The Role of Wnt Signaling in Squamous Cell Carcinoma.

Aberrant Wnt signaling pathway is a common feature of tumors and also plays important roles in tumor progression and metastasis of many cancer types. Various lines of evidence suggest that genetic defects affect Wnt pathway components, as well as epigenetic mechanisms that modulate the suppressors of Wnt pathway in oral squamous cell carcinoma. Recently, the newly discovered microRNAs are important molecular regulators in gene expression through transcription and translation repression. They play fundamental roles in a wide spectrum of biological functions, including cancer. In this review, we aim to accumulate recent research findings on the roles of Wnt/ß-catenin signaling and discuss how microRNAs affect Wnt/ß-catenin signaling in oral squamous cell carcinoma tumorigenesis. Apparently, investigations into the role of microRNAs with regard to the Wnt pathway in oral squamous cell carcinoma may help in the development of better strategies for tumor treatment.

Targeted endostatin-cytosine deaminase fusion gene therapy plus 5-fluorocytosine suppresses ovarian tumor growth.

There are currently no effective therapies for cancer patients with advanced ovarian cancer, therefore developing an efficient and safe strategy is urgent. To ensure cancer-specific targeting, efficient delivery, and efficacy, we developed an ovarian cancer-specific construct (Survivin-VISA-hEndoyCD) composed of the cancer specific promoter survivin in a transgene amplification vector (VISA; VP16-GAL4-WPRE integrated systemic amplifier) to express a secreted human endostatin-yeast cytosine deaminase fusion protein (hEndoyCD) for advanced ovarian cancer treatment. hEndoyCD contains an endostatin domain that has tumor-targeting ability for anti-angiogenesis and a cytosine deaminase domain that converts the prodrug 5-fluorocytosine (5-FC) into the chemotherapeutic drug, 5-fluorouracil. Survivin-VISA-hEndoyCD was found to be highly specific, selectively express secreted hEndoyCD from ovarian cancer cells, and induce cancer-cell killing in vitro and in vivo in the presence of 5-FC without affecting normal cells. In addition, Survivin-VISA-hEndoyCD plus 5-FC showed strong synergistic effects in combination with cisplatin in ovarian cancer cell lines. Intraperitoneal (i.p.) treatment with Survivin-VISA-hEndoyCD coupled with liposome attenuated tumor growth and prolonged survival in mice bearing advanced ovarian tumors. Importantly, there was virtually no severe toxicity when hEndoyCD is expressed by Survivin-VISA plus 5-FC compared with CMV plus 5-FC. Thus, the current study demonstrates an effective cancer-targeted gene therapy that is worthy of development in clinical trials for treating advanced ovarian cancer.

Axl promotes cell invasion by inducing MMP-9 activity through activation of NF-kappaB and Brg-1.

Activity of the Axl receptor tyrosine kinase is positively correlated with tumor metastasis; however, its detailed role in the mechanism of tumor invasion is still not completely understood. Here, we show that Axl enhances the expression of matrix metalloproteinase 9 (MMP-9), required for Axl-mediated invasion both in vitro and in vivo. We found that the highly selective MEK1/2 inhibitors U0126 and PD98059, and the expressed dominant-negative form of extracellular signal-regulated kinase (ERK), completely block Axl-mediated MMP-9 activation. In contrast, the phosphatidylinositol 3-kinase inhibitor LY294002 and wortmannin had little effect on activation. Interestingly, however, the Axl ligand Gas6 is not involved in Axl-mediated MMP-9 activation. Mutation of Glu59(Axl) and Thr77(Axl) dramatically reduced Gas6-Axl binding but continued to induce MMP-9 activation. In addition, overexpression of Axl-activated ERK and enhanced nuclear factor-kappaB (NF-kappaB) transactivation and brahma-related gene-1 (Brg-1) translocation. Exposure to the NF-kappaB inhibitor silibinin, which inhibits IkappaBalpha kinase activity, or overexpression of the dominant-negative mutant IkappaB and Brg-1 strikingly inhibited Axl-mediated MMP-9 activation. These data indicate that coordination of ERK signaling and NF-kappaB and Brg-1 activation are indispensable to regulation of Axl-dependent MMP-9 gene transcription. Together with previous data, our results provide a plausible mechanism for Axl-mediated tumor invasion and establish a functional link between the Axl and MMP-9 signaling pathways.

DNA methylation and histone modification regulate silencing of epithelial cell adhesion molecule for tumor invasion and progression.

Epithelial cell adhesion molecule (Ep-CAM) is believed to have a critical role in carcinogenesis and cell proliferation. However, the association of Ep-CAM with cancer invasion and progression is less clear. We found that Ep-CAM was highly expressed on low-invasive cells compared with highly invasive cells. Forced expression of Ep-CAM decreased cancer invasiveness, and silencing Ep-CAM expression elevated cancer invasiveness. Ep-CAM expression was associated with promoter methylation. Treatment with a demethylating agent, and/or the histone deacetylase inhibitor reactivated Ep-CAM expression in Ep-CAM-negative cells and inhibited cancer invasiveness. Using a promoter-reporter construct, we demonstrated methylation of the promoter fragment drive Ep-CAM-silenced transcription. Additionally, silenced Ep-CAM gene in cancer cells was enriched for hypermethylated histone 3 lysine 9. When unmethylated and active, this promoter was associated with acetylated histone 3 lysine 9. Furthermore, we observed an increased association of Ep-CAM promoter with repression components as tumor invasiveness increased. In cancer tissues, Ep-CAM expression significantly correlated with tumor progression and associated with promoter methylation. Our data support the idea that modulation of Ep-CAM plays a pivotal role in tumor invasion and progression. Moreover, aberrant DNA methylation of Ep-CAM is implicated in enhancing invasive/metastatic proclivity of tumors.

Involvement of Asp-Glu-Val-Asp-directed, caspase-mediated mitogen-activated protein kinase kinase 1 Cleavage, c-Jun N-terminal kinase activation, and subsequent Bcl-2 phosphorylation for paclitaxel-induced apoptosis in HL-60 cells.

Paclitaxel is a novel anticancer drug that has demonstrated efficacy toward treating several malignant tumor types. Here, we demonstrate that c-Jun NH(2)-terminal kinase (JNK), but not p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2, was persistently activated by paclitaxel or other microtubule-damaging agents within human leukemia HL-60 cells. Overexpression of a dominant-negative mutant, mitogen-activated protein kinase kinase 1 (MEKK1-DN) or treatment with JNK-specific antisense oligonucleotide prevented paclitaxel-induced JNK activation, Bcl-2 phosphorylation and apoptosis. Furthermore, we found that the full-length MEKK1 was cleaved to a 91-kDa carboxyl-terminal fragment at the earlier time of apoptosis induced by microtubule-damaging agents. This cleavage, however, occurred consistently with JNK activation and Bcl-2 phosphorylation, but preceded DNA fragmentation in cells in response to paclitaxel activity. The caspase inhibitor Ac-Asp-Glu-Val-Asp-CHO (DEVD-CHO), but not Ac-Tyr-Val-Ala-Asp-CHO (Ac-YVAD-CHO), effectively blocked MEKK1 cleavage, JNK activation, Bcl-2 phosphorylation, and subsequent apoptosis. Subcellular fractionation revealed that the 91-kDa C-terminal MEKK1 fragment was translocated to cytosol. Notably, the MEKK1 fragment could be coimmunoprecipitated with anti-JNK antibodies, suggesting that a signaling complex of C-terminal MEKK1/stress-activated protein kinase/extracellular-signal regulated kinase 1/JNK formed during apoptosis induced by microtubule-damaging agents. Taken together, our results suggest that disruption of cytoarchitecture by paclitaxel triggers a novel apoptosis-signaling pathway, wherein an active DEVD-directed caspase (DEVDase) initially cleaves MEKK1to generate a proapoptotic kinase fragment that is able to activate JNK and subsequent Bcl-2 phosphorylation, finally eliciting cell death.

Cytotoxicity of extractives from Taiwania cryptomerioides heartwood.

The cytotoxicity of the dominant lignans and sesquiterpenoids from Taiwania (Taiwania cryptomerioides Hayata) was investigated. Three human tumor cells including A-549 lung carcinoma. MCF-7 breast adenocarcinoma and HT-29 colon adenocarcinoma were selected to illustrate the structure-cytotoxicity relationships of Taiwania's dominant compounds. Taiwanin A, taiwanin E and dimethylmatairesinol exhibited significant cytotoxicity against three human tumor cells. Among them, taiwanin A possesses the strongest cytotoxic activity. In addition, the morphology-based evaluation, flow cytometric analysis, and DNA fragmentation assays demonstrated that the tumor cell death induced by taiwanin A was due to apoptosis.

Nitric oxide prevents apoptosis of human endothelial cells from high glucose exposure during early stage.

Hyperglycemia is a major cause of diabetic vascular disease. High glucose can induce reactive oxygen species (ROS) and nitric oxide (NO) generation, which can subsequently induce endothelial dysfunction. High glucose is also capable of triggering endothelial cell apoptosis. Little is known about the molecular mechanisms and the role of ROS and NO in high glucose-induced endothelial cell apoptosis. This study was designed to determine the involvement of ROS and NO in high glucose-induced endothelial cell apoptosis. Expression of endothelial nitric oxide synthase (eNOS) protein and apoptosis were studied in cultured human umbilical vein endothelial cells (HUVECs) exposed to control-level (5.5 mM) and high-level (33 mM) glucose at various periods (e.g., 2, 12, 24, 48 h). We also examined the effect of high glucose on H(2)O(2) production using flow cytometry. The results showed that eNOS protein expression was up-regulated by high glucose exposure for 2-6 h and gradually reduced after longer exposure in HUVECs. H(2)O(2) production and apoptosis, which can be reversed by vitamin C and NO donor (sodium nitroprusside), but enhanced by NOS inhibitor (N(G)-nitro-L-arginine methyl ether), were collated to a different time course (24-48 h) to HUVECs. These results provide the molecular basis for understanding that NO plays a protective role from apoptosis of HUVECs during the early stage (<24 h) of high glucose exposure, but in the late stage (>24 h), high glucose exposure leads to the imbalance of NO and ROS, resulting to the observed apoptosis. This may explain, at least in part, the impaired endothelial function and vascular complication of diabetic mellitus that would occur at late stages.

Suppression of apoptosis by Bcl-2 to enhance benzene metabolites-induced oxidative DNA damage and mutagenesis: A possible mechanism of carcinogenesis.

Apoptosis plays a crucial role in maintaining genomic integrity by selectively removing the most heavily damaged cells from the population. Under that premise, the dysregulation of apoptosis may result in an inappropriate survival of mutated cells. This study demonstrates that ectopic expression of Bcl-2 effectively suppresses benzene-active metabolites, 1,4-hydroquinone- and 1, 4-benzoquinone-induced apoptosis in human leukemic HL-60 cells, as evidenced by morphological changes and DNA fragmentation. Although reactive oxygen species production largely contributes to the benzene metabolites-induced apoptotic cell death, Bcl-2 fails to attenuate the benzene metabolites-elicited increase of reactive oxygen species in HL-60 cells, as confirmed by flow cytometry analysis. These data suggest that Bcl-2 prevents benzene metabolites-induced apoptosis at the downstream of oxidative damage events. This study also determines the level of 8-hydroxydeoxyguanosine (8-OH-dGua), an indicator for oxidative DNA damage, in neo- and Bcl-2-overexpressing HL-60 cells after treating with 1,4-hydroquinone or 1,4-benzoquinone. Interestingly, our results indicate that a majority of the 8-OH-dGua is efficiently removed in neo control cells within 3 to 6 h, whereas only 25 to 35% of 8-OH-dGua is repaired in Bcl-2 transfectants even for 24 h. Similarly, another oxidative DNA base, thymine glycol, failed to repair and was retained in genomic DNA of Bcl-2 transfectants. The above findings suggest that Bcl-2 may retain benzene metabolites-induced oxidative DNA damage in surviving cells. Indeed, the failure of repairing 8-OH-dGua and thymine glycol in benzene metabolites-treated Bcl-2 survivors increases the number of mutation frequencies at the hprt locus. Results in this study thus provide a novel benzene-induced carcinogenesis mechanism by which up-regulation of Bcl-2 protein may promote the susceptibility to benzene metabolites-induced mutagenesis by overriding apoptosis and attenuating DNA repair capacity.

Activation of c-Jun NH2-terminal kinase and subsequent CPP32/Yama during topoisomerase inhibitor beta-lapachone-induced apoptosis through an oxidation-dependent pathway.

Beta-lapachone (beta-Lap) has been found to inhibit DNA topoisomerases (Topos) by a mechanism distinct from that of other commonly known Topo inhibitors. Here, we demonstrated a pronounced elevation of H2O2 and O2- in human leukemia HL-60 cells treated with beta-Lap. Treatment with other Topo poisons, such as camptothecin (CPT), Vbeta-16, and GL331, did not have the same effect. On the other hand, antioxidant vitamin C (Vit C) treatment effectively antagonized beta-Lap-induced apoptosis. This suggested that a reactive oxygen species (ROS)-related pathway was involved in beta-Lap-induced apoptosis program. We also found that c-Jun NH2-terminal kinase (JNK) but not p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 was persistently activated in apoptosis induced by beta-Lap. Overexpression of a dominant-negative mutant mitogen-activated protein kinase kinase kinase 1 (MEKK1-DN) or treatment with JNK-specific antisense oligonucleotide or Vit C all prevented beta-Lap-induced JNK activation and the subsequent apoptosis. Only the expression of MEKK1-DN, not Vit C treatment, blocked the JNK activity induced by CPT, Vbeta-16, or GL331. These results confirm again that ROS acts as a mediator for JNK activation during beta-Lap-induced apoptosis. Furthermore, we found that beta-Lap can stimulate CPP32/Yama activity, which was, however, markedly inhibited by the MEKK1-DN expression or Vit C treatment. Again, CPT-induced CPP32/Yama activation can be abolished by MEKK1-DN but not by Vit C treatment. Taken together, these results indicate that beta-Lap but not other Topo inhibitors triggers apoptosis signaling, i.e., JNK and subsequent CPP32/Yama activation are mediated by the generation of ROS.

Involvement of hydrogen peroxide in topoisomerase inhibitor beta-lapachone-induced apoptosis and differentiation in human leukemia cells.

Beta-Lapachone a novel topoisomerase inhibitor, has been found to induce apoptosis in various human cancer cells. In this study we report that a dramatic elevation of hydrogen peroxide (H2O2) in human leukemia HL-60 cells following 1 microM beta-lapachone treatment and that this increase was effectively inhibited by treatment with antioxidant N-acetyl-L-cysteine (NAC), ascorbic acid, alpha-tocopherol. NAC strongly prevented beta-lapachone-induced apoptotic characteristics such as DNA fragmentation and apoptotic morphology. However, treatment of HL-60 cells with another topoisomerase inhibitor camptothecin (CPT) did not induce H2O2 production as compared to untreated cells. NAC also failed to block CPT-induced apoptosis. Correlated with these findings, we found that cancer cell lines K562, MCF-7, and SW620, contained high level of intracellular glutathione (GSH), were not elevated in H2O2 and were resistant to apoptosis after treatment with beta-lapachone. In contrast, cancer cell lines such as, HL-60, U937, and Molt-4 which have lower level of GSH, were readily increased of H2O2 and were sensitive to this drug. Furthermore, ectopic overexpression of Bcl-2 in HL-60 cells also attenuated beta-lapachone-induced H2O2 and conferred resistance to beta-lapachone-induced cell death. Beta-Lapachone at the concentration as low as 0.25 microM effectively induced HL-60 cells to undergo monocytic differentiation, as evidenced by CD14 antigenicity and alpha-naphthyl acetate esterase activity. Again, the beta-lapachone-induced monocytic differentiation was suppressed by NAC. These results suggest that intracellular H2O2 generation plays a crucial role in beta-lapachone-induced cell death and differentiation.

Inhibitors of poly(ADP-ribose) polymerase block nitric oxide-induced apoptosis but not differentiation in human leukemia HL-60 cells.

Previous studies have shown that chemically generated nitric oxide (NO) can induce human leukemia HL-60 cells to undergo monocytic differentiation. We show here that exposure of HL-60 cells to chemical NO generators induces cell death via apoptosis which was examined by morphological and biochemical criteria. The activation of poly(ADP-ribose) polymerase (pADPRp) was found to occur during the process of cell death. The NO-induced apoptosis of HL-60 cells was effectively prevented by the ADP-ribosylation inhibitors nicotinamide (NA) and 3-aminobenzamide (3-AB). Since NO not only induced apoptosis but also triggered differentiation under the same concentration, we thus examined whether pADPRp participated in monocytic differentiation. It is of interest to note that the NO-mediated monocytic differentiation was not affected by 3-AB or NA. These findings indicate that activation of pADPRp is specifically involved in NO-induced apoptosis but not differentiation.

Protein kinase FA/glycogen synthase kinase-3 alpha after heparin potentiation phosphorylates tau on sites abnormally phosphorylated in Alzheimer's disease brain.

Previously, we identified protein kinase FA/glycogen synthase kinase-3 alpha (GSK-3 alpha) as a brain microtubule-associated tau kinase that phosphorylates Ser235 and Ser404 of tau and causes its electrophoretic mobility shift in gels, a unique property characteristic of paired helical filament-associated pathological tau (PHF-tau) in Alzheimer's disease brains. In this study, we found that the activity of kinase FA/GSK-3 alpha towards phosphorylation of brain tau could be stimulated approximately fourfold by heparin. The phosphorylation molar ratio was increased simultaneously up to 9 mol of phosphates/mol of tau, resulting in a reduced mobility of tau with an apparent molecular mass shift to approximately 68 kDa in sodium dodecyl sulfate gels, which is very similar to that observed in Alzheimer-tau. Tryptic digestion of 32P-labelled tau, followed by HPLC and two-dimensional separation on TLC cellulose plates, revealed eight major phosphopeptides. Phosphoamino acid analysis together with sequential manual Edman degradation and protein sequence analysis further revealed that, in addition to Ser235 and Ser404, heparin generated Thr212, Thr231, Ser262, Ser324, and Ser356, the five extra phosphorylation sites in tau. As Ser235, Ser262, Ser324, Ser356, and Ser404 (particularly the site of Ser262) have been identified as five of the most potent sites in tau responsible for reducing microtubule binding possibly involved in neuronal degeneration, and Thr231, Ser235, Ser262, and Ser404 are four of the most well documented sites abnormally phosphorylated in Alzheimer-tau, the results provide initial evidence that protein kinase FA/GSK-3 alpha after heparin potentiation may represent one of the most potent systems possibly involved in the abnormal phosphorylation of PHF-tau in Alzheimer's disease brains.

Protein kinase FA/GSK-3 phosphorylates tau on Ser235-Pro and Ser404-Pro that are abnormally phosphorylated in Alzheimer's disease brain.

Previously, we identified protein kinase FA/glycogen synthase kinase-3 (GSK-3) as a microtubule-associated protein tau kinase that can incorporate 4 mol of phosphates into 1 mol of tau protein and cause its electrophoretic mobility shift in sodium dodecyl sulfate gels, a unique property characteristic of paired helical filament-associated pathological tau (PHF-tau) in Alzheimer's disease brains. In this report, we identified TPPKS(p)PSAAK and SPVVSGDTS(p)PR as two phosphorylation site sequences phosphorylated by kinase FA/GSK-3 in tau using peptide sequence analysis and sequential manual Edman degradation for radiosequencing. When mapping with human brain tau sequence, we further identified Ser235-Pro and Ser404-Pro as the two major phosphorylation sites according to the numbering of the longest tau isoform. Ser235 and Ser404 have been reported as two of the major abnormal phosphorylation sites in PHF-tau. Taken together, the results provide initial evidence that protein kinase FA/GSK-3 may represent one of the Ser-Pro motif-directed tau kinases involved in the abnormal phosphorylation of pathological PHF-tau in Alzheimer's disease brain.