Cancer is frequently considered to be a disease of the cell cycle. As such, it is not surprising that the deregulation of the cell cycle is one of the most frequent alterations during tumor development. Cell cycle progression is a highlyordered and tightly-regulated process that involves multiple checkpoints that assess extracellular growth signals, cell size, and DNA integrity. Cyclin-dependent kinases (CDKs) and their cyclin partners are positive regulators or accelerators that induce cell cycle progression; whereas, cyclindependent kinase inhibitors (CKIs) that act as brakes to stop cell cycle progression in response to regulatory signals are important negative regulators. Cancer originates from the abnormal expression or activation of positive regulators and functional suppression of negative regulators. Therefore, understanding the molecular mechanisms of the deregulation of cell cycle progression in cancer can provide important insights into how normal cells become tumorigenic, as well as how new cancer treatment strategies can be designed.

We have previously reported that ursolic acid, a pentacyclic triterpene acid, inhibited the invasion of HT1080 human ®brosarcoma cells by reducing the expression of matrix metalloproteinase-9. Since the chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid, we investigated whether ursolic acid acts through the glucocorticoid receptor. The expression of matrix metalloproteinase-9 is thought to be regulated similarly with matrix metalloproteinase-1 and matrix metalloproteinase-3 as containing common 2-O-tetradecanoylphorbol-acetate responsible region, where AP-1 proteins can bind. Dexamethasone has been studied to repress the 2-O-tetradecanoylphorbolacetate-induced expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 through a glucocorticoid receptor-mediated manner. In Northern blot analysis, we found that ursolic acid reduced the expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 induced by 2-O-tetradecanoylphorbol-acetate. Similarly, ursolic acid down-regulated 2-O-tetradecanoylphorbolacetate-induction of matrix metalloproteinase-9 gene in the same manner of dexamethasone. RU486, a potent glucocorticoid receptor antagonist, was used for identifying that ursolic acid-induced down-regulation of matrix metalloproteinase-9 expression is mediated by its binding to glucocorticoid receptor. The e ect of ursolic acid on the matrix metalloproteinase-9 expression was blocked by RU486, suggesting that ursolic acid acts via a glucocorticoid receptor in the regulation of matrix metalloproteinase-9. Western blot analysis and immunocytochemistry showed that ursolic acid increased glucocorticoid receptor fraction in the nucleus, although it decreased the synthesis of glucocorticoid receptor mRNA. In addition, ursolic acid did not decrease the expression of c-jun and DNAbinding activity of AP-1 to its cognate sequences. Taken together, we suggest that ursolic acid may induce the repression of matrix metalloproteinase-9 by stimulating the nuclear translocation of glucocorticoid receptor, and the translocated glucocorticoid receptor probably downmodulating the trans-activating function of AP-1 to 2-Otetradecanoylphorbol-acetate responsible element of matrix metalloproteinase-9 promoter region.

Backgroundβ-lapachone (β-lap) is a bioreductive agent that is activated by the two-electron reductase NAD(P)H quinone oxidoreductase 1 (NQO1). Although β-lap has been reported to induce apoptosis in various cancer types in an NQO1-dependent manner, the signaling pathways by which β-lap causes apoptosis are poorly understood.Methodology/Principal Findingsβ-lap-induced apoptosis and related molecular signaling pathways in NQO1-negative and NQO1-overexpressing MDA-MB-231 cells were investigated. Pharmacological inhibitors or siRNAs against factors involved in β-lap-induced apoptosis were used to clarify the roles played by such factors in β-lap-activated apoptotic signaling pathways. β-lap leads to clonogenic cell death and apoptosis in an NQO1- dependent manner. Treatment of NQO1-overexpressing MDA-MB-231 cells with β-lap causes rapid disruption of mitochondrial membrane potential, nuclear translocation of AIF and Endo G from mitochondria, and subsequent caspase-independent apoptotic cell death. siRNAs targeting AIF and Endo G effectively attenuate β-lap-induced clonogenic and apoptotic cell death. Moreover, β-lap induces cleavage of Bax, which accumulates in mitochondria, coinciding with the observed changes in mitochondria membrane potential. Pretreatment with Salubrinal (Sal), an endoplasmic reticulum (ER) stress inhibitor, efficiently attenuates JNK activation caused by β-lap, and subsequent mitochondria-mediated cell death. In addition, β-lap-induced generation and mitochondrial translocation of cleaved Bax are efficiently blocked by JNK inhibition.Conclusions/SignificanceOur results indicate that β-lap triggers induction of endoplasmic reticulum (ER) stress, thereby leading to JNK activation and mitochondria-mediated apoptosis. The signaling pathways that we revealed in this study may significantly contribute to an improvement of NQO1-directed tumor therapies.

NAD(P)H:quinone oxidoreductase (NQO1) has been reported to play an important role in cell death caused by beta-lapachone (beta-lap), 3,4-dihydro-22,2-dimethyl-2H-naphthol[1,22b]pyran-5,6-dione. This study investigated whether cisplatin (cis-diamminedichloroplatinum) sensitizes cancer cells to beta-lap by upregulating NQO1. The cytotoxicity of cisplatin and beta-lap alone or in combination against FSaII fibrosarcoma cells of C3H mice in vitro was determined with a clonogenic survival assay and assessment of gamma-H2AX foci formation, a hallmark of DNA double-strand breaks. The cellular sensitivity to beta-lap progressively increased during the 24 h after cisplatin treatment. The expression and enzymatic activity of NQO1 also increased during the 24 h after cisplatin treatment, and dicoumarol, an inhibitor of NQO1, was found to nullify the cisplatin-induced increase in beta-lap sensitivity. The role of NQO1 in the cell death caused by beta-lap alone or in combination with cisplatin was further elucidated using NQO1-positive and NQO1-negative MDA-MB-231 human breast cancer cells. Cisplatin increased the sensitivity of the NQO1-positive but not the NQO1-negative MDA-MB-231 cells to beta-lap treatment. Combined treatment with cisplatin and beta-lap suppressed the growth of FSaII tumors in the legs of C3H mice in a manner greater than additive. It is concluded that cisplatin markedly increases the sensitivity of cancer to beta-lap in vitro and in vivo by upregulating NQO1.

Histone deacetylase (HDAC) plays an important role in cancer onset and progression. Therefore, inhibition of HDAC offers potential as an effective cancer treatment regimen. CG200745, (E)-N(1)-(3-(dimethylamino)propyl)-N(8)-hydroxy-2-((naphthalene-1-loxy)methyl)oct-2-enediamide, is a novel HDAC inhibitor presently undergoing a phase I clinical trial. Enhancement of p53 acetylation by HDAC inhibitors induces cell cycle arrest, differentiation, and apoptosis in cancer cells. The purpose of the present study was to investigate the role of p53 acetylation in the cancer cell death caused by CG200745. CG200745-induced clonogenic cell death was 2-fold greater in RKO cells expressing wild-type p53 than in p53-deficient RC10.1 cells. CG200745 treatment was also cytotoxic to PC-3 human prostate cancer cells, which express wild-type p53. CG200745 increased acetylation of p53 lysine residues K320, K373, and K382. CG200745 induced the accumulation of p53, promoted p53-dependent transactivation, and enhanced the expression of MDM2 and p21(Waf1/Cip1) proteins, which are encoded by p53 target genes. An examination of CG200745 effects on p53 acetylation using cells transfected with various p53 mutants showed that cells expressing p53 K382R mutants were significantly resistant to CG200745-induced clonogenic cell death compared with wild-type p53 cells. Moreover, p53 transactivation in response to CG200745 was suppressed in all cells carrying mutant forms of p53, especially K382R. Taken together, these results suggest that acetylation of p53 at K382 plays an important role in CG200745-induced p53 transactivation and clonogenic cell death.

Backgroundβ-lapachone (β-lap), has been known to cause NQO1-dependnet death in cancer cells and sensitize cancer cells to ionizing radiation (IR). We investigated the mechanisms underlying the radiosensitization caused by β-lap.Methodology/Principal Findingsβ-lap enhanced the effect of IR to cause clonogenic cells in NQO1+-MDA-MB-231 cells but not in NQO1−-MDA-MB-231 cells. β-lap caused apoptosis only in NQO1+ cells and not in NQO1− cells and it markedly increased IR-induced apoptosis only in NQO1+ cells. Combined treatment of NQO1+ cells induced ROS generation, triggered ER stress and stimulated activation of ERK and JNK. Inhibition of ROS generation by NAC effectively attenuated the activation of ERK and JNK, induction of ER stress, and subsequent apoptosis. Importantly, inhibition of ERK abolished ROS generation and ER stress, whereas inhibition of JNK did not, indicating that positive feedback regulation between ERK activation and ROS generation triggers ER stress in response to combined treatment. Furthermore, prevention of ER stress completely blocked combination treatment-induced JNK activation and subsequent apoptotic cell death. In addition, combined treatment efficiently induced the mitochondrial translocation of cleaved Bax, disrupted mitochondrial membrane potential, and the nuclear translocation of AIF, all of which were efficiently blocked by a JNK inhibitor. Caspases 3, 8 and 9 were activated by combined treatment but inhibition of these caspases did not abolish apoptosis indicating caspase activation played a minor role in the induction of apoptosis.Conclusions/Significanceβ-lap causes NQO1-dependent radiosensitization of cancer cells. When NQO1+ cells are treated with combination of IR and β-lap, positive feedback regulation between ERK and ROS leads to ER stress causing JNK activation and mitochondrial translocation of cleaved Bax. The resultant decrease in mitochondrial membrane leads to translocation of AIF and apoptosis.

The purpose of the present investigation was to study the effects of ionizing radiation on endothelial cells derived from diverse normal tissues. We first compared the effects of radiation on clonogenic survival and tube formation of endothelial cells, and then investigated the molecular signaling pathways involved in endothelial cell survival and angiogenesis. Among the different endothelial cells studied, human hepatic sinusoidal endothelial cells (HHSECs) were the most radio-resistant and human dermal microvascular endothelial cells were the most radio-sensitive. The radio-resistance of HHSECs was related to adenosine monophosphate-activated protein kinase and p38 mitogen-activated protein kinase-mediated expression of MMP-2 and VEGFR-2, whereas the increased radio-sensitivity of HDMECs was related to extracellular signal-regulated kinase-mediated generation of angiostatin. These observations demonstrate that there are distinct differences in the radiation responses of normal endothelial cells obtained from diverse organs, which may provide important clues for protection of normal tissue from radiation exposure.

UHRF1 (ubiquitin-like, with PHD and RING finger domains 1) plays a crucial role in DNA methylation, chromatin remodeling and gene expression and is aberrantly upregulated in various types of human cancers. However, the precise role of UHRF1 in cancer remains controversial. In this study, we observed that hypoxia-induced downregulation of UHRF1 contributes to the induction of the epithelial-mesenchymal transition (EMT) in hepatocellular carcinoma cells. By negatively modulating UHRF1 expression, we further showed that UHRF1 deficiency in itself is sufficient to increase the migratory and invasive properties of cells via inducing EMT, increasing the tumorigenic capacity of cells and leading to the expansion of cancer stem-like cells. Epigenetic changes caused by UHRF1 deficiency triggered the upregulation of CXCR4, thereby activating AKT and JNK to increase the expression and secretion of IL-6. In addition, IL-6 readily activated the JAK/STAT3/Snail signaling axis, which subsequently contributed to UHRF1 deficiency-induced EMT. Our results collectively demonstrate that UHRF1 deficiency may play a pivotal role in the malignant alteration of cancer cells.