Bcl-2 Phosphorylation Research Articles

Noscapine induces apoptosis in human glioma cells by an apoptosis-inducing factor-dependent pathway

Previously, we identified noscapine as a small molecule inhibitor of the hypoxia-inducible factor-1 pathway in hypoxic human glioma cells and human umbilical vein endothelial cells. Noscapine is a nontoxic ingredient in cough medicine currently used in clinical trials for patients with non-Hodgkin's lymphoma or chronic lymphocytic leukemia to assess antitumor efficacy. Here, we have evaluated the sensitivity of four human glioma cell lines to noscapine-induced apoptosis. Noscapine was a potent inhibitor of proliferation and inducer of apoptosis. Induction of apoptosis was associated with activation of the c-jun N-terminal kinase signaling pathway concomitant with inactivation of the extracellular signal regulated kinase signaling pathway and phosphorylation of the antiapoptotic protein Bcl-2. Noscapine-induced apoptosis was associated with the release of mitochondrial proteins apoptosis-inducing factor (AIF) and/or cytochrome c. In some glioma cell lines, only AIF release occurred without cytochrome c release or poly (ADP-ribose) polymerase cleavage. Knock-down of AIF decreased noscapine-induced apoptosis. Our results suggest the potential importance of noscapine as a novel agent for use in patients with glioblastoma owing to its low toxicity profile and its potent anticancer activity.

Targeting Bcl-2-IP3 Receptor Interaction to Reverse Bcl-2's Inhibition of Apoptotic Calcium Signals

The antiapoptotic protein Bcl-2 inhibits Ca2+ release from the endoplasmic reticulum (ER). One proposed mechanism involves an interaction of Bcl-2 with the inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channel localized with Bcl-2 on the ER. Here we document Bcl-2-IP3R interaction within cells by FRET and identify a Bcl-2 interacting region in the regulatory and coupling domain of the IP3R. A peptide based on this IP3R sequence displaced Bcl-2 from the IP3R and reversed Bcl-2-mediated inhibition of IP3R channel activity in vitro, IP3-induced ER Ca2+ release in permeabilized cells, and cell-permeable IP3 ester-induced Ca2+ elevation in intact cells. This peptide also reversed Bcl-2's inhibition of T cell receptor-induced Ca2+ elevation and apoptosis. Thus, the interaction of Bcl-2 with IP3Rs contributes to the regulation of proapoptotic Ca2+ signals by Bcl-2, suggesting the Bcl-2-IP3R interaction as a potential therapeutic target in diseases associated with Bcl-2's inhibition of cell death.

17α-Estradiol arrests cell cycle progression at G 2/M and induces apoptotic cell death in human acute leukemia Jurkat T cells

A pharmacological dose (2.5–10 μM) of 17α-estradiol (17α-E 2) exerted a cytotoxic effect on human leukemias Jurkat T and U937 cells, which was not suppressed by the estrogen receptor (ER) antagonist ICI 182,780. Along with cytotoxicity in Jurkat T cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9, -3, and -8, PARP degradation, and DNA fragmentation were induced. The cytotoxicity of 17α-E 2 was not blocked by the anti-Fas neutralizing antibody ZB-4. While undergoing apoptosis, there was a remarkable accumulation of G 2/M cells with the upregulatoin of cdc2 kinase activity, which was reflected in the Thr56 phosphorylation of Bcl-2. Dephosphorylation at Tyr15 and phosphorylation at Thr161 of cdc2, and significant increase in the cyclin B1 level were underlying factors for the cdc2 kinase activation. Whereas the 17α-E 2-induced apoptosis was completely abrogated by overexpression of Bcl-2 or by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G 2/M cells significantly increased. The caspase-8 inhibitor z-IETD-fmk failed to influence 17α-E 2-mediated caspase-9 activation, but it markedly reduced caspase-3 activation and PARP degradation with the suppression of apoptosis, indicating the contribution of caspase-8; not as an upstream event of the mitochondrial cytochrome c release, but to caspase-3 activation. In the presence of hydroxyurea, which blocked the cell cycle progression at the G 1/S boundary, 17α-E 2 failed to induce the G 2/M arrest as well as apoptosis. These results demonstrate that the cytotoxicity of 17α-E 2 toward Jurkat T cells is attributable to apoptosis mainly induced in G 2/M-arrested cells, in an ER-independent manner, via a mitochondria-dependent caspase pathway regulated by Bcl-2.

Mechanism of 2‐methoxyestradiol‐induced apoptosis and growth arrest in human breast cancer cells

2-Methoxyestradiol, a well-known nonpolar endogenous metabolite of 17beta-estradiol, has been shown to selectively induce apoptosis in a number of cancer cell lines, but not in normal cells. The mechanism of 2-methoxyestradiol-induced apoptosis appears to vary considerably in different cell lines examined. In the present study, we systematically analyzed the mechanisms of 2-methoxyestradiol-induced apoptosis in the estrogen receptor-negative MDA-MB-435s human breast cancer cells. We found that 2-methoxyestradiol induced the activation of JNK, ERK, and p38 MAPKs. 2-methoxyestradiol-induced JNK activation was associated with the induction of apoptosis through the mitochondrial pathways as a result of increased phosphorylation (inactivation) of the anti-apoptotic Bcl-2 and Bcl-xL proteins. In comparison, 2-methoxyestradiol-induced activation of ERK and p38 in these cells was found to have a protective effect against 2-MeO-E(2)-induced apoptosis. Consistent with this observation, the presence of pharmacological inhibitor of ERK or p38 enhanced 2-methoxyestradiol-induced apoptosis. Mechanistically, inhibition of ERK and p38 activity was associated with activation of various caspases and PARP cleavage, and it also stabilized the pro-apoptotic proteins Bax and Bim, thereby preventing them from degradation during 2-methoxyestradiol treatment. These results suggest that ERK and p38 MAPKs may serve as viable targets for the sensitization of human breast cancer cells to 2-methoxyestradiol-induced apoptosis.

JNK1-Mediated Phosphorylation of Bcl-2 Regulates Starvation-Induced Autophagy

Starvation induces autophagy to preserve cellular homeostasis in virtually all eukaryotic organisms. However, the mechanisms by which starvation induces autophagy are not completely understood. In mammalian cells, the antiapoptotic protein, Bcl-2, binds to Beclin 1 during nonstarvation conditions and inhibits its autophagy function. Here we show that starvation induces phosphorylation of cellular Bcl-2 at residues T69, S70, and S87 of the nonstructured loop; Bcl-2 dissociation from Beclin 1; and autophagy activation. In contrast, viral Bcl-2, which lacks the phosphorylation site-containing nonstructured loop, fails to dissociate from Beclin 1 during starvation. Furthermore, the stress-activated signaling molecule, c-Jun N-terminal protein kinase 1 (JNK1), but not JNK2, mediates starvation-induced Bcl-2 phosphorylation, Bcl-2 dissociation from Beclin 1, and autophagy activation. Together, our findings demonstrate that JNK1-mediated multisite phosphorylation of Bcl-2 stimulates starvation-induced autophagy by disrupting the Bcl-2/Beclin 1 complex. These findings define a mechanism that cells use to regulate autophagic activity in response to nutrient status.

Eradication of metastatic HRBC resistant to trastuzumab and cetuximab after treatment with stealth nanoparticle formulation composed of clamp PNA against mRNA of sGCa1/b1,anti-MUC1 chimeric MAb,and vinorelbine tartrate

14640 Background: For HRBC,the unmet medical need is high due to the lack of treatment options.Overexpression of sGC,MUC1and bcl-2 causes immuno-and chemoresistance. Methods: We obtain surgically stage-IV HRBC cells from patients,characterised by upregulation of MUC1,sGCa1b1,and bcl-2.We synthesized antigene clamp PNA oligomers (DNA analogs),in which an N-C2- aminoethyl-glycine polyamide replaced PO4 ribose ring backbone,and methylene carbonyl linker connected nucleobases to central amine of N- C2-aminoethyl glycine.The 6 mer homopyrimidine triplex [(PNA)2/RNA)] hybridized to the 5’ end (Leader),and the 10 mer purine/pyrimidine duplex (PNA/RNA) hybridized to the 3’-end (Trailer) of the AUG start codon region on the mRNA of sGCa1/b1.The clampPNA anti-sGCa1/b1 was entrapped in the polar phase,and vinorelbine in the lipid phase, surrounded by a stealth polymer layer,and layer with linked chimeric MAbs against MUC1 of the nanoparticle formulation,termed as SEVINA-IV which was administered to xenograft animal models. Results: Post-treatment,downregulation of MUC1 released TKIs by blocking steric hindrance onto HER2/neu,and EGFR.Blocked phosphorylation of MUC1 inhibited Ras/Raf/Erk1/2/MAPK,PI3K/AKT,VEGF,and MMP-2.ADCC was induced.The clamp PNA formed Watson-crick double helices,and steric hindrance of the translation machinery downregulating sGCa1/b1 after assembly inhibition of the 80S ribosome initiation complex.Inhibited translation of NO blocked S-nitrosylation of cystein 118 leading to cGMP-dept downregulation of COX- 2,and NF-kB inducing p53 indpt apoptosis or type I PCD.PTEN was upregulated,and phosphorylation of pRb was blocked,inactivating mTOR,p70 S6K,and eIF-4E of the translational machinery,inhibiting cyclin D1 and ODC.Vinorelbine phosphorylated bcl-2 releasing beclin-1,and inducing type II PCD or autophagy.Also,TEM exhibited type III PCD/necrosis. Conclusion: SEVINA-IV immunochemogene Tx eradicated HRBC after phosphorylation of bcl-2,inhibition of MUC1,translation of sGCa1/b1,and downstream signaling pathways,circumventing resistance to trastuzumab,and cetuximab. No significant financial relationships to disclose.

Mebendazole induces apoptosis in distinct melanoma subtypes via Bcl-2 dependent mechanism

20008 Background: In a screen of 2,000 natural products and marketed drugs, we identified 10 compounds that inhibit growth of chemoresistant, metastatic melanoma cell lines, yet are largely non-toxic to spontaneously immortalized, non-tumorigenic melanocytes. Strikingly, 4 of the 10 compounds belong to the benzimidazole class of structurally related, tubulin-disrupting drugs. Based upon its history of safe oral use and favorable pharmacokinetic profile, mebendazole was prioritized to characterize its mechanism of growth inhibition in several cell lines representing a spectrum of melanoma biological subtypes. Methods: Growth inhibition was assessed by MTT assay in a panel of cell lines: melanocytes (melan-a), nodular melanoma (WM278, WM39), superficial spreading melanoma (WM35, WM1552C), and metastatic melanoma (M-14, SK-Mel-19, Yusac-2). Effects of mebendazole on tubulin structure were analyzed by immunofluorescence microscopy. The apoptotic response of mebendazole-treated cells was characterized by annexin V staining, western blotting, and immunoprecipitation. Bcl-2 protein levels were reduced by siRNA techniques. Results: Mebendazole inhibits growth and induces apoptosis in metastatic and superficial spreading melanoma cells with an IC50 of 0.32μM. However, mebendazole fails to significantly inhibit growth in melanocytes and nodular melanoma cells which display an IC50 of 6-fold higher (2.1 μM). After 18 hours of mebendazole treatment, microtubule structure is disrupted in all cell lines. The apoptosis regulatory protein Bcl-2, a key mediator of microtubule damage, is rapidly phosphorylated in mebendazole-sensitive melanoma cells, but not in resistant cells. Phosphorylation of Bcl-2 reduces its interaction with pro-apoptotic Bax by 46%, thereby promoting apoptosis in mebendazole-sensitive melanoma cells. Mebendazole-resistant cells can be sensitized through reduction of Bcl-2 protein levels. Conclusions: Our data identify Bcl-2 as a critical mediator of the differential cellular response to mebendazole treatment in various biological subtypes of melanoma. Our results also suggest that nodular melanoma may represent a distinct, chemoresistant biological subtype of melanoma. No significant financial relationships to disclose.

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 μg/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.

Cyclin-Dependent Kinase 5 Supports Neuronal Survival through Phosphorylation of Bcl-2

Accumulating evidence indicates that deregulation of cyclin-dependent kinase 5 (Cdk5) activity is associated with apoptosis in various neurodegenerative disease models. Interestingly, recent studies suggest that Cdk5 may also favor neuronal survival. Nonetheless, whether Cdk5 is directly required for neuronal survival during development remains enigmatic. In the current study, we established the pivotal role of Cdk5 in neuronal survival during development by demonstrating that reduction or absence of Cdk5 activity markedly exacerbated neuronal death in cultures and in vivo. Interestingly, the antiapoptotic protein Bcl-2 (B-cell lymphoma protein 2) was identified as a novel substrate of Cdk5. We found that Cdk5-mediated phosphorylation of Bcl-2 at Ser70 was required for the neuroprotective effect of Bcl-2. Intriguingly, inhibition of this phosphorylation conferred proapoptotic property to Bcl-2. Furthermore, overexpression of a Bcl-2 mutant lacking the Cdk5 phosphorylation site abolished the protective effect of Cdk5 re-expression in Cdk5(-/-) neurons, suggesting that Ser70 phosphorylation of Bcl-2 contributed to Cdk5-mediated neuronal survival. Our observations revealed that Cdk5-mediated Bcl-2 phosphorylation is pivotal for the antiapoptotic effect of Bcl-2 and contributes to the maintenance of neuronal survival by Cdk5. Our study has also identified Cdk5 as a regulator of Bcl-2 function in neuronal apoptosis.

The role of p38 MAPK and JNK in Arsenic trioxide‐induced mitochondrial cell death in human cervical cancer cells

Previously, we have shown that the release of AIF from mitochondria is required for As2O3-induced cell death in human cervical cancer cells, and that reactive oxygen species (ROS) is necessary for AIF release from mitochondria. In this study, we further investigated the role of MAPKs in ROS-mediated mitochondrial apoptotic cell death triggered by As2O3. As2O3-induced apoptotic cell death in HeLa cells was associated with activation and mitochondrial translocation of Bax, a marked phosphorylation of Bcl-2, reduction of Bcl-2 and Bax interaction, dissipation of mitochondrial membrane potential. Using small interfering RNA, reduced Bax expression effectively attenuated As2O3-induced mitochondrial membrane potential loss and apoptotic cell death. Moreover, the phosphorylation of Bcl-2 induced by As2O3 diminished its ability to bind to Bax. Treatment of cells with As2O3 activated both the p38 MAPK and JNK pathways. Mitochondrial translocation of Bax was completely suppressed in the presence of p38 MAPK inhibitor PD169316 or si-p38 MAPK. The As2O3-induced Bcl-2 phosphorylation was attenuated largely by JNK inhibition using SP600125 or si-JNK and to some extent by p38 MAPK inhibition with PD169316 or si-p38 MAPK. In addition, N-acetyl-L-cystein (NAC), a thiol-containing anti-oxidant, completely blocked As2O3-induced p38 MAPK and JNK activations, mitochondria translocation of Bax, and phosphorylation of Bcl-2. These results support a notion that ROS-mediated activations of p38 MAPK and JNK in response to As2O3 treatment signals activation of Bax and phosphorylation of Bcl-2, resulting in mitochondrial apoptotic cell death in human cervical cancer cells.

Pretreatment of docetaxel enhances TRAIL‐mediated apoptosis in prostate cancer cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity. TRAIL is known to induce apoptosis in cancer cells but spare most normal cells. In this study, we examined whether treatment of docetaxel (DTX) can enhance apoptotic cell death by TRAIL against androgen-independent prostate cancer (AIPC). The cell death effect of combinations of TRAIL and docetaxel on prostate cancer cell lines (androgen-dependent LNCaP and its derived androgen-independent, metastatic C4-2B) was evaluated by synergisms of apoptosis. Western blot assay and DNA fragmentation assay were used to study the underlying mechanisms of cell death and search for any mechanisms of enhancement of TRAIL induced apoptosis in the presence of docetaxel. In addition, we investigated the in vitro anti-tumor effects of combined docetaxel and TRAIL using MAP kinase inhibitors. Docetaxel itself could not induce apoptotic cell death in 24 h even in high concentration. Apoptotic cell death, however, was drastically enhanced by pretreatment of docetaxel 20 h before TRAIL treatment. Docetaxel enhanced the PARP-1 cleavage and caspases activation by TRAIL especially in androgen-independent, metastatic C4-2B cell line, mainly by phosphorylation of Bcl-2 by JNK activation. It appears that apoptotic cell death was protected by the JNK inhibitor SP600125. The results of our study show that pretreatment of docetaxel is able to enhance the apoptosis produced by TRAIL in prostate cancer cells, especially in hormone-refractory prostate cancer (HRPC).

Mitotic arrest-associated apoptosis induced by sodium arsenite in A375 melanoma cells is BUBR1-dependent

A375 human malignant melanoma cells undergo mitotic arrest-associated apoptosis when treated with pharmacological concentrations of sodium arsenite, a chemotherapeutic for acute promyelocytic leukemia. Our previous studies indicated that decreased arsenite sensitivity correlated with reduced mitotic spindle checkpoint function and reduced expression of the checkpoint protein BUBR1. In the current study, arsenite induced securin and cyclin B stabilization, BUBR1 phosphorylation, and spindle checkpoint activation. Arsenite also increased activating cyclin dependent kinase 1 (CDK1) Thr 161 phosphorylation but decreased inhibitory Tyr15 phosphorylation. Mitotic arrest resulted in apoptosis as indicated by colocalization of mitotic phospho-Histone H3 with active caspase 3. Apoptosis was associated with BCL-2 Ser70 phosphorylation. Inhibition of CDK1 with roscovitine in arsenite-treated mitotic cells inhibited spindle checkpoint maintenance as inferred from reduced BUBR1 phosphorylation, reduced cyclin B expression, and diminution of mitotic index. Roscovitine also reduced BCL-2 Ser70 phosphorylation and protected against apoptosis, suggesting mitotic arrest caused by hyperactivation of CDK1 directly or indirectly leads to BCL-2 phosphorylation and apoptosis. In addition, suppression of BUBR1 with siRNA prevented arsenite-induced mitotic arrest and apoptosis. These findings provide insight into the mechanism of arsenic's chemotherapeutic action and indicate a functional spindle checkpoint may be required for arsenic-sensitivity.

Ionizing radiation utilizes c‐Jun N‐terminal kinase for amplification of mitochondrial apoptotic cell death in human cervical cancer cells

Exposure of cells to ionizing radiation induces activation of multiple signaling pathways that play a critical role in controlling cell death. However, the basis for linkage between signaling pathways and the cell-death machinery in response to ionizing radiation remains unclear. Here we demonstrate that activation of c-Jun N-terminal kinase (JNK) is critical for amplification of mitochondrial cell death in human cervical cancer cells. Exposure of HeLa cells to radiation induced loss of mitochondrial membrane potential, release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, and apoptotic cell death. Radiation also induced transcriptional upregulation of Fas, caspase-8 activation, Bax and Bak activation, and phosphorylation and downregulation of Bcl-2. Inhibition of caspase-8 attenuated Bax and Bak activation, but did not affect phosphorylation and downregulation of Bcl-2. Expression of a mutant form of Bcl-2 (S70A-Bcl-2) completely attenuated radiation-induced Bcl-2 downregulation. Interestingly, inhibition of JNK clearly attenuated radiation-induced Bax and Bak activation, and Bcl-2 phosphorylation as well as Fas expression. In addition, dominant-negative form of c-Jun inhibited radiation-induced Fas expression and Bax and Bak activation. These results indicate that the JNK-c-Jun pathway is required for the transcriptional upregulation of Fas and subsequent activation of Bax and Bak, and that JNK, but not c-Jun, is directly associated with phosphorylation and downregulation of Bcl-2 in response to ionizing radiation. These results suggest that ionizing radiation can utilize JNK for amplification of mitochondrial apoptotic cell death in human cervical cancer cells.

Gα12 is targeted to the mitochondria and affects mitochondrial morphology and motility

G alpha12 constitutes, along with G alpha13, one of the four families of alpha subunits of heterotrimeric G proteins. We found that the N terminus of G alpha12, but not those of other G alpha subunits, contains a predicted mitochondrial targeting sequence. Using confocal microscopy and cell fractionation, we demonstrated that up to 40% of endogenous G alpha12 in human umbilical vein endothelial cells colocalize with mitochondrial markers. N-terminal sequence of G alpha12 fused to GFP efficiently targeted the fusion protein to mitochondria. G alpha12 with mutated mitochondrial targeting sequence was still located in mitochondria, suggesting the existence of additional mechanisms for mitochondrial localization. Lysophosphatidic acid, one of the known stimuli transduced by G alpha12/13, inhibited mitochondrial motility, while depletion of endogenous G alpha12 increased mitochondrial motility. G alpha12Q229L variants uncoupled from RhoGEFs (but not fully functional activated G alpha12Q229L) induced transformation of the mitochondrial network into punctate mitochondria and resulted in a loss of mitochondrial membrane potential. All examined G alpha12Q229L variants reduced phosphorylation of Bcl-2 at Ser-70, while only mutants unable to bind RhoGEFs also decreased cellular levels of Bcl-2. These G alpha12 mutants were also more efficient Hsp90 interactors. These findings are the first demonstration of a heterotrimeric G protein alpha subunit specifically targeted to mitochondria and involved in the control of mitochondrial morphology and dynamics.

Additive neuroprotection of GABA A and GABA B receptor agonists in cerebral ischemic injury via PI-3K/Akt pathway inhibiting the ASK1-JNK cascade

Co-activation of GABA A and GABA B receptors results in neuroprotection during in vitro ischemia. However, it is unclear whether this mode of action is responsible for its neuroprotective effects in animal models of ischemia in vivo, and the precise mechanisms are also unknown. This study compared the neuroprotective efficacies of muscimol, a GABA A receptor agonist, and a GABA B receptor agonist baclofen in rat brain ischemia. The additive neuroprotection could be obtained in the hippocampal CA1 pyramidal cells prominently when muscimol and baclofen were co-applied. In particular, our study showed that co-activation of GABA A and GABA B receptors could strongly increase Akt activation and inhibit ASK1 activation by phosphorylation of serine 83 of ASK1. PI-3K inhibitor LY294002 reversed the increasing Akt activation and ASK1 (S83) phosphorylation. Moreover, MKK4/MKK7-JNK signaling activation was inhibited during ischemia/reperfusion (I/R) by co-treatment of muscimol with baclofen. JNK substrate, Bcl-2 and c-jun phosphorylation were also attenuated. Our results indicated that co-activation of GABA A receptor and GABA B receptor exerted neuroprotective effect via PI-3K/Akt pathway, which could inhibit the ASK1-c-Jun N-terminal protein kinase (JNK) cascade.

Laminar shear stress stimulates vascular smooth muscle cell apoptosis via the Akt pathway

Vascular smooth muscle cells (SMC) may be directly exposed to blood flow after an endothelial-denuding injury. It is not known whether direct exposure of SMC to shear stress reduces SMC turnover and contributes to the low rate of restenosis after most vascular interventions. This study examines if laminar shear stress inhibits SMC proliferation or stimulates apoptosis. Bovine aortic SMC were exposed to arterial magnitudes of laminar shear stress (11 dynes/cm(2)) for up to 24 h and compared to control SMC (0 dynes/cm(2)). SMC density was assessed by cell counting, DNA synthesis by (3)[H]-thymidine incorporation, and apoptosis by TUNEL staining. Akt, caspase, bax, and bcl-2 phosphorylation were assessed by Western blotting; caspase activity was also measured with an in vitro assay. Analysis of variance was used to compare groups. SMC exposed to laminar shear stress had a 38% decrease in cell number (n = 4, P = 0.03), 54% reduction in (3)[H]-thymidine incorporation (n = 3, P = 0.003), and 15-fold increase in TUNEL staining (n = 4, P < 0.0001). Akt phosphorylation was reduced by 67% (n = 3, P < 0.0001), whereas bax/bcl-2 phosphorylation was increased by 1.8-fold (n = 3, P = 0.01). Caspase-3 activity was increased threefold (n = 5, P = 0.03). Pretreatment of cells with ZVAD-fmk or wortmannin resulted in 42% increased cell retention (n = 3, P < 0.01) and a fourfold increase in apoptosis (n = 3, P < 0.04), respectively. Cells transduced with constitutively-active Akt had twofold decreased apoptosis (n = 3, P < 0.002). SMC exposed to laminar shear stress have decreased proliferation and increased apoptosis, mediated by the Akt pathway. These results suggest that augmentation of SMC apoptosis may be an alternative strategy to inhibit restenosis after vascular injury.

Overexpression of phospho-Akt correlates with phosphorylation of EGF receptor, FKHR and BAD in nasopharyngeal carcinoma

The Akt pathway is one of the most common molecular alterations in various human malignancies. However, its involvement in nasopharyngeal carcinoma (NPC) tumorigenesis has not been well established. In this study, the status of Akt activation and expression of its upstream and downstream molecules was investigated in 64 NPC and 38 non-malignant nasopharyngeal tissues by immunohistochemistry. The hotspot mutations of PIK3CA, encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), were also determined in 25 of these NPC tissues. No hotspot mutations were found in any of the samples tested. Akt was activated in 27 (42.2%) and 23 (35.9%) NPCs, as indicated by p-Akt (Thr308) and p-Akt (Ser473) immunoreactivity, respectively. PTEN loss did not correlate statistically with activated Akt. However, a positive correlation was observed between activated Akt and phospho-epidermal growth factor receptor (p-EGFR), suggesting that the EGFR signaling might be one of the upstream regulators of the Akt pathway. The phosphorylation of forkhead (FKHR) and Bcl-2 associated death domain (BAD), but not mammalian target of rapamycin and glycogen synthase kinase-3beta, was significantly correlated with Akt activation. This implies that Akt promotes cell proliferation (as estimated by Ki-67) and survival, at least, through the inactivation of FKHR and BAD in NPC. Our data revealed that the EGFR/PI3K/Akt signaling pathway is important in NPC pathogenesis and that PIK3CA hotspot mutations are rare in NPC.

Bcl-2 Family Proteins Were Involved in Pseudolaric Acid B–Induced Autophagy in Murine Fibrosarcoma L929 Cells

Pseudolaric acid B (PAB) exerted cytostatic activity on murine fibrosarcoma L929 cells. The cytostatic mechanism of PAB on L929 cells was investigated in this paper. At 36 h, after 80 μM PAB treatment, the inhibitory ratio was 65.37 ± 4.12%, and the MDC staining ratio was strongest in L929 cells. 3-Methyladenine (3-MA), an inhibitor of autophagy, inhibited the generation of autolysosomes induced by PAB. The expression of autophagy-associated Beclin 1 protein was up-regulated, and microtubule-associated protein light chain 3 I (LC3 I) was cleaved into LC3 II after 80 μM PAB treatment from 12 h. Therefore, it was concluded that PAB exerted a cytostatic effect on L929 cells through autophagy. However, 80 μM PAB treatment did not induce apoptotic body formation, but 3 mM 3-MA promoted apoptosis, so autophagy might inhibit apoptosis. PAB had no effect on mitochondrial membrane potential (MMP), but upregulated the expressions of Bax and Bcl-2. Immunoprecipitation analysis showed that PAB inhibited Bcl-2 binding with Beclin 1. Additionally, PAB inhibited the localization of Bax in mitochondria, but Bcl-2 still was in the mitochondria to sustain MMP. Meanwhile PAB promoted the phosphorylation of cytoplasmic Bcl-2. Therefore the phosphorylation of Bcl-2 in the cytoplasm and the mitochondrial location of Bcl-2 might be the reasons why PAB inhibited the binding of Bcl-2 and Beclin 1.

Combination of all‐trans retinoic acid and paclitaxel‐induced differentiation and apoptosis in human glioblastoma U87MG xenografts in nude mice

Glioblastoma, which is the most malignant brain tumor, remains incurable and almost always causes death. As a new treatment strategy, the combination of all-trans retinoic acid (ATRA) and paclitaxel was explored for controlling the growth of glioblastoma U87MG xenografts. Human glioblastoma U87MG xenografts were developed in athymic nude mice for treatments with ATRA, paclitaxel, and ATRA plus paclitaxel. The efficacy of treatments in controlling tumor growth was assessed by histologic examination, Western blot analysis, and immunofluorescent labelings. Astrocytic differentiation in U87MG xenografts was associated with increased GFAP expression and decreased telomerase expression. The combination of ATRA and paclitaxel was found to cause more apoptosis than paclitaxel alone. Apoptosis occurred with down-regulation of MEK-2 and overexpression of p-ERK, p-JNK, and p-p38 MAPK. Down-regulation of both Akt and p-Akt also favored the apoptotic process. Combination therapy activated the receptor-mediated pathway of apoptosis with induction of TNF-alpha, activation of caspase-8, and cleavage of Bid to tBid. Combination therapy also induced the mitochondria-mediated pathway of apoptosis with an increase in the Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and Smac/Diablo into the cytosol. In addition, combination therapy promoted phosphorylation of Bcl-2 for its inactivation and down-regulated NF-kappaB and BIRC proteins, indicating suppression of several cell survival factors. Western blot analysis demonstrated that activation of cysteine proteases such as calpain, caspase-12, caspase-9, and caspase-3 contributed to apoptosis. Immunofluorescent labelings confirmed overexpression of cysteine proteases in apoptosis. Treatment of U87MG xenografts with a combination of ATRA and paclitaxel induced differentiation and also multiple molecular mechanisms for apoptosis.

The contribution of c-Jun N-terminal kinase activation and subsequent Bcl-2 phosphorylation to apoptosis induction in human B-cells is dependent on the mode of action of specific stresses

The c-Jun N-terminal kinase (JNK) pathway can play paradoxical roles as either a pro-survival or a pro-cell death pathway depending on type of stress and cell type. The goal of the present study was to determine the role of JNK pathway signaling for regulating B-cell apoptosis in two important but contrasting situations—global proteotoxic damage, induced by arsenite and hyperthermia, versus specific microtubule inhibition, induced by the anti-cancer drug vincristine, using the EW36 B-cell line. This cell line over-expresses the Bcl-2 protein and is a useful model to identify treatments that can overcome multi-drug resistance in lymphoid cells. Exposure of EW36 B-cells to arsenite or lethal hyperthermia resulted in activation of the JNK pathway and induction of apoptosis. However, pharmacological inhibition of the JNK pathway did not inhibit apoptosis, indicating that JNK pathway activation is not required for apoptosis induction by these treatments. In contrast, vincristine treatment of EW36 B-cells resulted in JNK activation and apoptosis that was suppressed by JNK inhibition. A critical difference between the two types of stress treatments was that only vincristine-induced JNK activation resulted in phosphorylation of Bcl-2 at threonine-56, a modification that can block its anti-apoptotic function. Importantly, Bcl-2 phosphorylation was attenuated by JNK inhibition implicating JNK as the upstream kinase. Furthermore, arsenite and hyperthermia treatments activated a p53/p21 pathway associated with apoptosis induction, whereas vincristine did not activate this pathway. These results reveal two stress-activated pathways, one JNK-dependent and another JNK-independent, either of which can bypass Bcl-2 mediated resistance, resulting in cell death.