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.
Parkinson's disease; miR-7; miR-153; MPP+; neuron; neuroprotection; rapamycin; mTOR
The Vav family of Rho/Rac guanosine nucleotide exchange factors comprises three members in mammalian cells. Vav3 enhances androgen receptor (AR) activity during progression to androgen independence in prostate cancer. We examined Vav3 small interfering RNA (siRNA) effects on cell proliferation and apoptosis in docetaxel-treated LNCaP cells under chronic hypoxia (LNCaPH).
We examined individual and combined effects of Vav3 siRNA (si-Vav3) and docetaxel on cell growth and apoptosis under chronic hypoxia by cell proliferation, flow cytometric, DNA fragmentation, and immunoblot analyses. To clarify the molecular basis of si-Vav3- and docetaxel-induced apoptosis, we analyzed alterations in phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal-regulate kinase (ERK), c-jun N-terminal kinase (JNK), and AR pathways using kinase inhibitors in LNCaPH cells. The effects of si-Vav3/atelocollagen complex alone or in combination with docetaxel were assessed on xenografts in nude mice by tumor growth delay.
Vav3 overexpression was observed in LNCaPH compared with the expression under normoxia. Interrupting Vav3 signaling using siRNA enhanced docetaxel-induced cell growth suppression compared with that induced by docetaxel alone by inhibition of Akt and ERK phosphorylation, resulting in AR phosphorylation inhibition. In addition to increased B-cell lymphoma 2 (Bcl-2) phosphorylation through JNK signaling in response to docetaxel, si-Vav3 enhanced docetaxel-induced apoptosis, as characterized by the accumulation of sub-G1 phase cells and DNA fragmentation, through Bcl-xL/Bcl-2-associated death promoter (Bad) dephosphorylation, resulting in increased caspase-9, caspase-3, and cleaved poly(ADP-ribose) polymerase activation. Xenograft tumor growth was slightly inhibited by si-Vav3/atelocollagen complex injection and combined use of si-Vav3/atelocollagen complex and docetaxel produced a greater effect than docetaxel alone.
Interrupting Vav3 signaling enhances docetaxel-induced apoptosis in LNCaP cells under chronic hypoxia by inhibiting the PI3K/Akt, ERK, and AR signaling pathways. Therapy targeting Vav3 in combination with docetaxel may have practical implications for managing castration-resistant prostate cancer.
Vav3; Docetaxel; Androgen receptor; Prostate cancer; Chronic hypoxia
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.
Apoptosis; Caspase-3; Cell survival; Cytotoxicity; DNA fragmentation; Guanosine; 1-methyl–4-phenyl-pyridinium (MPP+); Parkinson’s disease; SH-SY5Y human neuroblastoma cells
Constitutive activation of extracellular signal regulated kinase (ERK)-Jun NH2-terminal kinase (JNK) signaling commonly occurs in tumors. The activation of ERK promotes cell proliferation, whereas that of JNK induces cell apoptosis. However, the apoptotic mechanism of ERK-JNK signaling in cancer is not well understood. Recently, we identified that apoptosis and activation of the JNK signaling pathway were induced after cordycepin treatment in human renal cancer, suggesting that JNK signaling might contribute to TK-10 cell apoptosis. We investigated the apoptotic effects of cordycepin by evaluating the activation of the ERK-JNK signaling pathway in renal cancer TK-10 cells. We found that cordycepin downregulated ERK and DUSP5, upregulated phosphorylated-JNK (p-JNK), and induced apoptosis. Moreover, we showed that siRNA-mediated inhibition of ERK downregulated DUSP5, whereas ERK overexpression upregulated DUSP5, and that DUSP5 knockdown by siRNA upregulated p-JNK. The JNK-specific inhibitor SP600125 upregulated nuclear translocation of β-catenin, and downregulated Dickkopf-1 (Dkk1), which has been shown to be a potent inhibitor of Wnt signaling. Dkk1 knockdown by siRNA upregulated nuclear β-catenin, suggesting the involvement of the Wnt/β-catenin signaling pathway. DUSP5 overexpression in TK-10 cells decreased p-JNK and increased nuclear β-catenin. The decreased Bax activation markedly protected against cordycepin-induced apoptosis. Bax subfamily proteins induced apoptosis through caspase-3. Taken together, we show that JNK signaling activation by cordycepin mediated ERK inhibition, which might have induced Bax translocation and caspase-3 activation via regulation of DUSP5 in TK-10 cells, thereby promoting the apoptosis of TK-10 cells. Targeting ERK-JNK signaling via the apoptotic effects of cordycepin could be a potential therapeutic strategy to treat renal cancer.
Cordycepin; ERK; DUSP5; JNK; apoptosis; TK-10
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.
dopaminergic cells; mitochondria; neuronal cell death; Parkinson’s disease; primary midbrain neurons; RNA interference
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.
Ursolic acid (UA), a pentacyclic triterpenoid, is known to have anti-tumor activity in various cancers including human non small cell lung cancer (NSCLC). However, the molecular mechanisms underlying the action of UA remain largely unknown.
Cell viability was measured by MTT assays. Apoptosis was analyzed with Annexin V-FITC/PI Apoptosis Detection Kit by Flow cytometry. Western blot analysis was performed to measure the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), DNMT1 [DNA (cytosine-5)-methyltransferase 1], enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and SP1. Exogenous expression of SP1 and DNMT1 was carried out by transient transfection assays.
We showed that UA inhibited the growth and induced apoptosis of NSCLC cells in the dose- and time-dependent fashion. Furthermore, we found that UA induced phosphorylation of SAPK/JNK and suppressed the protein expression of DNMT1 and EZH2. The inhibitor of SAPK/JNK (SP600125) blocked the UA-reduced expression of DNMT1 and EZH2. In addition, UA suppressed the expression of SP1 protein. Conversely, overexpression of SP1 reversed the effect of UA on DNMT1 and EZH2 expression, and feedback attenuated UA-induced phosphorylation of SAPK/JNK. Moreover, exogenous expression of DNMT1 antagonized the effect of UA on SAPK/JNK signaling, EZH2 protein expression, and NSCLC cell growth.
Our results show that UA inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of SP1; this in turn results in inhibition the expression of DNMT1 and EZH2. Overexpression of DNMT1 diminishes UA-reduced EZH2 protein expression. The negative feedback regulation of SAPK/JNK signaling by SP1 and DNMT1, and the reciprocal interaction of EZH2 and DNMT1 contribute to the overall effects of UA. This study leads to important new insights into the mechanisms by which UA controls growth of NSCLC cells.
Human lung cancer cells; SAPK/JNK; DNMT1; EZH2; Ursolic acid; SP1
Polyphyllin I (PPI), a bioactive phytochemical extracted from the Rhizoma of Paris polyphylla, has been reported to exhibit anti-cancer activity. However, the detailed mechanism underlying this remains to be elucidated.
Cell viability and cell cycle distribution were measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. The expression of enhancer of zeste homolog 2 (EZH2) mRNA was measured by quantitative real time PCR (qRT-PCR). Western blot analysis was performed to examine the phosphorylation and protein expression of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p65, DNA methyltransferase 1 (DNMT1) and EZH2. Exogenous expression of p65, DNMT1, and EZH2 were carried out by transient transfection assays. Promoter activity of EZH2 gene was determined using Secrete-Pair Dual Luminescence Assay Kit. A xenografted tumor model in nude mice and bioluminescent imaging system were used to further test the effect of PPI in vivo.
We showed that PPI significantly inhibited growth and induced cell cycle arrest of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Mechanistically, we found that PPI increased the phosphorylation of SAPK/JNK, reduced protein expression of p65 and DNMT1. The inhibitor of SAPK/JNK (SP600125) blocked the PPI-inhibited p65 and DNMT1 protein expression. Interestingly, exogenously expressed p65 overcame PPI-inhibited protein expression of DNMT1. Moreover, PPI reduced EZH2 protein, mRNA, and promoter activity; overexpression of EZH2 resisted the PPI-inhibited cell growth, and intriguingly, negative feedback regulation of SAPK/JNK signaling. Finally, exogenous expression of DNMT1 antagonized the PPI-suppressed EZH2 protein expression. Consistent with this, PPI inhibited tumor growth, protein expression levels of p65, DNMT1 and EZH2, and increased phosphorylation of SAPK/JNK in vivo.
Our results show that PPI inhibits growth of NSCLC cells through SAPK/JNK-mediated inhibition of p65 and DNMT1 protein levels, subsequently; this results in the reduction of EZH2 gene expression. The interactions among p65, DNMT1 and EZH2, and feedback regulation of SAPK/JNK by EZH2 converge on the overall responses of PPI. This study reveals a novel mechanism for regulating EZH2 gene in response to PPI and suggests a new strategy for NSCLC associated therapy.
PPI; NSCLC; SAPK/JNK; NF-kB/p65; EZH2; DNMT1
Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of protein phosphatase 2A (PP2Ac) formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells.
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.
SAPK2/p38; HSP27; F-actin; blebbing; apoptosis
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.
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.
G Proteins/Coupled Receptors (GPCR); Neurochemistry; Neurobiology/Neuroscience; Peptides/Neuropeptide; Receptors/Endocytosis; Signal Transduction
Previous studies from this laboratory have demonstrated a critical role of cytosolic phospholipase A2 (cPLA2) and arachidonic acid in angiotensin II (Ang II) AT2 receptor-mediated signal transduction in renal epithelium. In primary proximal tubular epithelial cells exposed to hydrogen peroxide (H2O2), both the selective cPLA2 inhibitors and the cPLA2 antisense oligonucleotides significantly attenuated H2O2-induced arachidonic acid liberation and activation of p38SAPK, ERK1/2, and Akt1. This H2O2-induced kinase activation was significantly attenuated by a Src kinase inhibitor PP2, or by transient transfection of carboxyl-terminal Src kinase (CSK) that maintained Src in the dormant form. Under basal conditions, Src coimmunoprecipitated with epidermal growth factor receptor (EGFR), while H2O2 increased EGFR phosphorylation in the complex. We observed that inhibition of EGFR kinase activity with AG1478 significantly attenuated H2O2-induced p38SAPK and ERK1/2 activation, but did not inhibit Akt1 activation. Furthermore, it seems that p38SAPK is upstream of ERK1/2 and Akt1, since a p38SAPK inhibitor SB203580 significantly blocked H2O2-induced activation of ERK1/2 and Akt1. Interestingly, overexpression of the dominant-negative p38SAPK isoform α inhibited ERK1/2 but not Akt1 activation. Our observations demonstrate that in these nontransformed cells, activation of cPLA2 is a converging point for oxidative stress and Ang II, which share common downstream signaling mechanisms including Src and EGFR. In addition, p38SAPK provides a positive input to both growth and antiapoptotic signaling pathways induced by acute oxidative stress.
Proximal tubular epithelial cells; Oxidative stress; Growth factor; Phospholipase A2; Signal transduction; Protein kinase; Free radical
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.
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.
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.
Treatment of pancreatic acinar cells by hydrogen sulphide has been shown to induce apoptosis. However, a potential role of mitogen-activated protein kinases (MAPKs) in this apoptotic pathway remains unknown. The present study examined the role of MAPKs in H2S-induced apoptosis in mouse pancreatic acinar cells. Pancreatic acinar cells were treated with 10 μM NaHS (a donor of H2S) for 3 hrs. For the evaluation of the role of MAPKs, PD98059, SP600125 and SB203580 were used as MAPKs inhibitors for ERK1/2, JNK1/2 and p38 MAPK, respectively. We observed activation of ERK1/2, JNK1/2 and p38 when pancreatic acini were exposed to H2S. Moreover, H2S-induced ERK1/2, JNK1/2 and p38 activation were blocked by pre-treatment with their corresponding inhibitor in a dose-dependent manner. H2S-induced apoptosis led to an increase in caspase 3 activity and this activity was attenuated when caspase 3 inhibitor were used. Also, the cleavage of caspase 3 correlated with that of poly-(ADP-ribose)-polymerase (PARP) cleavage. H2S treatment induced the release of cytochrome c, smac from mitochondria into the cytoplasm, translocation of Bax into mitochondria and decreased the protein level of Bcl-2. Inhibition of ERK1/2 using PD98059 caused further enhancement of apoptosis as evidenced by annexin V staining, while SP600125 and SB203580 abrogated H2S-induced apoptosis. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to H2S-induced apoptosis.
H2S; apoptosis; ERK; JNK; p38
Heteronemin is a bioactive marine sesterterpene isolated from the sponge Hyrtios sp. Previous reports have shown that heteronemin possesses anticancer activity. Here, heteronemin displayed cytotoxic effects against three human cancer cell lines (A549, ACHN, and A498) and exhibited potent activity in A498 human renal carcinoma cells, with an IC50 value of 1.57 μM by MTT assay and a GI50 value of 0.77 μM by SRB assay. Heteronemin initiates apoptotic cell death by downregulating Bcl-2 and Bcl-xL and upregulating Bax, leading to the disruption of the mitochondrial membrane potential and the release of cytochrome c from the mitochondria. These effects were associated with the activation of caspase-3/caspase-8/caspase-9, followed by PARP cleavage. Furthermore, heteronemin inhibited the phosphorylation of AKT signaling pathway and ERK and activated p38 and JNK. The specific inhibition of the p38 pathway by SB203580 or p38 siRNA treatment reversed the heteronemin-induced cytotoxicity and apoptotic signaling. Heteronemin also induced autophagy in A498 cells, and treatment with chloroquine (autophagy inhibitor) or SP600125 (JNK inhibitor) inhibited autophagy and increased heteronemin-induced cytotoxicity and apoptotic signaling. Taken together, this study proposes a novel treatment paradigm in which the combination of heteronemin and autophagy inhibitors leads to enhanced RCC cell apoptosis.
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.
Type II cGMP-dependent protein kinase; phosphatidylinositol-3-kinase/Akt-mediated signal transduction; apoptosis; gastric cancer cells
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.
apoptosis; FDH; Jun kinases; c-Jun; RNAi; PC-3 cells
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.
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.
Diallyl disulfide (DADS), a sulfur compound derived from garlic, has various biological properties, such as anticancer, antiangiogenic and anti-inflammatory effects. However, the mechanisms of action underlying the compound’s anticancer activity have not been fully elucidated. In this study, the apoptotic effects of DADS were investigated in DU145 human prostate carcinoma cells. Our results showed that DADS markedly inhibited the growth of the DU145 cells by induction of apoptosis. Apoptosis was accompanied by modulation of Bcl-2 and inhibitor of apoptosis protein (IAP) family proteins, depolarization of the mitochondrial membrane potential (MMP, ΔΨm) and proteolytic activation of caspases. We also found that the expression of death-receptor 4 (DR4) and Fas ligand (FasL) proteins was increased and that the level of intact Bid proteins was down-regulated by DADS. Moreover, treatment with DADS induced phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular-signal regulating kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK). A specific JNK inhibitor, SP600125, significantly blocked DADS-induced-apoptosis, whereas inhibitors of the ERK (PD98059) and p38 MAPK (SB203580) had no effect. The induction of apoptosis was also accompanied by inactivation of phosphatidylinositol 3-kinase (PI3K)/Akt and the PI3K inhibitor LY29004 significantly increased DADS-induced cell death. These findings provide evidence demonstrating that the proapoptotic effect of DADS is mediated through the activation of JNK and the inhibition of the PI3K/Akt signaling pathway in DU145 cells.
diallyl disulfide; apoptosis; MAPK; PI3K/Akt
Ginseng has been shown to promote hair growth in several recent studies. However, its effects on human hair follicles and its mechanisms of action have not been sufficiently elucidated. This study aimed to investigate the hair growth-promoting effects of red ginseng extract (RGE) and its ginsenosides. The proliferative activities of cultured human hair follicles treated with RGE and ginsenoside-Rb1 were assessed using Ki-67 immunostaining. Their effects on isolated human dermal papilla cells (hDPCs) were evaluated using cytotoxicity assays, immunoblot analysis of signaling proteins, and the determination of associated growth factors. We examined the ability of RGE and ginsenosides to protect hair matrix keratinocyte proliferation against dihydrotestosterone (DHT)-induced suppression and their effects on the expression of androgen receptor. The in vivo hair growth-promoting effect of RGE was also investigated in C57BL/6 mice. Both RGE and ginsenoside-Rb1 enhanced the proliferation of hair matrix keratinocytes. hDPCs treated with RGE or ginsenoside-Rb1 exhibited substantial cell proliferation and the associated phosphorylation of ERK and AKT. Moreover, RGE, ginsenoside-Rb1, and ginsenoside-Rg3 abrogated the DHT-induced suppression of hair matrix keratinocyte proliferation and the DHT-induced upregulation of the mRNA expression of androgen receptor in hDPCs. Murine experiments revealed that the subcutaneous injection of 3% RGE resulted in more rapid hair growth than the negative control. In conclusion, RGE and its ginsenosides may enhance hDPC proliferation, activate ERK and AKT signaling pathways in hDPCs, upregulate hair matrix keratinocyte proliferation, and inhibit the DHT-induced androgen receptor transcription. These results suggest that red ginseng may promote hair growth in humans.
cell growth; ginseng; ginsenosides; proliferation; signal transduction
Microbial stimuli and atmospheric particulate matter (PM) interact to amplify the release of inflammatory and immune-modulating cytokines. The basis of this interaction, however, is not known. Cultured human lung fibroblasts (HLF) were used to determine whether various protein kinase pathways were involved in the release of IL-6 following combined exposure to the PM-derived metal, Ni, and M. fermentans-derived macrophage-activating lipopeptide 2 (MALP-2), a toll-like receptor 2 agonist. Synergistic release of IL-6 by MALP-2 and NiSO4 was obvious after 8 h of co-stimulation and correlated with a late phase accumulation of IL-6 mRNA. Ni and MALP-2, alone or together, all lead to rapid and transient phosphorylations of ERK1/2 and JNK/SAPK of similar magnitude. p38 phosphorylation, however, was observed only after prolonged treatment of cells with both stimuli together. A constitutive level of PI3K-dependent Akt phosphorylation remained unchanged by Ni and/or MALP-2 exposure. IL-6 induced by Ni/MALP-2 co-exposure was partially dependent on activity of HIF-1α and COX-2 as shown by targeted knockdown using siRNA. IL-6 release in response to Ni/MALP-2 was partially sensitive to pharmacological inhibition of ERK1/2, p38, and PI3K signaling. The protein kinase inhibitors had minimal or no effects on Ni/MALP-2-induced accumulation of HIF-1α protein, however, COX-2 expression and, more markedly PGE2 production, were suppressed by LY294002, SB203580, and U0126. Thus, Ni/MALP-2 interactions involve multiple protein kinase pathways (ERK1/2, p38, and PI3K) that modulate events downstream from the early accumulation of HIF-1α to promote IL-6 gene expression directly or secondarily, through COX-2-derived autocrine products like PGE2.
airborne particulate-derived metals; mitogen-activated protein kinase; p38; phosphoinositide 3-kinase; cyclooxygenase; hypoxia-inducible factor-1α; innate immunity; cytokines
Hydrogen sulfide (H2S) has been proposed as a novel neuromodulator and neuroprotective agent. Cobalt chloride (CoCl2) is a well-known hypoxia mimetic agent. We have demonstrated that H2S protects against CoCl2-induced injuries in PC12 cells. However, whether the members of mitogen-activated protein kinases (MAPK), in particular, extracellular signal-regulated kinase1/2(ERK1/2) and p38MAPK are involved in the neuroprotection of H2S against chemical hypoxia-induced injuries of PC12 cells is not understood. We observed that CoCl2 induced expression of transcriptional factor hypoxia-inducible factor-1 alpha (HIF-1α), decreased cystathionine-β synthase (CBS, a synthase of H2S) expression, and increased generation of reactive oxygen species (ROS), leading to injuries of the cells, evidenced by decrease in cell viability, dissipation of mitochondrial membrane potential (MMP) , caspase-3 activation and apoptosis, which were attenuated by pretreatment with NaHS (a donor of H2S) or N-acetyl-L cystein (NAC), a ROS scavenger. CoCl2 rapidly activated ERK1/2, p38MAPK and C-Jun N-terminal kinase (JNK). Inhibition of ERK1/2 or p38MAPK or JNK with kinase inhibitors (U0126 or SB203580 or SP600125, respectively) or genetic silencing of ERK1/2 or p38MAPK by RNAi (Si-ERK1/2 or Si-p38MAPK) significantly prevented CoCl2-induced injuries. Pretreatment with NaHS or NAC inhibited not only CoCl2-induced ROS production, but also phosphorylation of ERK1/2 and p38MAPK. Thus, we demonstrated that a concurrent activation of ERK1/2, p38MAPK and JNK participates in CoCl2-induced injuries and that H2S protects PC12 cells against chemical hypoxia-induced injuries by inhibition of ROS-activated ERK1/2 and p38MAPK pathways. Our results suggest that inhibitors of ERK1/2, p38MAPK and JNK or antioxidants may be useful for preventing and treating hypoxia-induced neuronal injury.