Effects of gambogic acid on the regulation of nucleoporin Nup88 in U937 cells

Abstract

In order to investigate the anti-leukemia effects of gambogic acid (GA) and its relation to the regulation of nucleoporin Nup88 in U937 cells in vitro, the inhibitory effect of GA on the growth of U937 cells was examined by using MTT assay. Apoptosis was detected by Annexin-V FITC/PI double-labeled cytometry. Cell cycle regulation was studied by propidium iodide method. Both flow cytometry (FCM) and RT-PCR were employed to assess the expression of Nup88, and the localization of Nup88 was determined by confocal microscopy. The results indicated that GA had strong inhibitory effect on cell proliferation and apoptosis induction activity in U937 cells in vitro in a time-and dose-dependent manner. The 24-h IC₅₀ value was (1.019±0.134) mg/L. Moreover, GA induced arrest of U937 cells in G₀/G₁ phase. Over-expression of Nup88 was found in U937 cells, whereas GA could significantly down-regulate both the protein and mRNA levels of Nup88. Nup88 was diffusely distributed between nucleus and cytoplasm and was located at the cytoplasmic side of nuclear rim, and occasionally in cytoplasm. It is suggested that GA exerts its anti-leukemia effects by regulating the expression and distribution of nucleoporin Nup88. It promises to be new agent for the treatment of acute leukemia.

Keywords

gambogic acid / acute leukemia / apoptosis / Nup88

References

[1] WuZ. Q., GuoQ. L., YouQ. D., et al.. Growth Inhibitory Effect of GGAs on Experimental Tumor in Mice and Human Tumor Cell Cultured in vitro. Chin J Nat Med (Chinese), 2003, 1: 99-102

[2] GuoQ. L., YouQ. D., WuZ. Q., et al.. General gambogic acids inhibited growth of human hepatoma SMMC-7721 cells in vitro and in nude mice. Acta Pharmacol Sin, 2004, 25: 769-774

[3] LiuW., GuoQ. L., YouQ. D., et al.. Anticancer effect and apoptosis induction of gambogic acid in human gastric cancer line BGC-823. World J Gastroenterol, 2005, 11: 3655-3659

[4] CronshawJ. M., KrutchinskyA. N., ZhangW. Z., et al.. Proteomic analysis of the mammalian nuclear pore complex. J Cell Biol, 2002, 158: 915-927

[5] ZhangH., SchneiderJ., RosdahlI.. Expression of p16, p27, p53, p73 and Nup88 proteins in matched primary and metastatic melanoma cells. Int J Oncol, 2002, 21: 43-48

[6] GouldV. E., OrucevicA., ZentgrafH., et al.. Nup88 (karyoporin) in human malignant neoplasms and dysplasias: correlations of immunostaining of tissue sections, cytologic smears, and immunoblot analysis. Hum Pathol, 2002, 33: 536-544

[7] AgudoD., GomezE. F., MartinezA. F., et al.. Nup88 mRNA overexpression is associated with high aggressiveness of breast cancer. Int J Cancer, 2004, 109: 717-720

[8] LiZ., GuoQ. L., YouQ. D., et al.. Gambogic Acid Induces Apoptosis and Regulates Expressions of Bax and Bcl-2 Protein in Human Gastric Carcinoma MGC-803 Cells. Biol Pharm Bull, 2004, 27: 998-1003

[9] YuJ., GuoQ. L., YouQ. D., et al.. Repression of telomerase reverse transcriptase mRNA and hTERT promoter by inhibitory effects of oncogene c-myc in gambogic acid treated-human gastric carcinoma cells. Cancer Chemother Pharmacol, 2006, 58: 434-443

[10] KasibhatlaS., JessenK. A., MaliartchoukS., et al.. A role for transferrin receptor in triggering apoptosis when targeted with gambogic acid. Proc Nat Acad Sci, 2005, 102: 12095-12100

[11] PietenpolJ. A., StewartZ. A.. Cell cycle checkpoint signaling: cell cycle arrest versus apoptosis. Toxicology, 2002, 27: 475-481

[12] YuJ., GuoQ. L., YouQ. D., et al.. Gambogic acid-induced G₂/M phase cell-cycle arrest via disturbing CDK7-mediated phosphorylation of CDC2/p34 in human gastric carcinoma BGC-823 cells. Carcinogenesis, 2007, 28: 632-638

[13] ZhangH. Z., KasibhatlaS., WangY., et al.. Discovery, characterization and SAR of gambogic acid as a potent apoptosis inducer by a HTS assay. Bioorg Med Chem, 2004, 12: 309-317

[14] RothP., XylourgidisN., SabriN., et al.. The Drosophila nucleoporin DNup88 localizes DNup214 and CRM1 on the nuclear envelope and attenuates NES-mediated nuclear export. J Cell Biol, 2003, 163: 701-706

[15] LiuH. L., ChenY., CuiG.H., et al.. Deguelin regulates nuclear pore complex proteins Nup98 and Nup88 in U937 cells in vitro. Acta Pharmacol Sin, 2005, 26: 1265-1273