Statin effects on cholesterol micro-domains in brain plasma membranes

Recent epidemiological studies revealed inhibitors of the hydroxymethylglutaryl-coenzyme A reductase, so-called statins, to be effective in lowering the prevalence of Alzheimer's disease (AD). In vitro, statins strongly reduced the cellular amyloid beta-protein load by modulating the processing of the amyloid beta precursor protein. Both observations are probably linked to cellular cholesterol homeostasis in brain. So far, little is known about brain effects of statins. Recently, we could demonstrate that treatment of mice with the lipophilic compound lovastatin resulted in a discrete reduction of brain membrane cholesterol levels. To follow up these findings, we subsequently carried out a further in vivo study including lovastatin and simvastatin as lipophilic agents, as well as pravastatin as a hydrophilic compound, focussing on their efficiency to affect subcellular membrane cholesterol pools in synaptosomal plasma membranes of mice. In contrast to the hydrophilic pravastatin, the lipophilic lovastatin and simvastatin strongly reduced the levels of free cholesterol in SPM. Interestingly, lovastatin and pravastatin but not simvastatin significantly reduced cholesterol levels in the exofacial membrane leaflet. These changes were accompanied by modified membrane bulk fluidity. All three statins reduced the expression of the raft marker protein flotillin. Alterations in transbilayer cholesterol distribution have been suggested as the underlying mechanism that forces amyloidogenic processing of APP in AD. Thus, our data give some first insight in the mode of action of statins to reduce the prevalence of AD in clinical trials.     

Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 μM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 μM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 μM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway.     

Indole-3-carbinol induces apoptosis through p53 and activation of caspase-8 pathway in lung cancer A549 cells

Indole-3-carbinol (I3C) has anti-tumor effects in various cancer cell lines. However, the anti-tumor effect of I3C on human lung cancers has been rarely reported. We investigated the anti-tumor effects and its mechanism of I3C on human lung carcinoma A549 cell line. Treatment of the A549 cells with I3C significantly reduced cell proliferation, increased formations of fragmented DNA and apoptotic body, and induced cell cycle arrest at G0/G1 phase. I3C increased not only the protein levels of cyclin D1, phosphorylated p53, and p21 but also the expression of Fas mRNA. Cleavage of caspase-9, -8, -3 and PARP also was increased by I3C. Treatment with wortmannin significantly suppressed both I3C-induced Ser15 phosphorylation and accumulation of p53 protein. The inhibition of caspase-8 by z-IETD-FMK significantly decreased cleavage of procaspase-8,-3 and PARP in I3C-treated A549 cells. Taken together, these results demonstrate that I3C induces cell cycle arrest at G0/G1 through the activation of p-p53 at Ser 15 and induces caspase-8 mediated apoptosis via the Fas death receptor. This molecular mechanism for apoptotic effect of I3C on A549 lung carcinoma cells may be a first report and suggest that I3C may be a preventive and therapeutic agent against lung cancer.     

Hydroxymethylglutaryl-coenzyme A reductase inhibitors induce apoptosis in human cardiac myocytes in vitro

Recent findings have implicated hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors or statins, an established class of drugs for the treatment of hypercholesterolemia, in tissue remodeling in the heart. Statins induce apoptosis in different cell culture systems including rat neonatal cardiomyocytes. We investigated possible effects of different statins in vitro in human adult cardiac myocytes on the expression of proteins thought to be involved in the regulation of apoptosis such as Mcl-1, an inhibitor of apoptosis, Bax, an inducer of apoptosis, as well as on cytoplasmic histone-associated-DNA-fragments. Human adult cardiac myocytes (HACM) were treated with different statins at concentrations from 0.01 to 5 microM for up to 96 h. Whereas the lipophilic statin simvastatin at a concentration of 5 microM downregulated Mcl-1 mRNA by 49%, the hydrophilic pravastatin had no effect. Bax mRNA levels were not affected by neither of the statins. Simvastatin but not pravastatin reduced Mcl-1 protein expression whereas Bax protein was not detectable in HACM as determined by Western blotting. Simvastatin, atorvastatin and fluvastatin induced an up to seven-fold increase in histone-associated-DNA-fragments whereas pravastatin did not. Simvastatin up regulated histone-associated-DNA-fragments dose-dependently, and mevalonate and geranylgeranyl pyrophosphate reversed this effect to control levels. Our results show that lipophilic statins can induce a pro-apoptotic state in human adult cardiac myocytes in vitro. We speculate that, similar to findings in animal models, statins might be involved in the attenuation of cardiac hypertrophy and remodeling in humans by modulating the balance between cell survival and apoptosis.     

Cell-specific toxicity of fibrates in human embryonal rhabdomyosarcoma cells

The effects of a variety of fibrates on the cell viability were examined in human embryonal rhabdomyosarcoma cells (HRMSC). Five fibrates, including fenofibrate, clofibrate, gemfibrozil, bezafibrate and ciprofibrate, all concentration-dependently reduced the cell viability determined by the mitochondrial enzyme activity. The cell injury occurred time-dependently and was marked at 24-48 h. The toxic action of fibrates was specific to HRMSC, since bezafibrate did not induce any marked changes in the viability of human microvascular endothelial cells or arterial smooth muscle cells. Synergistic cell injury was observed after a combined treatment with bezafibrate and simvastatin, although simvastatin alone reduced the cell viability. The cell injury was characterized by a typical nuclear damage, as evidenced by Hoechst 33342 staining and deoxynucleotidyl transferase dUTP nick-end label-positive staining. Similar cell-specific injury was induced by 8(S)-hydroxyeicosatetraenoic acid, a potent peroxisome proliferator-activated receptor alpha (PPARalpha) agonist. Consistent with these data, a marked expression for PPARalpha mRNA was observed in HRMSC but not in the endothelial or smooth muscle cells. Therefore, it is suggested that fibrates cause a cell-specific injury in HRMSC via activation of PPARalpha. Moreover, our present cell injury model using HRMSC may be useful for elucidating the mechanisms of clinical rhabdomyolysis induced by lipid-lowering agents.     

Adenosine induces apoptosis in the human gastric cancer cells via an intrinsic pathway relevant to activation of AMP-activated protein kinase

Extracellular adenosine significantly reduced cell viability in a dose (0.1-20mM)- and treatment time (24-72h)-dependent manner in GT3-TKB cells, a human gastric cancer cell line. Nuclei of cells were reactive to Hoechst 33342, a marker of apoptosis, and an anti-single-stranded DNA. Adenosine-induced GT3-TKB cell death was significantly inhibited by dipyridamole, an inhibitor of adenosine transporter, and 5'-amino-5'-deoxyadenosine, an inhibitor of adenosine kinase, but the effect was not affected by theophylline, a broad inhibitor of adenosine receptors, 8-cyclopentyltheophylline, an inhibitor of A(1) adenosine receptors or 3,7-dimethyl-1-propargylxanthine, an inhibitor of A(2a) adenosine receptors. Adenosine had no effect on mitochondrial membrane potentials. The effect of adenosine on GT3-TKB cell death was not inhibited by a pancaspase inhibitor or inhibitors of caspase-1,-3,-4,-8, and -9. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of AMP-activated protein kinase (AMPK), significantly reduced GT3-TKB cell viability, but the AICAR action was not reinforced in the presence of adenosine. The results of the present study, thus, suggest that extracellular adenosine induces apoptosis in GT3-TKB cells by its uptake into cells and conversion to AMP followed by activation of AMPK, regardless of caspase activation linked to the mitochondria and the endoplasmic reticulum.     

Cytoprotective effects of CSTMP, a novel stilbene derivative, against H2O2-induced oxidative stress in human endothelial cells

A novel stilbene derivative, (E)-2-(2-chlorostyryl)-3,5,6-trimethylpyrazine (CSTMP), was designed and synthesized based on the pharmacophores of tetramethylpyrazine (TMP) and resveratrol (RES). In the present study, we investigated the protective effects of CSTMP on vascular endothelial cells under oxidative stress and elucidated its molecular mechanisms. The radical scavenging activity of CSTMP was assessed by the DPPH test. Human Umbilical Vein Endothelial Cells (HUVECs) were exposed to 150 μM hydrogen peroxide (H(2)O(2)) for 12 h, resulting in a decrease of cell viability assessed by the MTT assay and an increase of apoptotic cells assessed by the nuclear staining assay and flow cytometry. The activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and nitric oxide synthase (NOS) and the contents of malondialdehyde (MDA), reduced glutathione (GSH) and nitric oxide (NO) in cells were determined by commercial kits. The expression levels of pro-apoptotic factor caspase-3 and anti-apoptotic signal ERK1/2 were detected by western blot. The results showed that CSTMP had a moderate anti-oxidative effect against the DPPH test, which was less than RES. Co-incubation with CSTMP increased the cell viability, markedly reduced the LDH leakage from the cells and decreased the lipid peroxidation. These effects of CSTMP were accompanied by increasing activity of the endogenous antioxidant enzyme SOD, the level of GSH, the production of NO and cNOS activity. Moreover, CSTMP showed stronger effects on the inhibition of apoptosis, caspase-3 expression, and the activation of phosphorylated ERK1/2 compared to RES. Furthermore, CSTMP could inhibit the expression of phospho-JNK and phospho-p38 induced by H(2)O(2). These results suggest that CSTMP prevents H(2)O(2)-induced cell injury through anti-oxidation and anti-apoptosis via the MAPK and caspase-3 pathways.     

Differential response of MG132 cytotoxicity against small cell lung cancer cells to changes in cellular GSH contents

The effect of the depletion or oxidation of cellular GSH on cytotoxicity of MG132 was assessed. Viability loss and decrease in GSH contents in small cell lung cancer (SCLC) cells treated with MG132 was attenuated by caspase inhibitors (z-IETD.fmk, z-LEHD.fmk and z-DQMD.fmk). Thiol compounds (N-acetylcysteine and N-(2-mercaptopropionyl)glycine) and free radical scavengers reduced MG132-induced cell death. Antioxidants, including N-acetylcysteine, inhibited the MG132-induced nuclear damage, loss in mitochondrial transmembrane potential, cytosolic accumulation of cytochrome c and caspase-3 activation. Depletion of GSH due to buthionine sulfoxime did not affect the cell viability loss, ROS formation and GSH depletion due to MG132 in SCLC cells. A thiol oxidant monochloramine, p-chloromercuribenzoate and N-ethylmaleiamide also did not affect cytotoxicity of MG132. The results suggest that the toxicity of MG132 on SCLC cells is mediated by activation of caspase-8, -9 and -3. Removal of free radicals and recovery of GSH contents may attenuate MG132-induced apoptotic cell death. Nevertheless, depletion or oxidation of cellular GSH may not affect toxicity of MG132.     

Synthesis and antileukemic activity of new 3-(5-methylisoxazol-3-yl) and 3-(pyrimidin-2-yl)-2-styrylquinazolin-4(3H)-ones

3-(3-Methylisoxazol-5-yl) and 3-(pyrimidin-2-yl)-2-styrylquinazolin-4(3H)-ones 8a-l and 9a,c-e,h-l were synthesized by refluxing in acetic acid the corresponding 2-methylquinazolinones 6 and 8 with the opportune benzoic aldehyde for 12 h. The synthesized styrylquinazolinones 8a-l and 9a,c-e,h-l were tested in vitro for their antileukemic activity against L-1210 (murine leukemia), K-562 (human chronic myelogenous leukemia) and HL-60 (human leukemia) cell lines showing in some cases good activity.     

The immunosuppressant drug FK506 prevents Fas-induced apoptosis in human hepatocytes

FK506 is a potent immunosuppressive drug used for the prevention of graft rejection in organ transplantation. Experimental and clinical studies have shown correlations between apoptosis and graft rejection, and apoptosis also plays a role in cell death after ischemia-reperfusion injury in the rat liver. Fas-mediated apoptosis is very likely involved in allograft rejection and experimental evidence has shown a decrease of FasR expression in mouse hepatocytes produced by the drugs. On the basis of these findings we have investigated the protective effect of FK506 in comparison with cyclosporine A (CsA) on Fas-induced apoptosis, by analysing the activation of downstream effector caspases in human hepatocytes. Apoptosis was induced by treatment with agonistic antibodies against FasR, which resulted in a significant activation of caspase-3 after 12 h. Prevention of the downstream activation of the caspase cascade and apoptosis was observed when hepatocytes were pre-treated for 3 h with immunosuppressant drugs. A significant reduction (ca. 30-40%) of caspase-3 activation by 5 microM FK506 and CsA was observed. Along with less activation of caspase-3 a decrease of apoptotic DNA fragmentation was found. In addition, FK506 significantly reduced not only caspase-8 but also caspase-9 activation, to a similar extent as CsA, thus suggesting a protective effect at the mitochondrial level of this drug, as has already been reported for CsA. These effects of FK506 help to explain its strong anti-rejection properties and suggest promising benefits of pharmacological preconditioning on ischemia-reperfusion injury following liver transplantation.     

Lupeol protects against acetaminophen-induced oxidative stress and cell death in rat primary hepatocytes

Drug induced hepatotoxicity is a major problem where phytochemicals hold promise for its abrogation. This study was carried out to explore cytoprotective potential of lupeol, a triterpene, against acetaminophen (AAP)-induced toxicity in rat hepatocytes. AAP exposure significantly (p < 0.05) reduced cell viability, disturbed Bcl-2 family pro/anti-apoptotic protein balance, increased ROS production and altered redox homeostasis. It also induced mitochondria-mediated hepatocellular injury by significant mitochondrial depolarization, caspase-9/3 activation and subsequent DNA fragmentation. Our results suggest that lupeol pre-treatment effectively restored antioxidant enzyme levels, decreased lipid peroxidation, inhibited ROS generation and depolarization of mitochondria. Lupeol also attenuated mitochondria-mediated signaling pathway and DNA damage as evident from TUNEL assay and cell cycle studies leading to prevention of cytotoxicity. This study confirms the efficacy of lupeol, a food derived antioxidant, in abrogating ROS generation, maintaining redox balance and providing significant protection against mitochondria-mediated cell death during AAP-induced toxicity.     

Contributions of caspase-8 and -9 to liver injury from CYP2E1-produced metabolites of halogenated hydrocarbons

1.?Drug-induced liver injury is difficult to predict at the pre-clinical stage. This study aimed to clarify the roles of caspase-8 and -9 in CYP2E1 metabolite-induced liver injury in both rats and cell cultures in vitro treated with carbon tetrachloride (CCl₄), halothane or sevoflurane. The human hepatocarcinoma functional liver cell line was maintained in 3-dimensional culture alone or in co-culture with human acute monocytic leukemia cells.

2.?In vivo, laboratory indices of liver dysfunction and histology were normal after administration of sevoflurane. CCl₄ treatment increased blood AST/ALT levels, liver caspase-3 and -9 activities and liver malondialdehyde, accompanied by centrilobular hepatocyte necrosis. Halothane increased AST/ALT levels, caspase-3 and -8 activities (but not malondialdehyde) concomitant with widespread hepatotoxicity. In vitro, CCl₄ treatment increased caspase-9 activity and decreased both mitochondrial membrane potential (MMP) and cell viability. In co-culture, halothane increased caspase-8 activity and decreased MMP and cellular viability. There were no toxic responses in CYP2E1 knockdown in monoculture and co-culture.

3.?CYP2E1-inducing compounds play a pivotal role in halogenated hydrocarbon toxicity.

4.?Changes in hepatocyte caspase-8 and -9 activities could be novel biomarkers of metabolites causing DILI, and in pre-clinical development of new pharmaceuticals can predict nascent DILI in the clinical stage.     

Cytotoxic and xenoestrogenic effects via biotransformation of trans-anethole on isolated rat hepatocytes and cultured MCF-7 human breast cancer cells

The metabolism and action of trans-anethole (anethole) and the estrogen-like activity of the compound and its metabolites were studied in freshly isolated rat hepatocytes and cultured MCF-7 human breast cancer cells, respectively. The incubation of hepatocytes with anethole (0.25-2.0mM) caused a concentration- and time-dependent cell death accompanied by losses of cellular ATP and adenine nucleotide pools. Anethole at a weakly toxic level (0.5mM) was metabolized to 4-methoxycinnamic acid (4MCA), 4-hydroxy-1-propenylbenzene (4OHPB), and the monosulfate conjugate of 4OHPB; the levels of 4OHPB sulfate and 4MCA reached approximately 20 and 200 microM within 2 hr, respectively, whereas that of free unconjugated 4OHPB was less than approximately 0.5 microM. At a moderately toxic concentration (1.0mM), unconjugated 4OHPB reached approximately 10 microM, followed by abrupt loss of 3'-phosphoadenosine 5'-phosphosulphate (PAPS). Based on cell viability and adenine nucleotide levels, 4OHPB was more toxic than anethole and 4MCA. The addition of 2,6-dichloro-4-nitrophenol (50 microM), an inhibitor of sulfotransferase, enhanced the anethole-induced cytotoxicity associated with losses of ATP, PAPS, and 4OHPB sulfate, and symmetrically increased the unconjugated 4OHPB concentration. 4OHPB as well as diethylstilbestrol (DES) and bisphenol A (BPA), which are known xenoestrogenic compounds, competitively displaced 17beta-estradiol bound to the estrogen receptor alpha in a concentration-dependent manner; IC(50) values of these compounds were approximately 1 x 10(-5), 1 x 10(-8) and 5 x 10(-5)M, respectively. 4OHPB also caused a concentration (10(-8) to 10(-6)M)-dependent proliferation of MCF-7 cells, whereas neither anethole nor 4MCA (10(-9) to 10(-5)M) affected cell proliferation. However, at higher concentrations (>10(-4)M), 4OHPB rather than anethole and 4MCA was cytotoxic. These results suggest that the biotransformation of anethole induces a cytotoxic effect at higher concentrations in rat hepatocytes and an estrogenic effect at lower concentrations in MCF-7 cells based on the concentrations of the hydroxylated intermediate, 4OHPB.     

Synthetic 1,4-anthracenedione analogs induce cytochrome c release, caspase-9, -3, and -8 activities, poly(ADP-ribose) polymerase-1 cleavage and internucleosomal DNA fragmentation in HL-60 cells by a mechanism which involves caspase-2 activation but not Fas signaling

Synthetic analogs of 1,4-anthraquinone (AQ code number), a compound that mimics the antiproliferative effects of daunorubicin (daunomycin) in the nanomolar range in vitro but has the advantage of blocking nucleoside transport and retaining its efficacy in multidrug-resistant tumor cells, were tested for their ability to induce apoptosis in the HL-60 cell system. AQ10 and, especially, the new lead antiproliferative compounds AQ8 and AQ9 reduce the growth and integrity of wild-type, drug-sensitive, HL-60-S cells more effectively than AQ1, suggesting that various methyl group substituents at C6 may enhance the bioactivity of the parent compound. Internucleosomal DNA fragmentation, a late marker of apoptosis, is similarly induced in a biphasic manner by increasing concentrations of AQ8 and AQ9 at 24 hr. Poly(ADP-ribose) polymerase-1 (PARP-1) cleavage, an early event required for cells committed to apoptosis, is detected within 3-6 hr in HL-60-S cells treated with AQ9. In accord with the fact that the caspases 9 and 3 cascade is responsible for PARP-1 cleavage, the activities of initiator caspase-9 and effector caspase-3 are induced by AQ9 in the same time- and concentration-dependent manners and to the same maximal degrees in both the HL-60-S and multidrug-resistant HL-60-RV cell lines. Interestingly, a 1-hr pulse treatment is sufficient for AQ8 and AQ9 to maximally induce caspase-9 and -3 activities at 6 hr. The release of mitochondrial cytochrome c (Cyt c) is also detected within 3-6hr in HL-60-S cells treated with AQ9, a finding consistent with the fact that Cyt c is the apoptotic trigger that activates caspase-9. Moreover, AQ analogs induce Cyt c release, caspase-9 and -3 activities and PARP-1 cleavage in relation with their abilities to decrease tumor cell growth and integrity, AQ8 and AQ9 being consistently the most effective. Since apical caspases 2 and 8 may both act upstream of mitochondria to promote Cyt c release, it is significant to show that AQ9 maximally induces caspase-2 and -8 activities at 6 and 9 hr, respectively. During AQ8 treatment, the caspase-2 inhibitor benzyloxycarbonyl (z)-Val-Asp-Val-Ala-Asp (VDVAD)-fluoromethyl ketone (fmk) totally blocks caspase-9, -3, and -8 activations, whereas the caspase-8 inhibitor z-Ile-Glu-Thr-Asp-(IETD)-fmk does not prevent caspase-2, -9, and -3 activations, suggesting that AQ-induced caspase-2 activity is an upstream event critical for the activation of the downstream caspases 9 and 3 cascade, including the mitochondrial amplification loop through caspase-8. However, these caspase-2 and -8 inhibitors fail to alter AQ8-induced Cyt c release, suggesting that AQs might also target mitochondria independently from caspase activation. Furthermore, the antagonistic anti-Fas DX2 and ZB4 monoclonal antibodies (mAbs), which block the induction of Cyt c release and caspase-2, -8, and -9 activities by the agonistic anti-Fas CH11 mAb, and the neutralizing anti-Fas ligand (FasL) NOK-1 mAb all fail to inhibit AQ9-induced Cyt c release and caspase-2, -8, and -9 activities, suggesting that the FasL/Fas signaling pathway is not involved in the mechanism by which antiproliferative AQ analogs trigger apoptosis in HL-60 cells.     

Solamargine induces apoptosis and sensitizes breast cancer cells to cisplatin.

Solamargine (SM), a major steroidal alkaloid glycoside, was purified from Solanum incanum plant. SM exhibited the most cytotoxic effect comparing with that of cisplatin (cDDP), methotrexate (MTX), 5-fluorouracil (5-FU), epirubicin (EPI) and cyclophosphamide (CP) against human breast cancer cells. In this study, SM induces apoptosis of the breast cancer cells and the mechanism was characterized. SM up-regulated the expressions of external death receptors, such as tumor necrosis factor receptor I (TNFR-I), Fas receptor (Fas), TNFR-I-associated death domain (TRADD), and Fas-associated death domain (FADD). SM also enhanced the intrinsic ratio of Bax to Bcl-2 by up-regulating Bax and down-regulating Bcl-2 and Bcl-xL expressions. These effects resulted in the release of mitochondrial cytochrome c and activation of caspase-8, -9 and -3 in the cells, indicating that SM triggered extrinsic and intrinsic apoptotic pathways of breast cancer cells. Similar to function way of SM, cDDP causes cancer cell apoptosis though caspase-8/caspase-3 and Bax/cytochrome c pathways, but the resistance to cDDP is correlated with Bcl-2 and Bcl-xL overexpression. However, the overexpression of Bcl-2 and Bcl-xL can be broken through by SM. The combined treatment of SM and cDDP significantly reduced Bcl-2 and Bcl-xL expressions, and enhanced Bax, cytochrome c, caspase-9 and -3 expressions in breast cancer cells. Thus, the combined use of SM and cDDP may be effective in cDDP-resistant breast cancer.     

Implication of caspases and subcellular compartments in tert-butylhydroperoxide induced apoptosis

Oxidative stress has been implicated in many physiopathologies including neurodegenerative diseases, cancer, cardiovascular and respiratory diseases, and in mechanisms of action of environmental toxicants. tert-butylhydroperoxide (t-BHP) is an organic lipid hydroperoxide analogue, which is commonly used as a pro-oxidant for evaluating mechanisms involving oxidative stress in cells and tissues. This study investigates mechanisms of apoptosis induced by oxidative stress in hepatocytes, in particular, the involvement of caspases and subcellular compartments. Freshly isolated hepatocytes were exposed to 0.4 mM t-BHP during 1 h. A general caspase inhibitor, Boc-D-FMK, reduced t-BHP-induced apoptosis (chromatin condensation), confirming the involvement of caspases in apoptosis. A caspase-9 inhibitor, Z-LEHD-FMK, also reduced t-BHP-induced apoptosis, suggesting that caspase-9 plays a critical role in this process. Procaspase-9 underwent cleavage in mitochondria and translocation to the nucleus, where increased caspase-9 activity was detected. The caspase-9 substrates, caspase-3 and caspase-7, were not activated. Caspase-7 was translocated from the cytosol to the endoplasmic reticulum (ER), where it underwent processing; however, enzymatic activity of caspase-7 was inhibited by t-BHP. t-BHP caused cleavage of procaspase-12 at the ER and its subsequent translocation to the nucleus, where increased caspase-12 activity was found. t-BHP caused translocation of calpain from the cytosol to the ER. Calpain inhibition reduced chromatin condensation and caspase-12 activity in the nucleus, suggesting that calpain is involved in caspase-12 activation and apoptosis. This study demonstrates that caspase-9 and caspase-12 are activated in t-BHP-induced apoptosis in hepatocytes. We highlight the importance of subcellular compartments such as mitochondria, ER and nuclei in the apoptotic process.     

Comparison of the efficacies of five different statins on inhibition of human saphenous vein smooth muscle cell proliferation and invasion

Statins (HMG-CoA reductase inhibitors) exhibit beneficial effects on the vasculature independently of their cholesterol-lowering properties. These pleiotropic effects underlie the ability of statins to reduce intimal hyperplasia in saphenous vein (SV) bypass grafts by attenuating smooth muscle cell (SMC) invasion and proliferation. Although all statins can effectively lower cholesterol, the pleiotropic effects of individual statins may well differ. We therefore compared the concentration-dependent effects of 4 lipophilic statins (simvastatin, atorvastatin, fluvastatin, and lovastatin) and 1 hydrophilic statin (pravastatin) on the proliferation and invasion of SMC cultured from SV of 9 different patients undergoing coronary artery bypass grafting (CABG). The lipophilic statins inhibited SV-SMC proliferation over a 4-day period with an order of potency of fluvastatin > atorvastatin > simvastatin > lovastatin (IC50 range = 0.07 to 1.77 microM). Similarly, these statins also inhibited SV-SMC invasion through an artificial basement membrane barrier (fluvastatin > atorvastatin > simvastatin > lovastatin; IC50 range = 0.92 to 26.9 microM). In contrast, the hydrophilic pravastatin had no significant effect on SV-SMC proliferation at concentrations up to 10 microM, nor did it attenuate SV-SMC invasion (up to 30 microM). Our data provide strong evidence that individual statins possess differential pleiotropic effects on SV-SMC function. This may be of clinical relevance in the selection of individual statins for the treatment of CABG patients.     

Inhibition of cyclooxygenase-2 and induction of apoptosis in estrogen-nonresponsive breast cancer cells by Antrodia camphorata.

The objective of this study was to investigate the fermented culture broth of Antrodia camphorata (A. camphorata) to induce apoptosis and inhibit cyclooxygenase-2 (COX-2) in estrogen-nonresponsive (MDA-MB-231) human breast cancer cells. Treatment of the highly invasive MDA-MB-231 cells with A. camphorata (40-240 microg/ml) resulted in dose and time-dependent sequences of events marked by apoptosis, as evidenced by loss of cell viability, chromatin condensation, and internucleosomal DNA fragmentation. Apoptosis in the MDA-MB-231 cells was accompanied by release of cytochrome c, activation of caspase-3, -8, and -9, and specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). Although the A. camphorata-induced apoptosis was associated with a reduction in Bcl-2 protein levels, negligible Bax increase was observed. Furthermore, A. camphorata treatment inhibited COX-2 protein expression and prostaglandin E2 (PGE2) production in MDA-MB-231 cells. Analysis of the study data suggests that A. camphorata exerts growth inhibition on (highly invasive) estrogen-nonresponsive human breast cancer cells through apoptosis induction associated with COX-2 inhibition, and that it may possess anticancer properties potentially valuable for application in drug products.     

Zinc attenuates ethanol-induced Sertoli cell toxicity and apoptosis through caspase-3 mediated pathways

Ethanol enhances apoptosis in testicular germ cells. Zinc reduces ethanol-induced apoptosis of somatic cells through inhibition of caspase-mediated pathways. Little is known about the effects of ethanol on Sertoli cells and the effects of Zinc on ethanol-induced testicular injury. The hypothesis tested was that ethanol enhances apoptosis of Sertoli cells through up-regulation of caspase-3 and Zinc inhibits ethanol-induced effects. Cultured Sertoli cells (TM4) were exposed to ethanol (160 mM), Zinc (8 μM) and Zinc prior to ethanol for duration of 24 or 48 h and their effects on TM4 cell viability was then investigated by MTT assay. Caspase-3 mRNA expression was also investigated using real-time RT-PCR. Cell viability decreased and caspase-3 mRNA expresstion increased in cells exposed to ethanol, while exposure to Zinc showed opposite effects. Pretreatment with Zinc recovered ethanol-induced anti-proliferative effects and over-expression of caspase-3. Zinc reduced ethanol-induced Sertoli cell toxicity and apoptosis via caspase-3 mediated pathways.