Jin Hyen Baek

Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes.

Low oxygen tension influences tumor progression by enhancing angiogenesis; and histone deacetylases (HDAC) are implicated in alteration of chromatin assembly and tumorigenesis. Here we show induction of HDAC under hypoxia and elucidate a role for HDAC in the regulation of hypoxia-induced angiogenesis. Overexpressed wild-type HDAC1 downregulated expression of p53 and von Hippel–Lindau tumor suppressor genes and stimulated angiogenesis of human endothelial cells. A specific HDAC inhibitor, trichostatin A (TSA), upregulated p53 and von Hippel–Lindau expression and downregulated hypoxia-inducible factor-1α and vascular endothelial growth factor. TSA also blocked angiogenesis in vitro and in vivo. TSA specifically inhibited hypoxia-induced angiogenesis in the Lewis lung carcinoma model. These results indicate that hypoxia enhances HDAC function and that HDAC is closely involved in angiogenesis through suppression of hypoxia-responsive tumor suppressor genes.

Effects of three-dimensional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells.

Embryonic stem (ES) cells have the ability to self‐replicate and differentiate into cells from all three germ layers, holding great promise for tissue regeneration applications. However, controlling the differentiation of ES cells and obtaining homogenous cell populations still remains a challenge. We hypothesize that a supportive three‐dimensional (3D) environment provides ES cell‐derived cells an environment that more closely mimics chondrogenesis in vivo. In the present study, the chondrogenic differentiation capability of ES cell‐derived embryoid bodies (EBs) encapsulated in poly(ethylene glycol)‐based (PEG) hy‐drogels was examined and compared with the chondrogenic potential of EBs in conventional monolayer culture. PEG hydrogel‐encapsulated EBs and EBs in monolayer were cultured in vitro for up to 17 days in chondrogenic differentiation medium in the presence of transforming growth factor (TGF)‐β1 or bone morphogenic protein‐2. Gene expression and protein analyses indicated that EB‐PEG hydrogel culture upregulated cartilage‐relevant markers compared with a monolayer environment and induction of chondrocytic phenotype was stimulated with TGF‐β1. Histology of EBs in PEG hydrogel culture with TGF‐β1 demonstrated basophilic extracellular matrix deposition characteristic of neocartilage. These findings suggest that EB‐PEG hydrogel culture, with an appropriate growth factor, may provide a suitable environment for chondrogenic differentiation of intact ES cell‐derived EBs.

Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis

Low oxygen and nutrient depletion play critical roles in tumorigenesis, but little is known about how they interact to produce tumor survival and tumor malignancy. In the present study, we investigated the mechanism underlying hypoxia-modulated apoptosis of serum-deprived HepG2 cells. Our results showed that hypoxia blocked the apoptosis, which was accompanied with decreased Bax/Bcl-2 ratio, inhibited cytochrome c release, and reduced caspase-3 activity. More importantly, increased expressions of VEGF and its receptor-2 (KDR) under hypoxic/serum-deprived condition suggest that VEGF may act as a survival factor in a self-promoting manner. Data were further supported by results that recombinant human VEGF (rhVEGF) suppressed the serum deprivation-induced apoptosis, and anti-VEGF neutralizing antibody block anti-apoptotic activity of hypoxia. In addition, inhibitors of receptor tyrosine kinase blocked anti-apoptosis of hypoxia. Our study further showed that rhVEGF or hypoxia induced ERK phosphorylation in serum-deprived cells, and that a specific inhibitor of MAPK/ERK, PD98059 eliminated the anti-apoptotic activity of rhVEGF or hypoxia by increasing Bax/Bcl-2 ratio and caspase-3 activity. Our data led us to conclude that induction of ERK phosphorylation and decrease of Bax/Bcl-2 ratio by rhVEGF implies that hypoxia-induced VEGF prevents apoptosis of serum-deprived cells by activating the MAPK/ERK pathway. Taken together, we propose that hypoxia enhances survival of nutrient-depleted tumor cells by reducing susceptibility to apoptosis, which consequently leads to tumor malignancy.

APOPTOTIC ACTIVITY OF URSOLIC ACID MAY CORRELATE WITH THE INHIBITION OF INITIATION OF DNA REPLICATION

Ursolic acid (UA), a pentacyclic triterpene acid, has been reported to exhibit anti‐tumor activity. In this study, we investigated the pro‐apoptotic effect of UA on HepG2 human hepatoblastoma cells. Treatment with UA decreased the viability of HepG2 cells in a concentration‐ and time‐dependent manner. Furthermore, 30 μM of UA induced DNA fragmentation and subdiploid cells and enhanced the release of cytochrome c and the activation of caspase‐3. These results suggest that UA induces cell death through apoptosis, which may be mediated by cytochrome c‐dependent caspase‐3 activation. In addition, cell‐cycle analysis revealed that UA‐treated cells were arrested predominantly in the G0 and G1 phases with a concomitant decrease in the cell population of S phase. Moreover, expression of p21WAF1, a cell‐cycle regulator, was increased by UA, indicating that p21WAF1 might mediate UA‐induced cell‐cycle arrest. However, UA markedly inhibited SV40 DNA replication in the initiation stage in vitro and significantly reduced the DNA cleaving of topoisomerase I and the ssDNA binding activity of replication protein A. These results indicate that the inhibition of DNA replication by UA may result from blockade of the establishment of the replication fork during initiation stage, consequently contributing to UA‐induced cell‐cycle arrest. Taken together, we suggest that UA‐induced cell‐cycle arrest may be mediated by inhibition of DNA replication and the increase of p21WAF1 expression, which induces the release of cytochrome c and the activation of caspase‐3, leading to apoptosis of HepG2 cells. Int. J. Cancer 87:629–636, 2000.

Intracellular Ca2+ release mediates ursolic acid–induced apoptosis in human leukemic HL‐60 cells

The effect of ursolic acid (UA) on tumor cell apoptosis was investigated using HL‐60 human promyelocytic leukemia cells as a model cellular system. Treatment with UA resulted in a concentration‐dependent decreased cell viability assessed by MTT assay. UA also induced genomic DNA fragmentation, a hallmark of apoptosis, indicating that the mechanism by which UA induced cell death was through apoptosis. The intracellular Ca2+ level was increased by treatment with UA. Intracellular Ca2+ inhibitors, such as intracellular Ca2+‐release blockers (dantrolene, TMB‐8 and ruthenium red) and an intracellular Ca2+ chelator (BAPTA/AM), significantly blocked the UA‐induced increased intracellular Ca2+ concentration. These inhibitors also blocked the effects of UA on cell viability and apoptosis. These results suggest that enhanced intracellular Ca2+ signals may be involved in UA‐induced apoptosis in HL‐60 cells. Int. J. Cancer 73:725–728, 1997.

Overexpression of AQP5, a putative oncogene, promotes cell growth and transformation

Overexpression of several aquaporins has been reported in different types of human cancer but the role of AQPs in human carcinogenesis has not yet been clearly defined. Here, we demonstrate that ectopic expression of human AQP5 (hAQP5), a water channel expressed in lung, salivary glands, and kidney, induces many phenotypic changes characteristic of transformation both in vitro and in vivo. Furthermore, the cell proliferative ability of AQP5 appears to be dependent upon the phosphorylation of a cAMP-protein kinase (PKA) consensus site located in a cytoplasmic loop of AQP5. In addition, phosphorylation of the PKA consensus site was found to be phosphorylated preferentially in tumors. These findings altogether indicate that hAQP5 plays an important role in human carcinogenesis and, furthermore, provide an attractive therapeutic target.

Aberrant promoter methylation and tumor suppressive activity of the DFNA5 gene in colorectal carcinoma

To identify novel methylated gene promoters, we compared differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2′-deoxycytidine (5-aza-dC). Out of 1776 genes that were initially ‘absent (that is, silenced)’ by gene expression array analysis, we selected 163 genes that were increased after 5-aza-dC treatment in at least two of three CRC cell lines. The microarray results were confirmed by Reverse Transcription–PCR, and CpG island of the gene promoters were amplified and sequenced for examination of cancer-specific methylation. Among the genes identified, the deafness, autosomal dominant 5 gene, DFNA5, promoter was found to be methylated in primary tumor tissues with high frequency (65%, 65/100). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary CRC tissues from normal colon tissues (3%, 3/100). The mRNA expression of DFNA5 in four of five colon cancer tissues was significantly downregulated as compared to normal tissues. Moreover, forced expression of full-length DFNA5 in CRC cell lines markedly decreased the cell growth and colony-forming ability whereas knockdown of DFNA5 increased cell growth in culture. Our data implicate DFNA5 as a novel tumor suppressor gene in CRC and a valuable molecular marker for human cancer.

The N-methyl-D-aspartate receptor type 2A is frequently methylated in human colorectal carcinoma and suppresses cell growth.

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2′-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

Hypoxia-induced apoptosis in human hepatocellular carcinoma cells: a possible involvement of the 6-TG-sensitive protein kinase(s)-dependent signaling pathway

Apoptosis is a morphologically and biochemically distinct form of cell death which can be triggered by a variety of extracellular agents both during normal developments and in adult pathological states. However, the molecular mechanism of apoptotic cell death due to hypoxia has not been clearly elucidated. In this study, we investigated critical factors involved in hypoxia-induced apoptosis using HepG2, a human hepatocellular carcinoma cell line, as an experimental model. We found that 24 h of exposure of HepG2 cells to hypoxia induced apoptosis, for which de novo protein synthesis was required. Apoptosis was demonstrated by DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Hypoxia-induced apoptosis was associated with a marked induction of c-jun and c-fos messenger RNAs. Electromobility shift assay showed the increased DNA binding activity of AP-1 during hypoxia, suggesting that AP-1 may be involved in the induction of cell death by acting as a transcriptional regulator. A purine analogue, 6-thioguanine (6-TG), significantly blocked the induction of apoptosis by hypoxia. Moreover, the inductive effect of hypoxia on c-jun expression was also inhibited by 6-TG, whereas the levels of c-fos mRNA and its protein were rather strongly increased. Iodoacetamide (IAA), a non-specific inhibitor of ICE family proteases, also has an inhibitory effect on hypoxia-induced apoptosis. These results suggest that the 6-TG-sensitive protein kinase(s)-dependent signaling pathway may be involved in the apoptotic response of HepG2 cells exposed to hypoxia by increasing the level of c-jun and c-fos and the activity of AP-1 and/or by activating ICE family protease(s).