Preliminary study of biological mechanism and apoptotic effect induced by YM155 in SUDHL-4 cell line
ZHANG Wen-jun, GAO Qing-mei, QIN Wei, LU Hui-na, HUANG Bin-bin, XIU Bing, LIANG Ai-bin
Published 2012-03-13
Cite as Nat Med J China, 2012,92(10): 700-704. DOI: 10.3760/cma.j.issn.0376-2491.2012.10.017
Abstract
Objective To explore the apoptotic effect of follicular lymphoma and related mechanism induced by YM155 in vitro and provide laboratory rationales for the clinical treatment of follicular of lymphoma with YM155 in the future.Methods SUDHL-4 cells were cultured to logarithmic phase and transferred to 96-well plates.There were a series of YM155 concentration gradients:100,10,1,0.1 and 0 ng/ml and cultured for 24,48 and 72 h.After the addition of CCK-8 reagent for 2 h at each time point,optical density values were obtained from the cell growth inhibition curves depending on time and drug concentration and the half growth inhibition concentration ( IC50 ) values calculated.SUDHL-4 cells were cocultured with YM155 (1 ng/ml) for0,24,48 and72 h respectively.Then flow cytometry (FCM) was used to detect apoptosis.SUDHL-4 cell line was treated with YM155 for 24 and 48 h to extract the total RNA.The mRNA expressions of bcl-2,bcl-xl,bid,bax and survivin gene at the timepoint of 48 h and the survivin mRNA expression at 24 h were detected by reverse transcription-PCR(RT-PCR).The protein expressions of survivin,caspase-9,cleaved caspase-9,caspase-3 and cleaved caspase-3 were detected at each timepoint with Western blot respectively.Results SUDHL-4 cell line showed significant growth inhibition effect depending on time and dose.And the 24,48,72 h IC50 was 6.1,2.7 and 1.2 ng/ml respectively.SUDHL-4 cells stained AnnexinV-FITC and PI examined by FCM demonstrated that the proportion of AnnexinV-FITC positive cells gradually increased with time(17.3% ±2.1%,35.7% ±3.3%,54.6% ±4.3% vs 2.1% ± 0.3%,all P <0.05). And the results of real-time fluorescent PCR proved that YM155 decreased the expression of survivin gene obviously(24 h:0.72 ± 0.02,48 h:0.56 ± 0.01 vs 1.00,both P < 0.05 ) but had little effects on the gene expressions of bax,bid,bcl-2 and bcl-xl. The Western blot results further confirmed that the protein expressions of survivin and caspase-3 decreased with time while caspase-9 and cleaved caspase-9 showed no obvious changes. But cleaved caspase-3 increased significantly.Conclusions YM155 displays significant apoptotic effects in SUDHL-4 cell lines. The mechanism may be the direct activation of caspase-3 through the down-regulation of survivin.And the apoptotic pathway is probably not regulated by bcl-2 family.
Key words:
Lymphoma; Apoptosis; YM155
Contributor Information
ZHANG Wen-jun
Department of Hematology, Affiliated Tongji Hospital of Tongji University, Shanghai 200065, China
GAO Qing-mei
Department of Hematology, Affiliated Tongji Hospital of Tongji University, Shanghai 200065, China
QIN Wei
Department of Hematology, Affiliated Tongji Hospital of Tongji University, Shanghai 200065, China
LU Hui-na
Department of Hematology, Affiliated Tongji Hospital of Tongji University, Shanghai 200065, China
HUANG Bin-bin
Department of Hematology, Affiliated Tongji Hospital of Tongji University, Shanghai 200065, China
XIU Bing
Department of Hematology, Affiliated Tongji Hospital of Tongji University, Shanghai 200065, China
LIANG Ai-bin
Department of Hematology, Affiliated Tongji Hospital of Tongji University, Shanghai 200065, China