Xiuzhen Han

Hydrogen peroxide inhibits mTOR signaling by activation of AMPKα leading to apoptosis of neuronal cells

Oxidative stress results in apoptosis of neuronal cells, leading to neurodegenerative disorders. However, the underlying molecular mechanism remains to be elucidated. Here, we show that hydrogen peroxide (H2O2), a major oxidant generated when oxidative stress occurs, induced apoptosis of neuronal cells (PC12 cells and primary murine neurons), by inhibiting the mammalian target of rapamycin (mTOR)-mediated phosphorylation of ribosomal p70 S6 kinase (S6K1) and eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1). N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS), blocked H2O2 inhibition of mTOR signaling. Ectopic expression of wild-type (wt) mTOR, constitutively active S6K1 or downregulation of 4E-BP1 partially prevented H2O2 induction of apoptosis. Furthermore, we identified that H2O2 induction of ROS inhibited the upstream kinases, Akt and phosphoinositide-dependent kinase 1 (PDK1), but not the type I insulin-like growth factor receptor (IGFR), and activated the negative regulator, AMP-activated protein kinase α (AMPKα), but not the phosphatase and tensin homolog (PTEN) in the cells. Expression of a dominant negative AMPKα or downregulation of AMPKα1 conferred partial resistance to H2O2 inhibition of phosphorylation of S6K1 and 4E-BP1, as well as cell viability, indicating that H2O2 inhibition of mTOR signaling is at least in part through activation of AMPK. Our findings suggest that AMPK inhibitors may be exploited for prevention of H2O2-induced neurodegenerative diseases.

Calcium Signaling Is Involved in Cadmium-Induced Neuronal Apoptosis via Induction of Reactive Oxygen Species and Activation of MAPK/mTOR Network

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+]i) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+]i elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+]i elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+]i elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+]i, which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.

Rapamycin inhibits cytoskeleton reorganization and cell motility by suppressing RhoA expression and activity

The mammalian target of rapamycin (mTOR) functions in cells at least as two complexes, mTORC1 and mTORC2. Intensive studies have focused on the roles of mTOR in the regulation of cell proliferation, growth, and survival. Recently we found that rapamycin inhibits type I insulin-like growth factor (IGF-1)-stimulated lamellipodia formation and cell motility, indicating involvement of mTOR in regulating cell motility. This study was set to further elucidate the underlying mechanism. Here we show that rapamycin inhibited protein synthesis and activities of small GTPases (RhoA, Cdc42, and Rac1), crucial regulatory proteins for cell migration. Disruption of mTORC1 or mTORC2 by down-regulation of raptor or rictor, respectively, inhibited the activities of these proteins. However, only disruption of mTORC1 mimicked the effect of rapamycin, inhibiting their protein expression. Ectopic expression of rapamycin-resistant and constitutively active S6K1 partially prevented rapamycin inhibition of RhoA, Rac1, and Cdc42 expression, whereas expression of constitutively hypophosphorylated 4E-BP1 (4EBP1-5A) or down-regulation of S6K1 by RNA interference suppressed expression of the GTPases, suggesting that both mTORC1-mediated S6K1 and 4E-BP1 pathways are involved in protein synthesis of the GTPases. Expression of constitutively active RhoA, but not Cdc42 and Rac1, conferred resistance to rapamycin inhibition of IGF-1-stimulated lamellipodia formation and cell migration. The results suggest that rapamycin inhibits cell motility at least in part by down-regulation of RhoA protein expression and activity through mTORC1-mediated S6K1 and 4E-BP1-signaling pathways.

The urokinase plasminogen activator system in breast cancer invasion and metastasis

The urokinase plasminogen activator system, which is a serine protease family include urokinase-type plasminogen activator (uPA), the uPA receptor and plasminogen activator inhibitors (PAIs). uPA catalyzes the transformation of plasminogen to its active form plasmin, which is able to degrade the extracellular matrix (ECM) and basement membranes, directly or indirectly through activating pro-matrix metalloproteinases (pro-MMPs), promoting cancer cell metastasis and invasion. Both uPA and PAI-1 are poor prognosis markers in primary breast cancer. Evidence has been presented that the uPA system facilitates breast cancer metastasis by several different mechanisms, such as the Ras-ERK pathway and p38 MAPK pathway. This review focuses on uPA system, summarizes their biological effects, highlights the molecular mechanism and pathway, and discusses the role of uPA system in the prevention and treatment of human breast cancers.

Cryptotanshinone Inhibits Cancer Cell Proliferation by Suppressing Mammalian Target of Rapamycin-Mediated Cyclin D1 Expression and Rb Phosphorylation

Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, little is known about its anticancer mechanism. Here, we show that CPT inhibited cancer cell proliferation by arresting cells in G1-G0 phase of the cell cycle. This is associated with the inhibition of cyclin D1 expression and retinoblastoma (Rb) protein phosphorylation. Furthermore, we found that CPT inhibited the signaling pathway of the mammalian target of rapamycin (mTOR), a central regulator of cell proliferation. This is evidenced by the findings that CPT inhibited type I insulin-like growth factor I– or 10% fetal bovine serum–stimulated phosphorylation of mTOR, p70 S6 kinase 1, and eukaryotic initiation factor 4E binding protein 1 in a concentration- and time-dependent manner. Expression of constitutively active mTOR conferred resistance to CPT inhibition of cyclin D1 expression and Rb phosphorylation, as well as cell growth. The results suggest that CPT is a novel antiproliferative agent. Cancer Prev Res; 3(8); 1015–25. ©2010 AACR.

Naringenin-7-O-glucoside protects against doxorubicin-induced toxicity in H9c2 cardiomyocytes by induction of endogenous antioxidant enzymes.

Doxorubicin, a widely used chemotherapeutic agent, can give rise to severe cardiotoxicity that limits its clinical use by generation of reactive oxygen species (ROS) and apoptosis. Protection or alleviation of doxorubicin cardiotoxicity can be achieved by administration of natural phenolic compounds via activating endogenous defense systems and antiapoptosis. Naringenin-7-O-glucoside (NARG), isolated from Dracocephalum rupestre Hance, has been demonstrated to protect against cardiomyocyte apoptosis. In the present study, we investigated the effects of NARG on endogenous antioxidant enzymes against doxorubicin toxicity and the potential role of extracellular signal-regulated kinase (ERK) in regulation of NARG-induced Nrf2-dependent gene expression in H9c2 cardiomyocytes. The mRNA expression of glutamate-cysteine ligase modifier subunit (GCLM) and glutamate-cysteine ligase catalytic subunit (GCLC) was upregulated by NARG as detected by RT-PCR. NARG (10, 20, and 40microM) pretreatment increased NAD (P) H: quinone oxidoreductase (NQO1), ERK, and Nrf2 protein levels in cardiomyocytes as detected by Western blotting. These results suggest that NARG could prevent cardiomyocytes from doxorubicin-induced toxicity by induction of endogenous antioxidant enzymes via phosphorylation of ERK1/2 and nuclear translocation of Nrf2.

Curcumin inhibits protein phosphatases 2A and 5, leading to activation of mitogen-activated protein kinases and death in tumor cells

Curcumin can induce p53-independent apoptosis. However, the underlying mechanism remains to be defined. Here, we show that curcumin-induced apoptosis in a panel of tumor cells with mutant p53. Curcumin rapidly induced activation of the mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun N-terminal kinase (JNK). Inhibition of JNK (with SP600125) or Erk1/2 (with U0126) partially prevented curcumin-induced cell death in the cells. Similarly, expression of dominant negative c-Jun or downregulation of Erk1/2 in part attenuated curcumin-induced cell death. It appears that curcumin-induced activation of MAPKs and apoptosis was due to induction of reactive oxygen species (ROS), as pretreatment with N-acetyl-L-cysteine, a ROS scavenger, blocked these events. Furthermore, we found that curcumin-induced activation of MAPK pathways was related to inhibition of the serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5). Overexpression of PP2A or PP5 partially prevented curcumin-induced activation of JNK and Erk1/2 phosphorylation as well as cell death. The results suggest that curcumin induction of ROS activates MAPKs, at least partially by inhibiting PP2A and PP5, thereby leading to p53-independent apoptosis in tumor cells.

The antitumor activity of the fungicide ciclopirox

Ciclopirox olamine (CPX) is a synthetic antifungal agent clinically used to treat mycoses of the skin and nails. Here, we show that CPX inhibited tumor growth in human breast cancer MDA‐MB‐231 xenografts. To unveil the underlying mechanism, we further studied the antitumor activity of CPX in cell culture. The results indicate that CPX inhibited cell proliferation and induced apoptosis in human rhabdomyosarcoma (Rh30), breast carcinoma (MDA‐MB231) and colon adenocarcinoma (HT‐29) cells in a concentration‐dependent manner. By cell cycle analysis, CPX induced accumulation of cells in G1/G0 phase of the cell cycle. Concurrently, CPX downregulated cellular protein expression of cyclins (A, B1, D1 and E) and cyclin‐dependent kinases (CDK2 and CDK4) and upregulated expression of the CDK inhibitor p21Cip1, leading to hypophosphorylation of retinoblastoma protein. CPX also downregulated protein expression of Bcl‐xL and survivin and enhanced cleavages of Bcl‐2. Z‐VAD‐FMK, a pan‐caspase inhibitor, partially prevented CPX‐induced cell death, suggesting that CPX‐induced apoptosis of cancer cells is mediated at least in part through caspase‐dependent mechanism. The results indicate that CPX is a potential antitumor agent.

Rapamycin Inhibits IGF-1 Stimulated Cell Motility through PP2A Pathway

Serine/threonine (Ser/Thr) protein phosphatase 2A (PP2A) has been implicated as a novel component of the mammalian target of rapamycin (mTOR) signaling pathway. Recently we have demonstrated that mTOR regulates cell motility in part through p70 S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) pathways. Little is known about the role of PP2A in the mTOR-mediated cell motility. Here we show that rapamycin inhibited the basal or insulin-like growth factor 1 (IGF-1)-induced motility of human Ewing sarcoma (Rh1) and rhabdomyosarcoma (Rh30) cells. Treatment of the cells with rapamycin activated PP2A activity, and concurrently inhibited IGF-1 stimulated phosphorylation of Erk1/2. Inhibition of Erk1/2 with PD98059 did not significantly affect the basal mobility of the cells, but dramatically inhibited IGF-1-induced cell motility. Furthermore, inhibition of PP2A with okadaic acid significantly attenuated the inhibitory effect of rapamycin on IGF-1-stimulated phosphorylation of Erk1/2 as well as cell motility. Consistently, expression of dominant negative PP2A conferred resistance to IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. Expression of constitutively active MKK1 also attenuated rapamycin inhibition of IGF-1-stimulated phosphorylation of Erk1/2 and cell motility. The results suggest that rapamycin inhibits cell motility, in part by targeting PP2A-Erk1/2 pathway.

Protective effect of naringenin-7-O-glucoside against oxidative stress induced by doxorubicin in H9c2 cardiomyocytes.

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits its clinical use. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) and cardiomyocyte apoptosis appear to be involved in DOX cardiotoxicity. Protection or alleviation of DOX cardiotoxicity can be achieved by administration of natural phenolic compounds via activating endogenous defense systems and antiapoptosis. Naringenin-7-O-glucoside (NARG), isolated from Dracocephalum rupestre Hance, could protect from cardiomyocyte apoptosis and induce endogenous antioxidant enzymes against DOX toxicity, but the effects on intracellular ROS generation and cell membrane stability were not demonstrated. In the present study, we investigated the effects of NARG on H9c2 cell morphology, viability, lactate dehydrogenase (LDH) and creatine kinase (CK) leakage, glutathine peroxidase (GSH-Px) activity, intracellular Ca2+ concentration, and ROS generation. Compared with DOX alone treatment group, the morphological injury of the cells in groups treated by DOX plus NARG was alleviated, cell viability was increased, the amount of released LDH and CK was significantly decreased, the activity of GSH-Px was increased, the content of intracellular Ca2+ and ROS generation was lowered remarkably. These results suggest that NARG could prevent cardiomyocytes from DOX-induced toxicity by their property of stabilizing the cell membrane and reducing ROS generation.