Apoptotic effect and cell arrest of deoxyshikonin in human osteosarcoma cells through the p38 pathway

Osteosarcoma is the most common primary bone cancer that affects adolescents with early metastatic potential and drastically reduces their long-term survival rate if pulmonary metastases are detected at diagnosis. The natural naphthoquinol compound deoxyshikonin exhibits anticancer properties, so we hypothesized that it has an apoptotic effect on osteosarcoma U2OS and HOS cells and studied its mechanisms. After deoxyshikonin treatment, dose-dependent decreases in cell viability, induction of cell apoptosis and arrest in the sub-G1 phase of U2OS and HOS cells were observed. The increases in cleaved caspase 3 expression and the decreases in X-chromosome-linked IAP (XIAP) and cellular inhibitors of apoptosis 1 (cIAP-1) expressions after deoxyshikonin treatment in the human apoptosis array were identified in HOS cells, and dose-dependent expression changes of IAPs and cleaved caspase 3, 8 and 9 were verified by Western blotting in U2OS and HOS cells. Phosphorylation of extracellular signal-regulated protein kinases (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2 and p38 expressions in U2OS and HOS cells was also increased by deoxyshikonin in a dose-dependent manner. Subsequently, cotreatment with inhibitors of ERK (U0126), JNK (JNK-IN-8) and p38 (SB203580) was performed to show that p38 signalling is responsible for deoxyshikonin-induced apoptosis in U2OS and HOS cells, but not via the ERK and JNK pathways. These discoveries demonstrate that deoxyshikonin may be a possible chemotherapeutic candidate to induce cell arrest and apoptosis by activating extrinsic and intrinsic pathways through p38 for human osteosarcoma.     

Activation and signaling of the p38 MAP kinase pathway

The family members of the mitogen-activated protein (MAP) kinases mediate a wide variety of cellular behaviors in response to extracellular stimuli. One of the four main sub-groups, the p38 group of MAP kinases, serve as a nexus for signal transduction and play a vital role in numerous biological processes. In this review, we highlight the known characteristics and components of the p38 pathway along with the mechanism and consequences of p38 activation. We focus on the role of p38 as a signal transduction mediator and examine the evidence linking p38 to inflammation, cell cycle, cell death, development, cell differentiation, senescence and tumorigenesis in specific cell types. Upstream and downstream components of p38 are described and questions remaining to be answered are posed. Finally, we propose several directions for future research on p38.     

LukS-PV induces cell cycle arrest and apoptosis through p38/ERK MAPK signaling pathway in NSCLC cells

Non-small-cell lung cancer (NSCLC) accounts for nearly 85% of lung cancer cases. LukS-PV, one of the two components of Panton-Valentine leucocidin (PVL), is produced by Staphylococcus aureus. The present study showed that LukS-PV can induce apoptosis in human acute myeloid leukemia (AML) lines (THP-1 and HL-60). However, the role of LukS-PV in NSCLC is unclear. In this study, we treated NSCLC cell lines A549 and H460 and a normal lung cell line, 16HBE, with LukS-PV and investigated the biological roles of LukS-PV in NSCLC. Cells were treated with varying concentrations of LukS-PV and cell viability was evaluated by CCK8 and EdU assay. Flow cytometry was used to detect cell apoptosis and analyze the cell cycle, and the expression of apoptosis and cell cycle-associated proteins and genes were identified by western blotting analysis and qRT-polymerase chain reaction, respectively. We found that LukS-PV inhibited the proliferation of NSCLC cells but had little cytotoxicity in normal lung cells. LukS-PV induced NSCLC cell apoptosis and increased the BAX/BCL-2 ratio, triggering S-phase arrest in A549 and H460 cells while increasing P21 expression and decreasing CDK2, cyclin D1, and cyclin A2 expression. We also observed increased P-p38 and P-ERK in NSCLC cells treated with LukS-PV. Treatment of NSCLC with LukS-PV combined with p38 and ERK inhibitors reversed the pro-apoptotic and pro-cell cycle arrest effects of LukS-PV. Overall, these findings indicate that LukS-PV has anti-tumor effects in NSCLC and may contribute to the development of anti-cancer agents.     

Wip1 phosphatase positively modulates dendritic spine morphology and memory processes through the p38MAPK signaling pathway

Dendritic spine morphology is modulated by protein kinase p38, a mitogen-activated protein (MAPK), in the hippocampus. Protein p38MAPK is a substrate of wip1, a protein phosphatase. The role of wip1 in the central nervous system (CNS) has never been explored. Here, we report a novel function of wip1 in dendritic spine morphology and memory processes. Wip1 deficiency decreases dendritic spine size and density in pyramidal neurons of the hippocampal CA1 region. Simultaneously, impairments in object recognition tasks and contextual memory occur in wip1 deficient mice, but are reversed in wip1/p38 double mutant mice. Thus, our findings demonstrate that wip1 modulates dendritic morphology and memory processes through the p38MAPK signaling pathway. In addition to the well-characterized role of the wip1/p38MAPK in cell death and differentiation, we revealed the novel contribution of wip1 to cognition and dendritic spine morphology, which may suggest new approaches to treating neurodegenerative disorders.     

Wip1 phosphatase positively modulates dendritic spine morphology and memory processes through the p38MAPK signaling pathway

Dendritic spine morphology is modulated by protein kinase p38, a mitogen-activated protein (MAPK), in the hippocampus. Protein p38MAPK is a substrate of wip1, a protein phosphatase. The role of wip1 in the central nervous system (CNS) has never been explored. Here, we report a novel function of wip1 in dendritic spine morphology and memory processes. Wip1 deficiency decreases dendritic spine size and density in pyramidal neurons of the hippocampal CA1 region. Simultaneously, impairments in object recognition tasks and contextual memory occur in wip1 deficient mice, but are reversed in wip1/p38 double mutant mice. Thus, our findings demonstrate that wip1 modulates dendritic morphology and memory processes through the p38MAPK signaling pathway. In addition to the well-characterized role of the wip1/p38MAPK in cell death and differentiation, we revealed the novel contribution of wip1 to cognition and dendritic spine morphology, which may suggest new approaches to treating neurodegenerative disorders.     

siRNA-mediated NCAM1 gene silencing suppresses oxidative stress in pre-eclampsia by inhibiting the p38MAPK signaling pathway

Pre-eclampsia (PE), whose pathophysiology and etiology remain undefined, represents a leading consequence of fetal and maternal mortality and morbidity. Oxidative stress (OS) is recognized to involve in this disorder. In this study, we hypothesized that neural cell adhesion molecule 1 (NCAM1) gene silencing would suppress the OS in the pregnancy complicated by PE. Initially, clinical samples were collected for determination of NCAM1 expression in placental tissues and levels of OS products in blood. To assess the regulatory mechanism of NCAM1 knockdown on OS, we used small interfering RNA (siRNA) to silence NCAM1 expression in human umbilical vein endothelial cells (HUVECs). Next, cells were treated with or without hypoxia/reoxygenation to observe the level changes of OS products and p38 mitogen-activated protein kinase (p38MAPK) pathway-related genes. Finally, an evaluation of HUVEC migration and invasion abilities was conducted by wound-healing and transwell assays. Placenta of pregnancy with PE presented significantly increased NCAM1 expression in comparison to placenta of normal pregnancy. Meanwhile, enhanced OS in blood of pregnant women with PE was observed relative to women with normal pregnancy. siRNA-mediated knockdown of NCAM1 gene could inhibit the p38MAPK signaling pathway, repress OS, and promote cell migration and invasion in HUVECs, indicating that NCAM1 inhibition could reduce the influence of PE. Importantly, blocking the p38MAPK signaling pathway reversed the inhibitory role of NCAM1 gene silencing on PE. Collectively, this study defines potential role of NCAM1 gene silencing as a therapeutic target in PE through inhibiting OS and enhancing HUVEC migration and invasion by disrupting the p38MAPK signaling pathway.     

Matrine reduces the proliferation and invasion of colorectal cancer cells via reducing the activity of p38 signaling pathway

Matrine has been used in anti-inflammatory and anti-cancer therapies for a long time. However, the anti-metastatic effect and related mechanism（s） in colorectal cancer （CRC） are still unclear. In this study, we investigated whether the adminis- tration of matrine could inhibit the proliferation, motility, and invasion of human CRC cells via regulating p38 signal- ing pathway. Results showed that matrine inhibited migration and invasion of CRC cells in vitro and in vivo. Additionally, after being treated with matrine for 24 h, the expression levels of matrix metalloproteinase-2 （MMP-2） and MMP-9 as well as proteinase activity in CRC cells were reduced in a dose-dependent manner. Moreover, matrine reduced the phosphorylation level of p38 obviously. Combined treatment with p38 inhibitor （SB203580） and matrine resulted in a syn- ergistic reduction of invasion as well as MMP-2/-9 expression in CRC cells. It was also found that matrine inhibited the pro- liferation and metastasis of CRC tumor in vivo. In conclusion, p38 signaling pathway may involve in matrine＇s inhibitory effects on migration and invasion of CRC cells by reducing the expression of MMP-2/-9, suggesting that matrine may be a potential therapeutic agent for CRC.     

Signaling Crosstalk of FHIT,p53, and p38 in etoposide-induced apoptosis in MCF-7 cells

Fragile histidine trail (FHIT) is a tumor suppressor in response to DNA damage which has been deleted in various tumors. However, the signaling mechanisms and interactions of FHIT with regard to apoptotic proteins including p53 and p38 in the DNA damage-induced apoptosis are not well described. In the present study, we used etoposide-induced DNA damage in MCF-7 as a model to address these crosstalks. The time course study showed that the expression of FHIT, p53, and p38MAPK started after 1 hour following etoposide treatment. FHIT overexpression led to increase p53 expression, p38 activation, and augmented apoptosis following etoposide-induced DNA damage compared to wild-type cells. However, FHIT knockdown blocked p53 expression, delayed p38 activation, and completely inhibited etoposide-induced apoptosis. Inhibition of p38 activity prevented induction of p53, FHIT, and apoptosis in this model. Thus, activation of p38 upon etoposide treatment leads to increase in FHIT and p53 expression. In p53 knockdown MCF-7, the FHIT induction was hampered but p38 activation was induced in lower doses of etoposide. In p53 knockdown cells, inhibition of p38 induced FHIT expression and apoptosis. Our data demonstrated that the exposure of MCF-7 cells to etoposide increases apoptosis through a mechanism involving the activation of the p38-FHIT-p53 pathway. Moreover, our findings suggest signaling interaction for these pathways may represent a promising therapy for breast cancer.     

Genistein enhances TRAIL-induced apoptosis through inhibition of p38 MAPK signaling in human hepatocellular carcinoma Hep3B cells

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some carcinoma cancer cells. However, it was found that treatment with TRAIL in combination with nontoxic concentrations of genistein sensitized TRAIL-resistant human hepatocellular carcinoma Hep3B cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis were correlated with the induction of caspase activity that resulted in the cleavage of poly(ADP-ribose) polymerase (PARP). Both cell viability and the cleavage of PARP induced by combined treatment were significantly inhibited by caspase-3, -8 and -9 inhibitors, which demonstrates the important roles of caspases in the observed cytotoxic effects. Genistein treatment also triggered the inhibition of p38-β mitogen-activated protein kinase (MAPK) activation. Pretreatment with SB203580 resulted in significantly increased sub-G1 population and loss of mitochondrial membrane potential (MMP) in TRAIL-induced apoptosis. By contrast, overexpression of p38 MAPK protected apoptosis by co-treatment with genistein and TRAIL, suggesting that the p38 MAPK act as key regulators of apoptosis in response to treatment with a combination of genistein and TRAIL in human hepatocellular carcinoma Hep3B cells.     

Retracted: Effects of microRNA-494 on proliferation,migration, invasion,and apoptosis of medulloblastoma cells by mediating c-myc through the p38 MAPK signaling pathway

Medulloblastoma (MB) is the most prevalent brain tumor that occurs during childhood and originates from cerebellar granule cell precursors. Based on recent studies, the differential expression of several microRNAs is involved in MB, while the role of microRNA-494 (miR-494) in MB remains unclear. Therefore, we conducted this study to investigate the regulative role of miR-494 in MB cells via the p38 mitogen-activated protein kinase (MAPK) signaling pathway by mediating c-myc. In the current study, MB cells were collected and transfected with miR-494 mimic, miR-494 inhibitor, siRNA- c-myc, and miR-494 inhibitor + siRNA-c-myc. The expressions of miR-494, c-myc, p38 MAPK, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), interleukin-6 (IL-6), metadherin (MTDH), phosphatase and tensin homolog (PTEN) and survivin were determined. Cell proliferation, cell-cycle distribution, apoptosis, migration, and invasion were evaluated. The results revealed that there was a poor expression of miR-494 and high expression of c-myc in MB tissues. C-myc was determined as the target gene of miR-494. In response to miR-494 mimic, MB cells were found to have increased Bax and PTEN expressions, as well as cell number in G1 phase and cell apoptosis and decreased c-myc, p38 MAPK, Bcl-2, MTDH, IL-6, and survivin expression and cell number count in the S phase, cell proliferation, migration, and invasion. In conclusion, the results demonstrated that the upregulation of miR-494 results in the suppression of cell proliferation, migration, and invasion, while it promotes apoptosis of MB cells through the negative mediation of c-myc, which in turn inactivates the p38 MAPK pathway.     

p38γ regulates UV-induced checkpoint signaling and repair of UV-induced DNA damage

In eukaryotic cells, DNA damage triggers activation of checkpoint signaling pathways that coordinate cell cycle arrest and repair of damaged DNA. These DNA damage responses serve to maintain genome stability and prevent accumulation of genetic mutations and development of cancer. The p38 MAPK was previously implicated in cellular responses to several types of DNA damage. However, the role of each of the four p38 isoforms and the mechanism for their involvement in DNA damage responses remained poorly understood. In this study, we demonstrate that p38γ, but not the other p38 isoforms, contributes to the survival of UV-treated cells. Deletion of p38γ sensitizes cells to UV exposure, accompanied by prolonged S phase cell cycle arrest and increased rate of apoptosis. Further investigation reveal that p38γ is essential for the optimal activation of the checkpoint signaling caused by UV, and for the efficient repair of UV-induced DNA damage. These findings have established a novel role of p38γ in UV-induced DNA damage responses, and suggested that p38γ contributes to the ability of cells to cope with UV exposure by regulating the checkpoint signaling pathways and the repair of damaged DNA.     

Arsenic induced apoptosis in malignant melanoma cells is enhanced by menadione through ROS generation, p38 signaling and p53 activation

Introduction  Resistance to apoptosis is a prominent feature of melanoma. Pharmacological concentration of arsenic in combination with a widely known oxidant, menadione was explored in this study to synergistically sensitize malignant melanoma cells to apoptosis. The molecular mechanism of apoptosis and the signaling-pathways involved were thoroughly investigated. Materials methods and results  Menadione synergized NaAsO₂ to significantly increase ROS generation and facilitate the major apoptotic signaling events: alteration of mitochondrial membrane potential, cytochrome c release and anti-apoptotic protein Bcl-2 down-regulation and subsequent activation of caspase-9 and caspase-3 followed by poly-ADP-ribose polymerase-1 cleavage. Antioxidant N-acetyl-l-cysteine antagonized these events. Investigation of the signaling-pathway revealed significant suppression of AP-1 activity but not NF-κB upon NaAsO₂ and menadione application. An increase in p38 phosphorylation and p53 protein expression did also dictate the apoptotic response. Suppression of p38 activation with SB203580 and inhibition of p53 expression by siRNA attenuated apoptosis. Transfection of p53, in p53 null HCT cells augmented the apoptotic events. Moreover, the treatment also led to tumor size reduction in BALB/c mice developed by intra-dermal B16 mouse melanoma cell injection; however, it had no detectable pro-proliferative or pro-apoptotic effect on non-tumor keratinocytes, normal fibroblasts or PBMC. Conclusion  This study thus provides an insight into innovative mechanisms of melanoma sensitization, a proper cure against which is still elusive. Taken together, our data also provides the first evidence of arsenic activity accentuation by menadione through modulation of specific signaling-pathways.     

Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways

Background  

Pigmentation is one of the essential defense mechanisms against oxidative stress or UV irradiation; however, abnormal hyperpigmentation in human skin may pose a serious aesthetic problem. C-phycocyanin (Cpc) is a phycobiliprotein from spirulina and functions as an antioxidant and a light harvesting protein. Though it is known that spirulina has been used to reduce hyperpigmentation, little literature addresses the antimelanogenic mechanism of Cpc. Herein, we investigated the rationale for the Cpc-induced inhibitory mechanism on melanin synthesis in B16F10 melanoma cells.     

Histamine activates tyrosine hydroxylase in bovine adrenal chromaffin cells through a pathway that involves ERK1/2 but not p38 or JNK

In bovine adrenal chromaffin cells (BACC) histamine promotes a rapid increase in the intracellular levels of Ca2+ together with the release of catecholamines and the phosphorylation of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). In this study we investigated the role of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK1/2), stress activated protein kinase (p38) and Jun N-terminal kinases (JNK) on the histamine-induced activation and phosphorylation of TH. We found that in BACC histamine produced a rapid, long lasting and histamine type-1 (H1) receptor-dependent increase in the phosphorylation levels of ERK1/2, p38 and JNK which was accompanied by a H1 receptor-dependent increase in TH activity. This increase in TH activity was partially blocked by the MEK1/2 inhibitor U0126 but was unaffected by the p38 antagonist SB203580 or the JNK blocker JNKI1. To study the effect of MAPK inhibition on histamine-induced TH phosphorylation, we generated phospho-specific antibodies against the different phosphorylated forms of TH. Treatment with U0126 totally inhibited the histamine-induced phosphorylation of TH at Ser31, without affecting the phosphorylation of either Ser40 or Ser19. Neither SB203580 nor JNKI1 treatments produced any significant modification of the histamine-induced TH phosphorylation. Our data support the hypothesis that the up-regulation of the ERK1/2 pathway, but not that of p38 or JNK, promoted by histamine is involved in the phosphorylation of TH at Ser31 and that this phosphorylation event is required for the full activation of this enzyme.     

Pseudolaric acid B suppresses T lymphocyte activation through inhibition of NF‐κB signaling pathway and p38 phosphorylation

Pseudolaric acid B (PAB) is a major bioactive component of the medicinal plant Pseudolarix kaempferi. Traditional medicine practitioners in Asia have been using the roots of this plant to treat inflammatory and microbial skin diseases for centuries. In the current study, in vitro immunosuppressive effect of PAB and the underlying mechanisms have been investigated. The results showed that PAB dose‐dependently suppressed human T lymphocyte proliferation, IL‐2 production and CD25 expression induced by co‐stimulation of PMA plus ionomycin or of anti‐OKT‐3 plus anti‐CD28. Mechanistic studies showed that PAB significantly inhibited nuclear translocation of NF‐κB p65 and phosphorylation and degradation of IκB‐α evoked by co‐stimulation of PMA plus ionomycin. PAB could also suppress the phosphorylation of p38 in the MAPKs pathway. Based on these evidences, we conclude that PAB suppressed T lymphocyte activation through inhibition of NF‐κB and p38 signaling pathways; this would make PAB a strong candidate for further study as an anti‐inflammatory agent. J. Cell. Biochem. 108: 87–95, 2009. © 2009 Wiley‐Liss, Inc.