Effects Of PD98059 Research Articles

Inhibitory Effects of PD98059, SB203580, and SP600125 on α- and δ-granule Release and Intracellular Ca2+Levels in Human Platelets

Inhibitory Effects of PD98059, SB203580, and SP600125 on α- and δ-granule Release and Intracellular Ca²⁺Levels in Human Platelets

Effects of PD98059 and LY294002 on subcutaneous xenograft of human endometrial carcinoma in nude mice

To investigate the effects of signal pathway inhibitors PD98059 and LY294002 on cell proliferation, apoptosis, expressions of phosphorylated extracellular signal-regulared kinase (p-ERK) and phosphorylated protein kinase B (p-Akt) in endometrial carcinoma xenografts. Human endometrial carcinoma Ishikawa cells were cultured in vitro. The effects of PD98059 and LY294002 on proliferation, apoptosis, and cell cycle distribution of endometrial cancer cells were detected by monotetrazolium (MTT) assay and fluorescence-activated cell sorting technique. The models of xenografted tumor were established by the subcutaneous inoculation in 24 nude mice, and then they were randomly divided into 4 groups (n = 6), normal saline group, PD98059 group (PD group), LY294002 group (LY group) or PD98059 + LY294002 group (PD + LY group) by intraperitoneal injections, respectively. The anti-tumor efficacy was evaluated by measuring tumor volume and tumor growth status. The histopathological change of tumor specimens was observed using HE staining and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigen in nick and labeling method (TUNEL) testing and the expression levels of p-ERK and p-Akt were detected by immunohistochemistry method. (1) The proliferation of Ishikawa cells were suppressed after treated by PD98059 and(or) Y294002, in which A(570) values of cells decreased showing both time-dependent and concentration-dependent manner (LY294002: F(group) = 9.801, P = 0.002; F(time) = 10.398, P = 0.001. PD98059: F(group) = 8.213, P = 0.015; F(time) = 6.839, P = 0.036). Cell cycle distribution analysis revealed that percentage of Ishikawa cells at G(0)/G(1) phase (F(time) = 35.049, P = 0.004; F(group) = 32.024, P < 0.01) increased and percentage of S phase cells (F(time) = 7.789, P = 0.049; F(group) = 30.132, P < 0.01) decreased significantly. The percentage of apoptotic cells increased significantly among PD group, LY group and PD + LY group, in which there were significant difference [(63.3 ± 0.5)% vs (30.7 ± 20.1)% vs (40.8 ± 1.3)%; F = 621.059, P < 0.01]. (2) Compared with the control group, the increasing of transplanting tumor volume in the treated groups were obviously (F = 23.545, P < 0.01), and the inhibited rate of the tumor was higher in PD + LY group than that in PD group or LY group [(68 ± 9)% vs (32 ± 16)% or (38 ± 17)%; F = 10.283, P < 0.05]. (3) HE staining shown that there were different degrees of necrosis for endometrial carcinoma cell in different groups. The apoptosis of tumor cells were significantly increased in treated groups by TUNEL testing [(13.7 ± 1.5)%, (14.1 ± 1.2)%, (29.0 ± 1.8)%; F = 320.344, P < 0.01]. Immunohistochemistry results demonstrated that the expressions of p-ERK and p-Akt in treated groups were lower than that in control group, of which LY + PD group was the lowest one. The signal pathway inhibitors PD98059 and LY294002 could inhibit the growth of human endometrial carcinoma in vivo and in vitro, in which may induce cell apoptosis.

Chemotherapy resistance induced by interleukin-6 in ovarian cancer cells and its signal transduction pathways

To study the mechanism of chemotherapy resistance caused by interleukin-6 (IL-6) in ovarian cancer cells and its related signal pathways. Ovarian cancer cell lines A2780 (IL-6 receptor positive, while non-IL-6-expressing and cisplatin/paclitaxel-responsive) and SKOV3 cell lines (overexpressing of IL-6 receptor and IL-6 and cisplatin/paclitaxel-resistant) were suitable models for this study. The effect of exogenous (a short period of treatment with recombination IL-6) and endogenous IL-6 (by transfecting with plasmid encoding for sense IL-6) in A2780 cells or deleting of endogenous IL-6 expression in SKOV3 cells (by transfecting with plasmid encoding for antisense IL-6) on the sensitivity to cisplatin and paclitaxel was investigated. Meanwhile, the mechanism of chemotherapy resistance caused by IL-6 in ovarian cancer cells and its related signal pathways were also analyzed. We found that both exogenous and endogenous IL-6 induce cisplatin and paclitaxel resistance in non-IL-6-expressing A2780 cells (the resistance multiple to cisplatin and paclitaxel was: exogenous, 6.25 and 7.31; endogenous, 7.13-8.34 and 7.61-10.70), while deleting of endogenous IL-6 expression in IL-6-overexpressing SKOV3 cells promotes its sensitivity to anticancer drugs (the resistance multiple to cisplatin and paclitaxel was 0.15 and 0.10, 0.10 and 0.08). IL-6 significantly up-regulated the expression levels of mRNA and protein of drug resistance-associated genes, MDR1 and GST-π, and apoptosis-inhibiting genes, bcl-2, bcl-xL and XIAP in a dose-dependent manner in A2780 cells. In accordance with this finding, the mRNA and protein levels of MDR1 and GST-π enhanced in sense IL-6-transfected A2780 cells, and reduced in antisense IL-6-transfected SKOV3 cells compared with the corresponding parental and control vector-transfected cells, which had no difference. It was found that PD98059 [mitogen-activated protein kinase-extracellular signal-regulated kinase (MEK) inhibitor] and wortmannin [phosphatidylinositol 3-kinase (PI3K) inhibitor] significantly antagonized IL-6-induced phosphorylation of extracellular signal-regulated kinase (ERK) and protein kinase B (Akt), respectively, and both of them blocked IL-6-induced cisplatin and paclitaxel resistance and the inhibitory effects of PD98059 and wortmannin were dependent on its concentration. These data suggest that IL-6-induced chemoresistance may be associated with increase of both drug resistance-associated genes (MDR1 and GST-π) and apoptosis-inhibiting genes (bcl-2, bcl-xL and XIAP), and activation of MEK/ERK and PI3K/Akt. Therefore, modulation of IL-6 expression or its related signaling pathway may be a promising strategy of treatment for drug-resistant ovarian cancer.

The Effects of ERK1/2 Inhibitor on the Chondrogenesis of Bone Marrow– and Adipose Tissue–Derived Multipotent Mesenchymal Stromal Cells

This study tested the hypothesis that mitogen-activated protein kinase inhibitors suppress hypertrophy and enhance chondrogenesis during chondrogenesis of multipotent mesenchymal stromal cells (MSCs). The effects of PD98059 (an extracellular signal-regulated kinase-1/2 inhibitor) and SB203580 (a p38 inhibitor) were tested on bone marrow-derived MSCs (BMMSCs) and adipose tissue-derived MSCs (ATMSCs). In vitro pellet cultures were carried out using 2.5 x 10(5) MSCs in a chondrogenic medium containing 5 ng/mL of transforming growth factor-beta(2) (TGF-beta(2)) for BMMSCs, and 5 ng/mL of TGF-beta(2) and 100 ng/mL of bone morphogenetic protein-7 (BMP-7) for ATMSCs. From the 14th day of culture, the pellets were additionally treated with PD98059 or SB203580. After 14 more days of in vitro culture, pellets were harvested for analysis. PD98059 increased DNA content and glycosaminoglycan amount in BMMSCs and ATMSCs, whereas SB203580 had little effect. Collagen type I (COL1A1) mRNA decreased to almost a quarter in BMMSCs treated with PD98059. The mRNA levels of collagen type II (COL2A1) and SRY (sex determining region Y)-box 9 (SOX-9) increased several fold in both cells after PD98059 treatment, whereas SB203580 had only a slight effect. The gene expression of collagen type X (COL10A1) and runt-related transcription factor 2 (Runx-2) decreased by half after PD98059 treatment in BMMSCs, and decreased further in ATMSCs. SB203580 elevated COL10A1 and Runx-2 gene expression in both cell types. Safranin-O staining and immunohistochemistry generally mirrored findings from real-time PCR except for diminished expression of type I collagen in ATMSCS, and more pronounced decrease in type X collagen and Runx-2 in BMMSCs after PD98059 treatment. Our study demonstrated that PD98059 suppressed hypertrophy and promoted chondrogenesis of MSCs, and provides a ground for using them in cartilage tissue engineering.

Effects of MAPKK inhibitor PD98059 on the gravitropism of primary roots of maize

Gravity plays a fundamental role in plant growth and development, yet the molecular details of gravitropism is not fully understood. Here, we report the effects of PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase kinase, on the gravitropism of primary roots of maize. Unilateral application of PD98059 to horizontal roots led to different gravitropic growth. Placing PD98059-containing agar on the upper side of the root tips accelerated gravitropic curvature, whereas placing the agar on the lower side inhibited gravitropic curvature. However, no effect was detected when asymmetric application of PD98059 to vertical roots. Global application of maize primary root with PD98059 suppressed root gravitropism. Furthermore, the effects of H2O2 on horizontal root gravitropism and vertical root bending were compromised by pretreatment with PD98059. These results suggest an involvement of MAP kinase pathway(s) in gravitropism of maize roots.

Inhibition of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Decreases DNA Methylation in Colon Cancer Cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

Sustained Activation of Phosphatidylinositol 3-Kinase/Akt/Nuclear Factor κB Signaling Mediates G Protein-coupled δ-Opioid Receptor Gene Expression

Expression of the delta-opioid receptor gene (dor) is tightly controlled during neuronal differentiation and developmental stages. Such distinct temporal and spatial expression of dor during development suggests a role for the delta-opioid receptor in early developmental events. However, little is known about intracellular signaling pathways that control dor expression. A well established cell line model for the study of gene expression during neuronal differentiation is the rat adrenal pheochromocytoma PC12 cell line. Here we found that the constitutively activated TrkA/phosphatidylinositol 3-kinase/Akt (protein kinase B)/NF-kappaB survival cascade mediates dor expression during nerve growth factor (NGF)-induced differentiation of PC12h cells. Biochemical experiments showed that constitutive phosphorylation of Akt and IkappaBalpha correlates with NGF-induced dor expression. Overexpression of the transcriptional activator NF-kappaB/p65 increased dor promoter activity. Overexpression of the NF-kappaB signaling super inhibitor mutant IkappaBalpha (S32A/S36A) abolished the effect of p65 and blocked NGF-induced activation of NF-kappaB signaling, resulting in a significant reduction in dor promoter activity. Treatment with SN50, an NF-kappaB-specific nuclear translocation peptide inhibitor, inhibited the translocation of NF-kappaB, resulting in a reduction of dor mRNA. The gel shift assay supported the fact that there exists an NF-kappaB-binding site on the dor promoter. RNA interference experiments using NF-kappaB/p65 small interfering RNA confirmed that NF-kappaB signaling is required for dor expression. Our findings not only provide a new mechanistic explanation for NGF-induced dor expression but also shed some light on the molecular mechanism of the temporal and spatial expression of dor and the roles of the delta-opioid receptor during neuronal differentiation.

Possible Pathophysiological Roles of Mitogen‐Activated Protein Kinases (MAPKs) in Endometriosis

Endometriosis accompanies local inflammatory reactions in the peritoneal cavity. We examined the phosphorylation of mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated kinase (ERK), p38 MAPK (p38) and c-Jun N-terminal kinase (JNK) in endometriotic stromal cells, and their possible pathophysiological roles in endometriosis in relation to proinflammatory substances. Endometriotic stromal cells were isolated from endometriomas and were cultured for the experiments. Phosphorylation of MAPKs in endometriotic stromal cells treated with interleukin (IL)-1beta, tumor necrosis factor (TNF)alpha and H(2)O(2) were examined by Western blot analysis. Effects of PD98059, SB202190 and SP600125 (inhibitors of ERK, p38 and JNK, respectively) on IL-1beta-induced secretion of IL-6 and IL-8, and on IL-1beta-induced expression of cyclo-oxygenase-2 (COX-2) in endometriotic cells were studied. In addition, eutopic endometrial tissues were collected, and the phosphorylation rate of p38 in eutopic endometrial tissues and endometriotic tissues were determined. IL-1beta, TNFalpha and H(2)O(2) stimulated the phosphorylation of ERK, p38 and JNK, while the total amounts of proteins of the respective MAPKs were virtually the same compared with those in the unstimulated controls. Both SB202190 and SP600125 suppressed IL-1beta-induced secretion of IL-6 and IL-8, and PD98059 suppressed IL-1beta-induced secretion of IL-8. Both SB202190 and PD98059 suppressed IL-1beta-induced expression of COX-2 in endometriotic cells. The p38 phosphorylation rates in the endometriotic tissues were significantly higher than those in the eutopic endometrial tissues of the same patients. Given the current theory that inflammatory changes are involved in the progression of endometriosis, MAPKs could play as pivotal intracellular signal transducers in endometriotic cells, and thus have a pathophysiological role in the disease.

Differential effects of PD98059 and U0126 on osteogenesis and adipogenesis

PD98059 and U0126 are considered as specific inhibitors of the p42/44 mitogen-activated protein kinases (MAPK) pathway, which affects osteogenesis and adipogenesis. Here, we show unexpected differential effects of PD98059 and U0126 on osteogenesis and adipogenesis as well as on estrogen (E2)-induced actions in osteoprogenitor KS483 cells. PD98059 dose-dependently inhibited osteogenesis indicated by cellular alkaline phosphatase (ALP) activity and nodule formation, but stimulated adipogenesis shown by the number of adipocytes. In contrast, U0126 slightly decreased osteogenesis but had no effects on adipogenesis, although it inhibited p42/44 MAPK more potently than PD98059. Furthermore, PD98059, but not U0126, counteracted E2-induced osteogenesis and adipogenesis. Transfection experiments showed that PD98059, but not U0126, had estrogenic transcriptional activity. Interestingly, both PD98059 and U0126 potentiated E2-induced estrogenic transcriptional activity in KS483 cells, which is opposite to the response in MCF7 breast cancer cells. Our data indicate that the cross-talk between growth factors and estrogen receptor (ER)-mediated pathways in KS483 cells is different from that in MCF7 cells. In summary, the differential effects of PD98059 and U0126 indicate their actions are not exclusively due to an inhibition of MAPK pathway. Caution should be taken in the interpretation of the results obtained using these inhibitors.

Effects of PD98059 on proliferation of rat cultured hepatic stellate cells stimulated by acetaldehyde

AIM: To study the effects of PD98059, the specific blocking agent of MEK1, on the proliferation of hepatic stellate cells and expression of Proliferating Cell Nuclear Antigen in rat hepatic stellate cells (HSC). METHODS: HSC stimulated by acetaldehyde were cultured. The cell growth was evaluated by MTT colorimetric assay. Proliferating cell nuclear antigen (PCNA) was examined by immunocytochemical staining. RESULTS: PD98059 of 20 μmol/L had an inhibitory effect on proliferation of HSC (P <0.05, 0.109±0.020 vs 0.146±0.030), which was more obvious when cells exposed to PD98059 at 50 and 100 μmol /L (P <0.05, 0.081±0.010, 0.056±0.020 vs 0.146±0.030), and the expression of PCNA also showed a descending tendency with the increase of PD98059 concentration (P <0.05, 0.62±0.09, 0.47±0.04, 0.34±0.04 vs 0.74±0.05) CONCLUSION: PD98059 inhibits proliferation of HSC and expression of PCNA, which is correlated with the decreased activity of PCNA. Ma HD, Jiang MD, Zhong XF, Xie FW, Zeng WZ. Effects of PD98059 on proliferation of rat cultured hepatic stellate cells stimulated by acetaldehyde. Shijie Huaren Xiaohua Zazhi 2003;11(8):1182-1184 摘要 目的: 探讨特异性MEK1阻断剂PD98059对乙醛刺激的肝 星状细胞(hepatic stellate cell HSC) 增生及其细胞增生核

Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells.

The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells.

Listeria monocytogenes invasion of epithelial cells requires the MEK-1/ERK-2 mitogen-activated protein kinase pathway.

PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blocked Listeria monocytogenes invasion into HeLa epithelial cells. The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug. Previously, we reported that L. monocytogenes could activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P. Tang, I. Rosenshine, P. Cossart, and B. B. Finlay, Infect. Immun. 64:2359-2361, 1996). We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-type L. monocytogenes. Mutants lacking functional LLO (hly mutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times. Two inhibitors of L. monocytogenes invasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-type L. monocytogenes and hly mutants. However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation. These results suggest that MEK-1 and ERK-2 activities are essential for L. monocytogenes invasion into host epithelial cells. This is the first report to show that a MAP kinase pathway is required for bacterial invasion.