Tangeretin Induces Caspase Dependent Apoptosis and G2/M Arrest in the Erythroleukemia Cell Line K562 In Vitro.

Introduction

Tangeretin, a polymethoxyflavon present in citrus peel oil, was found to inhibit Erk-phosphorylation in T47-D breast cancer cells, induce cell death and block invasion in the chick heart invasion assay. Erk-phosphorylation has been implicated in the growth of bcr-abl transformed cell lines as a result of constitutive abl-activation. Here we investigate whether tangeretin can induce apoptosis and growth arrest in the bcr-abl+ erytroleukemia cell line K562, how it affects signal transduction pathways and the balance between pro- and anti-apoptotic proteins.

Methods

Tangeretin was dissolved in DMSO and used at concentrations up to 100 μM. K562 cells were cultured in RPMI 1640–10% FBS in vitro. Proliferation was followed by MTT test. Apoptosis, cell cycle analysis and bcl-2 expression was assessed by flow cytometry. PARP, Erk1/2, p38, Akt, JNK, p70^S6K, and P-p38, P-Akt, P-JNK, and P-p70^S6K, bcl-XL and mcl-1 were assessed by Western Blot.

Results

Tangeretin shows a time and concentration dependent effect on bcr-abl + K562 cells with a LD₅₀ of 50–70 μM. This effect is accompanied by a G₂/M arrest and a significant increase in the percentage of subG₀ cells and PARP cleavage at 24 hrs. In short term kinetics (30 minutes) tangeretin inhibits the phosphorylation of Erk. No effect on total Erk1/2, p38, Akt, JNK, p70^S6K, and P-p38, P-Akt, P-JNK, and P-p70^S6K could be observed. After 24 and 48 hours of treatment, tangeretin is capable of stimulating the phosphorylation of Erk and p70^S6K. At the same time activation of procaspase-3 and -9 and downregulation of the anti-apoptotic proteins Mcl-1 and Bcl-x_L were seen. Bcl-2 expression was analysed by flow cytometry and was also downregulated.

Conclusion

The citrus flavonoid tangeretin is capable of inducing apoptosis and growth arrest in bcr-abl positive K562 cells through activation of caspase-3 and -9 accompanied by a biphasic change in phosphorylation of Erk and p70^S6K.