Human Colon Carcinoma Caco-2 Cells Research Articles

3D Model Replicating the Intestinal Function to Evaluate Drug Permeability.

Animal models are essential in drug development but present many concerns in the practical and ethical sense. To avoid the unnecessary use of animals other models are used in the beginning of any scientific discovery, the in vitro models. The relevance of in vitro cell based culture models for studying intestinal drug absorption and transcytosis during early stages of drug development is undeniable. Several in vitro co-culture models have been described for this purpose, however excluding the integration of the complex intestinal architecture and neglecting different physiological mechanisms involved in the drug transport. 2-D cell cultures are the current standard, but despite their widely use, they no longer are considered the most trustworthy in vitro models since they do not mimic many aspects that happen in vivo. The simulation of a complete microenvironment capable of mimicking the intestinal mucosa requires therefore further investigation, particularly focused in addressing the abovementioned unmet needs. 3D models came as bridge between the in vitro and in vivo models. These models are proven to be influential of the drug effect in cells, being the most adequate to mimic the live tissue especially in drug development. Supported by the great amount of studies using simple and reductionist co-culture monolayers, and pushing forward an innovative model previously reported by our group, the present study aims to describe a sophisticated and highly reproducible in vitro 3D co-culture intestinal model. Here, the components are assembled in a multistage process into Transwell filters by co-culturing human colon carcinoma Caco-2 and mucus-producing HT29-MTX cells over a layer of collagen embedding intestinal myofibroblasts (CCD-18Co). The 3D co-culture intestinal model described herein represents a particularly powerful and versatile tool that recapitulates the intestinal functioning regarding mucus production, tightness of the different cell types, and the 3D architecture, bridging the gap between simple monolayer cultures of epithelial cells and the complex in vivo physiological conditions. Importantly, it shows tremendous potential in predicting intestinal absorption of orally administered drugs when delivered alone, or encapsulated into micro- and nanosystems, the current leading force of pharmaceutical technology research.

N-3 Polyunsaturated Fatty Acids Stimulate Bile Acid Detoxification in Human Cell Models.

Cholestasis is characterized by the accumulation of toxic bile acids (BAs) in liver cells. The present study aimed to evaluate the effects of n-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, on BA homeostasis and toxicity in human cell models. The effects of EPA and/or DHA on the expression of genes involved in the maintenance of BA homeostasis were analyzed in human hepatoma (HepG2) and colon carcinoma (Caco-2) cells, as well as in primary culture of human intestinal (InEpC) and renal (RPTEC) cells. Extracellular BA species were quantified in culture media using LC-MS/MS. BA-induced toxicity was evaluated using caspase-3 and flow cytometry assays. Gene expression analyses of HepG2 cells reveal that n-3 PUFAs reduce the expression of genes involved in BA synthesis (CYP7A1, CYP27A1) and uptake (NTCP), while activating genes encoding metabolic enzymes (SULT2A1) and excretion transporters (MRP2, MRP3). N-3 PUFAs also generate a less toxic BA pool and prevent the BA-dependent activation of apoptosis in HepG2 cells. Conclusion. The present study reveals that n-3 PUFAs stimulate BA detoxification.

In Vitro Bioavailability Study of an Antiviral Compound Enisamium Iodide.

An investigation into the biopharmaceutics classification and a study of the in vitro bioavailability (permeability and solubility) of the antiviral compound enisamium iodide (4-(benzylcarbamoyl)-1-methylpyridinium iodide) were carried out. The solubility of enisamium iodide was determined in four different buffers. Apparent intestinal permeability (Papp) of enisamium iodide was assessed using human colon carcinoma (Caco-2) cells at three concentrations. The solubility of enisamium iodide in four buffer solutions from pH 1.2 to 7.5 is about 60 mg/mL at 25 °C, and ranges from 130 to 150 mg/mL at 37 °C, depending on the pH. Based on these results, enisamium iodide can be classified as highly soluble. Enisamium iodide demonstrated low permeability in Caco-2 experiments in all tested concentrations of 10–100 μM with permeability coefficients between 0.2 × 10−6 cm s−1 and 0.3 × 10−6 cm s−1. These results indicate that enisamium iodide belongs to class III of the Biopharmaceutics Classification System (BCS) due to its high solubility and low permeability. The bioavailability of enisamium iodide needs to be confirmed in animal and human studies.

Coffee reduces KRAS expression in Caco-2 human colon carcinoma cells via regulation of miRNAs.

Previous epidemiological studies have demonstrated that moderate coffee consumption is associated with a lower risk of certain types of cancer, particularly colon cancer. To elucidate the molecular basis for this protective action, the effect of coffee on Caco-2 human colon carcinoma cells was investigated. Low concentrations of coffee (<5%) inhibited proliferation of Caco-2 cells without affecting cell viability. Coffee also reduced KRAS proto-oncogene, GTPase (KRAS) gene expression in a dose-dependent manner; however, caffeine, caffeic acid and chlorogenic acid, three major constituents of coffee, did not exhibit this effect. Increasing the duration of coffee bean roasting increased the reduction in KRAS expression, suggesting that the active constituents responsible for this effect emerged during the roasting process. MicroRNA (miR) analysis revealed that coffee induced the expression of miR-30c and miR-96, both of which target the KRAS gene. The results of the present study suggested that daily coffee consumption may reduce KRAS activity, thereby preventing the malignant growth of colon carcinoma cells.

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages

BackgroundBifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial–host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant.ResultsA putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages.ConclusionsOur results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.

Astilbin from Engelhardtia chrysolepis enhances intestinal barrier functions in Caco-2 cell monolayers

Astilbin, which is one of polyphenolic compounds isolated from the leaves of Engelhardtia chrysolepis HANCE (Chinese name, huang-qui), is available as the effective component in food and cosmetics because of its anti-oxidant and anti-inflammatory effects. The tight junction (TJ) proteins, which protect the body from foreign substances, are related to adhesion between a cell and a cell. Previously, the enhancement of TJ's functions induced by aglycones of flavonoids has been demonstrated, but the effects of the glycosides such as astilbin have not been observed yet. In this study, we investigated the effects of astilbin on the TJ's functions, and human colon carcinoma Caco-2 cell monolayers were used to evaluate the effects of astilbin on transepithelial electrical resistance (TER) value and the mRNA and proteins expressions of TJ-related molecules. Astilbin increased the TER value, mRNA expression levels of claudin-1 and ZO-2, and protein expression levels of occludin and ZO-2 in Caco-2 cells. Astilbin also increased the TER value in Caco-2 cells co-stimulated with TNF-α plus IFN-γ, and moreover upregulated the protein expression of TJ-related molecules in Caco-2 cells co-treated with TNF-α plus IFN-γ. These results suggest that astilbin can enhance the expressions of TJ-related molecules, leading to upregulation of the barrier functions in the intestinal cells.

Activated carbon as a carrier for amorphous drug delivery: Effect of drug characteristics and carrier wettability

Recent research on porous silica materials as drug carriers for amorphous and controlled drug delivery has shown promising results. However, due to contradictory literature reports on toxicity and high costs of production, it is important to explore alternative safe and inexpensive porous carriers. In this study, the potential of activated carbon (AC) as an amorphous drug carrier was investigated using paracetamol (PA) and ibuprofen (IBU) as model drugs. The solution impregnation method was used for drug loading, with loading efficiency determined by UV spectroscopy and drug release kinetics studied using USP II dissolution apparatus. The physical state of the drug in the complex was characterised using differential scanning calorimetry and X-ray diffractions techniques, whilst sites of drug adsorption were studied using Fourier transform infrared spectroscopy and N2 adsorption techniques. In addition, the cytotoxicity of AC on human colon carcinoma (Caco-2) cells was assessed using the MTT assay. Results presented here reveal that, for PA/AC and IBU/AC complexes, the saturation solubility of the drug in the loading solvent appears to have an effect on the drug loading efficiency and the physical state of the drug loaded, whilst drug release kinetics were affected by the wettability of the activated carbon particles. Furthermore, activated carbon microparticles exhibited very low cytotoxicity on Caco-2 cells at the concentrations tested (10–800μg/mL). This study, therefore, supports the potential of activated carbon as a carrier for amorphous drug delivery.

On the sulfation of O-desmethyltramadol by human cytosolic sulfotransferases.

Previous studies have demonstrated that sulfate conjugation is involved in the metabolism of the active metabolite of tramadol, O-desmethyltramadol (O-DMT). The current study aimed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of mediating the sulfation of O-DMT. The sulfation of O-DMT under metabolic conditions was demonstrated using HepG2 hepatoma cells and Caco-2 human colon carcinoma cells. O-DMT-sulfating activity of thirteen known human SULTs and four human organ specimens was examined using an established sulfotransferase assay. pH-Dependency and kinetic parameters were also analyzed using, respectively, buffers at different pHs and varying O-DMT concentrations in the assays. Of the thirteen human SULTs tested, only SULT1A3 and SULT1C4 were found to display O-DMT-sulfating activity, with different pH-dependency profiles. Kinetic analysis revealed that SULT1C4 was 60 times more catalytically efficient in mediating the sulfation of O-DMT than SULT1A3 at respective optimal pH. Of the four human organ specimens tested, the cytosol prepared from the small intestine showed much higher O-DMT-sulfating activity than cytosols prepared from liver, lung, and kidney. Both cultured HepG2 and Caco-2 cells were shown to be capable of sulfating O-DMT and releasing sulfated O-DMT into cultured media. SULT1A3 and SULT1C4 were the major SULTs responsible for the sulfation of O-DMT. Collectively, the results obtained provided a molecular basis underlying the sulfation of O-DMT and contributed to a better understanding about the pharmacokinetics and pharmacodynamics of tramadol in humans.

On the Molecular Basis Underlying the Metabolism of Tapentadol Through Sulfation.

Previous studies reported that tapentadol-sulfate represented one of the major metabolites of tapentadol excreted in urine. The current study aimed to identify the human cytosolic sulfotransferases (SULTs) that is(are) capable of sulfating tapentadol and to examine whether human cells and human organ specimens are capable of sulfating tapentadol. Thirteen human SULTs, previously expressed and purified, as well as human organ cytosols, were analyzed for tapentadol-sulfating activity using an established sulfotransferase assay. Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [35S]sulfate in the presence of different concentrations of tapentadol. Three of the thirteen human SULTs, SULT1A1, SULT1A3, and SULT1C4, were found to display sulfating activity toward tapentadol. Kinetic analysis revealed that SULT1A3 displayed the highest catalytic efficiency in mediating the sulfation of tapentadol, followed by SULT1A1 and SULT1C4. Using cultured HepG2 and Caco-2 cells, the generation and release of sulfated tapentadol under metabolic conditions was demonstrated. Moreover, of the four human organ specimens (kidney, liver, lung, and small intestine) tested, the cytosols prepared from small intestine and liver showed significant tapentadol-sulfating capacity (at 0.0203 and 0.0054nmol/min/mg, respectively). Taken together, the results derived from the current study provided a molecular basis underlying the sulfation of tapentadol in humans.

Sulfate conjugation of daphnetin by the human cytosolic sulfotransferases

Ethnopharmacological relevanceIn Turkey, daphnetin-containing Daphne oleoides is used as a folk medicine for treating rheumatic pain and lumbago. A daphnetin-containing traditional Chinese medicine tablet, named Zushima-Pian, is available in China for treating rheumatoid arthritis. The present study aimed to investigate the metabolism of daphnetin through sulfation in cultured human cells and to identify the human cytosolic sulfotransferase(s) (SULT(s)) that is(are) capable of mediating the sulfation of daphnetin. Materials and methodsCultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [35S]sulfate in the presence of different concentrations of daphnetin. Thirteen known human SULTs, previously expressed and purified, as well as cytosols of human kidney, liver, lung, and small intestine, were examined for daphnetin-sulfating activity using an established sulfotransferase assay. Results[35S]sulfated daphnetin was found to be generated and released by HepG2 cells and Caco-2 cells labeled with [35S] sulfate in the presence of daphnetin. Among the 13 known human SULTs, SULT1A1, SULT1A2, SULT1A3, SULT1B1, and SULT1C4 displayed significant sulfating activity toward daphnetin. Of the four human organ samples later tested, small intestine and liver cytosols displayed considerably higher daphnetin-sulfating activity than those of lung and kidney. ConclusionThe results derived from the present study showed unequivocally that daphnetin could be sulfated in cultured human cells and by purified human SULT enzymes as well as human organ cytosols. The information obtained provided a basis for further studies on the metabolism of daphnetin through sulfation in vivo.

Preparation and characterization of soluble branched ionic β-cyclodextrins and their inclusion complexes with triclosan

This study aims to synthesize, characterize and investigate the water solubility and cytotoxicity of branched anionic/cationic β-cyclodextrins (bβCDs) obtained by reaction with epichlorohydrin and chloroacetic acid or choline chloride, respectively, by a single step polycondensation reaction. Obtained ionic bβCDs were investigated as an attempt to comparatively study anionic and cationic bβCDs. Water solubility of both ionic derivatives was similar (400mg/mL) at neutral and basic pHs and remarkably higher than that of their neutral homologues. Additionally, a pH-dependent solubility of anionic bβCDs was observed. Cytotoxicity of ionic bβCDs was evaluated on Human colon carcinoma Caco-2 cells and high cell viability (>99%) was observed in the range of 0–100mg/mL for anionic and cationic samples, in the same range of that of neutral and parent β-CDs. Additionally, complexes formation capacity with triclosan, a poor water soluble antimicrobial agent, was confirmed by several techniques observing a complexation limit around 4mg/mL for both systems and higher stability constant for anionic bβCDs than cationic derivatives.

Human cytosolic sulfotransferase SULT1C4 mediates the sulfation of doxorubicin and epirubicin

Doxorubicin, an anthracycline, has been reported to be excreted in sulfate conjugated form. The current study aimed to identify the human cytosolic sulfotransferase(s) (SULT(s)) that is(are) capable of sulfating doxorubicin and its analog epirubicin, and to verify whether sulfation of doxorubicin and epirubicin may occur under metabolic conditions. A systematic analysis of thirteen known human SULTs, previously cloned, expressed, and purified, revealed SULT1C4 as the only human SULT capable of sulfating doxorubicin and epirubicin. Cultured HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells were labeled with [35S]sulfate in the presence of different concentrations of doxorubicin or epirubicin. Analysis of spent labeling media showed the generation and release of [35S]sulfated doxorubicin and epirubicin by HepG2 cells and Caco-2 cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed the expression of SULT1C4 in both HepG2 cells and Caco-2 cells. These results provided a molecular basis underlying the previous finding that sulfate-conjugated doxorubicin was excreted in the urine of patients treated with doxorubicin.

Human Cytosolic Sulfotransferase SULT1A3 Mediates the Sulfation of Dextrorphan.

Dextrorphan, an active metabolite of the antitussive dextromethorphan, has been shown to be subjected to sulfation by several zebrafish cytosolic sulfotransferases (SULTs). We were interested in finding out which of the human SULT(s) is(are) capable of catalyzing the sulfation of dextrorphan, and to verify whether sulfation of dextrorphan may occur in cultured human cells and human organ cytosols. Data from the enzymatic assays showed that, of all thirteen known human SULTs, SULT1A3 displayed the strongest dextrorphan-sulfating activity. Cell culture experiments using HepG2 human hepatoma cells and Caco-2 human colon carcinoma cells incubated with [(35)S]sulfate together with varying concentrations of dextrorphan revealed indeed the production and release of [(35)S]sulfated dextrorphan in a concentration-dependent manner. Additionally, significant dextrorphan-sulfating activity was detected in human liver, small intestine and lung cytosols. Taken together, these results provided a biochemical basis for the sulfation of dextrorphan in humans.

The Intestinal Transport of Bovine Milk Exosomes Is Mediated by Endocytosis in Human Colon Carcinoma Caco-2 Cells and Rat Small Intestinal IEC-6 Cells1

Background: MicroRNAs play essential roles in gene regulation. A substantial fraction of microRNAs in tissues and body fluids is encapsulated in exosomes, thereby conferring protection against degradation and a pathway for intestinal transport. MicroRNAs in cow milk are bioavailable in humans.Objective: This research assessed the transport mechanism of bovine milk exosomes, and therefore microRNAs, in human and rodent intestinal cells.Methods: The intestinal transport of bovine milk exosomes and microRNAs was assessed using fluorophore-labeled bovine milk exosomes in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. Transport kinetics and mechanisms were characterized using dose-response studies, inhibitors of vesicle transport, carbohydrate competitors, proteolysis of surface proteins on cells and exosomes, and transepithelial transport in transwell plates.Results: Exosome transport exhibited saturation kinetics at 37°C [Michaelis constant (Km) = 55.5 ± 48.6 μg exosomal protein/200 μL of media; maximal transport rate = 0.083 ± 0.057 ng of exosomal protein · 81,750 cells−1 · h−1] and decreased by 64% when transport was measured at 4°C, consistent with carrier-mediated transport in Caco-2 cells. Exosome uptake decreased by 61–85% under the following conditions compared with controls in Caco-2 cells: removal of exosome and cell surface proteins by proteinase K, inhibition of endocytosis and vesicle trafficking by synthetic inhibitors, and inhibition of glycoprotein binding by carbohydrate competitors. When milk exosomes, at a concentration of 5 times the Km, were added to the upper chamber in transwell plates, Caco-2 cells accumulated miR-29b and miR-200c in the lower chamber, and reverse transport was minor. Transport characteristics were similar in IEC-6 cells and Caco-2 cells, except that substrate affinity and transporter capacity were lower and higher, respectively.Conclusion: The uptake of bovine milk exosomes is mediated by endocytosis and depends on cell and exosome surface glycoproteins in human and rat intestinal cells.

Cytotoxic effect and mechanism inducing cell death of α-mangostin liposomes in various human carcinoma and normal cells.

The aims of this study were to develop α-mangostin liposomes as well as to evaluate their physicochemical properties and cytotoxic activity. α-Mangostin liposomes were prepared using the reverse-phase evaporation method with lipid composition of phosphatidylcholine to cholesterol at 7 : 3 molar ratios; their physicochemical properties and antiproliferative activity were assessed using an MTT assay in four human carcinoma cells [that is, human lung epithelial carcinoma (Calu-3), human colon carcinoma (HT-29), human breast carcinoma (MCF-7), and human colon carcinoma (Caco-2) cells], and two human normal cells [that is, human dermal fibroblasts (HDF) and human adult low-calcium elevated temperature (HaCaT) keratinocytes]. Determinations of morphological changes and oligonucleosomal DNA fragments were also carried out. The liposomal dispersions obtained were unilamellar vesicles as confirmed by cryotransmission and freeze-fracture electron microscopy with a particle size of 114 nm and a ζ potential of -2.56 mV. The P-NMR spectra showed that α-mangostin molecules orientated in the phospholipid bilayer membrane. The α-mangostin could appreciably be entrapped with an efficiency and loading of 81 and 4%, respectively. The antiproliferative activity of α-mangostin liposomes in various cancer and normal cells showed a dose-dependent inhibition in all treated cell lines. The antiproliferative effect of α-mangostin liposomes was found to be associated with apoptosis, with differences in sensitivity among the cell lines treated.

CSPP-L Associates with the Desmosome of Polarized Epithelial Cells and Is Required for Normal Spheroid Formation.

Deleterious mutations of the Centrosome/Spindle Pole associated Protein 1 gene, CSPP1, are causative for Joubert-syndrome and Joubert-related developmental disorders. These disorders are defined by a characteristic mal-development of the brain, but frequently involve renal and hepatic cyst formation. CSPP-L, the large protein isoform of CSPP1 localizes to microtubule ends of the mitotic mid-spindle and the ciliary axoneme, and is required for ciliogenesis. We here report the microtubule independent but Desmoplakin dependent localization of CSPP-L to Desmosomes in apical-basal polarized epithelial cells. Importantly, siRNA conferred depletion of CSPP-L or Desmoplakin promoted multi-lumen spheroid formation in 3D-cultures of non-ciliated human colon carcinoma Caco-2 cells. Multi-lumen spheroids of CSPP1 siRNA transfectants showed disrupted apical cell junction localization of the cytoskeleton organizing RhoGEF ECT2. Our results hence identify a novel, non-ciliary role for CSPP-L in epithelial morphogenesis.

Interdisciplinary approach to cell–biomaterial interactions: biocompatibility and cell friendly characteristics of RKKP glass–ceramic coatings on titanium

In this work, titanium (Ti) supports have been coated with glass–ceramic films for possible applications as biomedical implant materials in regenerative medicine. For the film preparation, a pulsed laser deposition (PLD) technique has been applied. The RKKP glass–ceramic material, used for coating deposition, was a sol–gel derived target of the following composition: Ca-19.4, P-4.6, Si-17.2, O-43.5, Na-1.7, Mg-1.3, F-7.2, K-0.2, La-0.8, Ta-4.1 (all in wt%). The prepared coatings were compact and uniform, characterised by a nanometric average surface roughness. The biocompatibility and cell-friendly properties of the RKKP glass–ceramic material have been tested. Cell metabolic activity and proliferation of human colon carcinoma CaCo-2 cells seeded on RKKP films showed the same exponential trend found in the control plastic substrates. By the phalloidin fluorescence analysis, no significant modifications in the actin distribution were revealed in cells grown on RKKP films. Moreover, in these cells a high mRNA expression of markers involved in protein synthesis, proliferation and differentiation, such as villin (VIL1), alkaline phosphatase (ALP1), β-actin (β-ACT), Ki67 and RPL34, was recorded. In conclusion, the findings, for the first time, demonstrated that the RKKP glass–ceramic material allows the adhesion, growth and differentiation of the CaCo-2 cell line.

Synthesis and Biological Activity of Reversed Pyrimidine Nucleosides

An efficient approach to reversed nucleosides which enables their synthesis in gram quantities is described. N-1′-Pyrimidine reversed nucleosides were prepared by treating of the sodium salt of pyrimidine bases with protected 5-tosyl ribose. Additionally, N-1′, N-3′-disubstituted reversed nucleosides were isolated in the condensation reactions with the 5-halogen pyrimidines. Using the Sonogashira coupling of 5′-iodouracil reversed nucleoside with ethynyltrimethyl silane gave 5′-ethynyl derivative which was further transformed into 5′-acetyl reversed nucleoside. Biological activity of deprotected reversed nucleosides was validated on the panel of six human carcinoma cell lines (HeLa, MIAPaCa2, Hep2, NCI-H358, CaCo-2, and HT-29). 5′-Iodouracil derivative displayed moderate growth inhibition activity against human colon carcinoma (CaCo-2) cells.

Spontaneously hyperactive MEK-Erk pathway mediates paradoxical facilitation of cell proliferation in mild hypoxia

BackgroundOxygen is important for common eukaryotic cells to generate ATP. Pathophysiological conditions such as ischemic diseases cause tissue hypoxia. In addition, oxygen availability in deep tissues is supposed to be far lower than surrounding atmosphere even in healthy animals, and the oxygen partial pressures in most normal tissues are estimated to be around 40–50mmHg, so-called mild hypoxia. Recent studies have demonstrated that mild hypoxia has distinct effects on living cells from severe hypoxia. For instance, mild hypoxia was reported to promote cell reprogramming. Although severe hypoxia is known to inhibit cell proliferation, mild hypoxia has been paradoxically demonstrated to increase cell proliferation. However, it has not been clarified by which molecular mechanisms mild hypoxia evokes the discontinuous increment of cell proliferation. MethodsWe established experimental conditions showing the opposite influences of mild and severe hypoxia on cell proliferation using undifferentiated Caco2 human colon carcinoma cells in order to clarify the underlying molecular mechanism. ResultsThe basal activity of Erk, which is a typical mediator of mitogenic signals, is spontaneously increased specifically in cells exposed to mild hypoxia, and inhibition of MEK, an upstream kinase of the Erk, completely inhibited the mild hypoxia-induced enhancement of cell proliferation. ConclusionsSpontaneous hyperactivation of the MEK-Erk pathway by mild hypoxia should be the plausible molecular mechanism of the paradoxical promotion of cell proliferation. General SignificanceOur findings will provide clues to the molecular basis of mild hypoxia-evoked phenomena such as cell reprogramming.

Amphirionin-2, a novel linear polyketide with potent cytotoxic activity from a marine dinoflagellate Amphidinium species

A novel linear polyketide, amphirionin-2 (1), with two unique hexahydrofuro[3,2-b]furan moieties has been isolated from the cultivated algal cells of a benthic dinoflagellate Amphidinium sp. (strain KCA09051). The structure was elucidated on the basis of detailed analyses of 2D NMR data, and the absolute configuration of C-5 was determined by using modified Mosher’s method. Amphirionin-2 (1) exhibited potent cytotoxic activity against human colon carcinoma Caco-2 cells and human lung adenocarcinoma A549 cells.