Reevesioside A, a Cardenolide Glycoside, Induces Anticancer Activity against Human Hormone-Refractory Prostate Cancers through Suppression of c-myc Expression and Induction of G1 Arrest of the Cell Cycle

<div><p>In the past decade, there has been a profound increase in the number of studies revealing that cardenolide glycosides display inhibitory activity on the growth of human cancer cells. The use of potential cardenolide glycosides may be a worthwhile approach in anticancer research. Reevesioside A, a cardenolide glycoside isolated from the root of <i>Reevesia formosana</i>, displayed potent anti-proliferative activity against human hormone-refractory prostate cancers. A good correlation (r² = 0.98) between the expression of Na⁺/K⁺-ATPase α₃ subunit and anti-proliferative activity suggested the critical role of the α₃ subunit. Reevesioside A induced G1 arrest of the cell cycle and subsequent apoptosis in a thymidine block-mediated synchronization model. The data were supported by the down-regulation of several related cell cycle regulators, including cyclin D1, cyclin E and CDC25A. Reevesioside A also caused a profound decrease of RB phosphorylation, leading to an increased association between RB and E2F1 and the subsequent suppression of E2F1 activity. The protein and mRNA levels of c-myc, which can activate expression of many downstream cell cycle regulators, were dramatically inhibited by reevesioside A. Transient transfection of c-myc inhibited the down-regulation of both cyclin D1 and cyclin E protein expression to reevesioside A action, suggesting that c-myc functioned as an upstream regulator. Flow cytometric analysis of JC-1 staining demonstrated that reevesioside A also induced the significant loss of mitochondrial membrane potential. In summary, the data suggest that reevesioside A inhibits c-myc expression and down-regulates the expression of CDC25A, cyclin D1 and cyclin E, leading to a profound decrease of RB phosphorylation. G1 arrest is, therefore, induced through E2F1 suppression. Consequently, reevesioside A causes mitochondrial damage and an ultimate apoptosis in human hormone-refractory prostate cancer cells.</p></div

Determination of functional involvement of c-myc.

<p>(A) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for various times. The cells were harvested and lysed for the detection of the indicated protein by Western blot analysis. The expression was quantified using the computerized image analysis system ImageQuant (Amersham Biosciences). The data are expressed as mean±SEM of three independent experiments. *** <i>P</i><0.001 compared with 100% control. (B) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for the indicated times. The cells were harvested for the determination of mRNA expression by RT-PCR. (C) PC-3 cells were transfected with the indicated plasmid. The cells were treated without or with reevesioside A (50 nM) for 6 hours. After treatment, the cells were harvested and lysed for the detection of the indicated protein by Western blot analysis.</p

Effect of reevesioside A on cell proliferation.

<p>Chemical structure of reevesioside A (A). The graded concentrations of reevesioside A were added to PC-3 and DU-145 cells for 48 hours (A) or a single concentration (50 nM) was added for 48 hours (B) or the indicated times (C). After the treatment, the cells were observed by microscopic examination (B) or the cells were fixed and stained for SRB assay (A) or labeled with CFSE for flow cytometric analysis. Data are expressed as mean±SEM of three to five determinations. ** <i>P</i><0.01 and *** <i>P</i><0.001 compared with the respective control. Arrowhead, cell apoptosis; star, cell differentiation; <i>bar</i>, 50 µm.</p

Effect of reevesioside A on the expression of several cell cycle regulators.

<p>(A) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for various times. Cells were harvested and lysed for the detection of the indicated protein expression by Western blot analysis. The expression was quantified using the computerized image analysis system ImageQuant (Amersham Biosciences). The data are expressed as mean±SEM of three to five independent experiments. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001 compared with 100% control. (B) After the treatment, the cells were harvested for immunoprecipitation assay. The protein expression was detected by Western blot analysis. Data are representative of three independent experiments.</p

Effect of reevesioside A on cell-cycle progression.

<p>(A) Synchronization of PC-3 cells was performed by thymidine block as described in the Materials and Methods section. Then, the cells were released in the absence (upper panel) or presence of 50 nM reevesioside A for the indicated times. Data are representative of five independent experiments. (B) DU-145 cells were incubated in serum-free medium for 48 hours (starvation) and then, 10% FBS was added in the absence or presence of reevesioside A for 18 hours. The cells were harvested for the detection of cell cycle population by flow cytometric analysis. Quantitative data are expressed as mean±SEM of five (A) or three (B) independent experiments. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001 compared with the respective control.</p

Determination of functional involvement of Akt.

<p>(A) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for various times. The cells were harvested and lysed for the detection of the indicated protein by Western blot analysis. (B) PC-3 cells were transfected with the indicated plasmid. Then, the cells were treated without or with reevesioside A (50 nM) for 24 hours. After treatment, the cells were harvested and lysed for the detection of the indicated protein by Western blot analysis. The expression was quantified using the computerized image analysis system ImageQuant (Amersham Biosciences). The data are expressed as mean±SEM of three independent experiments. * <i>P</i><0.05 and ** <i>P</i><0.01 compared with 100% control. WT-Akt, wild type Akt; CA-Akt, constitutively active Akt.</p

Correlation between Na⁺/K⁺-ATPase α₃ subunit and anti-proliferative activity.

<p>(A) The expression of Na⁺/K⁺-ATPase α₃ subunit was detected using Western blot analysis. The anti-proliferative IC₅₀ values were determined using SRB assays except for HL-60 cells by MTT assays. (B) PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for the indicated times. After the treatment, the cells were harvested for the detection of intracellular Ca²⁺ levels using flow cytometric analysis of the staining with fluo-3 AM. Data are expressed as mean±SEM of three independent experiments. * <i>P</i><0.05 compared with the control.</p

Effect of reevesioside A on mitochondrial membrane potential (Δψ_m).

<p>PC-3 cells were incubated in the absence or presence of reevesioside A (50 nM) for the indicated times. Cells were incubated with JC-1 for the detection of Δψ_m using flow cytometric analysis. The data are expressed as mean±SEM of three independent experiments. * <i>P</i><0.05 and *** <i>P</i><0.001 compared with the control.</p

Cephalochromin Induces G0/G1 Cell Cycle Arrest and Apoptosis in A549 Human Non-Small-Cell Lung Cancer Cells by Inflicting Mitochondrial Disruption

The fungus-derived compound cephalochromin, isolated from the fermented broth of <i>Cosmospora vilior</i> YMJ89051501, shows growth-inhibitory and apoptotic activity against human lung cancer A549 cells in a concentration-dependent manner with an IC₅₀ value of 2.8 μM at 48 h. Cephalochromin induced cell cycle arrest at the G0/G1 phase through down-regulation of cyclin D1, cyclin E, Cdk 2, and Cdk 4 expressions. Cephalochromin markedly increased the hypodiploid sub-G1 phase (apoptosis) of the cell cycle at 48 h as measured by flow cytometric analysis. Reactive oxygen species generation and loss of the mitochondrial membrane potential (MMP) were also markedly induced by cephalochromin. Moreover, the immunoblotting assays showed that cephalochromin reduced survivin and Bcl-xL expression and induced the activation of caspase-8, -9, and -3 and the cleavage of poly­(ADP-ribose) polymerase, indicating the involvement of a caspase signaling cascade. The caspase inhibitor Z-VAD-fmk significantly suppressed cephalochromin-induced apoptosis. Cephalochromin also triggered LC3 II, autophagic marker, expression. Taken together, this is the first report that cephalochromin induced an antiproliferative effect on human lung cancer cells through mitochondrial disruption and down-regulation of survivin, leading to cell cycle arrest at the G0/G1 phase, loss of MMP, and subsequently apoptotic cell death

Bioactive Diterpenes from <i>Callicarpa longissima</i>

Investigation of the leaves and twigs of <i>Callicarpa longissima</i> resulted in the isolation of four new compounds (<b>1</b>–<b>4</b>), callilongisins A–D, and five known compounds, ursolic acid, 3-oxoanticopalic acid, (<i>E</i>)-6β-hydroxylabda-8­(17),13-dien-15-oic acid, 5-hydroxy-3,6,7,4′-tetramethoxyflavone, and artemetin. Compounds <b>1</b>–<b>3</b> are 3,4-<i>seco</i>-abietane-type diterpenoids, and compound <b>4</b> is an analogue of a labdenoic-type diterpene. The structure of compound <b>1</b> was confirmed by X-ray crystallographic analysis. Cytotoxicity against a human prostate cancer cell line (PC3) and anti-inflammatory activities of the isolated compounds were evaluated