Propofol induces apoptosis and increases gemcitabine sensitivity in pancreatic cancer cells in vitro by inhibition of nuclear factor-κ B activity

AIM:To investigate the effect of propofol on human pancreatic cells and the molecular mechanism of propofol action.METHODS:We used the human pancreatic cancer cell line MIAPaCa-2 for in vitro studies measuring growth inhibition and degree of apoptotic cell death induced by propofol alone,gemcitabine alone,or propofol followed by gemcitabine.All experiments were conducted in triplicate and carried out on three or more separate occasions.Data were means of the three or more independent experiments±SE.Statistically significant differences were determined by two-tailed unpaired Student’s t test and defined as P<0.05.RESULTS:Pretreatment of cells with propofol for 24 h followed by gemcitabine resulted in 24%-75% growth inhibition compared with 6%-18%when gemcitabine was used alone.Overall growth inhibition was directly correlated with apoptotic cell death.We also showed that propofol potentiated gemcitabine-induced killing by downregulation of nuclear factor-κB(NF-κB).In contrast,NF-κB was upregulated when pancreatic cancer cells were exposed to gemcitabine alone,suggesting a potential mechanism of acquired chemoresistance.CONCLUSION:Inactivation of the NF-κB signaling pathway by propofol might abrogate gemcitabineinduced activation of NF-κB,resulting in chemosensitization of pancreatic tumors to gemcitabine.     

Mitochondrial protection by low doses of insulin-like growth factor- Ⅰ in experimental cirrhosis

AIM： To characterize the mitochondrial dysfunction in experimental cirrhosis and to study whether insulin-like growth factor-Ⅰ （IGF-Ⅰ ） therapy （4 wk） is able to induce beneficial effects on damaged mitochondria leading to cellular protection.
METHODS： Wistar rats were divided into three groups： Control group, untreated cirrhotic rats and cirrhotic rats treated with IGF-Ⅰ treatment （2 μg/1O0 g bw/d）. Mitochondrial function was analyzed by flow cytometry in isolated hepatic mitochondria, caspase 3 activation was assessed by Western blot and apoptosis by TUNEL in the three expedmental groups.
RESULTS： Untreated cirrhotic rats showed a mitochondrial dysfunction characterized by a significant reduction of mitochondrial membrane potential （in status 4 and 3）; an increase of intramitochondrial reactive oxigen species （ROS） generation and a significant reduction of ATPase activity. IGF-Ⅰ therapy normalized mitochondrial function by increasing the membrane potential and ATPase activity and reducing the intramitochondrial free radical production. Activity of the electron transport complexes Ⅰ and Ⅲ was increased in both cirrhotic groups. In addition, untreated cirrhotic rats showed an increase of caspase 3 activation and apoptosis. IGF- Ⅰ therapy reduced the expression of the active peptide of caspase 3 and resulted in reduced apoptosis.
CONCLUSION： These results show that IGF- Ⅰ exerts a mitochondrial protection in experimental cirrhosis leading to reduced apoptosis and increased ATP production.     

Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IκBα protein

AIM: NF-κB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection. In this study, we use pCMV-IκBαM vector to inhibit NF-κB activation and investigate the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells. METHODS: The NF-κB activity was detected with pNF-κB reporter gene and electrophoretic mobility shift assay. Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells. RESULTS: We could find that NF-κB activity is inhibited by over-expression of non-degraded IκBα protein. Expression of adhesion molecules like ICAM-1, VCAM-1, and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV-IκBαM vector. CONCLUSION: Our results indicate that the pCMV-IκBαM, which inhibit the activity of NF-κB through over-expression of non-degraded IκBα protein, can be used for gene therapy in diseases involving NF-κB activation abnormally like organ transplantation via inhibiting cell adhesion.     

Antioxidant activity of chito-oligosaccharides on pancreatic islet cells in streptozotocin-induced diabetes in rats

AIM： To investigate the antioxidant activity of chitooligosaccharides （COSs） on pancreatic islet cells in diabetic rats induced by streptozotocin.
METHODS： The antioxidant effect of COSs on pancreatic islet cells was detected under optical microscopy and with colorimetric assay and gel electrophoresis. The activities of glutathione peroxidase and superoxide dismutase, total antioxidant capacity, and content of malondialdehyde in serum and tissue slices of pancreas were examined after 60 d to determine the effect of COSs in streptozotocin-induced diabetes in rats.
RESULTS： COSs can prohibit the apoptosis of pancreatic islet cells. All concentrations of COSs can improve the capability of total antioxidant capacity and activity of superoxide dismutase and decrease the content of malondialdehyde drastically. Morphological investigation in the pancreas showed that COSs have resulted in the reduction of islets, loss of pancreatic cells, and nuclear pyknosis of pancreatic cells.
CONCLUSION： COSs possess various biological activities and can be used in the treatment of diabetes mellitus.     

Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IκBα protein

AIM: NE-κB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection.In this study, we use pCMV-IκBαM vector to inhibit NE-κB activation and investigate the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells.METHODS: The NF-κB activity was detected with pNF-κB reporter gene and electrophoretic mobility shift assay.Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-I~BczMin in hibition of T cells adhesion to endothelial cells.RESULTS: We could find that NF-~B activity is inhibited by over-expression of non-degraded IκBα protein.Expression of adhesion molecules like ICAM-1, VCAM-1,and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV-IκBαM vector.CONCLUSION: Our results indicate that the pCMV-IκBαM, which inhibit the activity of NF-κB through over-expression of non-degraded IκBα protein, can be used for gene therapy in diseases involving NF-κB activation abnormally like organ transplantation via inhibiting cell adhesion.     

Effect of Tetrandrine on LPS-induced NF-κB activation in isolated pancreatic acinar cells of rat

AIM: To investigate the effect of Tetrandrine (Tet) on LPS-induced NF-κB activation and cell injury in pancreatic acinar cells and to explore the mechanism of Tetrandrine preventing LPS-induced acinar cell injury. METHODS: Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to LPS (10 mg/L), Tet (50μmol/L, 100μmol/L) or normal media. At different time point (30 min, 1 h, 4 h, 10 h) after treatment with the agents, cell viability was determined by MTT, the product and nuclear translocation of subunit p65 of NF-κB was visualized by immunofluorescence staining and nuclear protein was extracted to perform EMSA which was used to assay the NF-κB binding activity. RESULTS: LPS induced cell damage directly in a time dependent manner and Tet attenuated LPS-induced cell damage (50μmol/L, P < 0.05; 100μmol/L, P < 0.01). NF-κB p65 immunofluorescence staining in cytoplasm increased and began showing its nuclear translocation within 30 min and the peak was shown at 1 h of LPS 10 mg/L treatment. NF-κB DNA binding activity showed the same alteration pattern as p65 immunofluorescence staining. In Tet group, the immunofluorescence staining in cytoplasm and nuclear translocation of NF-κB were inhibited significantly. CONCLUSION: NF-κB activation is an important early event that may contribute to inflammatory responses and cell injury in pancreatic acinar cells. Tet possesses the protective effect on LPS-induced acinar cell injury by inhibiting NF-κB activation.     

Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

AIM： To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action.
METHODS： The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MI-I-） assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential （A~Pm） were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases （PI3K）.
RESULTS： Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time- and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 pmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901 cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt.
CONCLUSION： Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.     

Mitochondrial protection by low doses of insulin-like growth factor- Ⅰ in experimental cirrhosis

AIM:To characterize the mitochondrial dysfunction in experimental cirrhosis and to study whether insulin-like growth factor- I (IGF-I) therapy (4 wk) is able to in-duce beneficial effects on damaged mitochondria leading to cellular protection.METHODS:Wistar rats were divided into three groups:Control group,untreated cirrhotic rats and cirrhotic rats treated with IGF-I treatment (2 μg/100 g bw/d).Mitochondrial function was analyzed by flow cytometry in isolated hepatic mitochondria,caspase 3 activation was assessed by Western blot and apoptosis by TUNEL in the three experimental groups.RESULTS:Untreated cirrhotic rats showed a mitochondrial dysfunction characterized by a significant reduction of mitochondrial membrane potential (in status 4 and 3);an increase of intramitochondrial reactive oxigen species (ROS) generation and a significant reduction of ATPase activity.IGF-Ⅰ therapy normalized mitochondrial function by increasing the membrane potential and ATPase activity and reducing the intramitochondrial free radical production.Activity of the electron transport complexes Ⅰ and Ⅲ was increased in both cirrhotic groups.In addition,untreated cirrhotic rats showed an increase of caspase 3 activation and apoptosis.IGF- Ⅰ therapy reduced the expression of the active peptide of caspase 3 and resulted in reduced apoptosis.CONCLUSION:These results show that IGF- Ⅰ exerts a mitochondrial protection in experimental cirrhosis leading to reduced apoptosis and increased ATP production.     

Possible mechanism for the regulation of glucose on proliferation, inhibition and apoptosis of colon cancer cells induced by sodium butyrate

AIM： To study the effect of glucose on sodium butyrate- induced proliferation inhibition and apoptosis in HT-29 cell line, and explored its possible mechanisms.
METHODS： HT-29 cells were grown in RPMI-1640 medium supplemented with 10% fetal calf serum, and were allowed to adhere for 24 h, and then replaced with experimental medium. Cell survival rates were detected by MTr assay. Apoptosis was detected by TUNEL assay. Glucose transport protein 1 （GLUT1） and monocarboxylate transporter 1 （MCT1） mRNA expression was detected by RT-PCR. RESULTS： Low concentration of glucose induced apoptosis and regulated proliferation in HT-29 cell line, and glucose can obviously inhibit the effect of proliferation inhibition and apoptosis induced by sodium butyrate. Glucose also down-regulated the expression of MCT1mRNA （0.28 ± 0.07 vs 0.19± 0.10, P 〈 0.05）, and decreased the expression of GLUTlmRNA slightly （0.18 ± 0.04 vs 0.13 ± 0.03, P 〈 0.05）.
CONCLUSION： Glucose can regulate the effect of proliferation inhibition and apoptosis induced by sodium butyrate and this influence may be associated with the intracellular concentration of glucose and sodium butyrate.     

Influence of ellagic acid on prostate cancer cell proliferation: a caspase-dependent pathway

ObjectiveTo evaluate the effect of allagic acid treatment on the cell viability of human prostate cancer cells.MethodsEllagic acid (10-100 mol/L) treatment (48 h) of human prostate carcinoma PC3 cells was found to result in a dose-dependent inhibition of cell growth and apoptosis of PC3 cells as assessed by MTT assay, western blotting, flow cytometry and confocal microscopy.ResultsWe observed that ellagic acid treatment of PC3 cells resulted in a dose dependent inhibition of cell growth/cell viability. This ellagic acid caused cell growth inhibition was found to be accompanied by induction of apoptosis, as assessed by the cleavage of poly (ADP-ribose) polymerase (PARP) and morphological changes. Further, induction of apoptosis accompanied a decrease in the levels of antiapoptotic protein Bcl-2 and increase in proapoptotic protein Bax, thus shifting the Bax: Bcl-2 ratio in favor of apoptosis. Ellagic acid treatment of PC3 cells was also found to result in significant activation of caspases, as shown by the dose dependent decrease in the protein expression of procaspase?3, ?6, ?8 and ?9. This ellagic acid-mediated induction of apoptosis was significantly (80%-90%) inhibited by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (Z-VAD-FMK). Thus these data suggested an essential role of caspases in ellagic acid-mediated apoptosis of PC3 cells.ConclusionsIt is tempting to suggest that consumption of tropical pigmented fruits and vegetables could be an effective strategy to combat prostate cancer.     

Growth inhibition and apoptosis induction by alternol in pancreatic carcinoma cells

AIM: To investigate the effect of alternol on pancreatic cancer cells.METHODS: Pancreatic cancer cells PANC-1 and BxPC3 were treated with various concentrations of alternol for 24, 48 and 72 h. Cell proliferation was measured by cell counting. Cell cycle distribution and mitochondrial membrane potential were determined by flow cytometry. Apoptosis was determined by a TdT-mediated dUTP nick end labeling assay and Hoechst staining. Expression of caspase 3, Bcl-2, p53 and p21 was measured by western blotting.RESULTS: Alternol showed dose- and time-dependent inhibition of the proliferation of PANC-1 and BxPC3 cells in vitro. Alternol induced apoptosis and cell cycle arrest at S phase and decreased mitochondrial membrane potential. Alternol activated caspase 3, upregulated p53 and p21 expression, and downregulated Bcl-2 expression in a dose-dependent manner.CONCLUSION: Our results suggested that alternol is a candidate for treatment of pancreatic cancer.     

Ellagic Acid Inhibits Pancreatic Fibrosis in Male Wistar Bonn/Kobori Rats

The key pathological features of chronic pancreatitis are chronic inflammation, acinar atrophy, and pancreatic fibrosis. We have previously shown that ellagic acid, a plant-derived polyphenol found in fruits and nuts, inhibited activation of pancreatic stellate cells, a major profibrogenic cell type in the pancreas, in vitro. Here we examined whether ellagic acid inhibited the development of pancreatic fibrosis in vivo. Ellagic acid was administered orally in the diet to ten-week-old male Wistar Bonn/Kobori rats, an experimental model of spontaneous chronic pancreatitis, for ten weeks. Ellagic acid (100 mg/kg body weight/day) attenuated pancreatic inflammation and fibrosis. The protective effects were confirmed by an increase in pancreatic weight and decreases in myeloperoxidase activity (an index of neutrophil infiltration), collagen content, transforming growth factor-β1 expression, and the number of α-smooth muscle actin-positive cells (activated pancreatic stellate cells) and ED-1-positive cells (macrophages/monocytes). Ellagic acid inhibited the production of reactive oxygen species in pancreatic stellate cells in response to transforming growth factor-β1 or platelet-derived growth factor. Our results suggest that ellagic acid might be a candidate for treatment of chronic pancreatitis. N. Suzuki and K. Kikuta contributed equally to this work.     

Mechanistic Clues in the Protective Effect of Ellagic Acid Against Apoptosis and Decreased Mitochondrial Respiratory Enzyme Activities in Myocardial Infarcted Rats

Our previous study described the cardioprotective effects of ellagic acid in an isoproterenol-induced myocardial infarction model. In this study, we are reporting the mechanism of protective action of ellagic acid with respect to apoptosis and mitochondrial respiratory enzymes. Ellagic acid (7.5 and 15?mg/kg) was administered orally as a pretreatment for 10?days. Then, isoproterenol (100?mg/kg) was injected subcutaneously to rats at an interval of 24?h for 2?days. Myocardial infarction was quantified by planimetry. Apoptosis was measured by apoptotic gene expressions. The levels of mitochondrial respiratory enzymes were also measured. Isoproterenol-induced myocardial infarcted rats showed increased infarct size, a decrease in myocardial expression of the Bcl-2 gene and an increase in myocardial expression of the BAX gene. Fas ligand and caspases were markedly elevated along with compromised respiratory marker enzymes in isoproterenol-induced rats. Ellagic acid pretreatment reduced the infarct size, regulated apoptotic gene expressions and enhanced the activities of mitochondrial respiratory marker enzymes and cell viability, thereby protecting the myocardium against isoproterenol-induced myocardial infarction. The decreased infarct size associated with inhibited apoptosis and increased respiratory marker enzymes provide insight on the role of ellagic acid in antiapoptotic mechanism, and it may be the reason for its cardioprotective activity.     

Inhibition of N-methyl-N-nitrosourea-induced mutagenicity and DNA methylation by ellagic acid.

Ellagic acid, a naturally occurring plant phenol, inhibits the activity of the direct-acting mutagen N-methyl-N-nitrosourea (MeNU) in Salmonella typhimurium TA100. Ellagic acid at 0.10, 0.25, 0.50, and 1.00 mM inhibited the mutagenicity of MeNU (0.40 mM) by 3%, 13%, 45%, and 60%, respectively. Ellagic acid (3 mM) also inhibited the mutagenic activity of N,N-dimethylnitrosamine (25-200 mM) in the presence of pyrazole-induced rat liver fraction S-9. The effect of ellagic acid on DNA methylation was studied by incubating 0, 0.72, 1.32, 2.64, and 6.60 mM ellagic acid with DNA (0.9 mM nucleotide) and [3H]MeNU (0.66 mM). HPLC analysis of DNA hydrolysates showed that ellagic acid caused a dose-dependent 36-84% decrease in O6-methylguanine but only a 20% decrease in the 7-methylguanine adduct. Under conditions where methylation at the O6 position of guanine in double-stranded DNA was inhibited 65% by ellagic acid, no significant inhibition of either O6- or 7-methylguanine formation was detected in single-stranded DNA. Affinity-binding studies revealed that [3H]ellagic acid binds equally to double-stranded or single-stranded DNA but that poly(dA X dT) binds 1.5 times as much ellagic acid as does poly(dG X dC). The binding of ellagic acid to DNA is dependent on the concentration of both ellagic acid and DNA. The specific inhibition of O6-methylguanine formation only in double-stranded DNA and the relatively low inhibition of 7-methylguanine formation rule out the possibility that ellagic acid prevents DNA alkylation by scavenging the electrophilic intermediate generated in the hydrolysis of MeNU. The results suggest that ellagic acid inhibition of MeNU-induced mutagenicity is due to specific inhibition of methylation at the O6 position of guanine through an ellagic acid-duplex DNA affinity-binding mechanism.     

Extracellular matrix proteins protect pancreatic cancer cells from death via mitochondrial and nonmitochondrial pathways

BACKGROUND AND AIMS: Pancreatic cancer is a very aggressive malignancy. Normal cells die through apoptosis when detached from extracellular matrix (ECM), but the role of ECM in cancer cell survival is poorly understood. Here, we determined the effects of ECM proteins on death responses and underlying signaling pathways in human pancreatic cancer cells. METHODS: We measured apoptosis and necrosis, caspase activation, and mitochondrial dysfunction in MIA PaCa-2 and PANC-1 pancreatic carcinoma cells both detached and attached to ECM proteins. RESULTS: Detachment of pancreatic cancer cells from ECM did not induce classic apoptosis, as it does in normal cells, but induced necrosis and apoptosis associated with secondary necrosis. It caused a pronounced mitochondrial depolarization and release of cytochrome c and Smac/DIABLO. However, as different from normal cells, cytochrome c release did not result in downstream caspase activation. Executioner caspases were activated in detached pancreatic cancer cells independent of cytochrome c. Laminin and fibronectin, but not collagen I, markedly increased pancreatic cancer cell survival by inhibiting both mitochondrial dysfunction (leading to inhibition of necrosis) and caspase activity (leading to decreased apoptotic DNA fragmentation). CONCLUSIONS: ECM proteins greatly protect pancreatic cancer cells from death by mechanisms different from those operating in normal cells. The results suggest ECM proteins and their receptors as potential targets for treatment of pancreatic cancer.     

Ian4 is required for mitochondrial integrity and T cell survival

Apoptosis is a regulated cell death program controlled by extrinsic and intrinsic signaling pathways. The intrinsic pathway involves stress signals that activate pro-apoptotic members of the Bcl-2 family, inducing permeabilization of mitochondria and release of apoptogenic factors. These proteins localize to the outer mitochondrial membrane. Ian4, a mitochondrial outer membrane protein with GTP-binding activity, is normally present in thymocytes, T cells, and B cells. We and others have recently discovered that a mutation in the rat Ian4 gene results in severe T cell lymphopenia that is associated with the expression of autoimmune diabetes. The mechanism by which Ian4 controls T cell homeostasis is unknown. Here we show that the absence of Ian4 in T cells causes mitochondrial dysfunction, increased mitochondrial levels of stress-inducible chaperonins and a leucine-rich protein, and T cell-specific spontaneous apoptosis. T cell activation and caspase 8 inhibition both prevented apoptosis, whereas transfection of T cells with Ian4-specific small interfering RNA recapitulated the apoptotic phenotype. The findings establish Ian4 as a tissue-specific regulator of mitochondrial integrity.