JB6 Cl41 Research Articles

Role of NEK6 in Tumor Promoter-induced Transformation in JB6 C141 Mouse Skin Epidermal Cells

NEK6 (NIMA-related kinase 6) is a homologue of the Aspergillus nidulans protein NIMA (never in mitosis, gene A). We demonstrate that overexpression of NEK6 induces anchorage-independent transformation of JB6 Cl41 mouse epidermal cells. Tissue arrays and Western immunoblot analysis show that NEK6 is overexpressed in malignant tissues and several cancer cell lines. Our data also show that NEK6 interacts with STAT3, an oncogenic transcription factor, and phosphorylates STAT3 on Ser(727), which is important for transcriptional activation. Additional studies using NEK6 mutants suggested that the phosphorylation on both Ser(206) and Thr(210) of NEK6 is critical for STAT3 phosphorylation and anchorage-independent transformation of mouse epidermal cells. Notably, knockdown of NEK6 decreased colony formation and STAT3 Ser(727) phosphorylation. Based on our findings, the most likely mechanism that can account for this biological effect involves the activation of STAT3 through the phosphorylation on Ser(727). Because of the critical role that STAT3 plays in mediating oncogenesis, the stimulatory effects of NEK6 on STAT3 and cell transformation suggest that this family of serine/threonine kinases might represent a novel chemotherapeutic target.

Cancer‐preventive activities of secondary metabolites from leaves of the bilberry Vaccinium smallii A. Gray

Ten secondary metabolites including flavonoids (1-8), caffeic (9) and chlorogenic (10) acids were structurally identified from the extract of Sakhalin bilberry Vaccinium smallii leaves and studied in vitro as potential cancer-preventive agents. The results showed that compounds 1-10 inhibited EGF-induced neoplastic transformation of mouse JB6 Cl 41 P+ cells in soft agar with an inhibition concentration (INCC50) of 20-80 µm. Moreover, all these natural products were non-toxic against JB6 Cl 41 P+ cells up to a concentration of 200 µm.

P27 Suppresses Arsenite-induced Hsp27/Hsp70 Expression through Inhibiting JNK2/c-Jun- and HSF-1-dependent Pathways

p27 is an atypical tumor suppressor that can regulate the activity of cyclin-dependent kinases and G(0)-to-S phase transitions. More recent studies reveal that p27 may also exhibit its tumor-suppressive function through regulating many other essential cellular events. However, the molecular mechanisms underlying these anticancer effects of p27 are largely unknown. In this study, we found that depletion of p27 expression by either gene knock-out or knockdown approaches resulted in up-regulation of both Hsp27 and Hsp70 expression at mRNA- and promoter-derived transcription as well as protein levels upon arsenite exposure, indicating that p27 provides a negative signal for regulating the expression of Hsp27 and Hsp70. Consistently, arsenite-induced activation of JNK2/c-Jun and HSF-1 pathways was also markedly elevated in p27 knock-out (p27(-/-)) and knockdown (p27 shRNA) cells. Moreover, interference with the expression or function of JNK2, c-Jun, and HSF-1, but not JNK1, led to dramatic inhibition of arsenite-induced Hsp27 and Hsp70 expression. Collectively, our results demonstrate that p27 suppresses Hsp27 and Hsp70 expression at the transcriptional level specifically through JNK2/c-Jun- and HSF-1-dependent pathways upon arsenite exposure, which provides additional important molecular mechanisms for the tumor-suppressive function of p27.

Abstract 1227: Inhibition of the RSK2-ATF1 signaling axis by eriodictyol suppresses neoplastic cell transformation

Abstract The ribosomal S6 kinase 2 (RSK2), a member of the p90RSK (RSK) family of proteins, is a widely expressed serine/threonine kinase and its activation enhances cell proliferation. Here we report that activating transcription factor 1(ATF1) is a novel substrate of RSK2 and the RSK2-ATF1 signaling axis plays an important role in neoplastic cell transformation. RSK2 phosphorylates ATF1 at Ser63 and enhances the transactivation and transcriptional activities of ATF1. Computational modeling, high-through put screening and in vitro pull down assays demonstrated that eriodictiol, a flavanone found in fruits, binds with the N-terminal kinase domain and linker region of RSK2 and inhibits RSK2 N-terminal kinase activity. In a cell culture system, eriodictyol treatment suppressed phosphorylation of ATF1, but did not affect phosphorylation of RSK, MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol specifically inhibits RSK2 signaling. Furthermore, eriodictyol inhibited RSK2-mediated ATF1 transactivation activity and cell transformation induced by tumor promoters in JB6 Cl41 mouse skin epidermal cells. In a foci formation assay, knockdown of RSK2 or ATF1 suppressed foci formation compared with RasG12V, RasG12V/RSK2, RasG12V/ATF1 and RasG12V/RSK2/ATF1 expressing cells. In addition, eriodictyol treatment showed the same effect as RSK2 knockdown in foci formation. Taken together, these results indicated that the RSK2-ATF1 signaling axis plays an important role in neoplastic cell transformation and eriodictyol is a new natural compound for selectively inhibiting RSK2 kinase activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1227.

Extracellular Signal-Regulated Kinase 8–Mediated c-Jun Phosphorylation Increases Tumorigenesis of Human Colon Cancer

Extracellular signal-regulated kinase 8 (ERK8), a recently discovered member of the mitogen-activated protein kinase protein family, has been less studied than other family members, leaving its physiologic functions mostly unknown. The biological consequences of overexpression of ERK8 in JB6 Cl41 epidermal skin cells or knockdown of ERK8 in HCT15 colorectal cancer cells was studied. Kinase assays and transient transfection experiments were performed to study the signaling pathway between ERK8 and c-Jun. We found that ERK8 is relatively highly expressed in HCT15 human colorectal cancer cells and plays an important role in the promotion and progression of colorectal cancer. ERK8 promoted neoplastic transformation, and knockdown of ERK8 in HCT15 colorectal cancer cells reduced the tumorigenic properties of these cell lines. Furthermore, a direct interaction between ERK8 and c-Jun was shown. With epidermal growth factor treatment, overexpression of ERK8 in JB6 Cl41 cells caused an increased phosphorylation of c-Jun at Ser(63) and Ser(73), resulting in increased activator protein-1 transactivation. In contrast, knockdown of ERK8 in HCT15 colorectal cancer cells blocked c-Jun phosphorylation. The interaction between ERK8 and c-Jun seems to increase the tumorigenic properties of HCT15 colorectal cancer cells. Thus, ERK8-regulated signaling might serve as a potential therapeutic target in colorectal cancer.

Two new asterosaponins, archasterosides A and B, from the Vietnamese starfish Archaster typicus and their anticancer properties

New asterosaponins archasterosides A (1), B (2), and the known regularoside A (3) were isolated from the Vietnamese starfish Archaster typicus and structurally elucidated by extensive NMR techniques and chemical transformations. Compounds 1–3 showed moderate cytotoxic activities against HeLa and mouse JB6 P+ Cl41 cell lines. The most active, 2, induced basal AP-1- and p53-, but not NF-κB-transcriptional activations in JB6 Cl41 cells.

Cr(VI) induces mitochondrial-mediated and caspase-dependent apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41 cells

Cr(VI) compounds are known to cause serious toxic and carcinogenic effects. Cr(VI) exposure can lead to a severe damage to the skin, but the mechanisms involved in the Cr(VI)-mediated toxicity in the skin are unclear. The present study examined whether Cr(VI) induces cell death by apoptosis or necrosis using mouse skin epidermal cell line, JB6 Cl41 cells. We also investigated the cellular mechanisms of Cr(VI)-induced cell death. This study showed that Cr(VI) induced apoptotic cell death in a dose-dependent manner, as demonstrated by the appearance of cell shrinkage, the migration of cells into the sub-G1 phase, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cr(VI) treatment resulted in the increases of mitochondrial membrane depolarization and caspases activation. Electron spin resonance (ESR) and fluorescence analysis revealed that Cr(VI) increased intracellular levels of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion radical in dose-dependent manner. Blockage of p53 by si-RNA transfection suppressed mitochondrial changes of Bcl-2 family composition, mitochondrial membrane depolarization, caspase activation and PARP cleavage, leading to the inhibition of Cr(VI)-induced apoptosis. Further, catalase treatment prevented p53 phosphorylation stimulated by Cr(VI) with the concomitant inhibition of caspase activation. These results suggest that Cr(VI) induced a mitochondrial-mediated and caspase-dependent apoptosis in skin epidermal cells through activation of p53, which are mainly mediated by reactive oxidants generated by the chemical.

The Prolyl Isomerase Pin1 Enhances HER-2 Expression and Cellular Transformation via Its Interaction with Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Kinase 1

The HER-2 oncogene, a member of the erythroblastosis oncogene B (ERBB)-like oncogene family, has been shown to be amplified in many types of cancer, including breast cancer. However, the molecular mechanism of HER-2 overexpression is not completely understood. The phosphorylation of proteins on the serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the prolyl isomerase Pin1 and is a key signaling mechanism in cell proliferation and transformation. Here, we found that Pin1 interacts with mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) protein kinase 1, resulting in the induction of HER-2 expression. Pin1(-/-) mouse embryonic fibroblasts exhibited a decrease in epidermal growth factor (EGF)-induced MEK1/2 phosphorylation compared with Pin1(+/+) mouse embryonic fibroblast. In addition, a knockdown of Pin1 resulted in the inhibition of MEK1/2 phosphorylation induced by EGF in MCF-7 cells. Furthermore, PD98059, a specific inhibitor of MEK1/2, and Juglone, a potent Pin1 inhibitor, markedly suppressed the expression of activator protein-2alpha and the HER-2 promoter activity induced by EGF or 12-O-tetradecanoylphorbol-13-acetate in MCF-7 cells. Importantly, these inhibitors inhibited the neoplastic cell transformation induced by EGF in Pin1-overexpressing JB6 Cl41 cells, which showed enhanced cellular formation compared with the control cells. Therefore, Juglone and PD98059 inhibited the colony formation of MCF-7 breast cancer cells in soft agar. These results indicate that Pin1 amplifies EGF signaling in breast cancer cells through its interaction with MEK1 and then enhances HER-2 expression, suggesting that Pin1 plays an important role in the overexpression of HER-2 through Pin1-MEK1-activator protein-2alpha signaling in breast cancer.

Phenolic fraction of tobacco smoke condensate potentiates benzo[a]pyerene diol epoxide-induced cell transformation: Role of protein kinase C

In this study we separated weakly acidic phenolic components from other neutral, acidic and basic components of tobacco smoke condensate (TSC) and observed that phenolic fraction of TSC significantly increased the number of colonies of promotion-sensitive JB6 Cl41 cells that showed anchorage-independent growth on soft agar in response to BPDE (an ultimate carcinogen produced by metabolic activation of the PAH benzo[a]pyrene). Anchorage-independent cell growth is indicative of cell transformation resulting in acquisition of tumorigenic potential. In order to understand the underlying mechanism by which TSC phenolic fraction potentiates BPDE-induced tumorigenicity, we examined its effect on the activation of two transcription factors AP-1 and NF-κB which are known to be influenced by established tumor promoter TPA. BPDE treatment caused induction of both AP-1 and NF-κB activity as determined by luciferase reporter assay and only NF-κB induction in response to BPDE was significantly attenuated by TSC phenolic fraction whereas AP-1 induction remains unaltered. Attenuation of NF-κB activation by TSC phenolic fraction was associated with significant decrease of intracellular PKC substrate phosphorylation in BPDE treated cells. Non-specific PKC inhibitors staurosporine and bisindolylmaleimide II as well as inhibitors specific to conventional PKCs (Go6976) and PKC-δ (rottlerin) attenuated NF-κB activation in BPDE treated cells to a varying degree indicating a possible link between PKC down-regulation and the attenuation of NF-κB activity by TSC phenolic fraction. Treatment of cells with PKC inhibitors also potentiated anchorage-independent growth of BPDE treated cells on soft agar. Our data suggest a possible role of PKC down-regulation in potentiation of BPDE-induced tumorogenicity by TSC phenolic fraction.

24-Hydroxyursolic Acid from the Leaves of theDiospyros kaki(Persimmon) Induces Apoptosis by Activation of AMP-activated Protein Kinase

There are multiple lines of evidence that persimmon extract and its constituents have potent antitumor activity against human cancer cells. However, the molecular mechanisms of 24-hydroxyursolic acid, a triterpenoid found in persimmon, on antitumor activities are not yet understood. Here, we demonstrate that 24-hydroxyursolic acid inhibited cell proliferation, strongly activated AMP-activated protein kinase (AMPK) and mediated critical anticancer effects by inhibition of cyclooxygenase (COX-2) expression in HT-29 cells. In addition, 24-hydroxyursolic acid induced cellular apoptosis by activation of poly(ADP-ribose) polymerase (PARP), caspase-3, and phosphorylation of p53 at Ser15. It also strongly induced DNA fragmentation in HT-29 cells and thereby significantly inhibited colony formation of HT-29 cells in soft agar. In addition, 24-hydroxyursolic acid blocked the EGF-induced ERKs phosphorylation and led to the inhibition of AP-1 activity and cell transformation in JB6 CL41 cells. Collectively, these findings are the first to reveal a molecular basis for the anticarcinogenic action of 24-hydroxyursolic acid and might account for the reported chemopreventive and chemotherapic effects of persimmon extracts.

Abstract A45: The prolyl isomerase Pin1 interacts with a ribosomal protein S6 kinase to enhance insulin-induced AP-1 activity and cellular transformation

Abstract Phosphorylation of proteins on serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis/trans isomerase Pin1 and is a central signaling mechanism in cell proliferation and transformation. Although Pin1 is frequently overexpressed in hepatocellular carcinoma (HCC), the molecular mechanism of Pin1 in HCC has not been completely elucidated. Here, we show that Pin1 interacts with p70S6K in vitro and ex vivo. Overexpression of Pin1 resulted in enhanced p70S6K phosphorylation induced by insulin in SK-HEP-1 cells. In contrast, Pin1−/− mouse embryonic fibroblasts (MEFs) exhibited significantly decreased insulin-induced p70S6K phosphorylation compared to Pin1+/+ MEFs. Furthermore, Pin1 enhanced the insulin-induced ERK1/2 phosphorylation through its interaction with p70S6K, whereas the inhibition of p70S6K activity by rapamycin suppressed insulin-induced ERK1/2 phosphorylation in SK-HEP-1 cells. Hence, Pin1 affected AP-1 activity through p70S6K-ERK1/2 signaling in SK-HEP-1 cells. Most importantly, Pin1-overexpressing JB6 Cl41 cells enhanced neoplastic cell transformation promoted by insulin much more than GFP-overexpressing JB6 Cl41 control cells. These results imply that Pin1 amplifies insulin signaling in hepatocarcinoma cells through its interaction with p70S6K, suggesting that Pin1 plays an important role in insulin-induced tumorigenesis and is a potential therapeutic target in hepatocarcinoma. Citation Information: Cancer Res 2009;69(23 Suppl):A45.

The anticancer effects of actinoporin RTX-A from the sea anemone Heteractis crispa (= Radianthus macrodactylus)

Four isoforms of actinoporins were isolated in 2002–2004 from the tropical sea anemone Heteractis crispa (= Radianthus macrodactylus). Their potent hemolytic activities and effects on Ehrlich ascites carcinoma bearing mice were also studied. In this study, the individual actinoporin (RTX-A) demonstrated potential cancer-preventive activity at extremely low and non-cytotoxic concentrations. The substance suppressed the malignant transformation of mouse JB6 P + Cl41 cells stimulated by epidermal growth factor (EGF) in soft agar with the inhibition of number of the colonies C 50 (INCC 50) = 0.034 nM. Actinoporin RTX-A also was shown to inhibit the phenotype expression of HeLa human cancer cells with an INCC 50 = 0.03 nM. The cytotoxic effect of RTX-A against JB6 P + Cl41 cells and HeLa, THP-1, MDA-MB-231, and SNU-C4 human tumor cell lines was high (IC 50 = 0.57, 2.26, 1.11, 30.0 and 4.66 nM), but significantly less than their capacity to suppress tumor cell colony formation or phenotype expression. RTX-A also induced apoptosis and inhibited basal AP-1, NF-κB, and p53-dependent transcriptional activity in JB6 Cl41 cells. These results confirmed that actinoporin RTX-A from H. crispa, at least partially, might exhibit cancer-preventive and anticancer cytotoxic properties through the induction of p53-independent apoptosis and inhibition of the oncogenic AP-1 and NF-κB nuclear factors activity.

Cadmium Induces Intracellular Ca2+- and H2O2-Dependent Apoptosis through JNK- and p53-Mediated Pathways in Skin Epidermal Cell line

Cadmium is a toxic heavy metal and has been widely used in industry. The skin is an important target for this metal. The mechanisms by which cadmium leads to damage to the skin are unclear at present. The aims of this study were to examine whether cadmium induces apoptosis in mouse skin epidermal cell line, JB6 Cl41 cells, and to investigate the cellular mechanisms by which cadmium causes cytotoxicity in the cells. The present study showed that cadmium induced cell death by apoptosis in a dose-dependent manner, as proven by the appearance of cell shrinkage, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cadmium-induced apoptosis involved a mitochondria-mediated mechanism but not caspase-dependent pathway in that the critical apoptotic events induced by cadmium, such as the decrease of Bcl-2/Bcl-xL, the increase of GADD45alpha, and the nuclear translocation of apoptosis inducing factor, were not affected by the inhibition of executive caspases. In contrast, blockage of p53 and JNK by pharmacological inhibitors or small interference RNA transfection suppressed the cadmium-induced apoptosis with the concomitant inhibition of antiapoptotic Bcl-2 family proteins and GADD45alpha, respectively. Furthermore, the activation of p53 and JNK and their downstream proteins in cadmium-exposed cells were inhibited by individual treatment with catalase and Bapta-acetoxymethyl. These results suggest that cadmium induces apoptosis via the activation of JNK- and p53-mediated signaling, where calcium ion and hydrogen peroxide act as the pivotal mediators of the apoptotic signaling.

The prolyl isomerase Pin1 interacts with a ribosomal protein S6 kinase to enhance insulin-induced AP-1 activity and cellular transformation

Phosphorylation of proteins on serine or threonine residues that immediately precede proline (pSer/Thr-Pro) is specifically catalyzed by the peptidyl-prolyl cis-trans isomerase Pin1 and is a central signaling mechanism in cell proliferation and transformation. Although Pin1 is frequently overexpressed in hepatocellular carcinoma (HCC), the molecular mechanism of Pin1 in HCC has not been completely elucidated. Here, we show that Pin1 interacts with p70S6K in vitro and ex vivo. Overexpression of Pin1 resulted in enhanced p70S6K phosphorylation induced by insulin in SK-HEP-1 cells. In contrast, Pin1(-/-) mouse embryonic fibroblasts (MEFs) exhibited significantly decreased insulin-induced p70S6K phosphorylation compared with Pin1(+/+) MEFs. Furthermore, Pin1 enhanced the insulin-induced extracellular signal-regulated protein kinase (ERK)1/2 phosphorylation through its interaction with p70S6K, whereas the inhibition of p70S6K activity by rapamycin suppressed insulin-induced ERK1/2 phosphorylation in SK-HEP-1 cells. Hence, Pin1 affected activator protein-1 activity through p70S6K-ERK1/2 signaling in SK-HEP-1 cells. Most importantly, Pin1-overexpressing JB6 Cl41 cells enhanced neoplastic cell transformation promoted by insulin much more than green fluorescent protein-overexpressing JB6 Cl41 control cells. These results imply that Pin1 amplifies insulin signaling in hepatocarcinoma cells through its interaction with p70S6K, suggesting that Pin1 plays an important role in insulin-induced tumorigenesis and is a potential therapeutic target in hepatocarcinoma.

Cyclin-Dependent Kinase-3–Mediated c-Jun Phosphorylation at Ser63 and Ser73 Enhances Cell Transformation

c-Jun is a component of the activator protein-1 (AP-1) complex, which plays a crucial role in the regulation of gene expression, cell proliferation, and cell transformation, as well as cancer development. Herein, we found that cyclin-dependent kinase (Cdk)-3, but not Cdk2 or c-Jun NH(2)-terminal kinase, is a novel kinase of c-Jun induced by stimulation with growth factors such as epidermal growth factor (EGF). Cdk3 was shown to phosphorylate c-Jun at Ser63 and Ser73 in vitro and ex vivo. EGF-induced Cdk3 activation caused c-Jun phosphorylation at Ser63 and Ser73, resulting in increased AP-1 transactivation. Ectopic expression of Cdk3 resulted in anchorage-independent cell transformation of JB6 Cl41 cells induced by EGF and foci formation stimulated by constitutively active Ras (Ras(G12V)), which was mediated by AP-1 in NIH3T3 cells. These results showed that the Cdk3/c-Jun signaling axis plays an important role in EGF-stimulated cell proliferation and cell transformation.

C-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.

Proapoptotic and Anticarcinogenic Activities of Leviusculoside G from the Starfish Henricia leviuscula and Probable Molecular Mechanism

Proapoptotic and anticarcinogenic properties of leviusculoside G (LSG) isolated from the starfish Henricia leviuscula and a probable mechanism of these effects were investigated in human leukemia HL-60, THP-1, and mouse skin JB6 Cl41 cells, using a variety of assessments, including a cell viability (MTS) assay, flow cytometry, anchorage-independent soft agar assay, luciferase assay, and western-blotting. Application of LSG at nontoxic doses induced apoptosis in cancer cells and decreased cell transformation. This compound exerted its actions, at least in part, through the induction of p53-dependent apoptosis and inhibition of AP-1, NF-κB, and ERKs activities.

Cyclin-Dependent Kinase 3–Mediated Activating Transcription Factor 1 Phosphorylation Enhances Cell Transformation

Cyclin-dependent kinase (cdk)-3, a member of the cdk family of kinases, plays a critical role in cell cycle regulation and is involved in G(0)-G(1) and G(1)-S cell cycle transitions. However, the role of cdk3 in cell proliferation, as well as cell transformation, is not yet clearly understood. Here, we report that the protein expression level of cdk3 is higher in human cancer cell lines and human glioblastoma tissue compared with normal brain tissue. Furthermore, we found that cdk3 phosphorylates activating transcription factor 1 (ATF1) at serine 63 and enhances the transactivation and transcriptional activities of ATF1. Results also indicated that siRNA directed against cdk3 (si-cdk3) suppresses ATF1 activity, resulting in inhibition of proliferation and growth of human glioblastoma T98G cells in soft agar. Importantly, we showed that cdk3 enhances epidermal growth factor-induced transformation of JB6 Cl41 cells and si-cdk3 suppresses Ras(G12V)/cdk3/ATF1-induced foci formation in NIH3T3 cells. These results clearly showed that the cdk3-ATF1 signaling axis is critical for cell proliferation and transformation.

Mitogen- and Stress-Activated Kinase 1–Mediated Histone H3 Phosphorylation Is Crucial for Cell Transformation

Mitogen- and stress-activated kinase 1 (MSK1) belongs to a family of dual protein kinases that are activated by either extracellular signal-regulated kinase or p38 mitogen-activated protein kinases in response to stress or mitogenic extracellular stimuli. The physiologic role of MSK1 in malignant transformation and cancer development is not well understood. Here, we report that MSK1 is involved in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced or epidermal growth factor (EGF)-induced neoplastic transformation of JB6 Cl41 cells. H89, a potent inhibitor of MSK1, strongly suppressed TPA-induced or EGF-induced cell transformation. When cells overexpressing wild-type MSK1 were treated with TPA or EGF, colony formation increased substantially compared with untreated cells or cells that did not overexpress MSK1. In contrast, MSK1 COOH terminal or NH(2) terminal dead dominant negative mutants dramatically suppressed cell transformation. Introduction of small interfering RNA-MSK1 into JB6 Cl41 cells resulted in suppressed TPA-induced or EGF-induced cell transformation. In addition, cell proliferation was inhibited in MSK1 knockdown cells compared with MSK1 wild-type cells. In wild-type MSK1-overexpressing cells, activator protein (AP-1) activation increased after TPA or EGF stimulation, whereas AP-1 activation decreased in both MSK1 dominant-negative mutants and in MSK1 knockdown cells. Moreover, TPA-induced or EGF-induced phosphorylation of histone H3 at Ser(10) was increased in wild-type cells but the induced phosphorylation was abolished in MSK1 dominant-negative mutant or MSK1 knockdown cells. Thus, MSK1 is required for tumor promoter-induced cell transformation through its phosphorylation of histone H3 at Ser(10) and AP-1 activation.

Inhibitory effects of fucoidan on activation of epidermal growth factor receptor and cell transformation in JB6 Cl41 cells

Algal fucoidan is a marine sulfated polysaccharide with a wide variety of biological activities including anti-thrombotic, anti-inflammatory, and anti-tumor activities. In this study, we tested the hypothesis that fucoidan may suppress neoplastic cell transformation by inhibiting the phosphorylation of epidermal growth factor receptor (EGFR) in mouse epidermal JB6 Cl41 cells. Our results provided the first evidence that fucoidan from Laminaria guryanovae exerted a potent inhibitory effect on EGF-induced phosphorylation of EGFR. Consistent with its inhibitory action on phosphorylation of EGFR, fucoidan clearly suppressed the phosphorylation of extracellular signal-regulated kinase or c-jun N-terminal kinases induced by EGF. Moreover, EGF-induced the c-fos and c-jun transcriptional activities were inhibited by fucoidan, resulting to suppressing of activator protein-1 (AP-1) activity and cell transformation induced by EGF. Taken together, these results indicate that fucoidan might exert chemopreventive effects through the inhibition of phosphorylation of the EGFR.