PLK1 inhibition leads to mitotic arrest and triggers apoptosis in cholangiocarcinoma cells.

Cholangiocarcinoma (CCA) is a lethal cancer originating from the epithelial cells within the bile duct and ranks as the second most prevalent form of liver cancer in Thailand. Polo-like kinase 1 (PLK1), a protein serine/threonine kinase, regulates a number of steps in cell mitosis and is upregulated in several types of cancer, including CCA. Our previous study identified PLK1 as a biomarker of the C1 subtype, correlating with poor prognosis in intrahepatic CCA. The present study aimed to examine the effect of PLK1 inhibition on CCA cells. Different CCA cell lines developed from Thai patients, HuCCA1, KKU055, KKU100 and KKU213A, were treated with two PLK1 inhibitors, BI2536 and BI6727, and were transfected with small interfering RNA, followed by analysis of cell proliferation, cell cycle distribution and cell apoptosis. It was discovered that BI2536 and BI6727 inhibited cell proliferation and caused G2/M-phase arrest in CCA cells. Furthermore, the number of total apoptotic cells was increased in PLK1 inhibitor-treated CCA cells. The expression levels of mitotic proteins, aurora kinase A, phosphorylated PLK1 (T210) and cyclin B1, were augmented in PLK1-inhibited CCA cells. Additionally, inhibition of PLK1 led to increased DNA damage, as determined by the upregulated levels of γH2AX and increased cleavage of poly (ADP-ribose) polymerase, an apoptotic marker. These results suggested that inhibiting PLK1 prolonged mitotic arrest and subsequently triggered cell apoptosis. Validation of the antiproliferative effects of PLK1 inhibition was accomplished through silencing of the PLK1 gene. In conclusion, targeting PLK1 provided promising results for further study as a potential candidate for targeted therapy in CCA.

Capsaicin suppresses the migration and invasion of human nasopharyngeal carcinoma cells through the modulation of mTOR signaling pathway.

Nasopharyngeal carcinoma (NPC), a malignancy of the nasopharynx, is prevalent in Southeast Asia and Southern China. The prognosis of NPC is poor and local recurrence and metastasis often occur. Capsaicin (tran-8-methyl-N-vanillyl-6-nonenamide), a pungent constituent of hot chili peppers, shows anti-cancer activities such as anti-proliferation and anti-metastasis. Currently, the role of capsaicin in cell metastasis of NPC is not well understood. We tested whether capsaicin has anti-metastatic activity in NPC cell lines. Capsaicin suppressed cell proliferation in dose-dependent manner. Moreover, capsaicin inhibited cell metastasis as shown by wound healing assay and decreased the expressions of MMP-2 and MMP-9. In addition, the phosphorylation of mTOR was downregulated by capsaicin. Combination of capsaicin and rapamycin (mTOR inhibitor) treatments led to increasing of anti-growth and anti-metastatic activities. Therefore, capsaicin has potential to be used as an optional therapeutic drug for treatment of NPC.

Arsenite exposure potentiates apoptosis-inducing effects of tumor necrosis factor-alpha- through reactive oxygen species.

Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine released by immune cells during inflammation process. Sodium arsenite (NaAsO2) is an environmental toxic metal. The effects of excess NaAsO2 on TNF-α response and its intracellular signaling are not well understood. We hypothesized that NaAsO2 exposure might affect cellular response to TNF-α. Using HeLa cell model, we found that the combination of NaAsO2 and TNF-α clearly decreased cell viability and mitochondrial membrane potential, but increased percentage of early and late apoptotic cells and cleaved-poly (ADP-ribose) polymerase (PARP). Moreover, the combination prolonged the phosphorylation of mitogen-activated protein kinase (MAPK) members, including c-Jun-N-terminal kinase (JNK), p38, and extracellular signal related kinases (ERK), and increased intracellular reactive oxygen species (ROS), in comparison to treatment of NaAsO2 or TNF-α alone. We further investigated the role of ROS and MAPK signaling on this event by inhibiting ROS production and MAPK. An antioxidant N-acetylcysteine pretreatment diminished the apoptosis-inducing effect of NaAsO2 and TNF-α combination and also inhibited MAPK signaling. Using specific inhibitor of p38 (SB203580) and siRNA-p38 surprisingly increased cell apoptosis and this effect was not observed by JNK and ERK inhibition. This study suggests that p38 may possibly be a survival mediator in response to environmental toxicant-related inflammation. In conclusion, NaAsO2 exposure might amplify inflammation-related tissue injury by potentiating the apoptosis-inducing effect of TNF-α through ROS-dependent mechanism.

Effects of 5-Aza-2'-Deoxycytidine, Bromodeoxyuridine, Interferons and Hydrogen Peroxide on Cellular Senescence in Cholangiocarcinoma Cells.

Cellular senescence, a barrier to tumorigenesis, controls aberrant proliferation of cells. We here aimed to investigate cellular senescence in immortalized cholangiocyte and cholangiocarcinoma cell lines using five different inducing agents: 5-aza-2'deoxycytidine, bromodeoxyuridine, interferons (IFNß and IFNγ), and hydrogen peroxide. We analyzed senescence characteristics, colony formation ability, expression of genes involved in cell cycling and interferon signaling pathways, and protein levels. Treatment with all five agents decreased cell proliferation and induced cellular senescence in immortalized cholangiocyte and cholangiocarcinoma cell lines with different degrees of growth-inhibitory effects depending on cell type and origin. Bromodeoxyuridine gave the strongest stimulus to inhibit growth and induce senescence in most cell lines tested. Expression of p21 and interferon related genes was upregulated in most conditions. The fact that bromodeoxyuridine had the strongest effects on growth inhibition and senescence induction implies that senescence in cholangiocarcinoma cells is likely controlled by DNA damage response pathways relating to the p53/p21 signaling. In addition, interferon signaling pathways may partly regulate this mechanism in cholangiocarcinoma cells.

TAK1-mediated serine/threonine phosphorylation of epidermal growth factor receptor via p38/extracellular signal-regulated kinase: NF-{kappa}B-independent survival pathways in tumor necrosis factor alpha signaling.

The kinase TAK1, a mitogen-activated protein kinase kinase kinase (MAP3K), has been widely accepted as a key kinase activating NF-kappaB and MAPKs in tumor necrosis factor alpha (TNF-alpha) signaling. We have recently reported that TAK1 regulates the transient phosphorylation and endocytosis of epidermal growth factor receptor (EGFR) in a tyrosine kinase activity-independent manner. In the present study, we found that Thr-669 in the juxtamembrane domain and Ser-1046/1047 in the carboxyl-terminal regulatory domain were transiently phosphorylated in response to TNF-alpha. Experiments using chemical inhibitors and small interfering RNA demonstrated that TNF-alpha-mediated phosphorylation of Thr-669 and Ser-1046/7 were differently regulated via TAK1-extracellular signal-regulated kinase (ERK) and TAK1-p38 pathways, respectively. In addition, p38, but not ERK, was involved in the endocytosis of EGFR. Surprisingly, modified EGFR was essential to prevent apoptotic cellular responses; however, the EGFR pathway was independent of the NF-kappaB antiapoptotic pathway. These results demonstrated that TAK1 controls two different signaling pathways, IkappaB kinase-NF-kappaB and MAPK-EGFR, leading to the survival of cells exposed to the death signal from the TNF-alpha receptor.

Constitutive activation of TAK1 by HTLV-1 tax-dependent overexpression of TAB2 induces activation of JNK-ATF2 but not IKK-NF-kappaB.

HTLV-1 Tax oncoprotein induces persistent activation of the transcription factor NF-kappaB and CREB (cAMP-response element-binding protein)/ATF. Transforming growth factor-beta-activated kinase 1 (TAK1) has been shown to play a critical role in these transcription factors. Here, we found that TAK1 was constitutively activated in Tax-positive HTLV-1-transformed T cells. Tax induced persistent overexpression of TAK1-binding protein 2 (TAB2), but not TAB3, which is essential for TAK1 activation. Surprisingly, TAK1 was not involved in the activation of NF-kappaB. On the other hand, JNK and p38 mitogen-activated protein kinases were activated by TAK1. In addition, ATF2, but not CREB, was a target for the TAK1-JNK pathway, and p38 negatively regulated TAK1 activity through TAB1 phosphorylation. These results indicate that Tax-mediated TAK1 activation is important for the activation of ATF2 rather than NF-kappaB.

Transient suppression of ligand-mediated activation of epidermal growth factor receptor by tumor necrosis factor-alpha through the TAK1-p38 signaling pathway.

Epidermal growth factor receptor (EGFR) has been shown to be activated by specific ligands as well as other cellular stimuli including tumor necrosis factor-alpha (TNF-alpha). In the present study, we found that cellular stress suppressed ligand-mediated EGFR activity. Both TNF-alpha and osmotic stress rapidly induced phosphorylation of EGFR. This phosphorylation of EGFR and the activation of mitogen-activated protein kinases and NF-kappaB occurred independently of the shedding of extracellular membrane-bound EGFR ligands and intracellular EGFR tyrosine kinase activity. Transforming growth factor-beta-activated kinase 1 (TAK1) was involved in the TNF-alpha-induced signaling pathway to EGFR. In addition, experiments using chemical inhibitors and small interfering RNA demonstrated that p38 alpha is a common mediator for the cellular stress-induced phosphorylation of EGFR. Surprisingly, the modified EGFR was not able to respond to its extracellular ligand due to transient internalization through the clathrin-mediated mechanism. Furthermore, turnover of p38 activation led to dephosphorylation and recycling back to the cell surface of EGFR. These results demonstrated that TNF-alpha has opposite bifunctional activities in modulating the function of the EGFR.

Blockade of transforming growth factor-beta-activated kinase 1 activity enhances TRAIL-induced apoptosis through activation of a caspase cascade.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF-alpha ligand family that selectively induces apoptosis in a variety of tumor cells. To clarify the molecular mechanism of TRAIL-induced apoptosis, we focused on transforming growth factor-beta-activated kinase 1 (TAK1) mitogen-activated protein kinase (MAPK) kinase kinase, a key regulator of the TNF-alpha-induced activation of p65/RelA and c-Jun NH2-terminal kinase/p38 MAPKs. In human cervical carcinoma HeLa cells, TRAIL induced the delayed phosphorylation of endogenous TAK1 and its activator protein TAB1 and TAB2, which contrasted to the rapid response to TNF-alpha. Specific knockdown of TAK1 using small interfering RNA (siRNA) abrogated the TRAIL-induced activation of p65 and c-Jun NH2-terminal kinase/p38 MAPKs. TRAIL-induced apoptotic signals, including caspase-8, caspase-3, caspase-7, and poly(ADP-ribose) polymerase, were enhanced by TAK1 siRNA. Flow cytometry showed that the binding of Annexin V to cell surface was also synergistically increased by TRAIL in combination with TAK1 siRNA. In addition, pretreatment of cells with 5Z-7-oxozeaenol, a selective TAK1 kinase inhibitor, enhanced the TRAIL-induced cleavage of caspases and binding of Annexin V. The TAK1-mediated antiapoptotic effects were also observed in human lung adenocarcinoma A549 cells. In contrast, TAK1-deficient mouse embryonic fibroblasts are resistant to TRAIL-induced apoptosis, and treatment of control mouse embryonic fibroblasts with 5Z-7-oxozeaenol did not drastically promote the TRAIL-induced activation of a caspase cascade. These results suggest that TAK1 plays a critical role for TRAIL-induced apoptosis, and the blockade of TAK1 kinase will improve the chances of overcoming cancer.

Identification of TNF-alpha-responsive NF-kappaB p65-binding element in the distal promoter of the mouse serine protease inhibitor SerpinE2.

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.

TAK1-mediated transcriptional activation of CD28-responsive element and AP-1-binding site within the IL-2 promoter in Jurkat T cells.

We focused on the functional involvement of transforming growth factor-beta-activated kinase 1 (TAK1) in transcriptional regulation of interleukin-2 (IL-2) in T cells. Costimulation of Jurkat cells with 12-O-tetradecanoylphorbol-13-acetate and A23187 leads to a rapid phosphorylation of TAK1 and TAK1-binding protein 1 (TAB1), critical for TAK1 activation. A specific inhibitor of TAK1 blocked production of IL-2. In addition, overexpression of TAK1 and TAB1 induced secretion of IL-2. CD28-responsive element/activator protein-1-binding site (RE/AP) within the IL-2 promoter was a functional target for TAK1. The RE/AP-driven transcription was regulated by TAK1-mediated activation of the c-Jun NH2-terminal kinase, p38 and IkappaB kinase. These results indicate that TAK1 plays a critical role in T cell activation by controlling production of IL-2.

Vanillin suppresses in vitro invasion and in vivo metastasis of mouse breast cancer cells.

Vanillin, a food flavoring agent, has been reported to show anti-mutagenic activity and to inhibit chemical carcinogenesis. In this study, we examined the effect of vanillin on the growth and metastasis of 4T1 mammary adenocarcinoma cells in BALB/c mice. Mice orally administered with vanillin showed significantly reduced numbers of lung metastasized colonies compared to controls. In vitro studies revealed that vanillin, at concentrations that were not cytotoxic, inhibited invasion and migration of cancer cells and inhibited enzymatic activity of MMP-9 secreted by the cancer cells. Vanillin also showed growth inhibitory effect towards cancer cells in vitro. However, vanillic acid, a major metabolic product of vanillin in human and rat, was not active in these in vitro activity assays. Our findings suggest that vanillin has anti-metastatic potential by decreasing invasiveness of cancer cells. Since vanillin is generally regarded as safe, it may be of value in the development of anti-metastatic drugs for cancer treatment.

Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-beta-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2.

Transforming growth factor-beta-activated kinase 1 (TAK1) mitogen-activated protein kinase kinase kinase has been shown to be activated by cellular stresses including tumor necrosis factor-alpha (TNF-alpha). Here, we characterized the molecular mechanisms of cellular stress-induced TAK1 activation, focusing mainly on the phosphorylation of TAK1 at Thr-187 and Ser-192 in the activation loop. Thr-187 and Ser-192 are conserved among species from Caenorhabditis elegans to human, and their replacement with Ala resulted in inactivation of TAK1. Immunoblotting with a novel phospho-TAK1 antibody revealed that TNF-alpha significantly induced the phosphorylation of endogenous TAK1 at Thr-187, and subsequently the phosphorylated forms of TAK1 rapidly disappeared. Intermolecular autophosphorylation of Thr-187 was essential for TAK1 activation. RNA interference and overexpression experiments demonstrated that TAK1-binding protein TAB1 and TAB2 were involved in the phosphorylation of TAK1, but they regulated TAK1 phosphorylation differentially. Furthermore, SB203580 and p38alpha small interfering RNA enhanced TNF-alpha-induced Thr-187 phosphorylation as well as TAK1 kinase activity, indicating that the phosphorylation is affected by p38alpha/TAB1/TAB2-mediated feedback control of TAK1. These results indicate critical roles of Thr-187 phosphorylation in the stress-induced rapid and transient activation of TAK1 in a signaling complex containing TAB1 and TAB2.