Objective In order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells,the effects of PPARγ and POX-induced ROS were explored.Methods [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenylt...Objective In order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells,the effects of PPARγ and POX-induced ROS were explored.Methods [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay,Annexin V and PI staining using FACS,plasmid transfection,ROS formation detected by DCFH staining,RNA interference,RT-PCR RT-QPCR,and Western blotting analyses were employed to investigate the apoptotic effect of troglitazone and the potential role of PPARγ pathway and POX-induced ROS formation in HT29 cells.Results Troglitazone was found to inhibit the growth of HT29 cells by induction of apoptosis.During this process,mitochondria related pathways including ROS formation,POX expression and cytochrome c release increased,which were inhibited by pretreatment with GW9662,a specific antagonist of PPARγ.These results illustrated that POX upregulation and ROS formation in apoptosis induced by troglitazone was modulated in PPARγ-dependent pattern.Furthermore,the inhibition of ROS and apoptosis after POX siRNA used in troglitazone-treated HT29 cells indicated that POX be essential in the ROS formation and PPARγ-dependent apoptosis induced by troglitazone.Conclusion The findings from this study showed that troglitazone-induced apoptosis was mediated by POX-induced ROS formation,at least partly,via PPARγ activation.展开更多
Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR) in cancer cells.This study examined the effects of troglita...Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR) in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma(PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Western blotting,respectively.The SGC7901/VCR cells were treated with 0.1 mg/L vincristine(VCR) alone or in combination with 1,5,10 μmol/L troglitazone for 24 h.PPARγ was measured by electrophoretic mobility shift assay(EMSA).The intracellular concentration of Rhodamine123(Rh123,a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp.The cell cycle and apoptosis were measured by flow cytometry.The results showed that the P-gp was increasingly expressed in SGC7901,BGC823 and SGC7901/VCR cells in turn,suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp.In the SGC7901/VCR cells,the expression level of total PPARγ was increased,however,the protein level and activity of PPARγ in the nuclei of cells decreased significantly.Troglitazone elevated the PPARγ activity in SGC7901/VCR cells in a dose-dependent manner.Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner.We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner.Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner.It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity.Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells.It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.展开更多
AIM: To study the effect of troglitazone on primary culture human pterygium fibroblast (HPF). METHODS: Cell viability loss and apoptosis were quantified by cell counting kit-8, AnnexinV-FITC/PI double staining, caspas...AIM: To study the effect of troglitazone on primary culture human pterygium fibroblast (HPF). METHODS: Cell viability loss and apoptosis were quantified by cell counting kit-8, AnnexinV-FITC/PI double staining, caspases activity test and western blotting. Flow cytonnetry was used to detect mitochondrial membrane potential. RESULTS: Peroxisome proliferator-activated receptor gamma (PPAR-gamma) was positively expressed in pterygium specimens (n = 5). Troglitazone showed dose-dependent inhibition of cell survival, induced phospholipids redistribution, activated caspase-3, -9, and altered mitochondrial potential. Western blot assay demonstrated the increase of Bax/Bcl-2 protein ratio. CONCLUSION: Troglitazone induced apoptosis of HPF through a mitochondrial-dependent pathway.展开更多
To investigate whether peroxisome proliferators-activated receptor-γ (PPARγ) ligand Troglitazone can reduce endothelial injury and activation during storage of harvested saphenous vein grafts. Segments of human sa...To investigate whether peroxisome proliferators-activated receptor-γ (PPARγ) ligand Troglitazone can reduce endothelial injury and activation during storage of harvested saphenous vein grafts. Segments of human saphenous vein graft were collected from 9 patients undergoing coronary bypass surgery and then divided into two equal parts of control and test specimens, were stored in ei- ther heparinized blood (control group) or heparinized blood containing 20 μmol/L troglitazone (test group) for 1 h at room temperature. Tissue distribution and protein expression of VCAM-I, ICAM-I, and endothelial nitric oxide synthase (eNOS) were compared using immunohistochemistry and Western blot analysis. Myeloperoxidase (MPO) activity, a marker of neutrophil sequestration in human saphenous vein grafts, was also measured in each group. The expression of ICAM-1 (753±132 versus 7201±934; P〈0.01) , VCAM-1 (3731±294 versus 8292±793; P〈0.01), and MPO activity (1.52±0.42 U/g, 5.04±1.26 U/g P〈0.01) were significantly lower in test group. In contract, eNOS expression (7983±834 versus 3989±1008; P〈0.01) was significantly higher in test group. PPARγ ligand troglitazone might reduce endothelial injury during the storage period of human saphenous vein grafts.展开更多
Boiogito (BOT) ameliorates insulin resistance and diabetes in several animal models;however, the underlying mechanisms for these in vivo effects remain unclear. Thiazolidine derivatives, which are peroxisome prolifera...Boiogito (BOT) ameliorates insulin resistance and diabetes in several animal models;however, the underlying mechanisms for these in vivo effects remain unclear. Thiazolidine derivatives, which are peroxisome proliferator-activated receptor γ (PPARγ) agonists for the treatment of type II diabetes, promote adiponectin production by inducing adipocyte differentiation, thereby reducing insulin resistance. This study aimed to evaluate the effect of BOT on adipocyte differentiation using cultured human visceral preadipocytes (HVPAds) compared with the thiazolidine derivative troglitazone (TRG). We investigated the effects of BOT (0.125 - 1 mg/mL) and TRG (10 μM) on the differentiation of adipocytes treated with or without tumor necrosis factor-α (TNF-α: 5 ng/mL). On day 14 of culture, the following adipocyte differentiation marker levels were measured: intracellular lipids, extracellular (i.e., medium) adiponectin, and intracellular differentiation-related genes (PPARγ, CCAAT/enhancer binding protein, adiponectin, differentiation cluster 36, glucose transporter type 4). BOT and TRG increased factors associated with differentiation including lipid, adiponectin, and differentiation-related gene expression levels compared with the controls. The increases in these differentiation markers were inhibited by the PPARγ antagonist GW9662 (20 μM). Furthermore, TNF-α decreased all differentiation marker levels. The decreases in differentiation markers were inhibited by BOT and TRG;however, these inhibitory effects were blocked by GW9662. The results suggest that BOT increases the synthesis and secretion of adiponectin by promoting differentiation similar to TRG. This study is the first to demonstrate that adipocyte differentiation-promoting activity is a mechanism for the beneficial effects of BOT on diabetes and insulin resistance.展开更多
Mitochondrial fatty acid oxidation(mtFAO)is a key metabolic pathway required for energy production in the liver,in particular during periods of fasting.One major consequence of drug-induced impairment of mtFAO is hepa...Mitochondrial fatty acid oxidation(mtFAO)is a key metabolic pathway required for energy production in the liver,in particular during periods of fasting.One major consequence of drug-induced impairment of mtFAO is hepatic steatosis,which is characterized by an accumulation of triglycerides and other lipid species,such as acyl-carnitines.Actually,the severity of this liver lesion is dependent on the residual mitochondrial b-oxidation flux.Indeed,a severe inhibition of mtFAO leads to microvesicular steatosis,hypoglycemia and liver failure.In contrast,moderate impairment of mtFAO can cause macrovacuolar steatosis,which is a benign lesion in the short term.Because some drugs can induce both microvesicular and macrovacuolar steatosis,it is surmised that severe mitochondrial dysfunction could be favored in some patients by non-genetic factors(e.g.,high doses and polymedication),or genetic predispositions involving genes that encode proteins playing directly or indirectly a role in the mtFAO pathway.Example of drugs inducing steatosis include acetaminophen(APAP),amiodarone,ibuprofen,linezolid,nucleoside reverse transcriptase inhibitors,such as stavudine and didanosine,perhexiline,tamoxifen,tetracyclines,troglitazone and valproic acid.Because several previous articles reviewed in depth the mechanism(s)whereby most of these drugs are able to inhibit mtFAO and induce steatosis,the present review is rather focused on APAP,linezolid and troglitazone.These steatogenic drugs are indeed rarely discussed in the literature as regards their ability to impair mtFAO.展开更多
基金supported by the National Basic Research Program (973) of China (No. 2009CB421605, No.2008CB418102)the National Natural Science Foundation of China (No.20890112, No. 21077127)
文摘Objective In order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells,the effects of PPARγ and POX-induced ROS were explored.Methods [3-(4,5)-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay,Annexin V and PI staining using FACS,plasmid transfection,ROS formation detected by DCFH staining,RNA interference,RT-PCR RT-QPCR,and Western blotting analyses were employed to investigate the apoptotic effect of troglitazone and the potential role of PPARγ pathway and POX-induced ROS formation in HT29 cells.Results Troglitazone was found to inhibit the growth of HT29 cells by induction of apoptosis.During this process,mitochondria related pathways including ROS formation,POX expression and cytochrome c release increased,which were inhibited by pretreatment with GW9662,a specific antagonist of PPARγ.These results illustrated that POX upregulation and ROS formation in apoptosis induced by troglitazone was modulated in PPARγ-dependent pattern.Furthermore,the inhibition of ROS and apoptosis after POX siRNA used in troglitazone-treated HT29 cells indicated that POX be essential in the ROS formation and PPARγ-dependent apoptosis induced by troglitazone.Conclusion The findings from this study showed that troglitazone-induced apoptosis was mediated by POX-induced ROS formation,at least partly,via PPARγ activation.
基金supported by grants from Natural Sciences Foundation of Hubei Province (No.2007ABA065)Science and Technology Key Project of Health Bureau of Hubei Province (No.JX1B006)
文摘Over-expression of P-glycoprotein(P-gp),an ATP-dependent drug efflux pump,represents one of the major mechanisms that contribute to multidrug resistance(MDR) in cancer cells.This study examined the effects of troglitazone,a ligand of peroxisome proliferator-activated receptor gamma(PPARγ),on P-gp-mediated MDR in SGC7901/VCR cells(a vincristine-resistant human gastric cancer cell line).The expression of P-gp was detected by RT-PCR and Western blotting,respectively.The SGC7901/VCR cells were treated with 0.1 mg/L vincristine(VCR) alone or in combination with 1,5,10 μmol/L troglitazone for 24 h.PPARγ was measured by electrophoretic mobility shift assay(EMSA).The intracellular concentration of Rhodamine123(Rh123,a fluorescent P-gp substrate) was assayed to evaluate the activity of P-gp.The cell cycle and apoptosis were measured by flow cytometry.The results showed that the P-gp was increasingly expressed in SGC7901,BGC823 and SGC7901/VCR cells in turn,suggesting that MDR in the SGC7901/VCR cells was mediated by the increased expression of P-gp.In the SGC7901/VCR cells,the expression level of total PPARγ was increased,however,the protein level and activity of PPARγ in the nuclei of cells decreased significantly.Troglitazone elevated the PPARγ activity in SGC7901/VCR cells in a dose-dependent manner.Troglitazone decreased the P-gp expression and markedly enhanced the accumulation of Rh123 in SGC7901/VCR cells in a dose-dependent manner.We also found that troglitazone significantly increased the percentage of SGC7901/VCR cells in the G2/M phase and decreased the cell percentage in G1 and S phase in a dose-dependent manner.Troglitazone significantly increased the apoptotic rate of SGC7901/VCR cells treated by VCR or ADR in a dose-dependent manner.It was concluded that P-gp-overexpressed SGC7901/VCR cells have minor endogenous PPARγ activity.Elevation of the PPARγ activity by troglitazone can reverse P-gp-mediated MDR via down-regulating the expression and activity of P-gp in SGC7901/VCR cells.It was suggested that troglitazone can dramatically enhance the sensitivity of P-gp-mediated MDR cancer cells to chemotherapeutic agents.
基金National Natural Science Foundation of China (No.30872808)
文摘AIM: To study the effect of troglitazone on primary culture human pterygium fibroblast (HPF). METHODS: Cell viability loss and apoptosis were quantified by cell counting kit-8, AnnexinV-FITC/PI double staining, caspases activity test and western blotting. Flow cytonnetry was used to detect mitochondrial membrane potential. RESULTS: Peroxisome proliferator-activated receptor gamma (PPAR-gamma) was positively expressed in pterygium specimens (n = 5). Troglitazone showed dose-dependent inhibition of cell survival, induced phospholipids redistribution, activated caspase-3, -9, and altered mitochondrial potential. Western blot assay demonstrated the increase of Bax/Bcl-2 protein ratio. CONCLUSION: Troglitazone induced apoptosis of HPF through a mitochondrial-dependent pathway.
基金the Health Min-istry of Hubei Province (JX2B16).
文摘To investigate whether peroxisome proliferators-activated receptor-γ (PPARγ) ligand Troglitazone can reduce endothelial injury and activation during storage of harvested saphenous vein grafts. Segments of human saphenous vein graft were collected from 9 patients undergoing coronary bypass surgery and then divided into two equal parts of control and test specimens, were stored in ei- ther heparinized blood (control group) or heparinized blood containing 20 μmol/L troglitazone (test group) for 1 h at room temperature. Tissue distribution and protein expression of VCAM-I, ICAM-I, and endothelial nitric oxide synthase (eNOS) were compared using immunohistochemistry and Western blot analysis. Myeloperoxidase (MPO) activity, a marker of neutrophil sequestration in human saphenous vein grafts, was also measured in each group. The expression of ICAM-1 (753±132 versus 7201±934; P〈0.01) , VCAM-1 (3731±294 versus 8292±793; P〈0.01), and MPO activity (1.52±0.42 U/g, 5.04±1.26 U/g P〈0.01) were significantly lower in test group. In contract, eNOS expression (7983±834 versus 3989±1008; P〈0.01) was significantly higher in test group. PPARγ ligand troglitazone might reduce endothelial injury during the storage period of human saphenous vein grafts.
文摘Boiogito (BOT) ameliorates insulin resistance and diabetes in several animal models;however, the underlying mechanisms for these in vivo effects remain unclear. Thiazolidine derivatives, which are peroxisome proliferator-activated receptor γ (PPARγ) agonists for the treatment of type II diabetes, promote adiponectin production by inducing adipocyte differentiation, thereby reducing insulin resistance. This study aimed to evaluate the effect of BOT on adipocyte differentiation using cultured human visceral preadipocytes (HVPAds) compared with the thiazolidine derivative troglitazone (TRG). We investigated the effects of BOT (0.125 - 1 mg/mL) and TRG (10 μM) on the differentiation of adipocytes treated with or without tumor necrosis factor-α (TNF-α: 5 ng/mL). On day 14 of culture, the following adipocyte differentiation marker levels were measured: intracellular lipids, extracellular (i.e., medium) adiponectin, and intracellular differentiation-related genes (PPARγ, CCAAT/enhancer binding protein, adiponectin, differentiation cluster 36, glucose transporter type 4). BOT and TRG increased factors associated with differentiation including lipid, adiponectin, and differentiation-related gene expression levels compared with the controls. The increases in these differentiation markers were inhibited by the PPARγ antagonist GW9662 (20 μM). Furthermore, TNF-α decreased all differentiation marker levels. The decreases in differentiation markers were inhibited by BOT and TRG;however, these inhibitory effects were blocked by GW9662. The results suggest that BOT increases the synthesis and secretion of adiponectin by promoting differentiation similar to TRG. This study is the first to demonstrate that adipocyte differentiation-promoting activity is a mechanism for the beneficial effects of BOT on diabetes and insulin resistance.
文摘【目的】明确Src与胶原同源插头蛋白(Src homology and collagen homology,Shc)调控曲格列酮(troglitazone,TZ)引起的猪血管内皮(porcine aortic endothelial,PAE)细胞自噬的机制。【方法】我们先利用激光共聚焦显微镜、蛋白免疫杂交检测了TZ引起的PAE细胞自噬;然后通过siRNA干扰敲降Shc,转染wtShc、3mShc等质粒的方法确定了p52Shc参与自噬的调控;最后通过siRNA干扰敲降Ulk1得到最终结论。【结果】通过研究发现,敲降Shc,会增加细胞的自噬;而过量表达p52Shc抑制了TZ引起的细胞自噬;p52Shc抑制自噬与其自身的酪氨酸磷酸化位点Tyr239、Tyr240和Tyr317相关;同时发现,p52Shc能抑制自噬调节分子磷酸腺苷激活的蛋白激酶(AMP-activated protein kinase,AMPK)及其下游底物Unc51样激酶1(UNC-51-like kinase-1,Ulk1)的活性。【结论】Shc通过调控AMPK与Ulk1的磷酸化调节TZ引起的细胞自噬。
文摘Mitochondrial fatty acid oxidation(mtFAO)is a key metabolic pathway required for energy production in the liver,in particular during periods of fasting.One major consequence of drug-induced impairment of mtFAO is hepatic steatosis,which is characterized by an accumulation of triglycerides and other lipid species,such as acyl-carnitines.Actually,the severity of this liver lesion is dependent on the residual mitochondrial b-oxidation flux.Indeed,a severe inhibition of mtFAO leads to microvesicular steatosis,hypoglycemia and liver failure.In contrast,moderate impairment of mtFAO can cause macrovacuolar steatosis,which is a benign lesion in the short term.Because some drugs can induce both microvesicular and macrovacuolar steatosis,it is surmised that severe mitochondrial dysfunction could be favored in some patients by non-genetic factors(e.g.,high doses and polymedication),or genetic predispositions involving genes that encode proteins playing directly or indirectly a role in the mtFAO pathway.Example of drugs inducing steatosis include acetaminophen(APAP),amiodarone,ibuprofen,linezolid,nucleoside reverse transcriptase inhibitors,such as stavudine and didanosine,perhexiline,tamoxifen,tetracyclines,troglitazone and valproic acid.Because several previous articles reviewed in depth the mechanism(s)whereby most of these drugs are able to inhibit mtFAO and induce steatosis,the present review is rather focused on APAP,linezolid and troglitazone.These steatogenic drugs are indeed rarely discussed in the literature as regards their ability to impair mtFAO.