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1.  Heat shock protein 70 expression is associated with inhibition of renal tubule epithelial cell apoptosis during recovery from low-protein feeding 
Cell Stress & Chaperones  2006;11(4):309-324.
The cellular stress response can mediate cellular protection through expression of heat shock protein (Hsp70), which can interfere with the process of apoptotic cell death. Factors regulating renal epithelial cell apoptosis include angiotensin II. In the present study, we have examined the relationship between the Hsp70 expression and the apoptotic pathway in the kidneys from low-protein–fed rats (8% protein). The possible cytoprotective role of Hsp70 has been evaluated during low-protein feeding and after reincorporation of 24% protein in the diet. The effect of angiotensin II AT1 receptor inhibition has also been studied. Rats were fed with a low-protein (LP) diet (8% protein) for 14 days, and then the animals were recovered by means of a normal protein diet (24% protein) (RP) for 14, 21, and 30 days, and control rats received 24% protein (NP) in the diet. LP and NP rats treated with Losartan (10 mg/kg) were also evaluated. The following methods were performed on the kidneys: terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay for apoptosis, reverse transcriptase-polymerase chain reaction assay for AT1, Bax, and Bcl-2 messenger ribonucleic acid (mRNA) expression, and immunohistochemical and Western blot for Hsp70 and caspase 3 protein expression and activity. In the LP group, the cells of the medullary ducts (MDs) showed increased apoptosis associated with weak immunoreaction for Hsp70 and decreased Hsp70 protein levels. In these animals, enhanced proapoptotic ratio Bax/Bcl-2 linked to decreased procaspase 3 protein levels with increased caspase 3 activation were demonstrated. A cytoprotection attributed to Hsp70 could be noted in the RP rats after 21 days of reincorporation of the normal diet, and in the LP-fed group treated with Losartan. In these cases, the MD cells displayed decreased apoptosis and increased Hsp70 expression in colocalization staining, and high Hsp70 levels in cytosolic fraction. A decreased proapoptotic ratio Bax/Bcl-2, associated with increased Bcl-2 mRNA, was also observed. Our results provide evidence for an antiapoptotic, cytoprotective effect of Hsp70 in kidney MD cells of rats with LP intake, when the animals were recovered with 24% protein in diet and after angiotensin II AT1 receptor inhibition. Angiotensin II seems to play a role in the pathogenesis of tubule epithelial cell apoptosis during LP feeding.
PMCID: PMC1712679  PMID: 17278880
2.  Role of Bax in quercetin-induced apoptosis in human prostate cancer cells 
Biochemical Pharmacology  2008;75(12):2345-2355.
The aim of this study was to investigate the effect of quercetin, a flavonoid, on the apoptotic pathway in a human prostate cell line (LNCaP). We observed that treatment of cells for 24 h with quercetin induced cell death in a dose-dependent manner. A sustained inhibition of the major survival signal, Akt, occurred in quercetin-treated cells. Treatment of LNCaP cells with an apoptosis inducing concentration of quercetin (100 μM) resulted in a rapid decrease in the inhibitory Ser(473) phosphorylation of Akt leading to inhibition of its kinase activity. Quercetin treatment (100 μM) also caused a decrease in Ser(136) phosphorylation of Bad, which is a downstream target of Akt. Protein interaction assay revealed that during treatment with quercetin, Bcl-xL dissociated from Bax and then associated with Bad. Our results also show that quercetin decreases the Bcl-xL:Bax ratio and increases translocation and multimerization of Bax to the mitochondrial membrane. The translocation is accompanied by cytochrome c release, and procaspases-3, -8 and -9 cleavage and increased poly (ADP-ribose) polymerase (PARP) cleavage. Similar results were observed in human colon cancer HCT116Bax+/+ cell line, but not HCT116Bax−/− cell line. Interestingly, at similar concentrations (100 μM), quercetin treatment did not affect the viability or rate of apoptosis in normal human prostate epithelial cell line (PrEC) and rat prostate epithelial cell line (YPEN-1). Our results indicate that the apoptotic processes caused by quercetin are mediated by the dissociation of Bax from Bcl-xL and the activation of caspase families in human prostate cancer cells.
PMCID: PMC3266687  PMID: 18455702
Quercetin; apoptosis; caspase; PI3K/Akt pathway; cytochrome c; Bax; Bad; Bcl-xL
3.  Neurotoxic effect of lead on rats: Relationship to Apoptosis 
Lead toxicity has been subjected to intensive research work, but some aspects of its mechanism needs to be elucidated.
In the current study we aim to investigate the impact of lead toxicity on some different intermediates of apoptotic signaling pathway in experimental rats.
Design and methods
We measured caspase-8 and caspase-9 [by chemilumenescence], Bax and Bcl-2 [by ELISA] in Experimental rats, injected intraperitoneally with lead acetate for 7days at the dosage of 25, 50 and l00 mg/kg body weight and compared to control rats injected with deionized distilled water instead. instead.
Lead acetate significantly increased the levels of caspase 8, caspase 9 and Bax in liver, kidney and brain of experimental animals especially those with high doses. Meanwhile, caspase 8 and Bax significantly increased in brain tissue at low dose of lead, while Bcl-2 significantly increased only with advanced toxicity. Furthermore, Bax/bcl2 ratio was significantly high in kidney (p<0.05), liver (p<0.01) and brain (p<0.01) at higher doses of lead toxicity. However, brain tissues showed significant Bax/Bcl2 ratio (p<0.05) at low lead dose. A significant positive correlation was noticed between the blood level of lead and enzymatic level of caspase 8, caspase 9 and Bax in different tissues.
: we concluded that lead might have toxic effect through intrinsic and extrinsic induction of apoptotic pathway with prominent effect on brain tissue even at low dose.
PMCID: PMC3883608  PMID: 24421747
Lead toxicity; Rats; Apoptosis; Bcl-2; Bax; Caspase 8 and Caspase 9
4.  The Effects of Erdosteine and N-Acetylcysteine on Apoptotic and Antiapoptotic Markers in Pulmonary Epithelial Cells in Sepsis 
The Eurasian Journal of Medicine  2013;45(3):167-175.
This study investigated the frequency of apoptosis in rat pulmonary epithelial cells after the injection of an intraperitoneal endotoxin lipopolysaccharide (LPS), the effects of LPS on apoptotic (bax, caspase-3) and antiapoptotic (bcl-2) markers during lung damage, and the protective effects of two known antioxidant agents, erdosteine and N-acetylcysteine (NAC).
Materials and Methods:
Male Wistar rats were divided into the following six groups, which included nine rats each: two control groups, two LPS-treated groups, one erdosteine-treated group (150 mg/kg), and one NAC-treated group (150 mg/kg). LPS was injected intraperitoneally at a dosage of 20 mg/kg. Following LPS injection, the antioxidants were orally administered. The rats were sacrificed at 24 h after LPS administration. The levels of apoptosis in bronchiolar and alveolar cells were determined using the TUNEL-staining method. Immunohistochemical staining of cytoplasmic bax, caspase-3, and bcl-2 in the epithelial cells was performed.
Erdosteine and NAC significantly reduced the rate of LPS-induced pulmonary epithelial cell apoptosis. The effect of NAC on regulating apoptosis was weaker than that of erdosteine. Erdosteine and NAC significantly reduced the local induction of bax and caspase 3 and significantly increased the reduced local production of bcl-2.
These findings suggest that erdosteine and NΑC can effectively protect the lungs from the damaging effects of LPS.
PMCID: PMC4261429  PMID: 25610275
Apoptosis; epithelial cells; erdosteine; N-acetylcysteine; pathogenesis; sepsis
5.  An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats 
Cystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence.
We compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.
The proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins.
These results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.
PMCID: PMC2713246  PMID: 19570211
6.  Efficient Elimination of Cancer Cells by Deoxyglucose-ABT-263/737 Combination Therapy 
PLoS ONE  2011;6(9):e24102.
As single agents, ABT-263 and ABT-737 (ABT), molecular antagonists of the Bcl-2 family, bind tightly to Bcl-2, Bcl-xL and Bcl-w, but not to Mcl-1, and induce apoptosis only in limited cell types. The compound 2-deoxyglucose (2DG), in contrast, partially blocks glycolysis, slowing cell growth but rarely causing cell death. Injected into an animal, 2DG accumulates predominantly in tumors but does not harm other tissues. However, when cells that were highly resistant to ABT were pre-treated with 2DG for 3 hours, ABT became a potent inducer of apoptosis, rapidly releasing cytochrome c from the mitochondria and activating caspases at submicromolar concentrations in a Bak/Bax-dependent manner. Bak is normally sequestered in complexes with Mcl-1 and Bcl-xL. 2DG primes cells by interfering with Bak-Mcl-1 association, making it easier for ABT to dissociate Bak from Bcl-xL, freeing Bak to induce apoptosis. A highly active glucose transporter and Bid, as an agent of the mitochondrial apoptotic signal amplification loop, are necessary for efficient apoptosis induction in this system. This combination treatment of cancer-bearing mice was very effective against tumor xenograft from hormone-independent highly metastasized chemo-resistant human prostate cancer cells, suggesting that the combination treatment may provide a safe and effective alternative to genotoxin-based cancer therapies.
PMCID: PMC3176271  PMID: 21949692
Bcl-2 and bax are proteins with opposing roles in apoptosis regulation; yet abnormal expression of either has been associated with failure after radiotherapy (RT). In this study we examined bcl-2 and bax expression as predictive markers in men treated with radiotherapy ± androgen deprivation on Radiation Therapy Oncology Group (RTOG) protocol 86-10.
Experimental Design
Suitable archival diagnostic tissue was obtained from 119 (26%) patients for bcl-2 analysis and 104 (23%) patients for bax analysis. Cox proportional hazards multivariate analysis was used to determine the relationship of abnormal bcl-2 and bax expression to the end points of local failure, distant metastasis, cause-specific mortality, and overall mortality. Bcl-2 overexpression was classified as any tumor cell cytoplasmic staining and altered bax expression was classified as greater or lesser cytoplasmic staining intensity of tumor cells as compared with adjacent normal prostate epithelium.
The study cohort exhibited bcl-2 overexpression in 26% (n = 30) of cases and abnormal bax expression in 47% (n = 49) of cases. A borderline significant relationship was observed between abnormal bax expression and higher Gleason score (p = 0.08). In univariate and multivariate analyses, there was no statistically significant relationship seen between abnormal bcl-2 or bax expression and outcome.
Abnormal bcl-2 and bax expression were not related to any of the end points tested. The cohort examined was comprised of patients with locally advanced disease and it is possible that these markers may be of greater value in men with earlier-stage prostate cancer.
PMCID: PMC1950741  PMID: 16814949
Bcl-2; Bax; Androgen Deprivation; Radiation Therapy
8.  Anticancer activity of resveratrol on implanted human primary gastric carcinoma cells in nude mice 
AIM: To investigate the apoptosis of implanted primary gastric cancer cells in nude mice induced by resveratrol and the relation between this apoptosis and expression of bcl-2 and bax.
METHODS: A transplanted tumor model was established by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice. Resveratrol (500 mg/kg, 1000 mg/kg and 1500 mg/kg) was directly injected beside tumor body 6 times at an interval of 2 d. Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated. We observed the morphologic alterations by electron microscope, measured the apoptotic rate by TUNEL staining method, detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistoch-emical staining and PT-PCR.
RESULTS: Resveratrol could significantly inhibit carcinoma growth when it was injected near the carcinoma. An inhibitory effect was observed in all therapeutic groups and the inhibition rate of resveratrol at the dose of 500 mg/kg, 1000 mg/kg and 1500 mg/kg was 10.58%, 29.68% and 39.14%, respectively. Resveratrol induced implanted tumor cells to undergo apoptosis with apoptotic characteristics, including morphological changes of chromatin condensation, chromatin crescent formation, nucleus fragmentation. The inhibition rate of 0.2 mL of normal saline solution, 1 500 mg/kg DMSO, 500 mg/kg resveratrol, 1000 mg/kg resveratrol, and 1500 mg/kg resveratrol was 13.68±0.37%, 13.8±0.43%, 48.7±1.07%, 56.44±1.39% and 67±0.96%, respectively. The positive rate of bcl-2 protein of each group was 29.48±0.51%, 27.56±1.40%, 11.86±0.97%, 5.7±0.84% and 3.92±0.85%, respectively by immunohistochemical staining. The positive rate of bax protein of each group was 19.34±0.35%, 20.88±0.91%, 40.02±1.20%, 45.72±0.88% and 52.3±1.54%, respectively by immunohistochemical staining. The density of bcl-2 mRNA in 0.2 mL normal saline solution, 1500 mg/kg DMSO, 500 mg/kg resveratrol, 1000 mg/kg resveratrol, and 1500 mg/kg resveratrol decreased progressively and the density of bax mRNA in 0.2 mL normal saline solution, 1500 mg/kg DMSO, 500 mg/kg resveratrol, 1000 mg/kg resveratrol, and 1500 mg/kg increased progressively with elongation of time by RT-PCR.
CONCLUSION: Resveratrol is able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated by down-regulating apoptosis-regulated gene bcl-2 and up-regulating the expression of apoptosis-regulated gene bax.
PMCID: PMC4205418  PMID: 15633232
Gastric carcinoma; Resveratrol; Neoplasm Transplantation; Apoptosis
9.  Long-Lasting Effect of Infant Rats Endotoxemia on Heat Shock Protein 60 in the Pancreatic Acinar Cells: Involvement of Toll-Like Receptor 4 
Introduction. Lipopolysaccharide endotoxin (LPS) is responsible for septic shock and multiorgan failure, but pretreatment of rats with low doses of LPS reduced pancreatic acute damage. Aim. We investigated the effects of the endotoxemia induced in the early period of life on Toll-like receptor 4 (TLR4), heat shock protein 60 (HSP60) and proapoptotic Bax, caspase-9 and -3 or antiapoptotic Bcl-2 protein expression in the pancreatic acinar cells of adult animals. Material and Methods. Newborn rats (25 g) were injected with endotoxin (Escherichia coli) for 5 consecutive days. Two months later, pancreatic acinar cells were isolated from all groups of animals and subjected to caerulein stimulation (10−8 M). Protein expression was assessed employing Western blot. For detection of apoptosis we have employed DNA fragmentation ladder assay. Results. Preconditioning of newborn rats with LPS increased TLR4, Caspase-9 and -3 levels, but failed to affect basal expression of HSP60, Bax, and Bcl-2. Subsequent caerulein stimulation increased TLR4, Bcl-2, and caspases, but diminished HSP60 and Bax proteins in pancreatic acinar cells. Endotoxemia dose-dependently increased TLR4, Bax, HSP60, and both caspases protein signals in the pancreatic acini, further inhibiting antiapoptotic Bcl-2. Conclusions. Endotoxemia promoted the induction of HSP60 via TLR4 in the infant rats and participated in the LPS-dependent pancreatic tissue protection against acute damage.
PMCID: PMC3364569  PMID: 22685683
10.  Quercetin postconditioning attenuates myocardial ischemia/reperfusion injury in rats through the PI3K/Akt pathway 
Quercetin (Que), a plant-derived flavonoid, has multiple benefical actions on the cardiovascular system. The current study investigated whether Que postconditioning has any protective effects on myocardial ischemia/reperfusion (I/R) injury in vivo and its potential cardioprotective mechanisms. Male Sprague-Dawley rats were randomly allocated to 5 groups (20 animals/group): sham, I/R, Que postconditioning, Que+LY294002 [a phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway inhibitor], and LY294002+I/R. I/R was produced by 30-min coronary occlusion followed by 2-h reperfusion. At the end of reperfusion, myocardial infarct size and biochemical changes were compared. Apoptosis was evaluated by both TUNEL staining and measurement of activated caspase-3 immunoreactivity. The phosphorylation of Akt and protein expression of Bcl-2 and Bax were determined by Western blotting. Que postconditioning significantly reduced infarct size and serum levels of creatine kinase and lactate dehydrogenase compared with the I/R group (all P<0.05). Apoptotic cardiomyocytes and caspase-3 immunoreactivity were also suppressed in the Que postconditioning group compared with the I/R group (both P<0.05). Akt phosphorylation and Bcl-2 expression increased after Que postconditioning, but Bax expression decreased. These effects were inhibited by LY294002. The data indicate that Que postconditioning can induce cardioprotection by activating the PI3K/Akt signaling pathway and modulating the expression of Bcl-2 and Bax proteins.
PMCID: PMC3854307  PMID: 24068165
Ischemia and reperfusion; Quercetin; Postconditioning; PI3K/Akt
11.  1, 25-dihydroxyvitamin D3 decreases adriamycin-induced podocyte apoptosis and loss 
Background: Selective proteinuria is frequently observed in glomerular diseases characterized by podocyte injury. Although, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has potential therapeutic effects on chronic kidney diseases through decreasing podocyte loss, the mechanism underlying the beneficial effects of 1,25(OH)2D3 on podocytes remains still unknown. The present study tested the hypothesis that 1,25(OH)2D3 directly reduced podocyte apoptosis and loss.
Methods: Sprague-Dawley (SD) rats were randomly assigned into three groups: Adriamycin (ADR) group (n=15), ADR+1,25-(OH)2D3 group (n=16), and control group (n=16). Rats in ADR+1,25-(OH)2D3 group were treated with 1,25(OH)2D3 for 8 weeks. The number of podocytes and foot process width (FPW) were detected by transmission electron microscopy. The number of apoptotic podocytes per glomerulus and that of apoptotic nuclei and caspase-3 activity in cultured podocytes were determined by TUNEL staining. The average number of podocytes per glomerulus was quantified by immunohistochemistry. Expressions of p-Smad2/3, p-Smad1/5/8, Fas, Fas-Associated protein with Death Domain (FADD), Bax, and Bcl-2 proteins were examined by Western blot assay.
Results: Compared with control group, proteinuria, FPW, apoptotic podocytes, caspase-3 activity, the protein expressions of p-Smad2/3, Fas, FADD, and Bax were significantly increased, podocyte density, p-Smad1/5/8 and Bcl-2 expression were decreased in ADR group. 1,25(OH)2D3 significantly reduced proteinuria, FPW, caspase-3 activity, expressions of p-Smad2/3, Fas, FADD, and Bax and apoptosis of podocytes, but increased serum albumin, number of viable podocytes , p-Smad1/5/8 and Bcl-2 expression in ADR treated rats.
Conclusion: ADR-induced podocyte apoptosis was associated with the imbalance of p-Smad2/3, p-Smad1/5/8 the activity of caspase-3 and aberrant expressions of, Fas, FADD, Bax and Bcl-2. The beneficial effects of 1,25(OH)2D3 on podocytes may be attributable to inhibit podocyte apoptosis and the amelioration of podocytopenia.
PMCID: PMC2934728  PMID: 20827429
1; 25-dihydroxyvitamin D3; podocyte; proteinuria
12.  Ceftriaxone, a Beta-Lactam Antibiotic, Modulates Apoptosis Pathways and Oxidative Stress in a Rat Model of Neuropathic Pain 
BioMed Research International  2014;2014:937568.
Purpose. In our previous study, ceftriaxone, a beta-lactam antibiotic, elicited antinociceptive effects in the chronic constriction injury (CCI) of neuropathic pain. In this study, we assessed apoptosis and oxidative stress in the spinal cord of neuropathic rats treated with ceftriaxone. Methods. 45 male Wistar rats were divided as naïve, sham, normal saline-treated CCI rats, and CCI animals treated with the effective dose of ceftriaxone. Involvement of Bax, Bcl2, and caspases 3 and 9, important contributors of programmed cell death (apoptosis), was determined using western blotting at days 3 and 7. The markers of oxidative stress including malondialdehyde (MDA) and reduced glutathione (GSH) were measured on days 3 and 7. Results. Increased Bax/Bcl2 ratio and cleaved active forms of caspases 3 and 9 were observed in the spinal cord of CCI rats on day 3. Ceftriaxone attenuated the increased levels of Bax and cleaved forms of caspases 3 and 9, while it increased Bcl2 levels. Bax and active forms of caspases declined by day 7. Consequently, comparison among groups showed no difference at this time. CCI enhanced MDA and decreased GSH on days 3 and 7, while ceftriaxone protected against the CCI-induced oxidative stress. Conclusion. Our results suggest that ceftriaxone, an upregulator/activator of GLT1, could concomitantly reduce oxidative stress and apoptosis and producing its new analogs lacking antimicrobial activity may represent a novel approach for neuropathic pain treatment.
PMCID: PMC4084648  PMID: 25028668
13.  Protective effect of Curcumin on chemotherapy-induced intestinal dysfunction 
Objective: Chemotherapy is one of most important treatments for human cancers. However, side effects such as intestine dysfunction significantly impaired its clinical efficacy. This study aimed to investigate the protective effect of Curcumin on chemotherapy-induced intestinal dysfunction in rats. Methods: Sixty healthy Wistar rats were randomly divided into control group (normal saline), 5-FU group and 5-FU+Curcumin group. The weight, serum level of endotoxin, DAO and D-lactate were determined. The pathological change of intestinal mucosa structure was studied under light microscopy and electron microscopy. The expression of Bax, Bcl-2 and Caspase-3 were assessed by immunohistochemical staining. Results: The Curcumin intragastrically administrated obviously reduced 5-FU-induced weight-loss. 5-FU induced dramatic increase of serum endotoxin, D-lactate and D-Amino-Acid Oxidase (DAO) that were significantly reversed by Curcumin treatment. Meanwhile, 5-FU-induced-damage to intestinal mucosa structure was markedly recovered by Curcumin. The expression of Bax and Caspase-3 were dramatically increased after 5-FU treatment (p<0.01) and Curcumin treatment significantly reduced Bax expression (p<0.05) but had only a moderate effect on reducing caspase-3 expression (p>0.05). Interestingly, Bcl-2 expression was low in control group but increased after 5-FU treatment (p>0.05) and Curcumin treatment further stimulated Bcl-2 expression (p<0.05). Conclusions: Curcumin can significantly reverse chemotherapy-induced weight-loss, increase of serum endotoxin, D-lactate and DAO and damage to intestinal mucosa structure. Curcumin also reduced the expression of pro-apoptotic Bax but stimulated anti-apoptotic Bcl-2 to attenuate 5-FU-induced apoptosis of intestinal epithelial cells. The clinical administration of Curcumin may improve chemotherapy-induced intestinal dysfunction, thus increasing the clinical efficacy of chemotherapy.
PMCID: PMC3816802  PMID: 24228095
Curcumin; chemotherapy-induced; intestinal dysfunction; Bax; Bcl-2; 5-FU; ultrastructures
14.  Lenticular mitoprotection. Part B: GSK-3β and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen 
Molecular Vision  2013;19:2451-2467.
Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient that leads to mitochondrial membrane permeability transition (mMPT). This study emphasizes the role of glycogen synthase kinase 3beta (GSK-3β) in maintaining mitochondrial membrane potential, thus preventing mitochondrial depolarization (hereafter termed mitoprotection). Using 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), an inhibitor of GSK-3β, and drawing a distinction between it and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), an inhibitor of extracellular-signal-regulated kinase (ERK) phosphorylation, the means by which GSK-3β influences mitoprotection in cultured human lens epithelial (HLE-B3) cells and normal, secondary cultures of bovine lens epithelial cells, maintained in atmospheric oxygen, was investigated.
Virally transfected human lens epithelial cells (HLE-B3) and normal cultures of bovine lens epithelial cells were exposed to acute hypoxic conditions (about 1% O2) followed by exposure to atmospheric oxygen (about 21% O2). Specific antisera and western blot analysis was used to examine the state of phosphorylation of ERK and GSK-3β, as well as the phosphorylation of a downstream substrate of GSK-3β, glycogen synthase (GS, useful in monitoring GSK-3β activity). The potentiometric dye, 1H-benzimidazolium-5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-iodide (JC-1), was used to monitor mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Caspase-3 activation was scrutinized to determine whether mitochondrial depolarization inevitably leads to apoptosis.
Treatment of HLE-B3 cells with SB216763 (12 µM) inactivated GSK-3β activity as verified by the enzyme’s inability to phosphorylate its substrate, GS. SB216763-treated cells were not depolarized relative to the control cells as demonstrated with JC-1 fluorescent dye analysis. The HLE-B3 cells treated with UO126, which similarly blocked phosphorylation of GS, were nevertheless prone to mMPT relative to the control cells. Western blot analysis determined that Bcl-2-associated X (BAX) levels were unchanged for SB216763-treated or UO126-treated HLE-B3 cells when compared to their respective control cells. However, unlike the SB216763-treated cells, the UO126-treated cells showed a marked absence of Bcl-2, as well as phosphorylated Bcl-2 relative to the controls. UO126 treatment of bovine lens epithelial cells showed similar results with pBcl-2 levels, while the Bcl-2 content appeared unchanged relative to the control cells. HLE-B3 and normal bovine lens cell cultures showed susceptibility to mMPT associated with the loss of pBcl-2 by UO126 treatment.
Mitochondrial depolarization may occur by one of two key occurrences: interruption of the electrochemical gradient across the inner mitochondrial membrane resulting in mMPT or by disruption of the integrity of the inner or outer mitochondrial membrane. The latter scenario is generally tightly regulated by members of the Bcl-2 family of proteins. Inhibition of GSK-3β activity by SB216763 blocks mMPT by preventing the opening of the mitochondrial permeability transition pore. UO126, likewise, inhibits GSK-3β activity, but unlike SB216763, inhibition of ERK phosphorylation induces the loss of intracellular pBcl-2 levels under conditions where intracellular BAX levels remain constant. These results suggest that the lenticular mitoprotection normally afforded by the inactivation of GSK-3β activity may, however, be bypassed by a loss of pBcl-2, an anti-apoptotic member of the Bcl-2 family. Bcl-2 prevents the translocation of BAX to the mitochondrial outer membrane inhibiting depolarization by disrupting the normal electrochemical gradient leading to mMPT.
PMCID: PMC3850969  PMID: 24319338
15.  Evaluation of Bcl-2 Family Gene Expression and Caspase-3 Activity in Hippocampus STZ-Induced Diabetic Rats 
Experimental Diabetes Research  2008;2008:638467.
We assessed the expression of Bcl-2 family members at both mRNA and protein levels as well as the Caspase-3 activity, in order to investigate the occurrence of apoptosis in hippocampus of STZ-induced diabetic rats. We selected twenty-four Wistar rats; half of them were made diabetic by intraperitoneal injection of a single 60 mg/kg dose of streptozotocin (STZ, IP), while the others received normal saline and served as controls. The expressions of Bcl-2, Bcl-xL, and Bax mRNA and proteins were measured using RT-PCR and western blotting, respectively. Caspases-3 activity was determined by using the Caspase-3/CPP32 Fluorometric Assay Kit. The result showed that mRNA and protein levels of Bcl-2 and Bcl-xL were lower in hippocampus of diabetic group than that of the control group, whereas expressions of Bax in hippocampus of diabetic rats were higher than that of controls at both mRNA and protein levels (P < .01). Hyperglycemia was found to raise 6.9-fold hippocampal caspase-3 activity in diabetic group compared with control group (P < .001). Therefore, the induction of diabetes is associated with increased ratios of Bax/Bcl-2, Bax/Bcl-xL, and increased caspase-3 activity in hippocampus which shows that apoptosis is favored in hippocampal region.
PMCID: PMC2566751  PMID: 18923682
16.  Liguzinediol improved the heart function and inhibited myocardial cell apoptosis in rats with heart failure 
Acta Pharmacologica Sinica  2014;35(10):1257-1264.
Liguzinediol is a novel derivative of ligustrazine isolated from the traditional Chinese medicine Chuanxiong (Ligusticum wallichii Franch), and produces significant positive inotropic effect in isolated rat hearts. In this study we investigated the effects of liguzinediol on a rat model of heart failure.
To induce heart failure, male SD rats were injected with doxorubicin (DOX, 2 mg/kg, ip) once a week for 4 weeks. Then the rats were administered with liguzinediol (5, 10, 20 mg·kg−1·d−1, po) for 2 weeks. Hemodynamic examination was conducted to evaluate heart function. Myocardial cell apoptosis was examined morphologically. The expression of related genes and proteins were analyzed using immunohistochemical staining and Western blot assays, respectively.
Oral administration of liguzinediol dose-dependently improved the heart function in DOX-treated rats. Electron microscopy revealed that liguzinediol (10 mg·kg−1·d−1) markedly attenuated DOX-induced injury of cardiomyocytes, and decreased the number of apoptotic bodies in cardiomyocytes. Furthermore, liguzinediol significantly decreased Bax protein level, and increased Bcl-2 protein level in cardiomyocytes of DOX-treated rats, led to an increase in the ratio of Bcl-2/Bax. Moreover, liguzinediol significantly decreased the expression of both cleaved caspase-3 and NF-κB in cardiomyocytes of DOX-treated rats. Administration of digitalis (0.0225 mg·kg−1·d−1) also markedly improved the heart function and the morphology of cardiomyocytes in DOX-treated rats.
Liguzinediol improves the heart function and inhibits myocardial cell apoptosis in the rat model of heart failure, which is associated with regulating Bcl-2, Bax, caspase-3 and NF-κB expression.
PMCID: PMC4186991  PMID: 25220638
heart failure; cardiomyocyte; liguzinediol; apoptosis; caspases; Bcl-2; NF-κB; doxorubicin; Ligusticum wallichii
17.  Valproic acid inhibits neural progenitor cell death by activation of NF-κB signaling pathway and up-regulation of Bcl-XL 
At the beginning of neurogenesis, massive brain cell death occurs and more than 50% of cells are eliminated by apoptosis along with neuronal differentiation. However, few studies were conducted so far regarding the regulation of neural progenitor cells (NPCs) death during development. Because of the physiological role of cell death during development, aberration of normal apoptotic cell death is detrimental to normal organogenesis.
Apoptosis occurs in not only neuron but also in NPCs and neuroblast. When growth and survival signals such as EGF or LIF are removed, apoptosis is activated as well as the induction of differentiation. To investigate the regulation of cell death during developmental stage, it is essential to investigate the regulation of apoptosis of NPCs.
Neural progenitor cells were cultured from E14 embryonic brains of Sprague-Dawley rats. For in vivo VPA animal model, pregnant rats were treated with VPA (400 mg/kg S.C.) diluted with normal saline at E12. To analyze the cell death, we performed PI staining and PARP and caspase-3 cleavage assay. Expression level of proteins was investigated by Western blot and immunocytochemical assays. The level of mRNA expression was investigated by RT-PCR. Interaction of Bcl-XL gene promoter and NF-κB p65 was investigated by ChIP assay.
In this study, FACS analysis, PI staining and PARP and caspase-3 cleavage assay showed that VPA protects cultured NPCs from cell death after growth factor withdrawal both in basal and staurosporine- or hydrogen peroxide-stimulated conditions. The protective effect of prenatally injected VPA was also observed in E16 embryonic brain. Treatment of VPA decreased the level of IκBα and increased the nuclear translocation of NF-κB, which subsequently enhanced expression of anti-apoptotic protein Bcl-XL.
To the best of our knowledge, this is the first report to indicate the reduced death of NPCs by VPA at developmentally critical periods through the degradation of IκBα and the activation of NF-κB signaling. The reduced NPCs death might underlie the neurodevelopmental defects collectively called fetal valproate syndrome, which shows symptoms such as mental retardation and autism-like behavior.
PMCID: PMC3158748  PMID: 21722408
18.  Glutamine Treatment Attenuates Endoplasmic Reticulum Stress and Apoptosis in TNBS-Induced Colitis 
PLoS ONE  2012;7(11):e50407.
Endoplasmic reticulum (ER) stress and apoptotic cell death play an important role in the pathogenesis and perpetuation of inflammatory bowel disease (IBD). We aimed to explore the potential of glutamine to reduce ER stress and apoptosis in a rat model of experimental IBD. Colitis was induced in male Wistar rats by intracolonic administration of 30 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Glutamine (25 mg/dL) was given by rectal route daily for 2 d or 7 d. Both oxidative stress (TBARS concentration and oxidised/reduced glutathione ratio) and ER stress markers (CHOP, BiP, calpain-1 and caspase-12 expression) increased significantly within 48 h of TNBS instillation, and glutamine attenuated the extent of the changes. Glutamine also inhibited the significant increases of ATF6, ATF4 and spliced XBP-1 mRNA levels induced by TNBS instillation. TNBS-colitis resulted in a significant increase in p53 and cytochrome c expression, and a reduced Bcl-xL expression and Bax/Bcl-2 ratio. These effects were significantly inhibited by glutamine. Treatment with the amino acid also resulted in significant decreases of caspase-9, caspase-8 and caspase-3 activities. Double immunofluorescence staining showed co-localization of CHOP and cleaved caspase-3 in colon sections. Phospho-JNK and PARP-1 expression was also significantly higher in TNBS-treated rats, and treatment with glutamine significantly decreased JNK phosphorylation and PARP-1 proteolysis. To directly address the effect of glutamine on ER stress and apoptosis in epithelial cells, the ER stress inducers brefeldin A and tunicamycin were added to Caco-2 cells that were treated with glutamine (5 mM and 10 mM). The significant enhancement in PERK, ATF6 phosphorylated IRE1, BiP and cleaved caspase-3 expression induced by brefeldin A and tunicamycin was partly prevented by glutamine. Data obtained indicated that modulation of ER stress signalling and anti-apoptotic effects contribute to protection by glutamine against damage in TNBS-induced colitis.
PMCID: PMC3508929  PMID: 23209735
19.  Glycyrrhizic acid attenuates CCl4-induced hepatocyte apoptosis in rats via a p53-mediated pathway 
AIM: To investigate the effect of glycyrrhizic acid (GA) on carbon tetrachloride (CCl4)-induced hepatocyte apoptosis in rats via a p53-dependent mitochondrial pathway.
METHODS: Forty-five male Sprague-Dawley rats were randomly and equally divided into three groups, the control group, the CCl4 group, and the GA treatment group. To induce liver fibrosis in this model, rats were given a subcutaneous injection of a 40% solution of CCl4 in olive oil at a dose of 0.3 mL/100 g body weight biweekly for 8 wk, while controls received the same isovolumetric dose of olive oil by hypodermic injection, with an initial double-dose injection. In the GA group, rats were also treated with a 40% solution of CCl4 plus 0.2% GA solution in double distilled water by the intraperitoneal injection of 3 mL per rat three times a week from the first week following previously published methods, with modifications. Controls were given the same isovolumetric dose of double distilled water. Liver function parameters, such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined. Pathologic changes in the liver were detected by hematoxylin and eosin staining. Collagen fibers were evaluated by Sirius red staining. Hepatocyte apoptosis was investigated using the terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) assay and the cleaved caspase-3 immunohistochemistry assay. The expression levels of p53 and apoptosis-related proteins were evaluated by immunohistochemistry or Western blotting analysis.
RESULTS: After 8 wk of treatment, GA significantly reduced serum activity of ALT (from 526.7 ± 57.2 to 342 ± 44.8, P < 0.05) and AST (from 640 ± 33.7 to 462.8 ± 30.6, P < 0.05), attenuated the changes in liver histopathology and reduced the staging score (from 3.53 ± 0.74 to 3.00 ± 0.76, P < 0.05) in CCl4-treated rats. GA markedly reduced the positive area of Sirius red and the ratio of the hepatic fibrotic region (from 7.87% ± 0.66% to 3.68% ± 0.32%, P < 0.05) compared with the CCl4 group. GA also decreased the expression level of cleaved caspase-3 compared to the CCl4 group. TUNEL assay indicated that GA significantly diminished the number of TUNEL-positive cells compared with the CCl4 group (P < 0.05). GA treatment clearly decreased the level of p53 (P < 0.05) detected by immunohistochemistry and Western blotting analysis. Compared with the CCl4 group, we also found that GA reduced the Bax/Bcl-2 ratio (P < 0.05), the expression of cleaved caspase-3 (P < 0.05), cleaved caspase-9 (P < 0.05), and inhibited cytochrome C and second mitochondria-derived activator of caspases (Smac) release from mitochondria to cytoplasm, i.e., GA reduced the expression level of Smac, which inhibited c-IAP1 activity (P < 0.05), ultimately inhibiting the activity of caspase-3, according to Western blotting analysis. As a result, GA suppressed activation of the caspase cascades and prevented hepatocyte apoptosis.
CONCLUSION: GA can inhibit CCl4-induced hepatocyte apoptosis via a p53-dependent mitochondrial pathway to retard the progress of liver fibrosis in rats.
PMCID: PMC3699029  PMID: 23840116
P53; Apoptosis; Liver fibrosis; Glycyrrhizic acid; Mitochondria
20.  Silibinin, a natural flavonoid, modulates the early expression of chemoprevention biomarkers in a preclinical model of colon carcinogenesis 
International Journal of Oncology  2012;41(3):849-854.
The flavonolignan silibinin, the major biologically active compound of the milk thistle (Silybum marianum), has been shown to possess anticancer properties in a variety of epithelial cancers. The present study investigated the potential of silibinin as a chemopreventive agent in colon carcinogenesis. The rat azoxymethane (AOM)-induced colon carcinogenesis model was used because of its molecular and clinical similarities to sporadic human colorectal cancer. One week after AOM injection (post-initiation), Wistar rats received daily intragastric feeding of 300 mg silibinin/kg body weight per day until their sacrifice after 7 weeks of treatment. Silibinin-treated rats exhibited a 2-fold reduction in the number of AOM-induced hyperproliferative crypts and aberrant crypt foci in the colon compared to AOM-injected control rats receiving the vehicle. Silibinin-induced apoptosis in the colon mucosal cells was demonstrated by flow cytometry after propodium iodide staining and by colorimetric measurement of caspase-3 activity. Mechanisms involved in silibinin-induced apoptosis included the downregulation of the anti-apoptotic protein Bcl-2 and upregulation of the pro-apoptotic protein Bax, inverting the Bcl-2/Bax ratio to <1. This modulation already takes place at the mRNA expression level as shown by real-time RT-PCR. Furthermore, silibinin treatment significantly (P<0.01) decreased the genetic expression of biomarkers of the inflammatory response such as IL1β, TNFα and their downstream target MMP7, all of them shown to be upregulated during colon carcinogenesis. The downregulation of MMP7 protein was confirmed by western blot analysis. The present findings show the ability of silibinin to shift the disturbed balance between cell renewal and cell death in colon carcinogenesis in rats previously injected with the carcinogen AOM. Silibinin administered via intragastric feeding exhibited potent pro-apoptotic, anti-inflammatory and multi-targeted effects at the molecular level. The effective reduction of preneoplastic lesions by silibinin supports its use as a natural agent for colon cancer chemoprevention.
PMCID: PMC3582883  PMID: 22735354
colorectal cancer; aberrant crypt foci; inflammation; apoptosis
21.  Effect of Achyranthes Bidentata Polysaccharides on the Expression of BCL-2 and Bax in Hepatic Tissues after Exhaustive Exercise in Rats 
This study aims to assess the effects of Achyranthes bidentata polysaccharides (ABPS) on the expression of bcl-2 and bax in hepatic tissues after exhaustive exercise in order to provide theoretical support for the application of ABPS in the field of sports nutrition. Thirty male Sprague-Dawley rats were randomized into three groups, each consisting of 10 rats: Normal control group (NCG), Exhausting exercises control group (EECG), ABPS treated group (ATG). ABPS were fed orally by gastric intubation to rats of ABPS treated group (ATG) once daily for 7 days. Control animals (EECG and NCG) received the same amount of isotonic sodium chloride solution. Exhaustive exercise was performed on a rodent treadmill. The SP (streptavidin peroxidase) method for immunohistochemical staining was adopted to test the protein expression of bax and bcl-2 in the hepatic tissues of the rats. Exhausting exercises increased bax protein expression of hepatic tissues of rats and bax/bcl-2 ratio dramatically, but a decreased bcl-2 protein expression. In the rats fed ABPS orally by gastric intubation, the bax protein expression and bax/bcl-2 ratio obviously decreased, while bcl-2 protein expression increased. The result indicated that bax and bcl-2 co-regulated the exercise-induced hepatocyte apoptosis. Feeding ABPS orally by gastric intubation to rats can inhibit the hepatocyte apoptosis in exhaustive exercise.
PMCID: PMC3005393  PMID: 21731162
achyranthes bidentata polysaccharides; apoptosis; bcl-2; bax; exhaustive exercise
22.  The prognostic significance of apoptosis-associated proteins BCL-2, BAX and BCL-X in clinical nephroblastoma 
British Journal of Cancer  2001;85(10):1557-1563.
Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers in normal tissues. Various apoptosis-associated regulatory proteins, such as Bcl-2, Bax and Bcl-X, may contribute to the rate of apoptosis in neoplasia. The present study was performed to evaluate the prognostic value of these molecules in a group of 61 Wilms' tumours of chemotherapeutically pre-treated patients using an immunohistochemical approach. Generally, Bcl-2, Bax and for Bcl-X S/L were expressed in the blastemal and epithelial components of Wilms' tumour. Immunoreactive blastema cells were found in 53%, 41% and 38% of tumours for Bcl-2, Bax and for Bcl-X S/L, respectively. An increased expression of Bcl-2 was observed in the blastemal component of increasing pathological stages. In contrast, a gradual decline of Bax expression was observed in the blastemal component of tumours with increasing pathological stages. Also blastemal Bcl-X S/L expression decreased with stage. Univariate analysis showed that blastemal Bcl-2 expression and the Bcl-2/Bax ratio were indicative for clinical progression, whereas epithelial staining was of no prognostic value. Multivariate analysis showed that blastemal Bcl-2 expression is an independent prognostic marker for clinical progression besides stage. These findings demonstrate that alterations of the Bcl-2/Bax balance may influence the clinical outcome of Wilms' tumour patients by deregulation of programmed cell death. © 2001 Cancer Research Campaign
PMCID: PMC2363928  PMID: 11720445
Wilms’; tumour; Bcl-2; Bax; Bcl-X; prognosis
23.  Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice 
AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.
METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues.
RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 ± 12.51 vs 170.39 ± 25.29; 49.83 ± 11.46 vs 170.39 ± 25.29; 83.99 ± 24.63 vs 170.39 ± 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 ± 4.2 vs 23.4 ± 2.1 and 29.4 ± 3.4 vs 23.4 ± 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphological changes were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR.
CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.
PMCID: PMC4172720  PMID: 17659679
Bufalin; Hepatocellular carcinoma; Orthotopic transplantation; Nude mice; Model; Treatment; Apoptosis
24.  Doxazosin Induces Apoptosis of Benign and Malignant Prostate Cells via a Death Receptor–Mediated Pathway 
Cancer research  2006;66(1):464-472.
Quinazoline-based α1-adrenoceptor antagonists such as doxazosin and terazosin have been previously shown to induce apoptosis in prostate cancer cells via an α1-adrenoceptor–independent pathway, involving activation of transforming growth factor-β1 (TGF-β1) signaling. In this study, the molecular events initiating this apoptotic effect were further investigated in vitro using the human androgen-independent prostate cancer cells PC-3 and the human benign prostate epithelial cells BPH-1. Quantitative microarray assays were done in PC-3 and BPH-1 cells after treatment with doxazosin (25 μmol/L, 6 and 24 hours) to identify the early gene changes. Transient changes in the expression of several apoptosis regulators were identified, including up-regulation of Bax and Fas/CD95 and down-regulation of Bcl-xL and TRAMP/Apo3. Moreover, there were significant changes in the expression pattern of signaling components of the extracellular matrix such as integrins α2, αV, β1, and β8. Western blot analysis revealed activation of caspase-8 and caspase-3 within the first 6 to 12 hours of treatment with doxazosin in both PC-3 and BPH-1 cells. Doxazosin-induced apoptosis was blocked by specific caspase-8 inhibitors, supporting the functional involvement of caspase-8 in doxazosin-induced apoptosis. The effect of doxazosin on recruitment of Fas-associated death domain (FADD) and procaspase-8 to the Fas receptor was examined via analysis of death-inducing signaling complex formation. Doxazosin increased FADD recruitment and subsequent caspase-8 activation, implicating Fas-mediated apoptosis as the underlying mechanism of the effect of doxazosin in prostate cells. These results show that doxazosin exerts its apoptotic effects against benign and malignant prostate cells via a death receptor–mediated mechanism with a potential integrin contribution towards cell survival outcomes.
PMCID: PMC1850148  PMID: 16397262
25.  The effects of quercetin in cultured human RPE cells under oxidative stress and in Ccl2/Cx3cr1 double deficient mice 
Experimental eye research  2010;91(1):15-25.
Quercetin, a member of the flavonoid family, is one of the most prominent dietary antioxidants. This study investigates the mechanisms for the effects of quercetin on cultured human RPE cells and in Ccl2/Cx3cr1 double knock-out (DKO) mice, which spontaneously develop progressive retinal lesions mimicking age-related macular degeneration (AMD). In the in vitro experiment, cultured ARPE-19 cells were exposed to 1mM H2O2 with or without 50μM quercetin for 2 hours. Cellular viability, mitochondrial function, and apoptosis were assessed using crystal violet staining, MTT assay, and comet assay, respectively. Apoptotic molecular transcripts of BCL-2, BAX, FADD, CASPASE-3 and CASPASE-9 were measured by RQ-PCR. COX activity and nitric oxide (NO) level were determined in the supernatant of the culture medium. Quercetin treatment protected ARPE-19 cells from H2O2-induced oxidative injury, enhanced BCL-2 transcript levels, increased the BCL-2/BAX ratio, suppressed the transcription of pro-apoptotic factors such as BAX, FADD, CASPASE-3 and CASPASE-9, inhibited the transcription of inflammatory factors such as TNF-α, COX-2 and INOS, and decreased the levels of COX and NO in the culture medium. In the in vivo experiment, DKO and C57/B6 mice were treated with 25mg/kg/day quercetin by intraperitoneal injection daily for two months. Funduscopy was performed monthly. After two months, serum was collected to measure NADP+/NADPH, COX, PGE-2, and NO levels. The eyes were harvested for histology and A2E measurement. Ocular transcripts of Bcl-2, Bax, Cox-2, Inos, Tnf-α, Fas, FasL and Caspase-3 were detected by RQ-PCR. Quercetin treatment did not reverse the progression of retinal lesions in DKO mice funduscopically or histologically. Although quercetin treatment could recover systemic anti-oxidative capacity, suppress the systemic expression of NO, COX and PGE-2, and decrease ocular A2E levels, it could not effectively suppress the transcripts of the ocular inflammatory factors Tnf-α, Cox-2 and Inos, or the pro-apoptotic factors Fas, FasL and Caspase-3 in DKO mice. Our data demonstrate that quercetin can protect human RPE cells from oxidative stress in vitro via inhibition of pro-inflammatory molecules and direct inhibition of the intrinsic apoptosis pathway. However, quercetin (25mg/kg/day) does not improve the retinal AMD-like lesions in the Ccl2−/−/Cx3cr1−/− mice, likely due to its insufficient suppression of the inflammatory and apoptosis pathways in the eye.
PMCID: PMC2879439  PMID: 20361964
Quercetin; RPE; age-related macular degeneration; oxidative stress; inflammation; apoptosis; AMD mouse model

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