Fas-mediated autophagy requires JNK activation in HeLa cells

Fas has been reported to play an important role in apoptosis; however, Fas-mediated autophagy and its mechanism are still unclear. Here, we found that Fas agonistic antibody CH11-induced autophagy in HeLa cells, and inhibition of autophagy by 3-MA increased CH11-induced apoptosis. A Fas antagonistic antibody (UB2) suppressed both CH11-induced autophagy and apoptosis. In addition, the CH11-induced autophagy was blocked by JNK inhibitor (SP600125), but it was not affected by caspase 8 inhibitor (Z-IETD); whereas the CH11-induced apoptosis was increased by SP600125, and it was suppressed by Z-IETD. Further experiments confirmed that JNK was activated by CH11 dose-dependently, and the activation was suppressed when autophagy was blocked by 3-MA. Together, our results suggest that JNK, but not caspase 8, involves in Fas-mediated CH11-induced autophagy in HeLa cells, and this autophagy plays a protective role in CH11-induced cell death.     

PKCη confers protection against apoptosis by inhibiting the pro-apoptotic JNK activity in MCF-7 cells

Apoptosis is frequently regulated by different protein kinases including protein kinase C family enzymes. Both inhibitory and stimulatory effects were demonstrated for several of the different PKC isoforms. Here we show that the novel PKC isoform, PKCη, confers protection against apoptosis induced by the DNA damaging agents, UVC irradiation and the anti-cancer drug — Camptothecin, of the breast epithelial adenocarcinoma MCF-7 cells. The induced expression of PKCη in MCF-7 cells, under the control of the tetracycline-responsive promoter, resulted in increased cell survival and inhibition of cleavage of the apoptotic marker PARP-1. Activation of caspase-7 and 9 and the release of cytochrome c were also inhibited by the inducible expression of PKCη. Furthermore, JNK activity, required for apoptosis in MCF-7, as indicated by the inhibition of both caspase-7 cleavage and cytochrome c release from the mitochondria in the presence of the JNK inhibitor SP600125, was also suppressed by PKCη expression. Hence, in contrast to most PKC isoforms enhancing JNK activation, our studies show that PKCη is an anti-apoptotic protein, acting as a negative regulator of JNK activity. Thus, PKCη could represent a target for intervention aimed to reduce resistance to anti-cancer treatments.     

VMP1 related autophagy and apoptosis in colorectal cancer cells: VMP1 regulates cell death

Vacuole membrane protein 1 (VMP1) is an autophagy-related protein and identified as a key regulator of autophagy in recent years. In pancreatic cell lines, VMP1-dependent autophagy has been linked to positive regulation of apoptosis. However, there are no published reports on the role of VMP1 in autophagy and apoptosis in colorectal cancers. Therefore, to address this gap of knowledge, we decided to interrogate regulation of autophagy and apoptosis by VMP1. We have studied the induction of autophagy by starvation and rapamycin treatment in colorectal cell lines using electron microscopy, immunofluorescence, and immunoblotting. We found that starvation-induced autophagy correlated with an increase in VMP1 expression, that VMP1 interacted with BECLIN1, and that siRNA mediated down-regulation of VMP1-reduced autophagy. Next, we examined the relationship between VMP1-dependent autophagy and apoptosis and found that VMP1 down-regulation sensitizes cells to apoptosis and that agents that induce apoptosis down-regulate VMP1. In conclusion, similar to its reported role in other cell types, VMP1 is an important regulator of autophagy in colorectal cell lines. However, in contrast to its role in pancreatic cell lines, in colorectal cancer cells, VMP1-dependent autophagy appears to be pro-survival rather than pro-cell death.     

PKC and Raf-1 inhibition-related apoptotic signalling in N2a cells

In this study, a neuroblastoma N2a cell line was applied to investigate mechanisms of apoptosis induced either by selective inhibition of protein kinase C (PKC) by low amounts of staurosporine (STS(10) ) or by inhibition PI3-K after wortmannin (WM) treatment. We present evidence that, in the absence of serum in the medium, decreased phosphorylation of Raf-1 and BAD112, as well as Akt and BAD136, proteins and their translocation to mitochondria coincided with STS10 - or WM-induced apoptosis, respectively. Concomitantly, release of cytochrome c into the cytosol indicated a BCL-2-dependent mode of cell death after both treatments. Furthermore, in typical 'gain of function' experiments, cells with overexpression of permanently active Raf-1 or Akt transgenes displayed a significantly higher and independent resistance to either STS10 or WM. Thus, our results indicate that PKC/Raf-1/BAD112, as well as PI3-K/Akt/BAD136 signalling pathways, are both necessary for N2a cell survival and thus are unable to functionally substitute for each other as long as the cells do not receive additional signal(s) derived from serum. However, in the presence of serum, undefined trophic signal(s) can stimulate cross-talk between these two pathways at a level upstream from Raf-1 and Akt phosphorylation. In this case, only simultaneous inhibition of PKC and PI3-K is able to induce apoptosis.     

Mechanisms of autophagy and apoptosis: Recent developments in breast cancer cells

Autophagy,the pathway whereby cell components are degraded by lysosomes,is involved in the cell response to environmental stresses,such as nutrient deprivation,hypoxia or exposition to chemotherapeutic agents.Under these conditions,which are reminiscent of certain phases of tumor development,autophagy either promotes cell survival or induces cell death. This strengthens the possibility that autophagy could be an important target in cancer therapy,as has been proposed.Here,we describe the regulation of survival and death by autophagy and apoptosis,especially in cultured breast cancer cells.In particular,we discuss whether autophagy represents an apoptosis-independent process and/or if they share common pathways. We believe that understanding in detail the molecular mechanisms that underlie the relationships between autophagy and apoptosis in breast cancer cells could improve the available treatments for this disease.     

Cytotoxic constituents from Celastrus paniculatus induce apoptosis and autophagy in breast cancer cells

Celastrus paniculatus is a traditional medicinal plant with diverse pharmacological activities. To identify its bioactive constituents, three new β-dihydroagarofuranoid sesquiterpenes were isolated from the whole plant, of which the major constituent is (1α,2α,8β,9β)-1,8-bis(acetyloxy)-2,9-bis(benzoyloxy)-14-hydroxy-β-dihydroagarofuran. It was assessed for its antiproliferative activity, and it suppressed the viability of MCF-7 breast cancer cells with an IC₅₀ of 17 ± 1 μM. This growth inhibition was, in part, attributable to apoptosis. Moreover, this drug treatment led to LC3B-II accumulation, indicative of autophagy. Western blot analysis established its ability to target a broad range of signaling effectors related to survival and cell cycle progression, including Akt, NF-κB, p53, and MAP kinases. In addition, flow cytometry analysis indicates increased reactive oxygen species production in response to this compound. Taken together, these findings suggest a pleiotropic mode of mechanism that underlies the antiproliferative activity of this compound in MCF-7 breast cancer cells.     

Activation of multiple signal transduction pathways by glucocorticoids: protection of ovarian follicular cells against apoptosis

We have recently demonstrated that glucocorticoids protect against serum-deprivation, cAMP-, TNFalpha-, and p53-induced apoptosis in ovarian follicular cells involved in up-regulation of Bcl-2. We demonstrated that dexamethasone, which enhances steroidogenesis by up-regulation of the p450scc enzyme system, stimulates the MAPK cascade by phosphorylation of ERK1, ERK2 as well as by Akt phosphorylation within 1-5min with no effect on p38 MAPK phosphorylation. Moreover, glucocorticoids enhance expression of connexin 43, formation of gap junctions, expression of cadherins, and formation of adherence junctions within 24h of hormone stimulation of ovarian granulosa cells. It is suggested that the protective effects of glucocorticoids against apoptosis are mediated by both genomic and non-genomic mechanisms. Moreover, for the first time we show that protein phosphorylation, cell-cell contact, and intracellular communication are important mediators in glucocorticoid protection against apoptosis in ovarian follicular cells.     

Involvement of apoptosis and autophagy in the death of RPMI 8226 multiple myeloma cells by two enantiomeric sigma receptor ligands

Over-expression of σ receptors by many tumor cell lines makes ligands for these receptors attractive as potential chemotherapeutic drugs. Enantiomeric piperazines (S)-4 and (R)-4 were prepared as potential σ-receptor ligands in a chiral pool synthesis starting from (S)- and (R)-aspartate. Both compounds showed high affinities for the σ₁ and σ₂ receptors. In the human multiple myeloma cell line RPMI 8226, a line expressing high levels of σ receptors, both compounds inhibited cell proliferation with IC₅₀ values in the low μM range. No chiral differentiation between either the σ receptor binding affinity or the cytotoxicity of the two enantiomers was observed. Both compounds induced apoptosis, which was evidenced by nuclear condensation, binding of annexin-V to phosphatidylserine in the outer leaf of the cell membrane, cleavage products of poly(ADP-ribose) polymerase-1 (PARP-1) and caspase-8 as well as the expression of bcl₂ family members bax, bad and bid. However, apoptosis appeared to be caspase independent. Increased levels of the phosphorylated form of the microtubule associated protein light chain 3-II (LC3-II), an autophagosome marker, gave evidence that both compounds induced autophagy. However, further data (e.g., treatment with wortmannin) indicate that autophagy is incomplete and not cytoprotective. Lipid peroxidation (LPO) was observed in RPMI 8226 cells treated with the two compounds, and the lipid antioxidant α-tocopherol attenuated LPO. Interestingly, α-tocopherol reduced significantly both apoptosis and autophagy induced by the compounds. These results provide evidence that, by initiating LPO and changes in mitochondrial membrane potential, both compounds induce apoptosis and autophagy in RPMI 8226 cells.     

Antisense BAG-1 sensitizes HeLa cells to apoptosis by multiple pathways

To study the mechanism of action of BAG-1 in drug-induced apoptosis, we constructed an antisense BAG-1 vector and established a stably transfected cell line from BAG-1-over-expressing HeLa cells. Reduced BAG-1 protein was confirmed by Western blot. Treatment of the antisense BAG-1-transfected cells with the anti-cancer drugs staurosporine, paclitaxel, all-trans retinoic acid (ATRA), and N-(4-hydroxyphenyl) retinamide (4-HPR) resulted in significantly enhanced apoptosis and reduced cell viability relative to vector-transfected cells. While the expression of p53 was increased, the level of Bcl-2 and Bax was decreased. Cells underexpressing BAG-1 had reduced cytosolic cytochrome c level. Treatment with staurosporine and paclitaxel resulted in increased cytochrome c release from mitochondria, whereas there was no change induced by treatment with ATRA and 4-HPR. Our experiments suggest that BAG-1 inhibits anti-cancer drug-induced apoptosis through apoptosis regulation pathways that may involve the mitochondrial Bcl-2/Bax ratio, p53, and differential anti-cancer drug-mediated cytochrome c release.     

Dihydroartemisinin induces apoptosis in human gastric cancer cell line BGC-823 through activation of JNK1/2 and p38 MAPK signaling pathways

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, is associated with a broad range of biological properties including antitumor activity. However, the effect of DHA on gastric cancer has not been clearly clarified. The aim of this study was to investigate the role and mechanism of DHA in human gastric cancer cell line BGC-823. Cell viability was assessed by MTT assay. Cell apoptosis was analyzed with flow cytometry. The expressions of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and their phosphorylated forms as well as apoptosis related proteins were examined by western blot analysis. The results demonstrated that DHA inhibited cell viability of BGC-823 cells in a dose- and time-dependent manner. DHA treatment upregulated the expression of Bax, cleaved caspase-3 and -9, and degraded form of PARP, and downregulated the Bcl-2 expression and Bcl-2/Bax ratio. Meanwhile, DHA increased the phosphorylation of ERK1/2, JNK1/2 and p38 MAPK. Synthetic inhibitors of JNK1/2 or p38 MAPK kinase activity, but not inhibitor of ERK1/2, significantly abolished the DHA-induced activation of caspase-3 and -9. In vivo tumor-suppression assay further indicated that DHA displayed significant inhibitory effect on BGC-823 xenografts in tumor growth. These results indicate that DHA induces apoptosis of BGC-823 cells through JNK1/2 and p38 MAPK signaling pathways and DHA could serve as a potential additional chemotherapeutic agent for treatment of gastric cancer.     

The hierarchical relationship between MAPK signaling and ROS generation in human leukemia cells undergoing apoptosis in response to the proteasome inhibitor Bortezomib

The hierarchy of events accompanying induction of apoptosis by the proteasome inhibitor Bortezomib was investigated in Jurkat lymphoblastic and U937 myelomonocytic leukemia cells. Treatment of Jurkat or U937 cells with Bortezomib resulted in activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK (mitogen-activated protein kinase), inactivation of extracellular signal-regulating kinase 1/2 (ERK1/2), cytochrome c release, caspase-9, -3, and -8 activation, and apoptosis. Bortezomib-mediated cytochrome c release and caspase activation were blocked by the pharmacologic JNK inhibitor SP600125, but lethality was not diminished by the p38 MAPK inhibitor SB203580. Inducible expression of a constitutively active MEK1 construct blocked Bortezomib-mediated ERK1/2 inactivation, significantly attenuated Bortezomib lethality, and unexpectedly prevented JNK activation. Conversely, pharmacologic MEK/ERK1/2 inhibition promoted Bortezomib-mediated JNK activation and apoptosis. Lastly, the antioxidant N-acetyl-l-cysteine (LNAC) attenuated Bortezomib-mediated reactive oxygen species (ROS) generation, ERK inactivation, JNK activation, mitochondrial dysfunction, and apoptosis. In contrast, enforced MEK1 and ERK1/2 activation or JNK inhibition did not modify Bortezomib-induced ROS production. Together, these findings suggest that in human leukemia cells, Bortezomib-induced oxidative injury operates at a proximal point in the cell death cascade to antagonize cytoprotective ERK1/2 signaling, promote activation of the stress-related JNK pathway, and to trigger mitochondrial dysfunction, caspase activation, and apoptosis. They also suggest the presence of a feedback loop wherein Bortezomib-mediated ERK1/2 inactivation contributes to JNK activation, thereby amplifying the cell death process.     

Death-associated protein kinase 2: Regulator of apoptosis,autophagy and inflammation

Death-associated protein kinase 2 (DAPK2/DRP-1) belongs to a family of five related serine/threonine kinases that mediate a range of cellular processes, including membrane blebbing, apoptosis, and autophagy, and possess tumour suppressive functions. The three most conserved family members DAPK1/DAPK, DAPK2 and DAPK3/ZIPK share a high degree of homology in their catalytic domain, but differ significantly in their extra-catalytic structures and tissue-expression profiles. Hence, each orthologue binds to various unique interaction partners, localizes to different subcellular regions and controls some dissimilar cellular functions. In recent years, mechanistic studies have broadened our knowledge of the molecular mechanisms that activate DAPK2 and that execute DAPK2-mediated apoptosis, autophagy and inflammation. In this “molecules in focus” review on DAPK2, the structure, modes of regulation and various cellular functions of DAPK2 will be summarized and discussed.     

Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy

The present study was performed to examine the induction of apoptotic cell death and autophagy by blue LED irradiation, and the contribution of autophagy to apoptosis in B cell lymphoma A20 and RAMOS cells exposed to blue LED. Irradiation with blue LED reduced cell viability and induced apoptotic cell death, as indicated by exposure of phosphatidylserine on the plasma outside membrane and fragmentation of DNA. Furthermore, the mitochondrial membrane potential increased, and apoptotic proteins (PARP, caspase 3, Bax, and bcl-2) were observed. In addition, the level of intracellular superoxide anion (O₂⁻) gradually increased. Interestingly the formation of autophagosomes and level of LC3-II were increased in blue LED-irradiated A20 and RAMOS cells, but inhibited after pretreatment with 3-methyladenine (3-MA), widely used as an autophagy inhibitor. Inhibition of the autophagic process by pretreatment with 3-MA blocked blue LED irradiation-induced caspase-3 activation. Moreover, a significant reduction of both the early and late phases of apoptosis after transfection with ATG5 and beclin 1 siRNAs was shown by the annexin V/PI staining, indicating a crucial role of autophagy in blue LED-induced apoptosis in cells. Additionally, the survival rate of mice irradiated with blue LED after injection with A20 cells increased compared to the control group. Our data demonstrate that blue LED irradiation induces apoptosis via the mitochondrial-mediated pathway, in conjunction with autophagy. Further studies are needed to elucidate the precise mechanism of blue LED-induced immune cell death.     

MAPK signal pathways in the regulation of cell proliferation in mammalian cells

INTRODUCTIONMitogen-activated protein kinaJse (MAPK) cas-cades have been shown to play a key role in trans-duction extracellular signals to cellular responses. Inmammalian cells, three MAPK families have beenclearly characterized: namely classical MAPK (alsoknown as ERK), C-Jun N-terminal kinse/ stress-activated protein kinase (JNK/SAPK) and p38 ki-nase. MAP kinases lie within protein kinase cas-cades. Each cascade consists of no fewer than threeenzymes that are activated in se…     

Synthesis of novel pyrazole carboxamide derivatives and discovery of modulators for apoptosis or autophagy in A549 lung cancer cells

A series of novel 3-aryl-1-arylmethyl-1H-pyrazole-5-carboxamide derivatives 3a–l, were synthesized by the reaction of 3-aryl-1-arylmethyl-1H-pyrazole-5-carbonyl chloride with substituted amine in excellent yields. The compounds 3e–h could suppress A549 lung cancer cell growth. More interestingly, compounds 3e and 3f might inhibit the A549 cell growth by inducing apoptosis; whereas compounds 3g and 3h with fluorine group might inhibit the A549 cell growth by inducing autophagy.     

Redox signaling: Potential arbitrator of autophagy and apoptosis in therapeutic response

Redox signaling plays important roles in the regulation of cell death and survival in response to cancer therapy. Autophagy and apoptosis are discrete cellular processes mediated by distinct groups of regulatory and executioner molecules, and both are thought to be cellular responses to various stress conditions including oxidative stress, therefore controlling cell fate. Basic levels of reactive oxygen species (ROS) may function as signals to promote cell proliferation and survival, whereas increase of ROS can induce autophagy and apoptosis by damaging cellular components. Growing evidence in recent years argues for ROS that below detrimental levels acting as intracellular signal transducers that regulate autophagy and apoptosis. ROS-regulated autophagy and apoptosis can cross-talk with each other. However, how redox signaling determines different cell fates by regulating autophagy and apoptosis remains unclear. In this review, we will focus on understanding the delicate molecular mechanism by which autophagy and apoptosis are finely orchestrated by redox signaling and discuss how this understanding can be used to develop strategies for the treatment of cancer.     

Inhibition of autophagy enhances effects of PF-04691502 on apoptosis and DNA damage of lung cancer cells

Autophagy modulation has been considered as a potential therapeutic strategy for lung diseases. The PI3K-Akt-mTOR pathway may be one of the main targets for regulation of autophagy. We previously reported that a PI3 K/mTOR dual inhibitor PF-04691502 suppressed hepatoma cells growth in vitro. However, it is still unclear whether PF-04691502 induces autophagy and its roles in DNA damage and cell death in human lung cancer cells. In this study, we investigate the effects of PF-04691502 on the autophagy and its correlation with cell apoptosis and DNA damage in non-small-cell lung cancer (NSCLC) cell lines. PF-04691502 efficiently inhibited the phosphorylation of Akt and showed dose-dependent cytotoxicity in A549 and H1299 cells. PF-04691502 also triggered apoptosis and the cleavage of caspase-3 and PARP. Phosphorylated histone H2AX (γ-H2AX), a hallmark of DNA damage response, was dramatically induced by PF-04691502 treatment. By exposure to PF-04691502, A549 cells acquired a senescent-like phenotype with an increase in the level of β-galactosidase. Furthermore, PF-04691502 enhanced the expression of LC3-II in a concentration-dependent manner. More interestingly, effects of PF-04691502 on toxicity and DNA damage were remarkably increased by co-treatment with an autophagy inhibitor, chloroquine (CQ), in human lung cancer cells. These data suggest that a strategy of blocking autophagy to enhance the activity of PI3 K/mTOR inhibitors warrants further attention in treatment of NSCLC cells.     

High dose of extracellular ATP switched autophagy to apoptosis in anchorage-dependent and anchorage-independent hepatoma cells

Extracellular adenosine triphosphate (eATP) transduces purinergic signal and plays an important regulatory role in many biological processes, including tumor cell growth and cell death. A large amount of eATP exists in the fast-growing tumor center and inflammatory tumor microenvironment. Tumor cells could acquire anoikis resistance and anchorage independence in tumor microenvironment and further cause metastatic lesion. Whether such a high amount of eATP has any effect on the anchored and non-anchored tumor cells in tumor microenvironment has not been elucidated and is investigated in this study. Our data showed that autophagy helped hepatoma cells to maintain survival under the treatment of no more than 1 mM of eATP. Only when eATP concentration reached a relatively high level (2.5 mM), cell organelle could not be further maintained by autophagy, and apoptosis and cell death occurred. In hepatoma cells under treatment of 2.5 mM of eATP, an AMP-activated protein kinase (AMPK) pathway was dramatically activated while mTOR signaling pathway was suppressed in coordination with apoptosis. Further investigation showed that the AMPK/mTOR axis played a key role in tipping the balance between autophagy-mediated cell survival and apoptosis-induced cell death under the treatment of eATP. This work provides evidence to explain how hepatoma cells escape from eATP-induced cytotoxicity as well as offers an important clue to consider effective manipulation of cancer.     

Sonodynamic therapy induces the interplay between apoptosis and autophagy in K562 cells through ROS

Sonodynamic therapy (SDT) is a relatively new approach in the treatment of various cancers including leukemia cells. The aim of this study is to investigate the occurrence of apoptosis and autophagy after treated by protoporphyrin IX (PpIX)-mediated SDT (PpIX-SDT) on human leukemia K562 cells as well as the relationship between them. Firstly, mitochondrial-dependent apoptosis was observed through morphological observation and biochemical analysis. Meanwhile, SDT was shown to induce autophagy in K562 cells, which caused an increase in EGFP-LC3 puncta cells, a conversion of LC3 II/I, formation of acidic vesicular organelles (AVOs) and co-localization between LC3 and LAMP2 (a lysosome marker). Besides, pretreatment with autophagy inhibitor 3-MA or bafilomycin A1 was shown to provide protection against autophagy and to enhance SDT-induced apoptosis and necrosis, while the apoptosis suppressor z-VAD-fmk failed to affect formation of autophagic vacuoles or partially prevented SDT-induced cytotoxicity, which suggested that SDT-induced autophagy functioned as a survival mechanism. Additionally, this study reported apparent apoptosis and autophagy with dependence on intracellular reactive oxygen species (ROS) production. Preliminary data showed that ROS scavenger N-acetylcysteine (NAC) effectively blocked the SDT induced accumulation of ROS, reversed sono-damage, cell apoptosis and autophagy. Taken together, these data indicate that autophagy may be cytoprotective in our experimental system, and the ROS caused by PpIX-SDT treatment may play an important role in initiating apoptosis and autophagy.