DDTD, an isoflavone derivative, induces cell apoptosis through the reactive oxygen species/apoptosis signal-regulating kinase 1 pathway in human osteosarcoma cells

Osteosarcoma is the most common primary bone tumor associated with childhood and adolescence. In the present study, we investigated the anticancer effect of a new isoflavone derivative, 3',4'-dichloro-3-(3,4-dichlorophenylacetyl)-2,4,6-trihydroxydeoxybenzoin (DDTD) in human osteosarcoma cells. DDTD induced cell apoptosis in human osteosarcoma cell lines (including: U2OS, MG-63, Saos2 and ROS 17/2.8). We found that the accumulation of reactive oxygen species is a critical mediator in DDTD-induced cell death. DDTD induced apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation and its dissociation from 14-3-3. Treatment of osteosarcoma cells with DDTD induced p38 and p53 phosphorylation. Transfection with ASK1, mitogen activated protein kinase (MAPK) kinase (MKK)3/6, and p38 small interfering RNA (siRNA) antagonized the DDTD-induced cell apoptosis. DDTD also triggered the mitochondrial apoptotic pathway, as indicated by a change in Bax/Bcl2 ratio and Caspase-9 activation. Bax knockdown using a Bax siRNA strategy reduced Bax expression and subsequent cell death. In addition, transfection of cells with ASK1, MKK3/6, and p38 siRNA reduced DDTD-induced p38 activation, p53 phosphorylation and Bax expression. These results suggest that DDTD generates reactive oxygen species and activates the ASK1-MKK3/6-p38-p53-Bax pathway to cause osteosarcoma cell death.     

The upstream regulation of p38 mitogen-activated protein kinase phosphorylation by arachidonic acid in rat neutrophils

The signal transduction pathways activated by arachidonic acid that lead to p38 mitogen‐activated protein kinase (MAPK) activation in neutrophils remains unclear. In this study, selective inhibitors of several signalling pathways were utilized to investigate the mechanisms of activation of p38 MAPK by arachidonic acid in rat neutrophils. Stimulation of p38 MAPK phosphorylation by arachidonic acid and its trifluoromethyl ketone analogue AACOCF₃ was transient, peaking at 1 min, and was concentration‐dependent. Arachidonic acid‐stimulated p38 MAPK phosphorylation was attenuated in cells pretreated with the G_i/o inhibitor (pertussis toxin), but not with the dual cyclooxygenase/lipoxygenase inhibitor (BW755C) or the leukotriene biosynthesis inhibitor (MK886). Tyrosine kinase inhibitor (genistein), but not the extracellular signal‐regulated kinase kinase inhibitors (PD98059 and U0126), attenuated the phosphorylation of p38 MAPK by arachidonic acid. Phosphoinositide 3‐kinase inhibitors (wortmannin and LY294002) did not affect the arachidonic acid‐induced response. After pretreatment of the cells with protein kinase C inhibitors (Gö6976, Gö6983 and GF109203X), only Gö6976 significantly attenuated the phosphorylation of p38 MAPK by arachidonic acid. In addition, phosphorylation of p38 MAPK by arachidonic acid was greatly attenuated by the phospholipase C inhibitor (U73122) and the Ca²⁺ chelator BAPTA ((1,2‐bis‐o‐amino‐phenoxy)‐ethane‐N,N,N′,N′‐tetraacetic acid), but not altered by the nitric oxide synthase inhibitor, N‐nitro‐l ‐arginine methyl ester. Arachidonic acid did not cause an increase in cellular cyclic GMP level. This study revealed the involvement of pertussis toxin‐sensitive G protein, non‐receptor tyrosine kinase, phospholipase C/Ca²⁺, and probably Ca²⁺‐dependent protein kinase C in arachidonic acid‐stimulated p38 MAPK activation.     

Apoptosis induced by para‐phenylenediamine involves formation of ROS and activation of p38 and JNK in chang liver cells

para‐phenylenediamine (p‐PD) is a suspected carcinogen, but it has been widely used as a component in permanent hair dyes. In this study, the mechanism of p‐PD‐induced cell death in normal Chang liver cells was investigated. The results demonstrated that p‐PD decreased cell viability in a dose‐dependent manner. Cell death via apoptosis was confirmed by enhanced DNA damage and increased cell number in the sub‐G1 phase of the cell cycle, using Hoechst 33258 dye staining and flow cytometry analysis. Apoptosis via reactive oxygen species generation was detected by the dichlorofluorescin diacetate staining method. Mitogen‐activated protein kinase (MAPK) activation was assessed by western blot analysis and revealed that p‐PD activated not only stress‐activated protein kinase (SAPK)/c‐Jun N‐terminal kinases (JNK) and p38 MAPK but also extracellular signal‐regulated kinase (ERK). Cytotoxicity and apoptosis induced by p‐PD were markedly enhanced by ERK activation and selectively inhibited by ERK inhibitor PD98059, thus indicating a negative role of ERK. In contrast, inhibition of p38 MAPK activity with the p38‐specific inhibitor SB203580 moderately inhibited cytotoxicity and apoptosis induction by p‐PD. Similarly, SP600125, an inhibitor of SAPK/JNK, moderately inhibited cytotoxicity and apoptosis induced by p‐PD, thus implying that p38 MAPK and SAPK/JNK had a partial role in p‐PD‐induced apoptosis. Western blot analysis revealed that p‐PD significantly increased phosphorylation of p38 and SAPK/JNK and decreased phosphorylation of ERK. In conclusion, the results demonstrated that SAPK/JNK and p38 cooperatively participate in apoptosis induced by p‐PD and that a decreased ERK signal contributes to growth inhibition or apoptosis. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 981–990, 2014.     

Ceramide induces apoptosis in human lung adenocarcinoma A549 cells through mitogen-activated protein kinases

AIM: To provide experimental data for further research on the signal transduction of apoptosis in lung adenocarcinoma cells, we examined the effects of exogenous C2-ceramide administration on several members of the mitogen-activated protein kinase (MAPK) superfamily and caspase-3 in A549 cells. METHODS: Cell viability and apoptosis were analyzed by cell counting kit-8 assay and flow cytometry. Various MAPK and caspase-3 proteins were detected by Western blotting. RESULTS: C2-ceramide selectively altered the phosphorylation state of members of the MAPK superfamily, causing hyperphosphorylation of mitogen-activated protein kinase kinase (MEK)1/2 and the p38 MAPK, but not affecting the phosphorylation of extracellular signal-regulated kinase 1/2 and the c-Jun N-terminal kinase. SB-203580 (a p38 MAPK inhibitor) and p38 siRNA, but not U0126 (a MEK inhibitor), partially rescued cell death induced by C2-ceramide. C2-ceramide promoted the activation of caspase-3. CONCLUSION: Exogenous C2-ceramide induced apoptosis in human lung adenocarcinoma A549 cells. The activation of MAPK and caspase-3 were involved in the mechanisms of C2-ceramide-induced apoptosis in A549 cells.     

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.     

Berberine induces apoptosis in SW620 human colonic carcinoma cells through generation of reactive oxygen species and activation of JNK/p38 MAPK and FasL

Berberine is the major constituent of Coptidis Rhizoma with multiple pharmacological activities, including anti-inflammation, promotion of apoptosis and anticancer potential effect. Mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) may contribute to the causal relationship between tumorigenesis and pro-apoptotic function. Berberine is studied for the mechanism of its action in apoptotic pathway in human colonic carcinoma cell. Treatment of SW620 cells with 50 μM berberine resulted in activation of the caspase 3 and caspase 8, cleavage of poly ADP-ribose polymerase (PARP) and the release of cytochrome c; whereas, the expression of BID and anti-apoptosis factor c-IAP1, Bcl-2, and Bcl-_XL were decreased markedly. Berberine-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of JNK and p38 MAPK, as well as generation of the ROS. Furthermore, the induction of apoptosis was alleviated by inhibitors specific for JNK and p38. In addition, there was an increase in the cellular levels of phospho-c-Jun, FasL and t-BID in the berberine-induced apoptosis via the activation of JNK and p38 signaling modules. NAC administration, a scavenger of ROS, reversed berberine-induced apoptosis effects via inhibition of JNK, p38 and c-jun activation, and FasL and t-BID expression. These results leads us to speculate that berberine may play an apoptotic cascade in SW620 cells by activation of the JNK/p38 pathway and induction of ROS production, providing a new mechanism for berberine-induced cell death in human colon cancer cells.     

Thimerosal-induced apoptosis in human SCM1 gastric cancer cells: activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca2+]i elevation.

Thimerosal is a mercury-containing preservative in some vaccines. The effect of thimerosal on human gastric cancer cells is unknown. This study shows that in cultured human gastric cancer cells (SCM1), thimerosal reduced cell viability in a concentration- and time-dependent manner. Thimerosal caused apoptosis as assessed by propidium iodide-stained cells and caspase-3 activation. Although immunoblotting data revealed that thimerosal could activate the phosphorylation of extracellular signal-regulated kinase, c-Jun NH2-terminal protein kinase, and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Thimerosal also induced [Ca2+](i) increases via Ca2+ influx from the extracellular space. However, pretreatment with (bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate)/AM, a Ca2+ chelator, to prevent thimerosal-induced [Ca2+](i) increases did not protect cells from death. The results suggest that in SCM1 cells, thimerosal caused Ca2+-independent apoptosis via phosphorylating p38 MAPK resulting in caspase-3 activation.     

Involvement of the extracellular signal-regulated protein kinase (ERK) pathway in the induction of apoptosis by cadmium chloride in CCRF-CEM cells

When CCRF-CEM cells were incubated with 5–40 μM CdCl_2, apoptosis was observed most clearly at 10 μM. Prior to the development of apoptosis, mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, were activated with different sensitivity to CdCl₂ exposure. ERK and p38 MAPK were phosphorylated with incubation of 1 μM CdCl_2, but higher than 20 μM CdCl₂ was required for the clear phosphorylation of JNK. In the time–course study, ERK and p38 MAPK were phosphorylated earlier than JNK after CdCl₂ exposure. The in vitro activities of MAPKs also increased in response to CdCl₂ exposure. Pretreatment with an intracellular Ca²⁺ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), suppressed almost completely CdCl₂-induced phosphorylation of JNK and p38 MAPK, but not ERK phosphorylation, indicating that the activation of JNK and p38 MAPK depends on the intracellular Ca²⁺ but that of ERK does not. On the other hand, treatment with a MAPK/ERK kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), suppressed CdCl₂-induced ERK activation and the apoptosis as well. The inhibition of p38 MAPK activity with SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]1H-imidazole) did not protect cells from apoptosis. The present results showed that the activation of ERK, JNK, and p38 MAPK is differently regulated in CCRF-CEM cells exposed to CdCl_2, and that the ERK pathway seems to be responsible for the induction of apoptosis by CdCl₂ exposure in this human T cell line.     

Desipramine-induced Ca-independent apoptosis in Mg63 human osteosarcoma cells: dependence on P38 mitogen-activated protein kinase-regulated activation of caspase 3

1. It has been shown that the antidepressant desipramine is able to induce increases in [Ca(2+)](i) and cell death in MG63 human osteosacroma cells, but whether apoptosis is involved is unclear. In the present study, the effect of desipramine on apoptosis and the underlying mechanisms were explored. It was demonstrated that desipramine induced cell death in a concentration- and time-dependent manner. 2. Cells treated with 100-800 mmol/L desipramine showed typical apoptotic features, including an increase in sub-diploid nuclei and activation of caspase 3, indicating that these cells underwent apoptosis. Immunoblotting revealed that 100 mmol/L desipramine activated extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Although pretreatment of cells with 20 mmol/L PD98059 (an ERK inhibitor) or 20 mmol/L SP600125 (an inhibitor of JNK) did not inhibit cell death, the addition of 20 mmol/L SB203580 (a p38 MAPK inhibitor) partially rescued cells from apoptosis. Desipramine-induced caspase 3 activation required p38 MAPK activation. 3. Pretreatment of cells with BAPTA/AM (20 mmol/L) to prevent desipramine-induced increases in [Ca(2+)](i) did not protect cells from death. 4. The results of the present study suggest that, in MG63 human osteosarcoma cells, desipramine causes Ca(2+)-independent apoptosis by inducing p38 MAPK-associated activation of caspase 3.     

Sequence-dependent potentiation of paclitaxel-mediated apoptosis in human leukemia cells by inhibitors of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase pathway.

Effects of inhibitors of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase (MEK/MAPK) cascade have been examined in relation to paclitaxel-induced apoptosis in human monocytic leukemia cells (U937). Cells treated with paclitaxel (250 nm; 6 h) followed by PD98059 [corrected] exhibited a significant increase in mitochondrial dysfunction (e.g., cytochrome c release), caspase activation, poly ADP-ribose polymerase cleavage, and apoptosis, whereas pretreatment of cells with PD98059 reduced lethality. Similar results were obtained with other MEK/MAPK inhibitors (e.g., U0126 and PD184352). Subsequent exposure of paclitaxel-treated cells to PD98059 did not enhance dephosphorylation/activation of p34(cdc2) but diminished expression of the antiapoptotic protein Mcl-1. The caspase inhibitor ZVAD-fmk opposed potentiation of paclitaxel-induced loss of mitochondrial membrane potential (Deltapsi(m)) and apoptosis by PD98059, but not cytochrome c release. Paclitaxel treatment induced sustained phosphorylation/activation of MAPK, an effect prevented by subsequent, but not prior, exposure to PD98059. Paclitaxel treatment also induced c-Jun N-terminal kinase phosphorylation, but this effect was enhanced only slightly by subsequent PD98059 administration. Although paclitaxel alone failed to induce p38 MAPK activation, subsequent (but not prior) exposure to PD98059 induced a dramatic increase in p38 MAPK phosphorylation. Moreover, coadministration of the p38 MAPK inhibitors SB203580 and SB202190 abrogated the increase in paclitaxel-mediated apoptosis induced by PD98059. Finally, subsequent PD98059 exposure increased, whereas prior exposure decreased inhibition of clonogenicity by paclitaxel. Together, these findings suggest that subsequent exposure of paclitaxel-treated U937 cells to MEK/MAPK inhibitors induces perturbations in signaling pathways, particularly the p42/44 MAPK and p38 MAPK cascades, that lower the threshold for mitochondrial injury and induction of cell death.     

Nonylphenol‐induced apoptotic pathways in SCM1 human gastric cancer cells

Environmental chemicals may affect human health by disrupting endocrine function. Many endocrine disrupting chemicals (EDCs) are estrogen‐like molecules that are classified as xenoestrogens (XEs). One XE, nonylphenol, is used as a surfactant or plasticizer and exhibits biotoxicity when accumulated in the body via the food chain. The aim of the present study was to clarify the role of nonylphenol‐induced SCM1 apoptosis by measuring cultured human gastric cancer cell (SCM1) death. Using WST‐1 reduction and propidium iodide‐staining assays, nonylphenol treatment was found to activate caspase‐3 and mitogen‐activated protein kinases (MAPKs), major markers in apoptotic pathways. Nonylphenol also activated the phosphorylation of extracellular signal‐regulated kinase (ERK), c‐Jun NH_2‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (p38 MAPK). However, only SB203580 (a p38MAPK inhibitor) partially inhibited nonylphenol‐induced apoptosis. Nonylphenol induced a [Ca²⁺]_i rise by causing extracellular Ca²⁺ influx and intracellular Ca²⁺ release from the endoplasmic reticulum, and its effects on SCM1 cell death were prevented by pretreatment with the Ca²⁺ chelator BAPTA/AM. These results suggest that nonylphenol caused Ca²⁺‐dependent apoptosis via the activation of p38 MAPK‐associated caspase‐3 in SCM1 cells. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.     

The role of p38 MAPK activation in auranofin-induced apoptosis of human promyelocytic leukaemia HL-60 cells

In a previous study, we reported an antileukaemic activity of auranofin (AF), demonstrating its dual effects: on the induction of apoptotic cell death and its synergistic action with retinoic acid on cell differentiation. In this study, we investigated the downstream signalling events of AF-induced apoptosis to determine the molecular mechanisms of AF activity. Treatment of HL-60 cells with AF induced apoptosis in a concentration- and time-dependent manner. Western blot analysis showed that AF-induced apoptosis was accompanied by the activation of caspase-8, caspase-9, and caspase-3, and the release of cytochrome c from the mitochondria. The phosphorylation and kinase activities of p38 mitogen-activated protein kinase (p38 MAPK) increased gradually until 12 h after AF (2 microM) treatment, and p38 MAPK was also activated concentration-dependently. Pretreatment with SB203580, a specific inhibitor of p38 MAPK, significantly blocked DNA fragmentation and the cleavage of procaspase-8, procaspase-3, and poly-ADP-ribose polymerase (PARP), whereas SB203580 alone had no effect. Reactive oxygen species (ROS) were also detected within 1 h after AF treatment, and the antioxidant N-acetyl-L-cysteine (NAC) effectively protected the cells from apoptosis by inhibiting the phosphorylation of p38 MAPK and the activation of caspases. These results suggest that ROS generation and the subsequent activation of p38 MAPK are essential for the proapoptotic effects of AF in human promyelocytic leukaemia HL-60 cells.     

p38 MAP激酶在山冈橐吾碱肝细胞毒性中的作用

目的探讨丝裂原活化蛋白激酶p38 (p38MAP激酶)在山冈橐吾碱肝细胞毒性中的作用。方法采用western杂交方法观察山冈橐吾碱(100μmol·L~(-1),分别作用1,5,15,30及60 min)对p38MAP激酶激活的影响;10μmol·L~(-1) p38MAP激酶抑制剂SKF86002预处理15 min后,分别观察其对山冈橐吾碱(100μmol·L~(-1))诱导p38MAP激酶磷酸化(30 min,western杂交法)及细胞毒性(MTT法,36及48 h;台盼蓝染色法,36 h)的影响。结果western blot结果显示,100μmol·L~(-1)山冈橐吾碱明显诱导p38 MAP激酶的磷酸化激活,5～30 min时处于较高水平;SKF86002预处理可以明显抑制山冈橐吾碱诱导的p38 MAP激酶磷酸化;MTT染色法和台盼蓝染色实验均发现SKF86002预处理能部分降低山冈橐吾碱诱导的肝细胞毒性[MTT,36 h:(0.210±0.008) vs (0.170±0.003),48 h:(0.33±0.03) vs (0.200±0.003);台盼蓝染色(80±2)% vs (72±7)%;n=8,P<0.05]。结论p38MAP激酶可能参与了山冈橐吾碱诱导的肝细胞毒性作用。     

Bufalin‐inhibited migration and invasion in human osteosarcoma U‐2 OS cells is carried out by suppression of the matrix metalloproteinase‐2, ERK,and JNK signaling pathways

Bufalin has been shown to exhibit multiple pharmacological activities, including induction of apoptosis in many types of cancer cell lines. Osteosarcoma is a type of cancer which is difficult to treat and the purpose of this study was to investigate the effects of bufalin on the migration and invasion of human osteosarcoma U‐2 OS cells. The wound healing assay and Boyden chamber transwell assay were used for examining the migration of U‐2 OS cells. Western blotting and gelatin zymography assays were used for theexpression and activities of metalloproteinase (MMP)‐2, MMP‐7 or MMP‐9 levels. Western blotting analysis also was used for measuring the levels of growth factor receptor‐bound protein 2 (GRB2), son of sevenless homolog 1 (SOS1), c‐Jun N‐terminal kinases 1/2 (JNK1/2), extracellular signal‐regulated kinase 1/2 (ERK1/2), and p38 in bufalin‐treated U‐2 OS cells. Bufalin inhibited the cell migration and invasion of U‐2 OS cells in vitro. Moreover, bufalin reduced MMP‐2 and MMP‐9 enzyme activities of U‐2 OS cells. Bufalin also suppressed the protein level of MMP‐2 and reduced the levels of mitogen‐activated protein kinases (MAPKs) such as JNK1/2 and ERK1/2 signals in U‐2 OS cells. Our results suggest that signaling pathways for bufalin‐inhibited migration and invasion of U‐2 OS cells might be mediated through blocking MAPK signaling and resulting in the inhibition of MMP‐2. Bufalin could be a useful agent to develop as a novel antitumor agent by virtue of its ability to inhibit tumor cell migration and invasion. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 21–29, 2014.     

Naringin Inhibits ROS‐activated MAPK Pathway in High Glucose‐induced Injuries in H9c2 Cardiac Cells

Naringin, an active flavonoid isolated from citrus fruit extracts, exhibits biological and pharmacological properties, such as antioxidant activity and antidiabetic effect. Mitogen‐activated protein kinase (MAPK) signalling pathway has been shown to participate in hyperglycaemia‐induced injury. The present study tested the hypothesis that naringin protects against high glucose (HG)‐induced injuries by inhibiting MAPK pathway in H9c2 cardiac cells. To examine this, the cells were treated with 35 mM glucose (HG) for 24 hr to establish a HG‐induced cardiomyocyte injury model. The cells were pre‐treated with 80 μM naringin for 2 hr before exposure to HG. The findings of this study showed that exposure of H9c2 cells to HG for 24 hr markedly induced injuries, as evidenced by a decrease in cell viability, increases in apoptotic cells and reactive oxygen species (ROS) production, as well as dissipation of mitochondrial membrance potential (MMP). These injuries were significantly attenuated by the pre‐treatment of cells with either naringin or SB203580 (a selective inhibitor of p38 MAPK) or U0126 (a selective inhibitor of extracellular signal regulated kinase 1/2, ERK1/2) or SP600125 (a selective inhibitor of c‐jun N‐termanal kinase, JNK) before exposure to HG, respectively. Furthermore, exposure of cells to HG increased the phosphorylation of p38 MAPK, ERK1/2 and JNK. The increased activation of MAPK pathway was ameliorated by pre‐treatment with either naringin or N‐acetyl‐L‐cysteine (NAC), a ROS scavenger, which also reduced HG‐induced cytotoxicity and apoptosis, leading to increase in cell viability and decrease in apoptotic cells. In conclusion, our findings provide new evidence for the first time that naringin protects against HG‐induced injuries by inhibiting the activation of MAPK (p38 MAPK, ERK1/2 and JNK) and oxidative stress in H9c2 cells.     

Participation of various kinases in staurosporine induced apoptosis of RAW 264.7 cells

Staurosporine induced apoptosis of RAW 264.7 cells, a mouse macrophage-like cell line, as determined by DNA fragmentation, the increase of annexin V-stained cells, and the cleavage of poly(ADP-ribose)polymerase (PARP), a substrate of caspase. Analysis of the increase in the percentage of sub-G(1) cells revealed that the DNA fragmentation occurred in a time- and concentration-dependent manner at 0.021-2.1 microM of staurosporine. Staurosporine induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) but suppressed spontaneous phosphorylation of p44/42 MAPK. The p38 MAPK inhibitor SB203580, the MAPK/extracellular signal-regulated kinase kinase inhibitor PD98059 and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 potentiated the staurosporine-induced PARP cleavage and DNA fragmentation. The protein kinase A (PKA) inhibitor H-89 potentiated the staurosporine-induced DNA fragmentation without potentiating the PARP cleavage. In contrast, the protein kinase C (PKC) inhibitor Ro-31-8425 suppressed the PARP cleavage and DNA fragmentation. These findings suggested that staurosporine induces apoptosis via the caspase cascade in RAW 264.7 cells. The staurosporine-induced apoptosis is positively regulated by PKC, negatively regulated by p38 MAPK, p44/42 MAPK and PI3K via the caspase cascade, and negatively regulated by PKA without regulation of caspase activation.     

Involvement of reactive oxygen species and stress-activated MAPKs in satratoxin H-induced apoptosis

Satratoxins, members of the trichothecene mycotoxin family, have been known to be harmful to health. However, the mechanisms underlying the toxicity still remain unclear. The present study is undertaken to elucidate the mechanisms of the satratoxin H-induced cytotoxicity in PC12 cells. Satratoxin H caused cytotoxicity, which was reflected from apoptosis determined by chromatin staining and flow cytometry. Satratoxin H stimulated the phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Pre-incubation with SB203580, a p38 MAPK inhibitor, or SP600125, a JNK inhibitor, but not PD98059, an ERK inhibitor, reduced satratoxin-induced cytotoxicity. Co-incubation of cells with glutathione, N-acetyl-L-cysteine or glutathione reductase inhibited cytotoxicity and the phosphorylation of p38 MAPK induced by satratoxin H. Our data suggest that satratoxin H-induced apoptosis in PC12 cells is dependent on the activation of p38 MAPK/JNK and the increase in reactive oxygen species.