Uwe Wenzel

Resveratrol Induces Apoptosis through ROS-Dependent Mitochondria Pathway in HT-29 Human Colorectal Carcinoma Cells

trans-Resveratrol is a polyphenol found in blueberries, grapes, and wine with cancer chemopreventive properties. The low bioavailability of this compound enhances its concentration in the luminal content and becomes a potential chemopreventive agent against colon cancer. In the present study, the antiproliferative and pro-apoptotic effects on the human colorectal carcinoma HT-29 cells as well as the mechanisms underlying these effects were examined. Proliferation, cytotoxicity, and apoptosis were measured by fluorescence-based techniques. Studies of dose-dependent effects of trans-resveratrol showed antiproliferative activity with an EC 50 value of 78.9 +/- 5.4 microM. Caspase-3 was activated in a dose-dependent manner after incubation for 24 h giving an EC 50 value of 276.1 +/- 1.7 microM. Apoptosis was also confirmed with microscopic observation of changes in membrane permeability and detection of DNA fragmentation. The activity of trans-resveratrol on the mitochondria apoptosis pathway was evidenced by the production of superoxide anions in the mitochondria of cells undergoing apoptosis. In conclusion, trans-resveratrol inhibits cell proliferation without cytotoxicity and induces apoptosis in HT-29. Results of the present study provide evidence demonstrating the antitumor effect of trans-resveratrol via a ROS-dependent apoptosis pathway in colorectal carcinoma.

α-lipoic acid induces apoptosis in human colon cancer cells by increasing mitochondrial respiration with a concomitant O2−.-generation

The antioxidant α-lipoic acid (ALA) has been shown to affect a variety of biological processes associated with oxidative stress including cancer. We determined in HT-29 human colon cancer cells whether ALA is able to affect apoptosis, as an important parameter disregulated in tumour development. Exposure of cells to ALA or its reduced form dihydrolipoic acid (DHLA) for 24 h dose dependently increased caspase-3-like activity and was associated with DNA-fragmentation. DHLA but not ALA was able to scavenge cytosolic O2−. in HT-29 cells whereas both compounds increased O2− .-generation inside mitochondria. Increased mitochondrial O2− .-production was preceded by an increased influx of lactate or pyruvate into mitochondria and resulted in the down-regulation of the anti-apoptotic protein bcl-XL. Mitochondrial O2−.-generation and apoptosis induced by ALA and DHLA could be prevented by the O2− .-scavenger benzoquinone. Moreover, when the lactate/pyruvate transporter was inhibited by 5-nitro-2-(3-phenylpropylamino) benzoate, ALA- and DHLA-induced mitochondrial ROS-production and apoptosis were blocked. In contrast to HT-29 cells, no apoptosis was observed in non-transformed human colonocytes in response to ALA or DHLA addition. In conclusion, our study provides evidence that ALA and DHLA can effectively induce apoptosis in human colon cancer cells by a prooxidant mechanism that is initiated by an increased uptake of oxidizable substrates into mitochondria.

Antiproliferative and apoptosis-inducing effects of maslinic and oleanolic acids, two pentacyclic triterpenes from olives, on HT-29 colon cancer cells.

We have previously reported the anticarcinogenic effects of an olive fruit extract composed of pentacyclic triterpenes, the main components of which are maslinic acid (73.25%) and oleanolic acid (25.75%). Here we examined the effects of the individual components on proliferation, necrosis and apoptosis rates by fluorescence-based techniques in human HT-29 colon cancer cells. Oleanolic acid showed moderate antiproliferative activity, with an ec50 of 160.6 (se 10.6) micromol/l, and moderate cytotoxicity at high concentrations ( > or = 250 micromol/l). On the other hand, maslinic acid inhibited cell growth with an ec50 of 101.2 (se 7.8) micromol/l, without necrotic effects. Oleanolic acid, which lacks a hydroxyl group at the carbon 2 position, failed to activate caspase-3 as a prime apoptosis protease. In contrast, maslinic acid increased caspase-3-like activity at 10, 25 and 50 micromol/l by 3-, 3.5- and 5-fold over control cells, respectively. The detection of ROS in the mitochondria, which serve as pro-apoptotic signal, evidenced the different bioactivity of the two triterpenes. Confocal microscopy analysis revealed that maslinic acid generated superoxide anions while oleanolic acid-treated cells did not differ from the control. Completion of apoptosis by maslinic acid was confirmed microscopically by the increase in plasma membrane permeability, and detection of DNA fragmentation. In conclusion, the anticancer activity observed for olive fruit extracts seems to originate from maslinic acid but not from oleanolic acid. Maslinic acid therefore is a promising new compound for the chemoprevention of colon cancers.

Caenorhabditis elegans lifespan extension caused by treatment with an orally active ROS-generator is dependent on DAF-16 and SIR-2.1

In Caenorhabditiselegans pretreatment with juglone, a generator of reactive oxygen species (ROS) provides a subsequently increased ROS-resistance. We investigated whether juglone at low or high concentrations when provided via the oral route in a liquid axenic medium affects normal lifespan of C.elegans. High juglone concentrations led to premature death, low concentrations were tolerated well and caused a prolongation of lifespan. Lifespan extension under moderate oxidative stress was associated with increased expression of small heat-shock protein HSP-16.2, enhanced glutathione levels, and nuclear translocation of DAF-16. Silencing or deletion of DAF-16 prevented the juglone-induced adaptations. RNA-interference for SIR-2.1 had the same effects as the deletion of DAF-16 but did not affect nuclear accumulation of DAF-16. Our studies demonstrate that DAF-16- and SIR-2.1-dependent alterations in gene expression after a ROS challenge lead to a lifespan extension in C.elegans as long as the stressor concentration does not exceed the saturable protective capacity.

Stereoselective uptake of β-lactam antibiotics by the intestinal peptide transporter

1 The stereoselective transport of β‐lactam antibiotics has been investigated in the human intestinal epithelial cell line, Caco‐2, by use of D‐ and L‐enantiomers of cephalexin and loracarbef as substrates. 2 The L‐isomers of cephalexin, loracarbef and dipeptides displayed a higher affinity for the oligopeptide/H+‐symporter in Caco‐2 cells than the D‐isomers. This was demonstrated by inhibition of the influx of the β‐lactam, [3H]‐cefadroxil. 3 By measurement of the substrate‐induced intracellular acidification in Caco‐2 cells loaded with the pH‐sensitive fluorescent dye BCECF (2′,7′‐bis(2‐carboxyethyl)‐5‐(6)‐carboxy‐fluorescein), it was demonstrated for the first time that L‐isomers of β‐lactams not only bind to the peptide transporter with high affinity but are indeed transported. 4 Efficient proton‐coupled transport of L‐β‐lactam antibiotics was also shown to occur in Xenopus laevis oocytes expressing the cloned peptide transporter PepTl from rabbit small intestine. 5 Both cell systems therefore express a stereoselective transport pathway for β‐lactam antibiotics with very similar characteristics and may prove useful for screening rapidly the oral availability of peptide‐derived drugs.

Transport of Charged Dipeptides by the Intestinal H+/Peptide Symporter PepT1 Expressed in Xenopus laevis Oocytes

Abstract. The cloned intestinal peptide transporter is capable of electrogenic H+-coupled cotransport of neutral di- and tripeptides and selected peptide mimetics. Since the mechanism by which PepT1 transports substrates that carry a net negative or positive charge at neutral pH is poorly understood, we determined in Xenopus oocytes expressing PepT1 the characteristics of transport of differently charged glycylpeptides. Transport function of PepT1 was assessed by flux studies employing a radiolabeled dipeptide and by the two-electrode voltage-clamp-technique. Our studies show, that the transporter is capable of translocating all substrates by an electrogenic process that follows Michaelis Menten kinetics. Whereas the apparent K0.5 value of a zwitterionic substrate is only moderately affected by alterations in pH or membrane potential, K0.5 values of charged substrates are strongly dependent on both, pH and membrane potential. Whereas the affinity of the anionic dipeptide increased dramatically by lowering the pH, a cationic substrate shows only a weak affinity for PepT1 at all pH values (5.5–8.0). The driving force for uptake is provided mainly by the inside negative transmembrane electrical potential. In addition, affinity for proton interaction with PepT1 was found to depend on membrane potential and proton binding subsequently affects the substrate affinity. Furthermore, our studies suggest, that uptake of the zwitterionic form of a charged substrate contributes to overall transport and that consequently the stoichiometry of the flux-coupling ratios for peptide: H+/H3O+ cotransport may vary depending on pH.

Feeding a ROS-generator to Caenorhabditis elegans leads to increased expression of small heat shock protein HSP-16.2 and hormesis

Reactive oxygen species (ROS) are thought to be a driving force in the aging process. In transgenic Caenorhabditis elegans expressing green fluorescent protein (GFP) under control of the hsp-16.2 promoter (CL2070) 100 μM of the ROS-generator juglone induced GFP-expression. This was associated with translocation of DAF-16 to the nucleus as visualized in a transgenic strain expressing a DAF-16::GFP fusion protein (TJ356) and with increased cellular levels of reduced glutathione. RNA-interference for DAF-16 in CL2070 blocked the juglone-induced HSP-16.2 expression and the increase in glutathione levels. Higher concentrations of juglone did not further increase the adaptive responses but caused premature death, indicating hormetic adaptations unless the stressor exceeds the intrinsic protective capacity. The addition of the ROS-scavenger ascorbic acid finally blocked lifespan reductions and all of the adaptations to juglone stressing that ROS are indeed the molecular species that require protective response.

Nitric oxide suppresses apoptosis in human colon cancer cells by scavenging mitochondrial superoxide anions

In cells lacking a functional p53 tumor suppressor protein, the endogenous free radical nitric oxide (NO) appears to inhibit apoptosis, and thereby promotes growth of cancer cells. In order to elucidate the underlying mechanisms on a molecular basis, we used HT‐29 human colon carcinoma cells, carrying a p53 loss‐of‐function mutation, and examined the effects of NO on apoptosis when induced either by the flavonoid flavone or by the chemotherapeutic drug camptothecin (CPT), which is able to scavenge NO by superoxide anion production. Caspase‐3 activation as well as nuclear fragmentation, both indicative of apoptosis, were dose dependently inhibited by the NO‐liberating agents sodium nitroprusside (SNP) or S‐nitroso‐N‐acetyl‐D,L‐penicillamine (SNAP) when apoptosis was initiated by flavone with only minor effects on apoptosis when initiated by camptothecin. The transcript levels of 9 apoptosis‐related genes were assessed and NO liberation was shown to completely and specifically prevent the flavone‐induced but not camptothecin‐induced decrease in bcl‐XL mRNA levels. These results were also confirmed at the protein level. The effects of NO on the mitochondrial apoptosis pathway were further evidenced by the scavenging of superoxide anions as produced in mitochondria of cells undergoing apoptosis. Scavenging of mitochondrial superoxide anions by NO prevents the downregulation of bcl‐XL, the depolarization of the mitochondrial membrane potential, the cytochrome c release and finally the activation of caspase‐3. In conclusion, NO effectively inhibits apoptosis by scavenging superoxide anions generated in the mitochondria of p53 mutant cells and thereby prevents the downregulation of the antiapoptotic factor bcl‐XL, which controls the mitochondrial apoptosis pathway.

Electrophysiological analysis of the function of the mammalian renal peptide transporter expressed in Xenopus laevis oocytes.

1 To gain information on the mode of operation of the renal proton‐coupled peptide transporter PepT2, voltage clamp studies were performed in Xenopus laevis oocytes expressing the rabbit renal PepT2. 2 Using differently charged glycyl‐dipeptides we show that PepT2 translocates these dipeptides by an electrogenic pH‐dependent process that is essentially independent of the substrate net charge. The apparent substrate affinities are in the micromolar range (2–50 μm) between pH 5.5 and 7.4 and membrane potentials of ±0 to −50 mV. 3 Maximal substrate‐evoked inward currents (Imax) are affected by membrane voltage (Vm) and extracellular pH (pH0). Potential‐dependent interactions of H+/H3O+ with PepT2 seem to be mediated by a single low affinity binding site and PepT2 remains pH dependent at all voltages. 4 The effects of voltage on apparent Imax and substrate affinity display an inverse relationship. As Vm is altered from –50 to –150 mV substrate affinities decrease 10‐ to 50‐fold whereas apparent Imax increases almost 10‐fold. 5 Even at saturating H+/H3O+ and dipeptide concentrations the I–V curves did not show saturation at negative membrane potentials, suggesting that other steps in the reaction cycle and not the ligand affinity changes are rate limiting. These are possibly the conformational changes of the empty and/or loaded transporters. 6 These findings demonstrate that not only substrate affinities but also other kinetic characteristics of PepT2 differ markedly from those of the intestinal peptide transporter isoform PepT1.

Melatonin potentiates flavone-induced apoptosis in human colon cancer cells by increasing the level of glycolytic end products

Melatonin is a natural compound synthesized by a variety of organs. It has been described to possess cell protecting activity in normal cells but was shown to induce apoptotic cell death in cancer cells. We determined to which extent and based on which molecular mechanisms melatonin is able to cause apoptosis in HT‐29 human colon cancer cells. Induction of apoptosis was assessed by caspase‐3‐like activity, nuclear fragmentation and chromatin condensation. Melatonin, when given alone at a concentration of 1 mM, did not affect any of the apoptosis markers. It potentiated apoptosis induced by the flavonoid flavone significantly. Whereas flavone alone at a concentration of 150 μM led to a 8‐fold increase in caspase‐3‐like activity associated with around 40% of cells displaying DNA‐fragmentation, a combination of flavone and melatonin increased caspase‐3‐like activity 30‐fold and 80% of cells exhibited fragmentation of DNA when compared to untreated controls. Melatonin caused an increase in cytosolic lactate levels that most likely allows the flavone‐induced activation of the mitochondrial pyruvate/lactate importer to deliver more substrates to mitochondrial respiration. The subsequent increased production of mitochondrial O2− · in the presence of flavone was further increased by melatonin. Scavenging mitochondrial O2−. by benzoquinone or blocking the lactate/pyruvate transporter by 5‐nitro‐2‐(3‐phenylpropylamino) benzoate inhibited mitochondrial O2−.‐generation and apoptosis execution mediated by flavone and melatonin. Our study provides evidence that melatonin potentiates flavone‐induced apoptosis in HT‐29 human colon cancer cells by enhancing the level of oxidizable substrates that can be transported into mitochondria in the presence of flavone.