Anti-Leukemic Effect of a Synthetic Compound, (±)Trans-Dihydronarciclasione (HYU-01) Via Cell Cycle Arrest and Apoptosis

Over the past several decades, there has been considerable effort in the synthesis of narciclasine, lycoricidine, and pancratistatin. These naturally occurring isocarbostryls are known to have potent antitumoral and antiviral effects. Among them, trans-dihydronarciclasine isolated from the Chinese medicinal plant, Zephyranthes candida, exhibits even higher potency than pancratistatin against several human cancer cell lines and murine P388 lymphocytic leukemia cell line. However, much remains to be known about antitumoral mechanism of this natural product. In addition, the effect of transdihydronarciclasine in human acute myeloid leukemia (AML) has been not elucidated. The present study was undertaken to investigate the effect of novel synthetic (±)trans-dihydronarciclasine compound (code name; HYU-01) in human acute myeloid leukemia (AML). Treatment of HYU-01 for 72 hr inhibited the proliferation of human AML cell lines as well as primary leukemic blasts from AML patients in a dose-dependent manner with IC₅₀ ranging from 1×10⁻⁷M to 5×10⁻⁸M. To address the mechanism of the antiproliferative effect of HYU-01, cell cycle analysis was performed in HL-60 cells. DNA flow cytometric analysis indicated that HYU-01 (2.5×10⁻⁷M) efficiently induced G₁ arrest. Analysis of cell cycle-related proteins demonstrated that expression levels of CDK2, CDK4, CDK6, cyclin E and cyclin A were decreased in a time-dependent manner, and expression of cyclin D1 was up-regulated. In contrast, the level of cyclin B was not altered. In addition, HYU-01 (2.5×10⁻⁷M, 72 hr) increased the expression level of the CDKI p27^kip1 and markedly enhanced the binding of p27 with CDK2, CDK4, and CDK6 compared to HYU-01-untreated cells. Furthermore, the activity of CDK2-associated kinase was decreased, which resulted in the hypophosphorylation of Rb protein. HYU- 01 also induced the apoptosis in HL-60 cells. The apoptotic process was associated with increased Bax and decreased Bid, Bcl-X_L and poly(ADP-ribose) polymerase (PARP), primary leukemic blasts from AML patients in a dose-dependent manner with IC₅₀ ranging from 1×10⁻⁷M to 5×10⁻⁸M. To address the mechanism of the antiproliferative effect of HYU-01, cell cycle analysis was performed in HL-60 cells. DNA flow cytometric analysis indicated that HYU-01 (2.5×10⁻⁷M) efficiently induced G₁ arrest. Analysis of cell cycle-related proteins demonstrated that expression levels of CDK2, CDK4, CDK6, cyclin E and cyclin A were decreased in a time-dependent manner, and expression of cyclin D1 was up-regulated. In contrast, the level of cyclin B was not altered. In addition, HYU-01 (2.5×10^{− 7}M, 72 hr) increased the expression level of the CDKI p27^kip1 and markedly enhanced the binding of p27 with CDK2, CDK4, and CDK6 compared to HYU-01-untreated cells. Furthermore, the activity of CDK2-associated kinase was decreased, which resulted in the hypophosphorylation of Rb protein. HYU-01 also induced the apoptosis in HL-60 cells. The apoptotic process was associated with increased Bax and decreased Bid, Bcl-X_L and poly(ADP-ribose) polymerase (PARP), and activation of caspase-8, -9, and -3, and release of cytochrome C from mitochondria into cytosol. In addition, the apoptosis by HYU-01 was accompanied with the down-regulation of ERK and P90RSK. These results suggest that HYU-01 inhibit the proliferation of AML cells via triggering the apoptosis as well as the induction of p27 and the reduction of CDK2 activity leading to G₁ arrest.