Eon-Gi Sung

Berberine sensitizes TRAIL-induced apoptosis through proteasome-mediated downregulation of c-FLIP and Mcl-1 proteins.

Berberine (BBR) is an isoquinoline alkaloid which has a wide spectrum of clinical applications including anti-tumor, anti-microbial and anti-inflammatory activities. In this study, we showed that co-treatment with subtoxic doses of BBR and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis in human renal cancer cells, Caki cells, but not in normal tubular kidney cells. Treatment of Caki cells with BBR resulted in downregulation of c-FLIP and Mcl-1 proteins in a dose-dependent manner. The BBR-induced downregulation of c-FLIP and Mcl-1 proteins were involved in proteasome dependent pathways, which was confirmed by the result that pre-treatment with the proteasome inhibitor MG132 inhibited berberine-induced downregulation of the c-FLIP and Mcl-1 proteins. Pretreatment with N-acetyl-L-cysteine (NAC) significantly inhibited the cell death induced by the combined treatment with BBR and TRAIL as well as recovered the expression levels of c-FLIP and Mcl-1 downregulated by treatment with BBR. These results suggested that BBR-stimulated TRAIL-induced apoptosis is dependent on the generation of reactive oxygen species through the downregulation of c-FLIP and Mcl-1 proteins. In conclusion, this study demonstrates that BBR enhances TRAIL-induced apoptosis in human renal cancer cells by ROS-mediated c-FLIP and Mcl-1 down-regulation.

Evidence of neuronal degeneration in C57Bl/6 mice infected with the LP-BM5 leukemia retrovirus mixture

Mice infected with LP-BM5 develop a severe immunodeficiency accompanied by learning and memory deficits, gliosis, and neurotransmitter abnormalities. The neurochemical alterations are consistent with elevated excitotoxin levels, suggesting that infected mice may incur neuronal damage. Although the number of neocortical neurons was unchanged in mice 12 wk after LP-BM5 infection, the expression of cytoskeletal proteins declined, particularly in the frontal and parietal cortex as indicated by MAP2, NF-200, and synaptophysin immunoreactivity. In contrast, the number of striatal neurons decreased 19%. The remaining neurons were smaller, with fewer synaptic boutons, and showed decreased synaptophysin and NF-200, immunoreactivity. Immunoblots of cortex and striatum confirmed decreases in MAP2, NF-200 and synaptophysin expression. Finally, although NCAM expression decreased in the striatum, it increased in the cortex. These results indicate that LP-BM5-infected mice sustain significant neuronal damage, which may contribute to their behavioral deficits. Moreover, the increase in cortical NCAM expression suggests active synaptic remodeling to compensate for the persistent excitotoxic environment in these mice, whereas striatal neurons degenerate. These concurrent degenerative and compensatory processes may also occur in the brains of patients with AIDS dementia complex (ADC), who suffer extensive degeneration of the basal ganglia and cerebral cortex.

Dioscin induces caspase-independent apoptosis through activation of apoptosis-inducing factor in breast cancer cells

Dioscin, a saponin extracted from the roots of Polygonatum zanlanscianense, shows several bioactivities such as antitumor, antifungal, and antiviral properties. Although, dioscin is already known to induce cell death in variety cancer cells, the molecular basis for dioscin-induced cell death was not definitely known in cancer cells. In this study, we found that dioscin treatment induced cell death in dose-dependent manner in breast cancer cells such as MDA-MB-231, MDA-MB-453, and T47D cells. Dioscin decreased expressions of Bcl-2 and cIAP-1 proteins, which were down-regulated at the transcriptional level. Conversely, Mcl-1 protein level was down-regulated by facilitating ubiquitin/proteasome-mediated Mcl-1 degradation in dioscin-treated cells. Pretreatment with z-VAD fails to attenuate dioscin-induced cell death as well as caspase-mediated events such as cleavages of procaspase-3 and PARP. In addition, dioscin treatment increased the population of annexin V positive cells and induced DNA fragmentation in a dose-dependent manner in MDA-MB-231 cells. Furthermore, apoptosis inducing factor (AIF) was released from the mitochondria and translocated to the nucleus. Suppression in AIF expression by siRNA reduced dioscin-induced apoptosis in MDA-MB-231 cells. Taken together, our results demonstrate that dioscin-induced cell death was mediated via AIF-facilitating caspase-independent pathway as well as down-regulating anti-apoptotic proteins such as Bcl-2, cIAP-1, and Mcl-1 in breast cancer cells.

Inhibition of c-FLIPL expression by miRNA-708 increases the sensitivity of renal cancer cells to anti-cancer drugs

Dysregulation of the anti-apoptotic protein, cellular FLICE-like inhibitory protein (c-FLIP), has been associated with tumorigenesis and chemoresistance in various human cancers. Therefore, c-FLIP is an excellent target for therapeutic intervention. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumorigenesis, tumor suppression, and resistance or sensitivity to anti-cancer drugs. However, whether miRNAs can suppress c-FLIPL expression in cancer cells is unclear. The aim of this study was to identify miRNAs that could inhibit the growth of renal cancer cells and induce cell death by inhibiting c-FLIPL expression. We found that MiRNA-708 and c-FLIPL expression were inversely correlated. While c-FLIPL expression was upregulated, miRNA-708 was rarely expressed in renal cancer cells. Luciferase reporter assays demonstrated that miRNA-708 negatively regulated c-FLIPL expression by binding to the miRNA-708 binding site in the 3′ untranslated region (3′UTR) of c-FLIPL. Ectopic expression of miRNA-708 increased the accumulation of sub-G1 populations and cleavage of procaspase-3 and PARP, which could be prevented by pretreatment with the pan-caspase inhibitor, Z-VAD. Ectopic expression of miRNA-708 also increased the sensitivity to various apoptotic stimuli such as tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin (Dox), and thapsigargin in Caki cells. Interestingly, miRNA-708 specifically repressed c-FLIPL without any change in c-FLIPs expression. In contrast, inhibition of endogenous miRNA-708 using antago-miRNAs resulted in an increase in c-FLIPL protein expression. The expression of c-FLIPL was upregulated in renal cell carcinoma (RCC) tissues compared to normal tissues. In contrast, miRNA-708 expression was reduced in RCC tissues. Finally, miRNA-708 enhanced the tumor-suppressive effect of Dox in a xenograft model of human RCC. In conclusion, miRNA-708 acts as a tumor suppressor because it negatively regulates the anti-apoptotic protein c-FLIPL and regulates the sensitivity of renal cancer cells to various apoptotic stimuli.

Gambogic acid induces apoptosis and sensitizes TRAIL-mediated apoptosis through downregulation of cFLIPL in renal carcinoma Caki cells

Gambogic acid (GA) is a natural compound derived from brownish gamboge resin that shows a range of bioactivity, such as antitumor and antimicrobial properties. Although, GA is already known to induce cell death in a variety of cancer cells, the molecular basis for GA-induced cell death in renal cancer cells is unclear. In this study, a treatment with GA induced cell death in human renal carcinoma Caki cells in a dose-dependent manner. Treatment of Caki cells with GA decreased the levels of antiapoptotic proteins, such as Bcl-2 and XIAP in a dose-dependent manner. In addition, GA decreased the expression of the cFLIPL protein, which was downregulated at the transcriptional level without any change in the levels of cFLIPs expression. z-VAD (pan-caspase inhibitor) partially blocked GA-mediated cell death. GA-induced apoptotic cell death in Caki cells is mediated partly by the AIF translocation from the mitochondria into the nucleus via a caspase-independent pathway. In contrast, N-acetylcysteine (NAC), a ROS scavenger, had no effect on GA-induced cell death. The restoration of cFLIPL attenuated GA-induced cell death in Caki cells. Furthermore, a sub-toxic dose of GA sensitized TRAIL-mediated apoptosis in Caki cells. Pretreatment with z-VAD completely blocked GA plus TRAIL-mediated apoptosis. On the contrary, pretreatment with NAC partially inhibited GA plus TRAIL-induced apoptosis. Our findings suggested that GA induces apoptosis via the downregulation of cFLIPL and sensitized TRAIL-mediated apoptosis in Caki cells.

Propofol attenuates Kupffer cell activation during hypoxia-reoxygenation.

Objectif Determiner si le propofol diminue ľactivation des cellules de Kupffer (CK), protegeant ainsi les cellules contre une lesion ďhypoxie-reoxygenation par la modulation du calcium intracellulaire ([Ca2+]i).PurposeWe undertook a study to determine whether propofol may attenuate Kupffer cell (KC) activation, thus protecting the cells against hypoxia-reoxygenation injury through the modulation of intracellular calcium ([Ca2+]i).Methods[Ca2+]i, the expression of tumour necrosis factor (TNF)-α mRNA, and KC viability were measured in response to hypoxia-reoxygenation following pretreatment with propofol 0.5 and 5 μg·mL-1 (Groups P1 and P2, respectively) or without propofol (Group HRC). KCs were isolated and cultured from male Sprague-Dawley rats. KCs were incubated under an atmosphere of hypoxia (95% N2 + 5% CO2) for 60 min with subsequent 120 min reoxygenation (95% air + 5% CO2). [Ca2+]i for the first 12 min after reoxygenation, TNF-α mRNA, and KC viability at the end of reoxygenation in groups P1 and P2 were compared with those of HRC.ResultsThe increase of [Ca2+]i from the baseline was attenuated in P1 (96.6 ± 6.9%) and P2 (96.1 ± 5.4%) compared with HRC (143.8 ± 11.5%), (P < 0.001), with no difference between P1 and P2. The expression of TNF-α mRNA increased only in HRC during hypoxia-reoxygenation. KC viability increased in P1 (97.5 ± 2.6%) and P2 (94.6 ± 2.9%), compared with HRC (89.9 ± 1.4%), (P < 0.005), with no difference between P1 and P2.ConclusionThe results indicate that propofol attenuates KC activation and protects KC from hypoxia-reoxygenation injury at clinically relevant concentrations. This attenuation seems to result from inhibition of [Ca2+]i increase in KC.RésuméObjectifDéterminer si le propofol diminue ľactivation des cellules de Kupffer (CK), protégeant ainsi les cellules contre une lésion ďhypoxie-réoxygénation par la modulation du calcium intracellulaire ([Ca2+]i).MéthodeLe [Ca2+]i, expression du facteur de nécrose tumorale (FNT)-α ARNm, et la viabilité des CK ont été mesurés en réponse à ľhypoxie-réoxygénation suivant le prétraitement avec 0,5 et 5 μg·mL-1 de propofol (Groupes P1 et P2, respectivement) ou sans propofol (Groupe HRC). Des CK de rats Sprague-Dawley ont été isolées et mises en culture. Elles ont été incubées sous une atmosphère ďhypoxie (95 % N2 + 5 % de CO2) pendant 60 min et 120 min supplémentaires de réoxygénation (95 % ďair + 5 % CO2). Le [Ca2+]i pendant les 12 premières minutes après la réoxygénation, le FNT-α ARNm et la viabilité des CK à la fin de la réoxygénation dans les groupes P1 et P2 ont été comparés avec ceux du groupe HRC.RésultatsĽaugmentation du [Ca2+]i, au-dessus des mesures de base, a été réduite dans les groupes P1 (96,6 ± 6,9 %) et P2 (96,1 ± 5,4 %) comparés au groupe HRC (143,8 ± 11,5 %), (P < 0,001), sans différence entre P1 et P2. ľexpression du FNT-α ARNm a augmenté seulement dans le groupe HRC pendant ľhypoxie-réoxygénation. La viabilité des CK a augmenté dans les groupes P1 (97,5 ± 2,6 %) et P2 (94,6 ± 2,9 %), comparés au groupe HRC (89,9 ± 1,4 %), (P < 0,005) sans différence entre P1 et P2.ConclusionLe propofol diminue ľactivation des CK et protège ďune lésion liée à ľhypoxie-réoxygénation des CK selon des concentrations cliniquement significatives. Cette baisse semble résulter de ľinhibition de la hausse de [Ca2+]i dans les CK.

Methylglyoxal‐induced apoptosis is dependent on the suppression of c‐FLIPL expression via down‐regulation of p65 in endothelial cells

Methylglyoxal (MGO) is a reactive dicarbonyl metabolite of glucose, and its plasma levels are elevated in patients with diabetes. Studies have shown that MGO combines with the amino and sulphhydryl groups of proteins to form stable advanced glycation end products (AGEs), which are associated with vascular endothelial cell (EC) injury and may contribute to the progression of atherosclerosis. In this study, MGO induced apoptosis in a dose‐dependent manner in HUVECs, which was attenuated by pre‐treatment with z‐VAD, a pan caspase inhibitor. Treatment with MGO increased ROS levels, followed by dose‐dependent down‐regulation of c‐FLIPL. In addition, pre‐treatment with the ROS scavenger NAC prevented the MGO‐induced down‐regulation of p65 and c‐FLIPL, and the forced expression of c‐FLIPL attenuated MGO‐mediated apoptosis. Furthermore, MGO‐induced apoptotic cell death in endothelium isolated from mouse aortas. Finally, MGO was found to induce apoptosis by down‐regulating p65 expression at both the transcriptional and posttranslational levels, and thus, to inhibit c‐FLIPL mRNA expression by suppressing NF‐κB transcriptional activity. Collectively, this study showed that MGO‐induced apoptosis is dependent on c‐FLIPL down‐regulation via ROS‐mediated down‐regulation of p65 expression in endothelial cells.

Le propofol diminue ľactivation des cellules de Kupffer pendant ľhypoxie-réoxygénation

Objectif Determiner si le propofol diminue ľactivation des cellules de Kupffer (CK), protegeant ainsi les cellules contre une lesion ďhypoxie-reoxygenation par la modulation du calcium intracellulaire ([Ca2+]i).PurposeWe undertook a study to determine whether propofol may attenuate Kupffer cell (KC) activation, thus protecting the cells against hypoxia-reoxygenation injury through the modulation of intracellular calcium ([Ca2+]i).Methods[Ca2+]i, the expression of tumour necrosis factor (TNF)-α mRNA, and KC viability were measured in response to hypoxia-reoxygenation following pretreatment with propofol 0.5 and 5 μg·mL-1 (Groups P1 and P2, respectively) or without propofol (Group HRC). KCs were isolated and cultured from male Sprague-Dawley rats. KCs were incubated under an atmosphere of hypoxia (95% N2 + 5% CO2) for 60 min with subsequent 120 min reoxygenation (95% air + 5% CO2). [Ca2+]i for the first 12 min after reoxygenation, TNF-α mRNA, and KC viability at the end of reoxygenation in groups P1 and P2 were compared with those of HRC.ResultsThe increase of [Ca2+]i from the baseline was attenuated in P1 (96.6 ± 6.9%) and P2 (96.1 ± 5.4%) compared with HRC (143.8 ± 11.5%), (P < 0.001), with no difference between P1 and P2. The expression of TNF-α mRNA increased only in HRC during hypoxia-reoxygenation. KC viability increased in P1 (97.5 ± 2.6%) and P2 (94.6 ± 2.9%), compared with HRC (89.9 ± 1.4%), (P < 0.005), with no difference between P1 and P2.ConclusionThe results indicate that propofol attenuates KC activation and protects KC from hypoxia-reoxygenation injury at clinically relevant concentrations. This attenuation seems to result from inhibition of [Ca2+]i increase in KC.RésuméObjectifDéterminer si le propofol diminue ľactivation des cellules de Kupffer (CK), protégeant ainsi les cellules contre une lésion ďhypoxie-réoxygénation par la modulation du calcium intracellulaire ([Ca2+]i).MéthodeLe [Ca2+]i, expression du facteur de nécrose tumorale (FNT)-α ARNm, et la viabilité des CK ont été mesurés en réponse à ľhypoxie-réoxygénation suivant le prétraitement avec 0,5 et 5 μg·mL-1 de propofol (Groupes P1 et P2, respectivement) ou sans propofol (Groupe HRC). Des CK de rats Sprague-Dawley ont été isolées et mises en culture. Elles ont été incubées sous une atmosphère ďhypoxie (95 % N2 + 5 % de CO2) pendant 60 min et 120 min supplémentaires de réoxygénation (95 % ďair + 5 % CO2). Le [Ca2+]i pendant les 12 premières minutes après la réoxygénation, le FNT-α ARNm et la viabilité des CK à la fin de la réoxygénation dans les groupes P1 et P2 ont été comparés avec ceux du groupe HRC.RésultatsĽaugmentation du [Ca2+]i, au-dessus des mesures de base, a été réduite dans les groupes P1 (96,6 ± 6,9 %) et P2 (96,1 ± 5,4 %) comparés au groupe HRC (143,8 ± 11,5 %), (P < 0,001), sans différence entre P1 et P2. ľexpression du FNT-α ARNm a augmenté seulement dans le groupe HRC pendant ľhypoxie-réoxygénation. La viabilité des CK a augmenté dans les groupes P1 (97,5 ± 2,6 %) et P2 (94,6 ± 2,9 %), comparés au groupe HRC (89,9 ± 1,4 %), (P < 0,005) sans différence entre P1 et P2.ConclusionLe propofol diminue ľactivation des CK et protège ďune lésion liée à ľhypoxie-réoxygénation des CK selon des concentrations cliniquement significatives. Cette baisse semble résulter de ľinhibition de la hausse de [Ca2+]i dans les CK.

Mesenchymal stem cells attenuate adriamycin-induced nephropathy by diminishing oxidative stress and inflammation via downregulation of the NF-kB

This study aimed to evaluate the molecular mechanism mitigating progress of chronic nephropathy by mesenchymal stem cells (MSCs).