pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH_i) was 7.21±0.07 (n=22) in HCO ₃ ^– -containing and 7.21±0.09 (n=12) in HCO ₃ ^– -free solution. HOE-694 (10 mol/l), a potent inhibitor of the Na⁺/H⁺ exchanger, did not affect control pH_i. As a means to acidify cells we used the NH ₄ ⁺ /NH₃ (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC ₃ ^– -free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3 ₃ ^– -containing solutions a combination of HOE-694 and 4,4-diisothiocyanatostilbene-2, 2-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na⁺-dependent in HCO ₃ ^– -containing and HCO ₃ ^– -free solutions. Removal of external Cl^– caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na⁺ was removed together with Cl^–. This alkalinization was faster in HCO ₃ ^– -containing than in HCO ₃ ^– -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO ₃ ^– /Cl^– exchanger and (c) a Na⁺-dependent HCC ₃ ^– transporter, probably the Na⁺-HCO ₃ ^– /Cl^– antiporter. Under HCO ₃ ^– — free conditions the Na⁺/H⁺ exchanger fully accounts for recovery from acid load, whereas in HCO ₃ ^– -containing solutions this is accomplished by the Na⁺/H⁺ exchanger and a Na⁺-dependent mechanism, which imports HCO ₃ ^– . Recovery from alkaline load is caused by the HCO ₃ ^– /Cl^– exchanger.This study was supported by DFG Gr 480/10     

Regulation of chloride channels in the human colon carcinoma cell line HT29.cl19A

Chloride (Cl^–) channels are important in the regulation of salt and water transport in secretory epithelial cells. A disturbed Cl^– secretion is the most consistent characteristic in the genetic disease cystic fibrosis. An outwardly rectifying Cl^– channel (OR) with a conductance of 25–50 pS had been proposed to play a major role in Cl^– secretion. Activation by Ca²⁺ and the protein kinases (PK) A and C (at less than 10 nM Ca²⁺) as well as inhibition by PKC (at 1 M Ca²⁺) has been reported. In the present study, we have identified and characterized the OR in HT29.cl19A human colon carcinoma cells. The OR displayed a conductance of 31±4 pS (n=25). Its open probability in 10 nM Ca²⁺ was voltage-dependent in 50% of the patches, starting from 0.2 at -70 mV to 0.8 at 70 mV. The spontaneous activation in excised inside-out patches at –60 mV was Ca²⁺-dependent and decreased from 29% in 1 mM Ca²⁺ to 2% in 10 nM Ca²⁺. Active OR were found in (a) 25% of patches exposed to 10 nM Ca²⁺, ATP and cAMP only, (b) 42% of the patches exposed to 10 nM Ca²⁺, ATP and the catalytic subunit of PKA (C_AK) and (c) 67% of the patches exposed to 1 mM Ca²⁺, ATP plus C_AK. Inhibition of voltage-activated channels by addition of PKC in 1 M or 1 mM Ca²⁺ was not observed. Attempts to activate the OR in cell-attached patches by increasing cAMP levels under different experimental conditions were unsuccessful. Our data suggest that the OR may not be as important in Cl^– secretion as has been thought.     

MDR-1-overexpression in HT 29 colon cancer cells grown in SCID mice

The multidrug-resistance 1 (MDR-1) P-glycoprotein (Pgp) is a transmembrane transporter system, which actively pumps cytotoxic drugs out of the cell. MDR-1 acquired in vitro differs from MDR-1 acquired in vivo, but has important consequences on the cellular phenotype and metastatic behavior. Here we report that the human colonic cancer cell line HT29 (MDR-1 negative) is more malignant than its MDR-1 overexpressing variant (HT29 MDR-1 positive). HT29 MDR-1 negative cells produce undifferentiated signet ring carcinomas when implanted subcutaneously into SCID mice, while HT29 MDR-1 positive cells form tumors with tubular structures, but without signet ring cells. Immunohistochemical proliferation marker analysis revealed that the MDR-1 positive cells proliferate much more slowly than the MDR-1 negative cells. MDR-1 overexpression results in a less differentiated phenotype at the cellular level (absence of mucin producing cells) but in a more differentiated phenotype at the tissue level (tubule formation). In addition, lectin binding patterns including that of Helix pomatia agglutinin (HPA), an indicator of metastatic potential, differed between the two cell lines. HT29 MDR-1 positive cells had less HPA binding sites than HT29 MDR-1 negative counterparts and metastasized less frequently in SCID mice. As slow proliferation, low degree of differentiation and multidrug-resistance is a hallmark of cancer stem cells and all were present in MDR-1 positive tumors, it is attractive to speculate that they represent a stem cell rich tumor. As shown by global gene expression analyses, genes involved, e.g. in cell adhesion, glycosylation and signal transduction, were deregulated in MDR-1 positive tumors compared to MDR-negative tumors. Overexpression of E-cadherin and carcinoembryonic antigen-related cell adhesion molecules 1 (CEACAM1) may provide clues to the mechanisms responsible for the reduced metastatic potential of MDR-1 overexpressing tumors. Since drug treatment shifted the cells towards a less metastatic phenotype in this in vivo model, it seems conceivable to achieve this using drug treatment also in a clinical situation.     

Identification of an organic anion transport system in the human colon carcinoma cell line HT29 clone 19A

The human colon carcinoma cell line HT29 c1.19A was studied for organic anion transporter activity by determining intracellular fluo-3 and fura-red accumulation and by measuring fluo-3 efflux. Modulators of organic anion transport systems were used to identify the transporters that are involved in dye extrusion. Addition of probenecid to the dye-loading medium, containing 10 microM fluo-3/AM and fura-red/AM, resulted in a dose-dependent increase in fluo-3 and fura-red accumulation in the cells. The increase in fluo-3 accumulation in the cells in the presence of probenecid was explained by the inhibitory effect of this compound on fluo-3 efflux. Fluo-3 efflux from the cells was also inhibited by sulfinpyrazone, another inhibitor of organic anion transport. Substrates of renal probenecid-sensitive organic anion exchange mechanisms as well as modulators of multidrug resistance associated protein (MRP) activity did not influence fluo-3 extrusion rates. However, reducing intracellular ATP contents completely blocked fluo-3 extrusion. Moreover, MK571, an inhibitor of MRP, significantly stimulated dye accumulation, whereas inhibitors of the multidrug resistance gene (MDR1) product Pglycoprotein, cyclosporin A and verapamil, did not. As probenecid inhibits fluo-3 efflux across the apical membrane of cells grown on permeable supports, we conclude that a probenecid-sensitive organic anion transporter is present in the apical membrane of HT29 c1.19A cells. This organic anion transport system differs from MDRI and MRP2.     

Characteristics of apical chloride channels in human colon cells (HT29)

Recent studies have demonstrated that active chloride secretion in mammalian colon and other epithelia, is dependent on the induction of an increase of apical chloride conductance. Since the physical characteristics of apical chloride channels in man have not been elucidated, patch clamp analysis of human colon cells (HT₂₉), in culture, was performed, after stimulation with db-cAMP 10^–4 mol/l. In excised inside out patches of apical membranes two types of channels were found. The smaller and less frequent channel had a mean conductance of 15±1 pS (n=9). This type of channel showed identical I/V curves in NaCl and KCl solutions. It was inhibited by a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The more frequently observed larger conductance channel was selective for anions and was impermeable to Na⁺ and K⁺. Regarding anion selectivity, the channel was similarly permeable to Cl^–, Br^–, I^–, and NO ₃ ^– , but was impermeable to gluconate. The channel was completely inhibited by the potent Cl^– channel blocker NPPB (10^–6 mol/l). This channel exhibited rectification: The conductance was 50±4 pS at positive clamp potentials (sign referred to bath with respect to pipette interior) and 32±3 (n=33) pS at negative voltages. Moreover, the open state probability was doubled when the clamp potential was increased from –20 to +20 mV. These results demonstrate the existence of chloride channels in the apical membrane of db-cAMP treated colonic carcinoma cells.     

生长因子受体结合蛋白-2抑制肠癌细胞HT29增殖

目的 研究Grb2 shRNA转染对肠癌细胞HT29增殖相关信号通路的影响,探讨以Grb2为肠癌治疗靶点的可行性.方法 应用shRNA慢病毒质粒系统,将Grb2 shRNA转染至293T细胞,获得5株不同序列Grb2 shRNA慢病毒颗粒,并感染肠癌HT29细胞,分别获得的5株感染Grb2 shRNA慢病毒颗粒的肠癌HT29细胞系为实验组(即感染Grb2 shRNA的细胞HT29/shGrb2-69、HT29/shGrb2-70、HT29/shGrb2-71、HT29/shGrb2-72、HT29/shGrb2-73),以eGFP shRNA慢病毒感染肠癌HT29细胞为阴性对照组.MTT法检测细胞增殖情况,RT-PCR检测Grb2 mRNA表达,Western blot检测Grb2、P42/44 ERK、磷酸化P42/44 ERK、Akt、磷酸化Akt(P-Akt)和STAT5等信号通路分子表达的改变.结果 感染shGrb2病毒72 h,HT29/shGrb2-69、HT29/shGrb2-73两株细胞在mRNA和蛋白水平均出现抑制现象.HT29/shGrb2-69、HT29/shGrb2-73细胞在感染后24h A值分别为0.176±0.045,0.186±0.013,感染48 h后为0.347±0.048、0.382±0.041均显著高于空白对照、阴性对照(P ＜0.001),72 h为0.934±0.038、0.983±0.205高于空白对照、阴性对照(P＜0.01);感染后72 h,phospho-P42/44 ERK、Akt、phospho-Akt明显下降,而ERK、STAT5表达不受影响.结论 在HT29细胞,Grb2 mRNA和蛋白水平上明显抑制,可诱导HT29细胞增殖和相关信号传导通路分子表达下调.     

Simultaneous recording of the cell membrane potential and properties of the cell attached membrane of HT29 colon carcinoma and CF-PAC cells

Reconciliation of the properties of excised single channels and whole cell conductances is one of the major problems in the interpretation of patch clamp data. To combine cell attached and whole cell recordings we have modified the nystatin technique. Low concentrations of nystatin (3 * 10^–5 mol/l) were added to the filling solution of the patch pipettes. This permeabilized the cell attached membrane partially and made it possible to measure the potential difference (PD) of the cell in current clamp mode. The input resistance (R₁) of the cell attached patch was only slightly decreased by nystatin and stayed in the G range, allowing for the simultaneous recording of single channel activity and the input conductance of the cell attached membrane. This technique was examined in HT₂₉ colon carcinoma and CF-PAC cells. In both cells it was shown that this method provides reliable PD measurements. The method was used then to test which type of Cl^– channel is activated by carbachol. The PD of HT₂₉ cells was depolarized by carbachol. The depolarization was mainly due to an increase in the Cl^– conductance of the cell membrane and was followed by a slight and transient hyperpolarization. No detectable Cl^– channels (conductance > 4–8 pS, 300 Hz) were activated in the cell attached membrane, but the input conductance (G₀) increased concomitantly with cell depolarization. These results suggest that carbachol induces the opening of very small conductance or very rapidly opening and closing Cl^– channels in these cells. The ell ttached ystatin (CAN) technique, as described here, may serve as a powerful modification of the single channel patch clamp technique.     

Caspase-9途径在丁酸钠诱导结肠癌细胞株HT-29凋亡中的作用

目的： 探讨caspase-9途径在丁酸钠(NaBt)诱导人结肠癌细胞株HT-29凋亡中的作用。方法： HT-29细胞体外培养至对数生长期,分别及联合给予5.0 mmol/L丁酸钠、20 μmol/L z-VAD-fmk、z-DEVD-fmk、z-IETD-fmk、z-LEHD-fmk处理24 h,并设空白对照。以Annexin V-FITC法联合PI染色,流式细胞术检测细胞凋亡,JC-1染色检测线粒体膜电位变化,caspase活性检测试剂盒检测caspase-3、caspase-8、caspase-9的活性。结果： (1)丁酸钠诱导的HT-29细胞凋亡［（35.40±0.70）%］可被z-VAD-fmk抑制［（1.33±0.59）%］,亦可被z-DEVD-fmk抑制［（1.40±0.52）%］,并可被z-LEHD-fmk抑制［（1.27±0.91）%］,均P<0.01;但是z-IETD-fmk不能够抑制该作用［（32.10±2.33）%］,P>0.05;(2)丁酸钠干预HT-29细胞后,线粒体膜电位降低(5.53±0.91),z-VAD-fmk、z-DEVD-fmk、及z-LEHD-fmk 能够阻断这种作用(9.80±1.15, 10.23±0.50, 10.33±1.02), P<0.05;而z-IETD-fmk未显示对该作用的改变（5.93±1.31）, P>0.05;(3)丁酸钠干预HT-29细胞后,caspase-3、caspase-9的活性增高2-3倍,caspase-8的活性无显著变化,P>0.05。结论： 丁酸钠主要是通过线粒体途径,激活caspase-9,启动细胞凋亡环节,从而激发下游的效应caspases,诱导HT-29细胞凋亡。     

Small and intermediate conductance chloride channels in HT29 cells

Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT₂₉ cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g _o) is caused by inhibition of small non-resolvable channels, which are impermeable to Na⁺ and SO₄ ^2–, but permeable to Cl^–. The regulation of these small Cl^–-conducting channels (g _o) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (P _o) of the ICOR or on the g_o) were observed. The P_o of the ICOR was reduced by 22% in a Ca²⁺-free solution. g _o was insensitive to changes in the Ca²⁺ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT₂₉ cells contain ICOR and small non-resolvable Cl^–-conducting channels which are similarly inhibited by C. I. Apart from a weak effect of Ca²⁺ on the ICOR, g _o and the ICOR do not seem to be directly controlled by Ca²⁺ or PKA. C. I. of normal and CF epithelia have a similar inhibitory potency on Cl^– channels.     

Agonist-induced intracellular Ca2+ transients in HT29 cells

In the present study we have investigated the mechanism of intracellular Ca²⁺ activity ([Ca²⁺]_i) changes in HT₂₉ cells induced by adenosine triphosphate (ATP), carbachol (CCH), and neurotensin (NT). [Ca²⁺]_i was measured with the fluorescent Ca²⁺ indicator fura-2 at the single-cell level or in small cell plaques with high time resolution (1–40Hz). ATP and CCH induced not only a dose-dependent [Ca²⁺]_i peak response, but also changes of the plateau phase. The [Ca²⁺]_i plateau was inversely dependent on the ATP concentration, whereas the CCH-induced [Ca²⁺]_i plateau increased at higher CCH concentrations. NT showed (from 10^–10 to 10^–7 mol/l) in most cases only a [Ca²⁺]_i spike lasting 2–3 min. The [Ca²⁺]_i plateau induced by ATP (10^–6 mol/l) and CCH (10^–5 mol/l) was abolished by reducing the Ca²⁺ activity in the bath from 10^–3 to 10^–4 mol/l (n=7). In Ca²⁺-free bathing solution the [Ca²⁺]_i peak value for all three agonists was not altered. Using fura-2 quenching by Mn²⁺ as an indicator of Ca²⁺ influx the [Ca²⁺]_i peak was always reached before Mn²⁺ influx started. Every agonist showed this delayed stimulation of the Ca²⁺ influx with a lag time of 23±1.5 s (n=15) indicating a similar mechanism in each case. Verapamil (10^–6–10^–4 mol/l) blocked dose dependently both phases (peak and plateau) of the CCH-induced [Ca²⁺]_i increase. Short pre-incubation with verapamil augmented the effect on the [Ca²⁺]_i peak, whereas no further influence on the plateau was observed. Ni²⁺ (10^–3 mol/l) reduced the plateau value by 70%.     

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in HT29 colonic carcinoma cells

Cl^– secretion in HT₂₉ cells is regulated by agonists such as carbachol, neurotensin and adenosine 5-triphosphate (ATP). These agonists induce Ca²⁺ store release as well as Ca²⁺ influx from the extracellular space. The increase in cytosolic Ca²⁺ enhances the Cl^– and K⁺ conductances of these cells. Removal of extracellular Ca²⁺ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca²⁺ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=6) inhibited ATP (0.1 mmol·l^–1) induced increases in whole-cell conductance (G _m). When Cl^– and K⁺ currents were inhibited by the presence of Cs₂SO₄ in the patch pipette and gluconate in the bath, ATP (0.1 mmol·l^–1) still induced a significant increase in G _m from 1.2±0.3 nS to 4.7±1 nS (n=24). This suggests that ATP induces a cation influx with a conductance of approximately 3–4 nS. This cation influx was inhibited by flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=9). When Ba²⁺ (5 mmol·l^–1) and 4,4-diisothiocyanatostilbene-2-2-disulphonic acid (DIDS, 0.1 mmol·l^–1) were added to the KCl/K-gluconate pipette solution to inhibit K⁺ and Cl^– currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol·l^–1) reduced the membrane current (I _m) significantly from 86±14 pA to 54±11 pA (n=13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5–10 mmol·l^–1 1,2-bis-(2-aminoethoxy)ethane-N,N,N,N-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA). The zero-current membrane voltage (V _m) and I _m (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30–120 s after membrane rupture. V _m depolarised significantly from –33±2 mV to –12±1 mV, and I _m fell significantly from 17±2 pA to 8.9±1.0 pA (n=71). This negative current, representing a cation inward current, was activated when Ca²⁺ stores were emptied and was reduced significantly (I _m) when Ca²⁺ and/or Na⁺ were removed from the bathing solution: removal of Ca²⁺ in the absence of Na⁺ caused a I _m of 5.0±1.2 pA (n=12); removal of Na⁺ in the absence of Ca²⁺ caused a I _m of 12.8±3.5 pA (n=4). The cation inward current was also reduced significantly by La³⁺, Gd³⁺, and flufenamate. We conclude that store depletion induces a Ca²⁺/Na⁺ influx current in these cells. With 145 mmol·l^–1 Na⁺ and 1 mmol·l^–1 Ca²⁺, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.     

Adhesion of HT-29 colon carcinoma cells to endothelial cells requires sequential events involving E-selectin and integrin beta4

HT-29 colon carcinoma cells attach to TNFalpha-activated human umbilical vein endothelial cells (HUVECs) by their specific binding to E-selectin. This interaction activates, in the cancer cells, the MAPK SAPK2/p38, which leads to their transendothelial migration (Laferrière et al., J Biol Chem 2001; 276: 33762). In this study, we investigated the role of E-selectin in activating integrins to modulate adhesion and regulate integrin-mediated events. Blocking the integrins from HT-29 cells (alpha2, alpha3, alpha6, alphav/beta5, beta1 and beta4) with specific antibodies revealed a role for beta4 integrin in their adhesion to TNFalpha-treated HUVEC. The beta4 integrin-dependent adhesion was maximal after 30 min, whereas the-E-selectin-dependent adhesion was maximal after 15 min. Integrin beta4 became quickly phosphorylated upon addition of HT-29 cells to endothelial cells and the effect was independent of the expression of E-selectin. Moreover, a recombinant E-selectin/Fc chimera did not induce the phosphorylation of beta4. The phosphorylation of beta4 is not required for adhesion since adhesion was not affected in HT-29 cells that express a truncated form of beta4 that is deleted from its cytoplasmic phosphorylatable domain. However, the expression of the non-phosphorylatable deletant of beta4 was associated with decreased transendothelial cell migration underscoring the key role for the cytoplasmic domain of beta4 in cell migration. We suggest: 1) that the adhesion of HT-29 cells to activated endothelial cells follows at least two essential sequential steps involving the binding of E-selectin to its receptor on carcinoma cells and then the binding of beta4 to its own receptor on endothelial cells; 2) that the phosphorylation of integrin beta4 contributes to enhance the motile potential of cancer cells and increase their trans-endothelial migration. Overall, our results indicate that the interaction of metastatic cancer cells with endothelial cells implies a specific sequence of signaling events that ultimately leads to an increase in their efficient transendothelial migration.     

Adhesion of HT-29 colon carcinoma cells to endothelial cells requires sequential events involving E-selectin and integrin β4

HT-29 colon carcinoma cells attach to TNFα-activated human umbilical vein endothelial cells (HUVECs) by their specific binding to E-selectin. This interaction activates, in the cancer cells, the MAPK SAPK2/p38, which leads to their transendothelial migration (Laferrière et al., J Biol Chem 2001; 276: 33762). In this study, we investigated the role of E-selectin in activating integrins to modulate adhesion and regulate integrin-mediated events. Blocking the integrins from HT-29 cells (α2, α3, α6, αvβ5, β1 and β4) with specific antibodies revealed a role for β4 integrin in their adhesion to TNFα-treated HUVEC. The β4 integrin-dependent adhesion was maximal after 30 min, whereas the-E-selectin-dependent adhesion was maximal after 15 min. Integrin β4 became quickly phosphorylated upon addition of HT-29 cells to endothelial cells and the effect was independent of the expression of E-selectin. Moreover, a recombinant E-selectin/Fc chimera did not induce the phosphorylation of β4. The phosphorylation of β4 is not required for adhesion since adhesion was not affected in HT-29 cells that express a truncated form of β4 that is deleted from its cytoplasmic phosphorylatable domain. However, the expression of the non-phosphorylatable deletant of β4 was associated with decreased transendothelial cell migration underscoring the key role for the cytoplasmic domain of β4 in cell migration. We suggest: 1) that the adhesion of HT-29 cells to activated endothelial cells follows at least two essential sequential steps involving the binding of E-selectin to its receptor on carcinoma cells and then the binding of β4 to its own receptor on endothelial cells; 2) that the phosphorylation of integrin β4 contributes to enhance the motile potential of cancer cells and increase their trans-endothelial migration. Overall, our results indicate that the interaction of metastatic cancer cells with endothelial cells implies a specific sequence of signaling events that ultimately leads to an increase in their efficient transendothelial migration. Abbreviations: ERK – extracellular signal-regulated kinase; GFP – green fluorescent protein; ICAM – intercellular adhesion molecules: JNK – c-Jun NH₂-terminal kinase; MAPK – mitogen-activated protein kinase; MAPKAP K2, MAP kinase-activated protein kinase 2; PI3K – phosphatidyl inositol 3-kinase; SAPK – stress-activated protein kinase; TNFα– tumor necrosis factor-α. This revised version was published online in July 2006 with corrections to the Cover Date.     

Anti-angiogenic effects of epigallocatechin-3-gallate in human skin

Epigallocatechin-3-gallate (EGCG) is the main polyphenol component of green tea. This compound exhibits antioxidant, immunomodulatory, photoprotective, anti-angiogenic, and anti-inflammatory properties. We conducted a small randomized, double blind, split face trial using a cream containing 2.5% w/w of EGCG. Four healthy volunteers with significant erythema and telangiectasia on the face applied EGCG cream to one side of the face, and vehicle control cream to the other, twice daily for six weeks. After six weeks, biopsies were taken from EGCG and vehicle treated sites. Immunohistochemistry was used to measure VEGF and HIF-1α. HIF-1α expression was decreased in EGCG treated sites, such that 28.4% of the epidermis showed positive staining in vehicle treated vs. 13.8% in EGCG treated sites (p<0.001). A similar decrease in VEGF expression was found (6.7% in EGCG vs. 11.0%in in vehicle-treated skin (p<0.005). EGCG topical treatments influence HIF-1α induction and VEGF expression and may serve as a potential agent in the prevention of telangiectasias.     

Characterization of SA-11 rotavirus receptorid structures on human colon carcinoma cell line HT-29

The involvement of different cell membrane components in the receptor structures for SA-11 rotavirus was investigated. As experimental model, the human enterocyte-like HT-29 cell line, was used because of its closer resemblance to the in vivo viral cellular target as compared to other in vitro systems. Rotavirus was incubated with whole membranes or their separated protein and lipid fractions before infection. Either isolated cell membranes or lipid components were capable of binding to the virus and to prevent infection, whereas proteins did not show any inhibitory activity. Among lipids, the glycolipid fraction was shown to impede rotaviral antigen synthesis with a dose-dependent relationship, whereas phospholipids failed to prevent viral infection. To confirm these findings, membranes and target cells were subjected to different enzymatic treatments prior to infection. In addition, HT-29 cells were also incubated with different lectins before infection. The blocking activity of membranes was inhibited by treatment with ceramide glycanase, neuraminidase, and beta-galactosidase but not by treatment with proteases or heat (100°C). Viral infection was prevented by preincubation of target cells with lectins specific for sialic acid and galactose or with ceramide glycanase, neuraminidase, and beta-galactosidase, whereas protease treatments were not active. The results of these experimental procedures indicate that glycolipids containing specific carbohydrate moieties, such as sialic acid and galactose, contribute to the SA-11 rotavirus receptor structure on HT-29 cells. © Wiley-Liss, Inc.     

Induction of oxidative DNA damage by Helicobacter pylori in HT29 cells

Infection with Helicobacter pylori has been associated with the development of gastric adenocarcinoma in humans. Several routes have been implicated, the main one being oxidative DNA damage resulting from chronic inflammation, which accompanies infection. However, DNA has been demonstrated in human cells after in vitro incubation with H. pylori sonicates. Using the fragment length analysis using restriction enzymes (FLARE) assay, this study investigates the DNA damaging potential of three clinical isolates of H. pylori on cultured HT29 cells. Significant amounts of oxidative DNA damage were detected in HT29 cells following a 72-hour incubation with each H. pylori isolate. As tumour induction is a known consequence of oxidative DNA damage, chronic infection with the organism may lead to the development of adenocarcinoma of the stomach.     

Response of HT29 cells to butyrate depends on time of exposure and glucose deprivation

The high level of alkaline phosphatase activity in HT29 cells induced after 2 or 5 days of butyrate treatment was decreased during their prolonged exposure (about 30 days) to this agent together with a decrease of sensitivity to apoptosis. However, an enormous additive effect on alkaline phosphatase activity was found after butyrate treatment of glucose-starved cells. In concert with this finding, the substructural analysis revealed a dense brush border, tendency to polarization and morphologically normal mitochondria. It can be concluded that prolonged butyrate treatment of HT29 cells attenuated their response to this agent. On the other hand, glucose deprivation, as another inductor of differentiation, was found to increase the sensitivity of HT29 cells to butyrate.