BAI, a 3-aminoindazole derivative, inhibits interleukin-1ß-induced expression of cyclooxygenase-2 in A549 human airway cells.

Cyclooxygenase (COX)-2 and its products, including PGE2, are key inflammatory mediators. In this study, we have assessed the pharmacological characteristics of BAI, a 3-aminoindazole derivative and a novel cyclin-dependent kinase (CDK) inhibitor, for regulation of COX-2 expression induced by interleukin (IL)-1ß in A549 human airway cells. Treatment with BAI strongly inhibited IL-1ß-induced expression of COX-2 at both the protein and mRNA levels. Results of luciferase experiments also revealed that BAI treatment reduced IL-1ß-induced COX-2 promoter activity. Distinctly, treatment with BAI did not affect IL-1ß-induced phospho-rylation of extracellular signal-regulated protein kinase-1/2 (ERK-1/2), p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal protein kinase-1/2 (JNK-1/2) and proteolysis of IκB-α, an inhibitor of nuclear factor (NF)-κB, but inhibited IL-1ß-induced phosphorylation of histone H1, a target for phosphorylation by CDKs. siRNA transfection experiments demonstrated that knockdown of CDK2 and CDK4 led to a slight reduction of IL-1ß-induced histone H1 phosphorylation but had no effect on IL-1ß-induced COX-2 expression. Interestingly, additional cell culture experiments showed the ability of BAI to repress the PMA-induced COX-2 expression in A549 cells and serum-dependent COX-2 expression in NCI-H292 cells, a human laryngeal cell line. Collectively, these results demonstrate firstly that BAI downregulates IL-1ß-induced COX-2 expression through transcriptional repression, which appears to be independent of CDK2, CDK4, MAPKs and NF-κB, in A549 cells. It is suggested that BAI may be a potential candidate for treatment of the airway inflammatory diseases where COX-2 overexpression is problematic.

Pinus densiflora leaf essential oil induces apoptosis via ROS generation and activation of caspases in YD-8 human oral cancer cells.

The leaf of Pinus (P.) densiflora, a pine tree widely distributed in Asian countries, has been used as a traditional medicine. In the present study, we investigated the anticancer activity of essential oil, extracted by steam distillation, from the leaf of P. densiflora in YD-8 human oral squamous cell carcinoma (OSCC) cells. Treatment of YD-8 cells with P. densiflora leaf essential oil (PLEO) at 60 µg/ml for 8 h strongly inhibited proliferation and survival and induced apoptosis. Notably, treatment with PLEO led to generation of ROS, activation of caspase-9, PARP cleavage, down-regulation of Bcl-2, and phosphorylation of ERK-1/2 and JNK-1/2 in YD-8 cells. Treatment with PLEO, however, did not affect the expression of Bax, XIAP and GRP78. Importantly, pharmaco-logical inhibition studies demonstrated that treatment with vitamin E (an anti-oxidant) or z-VAD-fmk (a pan-caspase inhibitor), but not with PD98059 (an ERK-1/2 inhibitor) or SP600125 (a JNK-1/2 inhibitor), strongly suppressed PLEO-induced apoptosis in YD-8 cells and reduction of their survival. Vitamin E treatment further blocked activation of caspase-9 and Bcl-2 down-regulation induced by PLEO. Thus, these results demonstrate firstly that PLEO has anti-proliferative, anti-survival and pro-apoptotic effects on YD-8 cells and the effects are largely due to the ROS-dependent activation of caspases.

Anti-cancer properties of glucosamine-hydrochloride in YD-8 human oral cancer cells: Induction of the caspase-dependent apoptosis and down-regulation of HIF-1α.

Evidence suggests anti-tumor activities of glucosamine-hydrochloride (GS-HCl). In the present study, we investigated anti-proliferative, growth suppressive and/or pro-apoptotic effects of GS-HCl on YD-8 human oral squamous cell carcinoma (OSCC) cells. Fundamentally, treatment with GS-HCl strongly inhibited proliferation and induced apoptosis in YD-8 cells, as determined by MTS and DNA fragmentation analyses. Of further note, as measured by Western analyses, GS-HCl treatment led to activation of caspase-3, cytosolic accumulation of cytochrome c, down-regulation of Mcl-1 and HIF-1α, up-regulation of GRP78, an indicator of ER stress, and generation of ROS in YD-8 cells. Importantly, results of pharmacological inhibition studies showed that treatment with z-VAD-fmk, a pan-caspase inhibitor, but not with vitamin E, an anti-oxidant strongly blocked the GS-HCl-induced apoptosis in YD-8 cells. Analyses of additional cell culture works further revealed that GS-HCl had a strong growth suppressive effect on not only YD-8 but also YD-10B and YD-38, two other human OSCC cell lines. These findings collectively demonstrate that GS-HCl has anti-proliferative, anti-survival, and pro-apoptotic effects on YD-8 cells and the effects appear to be mediated via mechanisms associated with the mitochondrial-dependent activation of caspases, down-regulation of Mcl-1, and induction of ER stress. Considering HIF-1α as a tumor angiogenic transcription factor, the ability of GS-HCl to down-regulate HIF-1α in YD-8 cells may further support its anti-cancer property. It is thus suggested that GS-HCl may be used as a potential anti-cancer drug against human OSCC.

Overexpression of cyclooxygenase-2 in NCI-H292 human alveolar epithelial carcinoma cells: roles of p38 MAPK, ERK-1/2, and PI3K/PKB signaling proteins.

Evidence suggests overexpression of COX-2 and its role in many human cancers, including lung. However, the regulatory mechanism underlying COX-2 overexpression in lung cancer is not fully understood. We herein investigated whether COX-2 is overexpressed in human airway cancer cell lines, including A549 (lung), Hep-2 (bronchial), and NCI-H292 (alveolar). When grown in cell culture medium containing 10% FBS (serum), of note, there was strong and transient induction of COX-2 protein and mRNA in NCI-H292 cells, but little or low COX-2 expression is seen in A549 or Hep-2 cells. Interestingly, strong and sustained activities of ERK-1/2, JNK-1/2, p38 MAPK, and PKB were also shown in NCI-H292 cells grown in presence of serum. Profoundly, results of pharmacological inhibition studies demonstrated that the serum-dependent COX-2 up-regulation in NCI-H292 cells is attributed to not only the p38 MAPK-, PI3K/PKB-, and ERK-1/2-mediated COX-2 transcriptional up-regulation but also the p38 MAPK- and ERK-1/2-mediated post-transcriptional COX-2 mRNA stabilization. Of further note, it was shown that the ERK-1/2 and PI3K/PKB (but not COX-2, p38 MAPK, and JNK-1/2) activities are necessary for growth of NCI-H292 cells. These findings collectively demonstrate for the first time that COX-2 expression is transiently up-regulated by serum addition in NCI-H292 cells and the serum-induced COX-2 expression is closely linked to the p38 MAPK-, ERK-1/2-, and PI3K/PKB-mediated COX-2 transcriptional and post-transcriptional up-regulation.

Aspirin induces apoptosis in YD-8 human oral squamous carcinoma cells through activation of caspases, down-regulation of Mcl-1, and inactivation of ERK-1/2 and AKT.

NSAIDs and COX-2 inhibitors show anti-cancer activities in many cancer cells. In this study, we investigated the effects of NSAIDs (aspirin or indomethacin) and COX-2 inhibitor (NS-398) on growth of YD-8 human oral squamous carcinoma cells. Interestingly, among drugs tested, aspirin showed strongest inhibitory effects on viability and survival of YD-8 cells. Profoundly, aspirin treatment resulted in severe cell shrinkage and nuclear DNA fragmentation in YD-8 cells, suggesting the aspirin-induced apoptosis in YD-8 cells. Data of Western blot further demonstrated that aspirin treatment caused activation of caspases, down-regulation of Mcl-1 protein, dephosphorylation of ERK-1/2 and AKT, and also IkappaB-alpha proteolysis-dependent NF-kappaB activation in YD-8 cells. Aspirin, however, had no effect on expressions of Bcl-2, XIAP, and HIAP-1 in YD-8 cells. Importantly, pretreatment with z-VAD-fmk, a pan-caspase inhibitor blocked the aspirin-induced apoptosis and Mcl-1 down-regulation in YD-8 cells. These findings collectively suggest that aspirin induces apoptosis in YD-8 cells and the induction may be correlated to activation of caspases, caspase-dependent Mcl-1 proteolysis, inactivation of ERK-1/2 and AKT, and activation of NF-kappaB. It is suggested that aspirin may be applied a potential anti-cancer drug against human oral squamous carcinoma.

Manganese-mediated up-regulation of HIF-1alpha protein in Hep2 human laryngeal epithelial cells via activation of the family of MAPKs.

High exposure of manganese is believed to be a risk factor for respiratory diseases. Evidence suggests that overexpression of HIF-1alpha transcription factor is linked to pulmonary inflammation and vascular change. In this study, we investigated the effect of manganese-chloride (manganese) on expression and activity of HIF-1alpha in various human airway cells, including Hep2 (laryngeal), H292 (bronchial), and A549 (lung). Profoundly, while manganese treatment led to low or little effect on induction of HIF-1alpha protein in H292 or A549 cells, it strongly induced HIF-1alpha protein expression in Hep2 cells. Mn treatment, however, did not induce HIF-1alpha mRNA expression in Hep2 cells. Luciferase experiments further demonstrated that manganese treatment increased the HRE-driven luciferase activity, suggesting that the induced HIF-1 is functional. Interestingly, manganese treatment also caused activation of p38 MAPK, JNK-1/2, ERK-1/2, and ATF-2, but not of PKB or NF-kappaB in Hep2 cells. Importantly, the manganese-mediated expression and activity of HIF-1alpha protein were largely blocked by treatment with the inhibitor of p38 MAPK (SB203580), JNK-1/2 (SP600125), or ERK-1/2 (PD98059), suggesting roles of these MAPKs in the manganese-induced HIF-1alpha protein expression and activity. Moreover, treatment with SP600125 or SB203580, but not PD98059, had partial inhibitory effects on the stability of HIF-1alpha protein induced by manganese, suggesting that p38 MAPK and JNK-1/2 also contribute to the Mn-mediated HIF-1alpha protein stability. These results suggest that manganese is able to up-regulate HIF-1alpha at the protein level in Hep2 cells and the up-regulation is largely dependent of activities of the family of MAPKs.

Differential down-regulation of COX-2 and MMP-13 in human skin fibroblasts by glucosamine-hydrochloride.

BACKGROUND: Evidence suggests anti-inflammatory effects of glucosamine (GS) on inflammatory diseases. COX-2 is an enzyme to produce prostaglandins. MMPs are the family of matrix metalloproteinases degradable of ECM. Excess expression of COX-2 or MMPs involves in skin inflammation. OBJECTIVE: We evaluated whether GS-HCl modulates expression of COX-2 and/or MMPs by IL-1beta or PMA in human skin fibroblasts (HSF) or keratinocytes (HaCaT). METHODS: HSF or HaCaT cells were exposed to IL-1beta or PMA without or with GS-HCl. COX-2 or MMPs protein and mRNA expression, respectively, were analyzed by Western blot and RT-PCR. MTS assay was utilized to assess the cytotoxicity of GS-HCl on HSF cells. RESULTS: In HSF cells, IL-1beta treatment induced COX-2 and MMP-13 expressions in association with activation of ERKs, p38 MAPK, JNKs, and NF-kappaB. PMA treatment also induced COX-2 and MMP-13 expressions in association with p38 MAPK activation. Of interest, treatment with GS-HCl (10mM) led to blockage of p38 MAPK activation, accumulation of 66kDa COX-2 protein variant (without affecting COX-2 mRNA expression), and transcriptional down-regulation of MMP-13 in the IL-1beta- or PMA-treated HSF cells. Distinctly, pharmacological inhibition of p38 MAPK with SB203580 was associated with transcriptional down-regulation of COX-2 and MMP-13 in the IL-1beta- or PMA-treated HSF cells. In addition, the GS-HCl-mediated COX-2 protein modification was observed in both endogenous and PMA-induced COX-2 in HaCaT cells. CONCLUSIONS: GS-HCl differentially down-regulates COX-2 and MMP-13 expression in the IL-1beta- or PMA-treated human skin fibroblasts via the p38 MAPK-independent COX-2 translational inhibition and the p38 MAPK-dependent MMP-13 transcriptional suppression, respectively.

Sanguinarine induces apoptosis in A549 human lung cancer cells primarily via cellular glutathione depletion.

Sanguinarine is a plant-derived benzophenanthridine alkaloid and has been shown to possess anti-tumor activities against various cancer cells. In this study, we investigated whether sanguinarine induces apoptosis in A549 human lung cancer cells. Treatment of A549 cells with sanguinarine induced apoptosis in a dose- and time-dependent manner. Treatment with sanguinarine led to activation of caspases and MAPKs as well as increased MKP-1 expression. Importantly, pretreatment with z-VAD-fmk, a pan caspase inhibitor suppressed the sanguinarine-induced apoptosis in A549 cells. Moreover, pretreatment with NAC, a sulfhydryl group-containing reducing agent strongly suppressed the apoptotic response and caspase activation to sanguinarine. However, the sanguinarine-mediated cytotoxicity in A549 cells was not protected by pharmacological inhibition of MAPKs or MKP-1 siRNA-mediated knockdown of MKP-1. These results collectively suggest that sanguinarine induces apoptosis in A549 cells through cellular glutathione depletion and the subsequent caspase activation.