Overexpression Of Bcl-2 Protein Research Articles

ABT-737 Induces Expression of the Death Receptor 5 and Sensitizes Human Cancer Cells to TRAIL-induced Apoptosis

Because Bcl-2 family members inhibit the ability of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to induce apoptosis, we investigated whether ABT-737, a small molecule Bcl-2 inhibitor, enhances TRAIL killing. We demonstrate that a combination of ABT-737 and TRAIL induced significant cell death in multiple cancer types, including renal, prostate, and lung cancers, although each agent individually had little activity in these tumor cells. All of these cell lines expressed the Mcl-1 protein that is known to block the activity of ABT-737 and TRAIL but did not block the synergy between these agents. However, Bax-deficient cell lines, including DU145 and HCT116 cells and those cell lines expressing low levels of TRAIL receptor, were resistant to apoptosis induced by these agents. To understand how ABT-737 functions to markedly increase TRAIL sensitivity, the levels of specific death-inducing signaling complex components were evaluated. Treatment with ABT-737 did not change the levels of c-FLIP, FADD, and caspase-8 but up-regulated the levels of the TRAIL receptor DR5. DR5 up-regulation induced by ABT-737 treatment occurred through a transcriptional mechanism, and mutagenesis studies demonstrated that the NF-kappaB site found in the DR5 promoter was essential for the ability of ABT-737 to increase the levels of this mRNA. Using luciferase reporter plasmids, ABT-737 was shown to stimulate NF-kappaB activity. Together, these results demonstrate that the ability of ABT-737 and TRAIL to induce apoptosis is mediated through activation of both the extrinsic and intrinsic pathways. Combinations of ABT-737 and TRAIL can be exploited therapeutically where antiapoptotic Bcl-2 family members drive tumor cell resistance to current anticancer therapies.

The antioxidant function of Bcl-2 preserves cytoskeletal stability of cells with defective respiratory complex I.

Human thyroid carcinoma XTC.UC1 cells harbor a homoplasmic frameshift mutation in the MT-ND1 subunit of respiratory complex I. When forced to use exclusively oxidative phosphorylation for energy production by inhibiting glycolysis, these cells triggered a caspase-independent cell death pathway, which was associated to a significant imbalance in glutathione homeostasis and a cleavage of the actin cytoskeleton. Overexpression of the anti-apoptotic Bcl-2 protein significantly increased the level of endogenous reduced glutathione, thus preventing its oxidation after the metabolic stress. Furthermore, Bcl-2 completely inhibited actin cleavage and increased cell adhesion, but was unable to improve cellular viability. Similar effects were obtained when XTC.UC1 cells were incubated with exogenous glutathione. We hence propose that Bcl-2 can safeguard cytoskeletal stability through an antioxidant function.

BCL-2 phosphorylation modulates sensitivity to the BH3 mimetic GX15-070 (Obatoclax) and reduces its synergistic interaction with bortezomib in chronic lymphocytic leukemia cells

Chronic lymphocytic leukemia (CLL) is a B-cell lymphoid neoplasm with deregulated apoptosis and overexpression of several antiapoptotic BCL-2 proteins. GX15-070/Obatoclax is a small-molecule BH3 mimetic compound that has shown activity against several hematologic malignancies and solid tumors. In the present work, we report that GX15-070 led to the disruption of BCL-2/BIM and MCL-1/BAK complexes in CLL cells, followed by the activation of the mitochondrial apoptotic pathway. CLL cells showed lower sensitivity to GX15-070 than primary mantle cell lymphoma (MCL) ones, in correlation with higher levels of phosphorylated BCL-2 at serine 70 residue (pBCL-2(Ser70)) in CLL cells. Decrease in BCL-2 phosphorylation by extracellular signal-regulated kinase (ERK)1/2 inhibition increased CLL sensitivity to GX15-070, while blocking BCL-2 dephosphorylation using a PP2A antagonist reduced the activity of this BH3 mimetic. GX15-070 activity was increased by cotreatment with the proteasome inhibitor bortezomib. However, as proteasome inhibition led to the accumulation of phosphorylated BCL-2, the degree of interaction between GX15-070 and bortezomib was regulated by basal pBCL-2(Ser70) levels. These results support the role of BCL-2 phosphorylation as a mechanism of resistance to BH3 mimetic compounds, and demonstrate that combination approaches including ERK inhibitors could enhance BH3 mimetics activity both alone or in combination with proteasome inhibitors.

Role of HuR in the regulation of bcl‐2 mRNA stability in human HL60 leukemia cells

High bcl‐2 expression in hematological tumors is frequently an obstacle to cancer chemotherapy. We have demonstrated that Bcl‐2 overexpression in some leukemia cells is a consequence of abnormal bcl‐2 mRNA stability. Previously, nucleolin was identified as a factor that stabilizes bcl‐2 mRNA in human HL60 leukemia cells. This interaction occurs via an AU‐rich element (ARE) in the 3’‐untranslated region of bcl‐2 mRNA. Our current study demonstrated that HuR also plays a role in the regulation of bcl‐2 mRNA stability in HL60 cells. Using EMSA we observed that HuR in HL60 cell extracts binds to the bcl‐2 mRNA ARE. Also, we found that such interactions occur in vivo. Taxol treatment induced proteolysis of cytoplasmic HuR, suggesting that modulation of HuR may play a role in the downregulation of bcl‐2 that precedes taxol‐induced apoptosis. Pull‐down assays indicate that nucleolin and HuR co‐immunoprecipitate in an RNA‐dependent manner, suggesting the two proteins are part of common bcl‐2 mRNP complexes. Moreover, nucleolin and HuR can bind independently and concurrently to bcl‐2 ARE RNA in vitro. This suggests HuR and nucleolin bind to separate, non‐overlapping sites on the bcl‐2 ARE. Sh‐RNA‐mediated knockdown of HuR in A431 epithelial cancer cells leads to reduced levels of bcl‐2 mRNA and protein. Taken together, our studies suggest that both HuR and nucleolin play a role in stabilization of bcl‐2 mRNA, which contributes to the overexpression of Bcl‐2 protein in some types of leukemia cells. Supported by NCI R01CA87553 (EKS) and Leukemia and Lymphoma Society Grant 6006‐06 (DJF).

Evaluation of TW-37, a pan Bcl-2 Proteins Small-Molecule Inhibitor, Against Spectrum of Human B-Cell Lines and Patient-Derived Samples.

Non-Hodgkin's lymphoma (NHL) tumors include a group of heterogeneous diseases with varying natural histories and responsiveness to therapy; nonetheless, overexpression of Bcl-2 protein is seen in more than 80% of NHL. Throughout the years our laboratory succeeded in establishing a panel of B-cell lines representing various maturational stages of NHL. In this study, we have utilized a structure-based strategy to design a new class of potent nonpeptidic small-molecule inhibitor (SMI) of Bcl-2 family. TW-37, a lead compound that was designed to target the BH3 binding groove of antiapopototic Bcl-2 proteins. It binds to Bcl-2, Bcl-XL and Mcl-1 with Ki values of 290 nM, 1110 nM and 260 nM, respectively. TW-37 showed significant antiproliferative effect against Pre-B-Acute Lymphoblastic Leukemia (WSU-pre-B-ALL), Diffuse Large Cell Lymphoma (WSU-DLCL2), Follicular Small Cleaved Cell Lymphoma (WSU-FSCCL), Waldenstrom's Macroglobulinemia (WSU-WM) and primary cells obtained from lymphoma patients, despite variations in their anti- and pro-apoptotic Bcl-2 proteins (Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, Bim, Bad, BUMA and Bok). The IC50 for TW-37 varied from 165 nM in the WSU-FSCCL to 300 nM in WSU-DLCL2 cells. Apoptosis was independent of proliferative status or pathological classification of B-cell tumor. TW-37 was able to block Bim-Bcl-XL and Bim-Mcl-1 eterodimerization and induces apoptosis via activation of caspases -9, -3, PARP and DNA fragmentation. Although cell lines and patient samples expressed multiple Bcl-2 family proteins at various levels, TW-37 induced apoptosis was only strongly associated with Bax:Mcl-1 ratio. TW-37 administered to tumor-bearing SCID mice led to significant tumor growth inhibition (T/C), tumor growth delay (T-C) and Log10kill, when used at its maximum tolerated dose (40 mg/kg x 3days) via tail vein. failed to induce changes in the Bcl-2 proteins levels suggests that assessment of baseline Bcl-2 family proteins can be used to prognosticate the response to drug. These findings indicate activity of TW-37 across the spectrum of human B-cell tumors and support the concept of targeting the Bcl-2 system as a therapeutic strategy in the treatment of B-cell lymphoma.

Modulation of tumor radiation response with G3139, a bcl-2 antisense oligonucleotide

Overexpression of anti-apoptotic bcl-2 protein has been found in hematological and solid tumors and has been associated with increased resistance against cytotoxic therapy. While bcl-2 antisense (AS) treatment combined with chemotherapy has been successfully tested in clinical trials, trials evaluating the combination of bcl-2 AS with radiotherapy have not yet been performed. The aim of this study was to investigate in vivo anti-tumor effects of a combined modality treatment scheme consisting of radiation and the bcl-2 targeted AS oligonucleotide (ODN) G3139 (Oblimersen Sodium). Two human colon carcinoma cell lines, SW620, bcl-2 positive and HT-29, bcl-2 negative, were grown as xenografts and compared in their response to combined bcl-2 AS/radiation treatment. G3139 potentiated the radiation response of bcl-2 positive SW620 tumors, but had no significant effect on bcl-2 negative HT-29 tumors assayed by tumor growth delay. The profound enhancement of SW620 tumor growth delay by G3139 did not translate into effects on tumor cure, as no significant effect of G3139 was found on SW620 radiocurability (TCD50 assay). The control ODN G3622 had no effect on SW620 radiation response, indicating an ODN sequence specific effect.

Preclinical Studies of TW-37, a New Nonpeptidic Small-Molecule Inhibitor of Bcl-2, in Diffuse Large Cell Lymphoma Xenograft Model Reveal Drug Action on Both Bcl-2 and Mcl-1

Overexpression of Bcl-2 protein has been observed in more than 80% of B-cell lymphomas, including diffuse large cell lymphoma (DLCL), the most common subtype of non-Hodgkin's lymphoma. We have previously employed the natural product (-)-gossypol to test its therapeutic potential as a small-molecule inhibitor of Bcl-2 for the treatment of B-cell lymphomas. Recently, we have used a structure-based strategy to design a new class of potent small-molecule inhibitor acting on Bcl-2. One such lead compound is the benzenesulfonyl derivative TW-37, which was designed to target the BH3-binding groove in Bcl-2 where proapoptotic Bcl-2 proteins, such as Bak, Bax, Bid, and Bim bind. In our fluorescence polarization-based binding assays using recombinant Bcl-2, Bcl-X(L), and Mcl-1 proteins, TW-37 binds to Bcl-2, Bcl-X(L), and Mcl-1 with K(i) values of 290, 1,110 and 260 nmol/L, respectively. Hence, TW-37 is a potent inhibitor of Bcl-2 and has >3-fold selectivity over Bcl-X(L). In vitro, TW-37 showed significant antiproliferative effect in a de novo chemoresistant WSU-DLCL(2) lymphoma cell line and primary cells obtained from a lymphoma patient with no effect on normal peripheral blood lymphocytes. Coimmunoprecipitation experiments showed that TW-37 disrupted heterodimer formation between Bax or truncated-Bid and antiapoptotic proteins in the order Mcl-1 > Bcl-2 >> Bcl-X(L). As expected, TW-37 caused apoptotic death. Pre-exposure of lymphoma cells to TW-37 significantly enhanced the killing effect of cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP) regimen. The maximum tolerated dose of TW-37 in severe combined immunodeficient (SCID) mice was 40 mg/kg for three i.v. injections when given alone and 20 mg/kg, x3 when given in combination with CHOP. Using WSU-DLCL(2)-SCID mouse xenograft model, the addition of TW-37 to CHOP resulted in more complete tumor inhibition compared with either CHOP or TW-37 alone. We conclude that the administration of TW-37, as a potent Bcl-2 and Mcl-1 inhibitor, to standard chemotherapy may prove an effective strategy in the treatment of B-cell lymphoma.

Bcl‐2 Expression in Oral Squamous Cell Carcinoma

Apoptosis is a genetically regulated process involved in tissue size regulation, morphogenesis, and elimination of genetically damaged cells. A pallet of genes is involved in the control of apoptosis, such as bcl-2 family whose oncogenic potential has been demonstrated in oral tumorigenesis. Different members of bcl-2 family may promote or inhibit apoptosis by synthesizing anti- and proapoptotic proteins. One of antiapoptotic proteins, bcl-2, with a crucial role in apoptosis regulation was the object of our study. By means of immunohistochemistry we estimated the level of overexpression of bcl-2 proteins in a series of the 26 formalin fixed, paraffin-embedded samples of oral squamous cell carcinoma (OSCC). Analyzed tumors originated from different sites of oral cavity; 7/26 belonged to stage II, 14/26 to stage III, and 5/26 to stage IV. Immunoreactivity was scored according to the percentage and intensity of positive cytoplasmic bcl-2 staining. All tumors had low percentage of positively stained bcl-2 cells, with mean values for lower/higher intensity of 8.3 +/- 2.5/34.4 +/- 7, 7.5 +/- 1.1/31.9 +/- 4.3, and 8.4 +/- 5.8/31.5 +/- 5.8 within stages II, III, and IV, respectively. Low level of bcl-2 expression in our sample seems to be associated with higher survival rate: 77% for the 5-year follow-up period. Comparing clinicopathologic and risk factors data within each and between three groups of analyzed tumors (lip-tongue P = 0.58, tongue-floor of the mouth, P = 0.21, lip-floor of the mouth, P = 0.50) there was no significant difference. However, our results suggest that the level of bcl-2 expression could be a valuable predictor of tumor behavior and disease outcome.

Structure−Activity Relationship Studies of Ethyl 2-Amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA 14-1), an Antagonist for Antiapoptotic Bcl-2 Proteins To Overcome Drug Resistance in Cancer

The structure-activity relationship studies of ethyl 2-amino-6-cyclopentyl-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (1, HA 14-1), an antagonist of the antiapoptotic Bcl-2 proteins, are reported. A series of analogues of 1 with varied functional groups at the 6-position of the chromene ring were synthesized. These candidates were evaluated for their binding interactions with three antiapoptotic proteins: Bcl-2, Bcl-XL, and Bcl-w. They were also assayed for their in vitro cytotoxicities against a set of Jurkat cells with varied levels of Bcl-2 and Bcl-XL proteins and a non-small-cell lung carcinoma cell line (NCI-H460). It was found that the 6-bromo of 1 was not essential for its bioactivity and the 6-position can accommodate a variety of alkyl groups. 1 and its analogues bind to all of the three antiapoptotic Bcl-2 proteins tested. Positive correlations were observed between the binding affinities of these candidates to the antiapoptotic Bcl-2 proteins and their in vitro cytotoxicities, suggesting that the antiapoptotic Bcl-2 proteins are likely to be the cellular targets of 1 and its analogues. (In this study, the binding interactions of the small molecules to antiapoptotic Bcl-2 proteins were studied by assaying their abilities to compete against a Bak peptide binding to the antiapoptotic Bcl-2 proteins. Inhibitory constants, instead of dissociation constants, were obtained in such assays. The term "binding affinity" is used in this article for simplicity.) The most active compound, 3g, had a >3-fold increase of binding affinity to the antiapoptotic Bcl-2 proteins and a >13-fold increase of in vitro cytotoxicity over 1. Though Jurkat cells with transgenic overexpression of Bcl-2 or Bcl-XL protein can develop resistance to standard cancer therapies, such cells failed to develop resistance to 1 based candidates. 1 also sensitizes Jurkat cells to cisplatin. These studies provide further support that 1 and its analogues function as antagonists for antiapoptotic Bcl-2 proteins and that they have the potential, either as a single agent or as a combination therapy with other anticancer agents, to treat cancers with the overexpression of antiapoptotic Bcl-2 proteins.

A phase I study of G3139 in combination with RICE chemotherapy in relapsed non-Hodgkin’s lymphoma (NHL)

17529 Background: Aggressive lymphomas are chemosensitive, but frequently relapse. Bcl-2 protein overexpression promotes tumor cell survival via anti-apoptosis, and is clinically correlated with shortened remission duration and decreased survival. The antisense oligonucleotide oblimersen sodium (G3139) downregulates bcl-2, has activity in NHL, and may be synergistic with chemotherapy. Methods: Pts had histologically confirmed aggressive NHL that relapsed after one prior anthracycline-based regimen. G3139 was given as a continuous infusion on Days 1–8 (168 hours) every 14 days, with stem cell collection following cycle 3. Standard RICE chemotherapy was given on Days 4–6, allowing 72 hrs pre- and 48 hrs post-chemo G3139. Dose-limiting toxicity (DLT) was defined as grade 3 or greater non-hematologic toxicity, grade 4 thrombocytopenia, duration of neutropenia <500/mL greater than 7 days despite growth factors, grade 3 hemolytic anemia, or prolonged severe anemia. Pharmacodynamic endpoints included measurement of bcl-2 protein in peripheral blood (FACS analysis) and tumor (IHC), and bcl-2 RT-PCR in blood and tumor. Results: 8 pts were enrolled. At dose level 1 (G3139 3 mg/kg/day), both enrolled pts had grade 4 thrombocytopenia. The next 6 patients were treated with G3139 2.5 mg/kg/day (Dose Level -1) without any DLT’s. There were no grade 3 or 4 non-hematologic toxicities. 7 evaluable pts had successful stem cell collections: median CD34+ 4.2 × 106/kg (range, 1.3–8.72 × 106/kg). Responses: 4 CR/CRu, 2 PR, 1 SD, 1 too early. There was no detectable decrease in bcl-2 protein in the blood using flow cytometry or RT-PCR. Conclusion: The maximum tolerated dose of G3139 given over 8 days in combination with RICE chemotherapy is 2.5 mg/kg/day. It is unlikely that this dose sufficiently suppresses bcl-2 levels. The protocol will thus be amended to shorten the G3139 infusion to 5 days with renewed efforts to escalate the dose. No significant financial relationships to disclose.

7-Bromoindirubin-3′-oxime induces caspase-independent cell death

Indirubin, an isomer of indigo, is a reported inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase-3 (GSK-3) as well as an agonist of the aryl hydrocarbon receptor (AhR). Indirubin is the active ingredient of a traditional Chinese medicinal recipe used against chronic myelocytic leukemia. Numerous indirubin analogs have been synthesized to optimize this promising kinase inhibitor scaffold. We report here on the cellular effects of 7-bromoindirubin-3'-oxime (7BIO). In contrast to its 5-bromo- and 6-bromo- isomers, and to indirubin-3'-oxime, 7BIO has only a marginal inhibitory activity towards CDKs and GSK-3. Unexpectedly, 7BIO triggers a rapid cell death process distinct from apoptosis. 7-Bromoindirubin-3'-oxime induces the appearance of large pycnotic nuclei, without classical features of apoptosis such as chromatin condensation and nuclear fragmentation. 7-Bromoindirubin-3'-oxime-induced cell death is not accompanied by cytochrome c release neither by any measurable effector caspase activation. Furthermore, the death process is not altered either by the presence of Q-VD-OPh, a broad-spectrum caspase inhibitor, or the overexpression of Bcl-2 and Bcl-XL proteins. Neither AhR nor p53 is required during 7BIO-induced cell death. Thus, in contrast to previously described indirubins, 7BIO triggers the activation of non-apoptotic cell death, possibly through necroptosis or autophagy. Although their molecular targets remain to be identified, 7-substituted indirubins may constitute a new class of potential antitumor compounds that would retain their activity in cells refractory to apoptosis.

Chemosensitizing multiple drug resistance of human carcinoma by bicyclol involves attenuated P-glycoprotein, GST-P and Bcl-2

Bicyclol, a second generation of synthetic hepatoprotectant being used in China for anti hepatitis therapy, shows chemosensitizing effect on reverting multiple drug resistance (MDR) of cytostatic agents in two established MDR carcinoma cell lines, vincristine resistant human stomatic epidermoid carcinoma VinRKB and adriamycin resistant human breast carcinoma AdrRMCF-7. The reversal rate of drug resistance was calculated from the changes of the IC50 of cell growth inhibition. Bicyclol at the concentration of 25, 50, 100 mM induced 2.8 7.3 and 20.7 fold, respectively, reversal of vincristine resistance in VinRKB cell. Bicyclol also reversed the cross-resistance of VinRKB cell to taxol and AdrRMCF-7 cell resistance to adriamycin at the similar range of potency. Further, Bicyclol recovered the reduced accumulation of adriamycin in AdrRMCF-7 cell partially to the level in drug-sensitive MCF-7 cell, indicate the inhibition of MDR related membrane efflux pump system. Overexpression of membrane p-glycoprotein coded by Mdr-1 genes, the most common efflux pump correlated to MDR, was found in both VinRKB and AdrRMCF-7 cells by Western blot and immunocytochemistry as compared with drug-sensitive cells. The p-glycoprotein was decreased to the levels in drug-sensitive cells when VinRKB and AdrRMCF-7 cells were treated with Bicyclol for 12-72 hours. Both VinRKB and AdrRMCF-7 cells showed increased GSH contents, and AdrRMCF-7 cell showed increased GST activity and the overexpression of Bcl-2 protein, by which molecules are tightly related to the MDR formation besides Mdr-1 p-glycoprotein. Bicyclol reduced the GSH contents, GST activities and Bcl-2 expression. All these data demonstrate that, by modifying the expressions of Mdr-1, GSH/GST and Bcl-2, Bicyclol increases the intracellular drug concentration and sensitizes the resistant cells to the anti-carcinoma agents.

A case of malignant pheochromocytoma with early intense uptake and immediate rapid washout of99mTc-tetrofosmin characterizing the overexpression of anti-apoptotic Bcl-2

Defective apoptotic program due to the overexpression of the anti-apoptotic Bcl-2 protein of the outer mitochondrial membrane may be a cause of the poor response of malignant pheochromocytoma to 131I-MIBG therapy. We report a case of malignant pheochromocytoma which showed early intense uptake and immediate rapid washout of 99mTc-tetrofosmin characterizing the overexpression of anti-apoptotic Bcl-2 and which was refractory to 131I-MIBG therapy.

Identification of Ebp1 as a component of cytoplasmicbcl-2mRNP (messenger ribonucleoprotein particle) complexes

The 3'-UTR (untranslated region) of bcl-2 mRNA contains an ARE (AU-rich element) that potentially regulates the stability of bcl-2 mRNA in a cell specific fashion. Previous studies have demonstrated that multiple proteins interact with bcl-2 mRNA in HL-60 (human leukaemia-60) cells, potentially contributing to the overexpression of Bcl-2 protein. Treatment of HL-60 cells with taxol or okadaic acid has been shown to induce destabilization of bcl-2 mRNA, which was associated with decreased binding of trans-acting factors to bcl-2 mRNA. Nucleolin has been identified as one of the bcl-2 mRNA-binding proteins [Sengupta, Bandyopadhyay, Fernandes and Spicer (2004) J. Biol. Chem. 279, 10855-10863]. In an effort to identify additional bcl-2 mRNA-binding proteins, two polypeptides of approx. 45 kDa and 60 kDa were isolated from HL-60 cells by ARE(bcl-2) (transcripts that contain bcl-2 AREs) RNA affinity chromatography. These proteins were identified as the human proliferation associated protein, Ebp1, and human DRBP76 (double stranded RNA-binding protein 76) respectively, by MALDI (matrix-assisted laser-desorption ionization)-MS. RNA electrophoretic mobility shift assays indicated that recombinant Ebp1 binds to ARE(bcl-2) RNA but not to the group 1 ARE present in GM-CSF (granulocyte macrophage-colony stimulating factor) mRNA in vitro. Antibody supershift assays demonstrated that Ebp1 is present in protein-ARE(bcl-2) RNA complexes formed with cytosolic HL-60 extracts. The interaction of Ebp1 with bcl-2 mRNA in HL-60 cells was also demonstrated by RNA co-immunoprecipitation assays. This interaction was not detected in extracts of taxol-treated HL-60 cells. Immunoprecipitation assays further revealed that Ebp1 co-precipitates with nucleolin from HL-60 cytoplasmic extracts. The observation that co-precipitation was decreased when extracts were treated with RNase suggests that Ebp1 and nucleolin are present in the same bcl-2 mRNP (messenger ribonucleoprotein particle) complexes. RNA-decay assays further demonstrated that Ebp1 decreased the rate of decay of beta-globin-ARE(bcl-2) transcripts in HL-60 cell extracts. Collectively, these results indicate a novel function for Ebp1 in contributing to the regulation of bcl-2 expression in HL-60 cells.

Effect of 3-aminobenzamine on Bcl-2, Bax and AIF localization in hippocampal neurons altered by ischemia-reperfusion injury. The immunocytochemical study

Poly(ADP-ribose) polymerase plays an important role in cell survival and death. Our previous histological and ultrastructural studies showed that PARP inhibitor 3-aminobenzamide (3-AB) protected neurons against death after ischemia. In this study we investigated the effect of 3-AB on the localization and expression of apoptosis inducing factor (AIF) and on two proteins from Bcl-2 family: Bcl-2 and Bax in hippocampal area CA1, on the 4th day after 3 min of forebrain ischemia in gerbils. Our results indicated that after ischemia AIF is preferentially translocated from the mitochondria to the cytoplasm and to the nucleus. Intravenous administration of 3-AB (30 mg/kg b.w.) prevents AIF translocation to the nucleus. AIF was mainly seen in the structurally unchanged mitochondria and Golgi complex. Moreover, after 3-AB administration overexpression of Bcl-2 protein was observed in mitochondrial membranes, rough endoplasmatic reticulum, Golgi complex, nuclear envelopes, and also in cytoplasm and in nucleus. These data suggest that inhibition of PARP activity may have a beneficial effect on hippocampal neurons through overexpression of Bcl-2 protein and suppression of AIF translocation to the nucleus.

Bcl-2 overexpression prevents calcium overload and subsequent apoptosis in dystrophic myotubes

Duchenne muscular dystrophy (DMD) is a lethal disease caused by the lack of the cytoskeletal protein dystrophin. Altered calcium homoeostasis and increased calcium concentrations in dystrophic fibres may be responsible for the degeneration of muscle occurring in DMD. In the present study, we used subsarcolemmal- and mitochondrial-targeted aequorin to study the effect of the antiapoptotic Bcl-2 protein overexpression on carbachol-induced near-plasma membrane and mitochondrial calcium responses in myotubes derived from control C57 and dystrophic (mdx) mice. We show that Bcl-2 overexpression decreases subsarcolemmal and mitochondrial calcium overload that occurs during activation of nicotinic acetylcholine receptors in dystrophic myotubes. Moreover, our results suggest that overexpressed Bcl-2 protein may prevent near-plasma membrane and mitochondrial calcium overload by inhibiting IP3Rs (inositol 1,4,5-trisphosphate receptors), which we have shown previously to be involved in abnormal calcium homoeostasis in dystrophic myotubes. Most likely as a consequence, the inhibition of IP3R function by Bcl-2 also inhibits calcium-dependent apoptosis in these cells.

Pomolic acid may overcome multidrug resistance mediated by overexpression of anti-apoptotic Bcl-2 proteins

Multidrug resistance (MDR) is multifactorial and may be mediated by overexpression of anti-apoptotic proteins. This paper investigated whether pomolic acid (PA) was able to overcome resistance mediated by overexpression of Bcl-2 or BcL-xL. The results obtained showed that overexpression of these proteins partially inhibited the PA-induced apoptosis, loss of mitochondrial membrane potential and caspase -3 and -9 activation observed in HL-60/neo. Since Bcl-2 transfected cell lines were shown to be quite resistant to a series of chemotherapeutic agents, the data presented call attention to the possible clinical significance of PA as an anti-MDR drug.

Overexpression of Bcl-2 Protein in Bronchoalveolar Lavage Lymphocytes and Macrophages in Sarcoidosis

Background and Objectives: Apoptosis, also known as programmed cell death, probably correlates with the pathophysiologic mechanisms of alveolitis in sarcoidosis. Our purpose was to investigate any changes in the expression of the antiapoptotic protein Bcl-2, one of the most important inhibiting factors of apoptosis, in bronchoalveolar lavage fluid (BALF) cell populations in patients with sarcoidosis. Subjects and Methods: Fiberoptic bronchoscopy with BAL was performed in 13 patients with active sarcoidosis (10 patients with stage I and 3 with stage II disease based on chest radiography). None of them was under treatment with corticosteroids. Cellular Bcl-2 expression was identified using an immunoperoxidase staining method. Ten normal subjects served as control group. Results: BALF lymphocytes and macrophages in sarcoid patients exhibited a significant increase in the expression of Bcl-2 compared with the control group (p < 0.001). A Bcl-2 expression of 80.7 ± 8.5% in the lymphocytes and 77.4 ± 8.9% in the macrophages was observed in sarcoidosis versus 32.2 ± 13.8% and 19.6% ± 7.6% in normal subjects, respectively. The percentages of Bcl-2 expression in the lymphocytes were positively correlated with BALF CD4/CD8 ratio values (p = 0.02, r = 0.63). Conclusions: Our results suggest that the antiapoptotic protein Bcl-2 is overexpressed in alveolar lymphocytes and macrophages, which characterize the alveolitis in sarcoidosis and could provide insights into the pathogenesis of the disease and prove useful as a marker of disease activity or response to therapy.

The Apoptotic Effect of Brucine from the Seed of Strychnos nux-vomica on Human Hepatoma Cells is Mediated via Bcl-2 and Ca2+ Involved Mitochondrial Pathway

In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on human hepatoma cells (HepG2) were screened by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca2+], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca2+]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.

Bcl-2 siRNA Induces Apoptosis and Enhances the Sensitivity to Arsenic Trioxide in NB4 Cells.

Bcl-2 is the prominent member of a family of proteins responsible for dysregulation of apoptosis, prevention of cancer-cell death, and resistance to chemotherapy and radiotherapy. Overexpression of Bcl-2 protein was shown to result in resistance to a variety of apoptosis-inducing signals in acute leukemia. Arsenic trioxide (As203)induces clinical remission in acute promyelocytic leukemia with minimal toxicity and apoptosis in APL-derived NB4 cells at low (0.5 to 2 μmol/L) concentration. Short interfering RNA (siRNA) has been evaluated as an attractive and effective tool for suppressing a target protein by specifically digesting its mRNA. In this study, we studied the effect of transfection with siRNA targeting Bcl-2 in NB4 cells in terms of proliferation and induction of apoptosis. Further,we investigated whether the Bcl-2 siRNA may enhance the sensitivity of NB4 cells to arsenic trioxide. Synthetic siRNA was transferred against Bcl-2 into NB4 cells using Oligofectamine transfection protocol in the presence or absence of arsenic trioxide. RT-PCR and Western blotting analysis of Bcl-2 expression in NB4 cells was performed after transfection. The inhibition of cell growth was assessed by a modified MTT assay, counting cell number. Apoptosis was determined by morphological observation and flow cytometric analysis. When synthetic Bcl-2 siRNA was delivered into NB4 cells, no Bcl-2 transcripts were detected at 24h, and Bcl-2 protein levels were reduced by approximately 72% for 48h after siRNA transfer. Bcl-2 siRNA treatment for 72h led to 36% apoptosis of cells,as compared to control siRNA treatment. Control siRNA had no significant effect on growth and apoptosis of cells. Simultaneous administration of As203 and Bcl-2 siRNA was able to reduce the mRNA and protein expression of Bcl-2 significantly compared to Bcl-2 siRNA alone demonstrating a synergistic effect of combined As203 and siRNA treatment (P<0.05). Proliferation in NB4 cells was strongly inhibited (about 86%) following combination treatment for 72h as opposed to only 40% after administration of 0.5μmol/L As203 alone. When NB4 cells were incubated with 0.5μmol/L As203 for 72h after siRNA transfer, the percentage of apoptotic cells significantly increased (P<0.05), compared with combined As203 and control siRNA treatment or As203 treatment alone, respectively. No significant difference on apoptosis was seen in cells treated with As203 alone or treated with As203 and control siRNA. These results show that Bcl-2 siRNA could induce apoptosis and enhance the sensitivity to arsenic trioxide in NB4 cells. Our results suggest that the combination of arsenic trioxide and Bcl-2 siRNA might be an effective anti-leukemia protocol.