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1.  miR-7 and miR-153 protect neurons against MPP+-induced cell death via upregulation of mTOR pathway 
Differential expression of microRNAs (miRs) in the brain of patients with neurodegenerative diseases suggests that they may have key regulatory roles in the development of these disorders. Two such miRs, miR-7, and miR-153 have recently been shown to target α-synuclein, a protein critically involved in the pathological process of Parkinson's disease. By using a well-established in culture Parkinson's disease model that of neurotoxin 1-Methyl-4-Phenyl-Pyridinium (MPP+), we examined whether miR-7 and miR-153 display neuroprotective properties. Herein, we demonstrate that treatment of cortical neurons with MPP+ induced a dose-dependent cell death with apoptotic characteristics. This was reflected in altered intracellular signaling characterized by increased levels of activated kinases p38MAPK and ERK1/2 and reduced levels of activated AKT, p70S6K, and SAPK/JNK. Overexpression of miR-7 or miR-153 by adenoviral transduction protected cortical neurons from MPP+-induced toxicity, restored neuronal viability and anti-apoptotic BCL-2 protein levels while attenuated activation of caspase-3. Moreover, both miR-7 and miR-153 interfered with MPP+-induced alterations in intracellular signaling pathways in a partially overlapping manner; specifically, they preserved activation of mTOR and SAPK/JNK signaling pathways in the MPP+-treated neurons, while miR-153 also attenuated MPP+-induced activation of p38MAPK. No major effects were observed in the rest of signaling cascades or proteins investigated. Furthermore, the neuroprotective effect of miR-7 and miR-153 was alleviated when MPP+ was co-administered with rapamycin. Taken together, our results suggest that miR-7 and miR-153 protect neurons from cell death by interfering with the MPP+-induced downregulation of mTOR signaling.
PMCID: PMC4080263  PMID: 25071443
Parkinson's disease; miR-7; miR-153; MPP+; neuron; neuroprotection; rapamycin; mTOR
2.  MPP+-induced cytotoxicity in neuroblastoma cells: Antagonism and reversal by guanosine 
Purinergic Signalling  2007;3(4):399-409.
Guanosine exerts neuroprotective effects in the central nervous system. Apoptosis, a morphological form of programmed cell death, is implicated in the pathophysiology of Parkinson’s disease (PD). MPP+, a dopaminergic neurotoxin, produces in vivo and in vitro cellular changes characteristic of PD, such as cytotoxicity, resulting in apoptosis. Undifferentiated human SH-SY5Y neuroblastoma cells had been used as an in vitro model of Parkinson’s disease. We investigated if extracellular guanosine affected MPP+-induced cytotoxicity and examined the molecular mechanisms mediating its effects. Exposure of neuroblastoma cells to MPP+ (10 μM–5 mM for 24–72 h) induced DNA fragmentation in a time-dependent manner (p < 0.05). Administration of guanosine (100 μM) before, concomitantly with or, importantly, after the addition of MPP+ abolished MPP+-induced DNA fragmentation. Addition of MPP+ (500 μM) to cells increased caspase-3 activity over 72 h (p < 0.05), and this was abolished by pre- or co-treatment with guanosine. Exposure of cells to pertussis toxin prior to MPP+ eliminated the anti-apoptotic effect of guanosine, indicating that this effect is dependent on a Gi protein-coupled receptor, most likely the putative guanosine receptor. The protection by guanosine was also abolished by the selective inhibitor of the enzyme PI-3-K/Akt/PKB (LY294002), confirming that this pathway plays a decisive role in this effect of guanosine. Neither MPP+ nor guanosine had any significant effect on α-synuclein expression. Thus, guanosine antagonizes and reverses MPP+-induced cytotoxicity of neuroblastoma cells via activation of the cell survival pathway, PI-3-K/Akt/PKB. Our results suggest that guanosine may be an effective pharmacological intervention in PD.
PMCID: PMC2072917  PMID: 18404453
Apoptosis; Caspase-3; Cell survival; Cytotoxicity; DNA fragmentation; Guanosine; 1-methyl–4-phenyl-pyridinium (MPP+); Parkinson’s disease; SH-SY5Y human neuroblastoma cells
3.  Apoptosis inducing factor mediates caspase-independent 1-methyl-4-phenylpyridinium toxicity in dopaminergic cells 
Journal of neurochemistry  2005;94(6):1685-1695.
Parkinson’s disease is a debilitating neurodegenerative disease characterized by loss of midbrain dopaminergic neurons. These neurons are particularly sensitive to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which causes parkinsonian syndromes in humans, monkeys and rodents. Although apoptotic cell death has been implicated in MPTP/MPP+ toxicity, several recent studies have challenged the role of caspase-dependent apoptosis in dopaminergic neurons. Using the midbrain-derived MN9D dopaminergic cell line, we found that MPP+ treatment resulted in an active form of cell death that could not be prevented by caspase inhibitors or over-expression of a dominant negative inhibitor of apoptotic protease activating factor 1/caspase-9. Apoptosis inducing factor (AIF) is a mitochondrial protein that may mediate caspase-independent forms of regulated cell death following its translocation to the nucleus. We found that MPP+ treatment elicited nuclear translocation of AIF accompanied by large-scale DNA fragmentation. To establish the role of AIF in MPP+ toxicity, we constructed a DNA vector encoding a short hairpin sequence targeted against AIF. Reduction of AIF expression by RNA interference inhibited large-scale DNA fragmentation and conferred significant protection against MPP+ toxicity. Studies of primary mouse midbrain cultures further supported a role for AIF in caspase-independent cell death in MPP+-treated dopaminergic neurons.
PMCID: PMC1868549  PMID: 16156740
dopaminergic cells; mitochondria; neuronal cell death; Parkinson’s disease; primary midbrain neurons; RNA interference
4.  Regulation of GNRH production by estrogen and bone morphogenetic proteins in GT1-7 hypothalamic cells 
The Journal of endocrinology  2009;203(1):87-97.
Recent studies have shown that bone morphogenetic proteins (BMPs) are important regulators in the pituitary–gonadal endocrine axis. We here investigated the effects of BMPs on GNRH production controlled by estrogen using murine GT1-7 hypothalamic neuron cells. GT1-7 cells expressed estrogen receptor α (ERα; ESR1 as listed in MGI Database), ERβ (ESR2 as listed in MGI Database), BMP receptors, SMADs, and a binding protein follistatin. Treatment with BMP2 and BMP4 had no effect on Gnrh mRNA expression; however, BMP6 and BMP7 significantly increased Gnrh mRNA expression as well as GnRH production by GT1-7 cells. Notably, the reduction of Gnrh expression caused by estradiol (E2) was restored by cotreatment with BMP2 and BMP4, whereas it was not affected by BMP6 or BMP7. E2 activated extracellular signal-regulated kinase (ERK) 1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) signaling but did not activate p38-mitogen-activated protein kinase (MAPK) signaling in GT1-7 cells. Inhibition of ERK1/ERK2 reversed the inhibitory effect of estrogen on Gnrh expression, whereas SAPK/JNK inhibition did not affect the E2 actions. Expression levels of Erα and Erβ were reduced by BMP2 and BMP4, but were increased by BMP6 and BMP7. Treatment with an ER antagonist inhibited the E2 effects on Gnrh suppression including reduction of E2-induced ERK phosphorylation, suggesting the involvement of genomic ER actions in Gnrh suppression. BMP2 and BMP4 also suppressed estrogen-induced phosphorylation of ERK1/ERK2 and SAPK/JNK signaling, suggesting that BMP2 and BMP4 downregulate estrogen effects by attenuating ER–MAPK signaling. Considering that BMP6 and BMP7 increased the expression of α1E-subunit of R-type calcium channel (Cacna1e), which is critical for GNRH secretion, it is possible that BMP6 and BMP7 directly stimulate GNRH release by GT1-7 cells. Collectively, a newly uncovered interaction of BMPs and ER may be involved in controlling hypothalamic GNRH production and secretion via an autocrine/paracrine mechanism.
PMCID: PMC2768486  PMID: 19635757
5.  Pituitary Adenylate Cyclase-activating Polypeptide (PACAP)/PAC1HOP1 Receptor Activation Coordinates Multiple Neurotrophic Signaling Pathways 
The Journal of Biological Chemistry  2010;285(13):9749-9761.
MAPK and Akt pathways are predominant mediators of trophic signaling for many neuronal systems. Among the vasoactive intestinal peptide/secretin/glucagon family of related peptides, pituitary adenylate cyclase-activating polypeptide (PACAP) binding to specific PAC1 receptor isoforms can engage multiple signaling pathways and promote neuroprotection through mechanisms that are not well understood. Using a primary sympathetic neuronal system, the current studies demonstrate that PACAP activation of PAC1HOP1 receptors engages both MAPK and Akt neurotrophic pathways in an integrated program to facilitate neuronal survival after growth factor withdrawal. PACAP not only stimulated prosurvival ERK1/2 and ERK5 activation but also abrogated SAPK/JNK and p38 MAPK signaling in parallel. In contrast to the potent and rapid effects of PACAP in ERK1/2 phosphorylation, PACAP stimulated Akt phosphorylation in a late phase of PAC1HOP1 receptor signaling. From inhibitor and immunoprecipitation analyses, the PACAP/PAC1HOP1 receptor-mediated Akt responses did not represent transactivation mechanisms but appeared to depend on Gαq/phosphatidylinositol 3-kinase γ activity and vesicular internalization pathways. Phosphatidylinositol 3-kinase γ-selective inhibitors blocked PACAP-stimulated Akt phosphorylation in primary neuronal cultures and in PAC1HOP1-overexpressing cell lines; RNA interference-mediated knockdown of the receptor effectors attenuated PACAP-mediated Akt activation. Similarly, perturbation of endocytic pathways also blocked Akt phosphorylation. Between ERK and Akt pathways, PACAP-stimulated Akt signaling was the primary cascade that attenuated cultured neuron apoptosis after growth factor withdrawal. The partitioning of PACAP-mediated Akt signaling in endosomes may be a key mechanism contributing to the high spatial and temporal specificity in signal transduction necessary for survival pathways.
PMCID: PMC2843224  PMID: 20093365
G Proteins/Coupled Receptors (GPCR); Neurochemistry; Neurobiology/Neuroscience; Peptides/Neuropeptide; Receptors/Endocytosis; Signal Transduction
6.  TRPC1-mediated Inhibition of 1-Methyl-4-phenylpyridinium Ion Neurotoxicity in Human SH-SY5Y Neuroblastoma Cells* 
The Journal of biological chemistry  2004;280(3):2132-2140.
Mammalian homologues of the Drosophila canonical transient receptor potential (TRP) proteins have been implicated to function as plasma membrane Ca2+ channels. This study examined the role of TRPC1 in human neuroblastoma (SH-SY5Y) cells. SH-SY5Y cells treated with an exogenous neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP+) significantly decreased TRPC1 protein levels. Confocal microscopy on SH-SY5Y cells treatment with MPP+ showed decreased plasma membrane staining of TRPC1. Importantly, overexpression of TRPC1 reduced neurotoxicity induced by MPP+. MPP+-induced α-synuclein expression was also suppressed by TRPC1 overexpression. Protection of SH-SY5Y cells against MPP+ was significantly decreased upon the overexpression of antisense TRPC1 cDNA construct or the addition of a nonspecific transient receptor potential channel blocker lanthanum. Activation of TRPC1 by thapsigargin or carbachol decreased MPP+ neurotoxicity, which was partially dependent on external Ca2+. Staining of SH-SY5Y cells with an apoptotic marker (YO-PRO-1) showed that TRPC1 protects SH-SY5Y neuronal cells against apoptosis. Further, TRPC1 overexpression inhibited cytochrome c release and decreased Bax and Apaf-1 protein levels. Interpretation of the above data suggests that reduction in the cell surface expression of TRPC1 following MPP+ treatment may be involved in dopaminergic neurodegeneration. Furthermore, TRPC1 may inhibit degenerative apoptotic signaling to provide neuroprotection against Parkinson’s disease-inducing agents.
PMCID: PMC3619406  PMID: 15542611
7.  Type II cGMP-dependent protein kinase inhibits epidermal growth factor-induced phosphatidylinositol-3-kinase/Akt signal transduction in gastric cancer cells 
Oncology Letters  2013;6(6):1723-1728.
Our previous study revealed that Type II cGMP-dependent protein kinase (PKG II) inhibits epidermal growth factor (EGF)-induced MAPK/ERK and MAPK/JNK-mediated signal transduction through the inhibition of the phosphorylation/activation of the EGF receptor (EGFR). As EGFR also mediates several other signal transduction pathways besides MAPK-mediated pathways, the present study was designed to investigate whether PKG II was able to inhibit EGF/EGFR-induced phosphatidylinositol-3-kinase (PI3K)/Akt-mediated signal transduction. The AGS human gastric cancer cell line was infected with adenoviral constructs encoding a cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II, and treated with 8-pCPT-cGMP to activate the enzyme. Western blotting was used to detect the phosphorylation/activation of the key components of the signal transduction pathway, including EGFR, PI3K, Akt, mTOR and NF-κB. The levels of apoptosis-related proteins, including Bax, Bcl-2, caspase 9 and DNA fragment factor (DFF), were also determined by western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining was used to detect the apoptosis of the AGS cells. The results revealed that EGF treatment increased the phosphorylation (activation) of EGFR, PI3K, Akt and mTOR, and increased the nuclear localization (activation) of NF-κB. EGF treatment also reduced the apoptosis of the AGS cells and increased the expression of the anti-apoptotic protein, Bcl-2, but had no effect on the expression of the pro-apoptotic protein, Bax, and did not alter the levels of caspase 9 and DFF. Increasing the PKG II activity of AGS cells by infecting them with Ad-PKG II and stimulating them with 8-pCPT-cGMP inhibited the EGF-induced activation of EGFR, PI3K, Akt, mTOR and NF-κB; caused an increase in caspase 9 breakdown (activation) and DFF levels; and reversed the anti-apoptotic effect of EGF. The results suggest that PKG II may also inhibit EGF-induced signal transduction of PI3K/Akt-mediated pathways, and further confirm that PKG II is able to block the activation of EGFR.
PMCID: PMC3834871  PMID: 24273605
Type II cGMP-dependent protein kinase; phosphatidylinositol-3-kinase/Akt-mediated signal transduction; apoptosis; gastric cancer cells
8.  FDH-induced JNK pathways diverge at the JNK substrate level in cells with different p53 status 
FDH (10-formyltetrahydrofolate dehydrogenase) suppresses cancer cell proliferation through p53 dependent apoptosis but also induces strong cytotoxicity in p53-deficient prostate cells. In the present study we have demonstrated that FDH induces apoptosis in PC-3 prostate cells through simultaneous activation of the JNK and ERK pathways with JNK phosphorylating c-Jun and ERK1/2 phosphorylating Elk-1. The JNK1/2 inhibitor SP600125 or ERK1/2 inhibitor PD98059 prevented phosphorylation of c-Jun and Elk-1, correspondingly and partially protected PC-3 cells from FDH-induced cytotoxicity. Combination of the two inhibitors produced an additive effect. The contribution from the JNK cascade to FDH-induced apoptosis was significantly stronger than from the ERK pathway. siRNA knockdown of JNK1/2 or “turning off” the downstream target c-Jun by either siRNA or expression of the dominant negative c-Jun mutant, TAM67, rescued PC-3 cells from FDH-induced apoptosis. The pull-down assays on immobilized c-Jun demonstrated that c-Jun is directly phosphorylated by JNK2 in FDH-expressing cells. Interestingly, the FDH-induced apoptosis in p53-proficient A549 cells also proceeds through activation of JNK1/2 but the down-stream target for JNK2 is p53 instead of c-Jun. Furthermore, in A549 cells FDH activates caspase 9 while in PC-3 cells it activates caspase 8. Our studies indicate that the JNK pathways are common downstream mechanism of FDH-induced cytotoxicity in different cell types while the endpoint target in the cascade is cell type specific. JNK activation in response to FDH was inhibited by high supplementation of reduced folate leucovorin, further indicating a functional connection between folate metabolism and MAPK pathways.
PMCID: PMC2632845  PMID: 19147541
apoptosis; FDH; Jun kinases; c-Jun; RNAi; PC-3 cells
9.  ABL-N-induced apoptosis in human breast cancer cells is partially mediated by c-Jun NH2-terminal kinase activation 
The present study was designed to determine the possibility of acetylbritannilactone (ABL) derivative 5-(5-(ethylperoxy)pentan-2-yl)-6-methyl-3-methylene-2-oxo-2,3,3a,4,7,7a-hexahydrobenzofuran-4-yl 2-(6-methoxynaphthalen-2-yl)propanoate (ABL-N) as a novel therapeutic agent in human breast cancers.
We investigated the effects of ABL-N on the induction of apoptosis in human breast cancer cells and further examined the underlying mechanisms. Moreover, tumor growth inhibition of ABL-N was done in xenograft models.
ABL-N induced the activation of caspase-3 in estrogen receptor (ER)-negative cell lines MDA-MB-231 and MDA-MB-468, as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP). Pretreatment of cells with pan-caspase inhibitor z-VAD-fmk or caspase-3-specific inhibitor z-DEVD-fmk inhibited ABL-N-induced apoptosis. ABL-N treatment also resulted in an increase in the expression of pro-apoptotic members (Bax and Bad) with a concomitant decrease in Bcl-2. Furthermore, c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase (p38) were activated in the apoptosis induced by ABL-N and JNK-specific inhibitor SP600125 and JNK small interfering RNA (siRNA) antagonized ABL-N-mediated apoptosis. However, the p38-specific inhibitor SB203580 had no effect upon these processes. Moreover, neither of the caspase inhibitors prevented ABL-N-induced JNK activation, indicating that JNK is upstream of caspases in ABL-N-initiated apoptosis. Additionally, in a nude mice xenograft experiment, ABL-N significantly inhibited the tumor growth of MDA-MB-231 cells.
ABL-N induces apoptosis in breast cancer cells through the activation of caspases and JNK signaling pathways. Moreover, ABL-N treatment causes a significant inhibition of tumor growth in vivo. Therefore, it is thought that ABL-N might be a potential drug for use in breast cancer prevention and intervention.
PMCID: PMC2880430  PMID: 20096139
10.  Contribution of persistent C-Jun N-terminal kinase activity to the survival of human vestibular schwannoma cells by suppression of accumulation of mitochondrial superoxides 
Neuro-Oncology  2011;13(9):961-973.
Vestibular schwannomas (VSs) result from inactivating mutations in the merlin tumor suppressor gene. The merlin protein suppresses a variety of progrowth kinase–signaling cascades, including extracellular regulated kinase/mitogen-activated protein kinase (ERK/MAPK), c-Jun N-terminal kinase (JNK), and phosphatidyl-inositol 3-kinase (PI3-K)/Akt. Recent studies indicate that ERKs and Akt are active in human VSs, and here we show that JNKs are also persistently active in human VS cells. With use of cultures of human VSs, we investigated the contribution of each of these signals to the proliferative and survival response of VS cells. Inhibition of ERK or Akt signaling reduced VS cell proliferation but did not increase apoptosis, whereas inhibition of JNK with SP600125, I-JIP, or siRNA knock-down reduced VS cell proliferation and survival by inducing apoptosis. By contrast, JNK activity promotes apoptosis in normal Schwann cells. Inhibition of JNK increased the fluorescence intensity of VS cells loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), a fluorescent probe for reactive oxygen species (ROS). Furthermore, ebselen, a ROS scavenger, rescued VS cells with suppressed JNK from apoptosis, suggesting that JNK activity protects VS cells from apoptosis by limiting accumulation of ROS. VS cultures treated with JNK inhibitors demonstrated significantly higher levels of MitoSOX Red fluorescence, implying that persistent JNK activity specifically suppresses superoxide production in the mitochondria. Overexpression of superoxide dismutase 2 (MnSOD; mitochondrial SOD) prevented apoptosis in VS cells with suppressed JNK signaling. Taken together, these results indicate that persistent JNK activity enhances VS cell survival, at least in part, by suppressing accumulation of mitochondrial superoxides.
PMCID: PMC3158009  PMID: 21697181
acoustic neuroma; apoptosis; cell proliferation; cell signaling; merlin; reactive oxygen species
11.  AT-101, a small molecule inhibitor of anti-apoptotic Bcl-2 family members, activates the SAPK/JNK pathway and enhances radiation-induced apoptosis 
Gossypol, a naturally occurring polyphenolic compound has been identified as a small molecule inhibitor of anti-apoptotic Bcl-2 family proteins. It induces apoptosis in a wide range of tumor cell lines and enhances chemotherapy- and radiation-induced cytotoxicity both in vitro and in vivo. Bcl-2 and related proteins are important inhibitors of apoptosis and frequently overexpressed in human tumors. Increased levels of these proteins confer radio- and chemoresistance and may be associated with poor prognosis. Consequently, inhibition of the anti-apoptotic functions of Bcl-2 family members represents a promising strategy to overcome resistance to anticancer therapies.
We tested the effect of (-)-gossypol, also denominated as AT-101, radiation and the combination of both on apoptosis induction in human leukemic cells, Jurkat T and U937. Because activation of the SAPK/JNK pathway is important for apoptosis induction by many different stress stimuli, and Bcl-XL is known to inhibit activation of SAPK/JNK, we also investigated the role of this signaling cascade in AT-101-induced apoptosis using a pharmacologic and genetic approach.
AT-101 induced apoptosis in a time- and dose-dependent fashion, with ED50 values of 1.9 and 2.4 μM in Jurkat T and U937 cells, respectively. Isobolographic analysis revealed a synergistic interaction between AT-101 and radiation, which also appeared to be sequence-dependent. Like radiation, AT-101 activated SAPK/JNK which was blocked by the kinase inhibitor SP600125. In cells overexpressing a dominant-negative mutant of c-Jun, AT-101-induced apoptosis was significantly reduced.
Our data show that AT-101 strongly enhances radiation-induced apoptosis in human leukemic cells and indicate a requirement for the SAPK/JNK pathway in AT-101-induced apoptosis. This type of apoptosis modulation may overcome treatment resistance and lead to the development of new effective combination therapies.
PMCID: PMC2771029  PMID: 19852810
12.  Ginsenoside Rb3 Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of JNK-Mediated NF-κB Pathway: A Mouse Cardiomyocyte Model 
PLoS ONE  2014;9(8):e103628.
Ginsenoside Rb3 is extracted from the plant Panax ginseng and plays important roles in cardiovascular diseases, including myocardial ischemia-reperfusion (I/R) injury. NF-κB is an important transcription factor involved in I/R injury. However, the underlying mechanism of ginsenoside Rb3 in myocardial I/R injury remains poorly understood. In the current study, a model of myocardial I/R injury was induced via oxygen and glucose deprivation (OGD) followed by reperfusion (OGD-Rep) in mouse cardiac myoblast H9c2 cells. Our data demonstrate that ginsenoside Rb3 suppresses OGD-Rep-induced cell apoptosis by the suppression of ROS generation. By detecting the NF-κB signaling pathway, we discover that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is closely related to the inhibition of NF-κB activity. Ginsenoside Rb3 inhibits the upregulation of phospho-IκB-α and nuclear translocation of NF-κB subunit p65 which are induced by ORD-Rep injury. In addition, the extract also inhibits the OGD-Rep-induced increase in the expression of inflammation-related factors, such as IL-6, TNF-α, monocyte chemotactic protein-1 (MCP-1), MMP-2 and MMP-9. However, LPS treatment alleviates the protective roles of ginsenoside Rb3 and activates the NF-κB pathway. Finally, the upstream factors of NF-κB were analyzed, including the Akt/Foxo3a and MAPK signaling pathways. We find that ginsenoside Rb3 pretreatment only decreases the phosphorylation of JNK induced by OGD-Rep injury, an indicator of the MAPK pathway. Importantly, an inhibitor of phospho-JNK, SP600125, protects against OGD-Rep induced apoptosis and inhibited NF-κB signaling pathway, similar to the roles of ginsenoside Rb3. Taken together, our results demonstrate that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is attributed to the inhibition of JNK-mediated NF-κB activation, suggesting that ginsenoside Rb3 has the potential to serve as a novel therapeutic agent for myocardial I/R injury.
PMCID: PMC4118887  PMID: 25084093
13.  The Anti-Apoptotic and Cardioprotective Effects of Salvianolic Acid A on Rat Cardiomyocytes following Ischemia/Reperfusion by DUSP-Mediated Regulation of the ERK1/2/JNK Pathway 
PLoS ONE  2014;9(7):e102292.
The purpose of this study was to observe the effects of salvianolic acid A (SAA) pretreatment on the myocardium during ischemia/reperfusion (I/R) and to illuminate the interrelationships among dual specificity protein phosphatase (DUSP) 2/4/16, ERK1/2 and JNK pathways during myocardial I/R, with the ultimate goal of elucidating how SAA exerts cardioprotection against I/R injury (IRI). Wistar rats were divided into the following six groups: control group (CON), I/R group, SAA+I/R group, ERK1/2 inhibitor PD098059+I/R group (PD+I/R), PD+SAA+I/R group, and JNK inhibitor SP600125+I/R group (SP+I/R). The cardioprotective effects of SAA on the myocardium during I/R were investigated with a Langendorff device. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), maximum rate of ventricular pressure rise and fall (±dp/dtmax), myocardial infarction areas (MIA), lactate dehydrogenase (LDH), and cardiomyocytes apoptosis were monitored. To determine the crosstalk betwee JNK and ERK1/2 via DUSP2/4/16 with SAA pretreatment, siRNA-DUSP2/4/16 were performed. The expression levels of Bcl-2, Bax, caspase 3, p-JNK, p-ERK1/2 and DUSP2/4/16 in cardiomyocytes were assayed by Western blot. Our results showed that LDH, MIA and cell apoptosis were decreased, and various parameters of heart function were improved by SAA pretreatment and SP application. In the I/R group, the expression levels of p-ERK1/2 and DUSP4/16 were not significantly different compared with the CON group, however, the protein expression levels of p-ERK1/2, Bcl-2 and DUSP4/16 were higher, while p-JNK, Bax, caspase 3 and DUSP2 levels were reduced among the SAA+I/R, PD+SAA+I/R and SP+I/R groups. The above indices were not significantly different between the SAA+I/R and SP+I/R groups. Compared with the SAA+I/R group, p-ERK1/2 was increased and p-JNK was decreased in the SAA+si-DUSP2+I/R, however, p-ERK was downregulated and p-JNK was upregulated in SAA+si-DUSP4+I/R group. SAA exerts an anti-apoptotic role against myocardial IRI by inhibiting DUSP2-mediated JNK dephosphorylation and activating DUSP4/16-mediated ERK1/2 phosphorylation.
PMCID: PMC4096914  PMID: 25019380
14.  Gefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires BIM and Can Be Enhanced by BH3 Mimetics 
PLoS Medicine  2007;4(10):e316.
The epidermal growth factor receptor (EGFR) plays a critical role in the control of cellular proliferation, differentiation, and survival. Abnormalities in EGF-EGFR signaling, such as mutations that render the EGFR hyperactive or cause overexpression of the wild-type receptor, have been found in a broad range of cancers, including carcinomas of the lung, breast, and colon. EGFR inhibitors such as gefitinib have proven successful in the treatment of certain cancers, particularly non-small cell lung cancers (NSCLCs) harboring activating mutations within the EGFR gene, but the molecular mechanisms leading to tumor regression remain unknown. Therefore, we wished to delineate these mechanisms.
Methods and Findings
We performed biochemical and genetic studies to investigate the mechanisms by which inhibitors of EGFR tyrosine kinase activity, such as gefitinib, inhibit the growth of human NSCLCs. We found that gefitinib triggered intrinsic (also called “mitochondrial”) apoptosis signaling, involving the activation of BAX and mitochondrial release of cytochrome c, ultimately unleashing the caspase cascade. Gefitinib caused a rapid increase in the level of the proapoptotic BH3-only protein BIM (also called BCL2-like 11) through both transcriptional and post-translational mechanisms. Experiments with pharmacological inhibitors indicated that blockade of MEK–ERK1/2 (mitogen-activated protein kinase kinase–extracellular signal-regulated protein kinase 1/2) signaling, but not blockade of PI3K (phosphatidylinositol 3-kinase), JNK (c-Jun N-terminal kinase or mitogen-activated protein kinase 8), or AKT (protein kinase B), was critical for BIM activation. Using RNA interference, we demonstrated that BIM is essential for gefitinib-induced killing of NSCLC cells. Moreover, we found that gefitinib-induced apoptosis is enhanced by addition of the BH3 mimetic ABT-737.
Inhibitors of the EGFR tyrosine kinase have proven useful in the therapy of certain cancers, in particular NSCLCs possessing activating mutations in the EGFR kinase domain, but the mechanisms of tumor cell killing are still unclear. In this paper, we demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR–MEK–ERK signaling cascade is critical for BIM activation. Moreover, we demonstrate that addition of a BH3 mimetic significantly enhances killing of NSCLC cells by the EGFR tyrosine kinase inhibitor gefitinib. It appears likely that this approach represents a paradigm shared by many, and perhaps all, oncogenic tyrosine kinases and suggests a powerful new strategy for cancer therapy.
Andreas Strasser and colleagues demonstrate that activation of the proapoptotic BH3-only protein BIM is essential for tumor cell killing and that shutdown of the EGFR−MEK−ERK signaling cascade is critical for BIM activation.
Editors' Summary
Normally, cell division (which produces new cells) and cell death are finely balanced to keep the human body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—life-threatening, disorganized masses of cells. One protein with a critical role in cell division that is often mutated in tumors is the epidermal growth factor receptor (EGFR). In normal cells, protein messengers bind to EGFR and activate its tyrosine kinase. This enzyme then adds phosphate groups to tyrosine (an amino acid) in proteins that form part of signaling cascades (for example, the MEK–ERK signaling cascade) that tell the cell to divide. In cancers that have mutations in EGFR, signaling is overactive so the cancer cells divide much more than they should. Some non-small cell lung cancers (NSCLC, the commonest type of lung cancer), for example, have activating mutations within the EGFR tyrosine kinase. Treatment with EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib induces the cells in these tumors to stop growing and die. This cell death causes tumor shrinkage (regression) and increases the life expectancy of patients with this type of NSCLC.
Why Was This Study Done?
Unfortunately, treatment with TKIs rarely cures NSCLC, so it would be useful to find a way to augment the effect that TKIs have on cancer cells. To do this, the molecular mechanisms that cause cancer-cell death and tumor regression in response to these drugs need to be fully understood. In this study, the researchers have used a combination of biochemical and genetic approaches to investigate how gefitinib kills NSCLC cells with mutated EGFR.
What Did the Researchers Do and Find?
The researchers first measured the sensitivity of NSCLC cell lines (tumor cells that grow indefinitely in dishes) to gefitinib-induced apoptosis. Gefitinib caused extensive apoptosis in two cell lines expressing mutant EGFR but not in one expressing normal EGFR. Next, they investigated the mechanism of gefitinib-induced apoptosis in the most sensitive cell line (H3255). Apoptosis is activated via two major pathways. Hallmarks of the “intrinsic” pathway include activation of a protein called BAX and cytochrome c release from subcellular compartments known as mitochondria. Gefitinib treatment induced both these events in H3255 cells. BAX (a proapoptotic member of the BCL-2 family of proteins) is activated when proapoptotic BH3-only BCL-2 proteins (for example, BIM; “BH3-only” describes the structure of these proteins) bind to antiapoptotic BCL2 proteins. Gefitinib treatment rapidly increased BIM activity in H3255 and HCC827 cells (but not in gefitinib-resistant cells) by increasing the production of BIM protein and the removal of phosphate groups from it, which increases BIM activity. Pharmacological blockade of the MEK–ERK signaling cascade, but not of other EGFR signaling cascades, also caused the accumulation of BIM. By contrast, blocking BIM expression using a technique called RNA interference reduced gefitinib-induced apoptosis. Finally, a combination of gefitinib and a BH3-mimicking compound called ABT-737 (which, like BIM, binds to antiapoptotic BCL-2 proteins) caused more apoptosis than gefitinib alone.
What Do These Findings Mean?
These findings (and those reported by Gong et al. and Costa et al.) indicate that activation of the proapoptotic BH3-only protein BIM is essential for gefitinib-induced killing of NSCLC cells that carry EGFR tyrosine kinase mutations. They also show that inhibition of the EGFR–MEK–ERK signaling cascade by gefitinib is essential for BIM activation. Because these findings come from studies on NSCLC cell lines, they need confirming in freshly isolated tumor cells and in tumors growing in people. However, the demonstration that a compound that mimics BH3 action enhances gefitinib-induced killing of NSCLC cells suggests that combinations of TKIs and drugs that affect the intrinsic pathway of apoptosis activation might provide a powerful strategy for treating cancers in which tyrosine kinase mutations drive tumor growth.
Additional Information.
Please access these Web sites via the online version of this summary at
A perspective by Ingo Mellinghoff discusses this article and two related research articles
Wikipedia pages on epidermal growth factor receptor, apoptosis, and BCL2 proteins (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
CancerQuest provides information on all aspects of cancer from Emory University (in several languages)
US National Cancer Institute information for patients and professionals on lung cancer (in English and Spanish)
Information for patients from Cancer Research UK on lung cancer including information on treatment with TKIs
Information for patients from Cancerbackup on erlotinib and gefitinib
PMCID: PMC2043013  PMID: 17973573
15.  LPS-induced apoptosis in Transformed Bovine Brain Endothelial Cells (TBBEC) and Human Dermal Microvessel Endothelial Cells (HMEC): the role of JNK 
Stimulation of transformed bovine brain endothelial cells (TBBEC) with lipopolysaccharide (LPS) leads to apoptosis while human microvessel endothelial cells (HMEC) needs the presence of cycloheximide (CHX) with LPS to induce apoptosis. To investigate the molecular mechanism of LPS-induced apoptosis in HMEC or TBBEC, we analyzed the involvement of mitogen activated protein kinases (MAPKs) and PI3K in TBBEC and HMEC. LPS-induced apoptosis in TBBEC was hallmarked by the activation caspase-3, -6, and -8 after the stimulation of LPS, followed by poly-ADP ribose polymerase (PARP) cleavage and lactate dehydrogenase (LDH) release. We also observed DNA cleavage determined by terminal deoxy transferase (TdT)-mediated dUTP nick and labeling [TUNEL] staining in TBBEC treated with LPS. Herbimycin A, a tyrosine kinase inhibitor, and SP600125, a c-jun N-terminal kinase (JNK) inhibitor, suppressed the activation of caspases and LDH release. Moreover, a phosphatidyl-inositol 3-phosphate (PI3K) inhibitor (LY294002) suppressed activation of caspases, and combined treatment with both SP600125 and LY294002 completely inhibited the activation of caspases. These results suggest that JNK signaling pathway through the tyrosine kinase and PI3K pathways are involved in the induction of apoptosis in LPS-treated TBBEC. On the other hand, we observed sustained JNK activation in HMEC treated with LPS and CHX, and neither Erk1/2 nor AKT were activated. The addition of SP600125 suppressed phosphorylation of JNK and the activation of caspase-3 in HMEC treated with LPS and CHX. These results suggest that JNK plays an important role in the induction of apoptosis in endothelial cells.
PMCID: PMC3057198  PMID: 19454725
16.  Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis. 
Molecular and Cellular Biology  1997;17(9):5317-5327.
Resistance to stress-induced apoptosis was examined in cells in which the expression of hsp70 was either constitutively elevated or inducible by a tetracycline-regulated transactivator. Heat-induced apoptosis was blocked in hsp70-expressing cells, and this was associated with reduced cleavage of the common death substrate protein poly(ADP-ribose) polymerase (PARP). Heat-induced cell death was correlated with the activation of the stress-activated protein kinase SAPK/JNK (c-Jun N-terminal kinase). Activation of SAPK/JNK was strongly inhibited in cells in which hsp70 was induced to a high level, indicating that hsp70 is able to block apoptosis by inhibiting signaling events upstream of SAPK/JNK activation. In contrast, SAPK/JNK activation was not inhibited by heat shock in cells with constitutively elevated levels of hsp70. Cells that constitutively overexpress hsp70 resist apoptosis induced by ceramide, a lipid signaling molecule that is generated by apoptosis-inducing treatments and is linked to SAPK/JNK activation. Similar to heat stress, resistance to ceramide-induced apoptosis occurs in spite of strong SAPK/JNK activation. Therefore, hsp70 is also able to inhibit apoptosis at some point downstream of SAPK/JNK activation. Since PARP cleavage is prevented in both cell lines, these results suggest that hsp70 is able to prevent the effector steps of apoptotic cell death. Processing of the CED-3-related protease caspase-3 (CPP32/Yama/apopain) is inhibited in hsp70-expressing cells; however, the activity of the mature enzyme is not affected by hsp70 in vitro. Caspase processing may represent a critical heat-sensitive target leading to cell death that is inhibited by the chaperoning function of hsp70. The inhibition of SAPK/JNK signaling and apoptotic protease effector steps by hsp70 likely contributes to the resistance to stress-induced apoptosis seen in transiently induced thermotolerance.
PMCID: PMC232382  PMID: 9271409
17.  Chikusetsu Saponin V Attenuates MPP+-Induced Neurotoxicity in SH-SY5Y Cells via Regulation of Sirt1/Mn-SOD and GRP78/Caspase-12 Pathways 
Studies have shown that saponins from Panax japonicus (SPJ) possess neuroprotective effects. However, whether Chikusetsu saponin V (CsV), the most abundant member of SPJ, can exert neuroprotective effects against 1-methyl-4-phenylpyridinium ion (MPP+)-induced cytotoxicity is not known. In this study, we aimed to investigate the neuroprotective effects of CsV on MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells and explore its possible mechanisms. Our results show that CsV attenuates MPP+-induced cytotoxicity, inhibits ROS accumulation, and increases mitochondrial membrane potential dose-dependently. We also found that levels of Sirt1 protein and Mn-SOD mRNA significantly decreased in MPP+-treated group but were restored with CsV treatment in a dose-dependent manner. Furthermore, GRP78 protein and Caspase-12 mRNA levels were elevated by MPP+ exposure but reversed by CsV treatment. CsV inhibited the MPP+-induced downregulation of Bcl-2 and up-regulation of Bax in a dose-dependent manner and, thus, increased the ratio of Bcl-2/Bax. Overall, these results suggest that Sirt1/Mn-SOD and GRP78/Caspase-12 pathways might be involved in the CsV-mediated neuroprotective effects.
PMCID: PMC4159789  PMID: 25073091
Chikusetsu saponin V; MPP+; Sirt1; GRP78
18.  Late Activation of Stress Kinases (SAPK/JNK) by Genotoxins Requires the DNA Repair Proteins DNA-PKcs and CSB 
Molecular Biology of the Cell  2006;17(2):851-861.
Although genotoxic agents are powerful inducers of stress kinases (SAPK/JNK), the contribution of DNA damage itself to this response is unknown. Therefore, SAPK/JNK activation of cells harboring specific defects in DNA damage-recognition mechanisms was studied. Dual phosphorylation of SAPK/JNK by the genotoxin methyl methanesulfonate (MMS) occurred in two waves. The early response (≤2 h after exposure) was similar in cells knockout for ATM, PARP, p53, and CSB or defective in DNA-PKcs compared with wild-type cells. The late response however (≥4 h), was drastically reduced in DNA-PKcs and Cockayne's syndrome B (CSB)-deficient cells. Similar results were obtained with human cells lacking DNA-PKcs and CSB. Activation of SAPK/JNK by MMS was not affected upon inhibition of base excision repair (BER), indicating base damage itself does not signal to SAPK/JNK. Because SAPK/JNK activation was attenuated in nongrowing cells, DNA replication-dependent processing of lesions, involving DNA-PKcs and CSB, appears to be required. DNA-PKcs coprecipitates with SEK1/MKK4 and SAPK/JNK, supporting a role of DNA-PKcs in SAPK/JNK activation. In this process, Rho GTPases are involved since inhibition of Rho impairs MMS-induced signaling to SAPK/JNK. The data show that sensing of DNA damage by DNA-PKcs and CSB causes a delayed SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK.
PMCID: PMC1356594  PMID: 16319174
19.  Involvment of Cytosolic and Mitochondrial GSK-3β in Mitochondrial Dysfunction and Neuronal Cell Death of MPTP/MPP+-Treated Neurons 
PLoS ONE  2009;4(5):e5491.
Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson's disease (PD). 1-methyl-4-phenylpyridinium iodide (MPP+), the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3β (GSK-3β), a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3β in modulating MPP+-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP+ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3β, evidenced by the increased level of the active form of the kinase, i.e. GSK-3β phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3β partially localized within mitochondria in both neuronal cell models. Moreover, MPP+ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3β labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP+ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3β activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP+-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3β is a critical mediator of MPTP/MPP+-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3β activity might provide protection against mitochondrial stress-induced cell death.
PMCID: PMC2675062  PMID: 19430525
20.  SAPK2/p38-dependent F-Actin Reorganization Regulates Early Membrane Blebbing during Stress-induced Apoptosis  
The Journal of Cell Biology  1998;143(5):1361-1373.
In endothelial cells, H2O2 induces the rapid formation of focal adhesion complexes at the ventral face of the cells and a major reorganization of the actin cytoskeleton into dense transcytoplasmic stress fibers. This change in actin dynamics results from the activation of the mitogen-activated protein (MAP) kinase stress-activated protein kinase-2/p38 (SAPK2/p38), which, via MAP kinase-activated protein (MAPKAP) kinase-2/3, leads to the phosphorylation of the actin polymerization modulator heat shock protein of 27 kD (HSP27). Here we show that the concomitant activation of the extracellular signal-regulated kinase (ERK) MAP kinase pathway by H2O2 accomplishes an essential survival function during this process. When the activation of ERK was blocked with PD098059, the focal adhesion complexes formed under the plasma membrane, and the actin polymerization activity led to a rapid and intense membrane blebbing. The blebs were delimited by a thin F-actin ring and contained enhanced levels of HSP27. Later, the cells displayed hallmarks of apoptosis, such as DEVD protease activities and internucleosomal DNA fragmentation. Bleb formation but not apoptosis was blocked by extremely low concentrations of the actin polymerization inhibitor cytochalasin D or by the SAPK2 inhibitor SB203580, indicating that the two processes are not in the same linear cascade. The role of HSP27 in mediating membrane blebbing was assessed in fibroblastic cells. In control fibroblasts expressing a low level of endogenous HSP27 or in fibroblasts expressing a high level of a nonphosphorylatable HSP27, H2O2 did not induce F-actin accumulation, nor did it generate membrane blebbing activity in the presence or absence of PD098059. In contrast, in fibroblasts that expressed wild-type HSP27 to a level similar to that found in endothelial cells, H2O2 induced accumulation of F-actin and caused bleb formation when the ERK pathway was inhibited. Cis-platinum, which activated SAPK2 but induced little ERK activity, also induced membrane blebbing that was dependent on the expression of HSP27. In these cells, membrane blebbing was not followed by caspase activation or DNA fragmentation. We conclude that the HSP27-dependent actin polymerization–generating activity of SAPK2 associated with a misassembly of the focal adhesions is responsible for induction of membrane blebbing by stressing agents.
PMCID: PMC2133090  PMID: 9832563
SAPK2/p38; HSP27; F-actin; blebbing; apoptosis
21.  Suppression of MMP-2 Attenuates TNF-α Induced NF-κB Activation and Leads to JNK Mediated Cell Death in Glioma 
PLoS ONE  2011;6(5):e19341.
Abrogation of apoptosis for prolonged cell survival is essential in cancer progression. In our previous studies, we showed the MMP-2 downregulation induced apoptosis in cancer cell lines. Here, we attempt to investigate the exact molecular mechanism of how MMP-2 depletion leads to apoptosis in glioma xenograft cell lines.
Methodology/Principal Findings
MMP-2 transcriptional suppression by MMP-2siRNA (pM) induces apoptosis associated with PARP, caspase-8 and -3 cleavage in human glioma xenograft cells 4910 and 5310. Western blotting and cytokine array showed significant decrease in the cellular and secreted levels of TNF-α with concomitant reduction in TNFR1, TRADD, TRAF2, RIP, IKKβ and pIκBα expression levels resulting in inhibition of p65 phosphorylation and nuclear translocation in pM-treated cells when compared to mock and pSV controls. In addition MMP-2 suppression led to elevated Fas-L, Fas and FADD expression levels along with increased p38 and JNK phosphorylation. The JNK-activity assay showed prolonged JNK activation in pM-transfected cells. Specific inhibition of p38 with SB203580 did not show any effect whereas inhibition of JNK phosphorylation with SP600125 notably reversed pM-induced cleavage of PARP, caspase-8 and -3, demonstrating a significant role of JNK in pM-induced cell death. Supplementation of rhMMP-2 counteracted the effect of pM by remarkably elevating TNF-α, TRADD, IKKβ and pIκBα expression and decreasing FADD, Fas-L, and phospho-JNK levels. The EMSA analysis indicated significant reversal of pM-inhibited NF-κB activity by rhMMP-2 treatment which rescued cells from pM-induced cell death. In vivo studies indicated that pM treatment diminished intracranial tumor growth and the immuno histochemical analysis showed decreased phospho-p65 and enhanced phospho-JNK levels that correlated with increased TUNEL-positive apoptotic cells in pM-treated tumor sections.
In summary, our study implies a role of MMP-2 in the regulation of TNF-α mediated constitutive NF-κB activation and Fas-mediated JNK mediated apoptosis in glioma xenograft cells in vitro and in vivo.
PMCID: PMC3087754  PMID: 21573233
22.  Fas Induces Cytoplasmic Apoptotic Responses and Activation of the MKK7-JNK/SAPK and MKK6-p38 Pathways Independent of CPP32-like Proteases  
The Journal of Cell Biology  1997;139(4):1005-1015.
IL-1β converting enzyme (ICE) family cysteine proteases are subdivided into three groups; ICE-, CPP32-, and Ich-1–like proteases. In Fas-induced apoptosis, activation of ICE-like proteases is followed by activation of CPP32-like proteases which is thought to be essential for execution of the cell death. It was recently reported that two subfamily members of the mitogen-activated protein kinase superfamily, JNK/SAPK and p38, are activated during Fas-induced apoptosis. Here, we have shown that MKK7, but not SEK1/ MKK4, is activated by Fas as an activator for JNK/ SAPK and that MKK6 is a major activator for p38 in Fas signaling. Then, to dissect various cellular responses induced by Fas, we used several peptide inhibitors for ICE family proteases in Fas-treated Jurkat cells and KB cells. While Z-VAD-FK which inhibited almost all the Fas-induced cellular responses blocked the activation of JNK/SAPK and p38, Ac-DEVD-CHO and Z-DEVD-FK, specific inhibitors for CPP32-like proteases, which inhibited the Fas-induced chromatin condensation and DNA fragmentation did not block the activation of JNK/SAPK and p38. Interestingly, these DEVD-type inhibitors did not block the Fas-induced morphological changes (cell shrinkage and surface blebbing), induction of Apo2.7 antigen, or the cell death (as assessed by the dye exclusion ability). These results suggest that the Fas-induced activation of the JNK/SAPK and p38 signaling pathways does not require CPP32-like proteases and that CPP32-like proteases, although essential for apoptotic nuclear events (such as chromatin condensation and DNA fragmentation), are not required for other apoptotic events in the cytoplasm or the cell death itself. Thus, the Fas signaling pathway diverges into multiple, separate processes, each of which may be responsible for part of the apoptotic cellular responses.
PMCID: PMC2139975  PMID: 9362518
23.  Inhibition of Caspase 3 Abrogates Lipopolysaccharide-Induced Nitric Oxide Production by Preventing Activation of NF-κB and c-Jun NH2-Terminal Kinase/Stress-Activated Protein Kinase in RAW 264.7 Murine Macrophage Cells 
Infection and Immunity  2001;69(3):1315-1321.
The effect of caspase inhibitors on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 267.4 murine macrophage cells was investigated. Pretreatment of RAW cells with a broad caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK), resulted in a striking reduction in LPS-induced NO production. Z-VAD-FMK inhibited LPS-induced NF-κB activation. Furthermore, it blocked phosphorylation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) but not that of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinases. Similarly, a caspase 3-specific inhibitor, Z-Asp-Glu-Val-Asp-fluoromethylketone, inhibited NO production, NF-κB activation, and JNK/SAPK phosphorylation in LPS-stimulated RAW cells. The attenuated NO production was due to inhibition of the expression of an inducible-type NO synthase (iNOS). The overexpression of the dominant negative mutant of JNK/SAPK and the addition of a JNK/SAPK inhibitor blocked iNOS expression but did not block LPS-induced caspase 3 activation. It was therefore suggested that the inhibition of caspase 3 might abrogate LPS-induced NO production by preventing the activation of NF-κB and JNK/SAPK. The caspase family, especially caspase 3, is likely to play an important role in the signal transduction for iNOS-mediated NO production in LPS-stimulated mouse macrophages.
PMCID: PMC98022  PMID: 11179293
24.  Activation of SAPK/JNK by camptothecin sensitizes androgen-independent prostate cancer cells to Fas-induced apoptosis 
British Journal of Cancer  2000;82(11):1827-1834.
We have previously shown that the androgen-independent prostate cancer cells DU145, despite expressing Fas and FasL, were resistant to anti-Fas-induced apoptosis, and that this resistance could be overcome by pretreating the cells with sublethal doses of camptothecin. Here, we provide evidence that SAPK/JNK activity is required for camptothecin sensitization to anti-Fas-induced apoptosis. Camptothecin, but not Fas ligation, was shown to activate SAPK/JNK in a time-dependent manner, and to induce c-Jun expression. The effects were more prominent in cells treated with both camptothecin and anti-Fas. The expression levels of MKP-1, a phosphatase which regulates SAPK/JNK and which has been implicated in prostate cancer resistance to apoptosis, remained unchanged. Inhibition of caspases had no effect on the SAPK/JNK activation, suggesting that this activation is an upstream event in the Fas-signalling pathway, and is independent of caspase activity. Antisense oligonucleotides targeted to JNK1 and JNK2 reversed the effect of camptothecin. These results suggest that stress kinase activation can significantly influence the fate of androgen-independent prostate cancer cells following Fas receptor ligation. © 2000 Cancer Research Campaign
PMCID: PMC2363235  PMID: 10839298
prostate cancer; apoptosis; Fas; camptothecin; SAPK/JNK
25.  N-(4-Hydroxyphenyl)retinamide-induced apoptosis triggered by reactive oxygen species is mediated by activation of MAPKs in head and neck squamous carcinoma cells 
Oncogene  2006;25(19):2785-2794.
N-(4-hydroxyphenyl)retinamide (4HPR), a synthetic retinoid effective in cancer chemoprevention and therapy, is thought to act via apoptosis induction resulting from increased reactive oxygen species (ROS) generation. As ROS can activate MAP kinases and protein kinase C (PKC), we examined the role of such enzymes in 4HPR-induced apoptosis in HNSCC UMSCC22B cells. 4HPR increased ROS level within 1 h and induced activation of caspase 3 and PARP cleavage within 24 h. Activation of MKK3/6 and MKK4, JNK, p38 and ERK was detected between 6 and 12 h, increased up to 24 h and preceded apoptosis. 4HPR-induced activation of these kinases was abrogated by the antioxidants BHA and vitamin C. SP600125, a JNK inhibitor, suppressed 4HPR-induced c-Jun phosphorylation, cytochrome c release from mitochondria and apoptosis. Suppression of JNK1 and JNK2 using siRNA decreased, whereas overexpression of wild type-JNK1 enhanced 4HPR-induced apoptosis. PD169316, a p38, inhibitor suppressed phosphorylation of Hsp27 and apoptosis. PD98059, an MEK1/2 inhibitor, also suppressed ERK1/2 activation and apoptosis induced by 4HPR. Likewise, PKC inhibitor GF109203X suppressed ERK and p38 phosphorylation and PARP cleavage. These data indicate that 4HPR-induced apoptosis is triggered by ROS increase, leading to the activation of the mitogen-activated protein serine/threonine kinases JNK, p38, PKC and ERK, and subsequent apoptosis.
PMCID: PMC1458365  PMID: 16407847
4HPR; apoptosis; ROS; MAPK; HNSCC

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