Figure 1.
Effects of STK295900 on proliferation of various human cancer cell lines.
(A) Chemical structure of STK295900. (B) Inhibitory effects of STK295900 on the proliferation of variety of cancer cell lines. Cells were seeded at 1−2×103 cells in 96 well plates and treated with various concentrations of STK295900 for 4 days. Cell growth was determined by MTT assay. Data were fitted with dose-response curve using Graphpad Prism software. Values represent the mean ± SD from a representative triplicate experiment.
Table 1.
Inhibitory effect of STK295900 on proliferation of various cancer cell lines.
Table 2.
Comparison of STK295900 with camptothecin, etoposide, and Hoechst 33342 for its effect on the proliferation of various cancer and non-cancer cell lines.
Figure 2.
STK295900 induces G2 phase arrest in HeLa cells.
(A) Flow cytometric analysis for cell cycle distribution. HeLa cells were treated with the indicated concentrations of STK295900 for 24 h. Treated cells were then stained with propidium iodide (PI) and processed for cell cycle analysis. The bar graph represents the mean percentage of each cell cycle phase ± SD from three independent experiments. * = p<0.05 versus the respective G1, S, or G2/M phase of DMSO-treated cells. (B) Mitotic index of STK295900. HeLa cells were treated with the indicated concentrations of STK295900 for 24 h. Cells were then stained with Hoechst 33342 and mitotic cells were counted. The bar graph shows mean ± SD from the representative of triplicate experiments. (C) Cell cycle related proteins expression. HeLa cells were treated with DMSO control, STK295900 (STK) 1 or 5 µM, camptothecin (CPT) 10 µM, etoposide (ETO) 10 µM, or nocodazole (NOC) 200 ng/ml for 24 h. Treated cells were lysed and subjected to immunoblot analyses with antibodies against cyclin A, cyclin B1, phospho-Histone H3 (S10), and Histone H3. β-actin was used as a loading control.
Figure 3.
STK295900 does not activate DNA damage checkpoint.
(A) G2/M transition regulated proteins expression. HeLa cells were treated with STK295900 1 or 5 µM, Camptothecin 10 µM, etoposide 10 µM, or nocodazole 200 ng/ml. After 24 h incubation, cell lysates were prepared for immunoblot analyses with antibodies against phospho-Cdk1 (T161), phospho-Cdk1 (T14), phospho-Cdk1 (Y15), Cdk1, Wee1, and Cdc25C. GAPDH was used as a loading control. (B) DNA damage-checkpoint related proteins. The same lysates used in (A) were subjected to immunoblot analyses with antibodies against phospho-ATM (S1981), ATM, phospho-ATR (S428), ATR, phospho-Chk1 (S345), Chk1, phospho-Chk2 (T68), Chk2, p53, and p21. β-actin was used as a loading control. (C) Immunofluorescence staining for γ-H2A.X. HeLa cells were treated with 1, 5, or 10 µM of STK295900 or 10 µM of ICRF-193, etoposide, and camptothecin for 24 h. Treated cells were then fixed and stained with anti-γ-H2A.X (middle panel). DNA from ICRF-193-, etoposide-, and camptothecin-treated cells was stained with Hoechst 33342 (bottom panel). Images were analyzed on a fluorescence microscope.
Figure 4.
STK295900 inhibits topoisomerases activities.
Supercoiled DNA relaxation assay for (A) topoisomerase 1 (Top 1) and (B) topoisomerase 2 (Top 2). Supercoiled pBR322 plasmid DNA was incubated at 37°C for 30 min with Top 1 enzyme (A) or Top 2α (B) in the presence of various concentrations of indicated compounds. DNA samples were separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide, and visualized by UV light. (−), supercoiled DNA alone; oc, open circular; sc, supercoiled. (C) Antagonistic effect of STK295900 in camptothecin-induced DNA damage. HeLa cells were pretreated with 10 µM of STK295900 for 30 min and then incubated with the indicated concentrations of camptothecin for 1 h. Treated cells were lysed and subjected to immunoblot analyses with antibody against γ-H2A.X. β-actin was used as a loading control. (D) Antagonistic effect of STK295900 on etoposide-induced DNA damage. HeLa cells were pretreated with STK295900 at 10, 20, 30, or 50 µM or ICRF-193 at 10 µM for 30 min. Cells were incubated with 10 µM of etoposide for another 1 h. Treated cells were then lysed and subjected to immunoblot analyses with antibody against γ-H2A.X. β-actin was used as a loading control.