Yeonjoo Jung

Class I Histone Deacetylase-Selective Novel Synthetic Inhibitors Potently Inhibit Human Tumor Proliferation

We have developed previously a class of synthetic hybrid histone deacetylase (HDAC) inhibitors, which were built from hydroxamic acid of trichostatin A and pyridyl ring of MS-275. In this study we evaluated the antitumor effects of these novel hybrid synthetic HDAC inhibitors, SK-7041 and SK-7068, on human cancer cells. Both SK-7041 and SK-7068 effectively inhibited cellular HDAC activity at nanomolar concentrations and induced the time-dependent hyperacetylation of histones H3 and H4. These HDAC inhibitors preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, as compared with the other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041 and SK-7068. We found that these compounds exhibited potent antiproliferative activity against various human cancer cells in vitro. Growth inhibition effect of SK-7041 and SK-7068 was related with the induction of aberrant mitosis and apoptosis in human gastric cancer cells. Both compounds induced the accumulation of cells at mitosis after 6 h of treatment, which was demonstrated by accumulation of tetraploid cells, lack of G2 cyclin/cyclin-dependent kinase inactivation, and higher mitotic index. After 12 h of treatment, apoptotic cells were increased through mitochondrial and caspase-mediated pathway. Finally, in vivo experiment showed that SK-7041 or SK-7068 was found to reduce the growth of implanted human tumors in nude mice. Therefore, based on isotype specificity and antitumor activity, SK-7041 and SK-7068 HDAC inhibitors are expected to be promising anticancer therapeutic agents and need additional clinical development.

Histone deacetylase inhibitor enhances 5-fluorouracil cytotoxicity by down-regulating thymidylate synthase in human cancer cells

Thymidylate synthase (TS) overexpression is a key determinant of 5-fluorouracil (5-FU) resistance in human cancer cells. TS is also acutely up-regulated with 5-FU treatment, and, thus, novel strategies targeting TS down-regulation seem to be promising in terms of modulating 5-FU resistance. Here, we report that histone deacetylase inhibitors can reverse 5-FU resistance by down-regulating TS. By using cDNA microarrays and validation experiments, we found that trichostatin A reduced the expression of both TS mRNA and TS protein. Cotreatment with trichostatin A and cycloheximide restored TS mRNA expression, suggesting that TS mRNA is repressed through new protein synthesis. On the other hand, TS protein expression was significantly reduced by lower doses of trichostatin A (50 nmol/L). Mechanistically, TS protein was found to interact with heat shock protein (Hsp) complex, and trichostatin A treatment induced chaperonic Hsp90 acetylation and subsequently enhanced Hsp70 binding to TS, which led to the proteasomal degradation of TS protein. Of note, combined treatment with low-dose trichostatin A and 5-FU enhanced 5-FU–mediated cytotoxicity in 5-FU–resistant cancer cells in accordance with TS protein down-regulation. We conclude that a combinatorial approach using histone deacetylase inhibitors may be useful at overcoming 5-FU resistance. [Mol Cancer Ther 2006;5(12):3085–95]

Potential advantages of DNA methyltransferase 1 (DNMT1)-targeted inhibition for cancer therapy

The deoxyribonucleic acid (DNA) methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) has been used as a drug in a part of cancer therapy. However, because of its incorporation into DNA during DNA synthesis, 5-aza-dC can cause DNA damage, mutagenesis, and cytotoxicity. In view of the adverse effects of 5-aza-dC, DNMT-targeted inhibition may be a more effective approach than treatment with 5-aza-dC. To address the possibility of DNMT-targeted cancer therapy, we compared the effects of treatment with small interfering ribonucleic acids (siRNAs) specific for DNMT1 or DNMT3b and treatment with 5-aza-dC on transcription, cell growth, and DNA damage in gastric cancer cells. We found that DNMT1-targeted inhibition induced the re-expression and reversed DNA methylation of five (CDKN2A, RASSF1A, HTLF, RUNX3, and AKAP12B) out of seven genes examined, and 5-aza-dC reactivated and demethylated all seven genes. In contrast, DNMT3b siRNAs did not show any effect. Furthermore, the double knockdown of DNMT1 and DNMT3b did not show a synergistic effect on gene re-expression and demethylation. In addition, DNMT1 siRNAs showed an inhibitory effect of cell proliferation in the cancer cells and the induction of cell death without evidence of DNA damage, whereas treatment with 5-aza-dC caused DNA damage as demonstrated by the comet assay. These results provide a rationale for the development of a DNMT1-targeted strategy as an effective epigenetic cancer therapy.

Inhibitors of histone deacetylases induce tumor-selective cytotoxicity through modulating Aurora-A kinase

The molecular basis of the antitumor selectivity of histone deacetylase inhibitors (HDIs) remains unclear. Centrosomal Aurora-A kinase regulates chromosomal segregation during mitosis. The overexpression or amplification of Aurora-A leads to genetic instability, and its inhibition has shown significant antitumor effects. In this paper, we report that structurally related hydroxamate LAQ824 and SK-7068 induce tumor-selective mitotic defects by depleting Aurora-A. We found that HDI-treated cancer cells, unlike nontransformed cells, exhibit defective mitotic spindles. After HDI, Aurora-A was selectively downregulated in cancer cells, whereas Aurora-B remained unchanged in both cancer and nontransformed cells. LAQ824 or SK-7068 treatment inhibited histone deacetylase (HDAC) 6 present in Aurora-A/heat shock protein (Hsp) 90 complex. Inhibition of HDAC6 acetylated Hsp90 and resulted in dissociation of acetylated Hsp90 from Aurora-A. As a result, Hsp70 binding to Aurora-A was enhanced in cancer cells, leading to proteasomal degradation of Aurora-A. Overall, these provide a novel molecular basis of tumor selectivity of HDI. LAQ824 and SK-7068 might be more effective HDIs in cancer cells with Aurora-A overexpression.

Gabexate mesilate inhibits colon cancer growth, invasion, and metastasis by reducing matrix metalloproteinases and angiogenesis

Gabexate mesilate (GM), a synthetic protease inhibitor, has an antiproteinase activity on various types of plasma serine proteases. However, its role on matrix metalloproteinases (MMPs) has not been identified. In this study, we investigated the effect of GM on MMPs and on the invasion and metastasis of human colon cancer cell lines and neoangiogenesis. The activities of MMPs secreted from these cells were significantly reduced by GM but unaffected by the serine protease inhibitor aprotinin. GM directly inhibited purified progelatinase A derived from T98G human glioblastoma cells. In vitro, GM significantly reduced the invasive ability of colon cancer cells but not cellular motility, whereas aprotinin affected neither. Liver metastatic ability and tumorigenic potential in nude mice were remarkably reduced on treatment with GM. Immunohistochemical analysis of GM-treated tumors in mice showed a marked increase in apoptosis and a significant reduction in tumor angiogenesis. Human umbilical vein endothelial cell proliferation, tube formation, and neoangiogenesis in the rabbit cornea and Matrigel implanted in mice were significantly inhibited by GM. These results suggest that GM is a novel inhibitor of MMPs and that it may inhibit the invasion and metastasis of human colon cancer cells by blocking MMPs and neoangiogenesis.

Gene silencing of TSPYL5 mediated by aberrant promoter methylation in gastric cancers

DNA methylation is crucial for normal development, but gene expression altered by DNA hypermethylation is often associated with human diseases, especially cancers. The gene TSPYL5, encoding testis-specific Y-like protein, was previously identified in microarray screens for genes induced by the inhibition of DNA methylation and histone deacetylation in glioma cell lines. The TSPYL5 showed a high frequency of DNA methylation-mediated silencing in both glioma cell lines and primary glial tumors. We now report that TSPYL5 is also inactivated by DNA methylation and could be a putative epigenetic target gene in gastric cancers. We found that the expression of TSPYL5 mRNA was frequently downregulated and inversely correlated with DNA methylation in seven out of nine gastric cancer cell lines. TSPYL5 mRNA expression was also restored after treating with a DNA methyltransferase inhibitor. In primary gastric tumors, methylation-specific PCR results in 23 of the 36 (63.9%) cases revealed that the hypermethylation at CpG islands of the TSPYL5 was detectable at a high frequency. Furthermore, TSPYL5 suppressed the growth of gastric cancer cells as demonstrated by a colony formation assay. Thus, strong associations between TSPYL5 expression and hypermethylation were observed, and aberrant methylation at a CpG island of TSPYL5 may play an important role in development of gastric cancers.

Cytotoxic effects of pemetrexed in gastric cancer cells.

Pemetrexed is a newly developed multitargeted antifolate with promising clinical activity in many solid tumors including gastric cancer. The aim of the present study was to evaluate the cytotoxicity of pemetrexed and its mode of interaction with cisplatin in gastric cancer cell lines, and to identify genes associated with sensitivity to pemetrexed. The cytotoxic activity of pemetrexed was assessed by tetrazolium‐based colorimetric assay (MTT assay) and the interaction between pemetrexed and cisplatin was evaluated by the isobologram method. Western immunoblotting and real time RT‐PCR analysis of thymidylate synthase (TS), folylpoly‐γ‐glutamate synthetase (FPGS) and reduced folate carrier (RFC1) were performed in order to determine whether sensitivity to pemetrexed would be predictable by protein or mRNA expression levels. Pemetrexed was more cytotoxic than 5‐fluorouracil, with IC50 between 17 and 310 nM in most of the gastric cancer cell lines examined and the pemetrexed/cisplatin combination resulted in additive or synergistic interaction. The protein expressions of TS, FPGS, and RFC1 were significantly associated with IC50 for 5‐fluorouracil, but no such association was found for pemetrexed chemosensitivity. The mRNA expressions of RFC1, FPGS and other target and resistance related genes revealed no significant association with pemetrexed sensitivity. In conclusion, pemetrexed is active against gastric cancer cell lines and the pemetrexed/cisplatin combination showed a synergistic or additive interaction, supporting its clinical use in gastric cancer. Drug sensitivity toward pemetrexed could not be predicted by the expressions of TS, RFC1, or FPGS and we suggest that it is determined by interactions between multiple genes. (Cancer Sci 2005; 96: 365–371)

The tumorigenic, invasive and metastatic potential of epithelial and round subpopulations of the SW480 human colon cancer cell line

It has been reported that the SW480 human colon cancer cell line consists of E-type and R-type cells. The long-term tumorigenic potential, invasive and metastatic properties of these subclones have not been characterized. E-type and R-type cells were subcloned using limiting dilution methods from parental SW480 cells. The cell growth rate was determined by MTT colorimetric assay, and colony forming efficiency was analyzed using Matrigel-coated plates. The activity of matrix metalloproteinase (MMP) and of urokinase plasminogen activator (uPA) was assessed by zymography. Invasive and locomotive ability was analyzed using transwell chambers. In situ apoptosis detection of these subclones was also performed. In vivo long-term tumorigenicity and nodal metastasis were evaluated using nude mice. E-type cells produced spontaneously regressive tumors in spite of invasion and lymph node metastasis. In contrast, R-type cells revealed progressively growing tumors without invasion or metastasis. E-type cells exhibited increased apoptosis and invasive and motile ability, as well as strong MMP-9 and -2 activity. Although phorbol 12-myristate 13-acetate treatment induced MMP-9 activity in E-type cells, it had no effect on R-type cells. These findings suggest that E- and R-type cells may have different biological properties in terms of colon cancer progression, regression, invasion and nodal metastasis, and might serve as a useful model for these studies.