Effects of KAAD-cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the growth and apoptosis of human squamous cell carcinoma cell line A431 / 中华皮肤科杂志

ABSTRACT

Objective To investigate the in vitro effects of KAAD-cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the growth and apoptosis of human squamous cell carcinoma cell line A431. Methods A431 cells were cultured and treated with KAAD-cyclopamine(0.5, 1, 2, 5 μmol/L).Then, MTT assay was used to detect the proliferation of A431 cells, and light microscopy to observe cell morphology at different time points with a 24-hour interval. Flow cytometry was used to assess cell cycle,and annexin-V/propidium iodide double staining to evaluate the apoptosis in these cells after 48 hours of treatment with KAAD-cyclopamine. Results KAAD-cyclopamine of 0.5, 1, 2 and 5 μmol/L inhibited the proliferation of A431 cells by (7.0±2.3)%, (20.6±2.8)%, (48.3±3.4)% and (61.6±3.3)%, respectively (F = 49.92, P＜0.01 ). Furthermore, in the presence of KAAD-cyclopamine of 5 μmol/L, on day 1, 2, 3, 4,and 5 the proliferation of A431 cells was suppressed by (18.5±2.6)%, (56.1±3.7)%, (65.4±2.8)%,(71.2±1.9)% and (75.9±3.0)%, respectively, the difference was significant among these time points(F =16.32, P＜0.01 ). Statistical analysis showed that KAAD-cyclopamine downregulated the growth of A431 cells in a dose-and time-dependent manner (r = 0.91, 0.86, P＜0.01 and 0.05, respectively). Light microscopy revealed typical morphological changes of cell damage in A431 cells. KAAD-cyclopamine in creased the percentage of cells in G1 phase from (51.8±2.9)% to(76.2±1.8)% (F = 26.34, P＜0.01 ), the proportion of hypoploid cells from (1.7±0.3 )% to (8.7±0.2)% (F = 6.32, P＜0.05 ), which suggested that KAAD-cyclopamine could arrest A431 cells in G1 phase of the cell cycle. The apoptosis ratio in KAAD-cyclopamine-treated cells was significantly higher than that in the untreated control [ (46.2±2.8)% vs (18.5±3.1 )%, F = 32.01, P＜0.01 ]. Tomatidine treatment did not affect the proliferation or apoptosis of A431 cells (both P＞0.05).Conclusion KAAD-cyclopamine can markedly suppress the proliferation and induce apoptosis of A431 cells.