 and P-gp in vivo in such a way as to provoke clinically relevant drug interactions. From the numerous perpetrator characteristics, telaprevir's inhibitor properties, especially of CYP3A4 and P-gp, appear to be the most relevant mechanism for drug interactions. The clinical relevance of the strong inducing effects on ABCG2 requires proper assessment.

Combined effects of C225 and 125-iodine seed radiation on colorectal cancer cells

BackgroundTo characterize the effect of combined treatment of the anti-epidermal growth factor receptor (EGFR) monoclonal antibody C225 and 125-iodine (125I) seed radiation in human colorectal cancer.MethodsWe treated LS180 cells with 125I continuous low dose rate radiation in the presence and absence of 100 nM C225. The clonogenic capacity, cellular proliferation, cell cycle distribution, apoptosis, and molecular pathways of the cells following the treatments were analyzed in vitro.ResultsThe sensitizer enhancement ratio of C225 was approximately 1.4. Treatment with C225 and radiation alone produced significant inhibition of cell growth, but combination therapy produced greater inhibition than either treatment administered alone. C225 increased the radiation-induced apoptosis and the fraction of γ-H2AX foci positive cells at 48 h after treatment. The Akt phosphorylation level was lower in the cells receiving the combination treatment than in the cells treated with radiation or C225 alone.ConclusionsThese findings indicate that C225 sensitizes LS180 cells to 125I seed radiation. Growth inhibition is mediated by inducing apoptosis and not cell cycle arrest. Additionally, we confirmed that C225 impairs DNA repair by reducing the cellular level of the DNA-PKcs and Ku70 proteins. Furthermore, the inhibition of Akt signaling activation may be responsible for the C225-mediated radiosensitization.

Effects of Decreased Vitamin D and Accumulated Uremic Toxin on Human CYP3A4 Activity in Patients with End-Stage Renal Disease

In patients with end-stage renal disease, not only renal clearance but also hepatic clearance is known to be impaired. For instance, the concentration of erythromycin, a substrate of cytochrome P450 3A4 (CYP3A4), has been reported to be elevated in patients with end-stage renal disease. The purpose of this study is to elucidate the reason for the decrease in hepatic clearance in patients with end-stage renal disease. Deproteinized pooled sera were used to assess the effects of low-molecular-weight uremic toxins on CYP3A4 activity in human liver microsomes and human LS180 cells. Four uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, hippuric acid, indole-3-acetic acid, and 3-indoxyl sulfate) present at high concentrations in uremic serum were also studied. Simultaneous treatment of uremic serum (less than 10%) or uremic toxins did not affect testosterone 6β-hydroxylation in human liver microsomes. On the other hand, pretreatment of each serum activates CYP3A4 in LS180 cells, and the increased CYP3A4 activity in uremic serum-treated cells was smaller than normal serum-treated cells. In addition, CYP3A4 and CYP24A1 mRNA levels also increased in LS180 cells exposed to normal serum, and this effect was reduced in uremic serum-treated cells and in cells exposed to uremic serum added to normal serum. Furthermore, addition of 1,25-dihydroxyvitamin D to uremic serum partially restored the serum effect on CYP3A4 expression. The present study suggests that the decrease of 1,25-dihydroxyvitamin D and the accumulation of uremic toxins contributed to the decreased hepatic clearance of CYP3A4 substrates in patients with end-stage renal disease.

Effect of Chinese herbs on CYP3A4 activity and expression in vitro

Ethnopharmacological relevanceTraditional Chinese Medicine (TCM) has become more popular among cancer patients in the Western world, who often use Chinese herbs as adjuvant therapy to reduce the adverse effects of conventional chemotherapy. However, pharmacokinetic (PK) interactions between Chinese herbs and anticancer drugs can occur and have dramatic consequences for these patients. Currently, only a few possible PK interactions between Chinese herbs and conventional Western drugs have been documented. Aim of the studySince the drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) contributes to most of the PK interactions with (anticancer) drugs, the effect of four Chinese herbs (Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus) on the activity and expression of CYP3A4 was investigated in vitro. Materials and methodsEthanol and water–ethanol extracts of the four Chinese herbs were prepared from raw material. CYP3A4 inhibition was assessed by the use of Supersomes™ in a fluorescence assay. Furthermore, CYP3A4 induction was evaluated in a human pregnane X receptor (hPXR)-mediated CYP3A4 reporter gene assay and a quantitative real time PCR assay, both in human colon adenocarcinoma-derived LS180 cells (LS180). ResultsExtracts of Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus inhibited CYP3A4 in human CYP3A4 Supersomes™ (IC50 values: 17–83µg/mL). Oldenlandia diffusa and Rehmannia glutinosa significantly induced PXR-mediated CYP3A4 (p<0.001). Oldenlandia diffusa also significantly induced CYP3A4 mRNA levels (p<0.001 at 250µg/mL). ConclusionsConcomitant use of Oldenlandia diffusa and Rehmannia glutinosa could result in induction of CYP3A4, leading to a reduced efficacy of drugs that are CYP3A4 substrates and have a narrow therapeutic window. Because of the possible enhanced toxicity caused by CYP3A4 inhibition, clinical effects of CYP3A4 inhibition by Astragalus propinquus and Codonopsis tangshen must also be taken into account. In conclusion, herb–drug interactions between Chinese herbs and various CYP3A4 substrates can occur. Further research to investigate the clinical relevance of the interactions caused by Oldenlandia diffusa, Codonopsis tangshen, Rehmannia glutinosa and Astragalus propinquus is required.

Cytotoxic activity assessment, QSAR and docking study of novel bis-carboxamide derivatives of 4-pyrones synthesized by Ugi four-component reaction

Fourteen novel bis-carboxamide derivatives of 4-pyrones were designed and synthesized via Ugi four-component reactions of 4-pyrone carbaldehydes, aromatic amines, isocyanides and carboxylic acids. The cytotoxic activity of synthesized derivatives was evaluated against LS180, MCF-7 and HL-60 cell lines using MTT reduction assay. Synthesized compounds demonstrated strong cytotoxic potential in HL-60 cell line. Compound 12n was the most potent derivative with IC50 values of 16.1, 9.1 and 13.8 μM in LS180, MCF-7 and HL-60 cells, respectively. The results of MLR-QSAR study indicated that topological property of these derivatives directly influenced the cytotoxic potential in HL-60 cell line. Docking study of compounds, conducted for ATP binding site of Src tyrosine kinase, demonstrated the key H-bond interaction with Met 347 of the hinge region.

Quinaldic acid inhibits proliferation and affects signaling pathways in human colon cancer cells - in vitro studies

Quinaldic acid is naturally occurring in the human organism analog of endogenous tryptophan metabolite kynurenic acid, with not fully elucidated impact on endogenous physiological processes. Considering high level of structural similarity between quinaldic acid and kynurenic acid, which antiproliferative activity towards colon and renal cancer cells was already stated, and its presence in the human organism, evaluation of their chemopreventive potential seems to be a priority. We reported for the first time that in higher milimolar concentrations quinaldic acid possessed antiproliferative activity against colon adenocarcinoma HT-29 and LS-180 cells with the IC50 of 0.5 mM. Moreover, it inhibited DNA synthesis in both cell lines (HT-29, IC50 = 2.7 mM; LS-180, IC50 = 4.3 mM). Morphological changes in colon cancer cells exposed to quinaldic acid were also observed. Western blot analysis revealed that molecular mechanism of antiproliferative action might be based on strict control of the expression and activation of signaling kinases. Summarizing, although biological function of quinaldic acid in colon has still not been fully elucidated, it may be a potential agents in colon cancer chemoprevention.

Determination of the human SULT1C3 mRNA sequence in LS180 cells

The human SULT1C subfamily consists of SULT1C2, SULT1C4, and one putative gene, SULT1C3, which has been identified by computational analysis of the human genome but has not been reported as expressed in any human tissue. An unusual feature of the predicted SULT1C3 gene is that it contains a duplication of the last two exons, with the upstream copies designated as exons 7a and 8a and the downstream copies designated as exons 7b and 8b. The NCBI reference sequence for SULT1C3 mRNA (NM_001008743) begins at the predicted translation start site, ends with exons 7b and 8b, and does not include exon 7a or 8a. We used primers corresponding to sequences in exons 6 and 7b to survey SULT1C3 expression in human tissues and amplified a largerthan‐expected RT‐PCR fragment from small and large intestinal mRNA as well as from LS180 human colorectal adenocarcinoma cells. The cloned fragment contained exons 7a and 8a in addition to the expected portion of exon 7b. We then used 5′‐and 3′‐RACE to determine the entire SULT1C3 mRNA sequence in LS180 cells. These analyses indicated that the SULT1C3 gene contains a non‐coding exon ~7.1 kb upstream of the first coding exon and that the major SULT1C3 transcript contains exons 7a and 8a rather than 7b and 8b. We conclude that SULT1C3 is expressed in human intestinal cells but that its 3′‐exons are not those indicated for the reference sequence. This work was supported by NIH grants HL050710 and ES005823.

Regulation of human cytosolic sulfotransferases (SULTs) 1C2 and 1C3 by nuclear signaling pathways in LS180 cells

Enzymes of the SULT superfamily catalyze the sulfate conjugation of a myriad of endogenous and xenobiotic substrates. Among the human SULT families, little is known about regulation of the SULT1C enzymes. We evaluated the effects of a panel of nuclear signaling activators on levels of SULT mRNA (1C2 and 1C3) and protein (1C2) in LS180 colon cancer cells. Treatment with GW3965 (LXR activator), GW4064 (FXR), or rifampicin (PXR) significantly increased SULT1C2 and 1C3 mRNA levels. Vitamin D3 treatment (VDR) selectively increased SULT1C2 mRNA content, while TCDD (Ah receptor) or rosiglitazone (PPARγ) increased SULT1C3 mRNA levels. SULT1C2 protein levels were moderately increased by GW3965, GW4064, and rifampicin treatments and strongly increased (~5‐fold) by Vitamin D3. To evaluate SULT1C2 transcriptional regulation, treatment effects were determined on reporter expression from a transiently transfected plasmid containing ~5kb of the SULT1C2 gene, corresponding mainly to intron 1. Reporter activity was increased 2‐, 6‐, and 5‐fold by GW3965, GW4064, and Vitamin D3 treatment, respectively (P&lt;0.05). These results indicate that SULT1C2 and 1C3 expression is regulated by several nuclear signaling pathways and suggest that regulatory elements for LXR, FXR, and VDR are contained within intron 1 of the SULT1C2 gene. This work was supported by NIH grants HL050710 and ES005823.