Abstract
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, p70S6K, and P-p38, P-Akt, P-JNK, and P-p70S6K, 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, p70S6K, and P-p38, P-Akt, P-JNK, and P-p70S6K could be observed. After 24 and 48 hours of treatment, tangeretin is capable of stimulating the phosphorylation of Erk and p70S6K. At the same time activation of procaspase-3 and -9 and downregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL 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 p70S6K.
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