Simaomicin α, a polycyclic xanthone, induces G₁ arrest with suppression of retinoblastoma protein phosphorylation

Abstract

Recent progress in cancer biology research has shown that abnormal proliferation in tumor cells can be attributed to aberrations in cell cycle regulation, especially in G₁ phase. During the course of searching for microbial metabolites that affect cell cycle distribution, we have found that simaomicin α, a polycyclic xanthone antibiotic, arrests the cell cycle at G₁ phase. Treatment of T-cell leukemia Jurkat cells with 3 nM simaomicin α induced an increase in the number of cells in G₁ and a decrease in those in G₂–M phase. Cell cycle aberrations induced by simaomicin α were also detected in colon adenocarcinoma HCT15 cells. Simaomicin α had antiproliferative activities in various tumor cell lines with 50% inhibitory concentration values in the range of 0.3–19 nM. Furthermore, simaomicin α induced an increase in cellular caspase-3 activity and DNA fragmentation, indicating that simaomicin α promotes apoptosis. The retinoblastoma protein phosphorylation status of simaomicin α-treated cell lysate was lower than that of control cells, suggesting that the target molecule of simaomicin α is in a pathway upstream of retinoblastoma protein phosphorylation. In the course of evaluating polycyclic xanthone antibiotics structurally related to simaomicin α, we also found that cervinomycin A1 stimulated accumulation of treated cells in G₁ phase. These results indicate that the polycyclic xanthones, including simaomicin α and cervinomycin A1, may be candidate cancer chemotherapeutic agents.

Figure 1

Structure of simaomicin α.

Figure 2

Effects of simaomicin α on cell cycle progression. (a,c) Jurkat cells and (b,d) HCT15 cells were incubated with simaomicin α (0, 1, 3, and 10 nM) for 20 h. After treatment, cells were stained with DNA‐staining solution, and DNA contents were determined using FACSCalibur. Data analysis was carried out using ModFit LT software for cell cycle profiles. Subpopulations of (c) Jurkat cells and (d) HCT15 cells treated with simaomicin α are shown; G₁ (), S (), and G₂–M (). (e) HCT15 cells were synchronized with colcemide for 12 h and released from M phase. Cells were incubated with or without 10 nM simaomicin α and harvested after various time periods. Measurements of DNA contents and data analysis were carried out as above. (f) G₁ populations of non‐treated HCT15 cells () and HCT15 cells treated with simaomicin α ().

Figure 3

Induction of apoptosis in simaomicin α‐treated cells. (a) Caspase‐3 activity in simaomicin α‐treated cells. HCT15 cells were treated with 0, 1, or 10 nM simaomicin α for 24 h before lysis. The cell lysates were incubated with Ac‐DEVD‐MCA at 37°C for 2 h. Caspase‐3 activity was determined by measuring fluorescence intensity (excitation 355 nm, emission 460 nm). (b) DNA fragmentation in simaomicin α‐treated cells. Jurkat cells were treated with 0, 0.1, 1, or 10 nM simaomicin α for 48 h. The DNA was extracted from each cell and electrophoresed on a 1.5% agarose gel.

Figure 4

Effect of simaomicin α on the phosphorylation of retinoblastoma protein (pRb). Jurkat cells were incubated with simaomicin α (0, 0.1, 0.3, 1, 3, or 10 nM) for 20 h, washed and lysed. Cell lysates were clarified by centrifugation. Ten micrograms of each cell lysate was separated by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis followed by immunoblotting with anti‐pRb, anti‐phospho‐pRb (Ser807/811), or anti‐β‐actin antibody.

Figure 5

Effects of polycyclic xanthones on cell cycle progression. (a) Structures of cervinomycin A1, xanthoquinodin A1, and xanthone. (b) Jurkat cells were incubated with simaomicin α (), cervinomycin A1 (), xanthoquinodin A1 (), or xanthone () at the indicated concentrations for 20 h. After treatment, cells were stained with DNA‐staining solution, and DNA contents were determined using FACSCalibur. Analysis of the G₁ population of cells was carried out using ModFit LT 2.0 software.