Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells

The antineoplastic agent paclitaxel (TaxolTM), a microtubule stabilizing agent, is known to arrest cells at the G2/M phase of the cell cycle and induce apoptosis. We and others have recently demonstrated that paclitaxel also activates the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) signal transduction pathway in various human cell types, however, no clear role has been established for JNK/SAPK in paclitaxel-induced apoptosis. To further examine the role of JNK/SAPK signaling cascades in apoptosis resulting from microtubular dysfunction induced by paclitaxel, we have coexpressed dominant negative (dn) mutants of signaling proteins of the JNK/SAPK pathway (Ras, ASK1, Rac, JNKK, and JNK) in human ovarian cancer cells with a selectable marker to analyze the apoptotic characteristics of cells expressing dn vectors following exposure to paclitaxel. Expression of these dn signaling proteins had no effect on Bcl-2 phosphorylation, yet inhibited apoptotic changes induced by paclitaxel up to 16 h after treatment. Coexpression of these dn signaling proteins had no protective effect after 48 h of paclitaxel treatment. Our data indicate that: (i) activated JNK/SAPK acts upstream of membrane changes and caspase-3 activation in paclitaxel-initiated apoptotic pathways, independently of cell cycle stage, (ii) activated JNK/SAPK is not responsible for paclitaxel-induced phosphorylation of Bcl-2, and (iii) apoptosis resulting from microtubule damage may comprise multiple mechanisms, including a JNK/SAPK-dependent early phase and a JNK/SAPK-independent late phase.     

Dissociation of Bax from a Bcl-2/Bax heterodimer triggered by phosphorylation of serine 70 of Bcl-2.

Serine 70 in the loop region of Bcl-2 is specifically phosphorylated by paclitaxel-treatment in tumor cells and BHK cells expressing Bcl-2. The phosphorylation of serine 70 of Bcl-2 (pS70-Bcl-2) peaks 24 to 48 h after paclitaxel treatment and accelerates apoptosis. Phosphorylation is effectively inhibited in the presence of actinomycin D or cycloheximide, which restore cell viability to the same level as control cells not expressing Bcl-2. These results indicate that paclitaxel-induced kinase(s) and/or its activator(s) are synthesized de novo and play an important role in paclitaxel-induced apoptosis by phosphorylating Bcl-2. In binding assays using the phosphorylation-specific antibody against pS70-Bcl-2, the induction of serine 70 phosphorylation 70 results in a loss of the binding ability of Bcl-2 to Bax, a pro-apoptotic partner, and induces subsequent cell death. When the pS70-Bcl-2 antibody was added to human breast cancer tissue, serine 70 phosphorylation was also detected, even prior to treatment with anticancer agents. Further study of breast cancers revealed 83% of tumors with high pS70-Bcl-2 expression responded to paclitaxel or docetaxel treatment, whereas 57% of those with low expression not respond. These findings suggest that pS70-Bcl-2 might be a predictive factor for prognosis and sensitivity to paclitaxel treatment for breast cancer.     

Ran suppresses paclitaxel-induced apoptosis in human glioblastoma cells

Yeast-based functional screening of a human glioblastoma cDNA library identified ras-related nuclear protein (Ran) as a novel suppressor of Bcl-2-associated X protein (Bax), a pro-apoptotic member of the Bcl-2 family of proteins. Yeast cells that expressed human Ran were resistant to Bax-induced cell death. In U373MG glioblastoma cells, stable overexpression of Ran significantly attenuated apoptotic cell death induced by the chemotherapeutic agent paclitaxel. FACS analysis demonstrated that Ran is involved in paclitaxel-induced cell cycle arrest. Stable overexpression of Ran also markedly inhibited the phosphorylation of Bcl-2 by paclitaxel, and inhibited the translocation of Bax, the release of cytochrome c and activation of caspase-3. Paclitaxel-induced phosphorylation of c-JUN N-terminal kinase (JNK), but not p38, extracellular signal-regulated kinase and Akt, was markedly suppressed in U373MG cells that stably expressed Ran. These results suggest that Ran suppresses paclitaxel-induced cell death through the downregulation of JNK-mediated signal pathways. Im Sun Woo and Han-Su Jang contributed equally to this work.     

Bim is reversibly phosphorylated but plays a limited role in paclitaxel cytotoxicity of breast cancer cell lines

The chemotherapeutic drug, paclitaxel, induces mitotic arrest and then activates the cellular apoptotic program. Although paclitaxel has been in clinical use for over 10 years for the treatment of breast, ovarian, and lung cancer, the molecular mechanisms of paclitaxel-induced cytotoxicity are ill defined. We decided to investigate the regulatory mechanism of the pro-apoptotic BH3-only protein Bim, which is known to play a role in paclitaxel cytotoxicity. We discovered that paclitaxel induces reversible phosphorylation of Bim. Bim initially displays enhanced phosphorylation during paclitaxel-induced mitotic arrest, and then undergoes de-phosphorylation as cells become apoptotic. This dynamic phosphorylation is dependent on mitotic checkpoint signaling. However, while these results suggest that reversible phosphorylation of Bim may contribute to the transmission of a mitotic checkpoint-to-apoptosis signal, we did not observe a strong correlation between Bim protein levels and cellular sensitivity to paclitaxel. Indeed, in contrast to the well-defined role of Bim in paclitaxel-induced cell death in mouse model cells, our depletion studies demonstrate that Bim is not absolutely required for paclitaxel cytotoxicity in breast cancer cell lines. Clearly it is imperative to define the contribution of Bim in paclitaxel-induced apoptosis of clinically relevant targets in order to rationally develop enhanced treatment strategies.     

Akt is upstream and MAPKs are downstream of NF-κB in paclitaxel-induced survival signaling events, which are down-regulated by curcumin contributing to their synergism

Paclitaxel is the most promising chemotherapeutic agent of plant origin despite its high cost and dose-limiting toxicity. Our earlier report has shown that cervical cancer cells can be sensitized by curcumin to paclitaxel-induced apoptosis through down-regulation of NF-κB and Akt. In the present study we have attempted to decipher the signaling pathways regulating the synergism of paclitaxel and curcumin. The study has clearly proved that Akt and NF-κB function successively in the sequence of paclitaxel induced signaling events where Akt is upstream of NF-κB. While inhibition of NF-κB led to complete inhibition of the synergism of paclitaxel and curcumin, inhibition of Akt brought about only partial reduction of the same, suggesting that, apart from Akt, there are other pathways induced by paclitaxel leading to NF-κB activation, which are also down-regulated by curcumin. Inactivation of NF-κB did not affect the activation of Akt and survivin, while that of Akt significantly inhibited NF-κB and completely inhibited up-regulation of survivin. Up-regulation of Cyclin-D1, Cox-2, XIAP and cIAP1 and phosphorylation of MAPKs, were completely inhibited on inactivation of NF-κB assigning a key regulatory role to NF-κB in the synergistic effect of paclitaxel and curcumin. While up-regulation of survivin by paclitaxel is regulated by Akt, independent of NF-κB, inactivation of neither Akt nor NF-κB produced any change in Bcl-2 level suggesting a distinct pathway for its action. As curcumin could effectively down-regulate all these survival signals induced by paclitaxel, we suggest it as a potent chemosensitizer to improve the therapeutic index of paclitaxel.     

The Cyclin-Dependent Kinase Inhibitor p21CIP1/WAF1 Blocks Paclitaxel-Induced G2M Arrest and Attenuates Mitochondrial Injury and Apoptosis in p53-Null Human Leukemia Cells

The functional significance of the cyclin-dependent kinase inhibitor (CDKI) p21Cip1/WAF1 in paclitaxel-mediated lethality was examined in p53-null human leukemia cells (U937 and Jurkat). In these cells, paclitaxel exposure failed to induce p21Cip1/Waf1 expression. Nevertheless, stable expression of U937 cells with a p21Cip1/WAF1 antisense construct blocked paclitaxel-induced G2M arrest and significantly, albeit modestly, increased mitochondrial injury, caspase activation, apoptosis, and loss of clonogenic potential. These protective effects were less than those observed in cells exposed to the antimetabolite ara-C. Consistent with these results, enforced expression of p21Cip1/WAF1 in Jurkat cells transfected with a construct driven by a doxycycline-responsive promoter increased the percentage of cells arrested in G2M, but attenuated paclitaxel-mediated mitochondrial injury and apoptosis. Unexpectedly, enforced expression of p21Cip1/WAF1 diminished paclitaxel-mediated inactivation of ERK, and reduced paclitaxel-induced activation of JNK as well as Bcl-2 phosphorylation. Together, these findings suggest that the CDKI p21Cip1/WAF1 modestly but significantly protects p53-null human leukemia cells from paclitaxel-mediated lethality, and raise the possibility that p21Cip1/WAF1-associated perturbations in signal transduction pathways as well as Bcl-2 phosphorylation status may play a role in this phenomenon.     

紫杉醇对NK-92MI细胞杀伤功能及凋亡的影响

探讨紫杉醇对人自然杀伤细胞系NK-92MI细胞株杀伤活性及凋亡的影响。用不同浓度的紫杉醇处理NK.92MI细胞后,测定NK-92MI细胞的增殖率和对靶细胞的杀伤作用;同时检测NK·92MI细胞凋亡率及凋亡相关基因Bcl-2、Ba】【的表达。结果表明,紫杉醇可以抑制NK-92MI细胞增殖并且降低其杀伤活性,机制可能是通过诱导NK.92MI细胞的凋亡,而后者与Bcll2/Ba】【基因表达增高有关。     

p27kip1 overexpression promotes paclitaxel-induced apoptosis in pRb-defective SaOs-2 cells

p27kip1 is a cyclin-dependent kinase (CDK) inhibitor, which controls several cellular processes in strict collaboration with pRb. We evaluated the role of p27kip1 in paclitaxel-induced apoptosis in the pRb-defective SaOs-2 cells. Following 48 h of exposure of SaOs-2 cells to 100 nM paclitaxel, we observed an increase in p27kip1 expression caused by the decrease of the ubiquitin-proteasome activity. Such increase was not observed in SaOs-2 cells treated with the caspase inhibitors Z-VAD-FMK, suggesting that p27kip1 enhancement at 48 h is strictly related to apoptosis. Finally, we demonstrated that SaOs-2 cells transiently overexpressing the p27kip1 protein are more susceptible to paclitaxel-induced apoptosis than SaOs-2 cells transiently transfected with the empty vector. Indeed, after 48 h of paclitaxel treatment, 41.8% of SaOs-2 cells transiently transfected with a pcDNA3-p27kip1 construct were Annexin V-positive compared to 30.6% of SaOs-2 cells transfected with the empty vector (P < 0.05). In conclusion, we demonstrated that transfection of the pRb-defective SaOs-2 cells with the p27kip1 gene via plasmid increases their susceptibility to paclitaxel-induced apoptosis. The promoting effect of p27kip1 overexpression on apoptosis makes p27kip1 and proteasomal inhibitors interesting tools for therapy in patients with pRb-defective cancers.     

Increased cyclin B1/CDC 2 kinase activity and phosphorylation of Bcl-2 associated with paclitaxel-induced apoptosis in human nasopharyngeal carcinoma cells

Paclitaxel is a potential cancer chemotherapeutic agent for ovary, breast, and head and neck cancers; its effects on nasopharyngeal carcinoma (NPC) have not been reported previously. This study investigated the cytotoxic mechanism of paclitaxel in two NPC cell lines, NPC-TW01 and NPC-TW04. NPC cells treated with pacli-taxel showed convoluted nuclei, condensed chromatin and decreased cellular and nuclear volume, and also exhibited genomic DNA degradation into multiple oligonucleosomal fragments, suggesting that pacli-taxel induced apoptosis in these cells. The effects of paclitaxel on apoptosis-related proteins including Bcl-2, Bax and CDC 2 were also detected. Although the levels of Bcl-2 and Bax were not changed in NPC cells following treatment with 5 nM-1 μM of paclitaxel, phosphorylation of Bcl-2 was significantly observed in the cells treated with 1 μM of paclitaxel for 12 hours. In addition, cyclin B1-associated CDC 2 kinase was highly activated in the NPC cells exposed to paclitaxel even at low (5 nM) concentration, and this result is associated with the finding that low concentration of paclitaxel is able to induce apoptosis in NPC cells.     

Role of JNK activation in apoptosis： A double-edged sword

JNK is a key regulator of many cellular events, including programmed cell death (apoptosis). In the absence of NF-κB activation, prolonged JNK activation contributes to TNF-α induced apoptosis. JNK is also essential for UV induced apoptosis. However, recent studies reveal that JNK can suppress apoptosis in IL-3-dependent hematopoietic cells via phosphorylation of the proapoptotic Bcl-2 family protein BAD. Thus, JNK has pro- or antiapoptotic functions, depending on cell type, nature of the death stimulus, duration of its activation and the activity of other signaling pathways.     

Multiple Signaling Pathways of the Insulin-Like Growth Factor 1 Receptor in Protection from Apoptosis

The type 1 insulin-like growth factor receptor (IGF-1R), activated by its ligands, protects several cell types from a variety of apoptotic injuries. The main signaling pathway for IGF-1R-mediated protection from apoptosis has been previously elucidated and rests on the activation of phosphatidylinositol 3-kinase, Akt/protein kinase B, and the phosphorylation and inactivation of BAD, a member of the Bcl-2 family of proteins. In 32D cells (a murine hemopoietic cell line devoid of insulin receptor substrate 1 [IRS-1]), the IGF-1R activates alternative pathways for protection from apoptosis induced by withdrawal of interleukin-3. One of these pathways leads to the activation of mitogen-activated protein kinase, while a third pathway results in the mitochondrial translocation of Raf and depends on the integrity of a group of serines in the C terminus of the receptor that are known to interact with 14.3.3 proteins. All three pathways, however, result in BAD phosphorylation. The presence of multiple antiapoptotic pathways may explain the remarkable efficacy of the IGF-1R in protecting cells from apoptosis.     

Requirement of the JNK-associated Bcl-2 pathway for human lactoferrin-induced apoptosis in the Jurkat leukemia T cell line

The cell proliferation of p53-deficient Jurkat T cells is controlled after prolonged exposure to human lactoferrin (Lf). However, the molecular mechanism by which Lf influences these cellular responses remains unclear. In this study, we demonstrate that Lf-induced apoptosis in Jurkat T cells occurs in a dose- and time-dependent manner via the regulation of c-Jun N-terminal kinase (JNK) activity. Jurkat cells exposed to Lf for 1 day, especially at concentrations in excess of 500 microg/ml, showed typical apoptosis, as indicated by decreased cell viability and increased Annexin V binding. Our results also showed that Lf induced the activation of caspase 9 and caspase 3 activation, as demonstrated by our detection of cleaved caspases and PARP. Lf-induced apoptosis did not influence Bcl-2 expression via an ERK1/2 phosphorylation pathway, but was rather associated with the level of Bcl-2 phosphorylation. The treatment of cells with the specific JNK inhibitor SP600125, but not the p38 MAPK inhibitor SB203580, revealed that the JNK-Bcl-2 signaling cascade is required for Lf-induced apoptosis. When JNK activation was abolished by SP600125, no Bcl-2 phosphorylation was detected, and the Lf-treated Jurkat cells did not undergo cell death. These findings indicate that Lf functions as a biological mediator of apoptosis in the human leukemia Jurkat T-cell line, via the JNK-associated Bcl-2 signaling pathway.     

PKC signaling prevents irradiation-induced apoptosis of primary human fibroblasts

Primary cells respond to irradiation by activation of the DNA damage response and cell cycle arrest, which eventually leads to senescence or apoptosis. It is not clear in detail which signaling pathways or networks regulate the induction of either apoptosis or senescence. Primary human fibroblasts are able to withstand high doses of irradiation and to prevent irradiation-induced apoptosis. However, the underlying regulatory basis for this phenotype is not well understood. Here, a kinetic network analysis based on reverse phase protein arrays (RPPAs) in combination with extensive western blot and cell culture analyses was employed to decipher the cytoplasmic and nuclear signaling networks and to identify possible antiapoptotic pathways. This analysis identified activation of known DNA damage response pathways (e.g., phosphorylation of MKK3/6, p38, MK2, Hsp27, p53 and Chk1) as well as of prosurvival (e.g., MEK-ERK, cAMP response element-binding protein (CREB), protein kinase C (PKC)) and antiapoptotic markers (e.g., Bad, Bcl-2). Interestingly, PKC family members were activated early upon irradiation, suggesting a regulatory function in the ionizing radiation (IR) response of these cells. Inhibition or downregulation of PKC in primary human fibroblasts caused IR-dependent downregulation of the identified prosurvival (CREB phosphorylation) and antiapoptotic (Bad phosphorylation, Bcl-2) markers and thus lead to a proliferation stop and to apoptosis. Taken together, our analysis suggests that cytoplasmic PKC signaling conditions IR-stressed MRC-5 and IMR-90 cells to prevent irradiation-induced apoptosis. These findings contribute to the understanding of the cellular and nuclear IR response and may thus eventually improve the efficacy of radiotherapy and help overcome tumor radioresistance.     

Insulin-like growth factor binding protein-3 mediates tumor necrosis factor-alpha-induced apoptosis: role of Bcl-2 phosphorylation.

The insulin-like growth factor (IGF)-independent effects of insulin-like growth factor binding protein-3 (IGFBP-3) to effect cellular apoptosis have now been described in various cellular systems. IGFBP-3 mediates transforming growth factor-beta-induced apoptosis. Other growth-inhibitory and apoptosis-inducing agents such as tumor necrosis factor-alpha (TNF-alpha) and the tumor suppressor gene p53 also induce IGFBP-3. In this report, we demonstrate the role of IGFBP-3 as a mediator of apoptosis induced by TNF-alpha and elucidate the process involved in its signaling mechanism. Treatment of PC-3 cells with TNF-alpha resulted in the induction of IGFBP-3 expression in a dose- and time-dependent fashion and also induced apoptosis. TNF-alpha-induced apoptosis was prevented by cotreatment with IGFBP-3 neutralizing antibodies or IGFBP-3-specific antisense thiolated oligonucleotides. Both IGFBP-3 and TNF-alpha treatment increased the levels of the inactive, serine phosphorylated form of the survival protein Bcl-2. The effect of TNF-alpha on Bcl-2 serine phosphorylation was blocked by IGFBP-3 antisense oligomers. These findings confirm that IGFBP-3 is essential for TNF-alpha-induced apoptosis in PC-3 cells and that this IGFBP-3 effect includes the inactivation of Bcl-2 through serine phosphorylation.     

Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform

Tumor stroma and growth factors provide a survival environment to tumor cells and can modulate their chemoresistance by dysregulating several signal pathways. In this study, we fabricated a three-dimensional (3D) microfluidic chip using polydimethylsiloxane (PDMS) to investigate the impact of hepatocyte growth factor (HGF) from cancer-associated fibroblasts (CAF) on the Met/PI3K/AKT activation, glucose regulatory protein (GRP78) expression and the paclitaxel-induced A549 cell apoptosis. With a concentration gradient generator, the assembled chip was able to reconstruct a tumor microenvironment in vitro. We found high levels of HGF in the supernatants of CAF and the CAF matrix from the supernatants of activated HFL1 fibroblasts or HGF enhanced the levels of Met, PI3K and AKT phosphorylation and GRP78 expression in A549 cells cultured in a 3D cell chamber, which was abrogated by anti-HGF. Inhibition of Met attenuated the CAF matrix-enhanced PI3K/AKT phosphorylation and GRP78 expression while inhibition of PI3K reduced GRP78 expression, but not Met phosphorylation in A549 cells. Inhibition of GRP78 failed to modulate the CAF matrix-enhanced Met/PI3K/AKT phosphorylation in A549 cells. Furthermore, inhibition of PI3K or GRP78 enhanced spontaneous and paclitaxel-induced A549 cell apoptosis. Moreover, treatment with the CAF matrix inhibited spontaneous and medium or high dose of paclitaxel-induced A549 cell apoptosis. Inhibition of PI3K or GRP78 attenuated the CAF matrix-mediated inhibition on paclitaxel-induced A549 cell apoptosis. Our data indicated that HGF in the CAF matrix activated the Met/PI3K/AKT and up-regulated GRP78 expression, promoting chemoresistance to paclitaxel-mediated apoptosis in A549 cells. Our findings suggest that the microfluidic system may represent an ideal platform for signaling research and drug screening.     

Genistein induces G2/M cell cycle arrest and apoptosis of human ovarian cancer cells via activation of DNA damage checkpoint pathways

Genistein is a major isoflavonoid in dietary soybean, commonly consumed in Asia. Genistein exerts inhibitory effects on the proliferation of various cancer cells and plays an important role in cancer prevention. However, the molecular and cellular mechanisms of genistein on human ovarian cancer cells are still little known. We show that exposure of human ovarian cancer HO-8910 cells to genistein induces DNA damage, and triggers G2/M phase arrest and apoptosis. Furthermore, we also found that checkpoint proteins ATM and ATR are phosphorylated and activated in the cells treated with genistein. It is also shown that genistein increases the phosphorylation and activation of Chk1 and Chk2, which results in the phosphorylation and inactivation of phosphatases Cdc25C and Cdc25A, and thereby the phosphorylation and inactivation of Cdc2 which arrests cells in G2/M phase. Moreover, genistein enhances the phosphorylation and activation of p53, while decreases the ratio of Bcl-2/Bax and Bcl-xL/Bax and the level of phosphorylated Akt, which result in cells undergoing apoptosis. These results demonstrate that genistein-activated ATM-Chk2-Cdc25 and ATR-Chk1-Cdc25 DNA damage checkpoint pathways can arrest ovarian cancer cells in G2/M phase, and induce apoptosis while the cellular DNA damage is too serious to be repaired. Thus, the antiproliferative, DNA damage-inducing and pro-apoptotic activities of genistein are probably responsible for its genotoxic effects on human ovarian cancer HO-8910 cells.     

Phosphorylation and proteasome-dependent degradation of Bcl-2 in mitotic-arrested cells after microtubule damage.

Treatment of NIH-OVCAR-3 cells with paclitaxel, a microtubule-stabilizing agent, induces mitotic arrest and apoptosis, but also Bcl-2 phosphorylation. We report here that Bcl-2 phosphorylation precedes Bcl-2 down-regulation and that both events are closely associated with mitotic arrest, but are not sufficient for paclitaxel to trigger apoptosis. Indeed, when paclitaxel-treated cells were induced to exit mitosis in the presence of 2-aminopurine, Bcl-2 phosphorylation and Bcl-2 down-regulation were both inhibited. In contrast, when apoptosis was inhibited by a caspase inhibitor or Bcl-2 over-expression, Bcl-2 phosphorylation and down-regulation still occurred. Furthermore, we show that Bcl-2 is degraded in mitosis by the proteasome-dependent pathway since Bcl-2 down-regulation is inhibited by proteasome inhibitors such as MG132, Lactacystin and LLnL. Taken together these results indicate that mitotic spindle damage results in post-translational modifications of Bcl-2 by phosphorylation and degradation.     

Blockade of TNFR1 signaling: A role of oscillatory fluid shear stress in osteoblasts

Fluid shear stress protects cells from TNF‐α‐induced apoptosis. Oscillatory fluid shear stress (OFSS) is generally perceived as physiologically relevant biophysical signal for bone cells. Here we identify several cellular mechanisms responsible for mediating the protective effects of OFSS against TNF‐α‐induced apoptosis in vitro. We found that exposure of MC3T3‐E1 osteoblast‐like cells to as little as 5 min of OFSS suppressed TNF‐α‐induced activation of caspase‐3, cleavage of PARP and phosphorylation of histone. In contrast, H₂O₂‐induced apoptosis was not inhibited by OFSS suggesting that OFSS might not be protecting cells from TNF‐α‐induced apoptosis via stimulation of global pro‐survival signaling pathways. In support of this speculation, OFSS inhibition of TNF‐α‐induced apoptosis was unaffected by inhibitors of several pro‐survival signaling pathways including pI3‐kinase (LY294002), MAPK/ERK kinase (PD98059 or U0126), intracellular Ca2+ release (U73122), NO production (L‐NAME), or protein synthesis (cycloheximide) that were applied to cells during exposure to OFSS and during TNF‐α treatment. However, TNF‐α‐induced phosphorylation and degradation of IκBα was blocked by pre‐exposure of cells to OFSS suggesting a more specific effect of OFSS on TNF‐α signaling. We therefore focused on the mechanism of OFSS regulation of TNF‐receptor 1 (TNFR1) signaling and found that OFSS (1) reduced the amount of receptor on the cell surface, (2) prevented the association of ubiquitinated RIP in TNFR1 complexes with TRADD and TRAF2, and (3) reduced TNF‐α‐induced IL‐8 promoter activity in the nucleus. We conclude that the anti‐apoptotic effect of OFSS is not mediated by activation of universal pro‐survival signaling pathways. Rather, OFSS inhibits TNF‐α‐induced pro‐apoptotic signaling which can be explained by the down‐regulation of TNFR1 on the cell surface and blockade of TNFR1 downstream signaling by OFSS. J. Cell. Physiol. 226: 1044–1051, 2011. © 2010 Wiley‐Liss, Inc.     

Concomitant Inhibition of MDM2 and Bcl-2 Protein Function Synergistically Induce Mitochondrial Apoptosis in AML

Disruption of Mdm2-p53 interaction activates p53 signaling, disrupts the balance ofantiapoptotic and proapoptotic Bcl-2 family proteins and induces apoptosis in acutemyeloid leukemia (AML). Overexpression of Bcl-2 may inhibit this effect. Thus,functional inactivation of antiapoptotic Bcl-2 proteins may enhance apoptogenic effects ofMdm2 inhibition. We here investigate the potential therapeutic utility of combinedtargeting of Mdm2 by Nutlin-3a and Bcl-2 by ABT-737, recently developed inhibitors ofprotein-protein interactions. Nutlin-3a and ABT-737 induced Bax conformational changeand mitochondrial apoptosis in AML cells in a strikingly synergistic fashion. Nutlin-3ainduced p53-mediated apoptosis predominantly in S and G₂/M cells, while cells in G₁ were protected through induction of p21. In contrast, ABT-737 induced apoptosis predominantly in G₁ , the cell cycle phase with the lowest Bcl-2 protein levels and Bcl-2/Bax ratios. In addition, Bcl-2 phosphorylation on Ser⁷⁰ was absent in G₁ but detectable in G₂/M, thus lower Bcl-2 levels and absence of Bcl-2 phosphorylation appeared to facilitate ABT-737-induced apoptosis of G₁ cells. The complementary effects of Nutlin-3a and ABT-737 in different cell cycle phases could, in part, account for their synergistic activity. Our data suggest that combined targeting of Mdm2 and Bcl-2 proteins could offer considerable therapeutic promise in AML.