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.     

ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.     

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.     

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.     

Calcium‐stimulated mitogen‐activated protein kinase activation elicits Bcl‐xL downregulation and Bak upregulation in notexin‐treated human neuroblastoma SK‐N‐SH cells

Notechis scutatus scutatus notexin induced apoptotic death of SK‐N‐SH cells accompanied with downregulation of Bcl‐xL, upregulation of Bak, mitochondrial depolarization, and ROS generation. Upon exposure to notexin, Ca²⁺‐mediated JNK and p38 MAPK activation were observed in SK‐N‐SH cells. Production of ROS was a downstream event followed by Ca²⁺‐mediated mitochondrial alteration. Notexin‐induced cell death, mitochondrial depolarization, and ROS generation were suppressed by SB202190 (p38 MAPK inhibitor) and SP600125 (JNK inhibitor). Moreover, phospho‐p38 MAPK and phospho‐JNK were proved to be involved in Bcl‐xL degradation, and overexpression of Bcl‐xL attenuated the cytotoxic effect of notexin. Bak upregulation was elicited by p38 MAPK‐mediated ATF‐2 activation and JNK‐mediated c‐Jun activation. Suppression of Bak upregulation by ATF‐2 siRNA or c‐Jun siRNA attenuated notexin‐evoked mitochondrial depolarization and rescued viability of notexin‐treated cells. Taken together, our data indicate that notexin‐induced apoptotic death of SK‐N‐SH cells is mediated through mitochondrial alteration triggering by Ca²⁺‐evoked p38 MAPK/ATF‐2 and JNK/c‐Jun signaling pathways. J. Cell. Physiol. 222:177–186, 2010. © 2009 Wiley‐Liss, Inc.     

Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes

The p38 MAPK pathway controls critical premitochondrial events culminating in apoptosis of UVB-irradiated human keratinocytes, but the upstream mediators of this stress signal are not completely defined. This study shows that in human keratinocytes exposed to UVB the generation of reactive oxygen species (ROS) acts as a mediator of apoptosis signal regulating kinase-1 (Ask-1), a redox-sensitive mitogen-activated protein kinase kinase kinase (MAP3K) regulating p38 MAPK and JNK cascades. The NADPH oxidase antagonist diphenylene iodonium chloride and the EGFR inhibitor AG1487 prevent UVB-mediated ROS generation, the activation of the Ask-1-p38 MAPK stress response pathway, and apoptosis, evidencing the link existing between the early plasma membrane-generated ROS and the activation of a lethal cascade initiated by Ask-1. Consistent with this, Ask-1 overexpression considerably sensitizes keratinocytes to UVB-induced mitochondrial apoptosis. Although the JNK pathway is also stimulated after UVB, the killing effect of Ask-1 overexpression is reverted by p38 MAPK inhibition, suggesting that Ask-1 exerts its lethal effects mainly through the p38 MAPK pathway. Moreover, p38alpha(-/-) murine embryonic fibroblasts are protected from UVB-induced apoptosis even if JNK activation is fully preserved. These results argue for an important role of the UVB-generated ROS as mediators of the Ask-1-p38 MAPK pathway that, by culminating in apoptosis, restrains the propagation of potentially mutagenic keratinocytes.     

Taiwan cobra phospholipase A2‐elicited JNK activation is responsible for autocrine fas‐mediated cell death and modulating Bcl‐2 and Bax protein expression in human leukemia K562 cells

Phospholipase A₂ (PLA₂) from Naja naja atra venom induced apoptotic death of human leukemia K562 cells. Degradation of procaspases, production of tBid, loss of mitochondrial membrane potential, Bcl‐2 degradation, mitochondrial translocation of Bax, and cytochrome c release were observed in PLA₂‐treated cells. Moreover, PLA₂ treatment increased Fas and FasL protein expression. Upon exposure to PLA₂, activation of p38 MAPK (mitogen‐activated protein kinase) and JNK (c‐Jun NH₂‐terminal kinase) was found in K562 cells. SB202190 (p38 MAPK inhibitor) pretreatment enhanced cytotoxic effect of PLA₂ and led to prolonged JNK activation, but failed to affect PLA₂‐induced upregulation of Fas and FasL protein expression. Sustained JNK activation aggravated caspase8/mitochondria‐dependent death pathway, downregulated Bcl‐2 expression and increased mitochondrial translocation of Bax. SP600125 (JNK inhibitor) abolished the cytotoxic effect of PLA₂ and PLA₂‐induced autocrine Fas death pathway. Transfection ASK1 siRNA and overexpression of dominant negative p38α MAPK proved that ASK1 pathway was responsible for PLA₂‐induced p38 MAPK and JNK activation and p38α MAPK activation suppressed dynamically persistent JNK activation. Downregulation of FADD abolished PLA₂‐induced procaspase‐8 degradation and rescued viability of PLA₂‐treated cells. Taken together, our results indicate that JNK‐mediated autocrine Fas/FasL apoptotic mechanism and modulation of Bcl‐2 family proteins are involved in PLA₂‐induced death of K562 cells. J. Cell. Biochem. 109: 245–254, 2010. © 2009 Wiley‐Liss, Inc.     

Induction of apoptosis by shikonin through a ROS/JNK-mediated process in Bcr/Abl-positive chronic myelogenous leukemia （CML） cells

This study examined the signaling events induced by shikonin that lead to the induction of apoptosis in Bcr/ Abl-positive chronic myelogenous leukemia （CML） cells （e.g., K562, LAMA84）. Treatment of K562 cells with shikonin （e.g., 0.5 pM） resulted in profound induction of apoptosis accompanied by rapid generation of reactive oxygen species （ROS）, striking activation of c-Jun-N-terminal kinase （JNK） and p38, marked release of the mitochondrial proteins cytochrome c and Smac/DIABLO, activation of caspase-9 and -3, and cleavage of PARP. Scavenging of ROS completely blocked all of the above-mentioned events （i.e., JNK and p38 phosphorylation, cytochrome c and Smac/DIABLO release, caspase and PARP cleavage, as well as the induction of apoptosis） following shikonin treatment. Inhibition of JNK and knock-down of JNK1 significantly attenuated cytochrome c release, caspase cleavage and apoptosis, but did not affect shikonin-mediated ROS production. Additionally, inhibition of caspase activation completely blocked shikonin-induced apoptosis, but did not appreciably modify shikonin-mediated cytochrome c release or ROS generation. Altogether, these findings demonstrate that shikonin-induced oxidative injury operates at a proximal point in apoptotic signaling cascades, and subsequently activates the stress-related JNK pathway, triggers mitochondrial dysfunction, cytochrome c release, and caspase activation, and leads to apoptosis. Our data also suggest that shikonin may be a promising agent for the treatment of CML, as a generator of ROS.     

Benzyl isothiocyanate targets mitochondrial respiratory chain to trigger reactive oxygen species-dependent apoptosis in human breast cancer cells

Benzyl isothiocyanate (BITC), a dietary cancer chemopreventive agent, causes apoptosis in MDA-MB-231 and MCF-7 human breast cancer cells, but the mechanism of cell death is not fully understood. We now demonstrate that the BITC-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) due to inhibition of complex III of the mitochondrial respiratory chain. The BITC-induced ROS production and apoptosis were significantly inhibited by overexpression of catalase and Cu,Zn-superoxide dismutase and pharmacological inhibition of the mitochondrial respiratory chain. The mitochondrial DNA-deficient Rho-0 variant of MDA-MB-231 cells was nearly completely resistant to BITC-mediated ROS generation and apoptosis. The Rho-0 MDA-MB-231 cells also resisted BITC-mediated mitochondrial translocation (activation) of Bax. Biochemical assays revealed inhibition of complex III activity in BITC-treated MDA-MB-231 cells as early as at 1 h of treatment. The BITC treatment caused activation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), which function upstream of Bax activation in apoptotic response to various stimuli. Pharmacological inhibition of both JNK and p38 MAPK conferred partial yet significant protection against BITC-induced apoptosis. Activation of JNK and p38 MAPK resulting from BITC exposure was abolished by overexpression of catalase. The BITC-mediated conformational change of Bax was markedly suppressed by ectopic expression of catalytically inactive mutant of JNK kinase 2 (JNKK2(AA)). Interestingly, a normal human mammary epithelial cell line was resistant to BITC-mediated ROS generation, JNK/p38 MAPK activation, and apoptosis. In conclusion, the present study indicates that the BITC-induced apoptosis in human breast cancer cells is initiated by mitochondria-derived ROS.     

Suppression of ERK signaling evokes autocrine Fas‐mediated death in arachidonic acid‐treated human chronic myeloid leukemia K562 cells

Arachidonic acid (AA)‐induced apoptotic death of K562 cells (human chronic myeloid leukemic cells) was characteristic of reactive oxygen species (ROS) generation and mitochondrial depolarization. N‐Acetylcysteine pretreatment rescued viability of AA‐treated cells and abolished mitochondrial depolarization. In contrast to no significant changes in phospho‐JNK and phospho‐ERK levels, AA evoked notable activation of p38 MAPK. Unlike that of JNK and p38 MAPK, ERK suppression further reduced the viability of AA‐treated cells. Increases in Fas/FasL protein expression, caspase‐8 activation, the production of tBid and the loss of mitochondrial membrane potential were noted with K562 cells that were treated with a combination of U0126 and AA. Down‐regulation of FADD attenuated U0126‐evoked degradation of procaspase‐8 and Bid. Abolition of p38 MAPK activation abrogated U0126‐elicited Fas/FasL up‐regulation in AA‐treated cells. U0126 pretreatment suppressed c‐Fos phosphorylation but increased p38 MAPK‐mediated c‐Jun phosphorylation. Knock‐down of c‐Fos and c‐Jun protein expression by siRNA suggested that c‐Fos counteracted the effect of c‐Jun on Fas/FasL up‐regulation. Taken together, our data indicate that AA induces the ROS/mitochondria‐dependent death pathway and blocks the ERK pathway which enhances the cytotoxicity of AA through additionally evoking an autocrine Fas‐mediated apoptotic mechanism in K562 cells. J. Cell. Physiol. 222: 625–634, 2010. © 2009 Wiley‐Liss, Inc.     

Induction of apoptosis by Shikonin through ROS-mediated intrinsic and extrinsic apoptotic pathways in primary effusion lymphoma

Primary effusion lymphoma (PEL) is an incurable non-Hodgkin's lymphoma and novel biology-based treatments are urgently needed in clinical settings. Shikonin (SHK), a napthoquinone derivative, has been used for the treatment of solid tumors. Here, we report that SHK is an effective agent for the treatment of PEL. Treatment with SHK results in significant reduction of proliferation in PEL cells and their rapid apoptosis in vitro. SHK-induced apoptosis of PEL cells is accompanied by the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (Δψm), an activation of c-Jun-N-terminal kinase (JNK), p38, as well as caspase-3, -8, and -9. Scavenging of ROS in the presence of N-acetylcysteine (NAC) almost blocks the loss of mitochondrial membrane Δψm, activation of JNK, cleavage of caspase-3, -9, and an induction of apoptosis in SHK treated PEL cells. SP600125, a specific inhibitor of JNK, also rescues a proportion of cells from the apoptotic effect of SHK. In addition, inhibition of caspase activation in the presence of pan-caspase inhibitor, Q-VD-OPh, blocks the SHK-inducing apoptosis, but doesn't completely inhibit SHK-mediated JNK activation. Therefore, ROS is an upstream trigger of SHK-induced caspase dependent apoptosis of PEL cells through disruption of mitochondrial membrane Δψm in an intrinsic pathway and an activation of JNK in an extrinsic pathway. In a PEL xenografted mouse model, SHK treatment suppresses PEL-mediated ascites formation without showing any significant adverse toxicity. These results suggested that SHK could be a potent anti-tumor agent for the treatment of PEL.     

Mixed lineage kinase 3 connects reactive oxygen species to c-Jun NH2-terminal kinase-induced mitochondrial apoptosis in genipin-treated PC3 human prostate cancer cells

It has been reported that genipin, the aglycone of geniposide, induces apoptotic cell death in human hepatoma cells via a NADPH oxidase-reactive oxygen species (ROS)-c-Jun NH(2)-terminal kinase (JNK)-dependent activation of mitochondrial pathway. This continuing work aimed to define that mixed lineage kinase 3 (MLK3) is a key mediator, which connect between ROS and JNK in genipin-induced cell death signaling. In PC3 human prostate cancer cells, genipin stimulated MLK3 activity in concentration- and time-dependent manner. The PC3 cells stably transfected with dominant-negative form of MLK3 was less susceptible to population of the sub-G1 apoptotic cells, activation of caspase, collapse of mitochondrial membrane potential, and release of cytochrome c triggered by genipin, suggesting a crucial role of MLK3 in genipin signaling to apoptotic cell death. Diphenyleneiodonium (DPI), a specific inhibitor of NADPH oxidase, markedly inhibited ROS generation and MLK3 phosphorylation in the genipin-treated cells. Pretreatment with SP0600125, a specific inhibitor of JNK but neither U0126, a specific inhibitor of MEK1/2 nor PD169316, a specific inhibitor of p38 suppressed genipin-induced apoptotic cell death. Notably, both the phosphorylation of JNK and induction of c-Jun induced by genipin were markedly inhibited in PC3-EGFP-MLK3 (K144R) cells expressing a dominant-negative MLK3 mutant. Taken together, our observations suggest genipin signaling to apoptosis of PC3 cells is mediated via activation of ROS-dependent MLK3, which leads to downstream activation of JNK.     

p38 mitogen-activated protein kinase is a key regulator of 5-phenylselenyl- and 5-methylselenyl-methyl-2'-deoxyuridine-induced apoptosis in human HL-60 cells

Two novel, modified thymidine nucleosides, 5-phenylselenyl-methyl-2'-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2'-deoxyuridine (MeSe-T), trigger reactive oxygen species (ROS) generation and DNA damage and thereby induce caspase-mediated apoptosis in human HL-60 cells; however, the mechanism leading to caspase activation and apoptotic cell death remains unclear. Therefore, we investigated the signaling molecules involved in nucleoside derivative-induced caspase activation and apoptosis in HL-60 cells. PhSe-T/MeSe-T treatment activated two mitogen-activated protein kinases (MAPKs), extracellular-receptor kinase (ERK) and p38, and induced the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2. In addition, the selective p38 inhibitor SB203580 suppressed PhSe-T/MeSe-T-induced apoptosis and activation of caspase-3, -9, -8, and -2, whereas the jun amino-terminal kinase (JNK) inhibitor SP600125 and the ERK inhibitor PD98059 had no effect. SB203580 and an ROS scavenger, tiron, inhibited PhSe-T/MeSe-T-induced histone H2AX phosphorylation, which is a DNA damage marker. Moreover, tiron inhibited PhSe-T/MeSe-T-induced phosphorylation of p38 and enhanced p38 MAP kinase activity, indicating a role for ROS in PhSe-T/MeSe-T-induced p38 activation. Taken together, our results suggest that PhSe-T/MeSe-T-induced apoptosis is mediated by the p38 pathway and that p38 serves as a link between ROS generation and DNA damage/caspase activation in HL-60 cells.     

The activation of the c-Jun N-terminal kinase and p38 mitogen-activated protein kinase signaling pathways protects HeLa cells from apoptosis following photodynamic therapy with hypericin

In this study, we elucidate signaling pathways induced by photodynamic therapy (PDT) with hypericin. We show that PDT rapidly activates JNK1 while irreversibly inhibiting ERK2 in several cancer cell lines. In HeLa cells, sustained PDT-induced JNK1 and p38 mitogen-activated protein kinase (MAPK) activations overlap the activation of a DEVD-directed caspase activity, poly(ADP-ribose) polymerase (PARP) cleavage, and the onset of apoptosis. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (zDEVD-fmk) protect cells against apoptosis and inhibit DEVD-specific caspase activity and PARP cleavage without affecting JNK1 and p38 MAPK activations. Conversely, stable overexpression of CrmA, the serpin-like inhibitor of caspase-1 and caspase-8, has no effect on PDT-induced PARP cleavage, apoptosis, or JNK1/p38 activations. Cell transfection with the dominant negative inhibitors of the c-Jun N-terminal kinase (JNK) pathway, SEK-AL and TAM-67, or pretreatment with the p38 MAPK inhibitor PD169316 enhances PDT-induced apoptosis. A similar increase in PDT-induced apoptosis was observed by expression of the dual specificity phosphatase MKP-1. The simultaneous inhibition of both stress kinases by pretreating cells with PD169316 after transfection with either TAM-67 or SEK-AL produces a more pronounced sensitizing effect. Cell pretreatment with the p38 inhibitor PD169316 causes faster kinetics of DEVD-caspase activation and PARP cleavage and strongly oversensitizes the cells to apoptosis following PDT. These observations indicate that the JNK1 and p38 MAPK pathways play an important role in cellular resistance against PDT-induced apoptosis with hypericin.     

Involvement of p38 mitogen-activated protein kinase in gemcitabine-induced apoptosis in human pancreatic cancer cells

In this study, we investigated the involvement of Akt and members of the mitogen-activated protein kinase (MAPK) superfamily, including ERK, JNK, and p38 MAPK, in gemcitabine-induced cytotoxicity in human pancreatic cancer cells. We found that gemcitabine induces apoptosis in PK-1 and PCI-43 human pancreatic cancer cell lines. Gemcitabine specifically activated p38 MAPK in a dose- and time-dependent manner. A selective p38 MAPK inhibitor, SB203580, significantly inhibited gemcitabine-induced apoptosis in both cell lines, suggesting that phosphorylation of p38 MAPK may play a key role in gemcitabine-induced apoptosis in pancreatic cancer cells. A selective JNK inhibitor, SP600125, failed to inhibit gemcitabine-induced apoptosis in both cell lines. MKK3/6, an upstream activator of p38 MAPK, was phosphorylated by gemcitabine, indicating that the MKK3/6-p38 MAPK signaling pathway is indeed involved in gemcitabine-induced apoptosis. Furthermore, gemcitabine-induced cleavage of the caspase substrate poly(ADP-ribose) polymerase was inhibited by pretreatment with SB203580, suggesting that activation of p38 MAPK by gemcitabine induces apoptosis through caspase signaling. These results together suggest that gemcitabine-induced apoptosis in human pancreatic cancer cells is mediated by the MKK3/6-p38 MAPK-caspase signaling pathway. Further, these results lead us to suggest that p38 MAPK should be investigated as a novel molecular target for human pancreatic cancer therapies.     

Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.     

Olaquindox-induced apoptosis is suppressed through p38 MAPK and ROS-mediated JNK pathways in HepG2 cells

We investigated mitogen-activated protein kinase (MAPK) pathways as well as reactive oxygen species (ROS) in olaquindox-induced apoptosis. Exposure of HepG2 cells to olaquindox resulted in the phosphorylation of p38 MAPK and c-Jun N-terminal kinases (JNK). To confirm the role of p38 MAPK and JNK, HepG2 cells were pretreated with MAPKs-specific inhibitors prior to olaquindox treatment. Olaquindox-induced apoptosis was significantly potentiated by the JNK inhibitor (SP600125) or the p38 MAPK inhibitor (SB203580). Furthermore, we observed that olaquindox treatment led to ROS generation and that olaquindox-induced apoptosis and ROS generation were both significantly reduced by the antioxidants, superoxide dismutase and catalase. In addition, the levels of phosphorylation of JNK, but not p38 MAPK, were significantly suppressed after pretreatment of the antioxidants, while inhibition of the activations of JNK or p38 MAPK had no effect on ROS generation. This result suggested that ROS may be the upstream mediator for the activation of JNK. Conclusively, our results suggested that apoptosis in response to olaquindox treatment in HepG2 cells might be suppressed through p38 MAPK and ROS–JNK pathways.     

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.     

Amplification loop cascade for increasing caspase activity induced by docetaxel

The hierarchy of events accompanying induction of apoptosis by the microtubule inhibitor docetaxel was investigated in HL-60 human leukemia cells. Treatment of HL-60 cells with docetaxel resulted in the production of reactive oxygen species (ROS), activation of caspase-3 (-like) protease, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) activation, bcl-2 phosphorylation and apoptosis. Docetaxel elicited ROS production from NADPH oxidase as demonstrated by specific oxidase inhibitor diphenylene iodonium (DPI). ROS mediated the caspase-3 activation and apoptosis in HL-60 cells. The caspase inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) effectively inhibited JNK/SAPK activation, bcl-2 phosphorylation and partially attenuated the ROS production induced by docetaxel. Docetaxel-induced bcl-2 phosphorylation was completely blocked by expression of dominant negative JNK or the JNK/SAPK inhibitor SP600125. Overexpression of bcl-2 partially prevented docetaxel-mediated ROS production and subsequent caspase-3 activation, thereby inhibiting apoptotic cell death. It is thus conferred that such sequent events as ROS production, caspase activation, JNK/SAPK activation, bcl-2 phosphorylation and the further generation of ROS should be parts of an amplification loop to increase caspase activity, thereby facilitating the apoptotic cell death process.     

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.