Inhibition of cyclin-dependent kinase activity and induction of apoptosis by preussin in human tumor cells

Abstract

In this paper, we report that (+)-preussin, a pyrrolidinol alkaloid originally identified as an antifungal agent, has growth-inhibitory and cytotoxic effects on human cancer cells. Preussin was found to be a potent inhibitor of cyclin E kinase (CDK2-cyclin E) in vitro (50% inhibitory concentration; approximately 500 nM) and to inhibit cell cycle progression into S phase. In agreement with these findings, the level of the cyclin-dependent kinase inhibitor p27(KIP-1) is increased in response to preussin treatment while the expression of both cyclin A and the transcription factor E2F-1 is down-regulated. Preussin also induces programmed cell death (apoptosis), which requires caspase activation and involves the release of cytochrome c from mitochondria. This induction of apoptosis is not blocked by high levels of Bcl-2, which usually confers resistance to chemotherapeutic agents. Taken together, our data indicate that preussin could be a promising lead compound for the development of a new class of potent antitumor drugs.

FIG. 1

Chemical structures of anisomycin and preussin. AcO, acetyloxy.

FIG. 2

Inhibition by preussin of human cancer cell growth. (A) Dose-dependent inhibition of HL-60 cell growth. Cells were incubated with increasing concentrations of preussin for 18 h. The number of remaining live cells was determined and plotted as a function of the drug concentration. (B) IC₅₀s for different human tumor cell lines. Cells were grown in microtiter plates, treated with preussin at increasing concentrations for 48 h, washed with PBS, refed with normal medium, and allowed to grow for another 48 h before staining with crystal violet. The dye remaining was measured in an enzyme-linked immunosorbent assay reader at 540 nm, and the IC₅₀s were calculated as described in Materials and Methods.

FIG. 3

Incorporation of [³⁵S]methionine by A549 cells exposed to increasing concentrations of anisomycin (■) or preussin (◊).

FIG. 4

Cell cycle distribution of A549 (A) and HL-60 (B) cells before and after 18 h of treatment with preussin.

FIG. 5

Inhibition of CDK2 activity by preussin in vitro. The activity of recombinant cyclin E-CDK2 was measured in the presence of increasing concentrations of preussin (◊), FP (○), anisomycin (■), or Cam (▴).

FIG. 6

Effect of preussin on the expression of the cell cycle regulators cyclin A, E2F-1, and p27 in A549 and HL-60 cells. Lanes: −, untreated cells; +, cells exposed to preussin for 18 h; DA, density arrested.

FIG. 7

Induction of apoptosis by preussin in HL-60 cells. (A) Cytological detection of apoptotic cells. HL-60 cells treated with increasing concentration of preussin were stained with Hoechst 33342 and propidium iodide. The percentage of live (□) or apoptotic (■) cells is indicated. (B) DNA fragmentation. The agarose gel shows DNA fragmentation (DNA ladder) in cells that were untreated control [con] or treated with 0.5, 1, or 2.5 μM preussin. A 100-bp ladder was loaded in lane M. (C) Annexin V binding. Untreated cells (Control) and cells treated with 500 nM or 2.5 μM preussin were incubated with fluorescein isothiocyanate-labeled annexin V and propidium iodide; this was followed by FACS analysis. The designated areas represent live cells (R1), annexin V-positive cells (early apoptosis; R2), and propidium iodide-positive cells (late apoptosis and necrosis; R3).

FIG. 8

Role of caspases and cytochrome c in the induction of apoptosis by preussin. (A) Effect of caspase inhibition on preussin-induced cell death. HL-60 cells were untreated (−) or treated with preussin (+) after a 1-h preincubation with (+) or without (−) zVAD-fmk (100 μM). The percentage of live (□), apoptotic (■), or necrotic (░⃞) cells is given. Shown are the average results of three independent experiments ± the standard deviation. (B) Immunoblot analysis of procaspases, PARP, and cytoplasmic cytochrome c. Extracts from HL-60 cells that were untreated (Con) or treated with 0.5, 1, or 5 μM preussin for 18 h were analyzed by immunoblotting using antibodies specific for the indicated proteins.

FIG. 9

Induction of cell death by preussin in cells expressing high levels of Bcl-2. (A) Immunoblot analysis of Bcl-2 levels in H69, SW2, and HL-60 cells. The same blot was reprobed with an α-actin antibody as a loading control. (B) Cell killing induced by 10 μM preussin, 500 μM 5′FU, 6 μM Cisp, 1.7 μM Dox, 2 μM Etop, or 500 nM Cam in H69, SW2, and HL-60 cells after 18 h of drug exposure, as determined in Fig. 7A.