Cryptotanshinone Prevents the Binding of S6K1 to mTOR/Raptor Leading to the Suppression of mTORC1-S6K1 Signaling Activity and Neoplastic Cell Transformation.

Cryptotanshinone is known for its inhibitory activity against tumorigenesis in various human cancer cells. However, exact mechanisms underlying the anticancer effects of cryptotanshinone are not fully elucidated. Here, we propose a plausible molecular mechanism, wherein cryptotanshinone represses rapamycin-sensitive mTORC1/S6K1 mediated cancer cell growth and cell transformation. We investigated the various effects of cryptotanshinone on the mTORC1/S6K1 axis, which is associated with the regulation of cell growth in response to nutritional and growth factor signals. We found that cryptotanshinone specifically inhibited the mTORC1-mediated phosphorylation of S6K1, which consequently suppressed the clonogenicity of SK-Hep1 cells and the neoplastic transformation of JB6 Cl41 cells induced by insulin-like growth factor-1. Finally, we observed that cryptotanshinone prevented S6K1 from binding to the Raptor/mTOR complex, rather than regulating mTOR and its upstream pathway. Overall, our findings provide a novel mechanism underlying anti-cancer effects cryptotanshinone targeting mTORC1 signaling, contributing to the development of anticancer agents involving metabolic cancer treatment.

Enhancing the in vitro anticancer activity of albendazole incorporated into chitosan-coated PLGA nanoparticles.

To improve the solubility and anticancer activity of albendazole (ABZ), chitosan (CS)-coated poly-dl-lactic-co-glycolic acid (PLGA) nanoparticles were developed. CS was used to coat ABZ-loaded PLGA nanoparticles to enhance both mucoadhesiveness and colloidal stability. CS-coated PLGA nanoparticles were prepared by suspending the nanoparticles in CS solution after solvent diffusion. The CS-coated PLGA nanoparticles were characterized, and ABZ release was studied in vitro from various formulations. The mucoadhesive properties and in vitro anticancer activities of CS-coated PLGA nanoparticles were investigated by measurement of zeta potentials and the MTT assay, respectively. Spherical nanoparticles below 500nm in diameter were successfully prepared; the particle size distribution was narrow. Complete encapsulation of ABZ in CS-coated PLGA nanoparticles was confirmed by SEM, FTIR, DSC, and XRD. The particle sizes of CS-coated PLGA nanoparticles were in the range of 260-480nm; the encapsulation efficiency was 43.4-54.6%; and the yield 58.5-67.8%. The zeta potential of CS-coated nanoparticles was above +27mV and stability was maintained for 4 weeks. At pH 7.4, the in vitro release of ABZ from nanoparticles (P188-5) was 200-fold higher than that from untreated ABZ; this persisted for 12h. Moreover, ABZ release from CS-coated PLGA nanoparticles (P188-CS0.5) was 1.5-fold higher than that from untreated ABZ at pH 1.2. Additionally, the ABZ-loaded CS-coated nanoparticles exhibited superior mucoadhesion and improved cytotoxicity. The results show that CS coating of PLGA nanoparticles may improve the anticancer effect and the mucoadhesive properties of ABZ-loaded nanoparticles.

ERK1 Directly Interacts With JNK1 Leading to Regulation of JNK1/c-Jun Activity and Cell Transformation.

ERK1 and ERK2 share a great deal of homology and have been presumed to have similar functions. Available antibodies recognize both isoforms making the elucidation of functional differences challenging. Mitogen-activated protein (MAP) kinase networks are commonly depicted in the literature as linear and sequential phosphorylation cascades; however, the activation of these pathways is not mutually exclusive. Little doubt exists that MAP kinases engage in crosstalk, but the extent or the direct effect of these "conversations" is unclear. Here, we report the possible points of direct interaction as "crosstalk" points between ERK1 and JNK1 and a potential mechanism for ERK1 function in repressing Ras/JNK-mediated cell transformation. ERK1, but not ERK2, directly interacts with and antagonizes JNK1 phosphorylation and activity, resulting in suppression of neoplastic cell transformation mediated by the Ras/JNK/c-Jun signaling pathway. Interestingly, ERK1 phosphorylation was increased in normal tissues compared to liver cancer tissues. Furthermore, predominant JNK/c-Jun activation was observed in liver cancer tissues. These phenomena can provide evidence for the existence of a functional association between ERK and JNK signaling pathways during in vivo tumorigenesis. Overall, our findings provide new evidence supporting the paradigm of an ERK1/JNK1 antagonistic interaction as a novel mechanism of trans-regulation between different MAP kinase signaling modules. J. Cell. Biochem. 118: 2357-2370, 2017. © 2017 Wiley Periodicals, Inc.

Anti-Inflammatory Effect of Ascochlorin in LPS-Stimulated RAW 264.7 Macrophage Cells Is Accompanied With the Down-Regulation of iNOS, COX-2 and Proinflammatory Cytokines Through NF-κB, ERK1/2, and p38 Signaling Pathway.

A natural compound C23 H32 O4 Cl, ascochlorin (ASC) isolated from an incomplete fungus, Ascochyta viciae has been known to have several biological activities as an antibiotic, antifungal, anti-cancer, anti-hypolipidemic, and anti-hypertension agent. In this study, anti-inflammatory activity has been investigated in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cells, since ASC has not been observed on the inflammatory events. The present study has clearly shown that ASC (1-50 µM) significantly suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2 ) and decreased the gene expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in a dose-dependent manner. Moreover, ASC inhibited the mRNA expression and the protein secretion of interleukin (IL)-1ß and IL-6 but not tumor necrosis factor (TNF)-α in LPS-stimulated RAW 264.7 macrophage cells. In addition, ASC suppressed nuclear translocation and DNA binding affinity of nuclear factor-κB (NF-κB). Furthermore, ASC down-regulated phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-p38. These results demonstrate that ASC exhibits anti-inflammatory effects in RAW 264.7 macrophage cells.

Prolyl isomerase PIN1 negatively regulates SGK1 stability to mediate tamoxifen resistance in breast cancer cells.

BACKGROUND/AIM: Endocrine therapies that inhibit oestrogen receptor (ER)-α signaling are the most common and effective treatment for ER-α-positive breast cancer. The present study aimed to elucidate the mechanisms by which down-regulation of serum- and glucocorticoid-inducible protein kinase-1 (SGK1) expression confers tamoxifen resistance in breast cancer. MATERIALS AND METHODS: SGK1 expression and the cytotoxic effects of combinatorial 4-hydroxy-tamoxifen (4-OHT) treatment with SGK1 overexpression were investigated by immunoblotting, bromodeoxyuridine incorporation, and soft agar assay. RESULTS: We showed that PIN1 down-regulates SGK1 expression through interaction with and ubiquitination of SGK1. PIN1 silencing in MCF7 cells increased SGK1 expression. In tamoxifen-resistant human breast cancer, immunohistochemical staining analysis showed an inverse correlation between SGK1 expression and severity of tamoxifen resistance. Importantly, 4-OHT in combination with overexpression of SGK1 increased cleavage of poly-(ADP-ribose) polymerase and DNA fragmentation to inhibit clonogenic growth of tamoxifen-resistant MCF7 (TAMR-MCF7) cells. CONCLUSION: We suggest that PIN1-mediated SGK1 ubiquitination is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

Combined delivery of the adiponectin gene and rosiglitazone using cationic lipid emulsions.

For the combined delivery of an insulin-sensitizing adipokine; i.e., the ADN gene, and the potent PPARγ agonist rosiglitazone, cationic lipid emulsions were formulated using the cationic lipid DOTAP, helper lipid DOPE, castor oil, Tween 20 and Tween 80. The effect of drug loading on the physicochemical characteristics of the cationic emulsion/DNA complexes was investigated. Complex formation between the cationic emulsion and negatively charged plasmid DNA was confirmed and protection from DNase was observed. The in vitro transfection efficiency and cytotoxicity were evaluated in HepG2 cells. The particle sizes of the cationic emulsion/DNA complex were in the range 230-540 nm and those of the rosiglitazone-loaded cationic emulsion/DNA complex were in the range 220-340 nm. Gel retardation of the complexes was observed when the complexation weight ratios of the cationic lipid to plasmid DNA exceeded 4:1 for both the drug-free and rosiglitazone-loaded complexes. Both complexes stabilized plasmid DNA against DNase. The ADN expression level increased dose-dependently when cells were transfected with the cationic emulsion/DNA complexes. The rosiglitazone-loaded cationic emulsion/DNA complexes showed higher cellular uptake in HepG2 cells depending on the rosiglitazone loading, but not depending on the type of plasmid DNA type such as pVAX/ADN, pCAG/ADN, or pVAX. The drug-loaded cationic emulsion/plasmid DNA complexes were less cytotoxic than free rosiglitazone. Therefore, a cationic emulsion could potentially serve as a co-delivery system for rosiglitazone and the adiponectin gene.

Exploring the Preparation of Albendazole-Loaded Chitosan-Tripolyphosphate Nanoparticles.

The objective of this study was to improve the solubility of albendazole and optimize the preparation of an oral nanoparticle formulation, using ß-cyclodextrin (ßCD) and chitosan-tripolyphosphate (TPP) nanoparticles. The solubility of albendazole in buffers, surfactants, and various concentrations of acetic acid solution was investigated. To determine drug loading, the cytotoxic effects of the albendazole concentration in human hepatocellular carcinoma cells (HepG2) were investigated. The formulations were prepared by mixing the drug solution in Tween 20 with the chitosan solution. TPP solution was added dropwise with sonication to produce a nanoparticle through ionic crosslinking. Then the particle size, polydispersity index, and zeta potential of the nanoparticles were investigated to obtain an optimal composition. The solubility of albendazole was greater in pH 2 buffer, Tween 20, and ßCD depending on the concentration of acetic acid. Drug loading was determined as 100 µg/mL based on the results of cell viability. The optimized ratio of Tween 20, chitosan/hydroxypropyl ßCD, and TPP was 2:5:1, which resulted in smaller particle size and proper zeta positive values of the zeta potential. The chitosan-TPP nanoparticles increased the drug solubility and had a small particle size with homogeneity in formulating albendazole as a potential anticancer agent.

Thermosensitive and mucoadhesive sol-gel composites of paclitaxel/dimethyl-ß-cyclodextrin for buccal delivery.

The purpose of this study was to develop a buccal paclitaxel delivery system using the thermosensitive polymer Pluronic F127 (PF127) and the mucoadhesive polymer polyethylene oxide (PEO). The anticancer agent paclitaxel is usually used to treat ovarian, breast, and non-small-cell lung cancer. To improve its aqueous solubility, paclitaxel was incorporated into an inclusion complex with (2,6-di-O-methyl)-ß-cyclodextrin (DMßCD). The formation of the paclitaxel inclusion complex was evaluated using various techniques, including x-ray diffractometry (XRD), Fourier-transform infrared (FT-IR) spectrophotometry, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM). Hydrogels were prepared using a cold method. Concentrations of 18, 20, and 23% (w/v) PF127 were dissolved in distilled water including paclitaxel and stored overnight in a refrigerator at 4 °C. PEO was added at concentrations of 0.1, 0.2, 0.4, 0.8, and 1% (w/v). Each formulation included paclitaxel (0.5 mg/mL). The sol-gel transition temperature of the hydrogels was measured using the tube-inverting method. Drug release from the hydrogels was measured using a Franz diffusion cell containing pH 7.4 phosphate-buffered solution (PBS) buffer at 37 °C. The cytotoxicity of each formulation was measured using the MTT assay with a human oral cancer cell (KB cell). The sol-gel transition temperature of the hydrogel decreased when PF127 was present and varied according to the presence of mucoadhesive polymers. The in vitro release was sustained and the release rate was slowed by the addition of the mucoadhesive polymer. The cytotoxicity of the blank formulation was low, although the drug-loaded hydrogel showed acceptable cytotoxicity. The results of our study suggest that the combination of a PF 127-based mucoadhesive hydrogel formulation and inclusion complexes improves the in vitro release and cytotoxic effect of paclitaxel.

Aglycon of rhizochalin from the Rhizochalina incrustata induces apoptosis via activation of AMP-activated protein kinase in HT-29 colon cancer cells.

Rhizochalin is a two-headed sphingolipid-like compound isolated from the sponge Rhizochalina incrustata. It has been reported that rhizocalin and its derivates have a chemopreventive and chemotherapeutic effect. However, the molecular mechanism of these effects is not understood. Here, we demonstrate that aglycon of rhizochalin (AglRhz) from the Rhizochalina incrustata induces AMP-activated protein kinase (AMPK) phosphorylation, and thereby inhibits mammalian target of rapamycin (mTOR)-p70S6 kinase-extracellular signal-regulated kinase (ERK) signaling and activator protein 1 (AP-1) activity via phosphorylation of Raptor in HT-29 cells. In addition, AglRhz induced activation of caspase-3 and poly(ADP-ribose) polymerase (PARP), and DNA fragmentation in HT-29 cells, leads to induction of apoptosis as well as suppression of tumorigenicity of HT-29 cells. Notably, AglRhz inhibits insulin-like growth factor (IGF)-1-induced AP-1 activity and cell transformation in JB6 Cl41 cells. Overall, our findings identify AMPK as an important target protein for mediating the anti-tumor properties of AglRhz in HT-29 colon cancer cells and have important implication for sponges, the most important marine source, in colon cancer.

Gd-complexes of 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-1,4,7,10-tetraacetic acid (DOTA) conjugates of tranexamates as a new class of blood-pool magnetic resonance imaging contrast agents.

Gd-complexes of the type [Gd(L)(H(2)O)]·xH(2)O (5a-c), where L is DOTA conjugates of tranexamic acid (4a) and tranexamic esters (4b,c), have been prepared as a new class of MRI blood-pool contrast agents (BPCAs). Thermodynamic stability (K(GdL)) and pharmacokinetic inertness of 5 compare well with or better than those of analogous MRI contrasting agents (CAs) such as Gd-DOTA and Gd-DTPA-BMA. Their R(1)-relaxivities are significantly higher than those of any of the clinically used MRI CAs. T(1)-weighted MR images of mice administered by 5c demonstrate high blood-pool effect with simultaneous contrast enhancement in liver. The structural uniqueness of 5c lies in the fact that it adopts macrocyclic DOTA instead of acyclic DTPA. In addition, 5c is nonionic and makes no resort to aromatic substituent(s) in the chelate backbone for the blood-pool enhancement. The nature of hepatobiliary uptake demonstrated by 5c may be explained in terms of lipophilicity of tranexamate in the chelate (4c). The cell cytotoxicity test shows no toxicity found with 5, suggesting their use as a practical MRI BPCAs.

The prolyl isomerase Pin1 induces LC-3 expression and mediates tamoxifen resistance in breast cancer.

Endocrine therapies, which inhibit estrogen receptor signaling, are the most common and effective treatments for estrogen receptoralpha-positive breast cancer. However, the utility of these agents is limited by the frequent development of resistance, and the precise mechanisms underlying endocrine therapy resistance remain incompletely understood. Here, we demonstrate that peptidyl-prolyl isomerase Pin1 is an important determinant of resistance to tamoxifen and show that Pin1 increases E2F-4- and Egr-1-driven expression of LC-3 as a result of an increased interaction with and phosphorylation of MEK1/2. In human tamoxifen-resistant breast cancer, our results show a significant correlation between Pin1 overexpression and high levels of LC-3. Promoter activity as well as expression levels of Pin1 were drastically higher in tamoxifen-resistant MCF7 cells than control MCF7 cells, as were levels of LC-3 mRNA and protein, an autophagy marker. Pin1(-/-) mouse embryonic fibroblasts showed lower 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MEK1/2 phosphorylation than Pin1(+/+) mouse embryonic fibroblasts. Silencing of Pin1 expression inhibited TPA-induced MEK1/2 phosphorylation in MCF7 cells. Moreover, PD98059, a specific inhibitor of MEK1/2, and juglone, a potent Pin1 inhibitor, significantly suppressed the TPA-induced expression of E2F-4 as well as Egr-1 transcription factors, which control LC-3 gene expression. Importantly, 4-hydroxy tamoxifen, when used in combination with silencing of Pin1 or LC-3, increased cleaved poly(ADP-ribose) polymerase and DNA fragmentation to inhibit cologenic growth of MCF7 cells. We therefore link the Pin1-MEK pathway and LC-3-mediated tamoxifen resistance and show the therapeutic potential of Pin1 in the treatment of tamoxifen-resistant breast cancer.

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.

Tpl-2 kinase downregulates the activity of p53 and enhances signaling pathways leading to activation of activator protein 1 induced by EGF.

Tumor progression locus-2 (Tpl-2) kinase is a member of the mitogen-activated protein kinase kinase kinase family that has been implicated in cellular transformation. The enhanced expression of this protein has been shown to activate both the mitogen-activated protein kinase and c-Jun N-terminal kinase pathways. However, the molecular mechanisms responsible for the oncogenic potential of Tpl-2 are still largely unknown. Here, we showed that Tpl-2 interacted with p53 both in vitro and ex vivo. The overexpression of Tpl-2 inhibited the epidermal growth factor (EGF)-induced p53 phosphorylation (Ser15) through upregulating the activity of protein phosphatase 2A, which interacted with p53 stimulated by EGF. Also, the EGF-induced p53 activity was suppressed in the Tpl-2 wild-type (WT)-transfected HEK 293 cells, but had no effect in the Tpl-2-mutant (S413A)-transfected cells. Furthermore, introduction of small interfering RNA-Tpl-2 into HEK 293 cells resulted in decreased cell viability compared with only adenovirus-p53-infected cells. In addition, the Tpl-2 WT, but not Tpl-2 mutant (S413A), showed increased EGF-induced c-fos promoter activity, followed by activator protein 1 (AP-1) transactivation activity, which was associated with the cell transformation prompted by the H-Ras-Tpl-2-AP-1 signaling axis. These results indicated that the Ser413 of Tpl-2 plays an important role in EGF-induced carcinogenesis as well as inactivation of the p53.

Effects of TGFbeta1 and extracts from Cervus korean TEMMINCK var. mantchuricus Swinhoe on acute and chronic arthritis in rats.

AIM OF THE STUDY: To elucidate the pharmacological activities of deer antler acupuncture and TGF61538;1 on the acute and chronic phases of rheumatoid arthritis diseases. MATERIALS AND METHODS: Polyarthritis rats were administered with TGF61538;1 and water extract of deer antler acupunture (DAA), prepared from the pilose antler of Cervus korean TEMMINCK var. mantchuricus Swinhoe. TGF61538; (0.1 to 2 61549;g/animal) and DAA (5-100 61549;g/kg animal) were initiated 1 day before an arthritogenic dose of streptococcal cell wall fragments to see the effects on the joint swelling and distortion during the acute phase and the chronic phase of the disease. Arthritic index suppression of rat arthritis model was examined by TGF61538; and DAA administrations. RESULTS: TGF61538;1 and DAA diminished the polyarthritis development in rats. TGF61538; and DAA eliminated the joint swelling and distortion observed during the acute phase and the chronic phase of the disease. The TGF61538; and DAA suppressed the arthritis progress when administration was begun after acute phase of arthritis. DISCUSSION: Consistent with the inhibition of inflammatory cell recruitment into the synovium, TGF61538;1 and DAA reversed the leukocytosis associated with the chronic phase of the arthritis, respectively.

Cot, a novel kinase of histone H3, induces cellular transformation through up-regulation of c-fos transcriptional activity.

Post-translational modification of histones is critical for gene expression, mitosis, cell growth, apoptosis, and cancer development. Thus, finding protein kinases that are responsible for the phosphorylation of histones at critical sites is considered an important step in understanding the process of histone modification. The serine/threonine kinase Cot is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family. We show here that Cot can phosphorylate histone H3 at Ser-10 in vivo and in vitro, and that the phosphorylation of histone H3 at Ser-10 is required for Cot-induced cell transformation. We found that activated Cot is recruited to the c-fos promoter resulting in increased activator protein-1 (AP-1) transactivation. The formation of the Cot-c-fos promoter complex was also apparent when histone H3 was phosphorylated at Ser-10. Furthermore, the use of dominant negative mutants of histone H3 revealed that Cot was required for phosphorylation of histone H3 at Ser-10 to induce neoplastic cell transformation. These results revealed an important function of Cot as a newly discovered histone H3 kinase. Moreover, the transforming ability of Cot results from the coordinated activation of histone H3, which ultimately converges on the regulation of the transcriptional activity of the c-fos promoter, followed by AP-1 transactivation activity.

Ribosomal S6 kinase 2 is a key regulator in tumor promoter induced cell transformation.

The ribosomal S6 kinase 2 (RSK2), a member of the p90(RSK) (RSK) family of proteins, is a widely expressed serine/threonine kinase that is activated by extracellular signal-regulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones. Its activation signaling enhances cell survival. However, the roles of RSK2 in cell transformation have not yet been elucidated. Here, we found that RSK2 is a critical serine/threonine kinase for the regulation of cell transformation. When cells were stimulated with tumor promoters, such as epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), phosphorylation of RSK was increased within 5 min. Cell proliferation was suppressed in RSK2(-/-) mouse embryonic fibroblasts (MEFs) compared with RSK2(+/+) MEFs. Moreover, RSK2(-/-) MEFs accumulated at the G(1) phase of the cell cycle under normal cell culture conditions as well as after stimulation with EGF or TPA. In the anchorage-independent cell transformation assay (soft agar), stable expression of RSK2 in JB6 cells significantly enhanced colony formation in either the presence or absence of tumor promoters. Furthermore, knockdown of RSK2 with small interfering RNA-RSK2 suppressed constitutively active Ras (Ras(G12V))-induced foci formation in NIH3T3 cells. In addition, kaempferol, an inhibitor of RSK2, suppressed EGF-induced colony formation of JB6 Cl41 cells in soft agar, which was associated with inhibition of histone H3 phosphorylation (Ser(10)). These results showed that RSK2 is a key regulator for cell transformation induced by tumor promoters such as EGF and TPA.

Bidirectional signals transduced by TOPK-ERK interaction increase tumorigenesis of HCT116 colorectal cancer cells.

BACKGROUND & AIMS: Aberrant activation of Ras and Raf in mitogen-activated protein kinase (MAPK) signaling has been linked with cancer. However, the role of MAPK kinases (MAPKKs or MEKs) in cancer is unclear, although constitutively activated MEK1, which does not exist in nature, is "oncogenic." Herein, we found that T-cell-originated protein kinase (TOPK), a member of the MAPKK protein family, is highly expressed in human colorectal cancer tissues and cell lines and plays an important role in the transformation of colorectal cancer. METHODS: The biologic consequences of overexpression or knockdown of TOPK in JB6 Cl41 and HCT116 colorectal cancer cells were studied in vitro and in vivo, respectively. Kinase assay or transient transfection experiments were performed to study the bidirectional signaling pathway between TOPK and extracellular signal-regulated kinase (ERK). RESULTS: TOPK was shown to promote transformation in vitro and in vivo, and knockdown of TOPK in HCT116 colorectal cancer cells reduced this cell lines' tumorigenic properties in vitro and in vivo. Furthermore, a positive feedback loop between TOPK and ERK2 was identified. With epidermal growth factor treatment, knockdown of either TOPK or ERK2 in HCT116 cells resulted in a decreased phosphorylation of ERK2 or TOPK, respectively, and knockdown of TOPK in HCT116 colorectal cancer cells blocked the phosphorylation of downstream substrates of ERK2. CONCLUSIONS: The positive feedback loop between TOPK and ERK2 increases tumorigenesis properties of HCT116 colorectal cancer cells, and TOPK-regulated signaling may serve as a potential therapeutic target in colorectal cancer.

T-lymphokine-activated killer cell-originated protein kinase functions as a positive regulator of c-Jun-NH2-kinase 1 signaling and H-Ras-induced cell transformation.

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is overexpressed in highly proliferating tumors such as leukemias and myelomas, and seems to play a key role in tumorigenesis or metastasis. However, the precise role and regulatory mechanism explaining the effects of TOPK on tumor cells still remain elusive. Here, we reported that TOPK regulates UVB-induced c-Jun-NH2-kinase 1 (JNK1) activity, and is essential for H-Ras-induced activator protein-1 activity and cell transformation. We showed that TOPK associated with and phosphorylated JNK1 following UVB irradiation in vitro or in vivo. Moreover, UVB-induced JNK1 activity was greatly augmented in mouse epidermal JB6 Cl41 cells that stably expressed TOPK cDNA. On the other hand, JNK1 activity was markedly attenuated by stable expression of small interfering RNA against TOPK in malignant melanoma RPMI 7951 cells. Interestingly, TOPK interacted with JNK-interacting protein 1 and caused an elevation of JNK-interacting protein 1 scaffolding activity, thereby enhancing JNK1 activity. Furthermore, JNK1 was required for TOPK-mediated activator protein-1 transcriptional activity and transformed foci induced by UVB or H-Ras. Taken together, these findings showed that TOPK positively modulated UVB-induced JNK1 activity and played a pivotal role in JNK1-mediated cell transformation induced by H-Ras. These studies might also provide a novel molecular mechanism for the role of TOPK in UVB-mediated skin carcinogenesis.

Roles of MMP/TIMP in regulating matrix swelling and cell migration during chick corneal development.

Tissue remodeling is central to embryonic development. Here, we used immunohistochemistry, Western blotting, and RT-PCR analysis to investigate the roles of matrix metalloproteinases (MMPs) and the related "a disintegrin and metalloproteinase" (ADAM) family proteinases in chick corneal development. While MMP-13 was expressed in developing chick corneas from embryonic day (ED) 5 to ED 10, its inhibitor, tissue inhibitors of metalloproteinase-1 (TIMP-1), was expressed from ED 18 to 2 days post-hatching (P2). Early MMP-13 activity may be associated with degradation of type IX collagen from the primary stroma, which loosens the collagen fibrils and facilitates neural crest (NC) cell migration. The membrane-bound and secreted forms of ADAM10 were both detected throughout corneal development, and active ADAM10 formed a cleavage complex with CD44v6, a CD44 splice variant that is a major cell surface adhesion molecule for hyaluronic acid (HA) and has been implicated in cell migration. Both CD44v6 and its ectodomain cleavage products were detected from ED 5 to ED 14, and a broad-spectrum MMP inhibitor blocked ectodomain cleavage in cultured stromal cells. These findings suggest that ADAM10 mediates CD44v6 cleavage in the developing cornea, facilitating NC cell-derived mesenchymal cell migration. Finally, we identified high levels of active membrane-type 3-MMP (MT3-MMP) in developing corneas at ED 7, ED 14, and ED 18. MT3-MMP takes part in MMP-2 activation and possibly also CD44v6 shedding, suggesting that this pathway may be involved in cell migration. These findings collectively show for the first time that multiple MMPs, ADAMs, and TIMPs appear to functionally interact during corneal development.

RSK2 mediates muscle cell differentiation through regulation of NFAT3.

RSK2, an ERK downstream kinase, is a novel mediator of skeletal muscle cell differentiation through its regulation of NFAT3 activity. We found that the N-terminal (amino acids (aa) 1-68) and C-terminal (aa 416-674) kinase domains of RSK2 directly interacted with nuclear localization signal 1, the Ser/Pro repeat, and the polyproline domains (aa 261-365) of NFAT3. Upon A23187 stimulation, RSK2 induced nuclear localization of NFAT3. RSK2 phosphorylated NFAT3 in vitro (Km=3.559 microM), and activation of NFAT3 by RSK2 enhanced the promoter activity of NFAT3 downstream target genes in vivo. Furthermore, nuclear accumulation of NFAT3 was attenuated markedly in RSK2-/- cells compared with wild-type RSK2+/+ cells. Notably, RSK2 and NFAT3 induced a significant differentiation of C2C12 myoblasts to multinucleated myotubes. Multinucleated myotube differentiation was inhibited by small interfering RNA against RSK2, ERK1/2, or NFAT3. These results demonstrate that RSK2 is an important kinase for NFAT3 in mediating myotube differentiation.