Oligosaccharides from human milk influence growth-related characteristics of intestinally transformed and non-transformed intestinal cells

Human milk oligosaccharides (HMO) are considered to influence the composition of the gut microflora in breastfed infants. We investigated direct effects of milk HMO fractions or individual oligosaccharides on proliferation, differentiation and apoptosis in transformed human intestinal cells (HT-29 and Caco-2) and non-transformed small intestinal epithelial crypt cells of fetal origin (human intestinal epithelial cells; HIEC). We observed growth inhibition induced by neutral and acidic HMO fractions in HT-29, Caco-2 and HIEC cells in a dose dependent manner. However, the effects varied between cell lines, i.e. HT-29 and Caco-2 cells were more sensitive than HIEC cells. In HT-29, all 16 individual neutral and acidic oligosaccharides except from the two fucosyllactoses had an inhibitory effect on cell growth. Regarding the induction of differentiation in HT-29 and HIEC cells a threshold concentration was observed at 7.5 mg/ml for neutral and acidic HMO fractions. Among individual oligosaccharides, only sialyllactoses induced differentiation in HT-29 and HIEC cells; no effect neither of fractions nor of individual oligosaccharides was found in Caco-2 cells. A strong induction of apoptosis was only detected in HT-29 and HIEC cells for neutral oligosaccharide but not for acidic fractions. HMO were shown to induce growth inhibition in intestinal cells through two different mechanisms, by suppressing cell cycle progression through induction of differentiation and/or by influencing apoptosis. As the development and maturation of digestive and absorptive processes depend on differentiation our experiments show that oligosaccharides are effective at influencing various stages in gastrointestinal development in vitro.     

Combination of isoliquiritigenin and tumor necrosis factor-related apoptosis-inducing ligand induces apoptosis in colon cancer HT29 cells

Objectives  Isoliquiritigenin is a chalcone derivative with potential in cancer chemoprevention. Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent, some cancer cells are resistant to TRAIL treatment. Current studies have tried to overcome TRAIL-resistant cancer cells. Here, we show for the first time that isoliquiritigenin overcomes TRAIL resistance in colon cancer HT29 cells. Methods  HT29 cells were treated with isoliquiritigenin and/or TRAIL, and apoptosis induction was detected by flow cytometry and fluorescence microscopy. Protein expression relating to the TRAIL pathway was analyzed by Western blotting. Results  A single treatment with isoliquiritigenin scarcely induced apoptosis in HT29 cells. Combined treatment with suboptimal concentrations of isoliquiritigenin and TRAIL markedly induced apoptosis, however. The effect was blocked by a pan-caspase inhibitor and a caspase-3, 8, 9, or 10 inhibitor, suggesting that the combination facilitates caspase-dependent apoptosis. Furthermore, the apoptosis induced by isoliquiritigenin and TRAIL was blocked by a dominant negative form of the TRAIL receptor. This result indicates that the combined effect is caused by specific interaction between TRAIL and its receptors. Isoliquiritigenin increased the amount of DR5 protein among TRAIL receptors. Isoliquiritigenin did not significantly increase levels of the Bcl-2 family proteins Bcl-2, Bcl-xL, and BAX. Conclusions  Our results suggest that isoliquiritigenin has the potential to overcome resistance to TRAIL in cancer cells and its chemopreventive effects may depend on TRAIL function.     

Intracellular mechanisms mediating tocotrienol-induced apoptosis in neoplastic mammary epithelial cells

Tocotrienols and tocopherols represent the two subgroups that make up the vitamin E family of compounds. However, tocotrienols display significantly more potent apoptotic activity in neoplastic mammary epithelial cells than tocopherols. Studies were conducted to determine the intracellular mechanism(s) mediating tocotrienol-induced apoptosis in neoplastic +SA mouse mammary epithelial cells in vitro. An initial step in apoptosis is the activation of 'initiator' caspases (caspase-8 or -9) that subsequently activate 'effector' caspases (caspase-3, -6 and -7) and induce apoptosis. Treatment with cytotoxic doses of alpha-tocotrienol (20 microM) resulted in a time-dependent increase in caspase-8 and caspase-3 activity. Combined treatment with specific caspase-8 or caspase-3 inhibitors completely blocked alpha-tocotrienol-induced apoptosis and caspase-8 or caspase-3 activity, respectively. In contrast, alpha-tocotrienol treatment had no effect on caspase-9 activation, and combined treatment with a specific caspase-9 inhibitor did not block alpha-tocotrienol-induced apoptosis in (+)SA cells. Since caspase-8 activation is associated with the activation of death receptors, such as Fas, tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL) receptors, studies were conducted to determine the exact death receptor(s) and ligand(s) involved in mediating tocotrienol-induced caspase-8 activation and apoptosis. Treatment with Fas-ligand (FasL), Fas-activating antibody, or TRAIL failed to induce cell death in (+)SA neoplastic mammary epithelial cells, suggesting that these cells are resistant to death receptor-induced apoptosis. Moreover, treatment with cytotoxic doses of alpha-tocotrienol did not alter the intracellular levels of Fas, FasL, or Fas-associated death domain (FADD) in these cells. Western blot analysis also showed that alpha-tocotrienol did not induce FasL or FADD translocation from the cytosolic to membrane fraction in these cells. Finally, treatment with Fas-blocking antibody did not reverse the tocotrienol-induced apoptosis in (+)SA cells. These data demonstrate that tocotrienol-induced caspase-8 activation and apoptosis is not mediated through death receptor activation in malignant (+)SA mammary epithelial cells. Resistance to death receptor-induced apoptosis has been shown to be associated with increased expression of apoptosis-inhibitory proteins, such as FLICE-inhibitory protein (FLIP), and enhanced signalling of the phosphatidylinositol 3-kinase (PI3K)/PI3K-dependent kinase (PDK)/Akt mitogenic pathway. Additional studies showed that treatment with cytotoxic doses of alpha-tocotrienol decreased total, membrane, and cytosolic levels of FLIP, and reduced phosphorylated PDK-1 (active) and phosphorylated-Akt (active) levels in these cells. In summary, these findings demonstrate that tocotrienol-induced caspase-8 activation and apoptosis in malignant (+)SA mammary epithelial cells is not mediated through the activation of death receptors, but appears to result from the suppression of the PI3K/PDK/Akt mitogenic signalling pathway, and subsequent reduction in intracellular FLIP expression.     

Antioxidative and Apoptotic Properties of Polyphenolic Extracts from Edible Part of Artichoke (Cynara scolymus L.) on Cultured Rat Hepatocytes and on Human Hepatoma Cells

Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H₂O₂generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N′-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.     

Z-Ajoene Induces Apoptosis of HL-60 Cells: Involvement of Bcl-2 Cleavage

Garlic organosulfur components exhibit antitumor activity, but the molecular mechanisms underlying these effects have not been well characterized. We showed that Z-ajoene, a sulfur-rich compound purified from garlic, induced time- and dose-dependent apoptosis in HL-60 cells. This process implied the activation of caspase-3 and the cleavage of the antiapoptotic protein Bcl-2. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-[OMe]-fluoromethylketone inhibited Bcl-2 cleavage and apoptosis induced by Z-ajoene. This effect was partially prevented by treatment of HL-60 cells with the antioxidant N-acetylcysteine. Hence, the transmission of apoptotic signal induced by Z-ajoene involved a reactive oxygen species-dependent pathway leading to caspase-dependent Bcl-2 cleavage.     

Z-ajoene induces apoptosis of HL-60 cells: involvement of Bcl-2 cleavage

Garlic organosulfur components exhibit antitumor activity, but the molecular mechanisms underlying these effects have not been well characterized. We showed that Z-ajoene, a sulfur-rich compound purified from garlic, induced time- and dose-dependent apoptosis in HL-60 cells. This process implied the activation of caspase-3 and the cleavage of the antiapoptotic protein Bcl-2. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-[OMe]-fluoromethylketone inhibited Bcl-2 cleavage and apoptosis induced by Z-ajoene. This effect was partially prevented by treatment of HL-60 cells with the antioxidant N-acetylcysteine. Hence, the transmission of apoptotic signal induced by Z-ajoene involved a reactive oxygen species-dependent pathway leading to caspase-dependent Bcl-2 cleavage.     

Padina arborescens extract protects high glucose-induced apoptosis in pancreatic β cells by reducing oxidative stress

BACKGROUND/OBJECTIVES

This study investigated whether Padina arborescens extract (PAE) protects INS-1 pancreatic β cells against glucotoxicity-induced apoptosis.

MATERIALS/METHODS

Assays, including cell viability, lipid peroxidation, generation of intracellular ROS, NO production, antioxidant enzyme activity and insulin secretion, were conducted. The expressions of Bax, Bcl-2, and caspase-3 proteins in INS-1 cells were evaluated by western blot analysis, and apoptosis/necrosis induced by high glucose was determined by analysis of FITC-Annexin V/PI staining.

RESULTS

Treatment with high concentrations of glucose induced INS-1 cell death, but PAE at concentrations of 25, 50 or 100 µg/ml significantly increased cell viability. The treatment with PAE dose dependently reduced the lipid peroxidation and increased the activities of antioxidant enzymes reduced by 30 mM glucose, while intracellular ROS levels increased under conditions of 30 mM glucose. PAE treatment improved the secretory responsiveness following stimulation with glucose. The results also demonstrated that glucotoxicity-induced apoptosis is associated with modulation of the Bax/Bcl-2 ratio. When INS-1 cells were stained with Annexin V/PI, we found that PAE reduced apoptosis by glucotoxicity.

CONCLUSIONS

In conclusion, the present study indicates that PAE protects against high glucose-induced apoptosis in pancreatic β cells by reducing oxidative stress.     

Expression of apoptosis-associated microRNAs in ethanol-induced acute gastric mucosal injury via JNK pathway

MicroRNAs (miRNAs) have been shown to be closely associated with cellular apoptosis, but their involvement in response to ethanol-induced gastric mucosal epithelial cell apoptosis remains largely unknown. The purpose of this study was to investigate the expression profile of apoptosis-associated miRNAs in ethanol-induced acute gastric mucosal injury and the mechanisms underlying injury. Gastric mucosal injury was induced in rats by oral administration of ethanol, and gastric tissues were collected for analysis of gastric ulcer index, apoptosis ratio, caspase-3 activity, and miRNAs expression. Cell cultures of human gastric mucosal epithelial cells (GES-1) were incubated with ethanol to induce apoptosis. Mimics or inhibitors of miRNAs or c-Jun N-terminal kinase (JNK) inhibitor were added to the cell culture medium. GES-1 cells were collected for analysis of apoptosis ratio, caspase-3 activity, miRNAs expression, and protein phosphorylation levels of JNK, p38 mitogen-activated protein kinase (p38MAPK), or extracellular signal-regulated kinase (ERK). In the animal experiments, gastric ulcer index, cellular apoptosis, and caspase-3 activity were significantly increased, accompanied by up-regulation of miR-145 and down-regulation of the microRNAs miR-17, miR-19a, miR-21, miR-181a, and miR-200c. In the human cell culture experiments, the anti-apoptotic effects of miR-19a and miR-21 or pro-apoptotic effect of miR-145 were confirmed by their corresponding mimics or inhibitor; the ethanol-induced GES-1 apoptosis as well as the changes in miRNAs expression were significantly attenuated in the presence of JNK inhibitor. These results demonstrated that miR-145, miR-19a, and miR-21 were the apoptosis-associated miRNAs in gastric mucosal epithelial cells. The regulation of expression of these 3 miRNAs in ethanol-induced GES-1 apoptosis involved the JNK pathway.     

Dual effect of ethanol on cell death in primary culture of human and rat hepatocytes

AIMS: In-vivo and in-vitro studies have shown that ethanol induces hepatocyte damage. The aim of the present study was to evaluate the effect of a broad range of ethanol concentrations on apoptosis and necrosis in primary culture of human and rat hepatocytes. METHODS: Human and rat hepatocytes were isolated from human hepatectomies and male Wistar rats (200-250 g) using the classical collagenase perfusion method. After stabilization of cell culture, ethanol (0-10 mmol/l) was administered and the parameters were measured 24 h after ethanol addition. Apoptosis was studied by DNA fragmentation, iodide propidium-DNA staining, caspase-3 activity and annexin V binding in hepatocytes. Necrosis was evaluated by lactate dehydrogenase (LDH) release. Malondialdehyde (MDA) and GSH/GSSG were used as parameters of oxidative stress. RESULTS: Ethanol enhanced dose-dependently all the parameters associated with apoptosis in human and rat hepatocytes. Low or high ethanol concentrations induced an opposite action against cell necrosis in cultured hepatocytes. Low concentrations of ethanol (1-2 mmol/l) reduced LDH release from human and rat hepatocytes. However, the highest ethanol concentration (10 mmol/l) induced a sharp increase in cell necrosis. The effect of ethanol on cell necrosis was related to lipid peroxidation in hepatocytes. CONCLUSIONS: Ethanol differentially regulates apoptosis or necrosis in cultured hepatocytes. Although ethanol exerted a dose-dependent induction of apoptosis, low ethanol concentrations were able to reduce basal lipid peroxidation and necrosis in hepatocytes. The highest ethanol concentration (10 mmol/l) induced apoptosis and necrosis in human and rat cultured hepatocytes.     

The protective effect of fermented milk kefir on radiation-induced apoptosis in colonic crypt cells of rats

To evaluate the effect of fermented milk kefir on X-ray-induced apoptosis in the colon of rats, we examined the apoptotic index, the mean number of apoptotic cells detected by H&E staining per crypt in the colon, in control rats and kefir-pretreated rats drinking kefir for 12 days before irradiation. Apoptotic cells were confirmed by TUNEL staining, and active caspase-3 expression was studied by immunohistochemistry. The cell position of apoptotic cells and active caspase-3 positive cells were examined. The apoptotic index of kefir-treated rats was significantly (p < 0.05) decreased 2 h after 1 Gy irradiation in comparison with control rats at crypt cell positions 1-3, 5-7, 13, and 15. Active caspase-3 expression in the kefir-treated rats was also significantly (p < 0.05) reduced in comparison with control rats 2 h after 1 Gy irradiation at crypt cell positions 1-4, 13, and 15. This study indicated that kefir protects colonic crypt cells against radiation-induced apoptosis, which was most pronounced in the stem cell region of the crypt. The antiapoptotic effect of fermented milk kefir was due to the inhibition of caspase-3 activation.     

Black rice extract protected HepG2 cells from oxidative stress-induced cell death via ERK1/2 and Akt activation

BACKGROUND/OBJECTIVES

The objective of this study was to evaluate the protective effect of black rice extract (BRE) on tert-butyl hydroperoxide (TBHP)-induced oxidative injury in HepG2 cells.

MATERIALS/METHODS

Methanolic extract from black rice was evaluated for the protective effect on TBHP-induced oxidative injury in HepG2 cells. Several biomarkers that modulate cell survival and death including reactive oxygen species (ROS), caspase-3 activity, and related cellular kinases were determined.

RESULTS

TBHP induced cell death and apoptosis by a rapid increase in ROS generation and caspase-3 activity. Moreover, TBHP-induced oxidative stress resulted in a transient ERK1/2 activation and a sustained increase of JNK1/2 activation. While, BRE pretreatment protects the cells against oxidative stress by reducing cell death, caspase-3 activity, and ROS generation and also by preventing ERKs deactivation and the prolonged JNKs activation. Moreover, pretreatment of BRE increased the activation of ERKs and Akt which are pro-survival signal proteins. However, this effect was blunted in the presence of ERKs and Akt inhibitors.

CONCLUSIONS

These results suggest that activation of ERKs and Akt pathway might be involved in the cytoprotective effect of BRE against oxidative stress. Our findings provide new insights into the cytoprotective effects and its possible mechanism of black rice against oxidative stress.     

Curcumin-Induced Apoptosis in PC3 Prostate Carcinoma Cells Is Caspase-Independent and Involves Cellular Ceramide Accumulation and Damage to Mitochondria

Curcumin, the principal curcuminoid of tumeric, has potent anticancer activity. To determine the mechanism of curcumin-induced cytotoxicity in prostate cancer cells, we exposed PC3 prostate carcinoma cells to 25 to 100 μ M curcumin for 24 to 72 h. Curcumin treatment of PC3 cells caused time- and dose-dependent induction of apoptosis and depletion of cellular reduced glutathione (GSH). Exogenous GSH and its precursor N-acetyl-cysteine, but not ascorbic acid (AA) or ebselen, decreased curcumin accumulation in PC3 cells and also prevented curcumin-induced DNA fragmentation. The failure of AA and ebselen to protect PC3 cells from curcumin-induced apoptosis argued against the involvement of reactive oxygen species; rather, GSH-mediated inhibition of curcumin-induced cytotoxicity was due to reduced curcumin accumulation in PC3 cells. Curcumin-treated PC3 cells showed apoptosis-inducing cellular ceramide accumulation and activation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Caspase-3, caspase-8, and caspase-9 were activated, and cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria following curcumin treatment. Interestingly, curcumin-induced apoptosis was not prevented by p38 MAPK, JNK, or caspase inhibition. We conclude that curcumin-induced cytotoxicity was due to cellular ceramide accumulation and damage to mitochondria that resulted in apoptosis mediated by AIF and other caspase-independent processes.     

Antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (Cynara scolymus L.) on cultured rat hepatocytes and on human hepatoma cells

Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.     

Substitutes for glutamine in proliferation of rat intestinal epithelial cells

OBJECTIVES: Glutamine (Gln) is important for intestinal epithelial proliferation. The purpose of this study was to determine whether glutamate (Glu), a mixture of nucleotide monophosphates, arginine, or glucosamine could support proliferation of rat intestinal crypt cells (IEC-6) in the absence of Gln. METHODS: Glu with added ammonia acetate, glucosamine, arginine, and nucleotide monophosphates were tested at concentrations that were isonitrogenous with respect to Gln. To determine whether de novo synthesis of Gln was affected by these nutrients, a duplicate set of treatment groups was also tested with 1.0 mM/L of methionine sulfoximine, an inhibitor of Gln synthetase. RESULTS: Gln + methionine sulfoximine-treated cells showed suboptimal proliferation below 0.6 mM/L but normal proliferation between 0.6 and 4.0 mM/L of Gln. In the absence of exogenous Gln, isonitrogenous concentrations of Glu, glucosamine, arginine, or nucleotide monophosphates yielded similar proliferation as Gln. Cells treated with Glu, glucosamine, arginine, or nucleotide monophosphate mixture showed a decrease in proliferation compared with cells treated with Gln across all treatment doses (P < 0.03). CONCLUSIONS: The importance of these results is that, in the presence of active Gln synthetase, these nutrients can maintain intestinal epithelial proliferation similar to that observed with Gln.     

Increased carnitine-dependent fatty acid uptake into mitochondria of human colon cancer cells induces apoptosis

Carnitine-dependent fatty acid import into mitochondria and beta-oxidation seem to be impaired in tumor cells. In the present study we show that a supply of palmitoylcarnitine together with L-carnitine potently induces apoptosis in HT-29 human colon cancer cells as a consequence of accelerated fatty acid oxidation. Caspase-3-like activities, measured by the cleavage rate of a fluorogenic tetrapeptide substrate and nuclear fragmentation determined after DNA labeling in fixed cells by fluorescence microscopy, served as indicators of apoptosis. Neither L-carnitine nor palmitoylcarnitine alone were able to increase caspase-3-like activities and DNA fragmentation, but when provided together, apoptosis occurred. That exogenous carnitine was indeed able to enhance fatty acid uptake into mitochondria was demonstrated by an increased influx of a fluorescent palmitic acid analog. Enhanced fatty acid availability in mitochondria led to an increased generation of O*2-, as detected by a O*2- -sensitive fluorogenic dye, indicating oxidation of delivered substrates. Benzoquinone, an O*2- scavenger, blocked O*2- generation and prevented apoptosis as initiated by the combination of palmitoylcarnitine and carnitine. The lack of effect of the ceramide synthesis inhibitor fumonisin on palmitoylcarnitine/carnitine-induced apoptosis further supports the notion that apoptotic cell death is specifically due to fatty acid oxidation. In contrast to HT-29 cells, nontransformed human colonocytes did not respond to exogenous palmitoylcarnitine/carnitine and no apoptosis was observed. In conclusion, our studies provide evidence that a limited mitochondrial fatty acid import in human colon cancer cells prevents high rates of mitochondrial O*2- production and protects colon cancer cells from apoptosis that can be overcome by an exogenous carnitine supply.     

Induction of apoptosis by the aqueous extract of Rubus coreanum in HT-29 human colon cancer cells

OBJECTIVES: The incompletely ripened fruit of Rubus coreanum (IRFRC) has been used in traditional herbal medicine to manage various diseases. To explore the possibility that IRFRC has chemopreventive effects, we examined whether or not extracts of IRFRC inhibits HT-29 cell growth and explored the mechanism for this effect. METHODS: We cultured HT-29 cells in the presence of the aqueous or ethanol extract of IRFRC. DNA synthesis was estimated by 5-bromo-2'-deoxyuridine incorporation. We measured apoptosis using a DNA fragmentation assay and Annexin V staining. We used western blot analyses to determine the cleavage of caspases and poly (adenosine diphosphate-ribose) polymerase. RESULTS: Aqueous extract of IRFRC substantially inhibited viable HT-29 cell number in a dose-dependent manner, whereas ethanol extract had only a minimal effect. Aqueous extract inhibited DNA synthesis and induced apoptosis of HT-29 cells in a dose-dependent manner. Aqueous extract induced cleavage of caspase-3, -7, and -9 and induced the activity of caspase-3 and cleavage of poly (adenosine diphosphate-ribose) polymerase. CONCLUSIONS: We have shown that aqueous extract of IRFRC inhibits cell proliferation and stimulates apoptosis in HT-29 cells, and that this may be mediated by its ability to activate the caspase-3 pathway. It remains to be determined whether the aqueous extract of IRFRC has chemopreventive activities in animal models.     

Cisplatin Upregulates Glutamine Transport in Human Intestinal Epithelial Cells

Background: Glutamine (GLN) prevents the intestinal mucosal injury induced by chemotherapy, although the mechanism of this protective action has not yet been elucidated. Amino acid transport across the plasma membrane is essential for supplying enterocytes with amino acids for cellular metabolism. It was hypothesized that chemotherapy stimulates GLN transport, which enables GLN to be used more efficiently as a metabolic fuel. Methods: A rat model was used to examine the effect of enteral GLN on intestinal mucosal injury induced by intraperitoneal injection of cisplatin (7.0 mg/kg of body weight). The effects of cisplatin on amino acid transport and the expression of messenger RNA and protein were evaluated by real‐time polymerase chain reaction and Western blot analysis, respectively, in the human intestinal epithelial cell line Caco‐2. The effects of cisplatin on glutaminase activity and intracellular glutathione were also studied. Results: GLN prevented mucosal atrophy induced by cisplatin in rats. In Caco‐2 cells, cisplatin significantly increased GLN transport and the expression of GLN transporter ASCT2 messenger RNA and protein. Leucine, but not glutamate, transport significantly increased in the cisplatin‐treated group due to the increase in LAT1 (leucine transporter) protein expression. Glutaminase activity and intracellular glutathione increased significantly in the cisplatin‐treated group. Conclusions: Bolus enteral GLN prevents intestinal mucosal injury induced by cisplatin in rats, as demonstrated by increased GLN transport and increased GLN transporter expression after cisplatin administration.     

Mechanism of activation of caspase cascade during beta-carotene-induced apoptosis in human tumor cells

In this study, we examined possible mechanisms of caspase activation during carotenoid-induced apoptosis in tumor cells. We found that beta-Carotene induces apoptosis by the activation of caspase-3 in human leukemia (HL-60), colon adenocarcinoma (HT-29) as well as melanoma (SK-MEL-2) cell lines. This activation is dose dependent and follows that of caspase-8 and caspase-9. Although caspase-8 cleavage is an early event, reaching its maximum activation at 3 h, caspase-9 reaches its maximum activation only at 6 h. The addition of IETD-CHO, a caspase-8-specific inhibitor, completely prevents beta-Carotene-induced apoptosis, whereas only a partial prevention was observed in the presence of LEHD-CHO, a caspase-9-specific inhibitor. beta-Carotene activates caspase-9 via cytochrome c release from mitochondria and loss of mitochondrial membrane potential (Dym). Concomitantly, a dose-dependent decrease in the antiapoptotic protein Bcl-2 and a dose-dependent increase in the cleaved form of BID (t-BID) are observed. Moreover, NF-kB activation is involved in beta-Carotene-induced caspase cascade. These results support a pharmacological role for beta-Carotene as a candidate antitumor agent and show a possible sequence of molecular events by which this molecule may induce apoptosis in tumor cells.     

Local glutathione redox status does not regulate ileal mucosal growth after massive small bowel resection in rats

Glutathione (GSH) concentration affects cell proliferation and apoptosis in intestinal and other cell lines in vitro. However, in vivo data on gut mucosal GSH redox status and cell turnover are limited. We investigated the effect of altered GSH redox status on the ileal mucosa in a rat model of short bowel syndrome following massive small bowel resection (SBR). Rats underwent 80% mid-jejunoileal resection (RX) or small bowel transection (TX; as operative controls), with administration of either saline or D, L-buthionine-sulfoximine (BSO), a specific inhibitor of cellular GSH synthesis. Ileal mucosal redox, morphology, and indices of cell proliferation and apoptosis were determined at different days after surgery. Ileal GSH redox status was assessed by GSH and GSH disulfide (GSSG) concentrations and the redox potential of GSH/GSSG (Eh). Ileal lipid peroxidation [free malondialdehyde (MDA)] was measured as an index of lipid peroxidation. BSO markedly decreased ileal mucosal GSH, oxidized GSH/GSSG Eh, and increased MDA content without inducing morphological damage as assessed by light or electron microscopy. As expected, SBR stimulated adaptive growth of ileal villus height and total mucosal height at 7 d after surgery, but this response was unaffected by BSO treatment despite a modest increase in crypt cell apoptosis. Ileal cell proliferation (crypt cell bromodeoxyuridine incorporation) increased at 2 d after SBR but was unaffected by BSO. Collectively, our in vivo data show that marked depletion of ileal GSH and oxidation of the GSH redox pool does not alter indices of ileal epithelial proliferation or SBR-induced ileal mucosal adaptive growth.