Biomedical subjects

Xingming Deng

Publications and source records attributed to Xingming Deng.

16 recordsLinked to original sources

PubMed2006

Suppression of cancer cell migration and invasion by protein phosphatase 2A through dephosphorylation of mu- and m-calpains.

The mu- and m-calpains are major members of the calpain family that play an essential role in regulating cell motility. We have recently discovered that nicotine-activated protein kinase C iota enhances calpain phosphorylation in association with enhanced calpain activity and accelerated migration and invasion of human lung cancer cells. Here we found that specific disruption of protein phosphatase 2A (PP2A) activity by expression of SV40 small tumor antigen up-regulates phosphorylation of mu- and m-calpains whereas C2-ceramide, a potent PP2A activator, reduces nicotine-induced calpain phosphorylation, suggesting that PP2A may function as a physiological calpain phosphatase. PP2A co-localizes and interacts with mu- and m-calpains. Purified, active PP2A directly dephosphorylates mu- and m-calpains in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses nicotine-stimulated phosphorylation of mu- and m-calpains, which is associated with inhibition of calpain activity, wound healing, cell migration, and invasion. By contrast, depletion of PP2A/C by RNA interference enhances calpain phosphorylation, calpain activity, cell migration, and invasion. Importantly, C2-ceramide-induced suppression of calpain phosphorylation results in decreased secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential clinic relevance in controlling metastasis of lung cancer. These findings reveal a novel role for PP2A as a physiological calpain phosphatase that not only directly dephosphorylates but also inactivates mu- and m-calpains, leading to suppression of migration and invasion of human lung cancer cells.

Calpain↗

PubMed2006

Protein phosphatase 2A enhances the proapoptotic function of Bax through dephosphorylation.

Bax is a major proapoptotic member of the Bcl2 family that is required for apoptotic cell death. We have recently discovered that Bax phosphorylation at serine 184 induced by nicotine through activation of protein kinase AKT abolishes its proapoptotic function in human lung cancer cells. Here we found that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor okadaic acid or specific disruption of PP2A activity by expression of SV40 small tumor antigen enhanced Bax phosphorylation, whereas C(2)-ceramide, a potent PP2A activator, reduced nicotine-induced Bax phosphorylation, suggesting that PP2A may function as a physiological Bax phosphatase. PP2A co-localized and interacted with Bax. Purified, active PP2A directly dephosphorylated Bax in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppressed nicotine-stimulated Bax phosphorylation in association with increased apoptotic cell death. By contrast, depletion of PP2A/C by RNA interference enhanced Bax phosphorylation and prolonged cell survival. Mechanistically C(2)-ceramide-induced Bax dephosphorylation caused a conformational change by exposure of the 6A7 epitope (amino acids 13-19) that is normally hidden at its N terminus that promoted the insertion of Bax into mitochondrial membranes and formation of Bax oligomers leading to cytochrome c release and apoptosis. In addition, PP2A directly disrupted the Bcl2/Bax association to liberate Bax from the heterodimer complex. Thus, PP2A may function as a physiological Bax regulatory phosphatase that not only dephosphorylates Bax but also activates its proapoptotic function.

Antigens, Polyomavirus Transforming↗

PubMed2006

Bcl2 suppresses DNA repair by enhancing c-Myc transcriptional activity.

Bcl2 and c-Myc are two major oncogenic proteins that can functionally promote DNA damage, genetic instability, and tumorigenesis. However, the mechanism(s) remains unclear. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most potent carcinogen contained in cigarette smoke that induces cellular DNA damage. Here we found that Bcl2 potently suppresses the repair of NNK-induced abasic sites of DNA lesions in association with increased c-Myc transcriptional activity. The Bcl2 BH4 domain (amino acids 6-31) was found to bind directly to c-Myc MBII domain (amino acids 106-143), and this interaction is required for Bcl2 to enhance c-Myc transcriptional activity and inhibit DNA repair. In addition to mitochondria, Bcl2 is also expressed in the nucleus, where it co-localizes with c-Myc. Expression of nuclear-targeted Bcl2 enhances c-Myc transcriptional activity with suppression of DNA repair but fails to prolong cell survival. Depletion of c-Myc expression from cells overexpressing Bcl2 significantly accelerates the repair of NNK-induced DNA damage, indicating that c-Myc may be essential for the Bcl2 effect on DNA repair. It is known that apurinic/apyrimidinic endonuclease (APE1) plays a crucial role in the repair of abasic sites of DNA lesions. That overexpression of Bcl2 results in up-regulation of c-Myc and down-regulation of APE1 suggests APE1 may function as the downstream target of Bcl2/c-Myc in the DNA repair machinery. Thus, Bcl2, in addition to its survival function, may also suppress DNA repair in a novel mechanism involving c-Myc and APE1, which may lead to an accumulation of DNA damage in living cells, genetic instability, and tumorigenesis.

Carcinogens↗

PubMed2006

Mechanisms of apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in acute myeloid leukemia.

BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

Animals↗

PubMed2006

Bcl2's flexible loop domain regulates p53 binding and survival.

p53 not only functions as a transcription factor but also has a direct, apoptogenic role at the mitochondria. We have discovered that DNA damage-induced p53-Bcl2 binding is associated with decreased Bcl2-Bax interaction and increased apoptotic cell death in a mechanism regulated by Bcl2's flexible loop regulatory domain (FLD), since purified p53 protein can disrupt the Bcl2/Bax complex by directly binding to a negative regulatory region of the FLD (amino acids [aa] 32 to 68). Deletion of the negative regulatory region (Delta32-68) abolishes Bcl2-p53 binding and enhances Bcl2's antiapoptotic function. Conversely, removal of a positive regulatory region (aa 69 to 87) of the FLD, which contains the Bcl2 phosphorylation site(s) T69, S70, and S87, enhances Bcl2-p53 binding and significantly abrogates Bcl2's survival activity. The phospho-mimetic T69E/S70E/S87E (EEE) but not the nonphosphorylatable T69A/S70A/S87A (AAA) Bcl2 mutant displays a reduced capacity to bind p53 and potently inhibits p53-induced cytochrome c release from isolated mitochondria. Furthermore, the FLD-only aa32-87 and aa32-68 peptides but not the aa69-87 peptide can directly bind p53 in vitro. p53-induced cytochrome c release occurs through a mechanism involving Bax's integral insertion into the outer mitochondrial membrane. Either DNA damage to cells or expression of p53 selectively targeted to the mitochondria results in Bcl2-p53 binding followed by exposure of Bcl2's BH3 domain in association with inactivation of Bcl2's antiapoptotic function, indicating a conformational change in Bcl2 can occur upon direct ligation of p53. Thus, Bcl2's FLD contains both positive and negative regulatory regions which functionally regulate Bcl2's antiapoptotic activity by affecting Bax or p53 binding.

Animals↗

PubMed2005

Protein kinase Ciota promotes nicotine-induced migration and invasion of cancer cells via phosphorylation of micro- and m-calpains.

Nicotine is a major component in cigarette smoke that activates the growth-promoting pathways to facilitate the development of lung cancer. However, it is not clear whether nicotine affects cell motility to facilitate tumor metastasis. Here we discovered that nicotine potently induces phosphorylation of both mu- and m-calpains via activation of protein kinase Ciota (PKCiota), which is associated with accelerated migration and invasion of human lung cancer cells. Purified PKCiota directly phosphorylates mu- and m-calpains in vitro. Overexpression of PKCiota results in increased phosphorylation of both mu- and m-calpains in vivo. Nicotine also induces activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the alpha(7) nicotinic acetylcholine receptor inhibitor alpha-bungarotoxin can block nicotine-induced calpain phosphorylation with suppression of calpain activity, wound healing, cell migration, and invasion, indicating that nicotine-induced calpain phosphorylation occurs, at least in part, through a signaling pathway involving the upstream alpha(7) nicotinic acetylcholine receptor. Intriguingly, depletion of PKCiota by RNA interference suppresses nicotine-induced calpain phosphorylation, calpain activity, cell migration, and invasion, indicating that PKCiota is a necessary component in nicotine-mediated cell motility signaling. Importantly, nicotine potently induces secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential to cleave substrates in the extracellular matrix. These findings reveal a novel role for PKCiota as a nicotine-activated, physiological calpain kinase that directly phosphorylates and activates calpains, leading to enhanced migration and invasion of human lung cancer cells.

Bungarotoxins↗

PubMed2005

Survival function of protein kinase C{iota} as a novel nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-activated bad kinase.

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen in cigarette smoke. NNK cannot only induce DNA damage but also promotes the survival of human lung cancer cells. Protein kinase C (PKC)iota is an atypical PKC isoform and plays an important role in cell survival, but the downstream survival substrate(s) is not yet identified. Bad, a proapoptotic BH3-only member of Bcl2 family, is co-expressed with PKCiota in both small cell lung cancer and non-small cell lung cancer cells. We discovered that NNK potently induces multisite Bad phosphorylation at Ser-112, Ser-136, and Ser-155 via activation of PKCiota in association with increased survival of human lung cancer cells. Purified, active PKCiota can directly phosphorylate both endogenous and recombinant Bad at these three sites and disrupt Bad/Bcl-XL binding in vitro. Overexpression of PKCiota results in an enhancement of Bad phosphorylation. NNK also stimulates activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the PKC inhibitor (staurosporine) or a Src-specific inhibitor (PP2) can block NNK-induced Bad phosphorylation and promote apoptotic cell death. The beta-adrenergic receptor inhibitor propranolol blocks both NNK-induced activation of PKCiota and Bad phosphorylation, indicating that NNK-induced Bad phosphorylation occurs at least in part through the upstream beta-adrenergic receptor. Mechanistically, NNK-induced Bad phosphorylation prevents its interaction with Bcl-XL. Because the specific depletion of PKCiota by RNA interference inhibits both NNK-induced Bad phosphorylation and survival, this confirms that PKCiota is a necessary component in NNK-mediated survival signaling. Collectively, these findings reveal a novel role for PKCiota as an NNK-activated physiological Bad kinase that can directly phosphorylate and inactivate this proapoptotic BH3-only protein, which leads to enhanced survival and chemoresistance of human lung cancer cells.

Carrier Proteins↗

PubMed2005

Nicotine inactivation of the proapoptotic function of Bax through phosphorylation.

Nicotine-induced cell survival is associated with chemoresistance of human lung cancer cells, but our understanding of the intracellular mechanism(s) is fragmentary. Bax is a major proapoptotic member of the Bcl2 family and a molecule required for apoptotic cell death. Growth factor (i.e. granulocyte-macrophage colony-stimulating factor)-induced phosphorylation of Bax has been reported to negatively regulate its proapoptotic function. Because Bax is ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells, nicotine may mimic growth factor(s) to regulate the activity of Bax. We found that nicotine potently induces Bax phosphorylation at Ser-184, which results in abrogation of the proapoptotic activity of Bax and increased cell survival. AKT, a known physiological Bax kinase, is activated by nicotine, co-localizes with Bax in the cytoplasm, and can directly phosphorylate Bax in vitro. Treatment of cells with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or specific depletion of AKT expression by RNA interference can block both nicotine-induced Bax phosphorylation and cell survival. Importantly, nicotine-induced Bax phosphorylation potently blocks stress-induced translocation of Bax from cytosol to mitochondria, impairs Bax insertion into mitochondrial membranes, and reduces the half-life of Bax protein (i.e. from 9-12 h to <6 h). Because knockdown of Bax expression by gene silencing results in prolonged cell survival following treatment with cisplatin in the absence or presence of nicotine, Bax may be an essential component in the nicotine survival signaling pathway. Thus, nicotine-induced survival and chemoresistance of human lung cancer cells may occur in a novel mechanism involving activation of PI3K/AKT that directly phosphorylates and inactivates the proapoptotic function of Bax.

Apoptosis↗

PubMed2004

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone induces phosphorylation of mu- and m-calpain in association with increased secretion, cell migration, and invasion.

Mounting evidence indicates that cigarette smoking not only promotes tumorigenesis but also may increase the spread of cancer cells in the body. However, the intracellular mechanism(s) by which cigarette smoking promotes metastasis of human lung cancer remains enigmatic. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important component in cigarette smoke and is formed by nitrosation of nicotine. mu- and m-calpain (calpain I and calpain II) are major members of the calpain family, which are ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells. Our findings indicated that NNK potently induces phosphorylation of both mu- and m-calpain in association with their activation and increased migration as well as invasion of lung cancer cells. Treatment of cells with PD98059 blocked phosphorylation of m- and mu-calpain and resulted in suppression of NNK-induced cell migration and invasion. p44 MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 were activated by NNK, co-localized with mu- and m-calpain in cytoplasm, and directly phosphorylated mu- and m-calpain in vitro. These findings suggest a role for the ERK1/2 kinases as NNK-activated physiological calpain kinases. Specific knock-down of mu- and/or m-calpain expression by RNA interference blocked NNK-stimulated migration and invasion, suggesting that mu- and m-calpain may act as required targets in a NNK-induced metastatic signaling pathway. Furthermore, NNK promotes secretion of active mu- and m-calpain from lung cancer cells through vesicles, which may have the potential to cleave substrates in the extracellular matrix. Thus, NNK-induced cell migration and invasion may occur, at least in part, through a novel mechanism involving phosphorylation of calpains that leads to their activation and secretion, which may contribute to metastasis and/or progression of lung cancer.

Blotting, Western↗

PubMed2004

Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone promotes functional cooperation of Bcl2 and c-Myc through phosphorylation in regulating cell survival and proliferation.

Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen contained in cigarette smoke. NNK significantly contributes to smoking-related lung cancer, but the molecular mechanism remains enigmatic. Bcl2 and c-Myc are two major oncogenic proteins that cooperatively promote tumor development. We report here that NNK simultaneously stimulates Bcl2 phosphorylation exclusively at Ser(70) and c-Myc at Thr(58) and Ser(62) through activation of both ERK1/2 and PKCalpha, which is required for NNK-induced survival and proliferation of human lung cancer cells. Treatment of cells with staurosporine or PD98059 blocks both Bcl2 and c-Myc phosphorylation and results in suppression of NNK-induced proliferation. Specific depletion of c-Myc expression by RNA interference retards G(1)/S cell cycle transition and blocks NNK-induced cell proliferation. Phosphorylation of Bcl2 at Ser(70) promotes a direct interaction between Bcl2 and c-Myc in the nucleus and on the outer mitochondrial membrane that significantly enhances the half-life of the c-Myc protein. Thus, NNK-induced functional cooperation of Bcl2 and c-Myc in promoting cell survival and proliferation may occur in a novel mechanism involving their phosphorylation, which may lead to development of human lung cancer and/or chemoresistance.

Antineoplastic Agents↗

PubMed2004

Nicotine induces multi-site phosphorylation of Bad in association with suppression of apoptosis.

Nicotine is an important component in cigarette smoke that can activate the growth-promoting pathways to facilitate the development of lung cancer. However, the intracellular mechanism(s) by which nicotine promotes survival of lung cancer cells remains enigmatic. Bad is a proapoptotic BH3-only member of the Bcl2 family and is expressed in both small cell lung cancer and non-small cell lung cancer cells. Here we report that nicotine potently induces Bad phosphorylation at Ser112, Ser136, and Ser155 in a mechanism involving activation of MAPKs ERK1/2, PI3K/AKT, and PKA in human lung cancer cells. Nicotine-induced multi-site phosphorylation of Bad results in sequestering Bad from mitochondria and subsequently interacting with 14-3-3 in the cytosol. Treatment of cells with PKC inhibitor (staurosporine), MEK-specific inhibitor (PD98059), PI3 kinase inhibitor (LY294002), or PKA inhibitor (H89) blocks the nicotine-induced Bad phosphorylation that is associated with enhanced apoptotic cell death. The fact that beta-adrenergic receptor inhibitor (propranolol) blocks nicotine-induced activation of ERK1/2, AKT, PKA, Bad phosphorylation, and cell survival suggests that nicotine-induced Bad phosphorylation may occur through the upstream beta-adrenergic receptors. The fact that specific knockdown of Bad expression by RNA interference using short interfering RNA enhances cell survival and that nicotine has no additional survival effect on these cells suggests that Bad may act as a required target of nicotine. Thus, nicotine-induced survival may occur in a mechanism through multi-site phosphorylation of Bad, which may lead to development of human lung cancer and/or chemoresistance.

Apoptosis↗

PubMed2003

Mono- and multisite phosphorylation enhances Bcl2's antiapoptotic function and inhibition of cell cycle entry functions.

Bcl2 functions to suppress apoptosis and retard cell cycle entry. Single-site phosphorylation at serine 70 (S70) is required for Bcl2's antiapoptotic function, and multisite phosphorylation at threonine 69 (T69), S70, and S87 has been reported to inactivate Bcl2. To address this apparent conflict and identify the regulatory role for Bcl2 phosphorylation in cell death and cell cycle control, a series of serine/threonine (S/T) --> glutamate/alanine (E/A) mutants including T69E/A, S70E/A, S87E/A, T69E/S70A/S87A (EAA), T69A/S70E/S87A (AEA), T69A/S70A/S87E (AAE), T69E/S70E/S87E (EEE), and T69A/S70A/S87A (AAA) was created to mimic or abrogate, respectively, either single-site or multisite phosphorylation. The survival and cell cycle status of cells expressing the phosphomimetic or nonphosphorylatable Bcl2 mutants were compared. Surprisingly, all of the E but not the A Bcl2 mutants potently enhance cell survival after stress and retard G(1)/S cell cycle transition. The EEE Bcl2 mutant is the most potent, indicating a possible cumulative advantage for multisite phosphorylation of Bcl2 in survival and retardation of G(1)/S transition functions. Because the E-containing Bcl2 mutants, but not the A-containing mutants, can more potently block cytochrome c release from mitochondria during apoptotic stress, even at times when steady-state expression levels are similar for all mutants, we conclude that phosphorylation at one or multiple sites within the flexible loop domain of Bcl2 not only stimulates antiapoptotic activity but also can regulate cell cycle entry.

Amino Acid Substitution↗

PubMed2003

Bcl2 retards G1/S cell cycle transition by regulating intracellular ROS.

Bcl2's antiapoptotic function is regulated by phosphorylation. Bcl2 also regulates cell cycle progression, but the molecular mechanism is unclear. Bcl2 is functionally expressed in mitochondria where it can act as an antioxidant that may regulate intracellular reactive oxygen species (ROS). Since ROS have been reported to act as second messengers in cell signaling, we tested whether Bcl2 phosphorylation regulates ROS and cell cycle progression. G1 --> S transition and ROS levels were measured in cells expressing either the gain of function phosphomimetic Bcl2 mutants S70E and T69E/S70E/S87E (EEE) or the nonphosphorylatable and survival-deficient mutants S70A and T69A/S70A/S87A (AAA). Expression of S70E and EEE but not the A-containing Bcl2 mutants retards G1 --> S transition by 35% to 50% and significantly slows cell growth in association with reduced levels of intracellular ROS. In addition to expression of the phosphomimetic Bcl2 mutants, either interleukin-3 withdrawal or treatment of cells with the antioxidant pyrrolidine dithiocarbamate (PDTC) also reduces intracellular ROS levels in association with up-regulation of p27 and accumulation of cells in G0/G1. Retardation of G1 --> S transition can be overridden by directly adding H2O2 to the cells in a mechanism that involves down-regulation of p27 and activation of Cdk2. Thus, Bcl2 may regulate G1 --> S transition by a novel signaling mechanism that couples regulation of intracellular ROS with p27 and Cdk2. Furthermore, phosphorylation of Bcl2 may functionally link its antiapoptotic, cell cycle retardation, and antioxidant properties.

Animals↗

PubMed2003

Cleavage of Bax to p18 Bax accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade p18 Bax.

Bax is cleaved by calpain at aspartate 33 (Asp33) to yield p18 Bax during stress-induced apoptosis. To assess the role of p18 Bax in apoptosis, an ecdysone-inducible expression system was generated. Similar levels of wild-type (WT) and noncleavable Asp33Ala (Asp-->Ala) Bax are induced in 293 cells while expression of N-terminal-deleted p18 (Delta1-33) Bax remains low (20% of full-length p21 Bax) due to a reduced half-life (2 hours versus 12 hours for p21 Bax) resulting from increased sensitivity to cathepsin-like proteolytic degradation. Expression of p18 Bax is enhanced to levels comparable to p21 Bax when induction is carried out in the presence of cathepsin inhibitors, Z-Phe-Gly-NHO-Bz or N-Acetyl-Leu-Leu-Met-CHO. Compared with WT Bax, expression of similar levels of p18 Bax and, surprisingly, Asp33Ala Bax more potently induces apoptosis as indicated by increased cytochrome c release, caspase-9/-3 activation, and DNA fragmentation, potentially due to their increased homo-oligomerization in mitochondrial membranes. Studies in A-549, U-937, K-562, and HL-60 cells confirm that inhibition of Bax cleavage results in 25% to 35% reduction of drug-induced apoptosis, while inhibition of p18 Bax degradation enhances apoptosis by 25% to 40%. Results indicate that although cleavage to p18 Bax is not required for Bax to initiate apoptosis, p18 Bax potently accelerates the apoptotic process.

Animals↗

PubMed2003

Phosphorylation by aurora B converts MgcRacGAP to a RhoGAP during cytokinesis.

Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. Here, we show that MgcRacGAP colocalizes with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. We also report that Aurora B phosphorylates MgcRacGAP on serine residues and that this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, previously known as a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B.

Aurora Kinase B↗

PubMed2002

A functional role for nicotine in Bcl2 phosphorylation and suppression of apoptosis.

Nicotine is not only a major component in tobacco but is also a survival agonist that inhibits apoptosis induced by diverse stimuli including chemotherapeutic drugs. However, the intracellular mechanism(s) involved in nicotine suppression of apoptosis is unclear. Bcl2 is a potent antiapoptotic protein and tumor promotor that is expressed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells. It is possible that nicotine may regulate Bcl2 to stimulate cell survival. Here we report that nicotine can induce Bcl2 phosphorylation exclusively at the serine 70 site in association with prolonged survival of SCLC H82 cells expressing wild-type but not the phosphorylation-deficient S70A mutant Bcl2 after treatment with chemotherapeutic agents (i.e. cisplatin or VP-16). Nicotine induces activation of PKC alpha and the MAPKs ERK1 and ERK2, which are physiological Bcl2 kinases. Furthermore, ET-18-OCH3, a specific phospholipase C (PLC) inhibitor, blocks nicotine-stimulated Bcl2 phosphorylation and promotes apoptosis, suggesting that PLC may be involved in nicotine activation of Bcl2 kinases. Using a genetic approach, the gain-of-function S70E mutant, which mimics Ser(70) site phosphorylation in the flexible loop domain, potently enhances chemoresistance in SCLC cells. Thus, nicotine-induced cell survival results, at least in part, from a mechanism that involves Bcl2 phosphorylation. Therefore, novel therapeutic strategies for lung cancer in which Bcl2 is expressed may be used to abrogate the anti-apoptotic activity of Bcl2 by inhibiting multiple upstream nicotine-activated pathways.

Antineoplastic Agents↗