Involvement of oxidative stress-activated JNK signaling in the methamphetamine-induced cell death of human SH-SY5Y cells

Methamphetamine (METH) is one of the most commonly abused drugs that may result in neurotoxic damage. Many lines of evidence have revealed that oxidative stress plays an important role in METH-induced neurotoxic effects. In a previous study, it was demonstrated in human neuroblastoma SH-SY5Y cells that enhanced oxidative stress was related to METH-induced apoptosis. To evaluate which of the three major mitogen-activated protein (MAP) kinase signaling pathways are involved in the process, namely the extracellular signal-related kinases (ERK), the p38 MAP kinases (p38) and the Jun-N-terminal kinases (JNK), we performed a time-course assessment. This indicated that METH induced an increase in the phosphorylation of ERK and JNK, but not of p38. Moreover, a JNK-specific inhibitor, SP600125, partially but significantly rescued METH-induced cell death, while PD98059 (an ERK kinase inhibitor) and SB203580 (a p38 inhibitor) had no protective effect. We also found that vitamin E (Vit E) prevented METH-induced JNK phosporylation and SP600125 inhibited METH-induced c-Jun phosphorylation. Furthermore, METH-activated caspase-3 activity was significantly repressed by Vit E and in SP600125 treated cells. We suggest that the oxidative stress-activated JNK signaling pathway is involved in METH-induced cell death.     

Roles of p38 and JNK mitogen-activated protein kinase pathways during cantharidin-induced apoptosis in U937 cells

Cantharidin is an active compound from blister beetles traditionally used for the treatment of cancer. It is known to exert its antitumor activity by inducing apoptosis in cancer cells. However, its signaling pathway still remains unclear. Therefore, we investigated the roles of the mitogen-activated protein kinases (MAPKs) and the tumor suppressor gene, p53, during cantharidin-induced apoptosis in U937 human leukemic cells. Cantharidin effectively activated ERK-1/2, p38 and JNK in U937 cells in a time- and dose-dependent manner. Cantharidin also exhibited a strong cytotoxicity and induced apoptosis in U937 cells. For the evaluation of the role of MAPKs, PD98059, SB202190 and SP600125 were used as MAPK inhibitors for ERK-1/2, p38 and JNK. PD98059 did not affect cantharidin-induced cytotoxicity and apoptosis, whereas SB202190 and SP600125 significantly interfered with cytotoxic and apoptotic activities induced by cantharidin. Cantharidin alone induced the apoptosis by phosphorylation of p53, up-regulation of downstream target genes, MDM2 and p21 and also cleaved caspase-3, whereas SB202190 and SP600125 caused the down-regulation of p53, MDM-2, p21 and cleaved caspase-3 after a co-treatment with cantharidin. Similarly, SB202190 and SP600125 significantly disturbed the caspase-3 activity after a co-treatment with cantharidin by colorimetric assay. Taken together, these results suggest that cantharidin can induce apoptosis by activation of p38 and JNK MAP kinase pathways associated with p53 and caspase-3.     

Oxidative stress-driven mechanisms of nordihydroguaiaretic acid-induced apoptosis in FL5.12 cells

Nordihydroguaiaretic acid (NDGA), a general lipoxygenase (LOX) enzyme inhibitor, induces apoptosis independently of its activity as a LOX inhibitor in murine pro-B lymphocytes (FL.12 cells) by a mechanism that is still not fully understood. Glutathione depletion, oxidative processes and mitochondrial depolarization appear to contribute to the apoptosis induced by NDGA. The current data demonstrate that NDGA (20 microM)-induced apoptosis in FL5.12 cells is partially protected by N-acetylcysteine (NAC) (10 mM) and dithiothreitol (DTT) (500 microM) pretreatment, confirming a role for oxidative processes. In addition, the treatment of FL5.12 cells with NDGA led to an increase in phosphorylation and activation of the MAP kinases ERK, JNK and p38. Although pretreatment with ERK inhibitors (PD98059 or U0126) abolished ERK phosphorylation in response to NDGA, neither inhibitor had any effect on NDGA-induced apoptosis. SP600125, a JNK inhibitor, did not have any effect on NDGA-induced phosphorylation of JNK nor apoptosis. Pretreatment with the p38 inhibitor SB202190 attenuated NDGA-induced apoptosis by 30% and also abolished p38 phosphorylation, compared to NDGA treatment alone. NAC, but not DTT, also decreased the phosphorylation of p38 and JNK supporting a role for oxidative processes in activating these kinases. Neither NAC nor DTT blocked the phosphorylation of ERK suggesting that this activation is not related to oxidative stress. The release of cytochrome c and activation of caspase-3 induced by NDGA were inhibited by NAC. SB202190 slightly attenuated caspase-3 activation and had no effect on the release of cytochrome c. These data suggest that several independent mechanisms, including oxidative reactions, activation of p38 kinase and cytochrome c release contribute to NDGA-induced apoptosis.     

Desipramine-induced apoptosis in human PC3 prostate cancer cells: activation of JNK kinase and caspase-3 pathways and a protective role of [Ca2+]i elevation

The antidepressant desipramine has been shown to induce a rise in cytosolic Ca2+ levels ([Ca2+]i) and cytotoxicity in human PC3 prostate cancer cells, but the mechanisms underlying its cytotoxic effect is unclear. Cell viability was examined by WST-1 assays. Apoptosis was assessed by propidium iodide staining and an increase in caspase-3 activation. Phosphorylation of protein kinases was analyzed by immunoblotting. Desipramine caused cell death via apoptosis in a concentration-dependent manner. Immunoblotting data revealed that desipramine activated the phosphorylation of c-Jun NH2-terminal kinase (JNK), but not extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). SP600125 (a selective JNK inhibitor) partially prevented cells from apoptosis. Pretreatment with BAPTA/AM, a Ca2+ chelator, to prevent desipramine-induced [Ca2+]i rises worsened desipramine-induced cytotoxicity. Immunoblotting data suggest that BAPTA/AM pretreatment enhanced desipramine-evoked JNK phosphorylation and caspase-3 cleavage. The results suggest that in PC3 cells, desipramine caused apoptosis via inducing JNK-associated caspase-3 activation, and [Ca2+]i rises may slow down or alleviate desipramine-induced cytotoxicity.     

Genipin-induced apoptosis in hepatoma cells is mediated by reactive oxygen species/c-Jun NH2-terminal kinase-dependent activation of mitochondrial pathway

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.     

Desferrioxamine (DFX) induces apoptosis through the p38-caspase8-Bid-Bax pathway in PHA-stimulated human lymphocytes

Desferrioxamine (DFX) induces apoptosis in human lymphocytes, although the mechanism leading to cell death is unclear. Therefore, we investigated the signaling pathways implicated in DFX-induced apoptosis in lymphocytes. DFX treatment activated caspase-9, caspase-3, and caspase-8. DFX-induced apoptosis was inhibited by both z-IETD-fmk and z-DEVD-fmk. DFX treatment also enhanced caspase-8 activity, Bid cleavage, and the conformational activation of Bax. DFX treatment activated two MAPKs, p38 and JNK, and induced the phosphorylation of two proteins in the p38 pathway, MKK3 and MKK6. DFX treatment also increased the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2, indicating that DFX promotes p38 activity. In addition, the selective p38 inhibitor SB203580 suppressed DFX-induced apoptosis and caspase-8 activation, whereas the JNK inhibitor, SP600125, and the ERK inhibitor, PD98059, had no effect. Our results suggest that DFX-induced apoptosis is mediated by the p38 pathway and a caspase-8-dependent Bid-Bax pathway in human lymphocytes.     

atRA-induced apoptosis of mouse embryonic palate mesenchymal cells involves activation of MAPK pathway

Our previous studies have shown that atRA treatment resulted in cell-cycle block and growth inhibition in mouse embryonic palatal mesenchymal (MEPM). In the current study, gestation day (GD) 13 MEPM cells were used to test the hypothesis that the growth inhibition by atRA is due to apoptosis. The effects of atRA on apoptosis were assessed by performing MTT assay, Cell Death Detection ELISA and flow cytometry, respectively. Data analysis confirmed that atRA treatment induced apoptosis-like cell death, as shown by decreased cell viability and increased fragmented DNA and sub-G1 fraction. atRA-induced apoptosis was associated with upregulation of bcl-2, translocation of bax protein to the mitochondria from the cytosol, activation of caspase-3 and cytochrome c release into cytosol. atRA-induced apoptosis was abrogated by z-DEVD-fmk, a caspase-3 specific inhibitor, and z-VAD-fmk, a general caspase inhibitor, suggesting that the atRA-induced cell death of MEPM cells occurs through the cytochrome c- and caspase-3-dependent pathways. In addition, atRA treatment caused a strong and sustained activation of c-Jun N-terminal kinase (JNK) and p38 kinase (p38), as well as an early but transient activation of extracellular signal-regulated kinase (ERK). Importantly, atRA-induced DNA fragmentation and capase-3 activation were prevented by pretreatment with the JNK inhibitor (SP600125) and the p38 MAPK inhibitor (SB202190), but not by pretreatment with MEK inhibitor (U0126). From these results, we suggest that mitogen-activated protein kinase-dependent pathways is involved in the atRA-induced apoptosis of MEPM cells.     

Involvement of c-jun N-terminal kinase in G2/M arrest and caspase-mediated apoptosis induced by cardiotoxin III (Naja naja atra) in K562 leukemia cells.

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, may have a potentiality as a structural template for rational drug design in killing cancer cells. Treatment of K562 cells with 0.3 microM of CTX III resulted in G2/M phase cell cycle arrest that was associated with a marked decline in protein levels of G2/M regulatory proteins including cyclin A, cyclin B1, Cdk2 and Cdc25C. In contrast to no effect on the phosphorylation of ERK, p38 MAPK and Akt, an activation of JNK was noted when K562 cells were exposed to CTX III. CTX III-mediated G2/M phase arrest and apoptosis were reduced by treatment with the JNK-specific inhibitor SP600125, but not by ERK and p38MAPK inhibitors. Further investigation showed that the specific JNK inhibitor, SP600125, reduced the activation of caspase-3, caspase-9, and reversed the decline in the expression of cyclin B1. Taken together, our data show for the first time that JNK, but not ERK, p38MAPK or Akt signaling, plays an important role in CTX III-mediated G2/M arrest and apoptosis in K562 cancer cells.     

SNP-induced apoptosis may be mediated with caspase inhibitor by JNK signaling pathways in rabbit articular chondrocytes

NO plays an important role in cartilage destruction by inducing apoptosis of chondrocytes. Here we investigated the role of c-Jun N-terminal kinase (JNK) signal transduction pathways in the apoptosis induced by NO donor sodium nitroprusside (SNP) in rabbit articular chondrocytes. We used Annexin V-FITC/PI flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) assay to detect apoptosis rate. The expressions of p38, NF-κB p65, caspase-3 and p53 genes at protein levels were measured by Western blotting assay. RT-PCR was performed to show the mRNA expression of caspase-3, and the activity of caspase-3 was also detected. To investigate the effect of JNK-specific inhibitor SP600125, chondrocytes were pretreated with SP600125 ahead of SNP treatment. Treatment with SNP accelerated apoptosis in a concentration dependent manner, while such acceleration was reduced by SP600125 pretreatment. Moreover, we found that SP600125 significantly decreased NO-induced NF-κB, p53, caspase-3 protein expressions and caspase-3 mRNA expression, as well as intracellular caspase-3 activity (P < 0.05). Collectively, these data suggest that JNK plays an important role through stimulating NF-κB, p53 and caspase-3 activation.     

Melatonin Induces Apoptotic Cell Death via p53 in LNCaP Cells

In this study, we examined whether melatonin promotes apoptotic cell death via p53 in prostate LNCaP cells. Melatonin treatment significantly curtailed the growth of LNCaP cells in a dose- and time-dependent manner. Melatonin treatment (0 to 3 mM) induced the fragmentation of poly(ADP-ribose) polymerase (PARP) and activation of caspase-3, caspase-8, and caspase-9. Moreover, melatonin markedly activated Bax expression and decreased Bcl-2 expression in dose increments. To investigate p53 and p21 expression, LNCaP cells were treated with 0 to 3 mM melatonin. Melatonin increased the expressions of p53, p21, and p27. Treatment with mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor), confirmed that the melatonin-induced apoptosis was p21-dependent, but ERK-independent. With the co-treatment of PD98059 and melatonin, the expression of p-p53, p21, and MDM2 did not decrease. These effects were opposite to the expression of p-p53, p21, and MDM2 observed with SP600125 and SB202190 treatments. Together, these results suggest that p53-dependent induction of JNK/p38 MAPK directly participates in apoptosis induced by melatonin.     

Dracorhodin perchlorate induces apoptosis in HL-60 cells

Dracorhodin perchlorate, an anthocyanin red pigment, induces human premyelocytic leukemia HL-60 cell death through apoptotic pathway. Caspase -1, -3, -8, -9, and -10 inhibitors partially reversed the cell death induced by dracorhodin perchlorate. Caspase-3 and -8 were activated followed to the degradation of caspase-3 substrates, inhibitor of caspase-activated DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP). Dracorhodin perchlorate up-regulated the expression ratio of mitochondrial proteins, Bax/Bcl-X_L. The cell death was accompanied with phosphorylation of ERK, JNK and p38 MAPK and partially reduced by MEK inhibitor (PD98059), JNK MAPK inhibitor (SP600125) and p38 MAPK inhibitor (SB 203580). Taken together, dracorhodin perchlorate-induced apoptosis in HL-60 cells via up-regulation of Bax, activation of caspases and ERK/p38/JNK MAPKs.     

Etoposide induces pancreatic β-cells cytotoxicity via the JNK/ERK/GSK-3 signaling-mediated mitochondria-dependent apoptosis pathway

Etoposide is widely used in the treatment of the different types of tumors such as pancreatic cancer. However, etoposide also causes several unwanted side-effects in normal viable cells, including pancreatic β-cells, which are vulnerable to chemical-induced injuries, and the molecular mechanisms underlying etoposide-induced apoptosis are still unclear. Here, the results showed that in RIN-m5F cells (a β-cell-derived cell line), the number of viable cells was significantly decreased after 24 h of etoposide treatment and underwent mitochondria-dependent apoptotic signals accompanied by mitochondrial dysfunction, and increases in the population of sub-G1 hypodiploid cells and apoptotic cells, caspase-3 activity, and the activation of caspase cascades. Etoposide also increased the phosphorylation levels of glycogen synthase kinase (GSK)-3α/β in treated RIN-m5F cells. Pretreatment with LiCl, a GSK-3 inhibitor, prevented etoposide-induced mitochondria-dependent apoptosis and GSK-3 protein phosphorylation in RIN-m5F cells. Furthermore, exposure of the cells to etoposide induced the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK)1/2 but not p38-MAPK, which was suppressed by the specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059), respectively. Additionally, pretreatment with both SP600125 and PD98059 effectively suppressed etoposide-induced β-cell cytotoxicity, apoptosis, and GSK-3 protein phosphorylation; however, LiCl did not reverse JNK and ERK1/2 phosphorylation. Taken together, these results suggest that etoposide is capable of causing cytotoxicity on pancreatic β-cells by inducing apoptosis through the JNK/ERK-mediated GSK-3 downstream-triggered mitochondria-dependent signaling pathway.     

Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells

Tetrandrine, which is isolated from Chinese herb Stephania tetrandrae, possesses anti-inflammatory, immunosuppressive, and cytoprotective properties. Though it was previously shown that tetrandrine causes a G1 blockade and apoptosis in various cell types, however, the mechanism by which tetrandrine initiates apoptosis remains poorly understood. In present study, we investigated the mechanisms of apoptosis induced by tetrandrine in U937 leukemia cells. Tetrandrine inhibited U937 cell growth by inducing apoptosis. After treatment of U937 cells with tetrandrine (10microM) for 24h, alteration of cell morphology, chromatin fragmentation, cytochrome c release, and caspase activation were observed. Tetrandrine also induced early oxidative stress, which resulted in activation of JNK, but not ERK and p38 MAPK. A broad-spectrum caspase inhibitor and antioxidants significantly blocked tetrandrine-induced caspase-3 activation. However, inhibition of the JNK activity with SP600125 did not block tetrandrine-induced apoptosis. Tetrandrine-induced apoptosis of U937 cells also required activity of PKC-delta, because pretreatment with a specific PKC-delta inhibitor greatly blocked tetrandrine-induced caspase-3 activation. In addition, the apoptotic response to tetrandrine was significantly attenuated in dominant-negative PKC-delta transfected MCF-7 cells, suggesting that PKC-delta plays an important role in tetrandrine-induced apoptosis and can induce caspase activation. These results suggest that tetrandrine induces oxidative stress, JNK activation, and caspase activation. However, JNK activation by ROS is not involved in the tetrandrine-induced apoptosis. In addition, tetrandrine induces caspase-dependent generation of a catalytically active fragment of PKC-delta, and this fragment also appears to play a role in the activation of caspases.     

Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways

Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H(2)O(2)), superoxide dismutase (SOD) (scavenger of O(2)(?-)), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H(2)O(2) or O(2)(?-) showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.     

Anandamide-induced Ca2+ elevation leading to p38 MAPK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

The effect of anandamide on human osteoblasts is unclear. This study examined the effect of anandamide on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca2+ levels in MG63 osteosarcoma cells. Anandamide at 50-200 microM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that anandamide induced expression of ERK, JNK and p38 MAPK. Anandamide-induced cell death and apoptosis were reversed by SB203580, but not by PD98059 and SP600125, suggesting that anandamide's action was via p38 MAPK, but not via ERK and JNK. Anandamide at 1-100 microM induced [Ca2+]i increases. Removal of extracellular Ca2+ decreased the anandamide response, indicating that anandamide induced Ca2+ influx and Ca2+ release. Chelation of intracellular Ca2+ with BAPTA reversed anandamide-induced cell death and p38 MAPK phosphorylation. Collectively, in MG63 cells, anandamide induced [Ca2+]i increases which evoked p38 MAPK phosphorylation. This p38 MAPK phosphorylation subsequently activated caspase-3 leading to apoptosis.     

Nanoparticle realgar powders induce apoptosis in u937 cells through caspase mapk and mitochondrial pathways

Nanoparticle realgar powders (NRP) inhibited U937 cell growth in a time and dose-dependent manner. U937 cells treated with NRP showed typical characteristics of apoptosis including the morphological changes and DNA fragmentation. Caspase family inhibitor (z-VAD-fmk), caspase-8, -9 inhibitor (z-IETD-fmk, Ac-LEHD-CHO, respectively) and caspase-3 inhibitor (z-DEVD-fmk) partially prevented NRP -induced apoptosis. Moreover, the classical substrates of caspase-3, poly-ADP ribose polymerase (PARP) was degraded after U937 cells treatment with NRP. In addition, NRP increased the ratio of Bax/Bcl-2 protein expression. Although p38 inhibitor (SB203580) and ERK inhibitor (PD98059) failed to block cell death, JNK inhibitor (SP600125) had marked inhibitory effects on NRP -induced apoptosis. Furthermore, the phosphorylation of JNK was up-regulated, suggesting that JNK was responsible for NRP -induced apoptosis in U937 cells. These results suggested that the caspase, mitochondria and MAPK signal pathways were involved in NRP-induced U937 apoptosis.     

Mycotoxin patulin activates the p38 kinase and JNK signaling pathways in human embryonic kidney cells.

Patulin (PAT), a mycotoxin mainly produced by Penicillium and Aspergillus, is frequently detected in moldy fruits and fruit products. Exposure of human embryonic kidney (HEK293) cells to PAT led to a dose- and time-dependent increase in the phosphorylation of two major mitogen-activated protein kinases (MAPKs), p38 kinase and c-Jun N-terminal kinase (JNK). The phosphorylated forms of MAPK kinase 4 (MKK4), c-Jun, and ATF-2 were also seen in PAT-treated cultures. The cell death caused by PAT was significantly reduced by the p38 kinase inhibitor, SB203580, but not by the JNK inhibitor, SP600125. Neither p38 kinase nor JNK played a role in the PAT-induced DNA damage. In PAT-treated cells, inactivation of double-stranded RNA-activated protein kinase R (PKR) by the inhibitor, adenine, markedly suppressed JNK and ERK phosphorylation. Treatment of HEK293 cells with PAT-cysteine adduct, a chemical derivative of PAT, showed no effect on MAPK signaling pathways, cell viability, or DNA integrity. These results indicate that PAT causes rapid activation of p38 kinase and JNK in HEK293 cells, but only the p38 kinase signaling pathway contributes to the PAT-induced cell death. PKR also plays a role in PAT-mediated MAPK activation.     

Involvement of intracellular oxidative stress-sensitive pathway in phloxine B-induced photocytotoxicity in human T lymphocytic leukemia cells

We investigated the molecular mechanisms underlying phloxine B (PhB)-induced photocytotoxicity in human T lymphocytic leukemia Jurkat cells. In addition to apoptosis-related biochemical events, photo-irradiated PhB generated intracellular reactive oxygen species (ROS), induced phosphorylation of c-Jun-N-terminal kinase (JNK) in an oxidative stress-dependent manner and up-regulated the gene expression of interferon (IFN)-γ, an inducer of diverse apoptosis-related molecules in activated T cells. PhB-induced apoptosis was significantly inhibited by N-acetyl-l-cysteine, but not by catalase, indicating that ROS generation occurred intracellularly, and by SP600125 and AG490, specific inhibitors of JNK and IFN-γ signaling, respectively, confirming their roles in the apoptotic pathway. IFN-γ up-regulation was also inhibited by SP600125, indicating that it was downstream of JNK activation. These results suggest that PhB-induced apoptosis in Jurkat cells partially involves the intracellular oxidative stress-sensitive and T cell-specific IFN-γ pathway. These data present a novel insight into the mechanisms of photocytotoxicity induced by artificial food colorants in human T lymphocytic leukemia cells.     

Acetoxycycloheximide (E-73) rapidly induces apoptosis mediated by the release of cytochrome c via activation of c-Jun N-terminal kinase

Cycloheximide (CHX) is an inhibitor of protein synthesis and commonly used to modulate death receptor-mediated apoptosis or to induce apoptosis in a number of normal and transformed cells. In this study we show that a close structural derivative of CHX, acetoxycycloheximide (E-73) induced rapid processing of procaspases and subsequent apoptosis with much higher efficacy than CHX in human leukemia Jurkat T cells. E-73 induced the release of cytochrome c from mitochondria even in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone. The Bcl-2 family protein Bcl-x(L) suppressed cytochrome c release as well as processing of procaspases-3, -8, and -9 in E-73-treated cells. In Jurkat T cells transfected with the caspase-8 modulator FLIP(L), E-73 still induced activation of procaspase-3 and subsequent apoptosis, suggesting that the caspase-8 activity is dispensable for apoptosis. In contrast to CHX, E-73 drastically induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 MAP kinase. Inhibitory profiles of small-molecular kinase inhibitors revealed that JNK activation was critical for induction of cytochrome c release in E-73-induced apoptosis. Thus, our present results demonstrate that E-73, unlike CHX, induces strong activation of the JNK pathway and triggers rapid apoptosis mediated by the release of cytochrome c.