Bak Expression Research Articles

Short-term anastrozole therapy reduces Ki-67 and progesterone receptor expression in invasive breast cancer: a prospective, placebo-controlled, double-blind trial

The objective of this study was to compare Ki-67, Bcl-2, Bax, Bak, ER, and PgR expression in postmenopausal women with ER-positive invasive breast cancer (IBC) before and after short-term hormone therapy (HT) with either tamoxifen or anastrozole in order to identify a possible biomarker profile associated with hormone resistance. Fifty-eight patients with palpable IBC were assigned to receive neoadjuvant therapy with either anastrozole, placebo, or tamoxifen for 26 days prior to surgery. Tissue microarray blocks were constructed from pre- and post-treatment biopsy samples and used for immunohistochemical analysis. Biomarker (Ki-67, Bcl-2, Bax, Bak, ER, and PgR) levels were assessed semiquantitatively using the Allred score. A statistical analysis was performed using general estimating equations (GEE) and analysis of variance (ANOVA) with a significance level of 0.05. There was a significant reduction in PgR scores from baseline (mean, 4.22) to post-treatment (mean, 1.94) in the anastrozole group, but only a non-significant trend toward an increase in PgR scores was found in the tamoxifen group. There was a significant reduction in Ki-67 scores from baseline (mean, 3.61) to post-treatment (mean, 2.56) in the anastrozole group (P = 0.01), but only a non-significant trend toward a reduction in Ki-67 scores was found in the tamoxifen group. There was a significant reduction in PgR and Ki-67 expression in the group treated with anastrozole. In the present study, the short-term HT was not associated with changes in apoptosis-related protein levels, regardless the type of drug used.

ZBP-89 enhances Bak expression and causes apoptosis in hepatocellular carcinoma cells

ZBP-89 can enhance tumor cells to death stimuli. However, the molecular mechanism leading to the inhibitory effect of ZBP-89 is unknown. In this study, 4 liver cell lines were used to screen for the target of ZBP-89 on cell death pathway. The identified Bak was further analyzed for its role in ZBP-89-mediated apoptosis. The result showed that ZBP-89 significantly and time-dependently induced apoptosis. It significantly upregulated the level of pro-apoptotic Bak. ZBP-89 targeted a region between -457 and -407 of human Bak promoter to stimulate Bak expression based on the findings of Bak promoter luciferase report gene assay and electrophoretic mobility shift assay. ZBP-89-induced Bak increase and ZBP-89-mediated apoptosis were markedly suppressed by Bak siRNA, confirming that Bak was specifically targeted by ZBP-89 to facilitate apoptosis. In conclusion, this study demonstrated that ZBP-89 significantly induced apoptosis of HCC cells via promoting Bak level.

Malignant mixed Mullerian tumors of the uterus: histopathological evaluation of cell cycle and apoptotic regulatory proteins

AimThe aim of our study was to evaluate survival outcomes in malignant mixed Mullerian tumors (MMMT) of the uterus with respect to the role of cell cycle and apoptotic regulatory proteins in the carcinomatous and sarcomatous components.Methods23 cases of uterine MMMT identified from the Saskatchewan Cancer Agency (1970-1999) were evaluated. Immunohistochemical expression of Bad, Mcl-1, bcl-x, bak, mdm2, bax, p16, p21, p53, p27, EMA, Bcl-2, Ki67 and PCNA was correlated with clinico-pathological data including survival outcomes.ResultsHistopathological examination confirmed malignant epithelial component with homologous (12 cases) and heterologous (11 cases) sarcomatous elements. P53 was strongly expressed (70-95%) in 15 cases and negative in 5 cases. The average survival in the p53+ve cases was 3.56 years as opposed to 8.94 years in p53-ve cases. Overexpression of p16 and Mcl-1 were observed in patients with longer survival outcomes (> 2 years). P16 and p21 were overexpressed in the carcinomatous and sarcomatous elements respectively. Cyclin-D1 was focally expressed only in the carcinomatous elements.ConclusionsOur study supports that a) cell cycle and apoptotic regulatory protein dysregulation is an important pathway for tumorigenesis and b) p53 is an important immunoprognostic marker in MMMT of the uterus.

Transforming Growth Factor-β Signaling Curbs Thymic Negative Selection Promoting Regulatory T Cell Development

Thymus-derived naturally occurring regulatory T (nTreg) cells are necessary for immunological self-tolerance. nTreg cell development is instructed by the T cell receptor and can be induced by agonist antigens that trigger T cell-negative selection. How T cell deletion is regulated so that nTreg cells are generated is unclear. Here we showed that transforming growth factor-beta (TGF-beta) signaling protected nTreg cells and antigen-stimulated conventional T cells from apoptosis. Enhanced apoptosis of TGF-beta receptor-deficient nTreg cells was associated with high expression of proapoptotic proteins Bim, Bax, and Bak and low expression of the antiapoptotic protein Bcl-2. Ablation of Bim in mice corrected the Treg cell development and homeostasis defects. Our results suggest that nTreg cell commitment is independent of TGF-beta signaling. Instead, TGF-beta promotes nTreg cell survival by antagonizing T cell negative selection. These findings reveal a critical function for TGF-beta in control of autoreactive T cell fates with important implications for understanding T cell self-tolerance mechanisms.

High immunohistochemical expression of p‐AKT predicts inferior survival in patients with diffuse large B‐cell lymphoma treated with immunochemotherapy

Chemotherapy and rituximab (R) is current standard therapy in diffuse large B-cell lymphoma (DLBCL), but a substantial proportion of patients still fail to reach sustained remission. In vitro studies have indicated that rituximab resistance could be accompanied by dysregulated apoptotic pathways, such as the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway, which can be constitutively activated in DLBCL. In this retrospective, immunohistochemical study on 106 patients treated with R-CHO(E)P (cyclophosphamide, doxorubicin, vincristine, prednisone, rituximab [+etoposide]), we investigated the prognostic role of proteins involved in different apoptotic pathways; phosphorylated AKT (p-AKT), bcl-2, MCL1, bcl-xL, Bax and Bak. High p-AKT expression (>108 cells/mm2, highest quartile, n=27) predicted worse progression-free (PFS) (P=0.02) and overall (OS) (P=0.01) survival, independent of International Prognostic Index and sex. Also bcl-2+ (cut-off 50%) predicted worse PFS (P=0.005) and OS (P=0.05) but after adjustment for clinical factors only the influence on PFS (P=0.03) remained significant. The prognostic impact of p-AKT overexpression was independent of bcl-2 status. MCL1, bcl-xL, Bax and Bak expression did not add any prognostic information. Our results suggest that high p-AKT expression predicts worse outcome, possibly indicating that inhibition of the activated PI3K/AKT pathway could be of clinical interest in DLBCL patients. In addition, bcl-2 status could have prognostic importance also in the era of immunochemotherapy.

Abstract 610: Metformin augments rituximab and chemotherapeutic agents-induced apoptosis via mTOR inhibition in rituximab-resistant B lymphoma cell lines

Abstract With elusive mechanisms, a subgroup of NHL patients who treated with rituximab in combination with cytotoxic chemotherapeutic agents becomes unresponsive. Patients who relapsed lymphoma are known to have poorer outcome when treated with rituximab compared with treated without rituximab. Since activation of mTOR pathway has been recently suggested for progression of NHL, especially in salvage setting, we'd like to examine the effect of metformin which inhibits mTOR pathway in rituximab-resistant NHL. Three lymphoma cell lines (Raji, Raji-2R, Raji-4RH) were used, which were kindly given by Dr Czuczman MS from Rosewell Park Cancer Institute (Ref. Czuczmana MS et al. Clin Cancer Res 2008;14:1561, 2008;14:1550). These cells were treated with cisplatin, adriamycin, vincristine and rituximab in the absence or presence of metformin for 24, 48, 72 hrs. Cell growth and proliferation was evaluated by MTT assay and cell cycle progression was determined FACS analysis. To characterize molecular mechanisms involved in metformin-induced inhibition of cell proliferation, the level of BAX, BAK, Bcl-2, Bcl-XL, Mcl-1, AMPK, pAMPK, pACC, mTOR, p70S6K and S6K was determined by western blot analysis. Compared with rituximab-sensitive cell line (RSCL) which is parental lymphoma cell, Raji, rituximab-resistant cell lines (RRCLs), that are Raji-2R and Raji-4RH, have shown decreased cytotoxicity in response to chemotherapeutic agents including cisplatin, adriamycin and vincristine as well as rituximab. However, cytotoxic effect of these chemotherapeutic agents and rituximab was improved in combination of metformin in both RSCL and RRCLs. To us surprise, a better synergistic inhibition of cell proliferation was found in RRCLs as a result of combination of metformin and chemotherapeutic agents or rituximab, suggesting that intracellular signaling pathway induced by metformin might not be overlapped to that by chemotherapeutic agent and rituximab. Notably, it has known that in RRCLs, the level of proapototic proteins, BAX, BAK, is relatively low compared to in RSCL decreased, but the expression of anti-apoptotic protein and AMPK activity is increased and dysregulation of mTOR pathway is found. Our data showed that metformin restored dysregulation of mTOR pathway in RRCLs, since activation of p70S6K and S6K posphorylation was decreased. At the same time, expression of BAX and BAK was increased and that of anti-apoptotic proteins was decreased. Metformin enhances rituximab-induced and chemotherapy-induced apoptosis via mTOR inhibition in rituximab-resistant NHL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 610.

OC-049 Bak expression regulatesHelicobacter felis-induced gastric epithelial cell apoptosis and the extent of gastric atrophy in vivo

<h3>Introduction</h3> Infection with <i>Helicobacter</i> bacteria leads to a chronic superficial gastritis. In some cases this may progress to atrophic gastritis and subsequently gastric adenocarcinoma. Apoptosis is a type of programmed cell death in multicellular organisms and defects in its regulation may contribute towards carcinogenesis. Bak is a pro-apoptotic member of the bcl-2 family of proteins which has been shown to play important roles in the regulation of γ-radiation induced apoptosis in the stomach1 and colonic carcinogenesis <i>in vivo</i>.2 We therefore hypothesise that bak-regulated apoptosis may influence the development of <i>Helicobacter</i>-induced gastric cancer and have investigated whether <i>H felis</i>-induced gastric apoptosis and gastric atrophy are altered in a bak-null mouse model. <h3>Methods</h3> An initial time-course to assess the consequences of <i>H felis</i> infection was performed using groups of 6 adult C57BL/6 male mice sacrificed 2, 4, 6, 9, 12 and 20 weeks after infection. Groups of 7 C57BL/6 male mice and 7 bak-null male mice (on a C57BL/6 genetic background) were subsequently infected with <i>H felis</i> for 6 weeks. Histological and immunohistochemical techniques were used to assess the extent of infection, inflammation, bak expression, atrophy (using anti-H⁺/K⁺ ATPase to label parietal cells), cell proliferation (using anti-Mib-1) and apoptosis (using anti-active caspase-3). <h3>Results</h3> <i>H felis</i> infection of C57BL/6 mice resulted in an increase in gastric bak expression at 2 weeks and a significant decrease in the number of parietal cells was observed at 6 weeks, which then returned to control levels by 20 weeks. Cell proliferation and apoptosis both increased with time following <i>H felis</i> infection. 6 weeks following <i>H felis</i> infection, bak-null mice showed significantly less gastric atrophy and apoptosis than C57BL/6 mice, but no significant changes in proliferation were observed. <h3>Conclusion</h3> Gastric epithelial bak expression, atrophy, proliferation and apoptosis were increased following <i>H felis</i> infection of wild-type mice. After 6 weeks of <i>H felis</i> infection, bak-null mice showed significantly less atrophy and less apoptosis compared to their wild-type counterparts. These data suggest that bak plays a significant role in modulating the short-term consequences of <i>Helicobacter</i> infection in the murine stomach. Further studies at later timepoints following <i>H felis</i> infection are therefore warranted to investigate whether bak expression also regulates the development of gastric carcinoma.

Different contribution of BH3-only proteins and caspases to doxorubicin-induced apoptosis in p53-deficient leukemia cells

Bcl-2 family proteins are key regulators of the intrinsic apoptotic pathway, either facilitating (Bax, Bak, BH3-only) or inhibiting (Bcl-2, Bcl-x L, Mcl-1, A1) mitochondrial release of apoptogenic factors. The role of caspases in this process is a matter of controversy. We have analyzed the relative contribution of caspases and Bcl-2 family of proteins in the induction phase of apoptosis triggered by doxorubicin in two p53-deficient leukemia cell lines, Jurkat and U937. First, we have found that caspases are dispensable for the induction phase of doxorubicin-induced apoptosis in both cell lines but they are needed to speed up the execution phase in Jurkat cells, not expressing Bax. Thus, down-regulation of Bak expression by siRNA significantly prevented doxorubicin-induced apoptosis in Jurkat but not in U937 cells. Reduction of Mcl-1 protein levels with siRNA increased sensitivity to apoptosis in both cell lines. Moreover, our results indicate that the contribution of BH3-only proteins to apoptosis is cell line specific. In Jurkat cells simultaneous silencing of Bim and PUMA was necessary to reduce doxorubicin-induced apoptosis. In U937 cells silencing of Bim or Noxa reduced sensitivity to doxorubicin. Immunoprecipitation experiments discarded an interaction between Mcl-1 and Bak in both cell lines and underscored the role of Bim and PUMA as mediators of Bax/Bak activation.

Antimicrobial peptaibols, novel suppressors of tumor cells, targeted calcium-mediated apoptosis and autophagy in human hepatocellular carcinoma cells

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers in the world which is highly chemoresistant to currently available chemotherapeutic agents. Thus, novel therapeutic targets are needed to be sought for the successful treatment of HCC. Peptaibols, a family of peptides synthesized non-ribosomally by the Trichoderma species and other fungi, exhibit antibiotic activities against bacteria and fungi. Few studies recently showed that peptaibols exerted cytotoxicity toward human lung epithelial and breast carcinoma cells. However, the mechanism involved in peptaibol-induced cell death remains poorly understood.ResultsHere, we showed that Trichokonin VI (TK VI), a peptaibol from Trichoderma pseudokoningii SMF2, induced growth inhibition of HCC cells in a dose-dependent manner. It did not obviously impair the viability of normal liver cells at lower concentration. Moreover, the suppression of cell viability resulted from the programmed cell death (PCD) with characteristics of apoptosis and autophagy. An influx of Ca2+ triggered the activation of μ-calpain and proceeded to the translocation of Bax to mitochondria and subsequent promotion of apoptosis. On the other hand, typically morphological characteristics consistent with autophagy were also observed by punctate distribution of MDC staining and the induction of LC3-II, including extensive autophagic vacuolization and enclosure of cell organelles by these autophagosomes. More significantly, specific depletion of Bak expression by small RNA interfering (siRNA) could partly attenuate TK VI-induced autophagy. However, siRNA against Bax led to increased autophagy.ConclusionTaken together, these findings showed for the first time that peptaibols were novel regulators involved in both apoptosis and autophagy, suggesting that the class of peptaibols might serve as potential suppressors of tumor cells.

Ligation of cell surface GRP78 with antibody directed against the COOH-terminal domain of GRP78 suppresses Ras/MAPK and PI 3-kinase/AKT signaling while promoting caspase activation in human prostate cancer cells

We have previously shown that treatment of prostate cancer and melanoma cells expressing GRP782 on their cell surface with antibody directed against the COOH-terminal domain of GRP78 up-regulates and activates p53 causing decreased cell proliferation and up-regulated apoptosis. In this report, we demonstrate that treatment of 1-LN prostate cancer cells with this antibody decreases cell surface expression of GRP78, AktThr308 and AktSer473 kinase activities and reduces phosphorylation of FOXO, and GSK3β. This treatment also suppresses activation of ERK1/2, p38 MAPK, and MKK3/6; however, it up-regulates MKK4 activity. JNK, as determined by its phosphorylation state, is subsequently activated, triggering apoptosis. Incubation of cells with antibody reduced levels of anti-apoptotic Bcl-2, while elevating pro-apoptotic BAD, BAX, and BAK expression as well as cleaved caspases-3, -7, -8, and -9. Silencing GRP78 or p53 gene expression by RNAi prior to antibody treatment abrogated these effects. We conclude that antibody directed against the COOH-terminal domain of GRP78 may prove useful as a pan suppressor of proliferative/survival signaling in cancer cells expressing GRP78 on their cell surface.

Abstract C4: TLN-4601 is a novel tumor selective pro-apoptotic agent acting independently of Bax, Bak or Bcl-2 expression

Abstract Background: TLN-4601 is a structurally novel farnesylated dibenzodiazepinone discovered through Thallion's proprietary DECIPHER® technology platform. The compound has demonstrated broad in vitro cytotoxicity and in vivo tumor growth inhibition. Phase I clinical testing has shown the compound to be safe and well tolerated, with early signs of anti-tumor activity. Although considerable research efforts have focused on the ability of TLN-4601 to inhibit the Ras/MAPK signalling pathway, the compound has also been shown to induce apoptosis through caspase activation. The objectives of the present work were to further elucidate the molecular events, pathways and temporal patterns associated with TLN-4601-induced apoptosis. Material and Methods: Protein extracts from Jurkat cells (a human T cell lymphoblast-like cell line), Jurkat cells with caspase 8 knocked-down, Jurkat cells over-expressing Bcl-2, mouse embryonic fibroblasts isolated from Bax/Bak double knock-out mice, and both quiescent and PHA-L stimulated human peripheral blood lymphocytes (PBLs) treated with TLN-4601 over different exposure times were analyzed by Western blot to evaluate the induction of apoptotic markers, including cytochrome c release, caspase activation and PARP cleavage. Apoptosis was assessed by DNA laddering and sub-G1 DNA content. The effect of TLN-4601 on cell viability were measured using commercial Caspase-3/7 activity and ATP assays. Results: TLN-4601 caused rapid apoptotic cell death in Jurkat cells as characterized by mitochondrial cytochrome c release, caspase activation, loss of cellular ATP and DNA laddering. Equal sensitivity of the wild-type and caspase 8 knocked-down Jurkat cells to TLN-4601 suggests that the extrinsic apoptotic pathway was not involved in mediating this activity. Interestingly, TLN-4601 exposure resulted in cytochrome c release independently of the apoptosis-inducing proteins Bax and Bak. Furthermore, the over-expression of the anti-apoptotic protein Bcl-2 did not affect the ability of TLN-4601 to induce cell death. Finally, a short exposure of TLN-4601 exhibited a preferential selectivity for triggering apoptosis in Jurkat cells as compared to either quiescent and PHA-L stimulated PBLs. Conclusions: TLN-4601 mediates rapid cell death via the intrinsic apoptotic pathway independent of Bax, Bak or Bcl-2 expression. Moreover, the differential sensitivity to TLN-4601 observed between Jurkat cells and PBLs highlights the potential for a usable therapeutic margin between cancer and normal cells. Taken together, these properties represent a very promising profile for an anti-cancer agent and suggest that further investigation of TLN-4601 in hematological malignancies is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C4.

Simultaneous Targeting of Bcl-2/Bcl-Xl and MEK/ERK Results in Highly Synergistic Induction of Apoptosis in Human AML, Both in Vitro and In Vivo.

Abstract 2757Poster Board II-733Previous experience from our group suggests that simultaneous inhibition of MEK-to-ERK signaling and interference with the anti-apoptotic activity of Bcl-2/Bcl-xL, using a variety of different agents, may result in synergistic anti-leukemic activity (Milella M, Blood 2002; Ricciardi MR, ASH 2004; Konopleva M, Cancer Cell 2006; Milella M, ASH 2008). Here, we screened AML cell lines (OCI-AML3, HL-60, MOLM-13, and U937) for the growth inhibitory/pro-apoptotic effects of the combination of the BH3 mimetic ABT-737 and the selective MEK inhibitor PD0325901. With the notable exception of U937 cells all the cell lines tested displayed highly synergistic growth inhibition/induction of apoptosis in response to the combination (CIs ranging from 0.01 to 0.43); the 25:1 and 1:2 ABT-737/PD0325901 ratios appeared to have optimal synergistic effects in OCI-AML3 and MOLM-13 cells, respectively. Cell growth inhibition was primarily due to the highly synergistic induction of apoptosis in sensitive cell line models. From a mechanistic standpoint, ABT-737 induced ERK activation and Mcl-1 protein expression, two putative resistance mechanisms, both of which were efficiently abrogated by co-treatment with PD0325901. In addition to the modulation of Mcl-1, both single agent PD0325901 and combined ABT-737/PD0325901 treatment rapidly (1-6 hrs) induced BimEL dephosphorylation and Bak expression, thereby contributing to Mcl-1 inactivation. To further assess the role of pro-apoptotic Bcl-2 family members in the observed proapoptotic synergism betweeen ABT-737 and PD0325901, we analyzed the effects of the combination in Bim-, Bak-, and Bax-KO MEFs, as well as in double (Bak/Bax) KO; while these experiments demonstrated that all three proapoptotic proteins play a role in apoptosis induction by combined ABT-737/PD0325901, siRNA-mediated silencing of either Bim or Bak clearly indicated Bim as the most important player in the AML cell line OCI-AML3. In addition to cell line models of leukemia, striking apoptosis induction (20-75% net apoptosis induction) was also observed with the combination of Bcl-2 and MEK inhibitors in ex vivo-cultured primary AML samples (n=8); most interestingly, the ABT-737/PD0325901 combination appeared to selectively kill leukemic stem cells, with < 20% of CD34+/CD38- cells surviving after exposure to relatively low doses of the combination (50 nM for each agent). Finally, we tested the combination of ABT-737 and the MEK inhibitor CI-1040 in nude mice injected with GFP/luciferase bearing MOLM-13 human leukemia cells. Two weeks after leukemia transplantation, mice were randomized and treated with liposomal ABT-737 (i.v. 20 mg/kg, qod for three weeks), CI1040 (i.p. 50 mg/kg qod for three weeks), ABT-737 in combination with CI1040 (ABT-737 + CI1040), or with empty liposomes (i.v.; control). Engraftment of MOLM-13 cells was shown by immunohistochemical detection of GFP-positive cells in the spleen of control mice five weeks after transplantation. Notably, while control and CI1040 treated mice demonstrated progressive increases in leukemia-derived bioluminescence, ABT-737 treated mice, and to a greater extent ABT-737 + CI1040 treated mice, appeared to resist tumor burden progression. In addition, quantitation of leukemia-derived bioluminescence demonstrated that ABT-737 + CI1040 treated mice had significantly (p<0.00001) lower leukemia burden than control mice or ABT-737 treated mice at all time points (7, 14 and 21 days of treatment). Overall our data demonstrate that an anti-apoptotic crosstalk between the Bcl-2 and the MEK/ERK pathway is operative in AML cells and could be exploited therapeutically by targeting both pathways simultaneously. The combination of BH3 mimetics (such as ABT-737) and MEK inhibitors warrants clinical testing as a novel therapeutic strategy for patients with AML. Disclosures:No relevant conflicts of interest to declare.

Apoptosis in normal ovaries of women with and without endometriosis

The aim of this study was to evaluate the factors predisposing to implants of endometriotic lesions in normal ovarian cortexes of women with and without endometriosis by assessing the expression of pro-apoptotic and anti-apoptotic factors and follicular density. Ovarian biopsies were performed during laparoscopy in 18 patients with endometrioma and in 10 healthy women. Detection of apoptosis was performed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay. p53 and BCL2 proteins were assessed by immunohistochemistry. Quantitative real-time polymerase chain reaction was performed to evaluate BAX , BAK , BCL2 , BCL-XL , survivin and beta-actin ( ACTB ) expression. The p53 protein was positive in a significantly higher number of secondary follicles, whereas the B-cell chronic lymphocytic leukaemia/lymphoma 2 (BCL2) protein was positive in all follicles in unaffected tissue of endometriotic women, compared with the controls. Overexpression of the BCL2 and survivin genes and a decreased BAX and BAK gene expression were observed in the endometriotic group although only the difference in survivin expression was significant (P = 0.016). The BCL2 / BAX ratio showed an increased value in the ovarian cortex in controls compared with endometriosis patients. In conclusion, the reduction of apoptosis in unaffected tissue in women with endometriosis suggests that they may be predisposed to develop endometriosis.

Inhibition of the ER Ca2+ pump forces multidrug-resistant cells deficient in Bak and Bax into necrosis

Tumor cells deficient in the proapoptotic proteins Bak and Bax are resistant to chemotherapeutic drugs. Here, we demonstrate that murine embryonic fibroblasts deficient for both Bak and Bax are, however, efficiently killed by thapsigargin, a specific inhibitor of ER Ca(2+) pumps that induces ER stress by depleting ER Ca(2+) stores. In the presence of Bak and Bax, thapsigargin eliminates cells by release of mitochondrial cytochrome c and subsequent caspase activation, which leads to the proteolytic inactivation of the molecular necrosis switch PARP-1 and results in apoptosis. By contrast, in the absence of Bak and Bax, a failure to activate caspases results in PARP-1-mediated ATP depletion. The subsequent necrosis is not prevented by autophagy as an alternative energy source. Moreover, in cells deficient for both Bak and Bax, thapsigargin induces permanent mitochondrial damage by Ca(2+) overload, permeability transition and membrane rupture. Thus, even though deficiency in Bak and Bax protects these cells against apoptosis, it does not compromise necrosis induced by SERCA inhibitors. Importantly, thapsigargin induces caspase-independent cell death also in colon and prostate carcinoma cells deficient in Bak and Bax expression. Therefore, targeted application of ER stressors such as thapsigargin might be a promising approach for the treatment of Bak- and Bax-deficient, drug-resistant tumors.

Atorvastatin mediates increases in intralesional BAX and BAK expression in human end-stage abdominal aortic aneurysms

Chronic apoptosis activation may participate in abdominal aortic aneurysm (AAA) expansion. Statin treatment slows AAA progression independent of cholesterol lowering. We hypothesized that Atorvastatin treatment alters apoptosis protein expression and activation in AAAs. Protein was isolated from the central and distal portions of end-stage human AAA tissue obtained during surgical repair from non-statin (NST) and Atorvastatin-treated (AT) patients. Expression was compared using immunoblots. Bcl-2 expression was unchanged but Bak (4-fold, p < 0.013) and Bax (3-fold, p < 0.035) expression was increased in AT (n = 12) versus NST (n = 15) patients. No cytochrome c release or caspase 3 activation was detected and Clusterin, GRP78, and BNIP1 expression was similar in NST and AT samples. Bcl-2 and Bax cDNA sequences from AAA tissue (n = 10) and the general population were identical. Thus, the increase in Bax and Bak in AT-treated AAAs did not activate the mitochondria or endoplasmic reticulum mediated apoptosis pathways. Bcl-2, Bax, and Bak have non-apoptosis related functions that include maintenance of endoplasmic reticulum (ER), homeostasis, and adaptation to stress. We speculate that Atorvastatin-mediated increases in Bax and Bak may positively affect their non-apoptosis related cell functions to account for the beneficial effect of statins to slow AAA expansion.

The inhibitory effects of siRNA targeting bak on apoptosis of dendritic cells induced by glioma and the mechanism

Objective To study the inhibitory effects of siRNA targeting bak on the apoptosis of dendritic cells(DC)2.4 that induced by glioma and to investigate the possible pathway of the apoptosis.Methods The vector expressing siRNA(A:229,B:310,C:579,D:NC)targeting bak was transfected into DC2.4 by the lipofectamine 2000,and the cells stably expressing the siRNA were selected by G418.The DC2.4 that stably expressing the siRNA were co-cultured with glioma(group B)Control group(group A)DC2.4 only,group c DC2.4 co-culture with glioma,and observe the activity of DC2.4.Investing the relategene(Caspase-3,Caspase-8,Cytc)expression.Results When co-culture with glioma,the DC2.4 apoptosis.When the vector were transfected into DC2.4,the expression of bak were degraded(Western blot:A:0.83,B:0.14,C:0.75,D:0.89.un-transfected:0.92;RT-PCR:A:0.42,B:0.08,C:0.39,D:0.46.untransfected:0.49,P ＜ 0.05).When the DC2.4 that stably expressing siRNA target bak co-culture with glioma,the expression of Caspase-8 was not change(a:0.78,b:0.75,c:0.77,P ＞ 0.05),but the expression of Caspase-3 and the cytochrome C in endochylema was less than the unexpression siRNA one(Caspase3:a:0.67,b:0.70,c:0.21;Cytc:a:0.70,b:0.71,c:0.17,P＜0.05).Conclusion The glioma can induce apoptosis of DC2.4.The pathway of apoptosis may be the mitochandrial pathway;The siRNA targeting bak can degrade the apoptosis of DC2.4. Key words: Glioma; Apoptosis; siRNA; Dendritic cell

Correlation of expression of pro-apoptotic proteins bax and bak with lymph node metastasis in colonic adenocarcinoma

e15042 Background: Colon cancer has been associated with disturbances in the regulation of apoptosis. Down regulation of Bax and Bak, members of the Bcl-2 family, are associated with tumor metastasis. Here we investigate how these two pro-apoptotic markers correlate with lymph node (LN) metastasis in colonic adenocarcinoma (CA). Methods: Paraffin-embedded sections from 100 cases of CA were constructed in tissue microarrays and subjected to immunohistochemistry (IHC) using the ABC method and antibodies for Bax and Bak. The IHC score is the product of intensity and percentage of positively stained cells, and ranged between 0 and 9. The total scores were divided into categories of “2 or less” and “3 or more.” These were than correlated with LN metastasis. Results: For Bax, 88 tumors were available while Bak was present in 100 tumors. In scoring Bax, 67 tumors had a score of ≤2 and 21 had a score of ≥3. Within the ≤2 category, 23 of 67 (34%) tumors had no LN metastases while 15 of 21 (71%) in the ≥3 category had no metastasis (p= 0.0029). For Bak staining, 23 had a score of ≤2 and 77 had a score of ≥3. Five of 23 (22%) tumors with a score ≤2 had no metastases and 35 of 77 (46%) with score ≥3 had no metastases (p= 0.0427). The LOGISTIC Procedure was used to show that a patient with a ≥3 Bax score has a 4.78 times odds ratio (95% C.I.: 1.64- 13.98) of having no metastases while a patient with a ≥3 Bak score has a 3 times odds ratio (95% C.I.: 1.01- 8.90) of having no metastases. Conclusions: We show correlation of the pro-apoptotic proteins Bax and Bak with decreased lymph node metastasis in CA. The findings suggest a role for these markers in predicting stage and patient survival in colonic adenocarcinoma. No significant financial relationships to disclose.

Stimulatory heterotrimeric GTP‐binding protein augments cisplatin‐induced apoptosis by upregulating Bak expression in human lung cancer cells

The present study aimed to investigate the effect of the stimulatory heterotrimeric GTP-binding (Gs) protein signaling system on cisplatin-induced apoptosis of lung cancer cells and its underlying mechanism as an attempt to develop a novel strategy to improve the therapeutic efficacy of cisplatin. Overexpression of the constitutively active alpha subunit of Gs (GalphasQL) in A549 human lung cancer cells increased cisplatin-induced apoptosis, and knockdown of Galphas with small hairpin RNA decreased the percentage of apoptotic cells. GalphasQL increased the expression of the proapoptotic proteins B-cell leukemia/lymphoma-2 genes (Bcl-2) homologous antagonist killer protein (Bak) and Bcl-2 associated X protein (Bax), and decreased the expression of the antiapoptotic proteins Bcl-2 and Bcl-Xlong protein. Knockdown of Bak blocked the augmentative effects of GalphasQL. GalphasQL decreased the degradation rate of the Bak protein, and increased Bak mRNA transcript levels. GalphasQL increased Bak-luciferase activity in a protein kinase A and cyclic AMP response element-dependent manner. GalphasQL also augmented cisplatin-induced apoptosis of H1299 human lung cancer cells that lack functional p53. From this study, it is concluded that Galphas augments cisplatin-induced apoptosis of lung cancer cells partially through upregulating Bak expression by increasing transcription and by decreasing the rate of protein degradation.

The additional loss of Bak and not the lack of the protein tyrosine kinase p56/Lck in one JCaM1.6 subclone caused pronounced apoptosis resistance in response to stimuli of the intrinsic pathway

Ionising radiation, hypoxia, and the cyclooxygenase-2 inhibitor Celecoxib are known agonists of the intrinsic apoptosis pathway that involves mitochondrial damage upstream of caspase activation. Mitochondrial integrity is regulated by the pro-apoptotic Bcl-2 protein family members Bak and Bax. Upstream of the mitochondria, many kinases and phosphatases control the apoptotic response. However, the role of the non-receptor tyrosine kinase p56/Lck during apoptosis is controversial. The present investigation demonstrate the existence of two JCaM1.6 subclones, one expressing and one deficient for Bak. The lack of p56/Lck expression in JCaM1.6 cells per se did hardly affect apoptosis induced by ionising radiation, hypoxia, or Celecoxib. Only the additional loss of Bak expression, as observed in one JCaM1.6 subclone, rendered the cells resistant. siRNA-mediated downregulation of Bak and p56/Lck mimicked the observed effects in the subclones. Earlier experiments performed with the Bak-negative clone might have lead to the wrong assumption that lack of p56/Lck alone, and not the additonal loss of Bak, was responsible for reduced sensitivity towards stimuli of the intrinsic apoptosis pathway.

ERK Inhibition by SU1498 Alters Expression of Bcl‐2 Family Proteins in Breast Cancer Cells

The extracellular signal‐regulated kinase (ERK) pathway is vital to the cell survival. We have demonstrated the ability of SU1498, an inhibitor of ERK kinase activity, to sensitize breast cancer cells with constitutively active ERK to apoptosis. To identify the mechanisms involve, the expression of the antiapoptotic Bcl‐2 and Bcl‐xL and the proapoptotic Bad, Bax, Bik and Bak were examined under apoptotic conditions. One breast cell line (MCF‐10A) and three breast cancer cell lines (MCF 7, MDA‐MB‐231 and MDA‐MB‐468) were used to examine the effects of SU1498 and serum starvation on the expression of the Bcl‐2 family proteins. The MCF‐10A and MCF7 cells exhibit normal ERK regulation while the MDA‐MB‐231 and MDA‐MB‐468 cells lines have a constitutively activated ERK. Across all the cell lines, the combined treatments resulted in either decreased expression, or no changes in the expression, of the antiapoptotic Bcl2 proteins. The expression of the proapoptotic proteins Bad or Bak were dramatically upregulated only in those cells with constitutive ERK activation, while they tended to demonstrate reduced expression in the cells lines with normal ERK regulation. These cell lines with upregulate Bad and/or Bak expression were also those most sensitive to SU1498 induced cell death. The ability of SU1498 to sensitize breast cancer cells to apoptosis by inhibition of ERK may have therapeutic uses.Supported by NSERC