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 increased 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.

Key words: Hedgehog signaling pathway, KAAD-cyclopamine, A431 cell, Apoptosis