Surfactin induces apoptosis in human breast cancer MCF-7 cells through a ROS/JNK-mediated mitochondrial/caspase pathway

Surfactin has been known to inhibit proliferation and induce apoptosis in cancer cells. However, the molecular mechanisms involved in surfactin-induced apoptosis remain poorly understood. The present study was undertaken to elucidate the underlying network of signaling events in surfactin-induced apoptosis of human breast cancer MCF-7 cells. In this study, surfactin caused reactive oxygen species (ROS) generation and the surfactin-induced cell death was prevented by antioxidants N-acetylcysteine (NAC) and catalase, suggesting involvement of ROS generation in surfactin-induced cell death. Surfactin induced a sustained activation of the phosphorylation of ERK1/2 and JNK, but not p38. Moreover, surfactin-induced cell death was reversed by PD98059 (an inhibitor of ERK1/2) and SP600125 (an inhibitor of JNK), but not by SB203580 (an inhibitor of p38). However, the phosphorylation of JNK rather than ERK1/2 activation by surfactin was blocked by NAC/catalase. These results suggest that the action of surfactin on MCF-7 cells was via ERK1/2 and JNK, but not via p38, and the ERK1/2 and JNK activation induce apoptosis through two independent signaling mechanisms. Surfactin triggered the mitochondrial/caspase apoptotic pathway indicated by enhanced Bax-to-Bcl-2 expression ratio, loss of mitochondrial membrane potential, cytochrome c release, and caspase cascade reaction. The NAC and SP600125 blocked these events induced by surfactin. Moreover, the general caspase inhibitor z-VAD-FMK inhibited the caspase-6 activity and exerted the protective effect against the surfactin-induced cell death. Taken together, these findings suggest that the surfactin induces apoptosis through a ROS/JNK-mediated mitochondrial/caspase pathway.     

Butyrate induces cell apoptosis through activation of JNK MAP kinase pathway in human colon cancer RKO cells

Butyrate has been shown to display anti-cancer activity through the induction of apoptosis in various cancer cells. However, the underlying mechanism involved in butyrate-induced apoptosis is still not fully understood. Here, we investigated the cytotoxicity mechanism of butyrate in human colon cancer RKO cells. The results showed that butyrate induced a strong growth inhibitory effect against RKO cells. Butyrate also effectively induced apoptosis in RKO cells, which was characterized by DNA fragmentation, nuclear staining of DAPI, and the activation of caspase-9 and caspase-3. The expression of anti-apoptotic protein Bcl-2 decreased, whereas the apoptotic protein Bax increased in a dose-dependent manner during butyrate-induced apoptosis. Moreover, treatment of RKO cells with butyrate induced a sustained activation of the phosphorylation of c-jun N-terminal kinase (JNK) in a dose- and time-dependent manner, and the pharmacological inhibition of JNK MAPK by SP600125 significantly abolished the butyrate-induced apoptosis in RKO cells. These results suggest that butyrate acts on RKO cells via the JNK but not the p38 pathway. Butyrate triggered the caspase apoptotic pathway, indicated by an enhanced Bax-to-Bcl-2 expression ratio and caspase cascade reaction, which was blocked by SP600125. Taken together, our data indicate that butyrate induces apoptosis through JNK MAPK activation in colon cancer RKO cells.     

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via,mitochondrial mediated caspase-dependent mechanism

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.     

Reactive oxygen species-mediated mitochondrial pathway is involved in Baohuoside I-induced apoptosis in human non-small cell lung cancer

Baohuoside I (also known as Icariside II) is a flavonoid isolated from Epimedium koreanum Nakai. Although Baohuoside I exhibits anti-inflammatory and anti-cancer activities, its molecular targets/pathways in human lung cancer cells are poorly understood. Therefore, in the present study, we investigated the usefulness of Baohuoside I as a potential apoptosis-inducing cytotoxic agent using human adenocarcinoma alveolar basal epithelial A549 cells as in vitro model. The apoptosis induced by Baohuoside I in A549 cells was confirmed by annexin V/propidium iodide double staining, cell cycle analysis and dUTP nick end labeling. Further research revealed that Baohuoside I accelerated apoptosis through the mitochondrial apoptotic pathway, involving the increment of BAX/Bcl-2 ratio, dissipation of mitochondrial membrane potential, transposition of cytochrome c, caspase 3 and caspase 9 activation, degradation of poly (ADP-ribose) polymerase and the over-production of reactive oxygen species (ROS). A pan-caspase inhibitor, Z-VAD-FMK, only partially prevented apoptosis induced by Baohuoside I, while NAC, a scavenger of ROS, diminished its effect more potently. In addition, the apoptotic effect of Baohuoside I was dependent on the activation of ROS downstream effectors, JNK and p38(MAPK), which could be almost abrogated by using inhibitors SB203580 (an inhibitor of p38(MAPK)) and SP600125 (an inhibitor of JNK). These findings suggested that Baohuoside I might exert its cytotoxic effect via the ROS/MAPK pathway.     

c-Jun N-terminal protein kinase signalling pathway mediates lovastatin-induced rat brain neuroblast apoptosis

We have previously shown that lovastatin, an HMG-CoA reductase inhibitor, induces apoptosis in rat brain neuroblasts. c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) are implicated in regulation of neuronal apoptosis. In this work, we investigated the role of JNK and p38 MAPK in neuroblast apoptosis induced by lovastatin. We found that lovastatin induced the activation of JNK, but not p38 MAPK. It also induced c-Jun phosphorylation with a subsequent increase in activator protein-1 (AP-1) binding, AP-1-mediated gene expression and BimEL protein levels. The effects of lovastatin were prevented by mevalonate. Pre-treatment with iJNK-I (a selective JNK inhibitor) prevented the effect of lovastatin on both neuroblast apoptosis and the activation of the JNK cascade. Furthermore, we found that the activation of the JNK signalling pathway triggered by lovastatin is accompanied by caspase-3 activation which is also inhibited by iJNK-I pre-treatment. Finally, a specific inhibitor of p38 MAPK, SB203580, had no effect on lovastatin-induced neuroblast apoptosis. Taken together, our data suggest that the activation of the JNK/c-Jun/BimEL signalling pathway plays a crucial role in lovastatin-induced neuroblast apoptosis. Our findings may also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.     

Cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells through activation of the proapoptotic Bcl-2 family proteins and MAPK pathway

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. However, the intracellular death signaling mechanisms by which Cin inhibits tumor cell growth are poorly understood. In this study, we investigated the effect of mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on the stress-responsive MAPK pathway induced by Cin in PLC/PRF/5 cells. Trypan blue staining assay indicated that Cin was cytotoxic to PLC/PRF/5 cells. Cin caused cell cycle perturbation (S-phase arrest) and triggered apoptosis as revealed by the externalization of annexin V-targeted phosphatidylserine and accumulation of sub-G1 peak. It down-regulated the Bcl-2 and Mcl-1 expression, and up-regulated Bax protein in a time-response manner. Treatment with 1 microM Cin resulted in an activation of caspase-8 and cleavage of Bid to its truncated form in a time-dependent pattern. JNK, ERK and p38 kinases in cells were activated and phosphorylated after Cin treatment. Pre-incubation with SP600125 and SB203580 markedly suppressed the effect of Cin-induced apoptosis, but not PD98059. Both SP600125 and SB203580 significantly prevented the phosphorylation of JNK and p38 proteins, but not ERK. These results conclude that Cin triggers apoptosis in PLC/PRF/5 cells could be through the activation of pro-apoptotic Bcl-2 family (Bax and Bid) proteins and MAPK signaling pathway.     

Synergistic activation of JNK/SAPK induced by TNF-alpha and IFN-gamma: apoptosis of pancreatic beta-cells via the p53 and ROS pathway

IFN-γ and TNF-α are major proinflammatory cytokines implicated in islet β-cell destruction, which results in type-1 diabetes; however, the underlying mechanism is not clear. Using pancreatic β-cell line MIN6N8 cells, co-treatment with TNF-α and IFN-γ, but neither cytokine alone, synergistically induced apoptosis, correlated with the activation of the JNK/SAPK, which resulted in the production of reactive oxidative species (ROS) and loss of mitochondrial transmembrane potential (ΔΨm). Additionally, cells transfected with wild-type JNK1 became more susceptible to apoptosis induced by TNF-α/IFN-γ through ROS production and loss of Δψm, while cascading apoptotic events were prevented in dominant-negative JNK1-transfected or JNK inhibitor SP600125-treated cells. As the antioxidant, N-acetyl-cysteine, failed to completely suppress apoptosis induced by TNF-α/IFN-γ, an additional pathway was considered to be involved. The level of p53 was significantly increased through synergistic activation of JNK by TNF-α/IFN-γ. Furthermore, the synergistic effect of TNF-α/IFN-γ on apoptosis and ROS production was further potentiated by the overexpression of wild-type p53, but not with mutant p53. This synergistic activation of JNK/SAPK by TNF-α/IFN-γ was also induced in insulin-expressing pancreatic islet cells, and increased ROS production and p53 level, which was significantly inhibited by SP600125. Collectively, these data demonstrate that TNF-α/IFN-γ synergistically activates JNK/SAPK, playing an important role in promoting apoptosis of pancreatic β-cell via activation of p53 pathway together with ROS.     

JNK2 is required for efficient T-cell activation and apoptosis but not for normal lymphocyte development

BACKGROUND: The Jun N-terminal kinase (JNK) signaling pathway has been implicated in cell proliferation and apoptosis, but its function seems to depend on the cell type and inducing signal. In T cells, JNK has been implicated in both antigen-induced activation and apoptosis. RESULTS: We generated mice lacking the JNK2 isozymes. The mutant mice were healthy and fertile but defective in peripheral T-cell activation induced by antibody to the CD3 component of the T-cell receptor (TCR) complex - proliferation and production of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) were reduced. The proliferation defect was restored by exogenous IL-2. B-cell activation was normal in the absence of JNK2. Activation-induced peripheral T-cell apoptosis was comparable between mutant and wild-type mice, but immature (CD4(+) CD8(+)) thymocytes lacking JNK2 were resistant to apoptosis induced by administration of anti-CD3 antibody in vivo. The lack of JNK2 also resulted in partial resistance of thymocytes to anti-CD3 antibody in vitro, but had little or no effect on apoptosis induced by anti-Fas antibody, dexamethasone or ultraviolet-C (UVC) radiation. CONCLUSIONS: JNK2 is essential for efficient activation of peripheral T cells but not B cells. Peripheral T-cell activation is probably required indirectly for induction of thymocyte apoptosis resulting from administration of anti-CD3 antibody in vivo. JNK2 functions in a cell-type-specific and stimulus-dependent manner, being required for apoptosis of immature thymocytes induced by anti-CD3 antibody but not for apoptosis induced by anti-Fas antibody, UVC or dexamethasone. JNK2 is not required for activation-induced cell death of mature T cells.     

Comparison of roles of three mitogen‐activated protein kinases induced by chromium(VI) and cadmium in non‐small‐cell lung carcinoma cells

Chromium(VI) Cr(VI) and cadmium (Cd) compounds are ubiquitous environmental carcinogens that have been associated with lung tumors and can induce apoptosis in various cell types. Three major mitogenactivation protein kinases (MAPKs), extracellular signalregulated kinase (ERK), cJUN Nterminal kinase (JNK) and p38, have been shown to regulate apoptosis. In this study we explore the abilities of Cr(VI) and Cd to activate JNK, p38 and ERK, including their roles in metalmediated growth inhibition and apoptosis in a human nonsmallcell lung carcinoma cell line, CL3. Exposure to K₂Cr₂O₇ markedly activated JNK and p38 and moderately activated ERK in a dose and timedependent manner. The activated p38 decreased markedly and rapidly and the activated JNK decreased gradually when Cr(VI) was removed from media. At low cytotoxic doses, CdCl₂ decreased ERK activity with concurrently transient activation of JNK, whereas at high cytotoxic doses it persistently activated all three MAPKs. The strength and duration of JNK and p38 activated by Cd were higher and longer than Cr(VI) did when compared at similar cytotoxic doses. In comparable experiment conditions Cd is a much stronger apoptotic inducer than Cr(VI) in CL3 cells. Crosstalk of MAPKs was observed in cells exposed to Cr(VI) but not Cd. Both metals could increase JNK activity through MKK7 but not MKK4. The Cdactivated JNK is involved in apoptosis, but the Cractivated JNK is not. PD98059, an inhibitor of the ERK upstream activators MKK1/2, greatly enhanced the cytotoxicity and apoptosis of cells treated with low Cd doses. SB202190, an inhibitor of p38, decreased the cytotoxicity and apoptosis induced by high Cd doses. Conversely, neither SB202190 nor PD98059 altered Cr(VI)induced cytotoxicity. The results suggest that JNK and p38 signals cooperatively participate in apoptosis induced by Cd and that the decreased ERK signal by low Cd doses contributes to growth inhibition or apoptosis. Oppositely, activation of ERK, JNK and p38 by Cr(VI) does not affect cytotoxicity.     

Aplidin induces apoptosis in human cancer cells via glutathione depletion and sustained activation of the epidermal growth factor receptor,Src, JNK,and p38 MAPK

We report that Aplidin, a novel antitumor agent of marine origin presently undergoing Phase II clinical trials, induced growth arrest and apoptosis in human MDA-MB-231 breast cancer cells at nanomolar concentrations. Aplidin induced a specific cellular stress response program, including sustained activation of the epidermal growth factor receptor (EGFR), the non-receptor protein-tyrosine kinase Src, and the serine/threonine kinases JNK and p38 MAPK. Aplidin-induced apoptosis was only partially blocked by the general caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone and was also sensitive to AG1478 (an EGFR inhibitor), PP2 (an Src inhibitor), and SB203580 (an inhibitor of JNK and p38 MAPK) in MDA-MB-231 cells. Supporting a role for EGFR in Aplidin action, EGFR-deficient mouse embryo fibroblasts underwent apoptosis upon treatment more slowly than wild-type EGFR fibroblasts and also showed delayed JNK and reduced p38 MAPK activation. N-Acetylcysteine and ebselen (but not other antioxidants such as diphenyleneiodonium, Tiron, catalase, ascorbic acid, and vitamin E) reduced EGFR activation by Aplidin. N-Acetylcysteine and PP2 also partially inhibited JNK and p38 MAPK activation. The intracellular level of GSH affected Aplidin action; pretreatment of cells with GSH or N-acetylcysteine inhibited, whereas GSH depletion caused, hyperinduction of EGFR, Src, JNK, and p38 MAPK. Remarkably, Aplidin also induced apoptosis and activated EGFR, JNK, and p38 MAPK in two cell lines (A-498 and ACHN) derived from human renal cancer, a neoplasia that is highly refractory to chemotherapy. These data provide a molecular basis for the anticancer activity of Aplidin.     

Cell line dependent involvement of ceramide in ultraviolet light-induced apoptosis

Ultraviolet light (UV) activates an acid sphingomyelinase (ASMase) pathway, which hydrolyzes sphingomyeline to ceramide. Ceramide has been found to be a second messenger, which activates the c-jun N-terminal kinase (JNK) that is required for apoptotic cell death. However, the role of ceramide in UV-induced JNK activation and apoptosis remains controversial. In this study, we examined the correlation among ceramide production, JNK activation and cell apoptosis after UV-irradiation in three cell lines: 293 (kidney), Jurkat (lymphocytes) and MCF-7 (breast) were used in this study. The ceramide production was analyzed using the diacylglycerol kinase assay method. The JNK activation was measured by Western blot analysis using an antibody specifically recognizing phosphorylated JNK. Cell apoptosis was determined by morphological change or flow cytometry. Our data show that UV-irradiation induces ceramide production in both 293 and Jurkat cells. Inhibition of ceramide production by desipramine (25–50 M) reduced UV-induced JNK activation in both 293 and Jurkat cells; and protects 293 cells from UV-induced apoptosis. However, inhibition of ceramide production does not prevent Jurkat cells from UV-induced apoptosis. In addition, our data demonstrates that UV-irradiation induces JNK activation and apoptosis of MCF-7 cells without production of detectable amounts of ceramide after UV-irradiation. These results suggest that UV-induced JNK activation and apoptosis can be mediated through a ceramide dependent or an independent pathway.     

K252a suppresses neuronal cells apoptosis through inhibiting the translocation of Bax to mitochondria induced by the MLK3/JNK signaling after transient global brain ischemia in rat hippocampal CA1 subregion

It is demonstrated that the c-Jun N-terminal kinase (JNK) signaling pathway plays a critical role in ischemic brain injury. Our previous studies have suggested that K252a can obviously inhibit JNK activation induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. Here, we further discussed the potential mechanism of ischemic brain injury induced by the activation of JNK after 15?min of transient global cerebral ischemia. As a result, through inhibiting phosphorylation of Bcl-2 (a cytosolic target of JNK) and 14-3-3 protein (a cytoplasmic anchor of Bax) induced by the activation of JNK, K252a decreased the release of Bax from Bcl-2/Bax and 14-3-3/Bax dimers, further attenuating the translocation of Bax from cytosol to mitochondria and the release of cytochrome c induced by ischemia/reperfusion, which related to mitochondria-mediated apoptosis. Importantly, pre-infusion of K2525a 20?min before ischemia showed neuroprotective effect against neuronal cells apoptosis. These findings imply that K252a induced neuroprotection against ischemia/reperfusion in rat hippocampal CA1 subregion via inhibiting the mitochondrial apoptosis pathway induced by JNK activation.     

HSP72 can protect cells from heat-induced apoptosis by accelerating the inactivation of stress kinase JNK

The major heat shock protein Hsp72 prevents heat-induced apoptosis. We have previously demonstrated that transiently expressed Hsp72 exerts its anti-apoptotic effect by suppressing the activity of stress-kinase JNK, an early component of the apoptotic pathway initiated by heat shock. On the other hand, constitutive expression of Hsp72 does not lead to suppression of heat-induced JNK activation, yet still efficiently prevents apoptosis. To address this apparent contradiction, we studied the effects of constitutively expressed Hsp72 on activation of JNK and apoptosis in Rat-1 fibroblasts. We found that the level of heat-induced apoptosis directly correlated with the duration rather than the magnitude of JNK activity following heat shock. Constitutively expressed Hsp72 strongly reduced the duration of JNK while it did not suppress initial JNK activation. These effects were due to Hsp72-mediated acceleration of JNK dephosphorylation. Addition of vanadate to inhibit JNK phosphatase activity completely prevented the anti-apoptotic action of Hsp72. Therefore, suppression of heat-induced apoptosis by Hsp72 could be fully accounted for by its effects on JNK activity.     

Sodium 4-phenylbutyrate induces apoptosis of human lung carcinoma cells through activating JNK pathway

Sodium 4-phenylbutyrate (PB) has been used in the therapy of urea cycle defects for many years. Recently, it has been shown to cause cellular differentiation, growth arrest, and apoptosis in certain malignancies. We have analyzed the effects of PB on human lung carcinoma cells. PB has distinct patterns of effects on different lung carcinoma cells, inducing apoptosis in NCI-H460 and NCI-H1792 cells, causing G1 arrest in A549 and SK-LU-1 cells, but having no effect on a non-transformed bronchial epithelial cell line HBE4-E6/E7. We investigated the role of MAP kinase family members, extracellular signal-regulated kinase (ERK), JNK, and p38 mitogen-activated protein kinase (MAPK), as well as other important cell survival signaling molecules in PB-induced apoptosis. We observed activation of JNK and ERK by PB in the lung cancer cells. JNK was activated only in the two apoptotic cells, whereas ERK was activated in both the apoptotic and the growth-arrested cells, demonstrating a correlation between apoptosis and activation of JNK in response to PB. Both JNK inhibitor and JNK RNA interference (RNAi) inhibited PB-induced apoptosis, whereas MEK inhibitor did not, supporting that apoptosis induced by PB is through activation of JNK. De novo protein synthesis is required for the PB-induced JNK activation and induction of apoptosis. However, the production of known upstream activators of JNK, namely Fas/Fas ligand, tumor necrosis factor (TNF)-alpha, TNF-beta, and TRAIL, are not altered by PB treatment. Therefore, PB activates JNK through an unidentified and cell type-specific mechanism. Understanding of this mechanism is of therapeutic value in treating cancer patients with PB.     

Requirement of c-Jun NH2-terminal kinase activation in interferon-alpha-induced apoptosis through upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma cells

Interferon alpha (IFN-alpha) inhibits growth, at least in part, through induction of apoptosis. However, the molecular mechanisms underlying IFN-alpha-induced apoptosis are not completely understood. In the present study, we found that IFN-alpha induced a sustained activation of c-Jun N-terminal kinase 1 (JNK1), but not extracellular kinases (ERKs), in Daudi B lymphoma cells, as assessed by Western blotting using phospho-specific antibodies. Several lines of evidence support the notion that the IFN-alpha-induced activation of JNK is responsible for IFN-alpha-induced apoptosis, at least in part, through upregulation of TNF-related apoptosis-inducing ligand (TRAIL). First, pretreatment of Daudi cells with a JNK inhibitor reduced IFN-alpha-induced upregulation of TRAIL and loss of mitochondrial membrane potential (DeltaPsim) and annexin-positive cells, which was assessed by flow cytometry. Second, a dominant-negative form of JNK1 (dnJNK1) also reduced these apoptotic events, while a constitutively active form of JNK1, MKK7-JNK1beta, enhanced them. Finally, treatment with IFN-alpha enhanced the promoter activity of the TRAIL gene, which was partially abrogated by either JNK inhibitor or dnJNK1, while it was moderately enhanced by MKK7-JNK1beta. These findings are useful for understanding molecular mechanisms of IFN-alpha-induced apoptosis and also for development of treatment modalities of some tumors with IFN-alpha.