Blockade of constitutively activated ERK signaling enhances cytotoxicity of microtubule-destabilizing agents in tumor cells

The extracellular signal-regulated kinase (ERK) signaling pathway is constitutively activated in many human tumor cell types. Given the cytoprotective role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although blockade of ERK signaling alone did not induce substantial cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the ERK pathway is constitutively activated. The synergistic activation of c-Jun NH₂-terminal kinase by the combination of an ERK pathway inhibitor and a microtubule-destabilizing agent appeared to be responsible, at least in part, for this effect. These results suggest that administration of the combination of an ERK pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells with constitutive activation of the ERK pathway.     

PARP-1-induced cell death through inhibition of the MEK/ERK pathway in MNNG-treated HeLa cells

Poly(ADP-ribose) polymerase-1 (PARP-1) hyper-activation promotes cell death but the signaling events downstream of PARP-1 activation are not fully identified. To gain further information on the implication of PARP-1 activation and PAR synthesis on signaling pathways influencing cell death, we exposed HeLa cells to the DNA alkylating agent N-methyl-N′-methyl-nitro-N-nitrosoguanidine (MNNG). We found that massive PAR synthesis leads to down-regulation of ERK1/2 phosphorylation, Bax translocation to the mitochondria, release of cytochrome c and AIF and subsequently cell death. Inhibition of massive PAR synthesis following MNNG exposure with the PARP inhibitor PJ34 prevented those events leading to cell survival, whereas inhibition of ERK1/2 phosphorylation by inhibiting MEK counteracted the cytoprotective effect of PJ34. Together, our results provide evidence that PARP-1-induced cell death by MNNG exposure in HeLa cells is mediated in part through inhibition of the MEK/ERK signaling pathway and that inhibition of massive PAR synthesis by PJ34, which promotes sustained activation of ERK1/2, leads to cytoprotection.     

Specific blockade of the ERK pathway inhibits the invasiveness of tumor cells: down-regulation of matrix metalloproteinase-3/-9/-14 and CD44

Elevated expression of matrix metalloproteinases (MMPs) is associated with increased metastatic potential in many tumor cells. As activation of the ERK pathway has been linked to the expression of MMP-9, we examined a possible correlation between ERK activation, MMP-9 expression, and invasive phenotype in human tumor cells. Activation state of the ERK pathway in tumor cells was well correlated with the invasive phenotype, which was determined by the ability of cells to invade through reconstituted extracellular matrix. Elevated expression of MMP-9 as well as of MMP-3, MMP-14, and CD44 was observed in tumor cells in which constitutive activation of the ERK pathway is detected. Blockade of the ERK pathway by treatment with PD184352, a specific and powerful inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), suppressed the expression of MMP-3, MMP-9, MMP-14, and CD44, and inhibited markedly the invasiveness of tumor cells. These results imply that, in addition to anti-proliferative effects, specific blockade of the ERK pathway is expected to result in anti-metastatic effects in tumor cells.     

2-Hydroxycurcuminoid induces apoptosis of human tumor cells through the reactive oxygen species-mitochondria pathway

2-Hydroxycinnamaldehyde (HCA) and curcumin have been reported to have antitumor effects against various human tumor cells in vitro and in vivo by generation of ROS. Aldehyde-free HCA analogs were synthesized based on the structure of curcumin, which we have called 2-hydroxycurcuminoids. The hydroxyl group of curcuminoids enhances the ability to generate ROS. 2-Hydroxycurcuminoid (HCC-7) strongly inhibited the growth of SW620 colon tumor cells with a GI₅₀ value of 7 μM, while the parent compounds, HCA and curcumin, displayed GI₅₀ values of 12 and 30 μM, respectively. HCC-7 was found to induce apoptosis through the reactive oxygen species-mitochondria pathway and cell cycle arrest at G2/M phase.     

Hematopoietic cell kinase enhances osteosarcoma development via the MEK/ERK pathway

Osteosarcoma (OS) is a sarcoma with high rates of pulmonary metastases and mortality. The mechanisms underlying tumour generation and development in OS are not well-understood. Haematopoietic cell kinase (HCK), a vital member of the Src family of kinase proteins, plays crucial roles in cancer progression and may act as an anticancer target; however, the mechanism by which HCK enhances OS development remains unexplored. Therefore, we investigated the role of HCK in OS development in vitro and in vivo. Downregulation of HCK attenuated OS cell proliferation, migration and invasion and increased OS cell apoptosis, whereas overexpression of HCK enhanced these processes. Mechanistically, HCK expression enhanced OS tumorigenesis via the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway; HCK upregulation increased the phosphorylation of MEK and ERK and promoted epithelial-mesenchymal transition, with a reduction in E-cadherin in vitro. Furthermore, HCK downregulation decreased the tumour volume and weight in mice transplanted with OS cells. In conclusion, HCK plays a crucial role in OS tumorigenesis, progression and metastasis via the MEK/ERK pathway, suggesting that HCK is a potential target for developing treatments for OS.     

Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner

Constitutive activation of the PI3 kinase/Akt pathway is associated with the neoplastic phenotype of a large number of human tumor cells. As the anti-apoptotic role of the PI3 kinase/Akt pathway has been established, we have examined whether specific blockade of this pathway sensitizes tumor cells to DNA-damaging agent-induced cytotoxicity by enhancing apoptotic cell death. Although a PI3 kinase inhibitor, LY294002, by itself does not induce apoptotic cell death, LY294002 selectively and markedly enhances the apoptosis-inducing efficacy of doxorubicin: such an enhanced cell death is only detected in tumor cells in which the PI3 kinase/Akt pathway is constitutively activated, and it is totally dependent on the functional p53 pathway. These results suggest that the combination of a PI3 kinase/Akt pathway inhibitor and doxorubicin provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the PI3 kinase/Akt pathway is constitutively activated and the p53 pathway is functional.     

The atypical pattern of cell death in B16F10 melanoma cells treated with TNP-470

TNP-470 is an acknowledged anti-angiogenic factor, and was studied clinically as an anti-cancer drug. We previously reported on an additional property of this molecule: the intracellular generation of reactive oxygen species in B16F10 melanoma cells. We showed that a massive generation of ROS occurred in the first few hours after treatment with TNP-470 and that this event was critical to subsequent cell death. In this study, we analyzed the process of cell death and noticed an atypical pattern of death markers. Some of these, such as DNA fragmentation or condensation of chromatin, were characteristic for programmed cell death, while others (the lack of phosphatidylserine flip-flop but permeability to propidium iodide, the maintenance of adhesion to the substratum, no change in mitochondrial transmembrane potential, no effect of the panspecific caspase inhibitor) rather suggested a necrotic outcome. We concluded that TNP-470 induced at least some pathways of programmed cell death. However, increasing damage to critical cell functions appears to cause a rapid switch into the necrotic mode. Our data is similar to that in other reports describing the action of ROS-generating agents. We hypothesize that this rapid programmed cell death/necrosis switch is a common scenario following free radical stress.     

Elevated expression of ERK 2 in human tumor cells chronically treated with PD98059

We examined the effect of chronic exposure of tumor cells to a mitogen-activated protein kinase/extracellular signal-regulated kinases (ERK) kinase inhibitor, PD98059, on cell proliferation was investigated. Human renal carcinoma cells (ACHN) and prostatic carcinoma cells (DU145) were cultured in the presence of PD98059 for more than 4 weeks (denoted ACHN (PD) cells and DU145 (PD) cells, respectively) and proliferation and signal transduction pathways were examined. PD98059 significantly inhibited the proliferation of parental cells. However, PD98059 failed to inhibit proliferation of ACHN (PD) and DU145 (PD) cells significantly. Expression of ERK 1 and 2 was elevated in these cells. These phenotypes were reversible. Downregulation of ERK 2, but not ERK 1, by small interfering RNA significantly inhibited the proliferation of ACHN (PD) and DU145 (PD) cells. Taken together, chronic exposure of tumor cells to PD98059 induced elevated expression of ERK 2, which was associated with decreased sensitivity of cellular proliferation to PD98059.     

Association of RDM1 with osteosarcoma progression via cell cycle and MEK/ERK signalling pathway regulation

RAD52 motif-containing 1 (RDM1), a key regulator of DNA double-strand break repair and recombination, has been reported to play an important role in the development of various human cancers, such as papillary thyroid carcinoma, neuroblastoma and lung cancer. However, the effect of RDM1 on osteosarcoma (OS) progression remains unclear. Here, this study mainly explored the connection between RDM1 and OS progression, as well as the underlying mechanism. It was found that RDM1 was highly expressed in OS cells compared with human osteoblast cells. Knockdown of RDM1 caused OS cell proliferation inhibition, cell apoptosis promotion and cell cycle arrest at G1 stage, whereas RDM1 overexpression resulted in the opposite phenotypes. Furthermore, RDM1 silencing leads to a significant decrease in tumour growth in xenograft mouse model. RDM1 also increased the protein levels of MEK 1/2 and ERK 1/2. All these findings suggest that RDM1 plays an oncogenic role in OS via stimulating cell cycle transition from G1 to S stage, and regulating MEK/ERK signalling pathway, providing a promising therapeutic factor for the treatment of OS.     

From nature to bedside: Pro-survival and cell death mechanisms as therapeutic targets in cancer treatment

Cell death is an important physiological regulator during development, tissue homeostasis and stress response but it is also a protective tumor suppressive mechanism. Tumor cells almost universally acquire the ability to evade cell death pathways that in normal cells act as a protective mechanism to remove damaged cells. As a result, a population of death-resistant cells with accumulating genetic and epigenetic abnormalities contributes to malignant transformation.     

Blockade of SOCE protects HT22 cells from hydrogen peroxide-induced apoptosis

Oxidative stress is an established event in the pathology of neurobiological diseases. Previous studies indicated that store-operated Ca²⁺ entry (SOCE) has been involved in oxidative stress. The present study was carried out to investigate the effects of SOCE inhibition on neuronal oxidative stress injury induced by hydrogen peroxide (H₂O₂) in HT22 cells, a murine hippocampal neuronal model. H₂O₂ insult induced significant intracellular Ca²⁺ overload, mitochondrial dysfunction and cell viability decrease. Inhibition of SOCE by pharmacological inhibitor and STIM1 RNAi significantly alleviated intracellular Ca²⁺ overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and eventually inhibited H₂O₂-induced cell apoptosis. These findings suggest that SOCE inhibition exhibited neuroprotection against oxidative stress induced by H₂O₂ and SOCE might be a useful therapeutic target in neurobiological disorders.     

A proteomic analysis of the effect of mapk pathway activation on l-glutamate-induced neuronal cell death

Oxidative stress has been implicated in the pathogenesis of neuronal degenerative diseases. It is also widely known that oxidative stress induces mitogen-activated protein kinase (MAPK) signaling cascades. In this study, we used proteomic analysis to investigate the role of the MAPK pathway in oxidative stress-induced neuronal cell death. The results demonstrated that several proteins, including eukaryotic translation elongation factor 2 (eEF2) and enolase I, showed a differential expression pattern during the neuronal cell death process, and this was MAPK pathway dependent. Several chaperone and cytoskeletal proteins including heat shock protein 70, calreticulin, vimentin, prolyl 4-hydroxylase β polypeptide, and transgelin 2 were up-or down-regulated, despite their expressions not depending on the MAPK pathway. These findings strongly suggest that the expressions of proteins which play protective roles are independent of the MAPK pathway. On the other hand, eEF2 and enolase I may be the downstream targets of the MAPK pathway.     

Involvement of MEK5/ERK5 signaling pathway in manganese-induced cell injury in dopaminergic MN9D cells

BackgroundOver-exposure to manganese (Mn) causes irreversible movement disorders with signs and symptoms similar, but not identical, to idiopathic Parkinson's disease (IPD). Recent data suggest that Mn toxicity occurs in dopaminergic (DA) neurons, although the mechanism remains elusive. This study was designed to investigate whether Mn interfered the apoptotic signaling transduction cascade in DA neurons.MethodsMouse midbrain dopaminergic MN9D cells were exposed to Mn in a concentration range of 0, 400, 800, or 1200 μM as designated as control, low, medium, and high exposure groups, respectively. The flow cytometry with Annexin V/PI double staining and immunohistochemistry were used to assess the apoptosis.ResultsData indicated that Mn exposure caused morphological alterations typical of apoptosis, increased apoptotic cells by 2–8 fold, and produced reactive oxidative species (ROS) by 1.5–2.2 fold as compared to controls (p < 0.05). Studies by qPCR and Western blot revealed that Mn exposure significantly increased the protein expression of extracellular signal-regulated kinase-5 (ERK5) and mitogen-activated ERK kinase-5 (MEK5) (p < 0.05). The presence of BIX02189, a specific inhibitor of MER/ERK, caused a much greater cytotoxicity, i.e., higher cell death, more ROS production, and worsened apoptosis, than did the treatment with Mn alone. Following Mn exposure, the expression of a downstream effector Bcl- 2 was reduced by 48 % while those of Bax and Caspase-3 were increased by 266.7 % and 90.1 %, respectively, as compared to controls (p < 0.05).ConclusionTaken together, these data provide the initial evidence that the signaling transduction cascade mediated by MEK5/ERK5 is responsible to Mn-induced cytotoxicity; Mn exposure, by suppressing anti-apoptotic function while facilitating pro-apoptotic activities, alters neuronal cell’s survival and functionally inhibits DA production by MN9D cells.     

Mitochondrial role in life and death of the cell

Mitochondria are the major ATP producer of the mammalian cell. Moreover, mitochondria are also the main intracellular source and target of reactive oxygen species (ROS) that are continually generated as by-products of aerobic metabolism in human cells. A low level of ROS generated from the respiratory chain was recently proposed to take part in the signaling from mitochondria to the nucleus. Several structural characteristics of mitochondria and the mitochondrial genome enable them to sense and respond to extracellular and intracellular signals or stresses in order to sustain the life of the cell. It has been established that mitochondrial respiratory function declines with age, and that defects in the respiratory chain increase the production of ROS and free radicals in mitochondria. Within a certain concentration range, ROS may induce stress responses of the cell by altering the expression of a number of genes in order to uphold energy metabolism to rescue the cell. However, beyond this threshold, ROS may elicit apoptosis by induction of mitochondrial membrane permeability transition and release of cytochrome c. Intensive research in the past few years has established that mitochondria play a pivotal role in the early phase of apoptosis in mammalian cells. In this article, the role of mitochondria in the determination of life and death of the cell is reviewed on the basis of recent findings gathered from this and other laboratories.     

Activation of NPFF2 receptor stimulates neurite outgrowth in Neuro 2A cells through activation of ERK signaling pathway

Neurite outgrowth is an important process in neural regeneration and plasticity, especially after neural injury, and recent evidence indicates that several G_αi/o protein-coupled receptors play an important role in neurite outgrowth. The neuropeptide (NP)FF system contains two G_αi/o protein-coupled receptors, NPFF₁ and NPFF₂ receptors, which are mainly distributed in the central nervous system. The aim of the present study was to determine whether the NPFF system is involved in neurite outgrowth in Neuro 2A cells. We showed that Neuro 2A cells endogenously expressed NPFF₂ receptor, and the NPFF₂ receptor agonist dNPA inhibited cyclic adenosine monophosphate (cAMP) production stimulated by forskolin in Neuro 2A cells. We also demonstrated that NPFF and dNPA dose-dependently induced neurite outgrowth in Neuro 2A cells, which was completely abolished by the NPFF receptor antagonist RF9. Pretreatment with mitogen-activated protein kinase inhibitors PD98059 and U0126 decreased dNPA-induced neurite outgrowth. In addition, dNPA increased phosphorylation of extracellular signal-regulated kinase (ERK) in Neuro 2A cells, which was completely antagonized by pretreatment with U0126. Our results suggest that activation of NPFF₂ receptor stimulates neurite outgrowth in Neuro 2A cells through activation of the ERK signaling pathway. Moreover, NPFF₂ receptor may be a potential therapeutic target for neural injury and degeneration in the future.     

Migration of F9 parietal endoderm cells is regulated by the ERK pathway

Cell migration is regulated by the action of many signaling pathways that are activated in specific regions of migrating cells. Extracellular regulated kinase 1/2 (ERK) signaling can modulate the migration of cells by controlling the turnover of focal adhesions and the dynamics of actin polymerization. Focal adhesion turnover is necessary for cell migration, and the formation of strong actin stress fibers and mature focal adhesions puts the brakes on cell migration. We used F9 wild-type and vinculin null (vin-/-) parietal endoderm (PE) outgrowth to study the role of the ERK signaling pathway in cell migration. Upon plating of F9 embryoid bodies (EBs) onto laminin-coated dishes, PE cells migrate away from the EBs, providing an in vitro model for studying directed migration of this embryonic cell type. Our results suggest that the ERK pathway regulates PE cell migration by affecting the formation of focal adhesions and lamellipodia through the action of myosin light chain kinase (MLCK).     

The reduced catalase expression in TrkA-induced cells leads to autophagic cell death via ROS accumulation

TrkA receptor activation is a pivotal process for neuronal cell differentiation and survival. However, its overactivation or removal of its ligand NGF tends to cause the cell death. Recently, we demonstrated that TrkA overexpression induces cell death via apoptosis. In this study we also show that the TrkA-mediated cell death is associated with autophagy. TrkA-induced cells revealed an increase of GFP-LC3 punctate formation, development of acidic vesicular organelles (AVO) and formation of autophagosomes, which were eventually blocked by the addition of some autophagy inhibitors such as 3-methyladenine, ammonium chloride or wortmannin. In addition, although expression of autophagy-related proteins such as LC3-II or Beclin-1 was subtly altered during the TrkA-mediated cell death, depletion of ATG5 or Beclin-1 substantially decreased cell death in TrkA-expressing cells. In particular, reactive oxygen species (ROS) were dramatically accumulated in TrkA-induced cells, and the high accumulation of ROS was released by treatment of autophagy inhibitors. Furthermore, addition of an antioxidant N-acetylcysteine promoted the survival of TrkA-expressing cells and suppressed AVO production in cells. We also showed that this ROS accumulation was closely associated with reduction of catalase expression. Taken together, TrkA overexpression causes ROS accumulation via reduced catalase expression, ultimately leading to autophagic cell death.