促凋亡基因Bad在胰腺癌中的调控及靶向治疗研究进展

胰腺癌是一种预后极差的恶性消化道肿瘤,5年生存率小于5%,这与胰腺癌细胞的凋亡异常有着密切的关系。Bad基因是Bcl-2家族中的一种促凋亡基因,被认为可以通过浓度依赖性方式替换Bcl-xL/Bax、Bcl-2/Bax二聚体中的Bax,从而达到促进细胞凋亡、防止胰腺癌的发生。Bad基因在胰腺癌中的调控主要以磷酸化/去磷酸化调控最为常见,研究显示,胰腺癌中Bad表达总量变化不明显,而在Ser112位点上的磷酸化为灭活的pBad112形式表达却明显增多。胰腺癌中过表达的14-3-3sigma、Pim-3、cAMP等均可诱导Bad蛋白磷酸化,而PKC、MAP4K3等可诱导其去磷酸化。同时,针对Bad的靶向治疗在胰腺癌中的应用研究已经有所进展,其中有研究发现人参皂苷Rg3、Cantharidin、Stemonamid等药物均可通过Bad途径促胰腺癌细胞凋亡。对胰腺癌中的Bad的深入研究,有利于了解其与胰腺癌发病机制的关系,为胰腺癌的靶向治疗提供新的方向。     

促凋亡基因Bad在胰腺癌中的调控及靶向治疗研究进展

胰腺癌是一种预后极差的恶性消化道肿瘤,5年生存率小于5%,这与胰腺癌细胞的凋亡异常有着密切的关系。Bad基因是Bcl-2家族中的一种促凋亡基因,被认为可以通过浓度依赖性方式替换Bcl-xL/Bax、Bcl-2/Bax二聚体中的Bax,从而达到促进细胞凋亡、防止胰腺癌的发生。Bad基因在胰腺癌中的调控主要以磷酸化/去磷酸化调控最为常见,研究显示,胰腺癌中Bad表达总量变化不明显,而在Ser112位点上的磷酸化为灭活的pBad112形式表达却明显增多。胰腺癌中过表达的14-3-3sigma、Pim-3、cAMP等均可诱导Bad蛋白磷酸化,而PKC、MAP4K3等可诱导其去磷酸化。同时,针对Bad的靶向治疗在胰腺癌中的应用研究已经有所进展,其中有研究发现人参皂苷Rg3、Cantharidin、Stemonamid等药物均可通过Bad途径促胰腺癌细胞凋亡。对胰腺癌中的Bad的深入研究,有利于了解其与胰腺癌发病机制的关系,为胰腺癌的靶向治疗提供新的方向。     

Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.     

Regulation of bad phosphorylation and association with Bcl-x(L) by the MAPK/Erk kinase.

Phosphorylation of the Bcl-2 family protein Bad may represent an important bridge between survival signaling by growth factor receptors and the prevention of apoptosis. Bad phosphorylation was examined following cytokine stimulation, which revealed phosphorylation on a critical residue, serine 112, in a MEK-dependent manner. Furthermore, Bad phosphorylation also increased on several sites distinct from serine 112 but could not be detected on serine 136, previously thought to be a protein kinase B/Akt-targeted residue. Serine 112 phosphorylation was shown to be absolutely required for dissociation of Bad from Bcl-x(L). These results demonstrate for the first time in mammalian cells the involvement of the Ras-MAPK pathway in the phosphorylation of Bad and the regulation of its function.     

Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).     

Activation of JNK1, RSK2, and MSK1 is involved in serine 112 phosphorylation of Bad by ultraviolet B radiation

The Bcl-2 family member Bad is a pro-apoptotic protein, and phosphorylation of Bad by cytokines and growth factors promotes cell survival in many cell types. Induction of apoptosis by UV radiation is well documented. However, little is known about UV activation of cell survival pathways. Here, we demonstrate that UVB induces Bad phosphorylation at serine 112 in JNK1, RSK2, and MSK1-dependent pathways. Inhibition of mitogen-activated protein (MAP) kinases including ERKs, JNKs, and p38 kinase by the use of their respective dominant negative mutant or a specific inhibitor for MEK1 or p38 kinase, PD98059 or SB202190, resulted in abrogation of UVB-induced phosphorylation of Bad at serine 112. Incubation of active MAP kinase members with Bad protein showed serine 112 phosphorylation of Bad by JNK1 only. However, activated RSK2 and MSK1, downstream kinases of ERKs and p38 kinase, respectively, also phosphorylated Bad at serine 112 in vitro. Cells from a Coffin-Lowry syndrome patient (deficient in RSK2) or expressing an N-terminal or C-terminal kinase-dead mutant of MSK1 were defective for UVB-induced serine 112 phosphorylation of Bad. Furthermore, MAP kinase pathway-dependent serine 112 phosphorylation was shown to be required for dissociation of Bad from Bcl-X(L). These data illustrated that UVB-induced phosphorylation of Bad at serine 112 was mediated through MAP kinase signaling pathways in which JNK1, RSK2, and MSK1 served as direct mediators.     

bFGF inhibits the activation of caspase-3 and apoptosis of P19 embryonal carcinoma cells during neuronal differentiation.

P19 embryonal carcinoma (EC) cells undergo apoptosis during neuronal differentiation induced by all-trans retinoic acid (RA). Caspase-3-like proteases are activated and involved in the apoptosis of P19 EC cells during neuronal differentiation.1 Recently it has been shown that growth factor signals protect against apoptosis by phosphorylation of Bad. Phosphorylated Bad, an apoptotic member of the Bcl-2 family, cannot bind to Bcl-xL and results in Bcl-xL homodimer formation and subsequent antiapoptotic activity. In the present study, we demonstrate that this system is used generally to protect against apoptosis during neuronal differentiation. Bcl-xL inhibited the activation of caspase-3-like proteases. Basic fibroblast growth factor (bFGF) inhibited more than 90% of the caspase-3-like activity, inhibited processing of caspase-3 into its active form, and inhibited DNA fragmentation. bFGF activated phosphatidyl-inositol-3-kinase (PI3K) and stimulated the phosphorylation of Bad. Phosphorylation was inhibited by wortmannin, an inhibitor of PI3K and its downstream target Akt. Thus, Bad is a target of the FGF receptor-mediated signals involved in the protection against activation of caspase-3.     

Rac1 inhibits apoptosis in human lymphoma cells by stimulating Bad phosphorylation on Ser-75

The small GTPase Rac1 has emerged as an important regulator of cell survival and apoptosis, but the mechanisms involved are not completely understood. In this report, constitutively active Rac1 is shown to stimulate the phosphorylation of the Bcl-2 family member Bad, thereby suppressing drug-induced caspase activation and apoptosis in human lymphoma cells. Rac1 activation leads to human Bad phosphorylation specifically at serine-75 (corresponding to murine serine-112) both in vivo and in vitro. Inhibition of constitutive and activated Rac1-induced Bad phosphorylation by a cell-permeable competitive peptide inhibitor representing this Bad phosphorylation site sensitizes lymphoma cells to drug-induced apoptosis. The data show further that endogenous protein kinase A is a primary catalyst of cellular Bad phosphorylation in response to Rac activation, while Akt is not involved. These findings define a mechanism by which active Rac1 promotes lymphoma cell survival and inhibits apoptosis in response to cancer chemotherapy drugs.     

Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases

The Bcl-2 homology 3 (BH3) domain is crucial for the death-inducing and dimerization properties of pro-apoptotic members of the Bcl-2 protein family, including Bak, Bax, and Bad. Here we report that synthetic peptides corresponding to the BH3 domain of Bak bind to Bcl-xL, antagonize its anti-apoptotic function, and rapidly induce apoptosis when delivered into intact cells via fusion to the Antennapedia homeoprotein internalization domain. Treatment of HeLa cells with the Antennapedia-BH3 fusion peptide resulted in peptide internalization and induction of apoptosis within 2-3 h, as indicated by caspase activation and subsequent poly(ADP-ribose) polymerase cleavage, as well as morphological characteristics of apoptosis. A point mutation within the BH3 peptide that blocks its ability to bind to Bcl-xL abolished its apoptotic activity, suggesting that interaction of the BH3 peptide with Bcl-2-related death suppressors, such as Bcl-xL, may be critical for its activity in cells. While overexpression of Bcl-xL can block BH3-induced apoptosis, treatment with BH3 peptides resensitized Bcl-xL-expressing cells to Fas-mediated apoptosis. BH3-induced apoptosis was blocked by caspase inhibitors, demonstrating a dependence on caspase activation, but was not accompanied by a dramatic early loss of mitochondrial membrane potential or detectable translocation of cytochrome c from mitochondria to cytosol. These findings demonstrate that the BH3 domain itself is capable of inducing apoptosis in whole cells, possibly by antagonizing the function of Bcl-2-related death suppressors.     

p21-activated kinase 1 phosphorylates the death agonist bad and protects cells from apoptosis

Bad is a critical regulatory component of the intrinsic cell death machinery that exerts its death-promoting effect upon heterodimerization with the antiapoptotic proteins Bcl-2 and Bcl-x(L). Growth factors promote cell survival through phosphorylation of Bad, resulting in its dissociation from Bcl-2 and Bcl-x(L) and its association with 14-3-3tau. Survival of interleukin 3 (IL-3)-dependent FL5.12 lymphoid progenitor cells is attenuated upon treatment with the Rho GTPase-inactivating toxin B from Clostridium difficile. p21-activated kinase 1 (PAK1) is activated by IL-3 in FL5.12 cells, and this activation is reduced by the phosphatidylinositol 3-kinase inhibitor LY294002. Overexpression of a constitutively active PAK mutant (PAK1-T423E) promoted cell survival of FL5.12 and NIH 3T3 cells, while overexpression of the autoinhibitory domain of PAK (amino acids 83 to 149) enhanced apoptosis. PAK phosphorylates Bad in vitro and in vivo on Ser112 and Ser136, resulting in a markedly reduced interaction between Bad and Bcl-2 or Bcl-x(L) and the increased association of Bad with 14-3-3tau. Our findings indicate that PAK inhibits the proapoptotic effects of Bad by direct phosphorylation and that PAK may play an important role in cell survival pathways.     

Merosin (laminin-2/4)-driven survival signaling: complex modulations of Bcl-2 homologs

We have shown previously that the promotion of myofiber survival by the basement membrane component merosin (laminin-2 [alpha2beta1gamma1]/laminin-4 [alpha2beta2gamma1]) is dependent on the activity of the tyrosine kinase Fyn, whereas myofiber anoikis induced by merosin deficiency is dependent on the stress-activated protein kinase p38alpha. To further understand such merosin-driven survival signaling, we analyzed the expression of five Bcl-2 homologs (Bcl-2, Bcl-X(L), Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1) in normal and merosin-deficient myotubes, with or without pharmacological inhibitors for Fyn and p38. Herein, we report that (1) merosin deficiency induces anoikis and causes decreased Bcl-2, Bcl-X(L), and Bag-1 levels, increased Bax and Bak levels, and decreased Bad phosphorylation; (2) Bcl-2, Bcl-X(L), Bag-1, and Bad phosphorylation are also decreased in anoikis-dying, Fyn-inhibited myotubes; (3) the inhibition of p38alpha in Fyn-inhibited and/or merosin-deficient myotubes protects against anoikis and increases Bcl-2 levels above normal, in addition to restoring Bad phosphorylation and Bag-1 levels to normal; (4) the overexpression of merosin in deficient myotubes also rescues from anoikis and increases Bcl-2 levels and Bad phosphorylation above normal, in addition to restoring Bcl-X(L), Bag-1, Bax, and Bak levels to normal; and (5) Bcl-2 overexpression is sufficient to rescue merosin-deficient myotubes from anoikis, even though the expression/phosphorylation levels of the other homologs analyzed are not restored to normal. These results indicate that merosin-driven myofiber survival signaling affects complex, differential modulations of individual Bcl-2 homologs. These further suggest that Bcl-2 can play a major role in suppressing myofiber anoikis.     

C5b-9 terminal complement complex protects oligodendrocytes from death by regulating Bad through phosphatidylinositol 3-kinase/Akt pathway

Apoptosis of oligodendrocytes is induced by serum growth factor deprivation. We showed that oligodendrocytes and progenitor cells respond to serum withdrawal by a rapid decline of Bcl-2 mRNA expression and caspase-3-dependent apoptotic death. Sublytic assembly of membrane-inserted terminal complement complexes consisting of C5b, C6, C7, C8, and C9 proteins (C5b-9) inhibits caspase-3 activation and apoptotic death of oligodendrocytes. In this study, we examined an involvement of the mitochondria in oligodendrocyte apoptosis and the role of C5b-9 on this process. Decreased phosphatidylinositol 3-kinase and Akt activities occurred in association with cytochrome c release and caspase-9 activation when cells were placed in defined medium. C5b-9 inhibited the mitochondrial pathway of apoptosis in oligodendrocytes, as shown by decreased cytochrome c release and inhibition of caspase-9 activation. Phosphatidylinositol 3-phosphate kinase and Akt activities were also induced by C5b-9, and the phosphatidylinositol 3-phosphate kinase inhibitor LY294002 reversed the protective effect of C5b-9. Phosphatidylinositol 3-phosphate kinase activity was also responsible for the phosphorylation of Bad at Ser112 and Ser136. This phosphorylation resulted in dissociation of Bad from the Bad/Bcl-xL complex in a G(i)alpha-dependent manner. The mitochondrial pathway of oligodendrocyte apoptosis is, therefore, inhibited by C5b-9 through post-translational regulation of Bad. This mechanism may be involved in the promotion of oligodendrocyte survival in inflammatory demyelinating disorders affecting the CNS.     

Down-regulation of CMTM8 induces epithelial-to-mesenchymal transition-like changes via c-MET/extracellular signal-regulated kinase (ERK) signaling

The acquisition of an invasive phenotype is a critical turning point for malignant tumor cells. CMTM8, a potential tumor suppressor, is frequently down-regulated in solid tumors, and its overexpression induces tumor cell apoptosis. Here, we identify a new role for CMTM8 in regulating tumor cell migration. Reducing CMTM8 expression in HepG2 hepatocellular carcinoma cells results in the acquisition of epithelial-to-mesenchymal transition (EMT) features, including a morphological change from organized epithelial sheets to scattered fibroblast-like shapes, reduction of the epithelial marker E-cadherin, and an increased invasive and migratory ability. These phenotypic changes are mediated in large part by the ERK-MAPK pathway, as the MEK inhibitor U0126 and shRNA-mediated knockdown of ERK2 significantly reversed these phenotypes. Hepatocyte growth factor binding to the c-MET receptor is known to induce EMT in HepG2 cells. We found that CMTM8 knockdown in HepG2 cells induced c-MET signaling and ERK activation. Inhibition of c-MET signaling with the small molecule inhibitor SU11274 or c-MET RNAi blocked the EMT-like changes following CMTM8 knockdown. CMTM8 overexpression in HepG2 cells inhibited hepatocyte growth factor-induced EMT-like morphological changes and cell motility. Down-regulation of CMTM8 also promoted an EMT-like change in MCF-10A cells, indicating a broader role for CMTM8 in regulating cellular transformation.     

Selective involvement of the PI3K/PKB/bad pathway in retinal cell death

The phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB)/Bad signal transduction pathway is engaged in the control of apoptosis in many different cell types, particularly through phosphorylation of the Bcl-2 family protein Bad. We examined the involvement of this pathway in the control of programmed cell death in the retina of developing rats. PKB is constitutively phosphorylated in retinal tissue in vitro, whereas Bad was dephosphorylated both in Ser112 and Ser136. Cell death induced by either the PI3K inhibitor LY294002, or the general kinase inhibitor 2-aminopurine, were followed by PKB dephosphorylation, but PKB was not modulated during cell death induced by the protein synthesis inhibitor anisomycin. Treatment of retinal tissue cultures with forskolin, which increases intracellular levels of cAMP, partially blocked apoptosis induced by both anisomycin and 2-aminopurine, but not by LY294002, whereas forskolin invariably induced phosphorylation of Bad on both Ser112 and Ser136. The data suggest that Bad may be engaged in survival pathways in the immature retina, but pathways other than PI3K/PKB/Bad, and phosphorylation sites other than Ser112 and Ser136 in the Bad protein control cell survival in retinal tissue.     

Activation of Bad trafficking is involved in the BCR-mediated apoptosis of immature B cells

The activity of Bad, a pro-apoptotic protein, is regulated by reversible phosphorylation. Moreover, sequestration of Bad within subcellular compartments may be a new mechanism of apoptosis regulation. In this study, we report that Bad interacts with 14-3-3 protein in WEHI-231 immature B cells. This association is disrupted following BCR stimulation in correlation with Bad translocation to mitochondria and apoptosis. Confocal microscopy was further used to examine the co-localization of Bad with lipid rafts in WEHI-231 and murineex vivoB cells. Bad was found colocalized to lipid rafts in freshly isolated mature B lymphocytes, in contrast to immature cells. Finally, co-immunoprecipitation experiments performed on WEHI-231 B cells revealed that PP1α interacts with Bcl-2 and Bad, and dissociation of the complex was found correlated with appearance of apoptosis. Bcl-2 seemed to be required to assemble the complex which may regulate Bad phosphorylation status and consequently cell survival. Collectively, present data outline the role of Bad trafficking in the BCR-mediated apoptosis and suggest that differences in intracellular Bad trafficking may be involved in the differential outcome of BCR signaling.     

Bcl-2-related protein family gene expression during oligodendroglial differentiation

Oligodendroglial lineage cells (OLC) vary in susceptibility to both necrosis and apoptosis depending on their developmental stages, which might be regulated by differential expression of Bcl-2-related genes. As an initial step to test this hypothesis, we examined the expression of 19 Bcl-2-related genes in purified cultures of rat oligodendroglial progenitors, immature and mature oligodendrocytes. All 'multidomain' anti-apoptotic members (Bcl-x, Bcl-2, Mcl-1, Bcl-w and Bcl2l10/Diva/Boo) except Bcl2a1/A1 are expressed in OLC. Semiquantitative and real-time RT-PCR revealed that Bcl-xL and Mcl-1 mRNAs are the dominant anti-apoptotic members and increase four- and twofold, respectively, with maturation. Bcl-2 mRNA is less abundant than Bcl-xL mRNA in progenitors and falls an additional 10-fold during differentiation. Bcl-w mRNA also increases, with significant changes in its splicing pattern, as OLC mature. Transfection studies demonstrated that Bcl-xL overexpression protects against kainate-induced excitotoxicity, whereas Bcl-2 overexpression does not. As for 'multidomain' pro-apoptotic members (Bax, Bad and Bok/Mtd), Bax and Bak are highly expressed throughout differentiation. Among 'BH3 domain-only' members examined (Bim, Biklk, DP5/Hrk, Bad, Bid, Noxa, Puma/Bbc3, Bmf, BNip3 and BNip3L), BNip3 and Bmf mRNAs increase markedly during differentiation. These results provide basic information to guide further studies on the roles for Bcl-2-related family proteins in OLC death.     

Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad.     

Regulation of Bcl-xL expression by H2O2 in cardiac myocytes

Oxidative stress promotes cardiac myocyte apoptosis through the mitochondrial death pathway. Since Bcl-2 family proteins are key regulators of apoptosis, we examined the effects of H2O2 on the expression of principal Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bad) in neonatal rat cardiac myocytes. Protein expression was assessed by immunoblotting. Bcl-2, Bax, and Bad were all down-regulated in myocytes exposed to 0.2 mm H2O2, a concentration that induces apoptosis. In contrast, although Bcl-xL levels initially declined, the protein was re-expressed from 4-6 h. Bcl-xL mRNA was up-regulated from 2 to 4 h in neonatal rat or mouse cardiac myocytes exposed to H2O2, consistent with the re-expression of protein. Four different untranslated first exons have been identified for the Bcl-x gene (exons 1, 1B, 1C, and 1D, where exon 1 is the most proximal and exon 1D the most distal to the coding region). All were detected in mouse or rat neonatal cardiac myocytes, but exon 1D was not expressed in adult mouse hearts. In neonatal mouse or rat cardiac myocytes, H2O2 induced the expression of exons 1B, 1C, and 1D, but not exon 1. These data demonstrate that the Bcl-x gene is selectively responsive to oxidative stress, and the response is mediated through distal promoter regions.