Experimental study of up-regulating PTEN gene on increasing the chemosensitivity in K562/ADM cells.
- Author:
Hong ZHANG
1
;
Liang-chun YANG
;
Li-zhi CAO
;
Ming-hua YANG
;
Min XIE
;
Shan ZHU
;
Pei ZENG
;
Yan YU
Author Information
- Publication Type:Journal Article
- MeSH: Apoptosis; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Gene Expression Regulation, Leukemic; Genetic Vectors; Humans; K562 Cells; Leukemia; drug therapy; PTEN Phosphohydrolase; genetics; metabolism; Transfection; Up-Regulation
- From: Chinese Journal of Hematology 2012;33(5):412-416
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo investigate the mechanism of decreasing the K562/ADM cells chemosensitivity by up-regulating expression of PTEN gene.
METHODSK562/ADM cells were transient transfected with pGFP-PTEN or vector. The level of PTEN in K562/ADM cells was assayed by Western blot and RT-PCR. Cell viability on K562/ADM was determined by MTT assay. Cell apoptosis by flow cytometry. Activity of caspase-3 by Caspase Colorimetric Assay Kit. The proteins expression of LC3-I/II, Beclin1, p-Akt, p-p70S6K by Western blot. The autophagic vacuoles by MDC stain and Electron microscopy.
RESULTS(1) The mRNA and protein levels of PTEN in K562/ADM cells transfected with pGFP-PTEN were significantly increased compared with the control (untreated and transfected with empty vector). (2) Enhanced expression of PTEN by gene transfection resulted in a reversal of resistance to ADM. Compared with empty vector group, cell viability decreased from (94.07 ± 2.6)% to (53.83 ± 4.2)%, the cell apoptotic rate increased from (11.89 ± 1.7)% to (43.69 ± 2.3)%, meanwhile, pretreated with caspase-3 inhibitor (Z-DEVE-FMK) didn't completely inhibit the cytotoxicity of ADM to K562/ADM cells. (3) After treated with ADM for 12 and 24 h, the activities of caspase-3 in PTEN-transfected K562/ADM cells increased compared with those in pGFP-transfected K562/ADM cells \[(2.27 ± 0.13) vs (1.19 ± 0.14)\] at 12h, \[(3.15 ± 0.08) vs (1.48 ± 0.05)\] at 24 h (P < 0.05). (4) The protein levels of LC3-II and Beclin1 in K562/ADM cells transfected with pGFP-PTEN were increased by 83% and 18% respectively, and the protein levels of p-Akt and p-p70S6K were declined by 96% and 87% respectively, compared with those in K562/ADM cells transfected with pGFP plasmid. (5) The upregulation of PTEN in K562/ADM cells improved the number of autophagic vacuoles compared with the empty vector group.
CONCLUSIONThe upregulation of PTEN expression increases the chemosensitivity of K562/ADM to ADM, which may related with the inhibition of PI3K/AKT/mTOR pathway induced by PTEN gene transfection.
