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Pemetrexed, a new-generation antifolate, has demonstrated promising single-agent activity in front- and second-line treatments of non-small cell lung cancer. However, the molecular mechanism of pemetrexed-mediated antitumor activity remains unclear. The current study shows that pemetrexed induced DNA damage and caspase-2, -3, -8, and -9 activation in A549 cells and that treatment with caspase inhibitors significantly abolished cell death, suggesting a caspase-dependent apoptotic mechanism. The molecular events of pemetrexed-mediated apoptosis was associated with the activation of ataxia telangiectasia mutated (ATM)/p53-dependent and -independent signaling pathways, which promoted intrinsic and extrinsic apoptosis by upregulating Bax, PUMA, Fas, DR4, and DR5 and activating the caspase signaling cascade. Supplementation with dTTP allowed normal S-phase progression and rescued apoptotic death in response to pemetrexed. Overall, our findings reveal that the decrease of thymidylate synthase and the increase of Bax, PUMA, Fas, DR4, and DR5 genes may serve as biomarkers for predicting responsiveness to pemetrexed., (Copyright © 2011 Wiley Periodicals, Inc.)

FBXW7, a component of E3 ubiquitin ligase, plays an important role in mitotic checkpoint, but its role remains unclear. Aurora B is a mitotic checkpoint kinase that plays a pivotal role in mitosis by ensuring correct chromosome segregation and normal progression through mitosis. Whether Aurora B and FBXW7 are coordinately regulated during mitosis is not known. Here, we show that FBXW7 is a negative regulator for Aurora B. Ectopic expression of FBXW7 can suppress the expression of Aurora B. Accordingly, FBXW7 deficiency leads to Aurora B elevation. Mechanistic studies show that all FBXW7 isoforms are negative regulators of Aurora B expression through ubiquitination-mediated protein degradation. Aurora B interacts with R465 and R505 residues of WD 40 domain of FBXW7. Significantly, inverse correlation between FBXW7 and Aurora B elevation is translated into the deregulation of mitosis. FBWX7 expression mitigates Aurora B-mediated cell growth and mitotic deregulation. In addition, FBXW7 reduces the percentage of multinucleated cells caused by Aurora B overexpression. These data suggest that FBXW7 is an important negative regulator of Aurora B, and that the loss or mutation of FBXW7 as seen in many types of cancer could lead to an abnormal elevation of Aurora B and result in deregulated mitosis, which accelerates cancer cell growth.

p53 plays an important role in mitotic checkpoint, but what its role is remains enigmatic. Aurora A is a Ser/Thr kinase involved in correcting progression of mitosis. Here, we show that p53 is a negative regulator for Aurora A. We found that p53 deficiency leads to Aurora A elevation. Ectopic expression of p53 or DNA damage-induced expression of p53 can suppress the expression of Aurora A. Mechanistic studies show that p53 is a negative regulator for Aurora A expression through both transcriptional and posttranslational regulation. p53 knockdown in cancer cells reduces the level of p21, which, in turn, increases the activity of CDK2 followed by induction of Rb1 hyperphosphorylation and its dissociation with transcriptional factor E2F3. E2F3 can bind to Aurora A gene promoter, potentiating Aurora A gene expression and p53 deficiency, enhancing the binding of E2F3 on Aurora A promoter. Also, p53 deficiency leads to decelerating Aurora A's turnover rate, due to the fact that p53 deficiency causes the downregulation of Fbw7α, a component of E3 ligase of Aurora A. Consistently, p53 knockdown-mediated Aurora A elevation is mitigated when Fbw7α is ectopically expressed. Thus, p53-mediated Aurora A degradation requires Fbw7α expression. Significantly, inverse correlation between p53 and Aurora A elevation is translated into the deregulation of centrosome amplification. p53 knockdown leads to high percentages of cells with abnormal amplification of centrosome. These data suggest that p53 is an important negative regulator of Aurora A, and that loss of p53 in many types of cancer could lead to abnormal elevation of Aurora A and dysregulated mitosis, which provides a growth advantage for cancer cells.

Introduction: In an attempt to search for genes with abnormal expression in cancers, Suppressed in Lung Cancer (SLAN, also known as KIAA0256) is found underexpressed in human lung cancer tissues by quantitative real-time PCR (Q-RT-PCR). The study set out to characterize SLAN protein and explore its cellular functions., Methods: SLAN or its specific short hairpin RNA, full length or various deletion mutants were overexpressed in 293T or lung cancer cell lines, and cell proliferation, cell cycle, mitosis progression, and spindle configuration were surveyed., Results: SLAN and its deletion mutants are localized to many subcellular locations such as endoplasmic reticulum (ER), nucleus, nucleolus, spindle pole and midbody, suggesting SLAN may function as a multifunctional protein. Overexpression of SLAN per se or its short hairpin RNAs (shRNAs) inhibits or accelerates cell proliferation through prolonging or shortening mitosis. Time-lapse microscopic recording reveals that cells overexpressing exogenous SLAN are arrested in mitosis or cannot undergo cytokinesis. SLAN 2-551 mutants drastically arrest cells in mitosis, where α- and γ-tubulin are disorganized. SLAN employs C-terminal to interact with Aurora-A, a key mitosis regulator and an oncogenic kinase associated with a wide range of human cancers. SLAN negatively regulates the activity of Aurora-A by directly inhibiting kinase activity in vitro or reducing the level of active Aurora-A in cells. SLAN is frequently reduced in lung cancer tissues overexpressing Aurora-A, arguing for the necessity to suppress SLAN during the Aurora-A-associated cancer formation., Conclusions: Taken together, we have identified a novel protein SLAN downregulated in lung caner, having multiple subcellular localization including spindle matrix and midbody, inhibiting cell proliferation and Aurora-A.

Mutations of the p53 tumor suppressor gene are the most common mutations found in human tumors. There is increasing evidence that suggests that p53 status is a determinant of chemosensitivity of tumor cells. We have previously demonstrated that p53 is a crucial regulator in mediating gefitinib-induced cell death, which upregulates apoptosis-related molecules. However, the mechanism of p53 involvement in cellular resistance to gefitinib remains unclear. In this study, we found that human non-small cell lung cancer cells, A549, with wild-type p53 exhibited a low level of Aurora-A expression and were sensitive to treatment with gefitinib. p53-knockdown A549 cells exhibited a high level of Aurora-A expression and were resistant to gefitinib-mediated apoptosis induction. In addition, the silencing of Aurora-A expression using an Aurora-A specific siRNA in p53-knockdown cells sensitized the A549 cancer cells to gefitinib-mediated apoptosis, suggesting a role for Aurora-A in gefitinib resistance. The activation of Aurora-A was accompanied by destabilization of IκBα and an increase in NF-κB transcriptional activity and was correlated with gefitinib resistance. Conversely, knockdown of Aurora-A with a siRNA stabilized IκB protein suppressed NF-κB activation and reduced gefitinib resistance. Additionally, ectopic expression of an active form of Aurora-A increased the degradation of IκB, the activation of NF-κB and the enhancement of gefitinib resistance in comparison with parental cells. These results suggest that Aurora-A is potentially involved in promoting gefitinib resistance via the activation of NF-κB pathway. Our findings also suggest that p53 not only stimulates apoptosis-related event but also inhibits the drug-resistance ability of Aurora-A, and consequently promotes the gefitinib-induced cellular apoptotic process., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by the activation of fibroblasts and the overproduction of extracellular matrix. Fibroblast resistance to apoptosis leads to increased fibrosis. Targeting fibroblasts with apoptotic agents represents a major therapeutic intervention for debilitating IPF. Gallic acid (3,4,5-trihydroxybenzoic acid), a naturally occurring plant phenol, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. However, the effects of gallic acid on lung fibroblasts have not been investigated. The aim of the present study is to determine the effects of gallic acid on primary cultured mouse fibroblasts. Our results showed that gallic acid induces the apoptotic death of fibroblasts via both intrinsic and extrinsic apoptotic pathways by the elevation of PUMA, Fas, and FasL protein levels. Moreover, intracellular reactive oxygen species (ROS) generation and 8-hydroxy-2'-deoxyguanosine production were observed in gallic acid-stimulated fibroblasts. Mechanistic studies showed that gallic acid induces early phosphorylation of p53(Ser18) and histone 2AX(Ser139) (H2AX) via ataxia telangiectasia mutated (ATM) activation in response to ROS-provoked DNA damage. When mouse lung fibroblasts were treated with caffeine, an ATM kinase inhibitor, the levels of p53, phosphorylated p53(Ser18), and cell death induced by gallic acid were significantly attenuated. Additionally, pretreatment with antioxidants drastically inhibited the gallic acid-induced 8-hydroxy-2'-deoxyguanosine (8-OH-dG) formation and phosphorylation of p53(Ser18) and ATM(Ser1981), as well as apoptosis. Our results provide the first evidence of the activation of ROS-dependent ATM/p53 signaling as a critical mechanism of gallic acid-induced cell death in primary cultured mouse lung fibroblasts.

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone), a natural anthraquinone compound isolated from the rhizome of rhubarb, has been reported to suppress tumor growth in many clinical situations. Here, we demonstrate that emodin induces apoptosis in human lung adenocarcinoma A549 cells by activating a reactive oxygen species-elicited ATM-p53-Bax signaling pathway. In response to emodin treatment, p53 protein increases in A549 cells, which in turn up-regulates Bax expression. Co-treating cells with either a p53 inhibitor or respectively knocking down the expression of p53 and Bax by shRNA extensively diminished emodin-induced cell viability, caspase 3 activation and the release of cytochrome c from the mitochondria, indicating the crucial role for p53/Bax in emodin-mediated cytotoxicity. Pre-treating cells with the antioxidant ascorbic acid not only prohibited the induction of reactive oxygen species by emodin, but also inhibited the up-regulation of p53. Upon emodin treatment, p53 is phosphorylated at Ser(15), which is accompanied by the ATM phosphorylation at Ser(1981). Both of these events could also be blocked by the presence of ascorbic acid. Moreover, knockdown of ATM by siRNA significantly reduced p53 phosphorylation and stabilization, indicating the upstream role of emodin-induced reactive oxygen species generation in ATM activation and following p53 phosphorylation and stabilization. Taken together, our results demonstrate that emodin-induced reactive oxygen species generation activates an ATM-p53-Bax-dependent signaling pathway, which consequently leads to mitochondria-dependent apoptotic cell death in human lung adenocarcinoma A549 cells.

Platelet-derived growth factor (PDGF) is released from vascular smooth muscle cells (VSMCs), endothelial cells, or macrophages after percutaneous coronary intervention and is related with neointimal proliferation and restenosis. Berberine is a well-known component of the Chinese herb medicine Huanglian (Coptis chinensis), and is capable of inhibiting growth and endogenous PDGF synthesis in VSMCs after in vitro mechanical injury. We analyzed the effects of berberine on VSMC growth, migration, and signaling events after exogenous PDGF stimulation in vitro in order to mimic a post-angioplasty PDGF shedding condition. Pretreatment of VSMCs with berberine inhibited PDGF-induced proliferation. Berberine significantly suppressed PDGF-stimulated Cyclin D1/D3 and Cyclin-dependent kinase (Cdk) gene expression. Moreover, berberine increased the activity of AMP-activated protein kinase (AMPK), which led to phosphorylation activation of p53 and increased protein levels of the Cdk inhibitor p21(Cip1). Compound C, an AMPK inhibitor, partly but significantly attenuated berberine-elicited growth inhibition. In addition, stimulation of VSMCs with PDGF led to a transient increase in GTP-bound, active form of Ras, Cdc42 and Rac1, as well as VSMC migration. However, pretreatment with berberine significantly inhibited PDGF-induced Ras, Cdc42 and Rac1 activation and cell migration. Co-treatment with farnesyl pyrophosphate and geranylgeranyl pyrophosphate drastically reversed berberine-mediated anti-proliferative and migratory effects in VSMCs. Based on these findings, we conclude that berberine inhibited PDGF-induced VSMC growth via activation of AMPK/p53/p21(Cip1) signaling while inactivating Ras/Rac1/Cyclin D/Cdks and suppressing PDGF-stimulated migration via inhibition of Rac1 and Cdc42. These observations offer a molecular explanation for the anti-proliferative and anti-migratory properties of berberine.

The c-Fos has been implicated in the regulation of gene expression under a variety of stimuli. It is known that c-Fos undergoes protein phosphorylation, which may subsequently modulate diverse functions in cells. However, less is known about the role and phosphorylation status of c-Fos during mitosis. Here, we showed that c-Fos exhibited an electrophoretic mobility up-shift as detected by SDS-PAGE during mitosis, which is an indication of protein phosphorylation. Aurora-A, but not Aurora-B or -C, serves as one of the kinases catalyzing the mitotic phosphorylation of c-Fos. The mobility up-shift was partially abolished by introducing siRNA or a catalytically inactive form of Aurora-A. Moreover, ectopic expression of the wild type, but not the catalytically inactive form of Aurora-A resulted in the alteration of c-Fos complex formation, suggesting Aurora-A is engaged in the regulation of c-Fos protein-protein interaction. These findings imply that c-Fos may undergo cell cycle dependent phosphorylation, in which some kinases including Aurora-A play a role in catalyzing the post translational modification of c-Fos.

The evolutionarily conserved Aurora family kinases, a family of mitotic serine/threonine kinases, has three members in humans (Aurora-A, -B and -C). Overexpression of Aurora family members, particularly Aurora-A, has been reported in many human cancers and cell lines. In this study, we present evidence based on comparative gene expression analysis via quantitative RT-PCR to delineate the relative contributions of these kinases in 60 cell lines and statistical analysis in five different human cancer microarray datasets. The analysis demonstrated the selective upregulation of these Aurora members in various cancers. In general, Aurora-A exhibited the highest expression levels, with substantially decreased quantities of the Aurora-C transcript detected relative to Aurora-A and -B. Moreover, to characterize the roles of each Aurora member, which share many similarities, we investigated the expression profiles of the family in normal tissues and a panel of different phases of the HeLa cell cycle. Finally, both Aurora-A and -B were overexpressed in a majority of esophageal tumor tissues in comparison to the normal variants. Taken together, the results show that each Aurora member exhibits distinct expression patterns, implying that they are engaged in different biological processes to accomplish more elaborate cell physiological functions in higher organisms.