Anisomycin-induced Apoptosis Research Articles

The inhibition of Bax activation-induced apoptosis by RasGRP2 via R-Ras-PI3K-Akt signaling pathway in the endothelial cells

Apoptosis of endothelial cells is a very important event in various diseases and angiogenesis. We recently reported that ras guanyl nucleotide releasing protein 2 (RasGRP2), which is a guanine nucleotide exchange factor, was expressed in the human umbilical vein endothelial cells (HUVECs) and that Rap1 activation by its overexpression inhibited apoptosis by suppressing tumor necrosis factor-α induced-reactive oxygen species (ROS) production. However, other signaling pathways and roles of RasGRP2 not mediated via Rap1 are not well understood. Therefore, we compared the Mock (M) and the RasGRP2-stable overexpression (R) immortalized HUVECs using BAM7 and anisomycin, which are apoptosis inducers. BAM7 and anisomycin induced apoptosis without causing ROS production, and such apoptosis was significantly increased in M cells, but not in R cells. RasGRP2 suppressed BAM7- and anisomycin-induced apoptosis, but not via the Rap1 pathway as observed using Rap1 knockdown. Furthermore, RasGRP2 activated not only Rap1 but also R-Ras, and suppressed apoptosis by activating R-Ras-phosphoinositide 3-kinase (PI3K)-Akt signaling pathway. The phosphorylation of Akt by RasGRP2 inhibited Bax translocation by promoting translocation of hexokinase-2 (HK-2) from cytoplasm to mitochondria. Taken together, it was suggested that RasGRP2 suppresses the Bax activation-induced apoptosis by promoting HK-2 translocation to mitochondria via R-Ras-PI3K-Akt signaling pathway.

Partial p53-dependence of anisomycin-induced apoptosis in PC12 cells.

The bacterial antibiotic anisomycin is known to induce apoptosis by activating several mitogen-activated protein kinases and by inhibiting protein synthesis. In this study, the influence of p53 protein on the apoptosis-inducing effect of anisomycin was investigated. The effect of protein synthesis-inhibiting concentration of anisomycin on apoptotic events was analyzed using Western blot, DNA fragmentation, and cell viability assays in wild-type PC12 and in mutant p53 protein expressing p143p53PC12 cells. Anisomycin stimulated the main apoptotic pathways in both cell lines, but p143p53PC12 cells showed lower sensitivity to the drug than their wild-type counterparts. Anisomycin caused the activation of the main stress kinases, phosphorylation of the p53 protein and the eukaryotic initiation factor eIF2α, proteolytic cleavage of protein kinase R, Bid, caspase-9 and -3. Furthermore, anisomycin treatment led to the activation of TRAIL and caspase-8, two proteins involved in the extrinsic apoptotic pathway. All these changes were stronger and more sustained in wtPC12 cells. In the presence of the dominant inhibitory p53 protein, p53- dependent genes involved in the regulation of apoptosis may be less transcribed and this can lead to the decrease of apoptotic processes in p143p53PC12 cells.

MicroRNA let-7c is essential for the anisomycin-elicited apoptosis in Jurkat T cells by linking JNK1/2 to AP-1/STAT1/STAT3 signaling.

Anisomycin, an antibiotic produced by Streptomyces griseolus, strongly induces apoptosis in various tumor cells in vitro, superior dramatically to adriamycin. The present study aims to elucidate its detailed mechanistic process. The results showed that anisomycin sufficiently promoted the apoptosis in human leukemic Jurkat T cells at a quite low dose. microRNA let-7c (let-7c) contributed to the anisomycin-induced apoptosis, which could be abrogated by the inactivation of JNK signaling. The let-7c over-expression and the addition of its mimics facilitated the activation of AP-1, STAT1 and Bim by linking JNK1/2 to AP-1/STAT1, but rather inhibited the activation of STAT3 and Bcl-xL by connecting JNK1/2 to STAT3, followed by the augmented apoptosis in the cells. The let-7c deficiency reduced the AP-1, STAT1 and Bim activities, and enhanced the STAT3 and Bcl-xL, alleviating the anisomycin-induced apoptosis. The knockdown of the bim gene repressed the anisomycin-boosted apoptosis through the attenuation of the active Bak and Bax. The findings indicate for the first time that miR let-7c is essential for the anisomycin-triggered apoptosis by linking JNK1/2 to AP-1/STAT1/STAT3/Bim/Bcl-xL/Bax/Bak signaling. This provides a novel insight into the mechanism by which anisomycin leads to the tumor cell apoptosis, potentially laying the foundations for its development and clinical application.

Activating Transcription Factor 2 and c-Jun–Mediated Induction of FoxP3 for Experimental Therapy of Mammary Tumor in the Mouse

FOXP3 is inactivated in breast cancer cells by a number of mechanisms, including somatic mutations, deletion, and epigenetic silencing. Because the mutation and deletion are usually heterozygous in the cancer samples, it is of interest to determine whether the gene can be induced for the purpose of cancer therapy. Here, we report that anisomycin, a potent activator of activating transcription factor (ATF) 2, and c-Jun-NH(2)-kinase, induces expression of FoxP3 in both normal and malignant mammary epithelial cells. The induction is mediated by ATF2 and c-Jun. Targeted mutation of ATF2 abrogates both constitutive and inducible expression of FoxP3 in normal epithelial cells. Both ATF2 and c-Jun interact with a novel enhancer in the intron 1 of the FoxP3 locus. Moreover, shRNA silencing of ATF2 and FoxP3 reveals an important role of ATF2-FoxP3 pathway in the anisomycin-induced apoptosis of breast cancer cells. A low dose of anisomycin was also remarkably effective in treating established mammary tumor in the mice. Our data showed that FoxP3 can be reactivated for cancer therapy.

NOD2 engagement induces proinflammatory cytokine production, but not apoptosis, in leukocytes isolated from patients with Crohn's disease.

NOD2/CARD15 is a member of the NACHT-LRR (NLR) family of proteins, which recognize the muramyl dipeptide motif from bacterial peptidoglycans. NOD2 has been shown to be involved in the pathogenesis of Crohn's disease. NLR proteins modulate inflammation and apoptosis, and several studies have implicated NOD2 in the induction of cytokines and inflammatory reactions. However, only scarce data are available regarding its role in apoptosis. Neutrophils and lymphocytes isolated from the blood from four Crohn's disease patients homozygous for the loss-of-function 3020insC NOD2 mutation were examined for spontaneous and anisomycin-induced apoptosis. They were compared with cells from healthy controls and Crohn's disease patients bearing the wild-type NOD2 allele. Cytokine production after stimulation of mononuclear cells (MNCs) with muramyl dipeptide was assessed by specific immunoassays. We observed that MNCs isolated from the blood of patients with Crohn's disease bearing the loss of function mutation in NOD2 displayed defective muramyl dipeptide-induced cytokine responses, but both granulocytes and lymphocytes from the same donors displayed normal apoptosis. NOD2 engagement by MDP mainly triggers cytokine activation and inflammatory reactions, but has negligible effects on cell apoptosis.

Phosphatidylinositol Ether Lipid Analogues That Inhibit AKT Also Independently Activate the Stress Kinase, p38α, through MKK3/6-independent and -dependent Mechanisms

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.

Reduced Levels of ATF-2 Predispose Mice to Mammary Tumors

Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia- and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2(-/-) mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia- and anisomycin-induced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2(-/-) and Atf-2(+/-) MEFs could not express a group of genes, such as Gadd45alpha, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2(-/-) MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2(-/-) MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2(+/-) mice were highly prone to mammary tumors that expressed reduced levels of Gadd45alpha and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45alpha.

Mitogen activated protein kinase‐dependent activation of c‐Jun and c‐Fos is required for neuronal differentiation but not for growth and stress response in PC12 cells

MAPK-dependent activation of AP-1 protein c-Jun is involved in PC12 cell differentiation and apoptosis. However, the role of other AP-1 proteins and their connection to MAPKs during growth, differentiation and apoptosis has remained elusive. Here we studied the activation of AP-1 proteins in response to ERK, JNK, and p38 signaling upon NGF, EGF and anisomycin exposures. All treatments caused different kinetics and strength of MAPK and AP-1 activities. NGF induced persistent ERK and AP-1 activities, whereas upon EGF and anisomycin exposures, their activities were only weakly and transiently induced. The sustained AP-1 activity was associated with concomitant c-Fos and c-Jun expression and phoshorylation, which were JNK and ERK dependent. While inhibition of the ERK, JNK, and p38 activities partially prevented AP-1 activity and suppressed differentiation, none of them was required for anisomycin-induced apoptosis. The importance of c-Fos and c-Jun as mediators of differentiation was demonstrated by the findings that the corresponding siRNAs suppressed NGF-induced neurite outgrowth. However, the capacity of c-Fos to promote differentiation required cooperation with Jun proteins. In contrast, Fra-2 expression was not required for the differentiation response. Together, the results show that sustained c-Jun and c-Fos activities mediate MAPK signaling and are essential for differentiation of PC12 cells.

Ha-Ras sensitizes transformed mouse skin cells to Anisomycin-induced apoptosis

Efforts have been made to develop a chemoprevention that selectively triggers apoptosis in malignant cancer cells. Here, we demonstrated that a mutated Ha-Ras activity is required in Anisomycin-induced apoptosis in transformed keratinocytes. Anisomycin stimulates JNK activity and apoptosis in oncogenic Ha-Ras positive cells, but not in normal keratinocytes. This effect was demonstrated in stably transfected cells with dominant negative Ha-Ras, that protected transformed cells, and oncogenic Ha-Ras that sensitized non-transformed cells to Anisomycin-induced apoptosis. Lastly, the treatment of cells with inhibitors of the JNK displayed resistance to Anisomycin induced apoptosis. These data suggests that the oncogenic Ha-Ras is important for Anisomycin-induced JNK activation and apoptosis in transformed keratinocytes.

Essential Role for Mitogen-activated Protein (MAP) Kinase Phosphatase-1 in Stress-responsive MAP Kinase and Cell Survival Signaling

Mitogen-activated protein kinase (MAPK) phosphatases (MKPs) constitute a family of 11 dual-specificity phosphatases that inactivate the MAPKs by dephosphorylation. Although the contribution of MAPKs to cell growth and cell death has been examined extensively, it remains unclear whether MKPs play an essential role in the regulation of these processes. To clarify the role of MKP-1, we determined the effects on the MAPKs and cell growth and death in primary fibroblasts derived from mice lacking MKP-1. Here we have shown that MKP-1 is critical for the inactivation of p38 MAPK and JNK following stimulation with serum, anisomycin, and osmotic stress. In addition, MKP-1 was identified as a critical negative regulator of the cAMP-mediated p38 MAPK pathway. MKP-1-deficient mouse embryonic fibroblasts (MEFs) displayed enhanced p38 MAPK activity and cAMP-response element-dependent transcriptional activation in response to forskolin. Surprisingly, MKP-1-deficient fibroblasts exhibited reduced cell growth compared with wild type MEFs as a result of enhanced cell death. The enhanced level of cell death in MKP-1-deficient MEFs was rescued by SB203580, an inhibitor of p38 MAPK. MKP-1-deficient MEFs were also sensitive to anisomycin-induced apoptosis. Collectively, these data demonstrate that MKP-1 promotes cell survival by attenuating stress-responsive MAPK-mediated apoptosis.

Inhibitors of protein phosphatases 1 and 2A differentially prevent intrinsic and extrinsic apoptosis pathways

Inhibitors of serine/threonine protein phosphatases can inhibit apoptosis. We investigated which protein phosphatases are critical for this protection using calyculin A, okadaic acid, and tautomycin. All three phosphatase inhibitors prevented anisomycin-induced apoptosis in leukemia cell models. In vitro, calyculin A does not discriminate between PP1 and PP2A, while okadaic acid and tautomycin are more selective for PP2A and PP1, respectively. Increased phosphorylation of endogenous marker proteins was used to define concentrations that inhibited each phosphatase in cells. Concentrations of each inhibitor that prevented anisomycin-induced apoptosis correlated with inhibition of PP2A. The inhibitors prevented Bax translocation to mitochondria, indicating inhibition upstream of mitochondria. Tautomycin and calyculin A, but not okadaic acid, also prevented apoptosis induced through the CD95/Fas death receptor, and this protection correlated with inhibition of PP1. The inhibitors prevented Fas receptor oligomerization, FADD recruitment, and caspase 8 activation. The differential effects of PP1 and PP2A in protection from death receptor and mitochondrial-mediated pathways of death, respectively, may help one to define critical steps in each pathway, and regulatory roles for serine/threonine phosphatases in apoptosis.

Effects of PACAP on in vitro and in vivo neuronal cell death, platelet aggregation, and production of reactive oxygen radicals

Pituitary adenylate cyclase activating polypeptide (PACAP) exerts neuroprotective effects in various in vitro and in vivo models of cerebral pathologies. It has been shown that PACAP protects neurons in rat models of both global and focal ischemia. In the present study, we investigated factors that may play a role in the neuroprotective effects of PACAP. PACAP strongly reduced the anisomycin-induced apoptosis of PC12 cells, which was abolished in a PKA-deficient PC12 cell line (A126). This effect was also observed in vivo, in permanent occlusion of the middle cerebral artery, where the number of TUNEL-positive neurons was significantly reduced in the ischemic core of PACAP-treated animals. Our results show that PACAP has a minor antioxidant effect in a non-cellular in vitro system, and has considerable antioxidant effects in an in vitro red blood cell filtration model. PACAP had no effect on platelet aggregation induced by collagen, ADP or epinephrine. Our results demonstrate that the effects of PACAP on delayed neuronal death may play a significant role in the reduction of the infarct size in vivo, but the antioxidant effect could only be observed at concentrations higher than that used in the model of focal ischemia.

IL-4 augments anisomycin-induced p38 activation via Akt pathway in a follicular dendritic cell (FDC)-like line

Follicular dendritic cells (FDCs) play pivotal roles in germinal center (GC) responses in secondary lymphoid follicles, and their functions are influenced by cytokines present in the GC. We investigated the functional effects of interleukin-4 (IL-4) using an established FDC-like line of HK cells. In spite of the activation of ERK1/2 and PI3K/Akt pathways by IL-4, which are implicated in the induction of cell proliferation and survival, IL-4 did not exhibit protective function on anisomycin-induced apoptosis of HK cells, but rather slightly enhanced it. This IL-4 effect correlated with the up-regulation of anisomycin-induced p38 signaling, which is attenuated by inhibition of the PI3K/Akt pathway. Expression of an active form of Akt increased anisomycin-elicited activation of p38 and its upstream kinase MKK3/6. Our data indicate a positive cross-talk between the p38 and PI3K/Akt pathways.

Formation of caspase-3 complexes and fragmentation of caspase-12 during anisomycin-induced apoptosis in AKR-2B cells without aggregation of Apaf-1

Treatment of AKR-2B fibroblasts with anisomycin (10 microM) led to a rapid disintegration of the cells (t1/2 = 5 h) which was complete after 24 h. Cell death was associated with typical hallmarks of apoptosis like membrane blebbing, exposure of phophatidylserine on the cell surface, nuclear condensation and specific cleavage of rRNA. However, there was no dissipation of the mitochondrial potential and no intranucleosomal fragmentation. By affinity labeling with YVK(-bio)D.aomk in combination with immunostaining against activated caspase-3 analyzed by 2-D gel electrophoresis it was shown that caspase-3 is the dominant executioner caspase. Gel filtration experiments of cytosolic extract analyzed by Western blotting revealed the formation of high-molecular-weight complexes of caspase-3 (600 kDa and 250 kDa, respectively), but there was no complex formation of Apaf-1. Anisomycin treatment led to a strong activation of the stress kinases p38 kinases and the jun kinases, that was not sufficient for the activation of caspase-3 which required much higher concentrations. By using the selective inhibitors SB 203580 for p38 kinases and SP 600125 for c-jun kinases, respectively, it is shown that activation of these kinases is not necessary for cell death induced by anisomycin in AKR-2B cells. Furthermore, we disclose the activation of caspase-12 in AKR-2B cells following the addition of anisomycin. Caspase-12 zymogen present as a cytosolic complex (> 600 kDa) is activated by anisomycin leading to an uncomplexed cleaved enzyme. Since anisomycin treatment did neither lead to stress of the endoplasmic reticulum nor to a breakdown of intracellular Ca(2+)-stores, alternative pathways involved in the activation of caspases are discussed.

C-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for mitoxantrone- and anisomycin-induced apoptosis in HL-60 cells

Programmed cell death, or apoptosis, has emerged as a common mechanism by which cells respond to chemotherapeutic drugs. However, the signaling mechanisms that mediate drug-induced apoptosis are still widely unknown. Mitogen-activated protein kinase (MAPK) signaling cascades trigger stimulus-specific responses in cells with ERK being associated with proliferation and differentiation, and JNK/SAPK and p38 mediating stress and apoptotic responses. Here, we found that mitoxantrone and anisomycin stimulated a dose- and time-dependent induction of JNK/SAPK activity, and to a lesser extent p38 activity, that preceded the appearance of apoptosis as measured by internucleosomal DNA fragmentation. These compounds did not induce ERK activity. We further demonstrated that p38 activity was not involved in the induction of apoptosis since the use of the p38 inhibitor, SB203580, did not prevent drug-induced apoptotic DNA fragmentation. Additionally, direct inhibition of JNK/SAPK signaling through the use of dominant-negative MKK4/SEK1 (SEK-AL) inhibited mitoxantrone- and anisomycin-induced apoptosis. These results suggest that mitoxantrone- and anisomycin-induced apoptosis is dependent on JNK/SAPK, but not p38, activity.