Molecular mechanisms of oridonin-induced apoptosis and autophagy in murine fibrosarcoma L929 cells

Apoptosis and autophagy are genetically regulated, evolutionarily-conserved processes that can jointly seal the fate of cancer cells; however, substantial gaps remain in our understanding of the molecular mechanisms that mediate the two cellular processes. In the present study, the exposure of murine fibrosarcoma L929 cells to oridonin led to the generation of intracellular reactive oxygen species (ROS) and, subsequently, the ROS triggered apoptosis by Bax translocation, cytochrome c release and ERK activations. In addition, oridonin induced autophagy in L929 cells, and the inhibition of autophagy by 3-MA or siRNA against LC3 and beclin 1 promoted oridonin-induced apoptosis. Furthermore, p38 and NF-κB were confirmed to have roles in inhibiting apoptosis but promoting autophagy. Moreover, the inhibition of autophagy could reduce oridonin-induced activation of p38. Finally, NF-κB activation was inhibited by blocking the p38 pathway. In conclusion, these findings indicate that oridonin-induced apoptosis can be regulated by ROS-mediated signaling pathways, and oridonin-induced autophagy may block apoptosis by up-regulating p38 and NFκB activation.     

Oridonin induces G2/M arrest and apoptosis via activating ERK-p53 apoptotic pathway and inhibiting PTK-Ras-Raf-JNK survival pathway in murine fibrosarcoma L929 cells

Oridonin was reported to induce L929 cell apoptosis via ROS-mediated mitochondrial and ERK pathways; however, the precise mechanisms by which oridonin induces cell death remain unclear. Herein, we found that oridonin treatment induced an increase in G₂/M phase cell percentage. And, G₂/M phase arrest was associated with down-regulation of cell cycle related cdc2, cdc25c and cyclinB levels, as well as up-regulation of p21 and p-cdc2 levels. In addition, we discovered that interruption of p53 activation decreased oridonin-induced apoptosis, and blocking ERK by specific inhibitors or siRNA suppressed oridonin-induced p53 activation. Moreover, inhibition of PTK, protein kinase C, Ras, Raf or JNK activation increased oridonin-induced apoptosis. Also, the level of Ras, Raf or JNK was down-regulated by oridonin, and the inhibition of PTK, Ras, Raf activation decreased p-JNK level. In conclusion, oridonin induces L929 cell G₂/M arrest and apoptosis, which is regulated by promoting ERK-p53 apoptotic pathway and suppressing PTK-mediated survival pathway.     

zVAD-induced autophagic cell death requires c-Src-dependent ERK and JNK activation and reactive oxygen species generation

The treatment of L929 fibrosarcoma cells with zVAD has been shown to induce necroptosis. However, whether autophagy is involved or not in this event remains controversial. In this study, we re-examined the role of autophagy in zVAD-induced cell death in L929 cells and further elucidated the signaling pathways triggered by caspase inhibition and contributing to autophagic death. First, we found that zVAD can stimulate LC3-II formation, autophagosome and autolysosome formation, and ROS accumulation. Antioxidants, beclin 1 or Atg5 silencing, and class III PtdIns3K inhibitors all effectively blocked ROS production and cell death, suggesting ROS accumulation downstream of autophagy contributes to cell necrosis. zVAD also stimulated PARP activation, and the PARP inhibitor DPQ can reduce zVAD-induced cell death, but did not affect ROS production, suggesting the increased ROS leads to PARP activation and cell death. Notably, our data also indicated the involvement of Src-dependent JNK and ERK in zVAD-induced ROS production and autophagic death. We found caspase 8 is associated with c-Src at the resting state, and upon zVAD treatment this association was decreased and accompanied by c-Src activation. In conclusion, we confirm the autophagic death in zVAD-treated L929 cells, and define a new molecular pathway in which Src-dependent ERK and JNK activation can link a signal from caspase inhibition to autophagy, which in turn induce ROS production and PARP activation, eventually leading to necroptosis. Thus, in addition to initiating proteolytic activity for cell apoptosis, inactivated caspase 8 also functions as a signaling molecule for autophagic death.     

I-κBα depletion by transglutaminase 2 and μ-calpain occurs in parallel with the ubiquitin-proteasome pathway

Transglutaminase 2 (TGase2) is a calcium-dependent, cross-linking enzyme that catalyzes iso-peptide bond formation between peptide-bound lysine and glutamine residues. TGase 2 can activate NF-κB through the polymerization-mediated depletion of I-κBα without IKK activation. This NF-κB activation mechanism is associated with drug resistance in cancer cells. However, the polymers cannot be detected in cells, while TGase 2 over-expression depletes free I-κBα, which raises the question of how the polymerized I-κBα can be metabolized in cells. Among proteasome, lysosome and calpain systems, calpain inhibition was found to effectively increase the accumulation of I-κBα polymers in MCF7 cells transfected with TGase 2, and induced high levels of I-κBα polymers as well in MDA-MB-231 breast cancer cells that naturally express a high level of TGase 2. Inhibition of calpain also boosted the level of I-κBα polymers in HEK-293 cells in case of TGase 2 transfection either with I-κBα or I-κBα mutant (S32A, S36A). Interestingly, the combined inhibition of calpain and the proteasome resulted in an increased accumulation of both I-κBα polymers and I-κBα, concurrent with an inhibition of NF-κB activity in MDA-MB-231 cells. This suggests that μ-calpain proteasome-dependent I-κBα polymer degradation may contribute to cancer progression through constitutive NF-κB activation.     

Reovirus-Induced Apoptosis Is Preceded by Increased Cellular Calpain Activity and Is Blocked by Calpain Inhibitors

The cellular pathways of apoptosis have not been fully characterized; however, calpain, a cytosolic calcium-activated cysteine protease, has been implicated in several forms of programmed cell death. Reoviruses induce apoptosis both in vitro and in vivo and serve as a model for studying virus-induced cell death. We investigated the potential role of calpain in reovirus-induced apoptosis in vitro by measuring calpain activity as well as evaluating the effects of calpain inhibitors. L929 cells were infected with reovirus type 3 Abney (T3A), and calpain activity, measured as cleavage of the fluorogenic calpain substrate Suc-Leu-Leu-Val-Tyr-AMC, was monitored. There was a 1.6-fold increase in calpain activity in T3A-infected cells compared to mock-infected cells; this increase was completely inhibited by preincubation with calpain inhibitor I (N-acetyl-leucyl-leucyl-norleucinal [aLLN]), an active-site inhibitor. Both aLLN and PD150606, a specific calpain inhibitor that interacts with the calcium-binding site, inhibited reovirus-induced apoptosis in L929 cells by 54 to 93%. Apoptosis induced by UV-inactivated reovirus was also reduced 65 to 69% by aLLN, indicating that inhibition of apoptosis by calpain inhibitors is independent of effects on viral replication. We conclude that calpain activation is a component of the regulatory cascade in reovirus-induced apoptosis.     

Involvement of PKC signal pathways in oridonin-induced autophagy in HeLa cells: A protective mechanism against apoptosis

Our previous studies showed that oridonin could induce both apoptosis and autophagy in HeLa cells, and this autophagy might be a protective mechanism against apoptosis. In this study, the roles of PKC signal pathways in oridonin-induced HeLa cell autophagy and apoptosis were further investigated. We found that inhibition of PKC significantly reduced oridonin-induced autophagy whereas markedly increased apoptosis, while pretreatment with PKC activator caused opposite results. Subsequently, the oridonin-induced autophagy was also suppressed by Raf-1 or JNK inhibition accompanied by the increase of apoptosis, but it was not affected by ERK or p38 inhibition. In addition, oridonin-induced protein levels of Raf-1, JNK and p-JNK were sharply downregulated by PKC inhibitor, and they were enhanced by PKC activator. Taken together, these results demonstrate that PKC enhances oridonin-induced autophagy against apoptosis through regulating its downstream factors Raf-1 and JNK in HeLa cells.     

Differential effects of the autophagy inhibitors 3-methyladenine and chloroquine on spontaneous and TNF-α-induced neutrophil apoptosis

Autophagy and apoptosis cooperate to modulate cell survival. Neutrophils are short-lived cells and apoptosis is considered to be the major mechanism of their death. In the present study, we addressed whether autophagy regulates neutrophil apoptosis and investigated the effects of autophagy inhibition on apoptosis of human neutrophils. We first showed that the established autophagy inhibitors 3-methyladenine (MA) and chloroquine (CQ) markedly accelerated spontaneous neutrophil apoptosis as was evidenced by phosphatidylserine exposure, DNA fragmentation and caspase-3 activation. Apoptosis induced by the autophagy inhibitors was completely abrogated by a pan-caspase inhibitor Q-VD-OPh. Unexpectedly, both MA and CQ significantly delayed neutrophil apoptosis induced by TNF-α, although the inhibitors did attenuate late pro-survival effect of the cytokine. The effect was specific for TNF-α because it was not observed in the presence of other inflammation-associated cytokines (IL-1β or IL-8). The autophagy inhibitors did not modulate surface expression of TNF-α receptors in the absence or presence of TNF-α. Both MA and CQ induced a marked down-regulation of a key anti-apoptotic protein Mcl-1 but did not affect significantly the levels of another anti-apoptotic protein Bcl-X(L). Finally, to confirm the effects of the pharmacological inhibition of autophagy by a genetic approach, we evaluated the consequences of siRNA-mediated autophagy suppression in neutrophil-like differentiated HL60 cells. Knockdown of ATG5 in the cells resulted in accelerated spontaneous apoptosis but attenuated TNF-α-induced apoptosis. Together, these data suggest that autophagy regulates neutrophil apoptosis in an inflammatory context-dependent manner and mediates the early pro-apoptotic effect of TNF-α in neutrophils.     

3-Formylchromone interacts with cysteine 38 in p65 protein and with cysteine 179 in IκBα kinase, leading to down-regulation of nuclear factor-κB (NF-κB)-regulated gene products and sensitization of tumor cells

3-Formylchromone (3-FC) has been associated with anticancer potential through a mechanism yet to be elucidated. Because of the critical role of NF-κB in tumorigenesis, we investigated the effect of this agent on the NF-κB activation pathway. Whether activated by inflammatory agents (such as TNF-α and endotoxin) or tumor promoters (such as phorbol ester and okadaic acid), 3-FC suppressed NF-κB activation. It also inhibited constitutive NF-κB expressed by most tumor cells. This activity correlated with sequential inhibition of IκBα kinase (IKK) activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and reporter gene expression. We found that 3-FC inhibited the direct binding of p65 to DNA, and this binding was reversed by a reducing agent, thus suggesting a role for the cysteine residue. Furthermore, mutation of Cys38 to Ser in p65 abolished this effect of the chromone. This result was confirmed by a docking study. 3-FC also inhibited IKK activation directly, and the reducing agent reversed this inhibition. Furthermore, mutation of Cys179 to Ala in IKK abolished the effect of the chromone. Suppression of NF-κB activation led to inhibition of anti-apoptotic (Bcl-2, Bcl-xL, survivin, and cIAP-1), proliferative (cyclin D1 and COX-2), invasive (MMP-9 and ICAM-1), and angiogenic (VEGF) gene products and sensitization of tumor cells to cytokines. Thus, this study shows that modification of cysteine residues in IKK and p65 by 3-FC leads to inhibition of the NF-κB activation pathway, suppression of anti-apoptotic gene products, and potentiation of apoptosis in tumor cells.     

Nitric oxide augments oridonin-induced efferocytosis by human histocytic lymphoma U937 cells via autophagy and the NF-κB-COX-2-IL-1β pathway

Abstract We previously demonstrated that oridonin-induced autophagy enhanced efferocytosis (phagocytosis of apoptotic cells) by macrophage-like U937 cells through activation of the inflammatory pathways. In this study, exposure of U937 cells to 2.5 μM oridonin caused up-regulation of inducible nitric oxide synthase (iNOS) expression and continuous endogenous generation of nitric oxide (NO), which was reversed by pre-treatment with the inhibitors of nitric oxide synthase 1400 W (dihydrochloride) or L-NAME (hydrochloride). NO donor sodium nitroprusside (SNP) and efferocytosis irritant lipopolysaccharide (LPS) could also exert NO generation and iNOS expression. Moreover, oridonin-induced stimulation of efferocytosis was significantly suppressed by 1400 W or L-NAME. In addition, 1400 W or L-NAME impaired oridonin-induced autophagy. Inhibition of autophagy with 3-methyladenine (3MA) or Beclin-1 siRNA attenuated the uptake of apoptotic cells with a slight increase in the production of NO. The pro-inflammatory cytokine interleukin-1β (IL-1β) has been reported to be involved in oridonin-induced efferocytosis in U937 cells and interact with NO to contribute to inflammatory responses. 1400 W or L-NAME blocked the secretion of IL-1β and the activation of NF-κB and COX-2. Provision of SNP or LPS in place of oridonin resulted in the similar enhancement of efferocytosis, autophagy, the release of IL-1β and the expression of signal protein. NO augmented the oridonin-induced efferocytosis by mediating autophagy and activating the NF-κB-COX-2-IL-1β pathway. Inhibition of NF-κB or COX-2 in turn decreased the production of NO and the expression of iNOS. There exists a positive feedback loop between NO generation and NF-κB-COX-2-IL-1β pathway.     

Polygonatum cyrtonema lectin induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras–Raf and PI3K–Akt signaling pathways

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding lectin, has been reported to display remarkable anti-proliferative and apoptosis-inducing activities toward a variety of cancer cells; however, the precise molecular mechanisms by which PCL induces cancer cell death are still elusive. In the current study, we found that PCL could induce apoptosis and autophagy in murine fibrosarcoma L929 cells. Subsequently, we demonstrated that inhibition of Ras could promote L929 cell death, suggesting that Ras–Raf signaling pathway plays the key negative regulator in PCL-induced apoptosis. And, we showed that Ras-Raf signaling pathway was also involved in PCL-induced autophagy as the negative regulator. In addition, we found that class I phosphatidylinositol 3-kinase (PI3K)–Akt signaling pathway could play the negative regulator in PCL-induced apoptosis and autophagy. Taken together, these results demonstrate that PCL induces murine fibrosarcoma L929 cell apoptosis and autophagy via blocking Ras-Raf and PI3K–Akt signaling pathways.     

Rapamycin generates anti-apoptotic human Th1/Tc1 cells via autophagy for induction of xenogeneic GVHD

Murine T cells exposed to rapamycin maintain flexibility towards Th1/Tc1 differentiation, thereby indicating that rapamycin promotion of regulatory T cells (Tregs) is conditional. The degree to which rapamycin might inhibit human Th1/Tc1 differentiation has not been evaluated. In the presence of rapamycin, T cell costimulation and polarization with IL-12 or IFN-α permitted human CD4+ and CD8+ T cell differentiation towards a Th1/Tc1 phenotype; activation of STAT1 and STAT4 pathways essential for Th1/Tc1 polarity was preserved during mTOR blockade but instead abrogated by PI3 kinase inhibition. Such rapamycin-resistant human Th1/Tc1 cells: (1) were generated through autophagy (increased LC3BII expression; phenotype reversion by autophagy inhibition via 3-MA or siRNA for Beclin1); (2) expressed anti-apoptotic bcl-2 family members (reduced Bax, Bak; increased phospho-Bad); (3) maintained mitochondrial membrane potentials; and (4) displayed reduced apoptosis. In vivo, type I polarized and rapamycin-resistant human T cells caused increased xenogeneic graft-versus-host disease (x-GVHD). Murine recipients of rapamycin-resistant human Th1/Tc1 cells had: (1) persistent T cell engraftment; (2) increased T cell cytokine and cytolytic effector function; and (3) T cell infiltration of skin, gut, and liver. Rapamycin therefore does not impair human T cell capacity for type I differentiation. Rather, rapamycin yields an anti-apoptotic Th1/Tc1 effector phenotype by promoting autophagy.     

Autophagy enhanced phagocytosis of apoptotic cells by oridonin-treated human histocytic lymphoma U937 cells

Macrophages rapidly engulf and remove apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune disease or inflammation. Recent developments reveal an important role in autophagy for clearance of apoptotic corpses. However, the relationship between autophagy and phagocytosis remains unclear. In this study we found that low doses of oridonin, an active diterpenoid, enhanced phagocytosis of apoptotic cells by human macrophage-like U937 cells, meanwhile it also induced autophagy in these U937 cells. Moreover, inhibition of extracellular signal-related kinase (ERK), nuclear factor-κB (NF-κB) and caspase-1 significantly suppressed oridonin-induced phagocytosis and autophagy. In addition, oridonin increased the protein levels of p-ERK, NF-κB, caspase-1 and pro IL-1β. Autophagic inhibitor 3-methyladenine (3-MA) decreased phagocytosis and the expression of ERK whereas increased the expression of NF-κB- and caspase-1-mediated IL-1β release. Beclin-1 (known as autophagic regulator) loss also led to the similar results. Pretreatment with autophagic agonist rapamycin caused opposite results. Autophagy-associated proteins, Beclin-1, LC3 and Atg4B, involved in this phagocytosis process. These results demonstrated that autophagy enhanced oridonin-induced phagocytosis through feedback regulation of ERK, NF-κB- and caspase-1-mediated IL-1β release.     

Autophagy is required for the activation of NFκB

It is well-established that the activation of the inhibitor of NFκB (IκBα) kinase (IKK) complex is required for autophagy induction by multiple stimuli. Here, we show that in autophagy-competent mouse embryonic fibroblasts (MEFs), distinct autophagic triggers, including starvation, mTOR inhibition with rapamycin and p53 inhibition with cyclic pifithrin α lead to the activation of IKK, followed by the phosphorylation-dependent degradation of IκBα and nuclear translocation of NFκB. Remarkably, the NFκB signaling pathway was blocked in MEFs lacking either the essential autophagy genes Atg5 or Atg7. In addition, we found that tumor necrosis factor α (TNFα)-induced NFκB nuclear translocation is abolished in both Atg5- and Atg7-deficient MEFs. Similarly, the depletion of essential autophagy modulators, including ATG5, ATG7, Beclin 1 and VPS34, by RNA interference inhibited TNFα-driven NFκB activation in two human cancer cell lines. In conclusion, it appears that, at least in some instances, autophagy is required for NFκB activation, highlighting an intimate crosstalk between these two stress response signaling pathways.     

Areca nut extract induced oxidative stress and upregulated hypoxia inducing factor leading to autophagy in oral cancer cells

Areca (betel) chewing was tightly linked to oral tumorigenesis in Asians. Areca nut was a recently confirmed group I carcinogen and a popular addictive substance used by Asians. While, the pathogenetic impact of areca on oral epithelial cells was still unclear. This study investigated the association between the induction of autophagy by areca nut extract (ANE) and the molecular regulation underlying this induction in oral cancer cells. Oral cancer cells were treated with ANE to insight the signaling changes underlying phenotypic alterations. The NFκB activation and reactive oxygen species (ROS) genesis were induced by ANE and the NF-κB activation could be the basis of the ROS genesis. Furthermore, p38 activation and upregulation of MKP-1 phosphatase occurred following ANE treatment. These effects can be inhibited by ROS blockers. ANE treatment induced autophagy among oral cancer cells, which was characterized by LC3-II accumulation, genesis of autophagosomes and the appearance of EGFP-LC3 puncta. This induction was mediated through the activation of p38, MKP-1 and HIF-1α. Knockdown of ANE-modulated HIF-1α expression reduced autophagy. Blockage of ANE-induced autophagy increased the proportion of oral cancer cells undergoing apoptotic death. This study identified for the first time that ANE modulates a signaling cascade that induces HIF-1α expression in oral cancer cells. The eventual induction of autophagy was beneficial to cell survival from ANE-induced apoptosis.     

BCL2L11/BIM

In response to toxic stimuli, BCL2L11 (also known as BIM), a BH3-only protein, is released from its interaction with dynein light chain 1 (DYNLL1 also known as LC8) and can induce apoptosis by inactivating anti-apoptotic BCL2 proteins and by activating BAX-BAK1. Recently, we discovered that BCL2L11 interacts with BECN1 (Beclin 1), and that this interaction is facilitated by DYNLL1. BCL2L11 recruits BECN1 to microtubules by bridging BECN1 and DYNLL1, thereby inhibiting autophagy. In starvation conditions, BCL2L11 is phosphorylated by MAPK8/JNK and this phosphorylation abolishes the BCL2L11-DYNLL1 interaction, allowing dissociation of BCL2L11 and BECN1, thereby ameliorating autophagy inhibition. This finding demonstrates a novel function of BIM beyond its roles in apoptosis, highlighting the crosstalk between autophagy and apoptosis, and suggests that BCL2L11’s dual effects in inhibiting autophagy and promoting apoptosis may have important roles in disease pathogenesis.     

Autophagy plays a protective role during zVAD-induced necrotic cell death

The aim of this study is to examine the role of autophagy in cell death by using a well-established system in which zVAD, a pan-caspase inhibitor, induces necrotic cell death in L929 murine fibrosarcoma cells. First, we observed the presence of autophagic hallmarks, including an increased number of autophagosomes and the accumulation of LC3-II in zVAD-treated L929 cells. Since the presence of such autophagic hallmarks could be the result of either increased flux of autophagy or blockage of autophagosome maturation (lysosomal fusion and degradation), we next tested the effect of rapamycin, a specific inhibitor for mTOR, and chloroquine, a lysosomal enzyme inhibitor, on zVAD-induced cell death. To our surprise, rapamycin, known to be an autophagy inducer, blocked zVAD-induced cell death, whereas chloroquine greatly sensitized zVAD-induced cell death in L929 cells. Moreover, similar results with rapamycin and chloroquine were also observed in U937 cells when challenged with zVAD. Consistently, induction of autophagy by serum starvation offered significant protection against zVAD-induced cell death, whereas knockdown of Atg5, Atg7 or Beclin 1 markedly sensitized zVAD-induced cell death in L929 cells. More importantly, Atg genes knockdown completely abolished the protective effect of serum starvation on zVAD-induced cell death. Finally, we demonstrated that zVAD was able to inhibit lysosomal enzyme cathepsin B activity, and subsequently blocked autophagosome maturation. Taken together, in contrast to the previous conception that zVAD induces autophagic cell death, here we provide compelling evidence suggesting that autophagy serves as a cell survival mechanism and suppression of autophagy via inhibition of lysosomal function contributes to zVAD-induced necrotic cell death.