In vitro induction of apoptosis of a cutaneous squamous cell carcinoma cell line, Colo-16 cells, by sirolimus / 中华皮肤科杂志

ABSTRACT

Objective To investigate the in vitro effect of sirolimus on the apoptosis of a cutaneous squamous cell carcinoma cell line, Colo-16 cells. Methods Cultured Colo-16 cells were treated with different concentrations (50, 100, 150, 200 nmol/L) of sirolimus for various durations ( 12, 24, 48, 72 hours). Subse-quently, cell proliferation was detected by MTT assay, and cell apoptosis by Annexin V-FITC and PI double staining. Morphological changes of the cells were observed with Hoechst 33258 fluorescent staining. Total RNA was extracted from Colo-16 cells treated with sirolimus for 48 hours, and subjected to reverse tran-scription (RT)-PCR for the detection of mRNA expression of B cell lymphoma/leukmia-2 (Bcl-2) and Bcl-2-associated X Protein (Bax). Results Sirolimus inhibited the proliferation of Colo-16 cells in a time-and dose-dependent fashion. The early apoptosis rate was 7.26%±0.26%, 8.34% ±0.19%, 9.86%±0.14%, 11.92% ±0.15% in Colo-16 cells treated with sirolimus of 50, 100, 150, and 200 nmol/L, respectively, signifi-candy higher than that in untreated cells (1.53%±0.09%, P < 0.05); a positive correlation was observed between the apoptosis rate and concentrations of sirolimus (r = 0.955, P = 0.000). Typical morphological changes of apoptosis, such as chromatin condensation and margination as well as nuclear fragmentation were observed by fluorescence staining. After treatment with sirolimus for 48 hours, a significant decrease was observed in the mRNA expression of Bcl-2, while an increase in that of Bax was noticed. Conclusion Sirolimus could induce Colo-16 cells apoptosis in vitro, which may be associated with the modulation of expression of apoptosis-regnlating genes, such as Bcl-2 and Bax.