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.     

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.     

Mitogen-activated protein kinases regulate Mycobacterium avium-induced tumor necrosis factor-alpha release from macrophages

Tumor necrosis factor-alpha (TNF-alpha) is one of the key cytokines elicited by host macrophages upon challenge with pathogenic mycobacteria. Infection of human peripheral blood mononuclear cells or the murine macrophage cell line J774A-1 with Mycobacterium avium induced activation of the mitogen-activated protein kinases (MAPKs) ERK1/2, p38 and c-Jun N-terminal kinase. U0126, an MEK-specific inhibitor, abrogated M. avium-induced TNF-alpha secretion. Transfection of cells with dominant-negative MEK1 led to the suppression of TNF-alpha release in M. avium-challenged macrophages. M. avium activated p38 MAPK and use of the p38 MAPK inhibitor, SB203580, revealed that the p38 signaling pathway negatively regulates activation of ERK1/2 and release of TNF-alpha. Taken together, these results provide evidence that M. avium-induced TNF-alpha release from macrophages depends on an interplay between the ERK1/2 and the p38 MAPK signaling pathways.     

MAVS-MKK7-JNK2 Defines a Novel Apoptotic Signaling Pathway during Viral Infection

Viral infection induces innate immunity and apoptosis. Apoptosis is an effective means to sacrifice virus-infected host cells and therefore restrict the spread of pathogens. However, the underlying mechanisms of this process are still poorly understood. Here, we show that the mitochondrial antiviral signaling protein (MAVS/VISA/Cardif/IPS-1) is critical for SeV (Sendai virus)-induced apoptosis. MAVS specifically activates c-Jun N-terminal kinase 2 (JNK2) but not other MAP kinases. Jnk2−/− cells, but not Jnk1−/− cells, are unable to initiate virus-induced apoptosis and SeV further fails to trigger apoptosis in MAPK kinase 7 (MKK7) knockout (Mkk7−/−) cells. Mechanistically, MAVS recruits MKK7 onto mitochondria via its 3D domain, which subsequently phosphorylates JNK2 and thus activates the apoptosis pathway. Consistently, Jnk2−/− mice, but not Jnk1−/− mice, display marked inflammatory injury in lung and liver after viral challenge. Collectively, we have identified a novel signaling pathway, involving MAVS-MKK7-JNK2, which mediates virus-induced apoptosis and highlights the indispensable role of mitochondrial outer membrane in host defenses.     

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.     

ASK1 regulates influenza virus infection-induced apoptotic cell death

Apoptosis occurs in influenza virus (IV)-infected cells. There are a number of mechanisms for the regulation of apoptosis. However, the molecular mechanism of IV infection-induced apoptosis is still controversial. Apoptosis signal-regulating kinase1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase (MAPKKK) that activates the SEK1-c-Jun N-terminal kinase (JNK) and MKK3/MKK6-p38 MAPK signaling cascades. ASK1 has been implicated in cytokine- and stress-induced apoptosis. Here, we show the following: (1) IV infection activated ASK1 and concomitantly phosphorylated JNK and p38 MAPK in human bronchial epithelial cells; (2) the activation of JNK and p38 MAPK but not extracellular-regulated kinase (ERK) in embryonic fibroblasts (MEFs) derived from ASK1 knockout mice (ASK1(-/-) MEFs) was depressed compared to MEFs derived from wild type mice (ASK1(+/+) MEFs); and (3) ASK1(-/-) MEFs were defective in IV infection-induced caspase-3 activation and cell death. These results indicate that apoptosis in IV-infected BEC is mediated through ASK1-dependent cascades.     

Cadmium Induces PC12 Cells Apoptosis via an Extracellular Signal-Regulated Kinase and c-Jun N-Terminal Kinase-Mediated Mitochondrial Apoptotic Pathway

To investigate the role of mitogen-activated protein kinase (MAPK) and downstream events in cadmium (Cd)-induced neuronal apoptosis executed via the mitochondrial apoptotic pathway, this study used the PC-12 cell line as a neuronal model. The result showed that Cd significantly decreased cell viability and the Bcl-2?/?Bax ratio and increased the percentage of apoptotic cells, release of cytochrome c, caspase-3, and poly(ADP-ribose) polymerase cleavage, and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G. In addition, exposure to Cd-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2?/?Bax ratio and cytochrome c release and suppressed caspase-3 and poly(ADP-ribose) polymerase cleavage and AIF and endonuclease G nuclear translocation. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathway played an important role in Cd-induced PC12 cells apoptosis.     

The role of mitogen-activated protein kinase in cadmium-induced primary rat cerebral cortical neurons apoptosis via a mitochondrial apoptotic pathway

Cadmium (Cd) is an extremely toxic metal capable of severely damaging several organs, including the brain. Studies have shown that Cd induces neuronal apoptosis partially by activating the mitogen-activated protein kinase (MAPK) pathways. However, the underlying mechanism of MAPK involving the mitochondrial apoptotic pathway in neurons remains unclear. In this study, primary rat cerebral cortical neurons were exposed to Cd, which significantly decreased cell viability and the B-cell lymphoma 2/Bcl-2 associate X protein (Bcl-2/Bax) ratio and increased the percentage of apoptotic cells, release of cytochrome c, cleavages of caspase-3 and poly (ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF). In addition, Cd induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK. Inhibition of ERK and JNK, but not p38 MAPK, partially protected the cells from Cd-induced apoptosis. ERK and JNK inhibition also blocked alteration of the Bcl-2/Bax ratio, release of cytochrome c, cleavages of caspase-3 and PARP, and nuclear translocation of AIF. Taken together, these data suggest that the ERK- and JNK-mediated mitochondrial apoptotic pathways play important roles in Cd-induced neuronal apoptosis.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway

IL-2 stimulates extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in various immune cell populations. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK kinase (MKK)/ERK and p38 MAPK pathways are necessary for IL-2 to activate NK cells. Using freshly isolated human NK cells, we established that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK generation, IFN-gamma secretion, and CD25 and CD69 expression. IL-2 induced ERK activation within 5 min. Treatment of NK cells with a specific inhibitor of MKK1/2, PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four sequelae, with inhibition of lymphokine-activated killing induction being least sensitive to MKK/ERK pathway blockade. Activation of p38 MAPK by IL-2 was not detected in NK cells. In contrast to what was observed by others in T lymphocytes, SB203850, a specific inhibitor of p38 MAPK, did not inhibit IL-2-activated NK functions. This data indicate that p38 MAPK activation was not required for IL-2 to activate NK cells for the four functions examined. These results reveal selective signaling differences between NK cells and T lymphocytes; in NK cells, the MKK/ERK pathway and not p38 MAPK plays a critical positive regulatory role during activation by IL-2.     

Roles of mitogen-activated protein kinase pathways during <Emphasis Type="Italic">Escherichia coli-</Emphasis>induced apoptosis in U937 cells

Escherichia coli (E. coli) infections play an important and growing role in the clinic. In the present study, we investigated the involvement of members of the mitogen-activated protein kinase (MAPK) superfamily, including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 MAPK, and caspase-3 and 9 activity in E. coli-induced apoptosis in human U937 cells. We found that E. coli induces apoptosis in U937 cell lines in a dose- and time-dependent manner, p38 MAPK and JNK were activated after 10 min of infection with E. coli. In contrast, ERK1/2 was down-regulated in a time-dependent manner. The levels of total (phosphorylation state-independent) p38 MAPK, JNK and ERK1/2 did not change in E. coli-infected U937 cells at all times examined. Moreover, exposure of U937 cells to E. coli led to caspase-3 and 9 activity. For the evaluation of the role of MAPKs, PD98059, SB203580 and SP600125 were used as MAPKs inhibitors for ERK1/2, p38 MAPK and JNK. Inhibition of ERK1/2 with PD98059 caused further enhancement in apoptosis and caspase-3 and 9 activity, while a selective p38 MAPK inhibitor, SB203580 and JNK inhibitor, SP600125 significantly inhibited E. coli-induced apoptosis and caspase-3 and 9 activity in U937 cells. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked E. coli-induced U937 apoptosis. Taken together, we have shown that E. coli increase p38 MAPK and JNK and decrease ERK1/2 phosphorylation and increase caspase-3 and 9 activity in U937 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.     

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.     

Paclitaxel induces prolonged activation of the Ras/MEK/ERK pathway independently of activating the programmed cell death machinery

Paclitaxel is a widely used chemotherapeutic agent and is known to induce programmed cell death (apoptosis) in a variety of cell types, but the precise underlying mechanisms are poorly understood. To elucidate these mechanisms, we challenged human esophageal squamous cancer cell lines with paclitaxel and investigated its effects upon signal transduction pathways. Physiologically relevant concentrations of paclitaxel (1-1,000 nm) induced apoptosis. All three mitogen-activated protein kinase (MAPK) family members, c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK) were activated upon paclitaxel treatment. Interestingly, JNK activation and p38 MAPK activation were delayed and peaked at 48 h, whereas ERK activity was sustained over 72 h. In addition, Ras activation and MAPK/ERK kinase (MEK) phosphorylation were observed in concordance with ERK activation. While ERK activation was completely ablated by MEK inhibitors, immunoprecipitation and Western blot analysis revealed that neither MEK-1 nor MEK-2 was involved, but instead another member of the MEK family may potentially participate. Although pretreatment with a general caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone rescued the cell death, it did not prevent Ras or ERK activation. Furthermore, inhibition of JNK, p38 MAPK, or MEK did not alter PARP cleavage and the cell death induced by paclitaxel. These results in aggregate suggest that the delayed activation of JNK, p38 MAPK, and ERK was not linked to activation of the cell death machinery.     

Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX–MAPK pathway

Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP–human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal–regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.     

Reduced Parasite Motility and Micronemal Protein Secretion by a p38 MAPK Inhibitor Leads to a Severe Impairment of Cell Invasion by the Apicomplexan Parasite Eimeria tenella

E. tenella infection is associated with a severe intestinal disease leading to high economic losses in poultry industry. Mitogen activated protein kinases (MAPKs) are implicated in early response to infection and are divided in three pathways: p38, extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK). Our objective was to determine the importance of these kinases on cell invasion by E. tenella. We evaluated the effect of specific inhibitors (ERK: PD98059, JNKII: SP600125, p38 MAPK: SB203580) on the invasion of epithelial cells. Incubation of SP600125 and SB203580 with epithelial cells and parasites significantly inhibited cell invasion with the highest degree of inhibition (90%) for SB203580. Silencing of the host p38α MAPK expression by siRNA led to only 20% decrease in cell invasion. In addition, when mammalian epithelial cells were pre-treated with SB203580, and washed prior infection, a 30% decrease in cell invasion was observed. This decrease was overcome when a p38 MAPK activator, anisomycin was added during infection. This suggests an active but limited role of the host p38 MAPK in this process. We next determined whether SB203580 has a direct effect on the parasite. Indeed, parasite motility and secretion of micronemal proteins (EtMIC1, 2, 3 and 5) that are involved in cell invasion were both decreased in the presence of the inhibitor. After chasing the inhibitor, parasite motility and secretion of micronemal proteins were restored and subsequently cell invasion. SB203580 inhibits cell invasion by acting partly on the host cell and mainly on the parasite.     

JNK and p38 MAPK are independently involved in tributyltin-mediated cell death in rainbow trout (Oncorhynchus mykiss) RTG-2 cells

Mitogen-activated protein kinases (MAPKs) are a family of Ser/Thr protein kinases that transmit various extracellular signals to the nucleus inducing gene expression, cell proliferation, and apoptosis. Recent studies have revealed that organotin compounds induce apoptosis and MAPK phosphorylation/activation in mammal cells. In this study, we elucidated the cytotoxic mechanism of tributyltin (TBT), a representative organotin compound, in rainbow trout (Oncorhynchus mykiss) RTG-2 cells. TBT treatment resulted in significant caspase activation, characteristic morphological changes, DNA fragmentation, and consequent apoptotic cell death in RTG-2 cells. TBT exposure induced the rapid and sustained accumulation of phosphorylated MAPKs, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAP kinase (p38 MAPK). Further analysis using pharmacological inhibitors against caspases and MAPKs showed that TBT also induced cell death in a caspase-independent manner and that p38 MAPK is involved in TBT-induced caspase-independent cell death, whereas JNK is involved in the caspase-dependent apoptotic pathway. Thus, TBT employs at least two independent signaling cascades to mediate cell death in RTG-2 cells. To our knowledge, this is the first study revealing the relationship between MAPK activation and TBT cytotoxicity in RTG-2 cells.     

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.     

Synthesis,antiproliferative and apoptosis induction potential activities of novel bis(indolyl)hydrazide-hydrazone derivatives

In recent years, indole-indazolyl hydrazide-hydrazone derivatives with strong cell growth inhibition and apoptosis induction characteristics are being strongly screened for their cancer chemo-preventive potential. In the present study, N-methyl and N,N-dimethyl bis(indolyl)hydrazide-hydrazone analog derivatives were designed, synthesized and allowed to evaluate for their anti-proliferative and apoptosis induction potential against cervical (HeLa), breast (MCF-7 and MDA-MB-231) and lung (A549) cancer cell lines relative to normal HEK293 cells. The MTT assay in conjunction with mitochondrial potential assays and the trypan blue dye exclusion were employed to ascertain the effects of the derivatives on the cancer cells. Further, mechanistic studies were conducted on compound 14a to understand the biochemical mechanisms and functional interactions with various signaling pathways triggered in HeLa and MCF-7 cells. Compound 14a induced apoptosis via caspase independent pathway through the participation of mitogen-activated protein kinases (MAPK) such as extracellular signal related kinase (ERK) and p38 as well as p53 pathways. It originates the activation of pro-apoptotic proteins such as Bak and Mcl-1s and also strongly induced the generation of reactive oxygen species. In downstream signaling pathway, activated p53 protein interacted with MAPK pathways, including SAPK/c-Jun N-terminal protein kinase (JNK), p38 and ERK kinases resulting in apoptotic cell death. The involvement of MAPK cascades such as p38, ERK and p38 on compound 14a induced apoptotic cell death was evidenced by the fact that the inclusion of specific inhibitors of p38, ERK1/2 and JNK MAPK (SB2035809, PD98059 and SP600125) prevented the compound 14a towards induced apoptosis. The results clearly showed that MAP kinase cascades were crucial for apoptotic response in compound 14a induced cellular killing and were dependent on p53 activity. Based on the results, compound 14a was identified as a promising candidate for cancer therapeutics and these findings furnish a basis for further in vivo experiments on anti-proliferative activity.