Inactivation of PI3k/Akt signaling pathway and activation of caspase-3 are involved in tanshinone I-induced apoptosis in myeloid leukemia cells in vitro.

Tanshinone I (Tan I), a diterpene quinone extracted from herbal medicine Salvia miltiorrhiza Bunge, has recently been reported to have antitumor effects. As the mechanism of its proapoptotic effects on human myeloid leukemia cells has not been extensively studied, we performed an in-depth evaluation of the effects of Tan I on apoptosis in human K562 and HL-60 cells. The results revealed that Tan I could inhibit the growth of leukemia cells and cause apoptosis in a time- and dose-dependent manner. Apoptosis was observed clearly by flow cytometry and Hoechst 33258 staining, as well as DNA fragmentation analysis. After treatment by Tan I for 48 h, the percentage of disruption of mitochondrial membrane potential (Δψm) was increased in a dose-dependent manner. Western blotting analysis demonstrated the cleavage of caspase-3 zymogen protein and a dose-dependent cleavage of poly-(ADP-ribose) polymerase. Tan I-induced apoptosis was accompanied by a significant decrease in survivin and an increase in Bax. Moreover, Tan I treatment remarkably downregulated the phosphorylation of both P85/PI3K and Akt in a time-dependent manner, and the PI3K/AKT-specific inhibitor (LY294002) mimicked the apoptosis-inducing effects of Tan I. We therefore conclude that the induction of apoptosis by Tan I in these leukemia cells is mainly related to the disruption of Δψm, the upregulation of Bax expression, and the activation of caspase-3. This process is highly correlated with the inactivation of PI3K/Akt/survivin signaling pathways. The results indicate that Tan I may serve as an effective adjunctive reagent in the treatment of leukemia.

[Pro-apoptotic effect of ciglitazone in leukemic HL-60 cells via PPARγ and P38 MAPK signaling pathway].

OBJECTIVE: To investigate the apoptosis-inducing effect of peroxisome proliferator-activated receptor γ (PPARγ) agonist ciglitazone (CGZ) on leukemic HL-60 cells and its mechanisms of action. METHODS: HL-60 cells in vitro culture medium were subject to different concentrations of CGZ (10-50 µmol/L) for 24, 48 and 72 h. MTT assay was used to detect the cell inhibitory rate and agarose gel electrophoresis to observe DNA fragmentation. Flow cytometry (FCM) and Annexin V/PI staining were used to detect CGZ and/or GW9662 (PPARγ antagonist)-induced cell apoptosis. The expression of PPARγ was examined by RT-PCR and Western blotting. The caspase-3 and protein levels in mitogen-activated protein kinase signaling pathways (MAPKs, p-P38, p-ERK and p-JNK) were also detected. RESULTS: CGZ (over 30 µmol/L) could inhibit the growth of HL-60 cells in both time- and dose-dependent manner. After treatment for 72 h, the cell growth inhibitory rate in 50 µmol/L CGZ (84% ± 11%) treated cells was found more higher than that in both 40 µmol/L and 30 µmol/L CGZ treated cells (72% ± 13%, 59% ± 13%, P < 0.01) and a typical DNA ladder was also observed by agarose gel electrophoresis. The expression of PPARγ was gradually up-regulated by CGZ treatment and could be down-regulated partially by PPARγ antagonist GW9662. The results also revealed that CGZ-induced cell apoptosis (49.7%, 72 h) could not be inhibited thoroughly by GW9662 (36.2%, control:3.2%). It indicated that the CGZ-induced cell apoptosis was partially PPARγ-independent. Western blotting showed a cleavage of caspase-3 zymogen protein and up-regulation of p-P38 protein. Thus it indicated that the activations of caspase-3 and P38 MAPK were involved in CGZ-induced cell apoptosis. CONCLUSION: CGZ inhibits cell growth by induction of cell apoptosis in HL-60 cells via PPARγ dependent and independent signaling pathways. The activations of caspase-3 and P38 MAPK may be one of the important mechanisms in CGZ in treated HL-60 cells.