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Recent studies have shown mitochondrial fragmentation during cell stress and have suggested a role for the morphological change in mitochondrial injury and ensuing apoptosis. However, the underlying mechanism remains elusive. Here we demonstrate that mitochondrial fragmentation facilitates Bax insertion and activation in mitochondria, resulting in the release of apoptogenic factors. In HeLa cells, overexpression of mitofusins attenuated mitochondrial fragmentation during cisplatin- and azide-induced cell injury, which was accompanied by less apoptosis and less cytochrome c release from mitochondria. Similar effects were shown by inhibiting the mitochondrial fission protein Drp1 with a dominant negative mutant (dn-Drp1). Mitofusins and dn-Drp1 did not seem to significantly affect Bax translocation/accumulation to mitochondria; however, they blocked Bax insertion and activation in mitochondrial membrane. Consistently, in rat kidney proximal tubular cells, small interfering RNA knockdown of Drp1 prevented mitochondrial fragmentation during azide-induced ATP depletion, which was accompanied by less Bax activation, insertion, and oligomerization in mitochondria. These cells released less cytochrome c and AIF from mitochondria and showed significantly lower apoptosis. Finally, mitofusin-null mouse embryonic fibroblasts (MEF) had fragmented mitochondria. These MEFs were more sensitive to cisplatin-induced Bax activation, release of cytochrome c, and apoptosis. Together, this study provides further support for a role of mitochondrial fragmentation in mitochondrial injury and apoptosis. Mechanistically, mitochondrial fragmentation may sensitize the cells to Bax insertion and activation in mitochondria, facilitating the release of apoptogenic factors and consequent apoptosis.
Proapoptotic Bax and Bak are the key B-cell lymphoma-2 family members mediating apoptosis through the intrinsic pathway. Cells doubly deficient for Bax and Bak are profoundly resistant to apoptotic stimuli originating from multiple stimuli. Here we describe mice in which Bax and Bak have been deleted specifically in T-cells using Lck-Cre. In these T cell-specific BaxBak-deficient mice, early T-cell progenitors accumulate in the thymus, with relative depletion of more mature T cells. In addition, bone marrow progenitor cells fail to progress to the double positive stage when cultured on OP9 stromal cells expressing the Notch ligand Delta-like 1, consistent with a critical role for Bax and Bak in early T-cell development. Over time, T cell-specific BaxBak-deficient mice progress to an aggressive T-cell lymphoblastic leukemia/lymphoma. Interestingly, quantitative real-time polymerase chain reaction analysis of BaxBak-deficient T-cell lymphomas does not display amplification of the Notch signal transduction pathway, commonly activated in T-cell leukemia in both mouse and man. Bax and Bak, key regulators of the intrinsic pathway of apoptosis, are thus required to prevent T-cell malignancy, and for normal T-cell differentiation, regulating early T-cell development at the stage of early T-lineage progenitor cells.
We examined whether betulin, a naturally abundant compound, has anticancer functions in human cancer cells. The results showed that betulin significantly inhibited cell viability in cervix carcinoma HeLa cells, hepatoma HepG2 cells, lung adenocarcinoma A549 cells, and breast cancer MCF-7 cells with IC(50) values ranging from 10 to 15 microg/mL. While betulin exhibited only moderate anticancer activity in other human cancer cells such as hepatoma SK-HEP-1 cells, prostate carcinoma PC-3, and lung carcinoma NCI-H460, with IC(50) values ranging from 20 to 60 microg/mL, it showed minor growth inhibition in human erythroleukemia K562 cells (IC(50) > 100 microg/mL). We further investigated the mechanism of anticancer activity by betulin, using HeLa cells as an experimental model. Betulin (10 microg/mL) induces apoptotic cell death, as evidenced by morphological characteristics such as membrane phosphatidylserine translocation, nuclear condensation/fragmentation, and apoptotic body formation. A kinetics analysis showed that the depolarization of mitochondrial membrane potential and the release of mitochondrial cytochrome c occurred as early as 30 min after treatment with betulin. Betulin, unlike its chemical derivative betulinic acid, did not directly trigger mitochondrial cytochrome c release in isolated mitochondria. Importantly, Bax and Bak were rapidly translocated to the mitochondria 30 min after betulin treatment. The sequential activation of caspase-9 and caspase-3/-7 and the cleavage of poly(ADP-ribose) polymerase (PARP) were observed behind those mitochondrial events. Furthermore, specific downregulation of either caspase-9, Bax, or Bak by siRNA effectively reduced PARP cleavage and caspase-3 activation. Taken together, the lines of evidence demonstrate that betulin triggers apoptosis of human cancer cells through the intrinsic apoptotic pathway.
PURPOSE - Development of new treatments is critical to effective protection against radiation-induced injury. We investigate the potential of developing small-molecule inhibitors of glycogen synthase kinase 3beta (GSK-3beta)-SB216763 or SB415286-as radioprotective agents to attenuate intestinal injury.
METHODS AND MATERIALS - A survival study was done by use of C57BL/6J mice to evaluate the radioprotective effect of GSK-3beta inhibitors. Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and immunohistochemical staining for Bax and Bcl-2 were used to assess apoptosis in the small intestines of the treated mice. A clonogenic survival study, apoptosis assays (staining with annexin V or 4',6-diamidino-2-phenylindole), and immunoblot analysis of beta-catenin, Bcl-2, Bax, and caspase 3 were done by use of Rat intestinal epithelial cell line IEC-6 cells.
RESULTS - Pretreatment with SB415286 significantly improved survival of mice irradiated with 8 and 12 Gy. Mice pretreated with SB216763 or SB415286 showed a significant reduction in TUNEL- and Bax-positive cells and an increase in Bcl-2-positive cells in intestinal crypts at 4 and/or 12 h after radiation with 4 and/or 8 Gy compared with radiation alone. Pretreatment of irradiated IEC-6 cells with GSK-3beta inhibitors significantly increased clonogenic survival compared with cells treated with radiation alone. This increase was due to the attenuation of radiation-induced apoptosis, as shown by annexin V and 4',6-diamidino-2-phenylindole assays, as well as immunoblot analysis of Bcl-2, Bax, and caspase 3.
CONCLUSIONS - Glycogen synthase kinase 3beta small-molecule inhibitors protect mouse intestine from radiation-induced damage in cell culture and in vivo and improve survival of mice. Molecular mechanisms of this protection involve attenuated radiation-induced apoptosis regulated by Bcl-2, Bax, and caspase 3. Therefore GSK-3beta inhibitors reduce deleterious consequences of intestinal irradiation and thereby improve quality of life during radiation therapy.
Copyright 2010 Elsevier Inc. All rights reserved.
Bax is a pro-apoptotic protein that mediates intrinsic cell-death signaling. Using a yeast-based functional screening approach, we identified interferon gamma receptor beta chain (IFNgammaR2) as a new Bax suppressor. IFNgammaR2 is a component of the IFNgamma receptor complex along with the IFNgammaR alpha chain (IFNgammaR1). Upon IFNgamma binding, a conformational change in the receptor complex occurs that activates the Jak2/STAT1 signaling cascade. We found that the C-terminal region (amino acids 296-337) of IFNgammaR2 (IFNgammaR2(296-337)) contains a novel Bax inhibitory domain. This portion does not contain the Jak2-binding domain; therefore, the antiapoptotic function of IFNgammaR2 is independent of JAK/STAT signaling. IFNgammaR2(296-337) rescued human cells from apoptosis induced by overexpression of Bax but not Bak. Overexpression of IFNgammaR2 (wild type and IFNgammaR2(296-337)) rescued cells from etoposide and staurosporine, which are known to induce Bax-mediated cell death. Interestingly, IFNgammaR2 inhibited apoptosis induced by the BH3-only protein Bim-EL, suggesting that IFNgammaR2 inhibits Bax activation through a BH3-only protein. Bax and IFNgammaR2 were co-immunoprecipitated from cell lysates prepared from HEK293 and DAMI cells. Furthermore, direct binding of purified recombinant proteins of Bax and IFNgammaR2 was also confirmed. Addition of recombinant Bcl-2 protein to cell lysates significantly reduced the interaction of IFNgammaR2 and Bax, suggesting that Bcl-2 and IFNgammaR2 bind a similar domain of Bax. We found that the C-terminal fragment (cytoplasmic domain) of IFNgammaR2 is expressed in human cancer cell lines of megakaryocytic cancer (DAMI), breast cancer (MDA-MD-468), and prostate cancer (PC3 cells). The presence of the C-terminal fragment of IFNgammaR2 may confer on cancer cells resistance to apoptotic stresses. Our discovery of the anti-Bax activity of the cytoplasmic domain of IFNgammaR2 may shed new light on the mechanism of how cell death is controlled by IFNgamma and Bax.
Diallyl disulfide (DADS), an important component of garlic (Allium sativum) derivative, has been demonstrated to exert a potential molecular target against human cancers. We investigated DADS-induced expressions of Apaf1, cystatin B, caspase-3 and FADD (fas-associated protein with death domain) in breast, prostate and lung cancer cells. These showed coincident data when further examined by quantitative reverse transcription-polymerase chain reaction and Western blot analysis. Furthermore, DADS induced a marked amount of Bax translocation, cytochrome c release and activation of caspase-3 and caspase-9. DADS-treated tumor cells triggered mitochondria-mediated signaling pathways that led to a significant increase in apoptosis induction. Further studies with caspase-3 and caspase-9 inhibitors (zDEVD-fmk and zLEHD-fmk, respectively) proved that DADS induces apoptosis through a caspase-3-dependent pathway. DADS is only an agent used in the study. The molecular mechanism presented therefore provides strong additional support to the hypothesis that DADS is a strong inducer of apoptosis through a Bax-triggered mitochondria-mediated and caspase-3-dependent pathway. This study shows clearly that DADS causes caspase-dependent apoptosis in human cancer cells through a Bax-triggered mitochondrial pathway. Therefore, the mitochondrial pathway might be the target for cancer chemoprevention and/or chemotherapy by DADS.
Published by Elsevier Inc.
BACKGROUND & AIMS - Helicobacter pylori infection disrupts the balance between gastric epithelial cell proliferation and apoptosis, which is likely to lower the threshold for the development of gastric adenocarcinoma. H pylori infection is associated with epidermal growth factor (EGF) receptor (EGFR) activation through metalloproteinase-dependent release of EGFR ligands in gastric epithelial cells. Because EGFR signaling regulates cell survival, we investigated whether activation of EGFR following H pylori infection promotes gastric epithelial survival.
METHODS - Mouse conditionally immortalized stomach epithelial cells (ImSt) and a human gastric epithelial cell line, AGS cells, as well as wild-type and kinase-defective EGFR (EGFRwa2) mice, were infected with the H pylori cag+ strain 7.13. Apoptosis, caspase activity, EGFR activation (phosphorylation), and EGFR downstream targets were analyzed.
RESULTS - Inhibiting EGFR kinase activity or decreasing EGFR expression significantly increased H pylori-induced apoptosis in ImSt. Blocking H pylori-induced EGFR activation with a heparin-binding (HB)-EGF neutralizing antibody or abrogating a disintegrin and matrix metalloproteinase-17 (ADAM-17) expression increased apoptosis of H pylori-infected AGS and ImSt, respectively. Conversely, pretreatment of ImSt with HB-EGF completely blocked H pylori-induced apoptosis. H pylori infection stimulated gastric epithelial cell apoptosis in EGFRwa2 but not in wild-type mice. Furthermore, H pylori-induced EGFR phosphorylation stimulated phosphotidylinositol-3'-kinase-dependent activation of the antiapoptotic factor Akt, increased expression of the antiapoptotic factor Bcl-2, and decreased expression of the proapoptotic factor Bax.
CONCLUSIONS - EGFR activation by H pylori infection has an antiapoptotic effect in gastric epithelial cells that appears to involve Akt signaling and Bcl family members. These findings provide important insights into the mechanisms of H pylori-associated tumorigenesis.
Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.
During hematopoiesis, myeloid cell leukemia-1 (MCL-1) mediates the survival of bone marrow progenitors and lymphocytes. However, its requirement during myeloid cell differentiation, development, and effector function is less clear. Lineage-specific deletion of MCL-1 in myeloid precursors results in neutropenia due to death during differentiation. The loss of mature neutrophils induced by Mcl-1 deletion was not rescued by genetic deletion of proapoptotic Bim and Puma or by exogenous cytokine treatment. However, blockade of intrinsic apoptosis by lineage-specific deletion of both multidomain proapoptotics Bax and Bak was capable of rescuing the neutropenia associated with Mcl-1 deletion. In the monocytic lineage, despite efficient Mcl-1 deletion, monocytes and macrophages undergo normal development. During the phagocytosis of extracellular bacteria, macrophages concomitantly increase the expression of both MCL-1 and BIM. However, Mcl-1-deficient macrophages exhibit increased sensitivity to death during bacterial phagocytosis that can be abolished by codeletion of Bim. These data suggest that MCL-1 may be necessary to antagonize BIM during macrophage effector responses. Thus, MCL-1 plays selective roles in myeloid development, being required for neutrophil development and setting the threshold for apoptosis during a macrophage effector response.
OBJECTIVE - Determine the molecular mechanism(s) behind tumor necrosis factor-alpha (TNFalpha)-induced loss of auditory hair cells and the ability of dexamethasone base (DXMb) to protect against TNFalpha ototoxicity.
METHODS - Hair cell counts: Three-day-old rat organ of Corti explants were cultured under three different conditions: 1) untreated-control; 2) TNFalpha (2 mug/ml); and 3) TNFalpha (2 mug/ml)+DXMb (70 mug/ml) for 4 days, fixed, and stained with FITC-phalloidin. Hair cells were counted in the basal and middle turns. Gene expression: total RNA was extracted from the three different groups of explants at 0, 12, 24 and 48 h. Using quantitative real-time RT-PCR, mRNAs were transcribed into cDNAs and amplification was performed using primers for rat ss-actin (housekeeping gene), TNFR1, Bcl-2, Bax, and Bcl-xl.
RESULTS - DXMb protected explant hair cells from TNFalpha-induced loss. Bax gene expression was greater in TNFalpha-exposed explants compared with TNFalpha+DXMb-treated explants at 48 h (P=0.023), confirmed by the increase in the Bax/Bcl-2 ratio at 48 h (P<0.001). These results correlated with increased TNFR1 expression at 24 h (P=0.038). DXMb otoprotection in TNFalpha-exposed cultures was accompanied by an up-regulation of Bcl-xl at both the 24 (P<0.001) and 48 h time points (P=0.030) and up-regulation of Bcl-2 expression at 24 h (P=0.018). DXMb treatment also prevented increases in the expression levels of Bax, TNFR1, and the Bax/Bcl-2 ratio that occurred in untreated TNFalpha-exposed explants.
CONCLUSIONS - TNFalpha's ototoxicity may be mediated through an up-regulation of Bax and TNFR1 expression as well as an increase in the Bax/Bcl-2 ratio. DXMb protects the organ of Corti against TNFalpha ototoxicity by up-regulating Bcl-2 and Bcl-xl expression and by inhibiting TNFalpha-induced increases in Bax, TNFR1, and the Bax/Bcl-2 ratio. These results support the use of local dexamethasone treatment to conserve hearing following a trauma.