Bortezomib (PS-341), a proteasome inhibitor, has been examined clinically for the treatment of multiple myeloma and several solid tumors. Bortezomib directly induces tumor cell death and has also been reported to inhibit tumor adaptation to hypoxia by functionally inhibiting hypoxia-inducible factor-1␣ (HIF-1␣). However, the mechanism underlying HIF-1 inhibition by bortezomib remains obscure. In the present study, we demonstrated that bortezomib attenuated the hypoxic induction of erythropoietin and vascular endothelial growth factor at subnanomolar concentrations in multiple myeloma and liver cancer cell lines, regardless of cytotoxic concentrations of bortezomib. Bortezomib repressed HIF-1␣ activity by inhibiting the recruitment of p300 coactivator. Specifically, bortezomib targeted HIF-1␣ C-terminal transactivation domain (CAD) but not the CAD lacking Asn803, which is a hydroxylation site by the factor inhibiting HIF-1 (FIH). Accordingly, this effect of bortezomib on CAD was augmented by FIH expression and abolished by FIH knock-down. Furthermore, bortezomib stimulated the interaction between CAD and FIH under hypoxic conditions, and FIH inhibition reversed the suppressions of erythropoietin and vascular endothelial growth factor by bortezomib. We propose that the mechanism underlying the inhibitory effects of bortezomib on tumor angiogenesis and hypoxic adaptation involves the repression of HIF-1␣ transcriptional activity by reinforcing the FIH-mediated inhibition of p300 recruitment. IntroductionHypoxia commonly develops in solid tumors because tumor growth outpaces vessel formation and because the blood supply is compromised due to aberrant vasculature formation. 1 Tumor hypoxia contributes to angiogenesis and modulates tumor energy metabolism, which are both essential required for tumor growth. 2 In multiple myeloma (MM), hypoxia is also an important environmental factor because bone marrow is intrinsically hypoxic in nature. 3 Thus, MM cells must survive and grow under such hypoxic conditions, and this requires the expressions of many genes essential for adaptation. Hypoxic adaptation is mainly provided by hypoxia-inducible factor-1 (HIF-1), which orchestrates cellular adaptation to hypoxia by transactivating about 60 genes. 4 HIF-1 is composed of HIF-1␣ and HIF-1␤/aryl hydrocarbon nuclear translocator (ARNT), 5 and of these, HIF-1␣ is the key protein that determines the presence of HIF-1 and transactivates genes. Under normoxic conditions, HIF-1␣ is hydroxylated at its Pro402 and Pro564 residues by HIF-1 prolyl hydroxylases (PHDs), and thus, targeted by von Hippel-Lindau protein (pVHL), ubiquitinated, and finally degraded by 26S proteasomes. [6][7][8][9][10] In addition, the C-terminal transactivation domain (CAD) of HIF-1␣ is hydroxylated at Asn803 by the factor inhibiting HIF-1 (FIH), which represses the transcriptional activity of HIF-1␣ by blocking the recruitment of p300 coactivator. 11,12 However, PHD and FIH activities depend on oxygen tension, and as a result HIF-1␣ is stabilized and activated...

SIRT1 is an NAD þ -dependent protein deacetylase induced by metabolic stresses, such as nutrition or oxygen deprivation. Although SIRT1 contributes to aging and metabolic disorders, its role in cancer progression and therapeutic responses remains controversial. Because hypoxia occurs widely in solid tumors, where it provokes drug resistance, we investigated the involvement of SIRT1 in hypoxia-induced chemoresistance. SIRT1 was downregulated in a panel of non-small cell lung carcinoma (NSCLC) cells exposed to hypoxia for 48 hours. The master metabolic kinase AMP-activated protein kinase (AMPK) was inactivated under the same conditions, likely due to attenuation of the SIRT1/LKB1-mediated AMPK activation process. Notably, hypoxic inactivation of this SIRT1-AMPK pathway led to cisplatin and doxorubicin resistance. Mechanistic investigations suggested that this pathway supported the cytotoxic response to cisplatin and doxorubicin by licensing an apoptotic process controlled by mitochondria. We confirmed the involvement of this pathway in a mouse xenograft model of human NSCLC. Furthermore, we demonstrated that a SIRT1 activator SRT1720 augmented the antitumor effects of cisplatin, and these effects could be blocked by administration of an AMPK inhibitor compound C. Taken together, our results offer preclinical proof-of-concept to target the SIRT1-AMPK pathway as a strategy to overcome hypoxia-induced chemoresistance in NSCLC. Cancer Res; 74(1); 298-308. Ó2013 AACR.

Runt-related transcription factor 2 (Runx2) transactivates many genes required for osteoblast differentiation. The role of N-a-acetyltransferase 10 (NAA10, arrest-defective-1), originally identified in yeast, remains poorly understood in mammals. Here we report a new NAA10 function in Runx2-mediated osteogenesis. Runx2 stabilizes NAA10 in osteoblasts during BMP-2-induced differentiation, and NAA10 in turn controls this differentiation by inhibiting Runx2. NAA10 delays bone healing in a rat calvarial defect model and bone development in neonatal mice. Mechanistically, NAA10 acetylates Runx2 at Lys225, and this acetylation inhibits Runx2-driven transcription by interfering with CBFb binding to Runx2. Our study suggests that NAA10 acts as a guard ensuring balanced osteogenesis by fine-tuning Runx2 signalling in a feedback manner. NAA10 inhibition could be considered a potential strategy for facilitating bone formation.

Sodium-dependent vitamin C transporters (SVCTs) is known to transport the reduced form of ascorbic acid into the cell, whereas the oxidized form of vitamin C (VC) is moved through a facilitative sugar transporter, such as glucose transporter (GLUT). With regard to the distribution of SVCT1 and -2 within the various organs, they were reported to be expressed in different types of cells. Especially in the central nervous system, only SVCT2 mRNA was expressed mainly in neurons and some types of neuroglial cells. However, data on the expression of SVCT proteins in the brain are scant. Therefore, we tried to develop comprehensive data on the distribution of SVCT proteins in adult rat brain by using immunohistochemical techniques for the first time. In our study, SVCT2 immunoreactivities (IRs) were intensely localized in the neurons of cerebral cortex, hippocampus, and Purkinje cells of cerebellum, and much weaker SVCT2 IRs were found in the other brain regions. Judging from double-immunohistochemical data, most of the cells expressing SVCT2 IRs were likely to be neurons or microglia, even though the cells in choroids plexus or ependymal cells around the ventricles also exhibited SVCT2 IRs. Complete mapping of the distribution of SVCT2 IRs was available by using a semiquantitative method. The subcellular localization of SVCT proteins is necessary for understanding the exact role of the protein, so the current overall mapping of SVCT IRs in the rat brain could be the basis for further studies on related subjects.

Hypoxia-inducible factor (HIF)-1 plays a key role in tumor promotion by inducing f60 genes required for tumor adaptation to hypoxia; thus, it is viewed as a target for cancer therapy. For this reason, YC-1, which downregulates HIF-1A and HIF-2A at the post-translational level, is being developed as a novel anticancer drug. We here found that YC-1 acts in a novel manner to inhibit HIF-1. In the Gal4 reporter system, which is not degraded by YC-1, YC-1 was found to significantly inactivate the COOH-terminal transactivation domain (CAD) of HIF-1A, whereas it failed to inactivate CAD(N803A) mutant. In coimmunoprecipitation assays, YC-1 stimulated factor inhibiting HIF (FIH) binding to CAD even in hypoxia, whereas it failed to increase the cellular levels of hydroxylated Asn 803 of CAD. It was also found that YC-1 prevented p300 recruitment by CAD in mammalian two-hybrid and coimmunoprecipitation assays. The involvement of FIH in YC-1-induced CAD inactivation was confirmed in EPO-enhancer and Gal4 reporter systems using FIH small interfering RNA and dimethyloxalylglycine FIH inhibitor. Indeed, FIH inhibition rescued HIF target gene expressions repressed by YC-1. In cancer cell lines other than Hep3B, YC-1 inhibits HIF-1A via the FIHdependent CAD inactivation as well as via the protein down-regulation. Given these results, we suggest that the functional inactivation of HIF-A contributes to the YC-1-induced deregulation of hypoxia-induced genes.

BackgroundThe tumor-associated calcium signal transducer (TACSTD) genes, originally designated epithelial cell adhesion molecule (EpCAM) and TROP2, represent true oncogenes. Little is known about EpCAM and TROP2 gene expression in non-small cell lung carcinoma (NSCLC). This study evaluated EpCAM and TROP2 protein expression and clinicopathologic significance in cases of NSCLC.MethodsTissue microarray blocks acquired from 164 cases of NSCLC, including 100 cases of adenocarcinoma (AdC) and 64 of squamous cell carcinoma (SCC), were examined by immunohistochemical staining for EpCAM, and TROP2. The results were correlated with clinicopathologic data.ResultsEpCAM and TROP2 were significantly overexpressed in SCC than in AdC (P < 0.01). In AdC, EpCAM overexpression was closely related to sex, histologic grade, pathologic T stage, pathologic N stage, and TNM stage, and TROP2 overexpression was only related to histologic grade (P < 0.05, respectively). In SCC, correlations were evident between EpCAM overexpression and TNM stage (P = 0.01), and between TROP2 overexpression and pathologic T stage (P = 0.02). EpCAM overexpression showed no significance with overall survival in AdC and SCC patients. However, TROP2 overexpression in AdC had a positive influence on overall survival (P = 0.02) and disease-free survival (P = 0.03). In particular, AdC patients with stage II or III showed better overall survival (P = 0.05) and disease-free survival (P = 0.04).ConclusionsWhile EpCAM and TROP2 show weak and non-complete membranous staining in normal bronchial epithelium and pneumocyte, their complete membranous expression in carcinoma suggests their role in carcinogenesis. EpCAM and TROP2 were more frequently overexpressed in SCC. EpCAM overexpression had no prognostic value in this study, but TROP2 overexpression showed better survival in AdC patients and might be a better prognostic marker in advanced stage AdC.

One-dimensional MnO2-based multiscale micronodules were fabricated through a facile electrodeposition and coating process to produce ultrahigh performance flexible asymmetric supercapacitors.

Joseon mummies have proved to be excellent subjects for scientific research on the health and disease statuses of pre-modern Korean peoples. Despite its academic significance, the origins of the Hoegwakmyo tomb in which the Joseon mummy was discovered have not yet been entirely revealed. Meanwhile, over the past several decades, there have been some reports on mummies and cultural artifacts preserved very well in the tombs of several Chinese dynasties (especially Song, Yuan, Ming and Qing). Although the Chinese tombs were very diverse in structure, we note that some graves among them were structurally very similar to Joseon Hoegwakmyo tombs. Before the Hoegwakmyo tomb in Korea, there were already similar tombs in China, inside which dead persons were mummified like the Joseon mummies. Considering that the Hoegwakmyo tomb of the Joseon Dynasty was established by the influence of the Confucian ideology, the Korean and Chinese mummies might share common cultural origins in history.