Cyclic AMP-Response Element Regulated Cell Cycle Arrests in Cancer Cells

Recently, we have demonstrated that trichosanthin (TCS), a promising agent for the treatment of cervical adenocarcinoma, inhibited HeLa cell proliferation through the PKC/MAPK/CREB signal pathway. Furthermore, TCS down-regulated Bcl-2 expression was abrogated by a decoy oligonucleotide (OGN) to the cyclic AMP-responsive element (CRE). The decoy OGN blocked the binding of CRE-binding protein (CREB) to Bcl-2. These results suggested that CRE-mediated gene expression may play a pivotal role in HeLa cell proliferation. However, little is known about the effect of TCS on cell cycle arrests, particularly, whether the genes involved in cell cycle were regulated by CRE. Our present study shows that the arrests of S, G1 and G2/M phases were accompanied by the significant down-regulation of cyclin A, D1 and CDK 2, 4 in HeLa cells, cyclin D1, E and CDK 2, 4 in Caski and C33a cells, and cyclin A, B1, E and CDK 2 in SW1990 cells. However, the cell cycle arrests were reversed via the significant up-regulation of cyclin A and D1, by the combined treatment of TCS and CRE. In conclusion, these data demonstrate for the first time that specific cell cycle arrests in cancer cells can be induced by TCS by inhibiting the binding of CREB to CRE on genes related to cell proliferation.     

CREB,a possible upstream regulator of Bcl-2 in trichosanthin-induced HeLa cell apoptosis

Our previous reports indicated that cyclic AMP response element-binding (CREB) protein was involved in the regulation of Bcl-2 expression in apoptotic HeLa cells induced by trichosanthin (TCS). Here we presented that blockade the binding site of CREB to Bcl-2 by a CRE decoy oligonucleotide abrogated the TCS-decreased Bcl-2 expression. Furthermore, overexpression of phosphorylated CREB (p-CREB) in cells transfected with p-CREB/GFP fusion construct resulted in an increase of Bcl-2 protein content, however, this increase was attenuated by TCS treatment. Therefore, this data supports the hypothesis that CREB is a possible upstream regulator of Bcl-2 in apoptotic HeLa cells induced by TCS. The study provides new insights into understanding the mechanism of TCS in the treatment of cervical cancer.     

PKC inhibition is involved in trichosanthin-induced apoptosis in human chronic myeloid leukemia cell line K562

Trichosanthin (TCS), a type I ribosome-inactivating protein, induces cell death in various cell types including several tumor cell lines. However, the mechanism remains largely uncharacterized. In this study, we investigated the possible mechanism underlying its cytotoxicity by using human chronic myeloid leukemia cell line K562. We found that TCS induced apoptosis in K562 cells in a time- and concentration-dependent manner and can be blocked by caspase-3 inhibitors. Interestingly, TCS treatment induced a transient elevation in intracellular calcium concentration and a slow increase in reactive oxygen species production, while calcium chelators and antioxidants had no obvious effect on TCS-induced apoptosis, suggesting that calcium changes and reactive oxygen species may not be involved in TCS-mediated apoptosis in K562 cells. Instead we found that TCS partly inhibited PKC activity. Indeed, the PKC activator, PMA, inhibited while the PKC inhibitor, calphostin c, enhanced TCS-induced apoptosis. These PKC modulators had similar effects on TCS-induced cleavage of caspase-3, and caspase-3 inhibitors prevented calphostin c-enhanced apoptosis induced by TCS. In summary, we conclude that TCS induces apoptosis in K562 cells partly via PKC inhibition and caspase-3 activation.     

Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.

Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during the rescue of immature B cells from apoptosis. It is possible that the CRE site is responsible for induction of bcl-2 expression in other cell types, particularly those in which protein kinase C is involved.     

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways

Trichosanthin (TCS), a traditional Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. However, the mechanisms remain obscure. The present study focused on various caspase pathways that may be involved in TCS-induced apoptosis in leukemia HL-60 cells. Key caspases in both intrinsic and extrinsic pathways including caspase-8, -9 and -3 were activated upon TCS treatment. Additionally, TCS treatment induced upregulation of BiP and CHOP and also activated caspase-4, which for the first time strongly supported the involvement of endoplasmic reticulum stress pathway in TCS-induced apoptosis. Interestingly, although caspase-8 was activated, Fas/Fas ligand pathway was not involved as evidenced by a lack of induction of Fas or Fas ligand and a lack of inhibitory effect of anti-Fas blocking antibody on TCS-induced apoptosis. Instead, caspase-8 was activated in a caspase-9 and -4 dependent manner. The involvement of mitochondria was demonstrated by the reduction of mitochondrial membrane potential and release of cytochrome c and Smac besides the activation of caspase-9. Further investigation confirmed that caspase-3 was the major executioner caspase downstream to caspase-9, -4 and -8. Taken together, our results suggested that TCS-induced apoptosis in HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways via caspase-3.     

Dexamethasone enhances trichosanthin-induced apoptosis in the HepG2 hepatoma cell line

AimsTrichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) with antitumor activities for various cancers. In this paper, we aimed to investigate whether dexamethasone, an important synthetic member of the glucocorticoid steroids, in combination with TCS can be a potential therapy in treating hepatoma.Main methodsCell viability was investigated using MTT assay, and apoptosis was evaluated with Hoechst 33258 staining. Western blot analysis was used to examine the changes in the expression levels of IκB-α, NF-κB p65 subunit and Cox-2. Additionally, we took advantage of dominant-negative IκB (IκB-DM) over-expression and chemical inhibitor PDTC to inhibit NF-κB activation.Key findingsOur results demonstrated that dexamethasone could enhance TCS-induced apoptosis in the hepatoma cell line HepG2, decreasing IC50 values from in excess of 200 μg/ml to 50 µg/ml. In addition, our results demonstrated that TCS could induce rapid degradation of IκB-α, nuclear translocation of NF-κB and decrease of COX-2 expression in HepG2 cells. Inhibition of NF-κB by biological (IκB-DM) or chemical inhibitor (PDTC) increased HepG2 cells' sensitivity to TCS, resulting in cell viability rate decreasing and apoptotic rate increasing. Simultaneously, dexamethasone increased the level of IκB-α protein and effectively inhibited TCS-induced degradation of IκB-α.SignificanceThese results suggest that dexamethasone could enhance trichosanthin-induced apoptosis in the HepG2, at least in part, by inhibiting the NF-κB signaling pathway and thus strengthening the antitumor effects of TCS, which highlights the possibility of combined drug application of TCS and dexamethasone in the clinical treatment of hepatoma.     

CREB Is One Component of the Binding Complex of the Ces-2/E2A-HLF Binding Element and Is an Integral Part of the Interleukin-3 Survival Signal

The Ces-2/E2A-HLF binding element (CBE) is recognized by Caenorhabditis elegans death specification gene product Ces-2 and human acute lymphocytic leukemia oncoprotein E2A-HLF. In an attempt to identify a cellular CBE-binding protein(s) that may be involved in apoptosis regulation in mammals, multiple nuclear binding complexes of CBE were identified in various mammalian cell lines and tissues by electrophoretic mobility shift assay. Cyclic AMP (cAMP)-responsive element (CRE)-binding protein (CREB) was present in one major CBE complex of Ba/F3 and TF-1 cells, and both in vitro-translated and Escherichia coli-synthesized CREB bound to CBE. Activation of CREB by cAMP-elevating chemicals or the catalytic subunit of protein kinase A (PKAc) resulted in induction of the CBE-driven reporter gene. Stimulation of Ba/F3 cells with interleukin-3 (IL-3) promptly induced phosphorylation of CREB at serine(133) partially via a PKA-dependent pathway. Consistently, Ba/F3 cell survival in the absence of IL-3 was prolonged by activation of PKA. Conversely, treatment of cells with a PKA inhibitor or expression of the dominant negative forms of the regulatory subunit type I of PKA and CREB overrode the survival activity of IL-3. Last, the bcl-2 gene was demonstrated to be one candidate cellular target of the CREB-containing CBE complex, as mutations in the CRE and CBE sites significantly reduced the IL-3 inducibility of the bcl-2 promoter. Together, our results suggest that CREB is one cellular counterpart of Ces-2/E2A-HLF and is part of IL-3 dependent apoptosis regulation in hematopoietic cells.     

Analysis of the Proenkephalin Second Messenger-Inducible Enhancer in Rat Striatal Cultures

Abstract: We have previously shown that in cell extracts from rat striatum, cyclic AMP response element (CRE) binding protein (CREB), rather than AP-1 proteins, preferentially interacts with the CRE-2 element of the proenkephalin second messenger-inducible enhancer, even under conditions in which AP-1 proteins are highly induced. Here we use primary striatal cultures to permit a more detailed analysis of CRE-2 function and protein binding in relevant neural cell types. By transfection we find that in primary striatal cultures, as in transformed cell lines, the CRE-1 and CRE-2 elements are required for significant induction by cyclic AMP. We report that cyclic AMP induction of the proenkephalin gene in striatal cultures is protein synthesis independent, excluding a role for newly synthesized proteins like c-Fos. We also show that cyclic AMP induces CREB phosphorylation and that phosphorylated CREB interacts strongly with CRE-2 and weakly with CRE-1. The predominant protein bound to CRE-1 is not CREB, however, and remains to be identified. Despite some prior predictions, we do not find a role for c-Fos in cyclic AMP regulation of proenkephalin gene expression in neurons.