ABSTRACT
Aim: To explore the role of apoptosis stimulating protein 2 of p53 (ASPP2) on L-NAME induced apoptosis of placental trophoblast cells by regulating glucose-regulated protein78(GRP78), and provide a theoretical basis for the study of clinical pregnancy-induced hypertension. Methods: The HTR-8/SVneo human placental trophoblast cells were cultured in vitro, and in the absence (control group) or presence of 100 μmol · L-1 L-NAME (L-NAME group) for 48 h. The effects of L-NAME on placental trophoblast cell apoptosis were tested using flow cytometry and AO/EB assay. The expressions of caspase-12, GRP78 and ASPP2 were detected by Western blot. The ASPP2 interference with adenovirus was used to transfect the cells, and the mRNA expression level of ASPP2 and the protein expression level of GRP78 were detected by qRT-PCR and Western blot, respectively. After treated with 100 (xrnol · L-1 L-NAME for 48 h, the protein expression of caspase-12 and GRP78 was detected by Western blot and immunofluorescence. Results: Compared with control group, the placental trophoblast cell apoptosis significantly increased in L-NAME group (P < 0. 05). AO/EB staining showed that compared with control group, the majority of cells in L-NAME group showed bright orange and the number of late apoptotic cells increased significantly. At the same time, caspase-12, GRP78 and ASPP2 protein expression increased (P < 0. 05, P < 0. 01). After interfering with ASPP2, caspase-12 and GRP78 protein expressions decreased (P < 0. 05). Conclusions: Down-regulation of ASPP2 could decrease GRP78 expression and inhibit L-NAME-induced apoptosis in placental trophoblast cells.
ABSTRACT
Objective To investigate the role of apoptosis stimulating protein 2 of p53 (ASPP2)in the apoptosis,cell cycle and autophagy of starvation-induced colorectal cancer HCT116 p53 +/+ (p53 wild-type) cell line.Methods Six groups were included:(1) control group; (2) green fluorescent protein adenovirus (rAd-GFP) infection group; (3)ASPP2 adenovirus (rAd-ASPP2) infection group; (4)starvation group; (5)rAd-GFP + starvation group; (6) rAd-ASPP2 + starvation group.HCT116 cells were infected with ASPP2 adenovirus (rAd-ASPP2),resulting ASPP2 gene over-expression.The apoptosis,autophagy and cell cycle changes were induced by culturing with serum-free medium for 24 h.Apoptosis was evaluated by Calcein/PI uptaking test,and autophagy was observed by counting the red fluorescent protein autophagy plasmid CFP-Lc3 which was transfected into cytoplasm.Cell cycle was detected by flow cytometry.Statistical analysis was performed by one-way analysis of variance (ANOVA).Results Over-expressed ASPP2 was found to significantly promote starvation-induced HCT116 apoptosis and autophagy.The cell apoptosis rate in rAd-GFP + starvation group was 10.00％ ± 1.42％,and 18.44％ ±2.06％ in rAd-ASPP2 + starvation group(q =9.548,P =0.000).The cell autophagy rate in rAd-GFP+ starvation group and rAd-ASPP2 + starvation group was 35.00％ ± 5.34％ and 57.61％ ± 6.06％ respectively(q =7.657,P =0.000).Over-expressed ASPP2 accelerated HCT116 G2/M arrest under starvation,but resulted in both G0/G1 and G2/M arrest without starvation.Conclusion These results suggest that ASPP2 can promote starvation-induced HCT116 p53 +/+ cells apoptosis and autophagy,and affect the cell cycle.