Tangeretin Synergizes with Imatinib in Inhibiting Growth of Bcr-Abl Positive K562 Erythroleukemia Cells in an Erk-Dependent Way.

Introduction

Tangeretin, a methoxyflavone derived from citrus peel oil, is a weak inhibitor of Erk-phosphorylation in MCF7/6 and T47-D breast cancer cells, capable of inducing apoptosis and growth arrest and blocking invasion in the chick heart invasion assay. Erk-phosphorylation has been implicated in the growth of bcr-abl transformed cell lines and growth advantage in Phi+-CML, both as a result of constitutive abl-activation and potentially directly through<S> </S>VEGF-R stimulation. Erk-P inhibitors have been demonstrated to synergize with imatinib. We therefore assessed the potential of tangeretin to inhibit Erk-phosphorylation, induce apoptosis and growth arrest and to synergize with imatinib in bcr-abl+ K562 erythroleukemia cells.

Methods

Tangeretin was dissolved in DMSO and studied in 0,1–100 μM range. K562 were cultured in 96 well plates in RPMI-10%FCS in vitro. Growth was assessed by MTT assay on 1 to 6 consecutive days. Apoptosis was assessed by Annexin V-PI staining and Western Blot of PARP cleavage. Differentiation of K562 was studied by cytological assessment of cytospins, and flow cytometry of CD33 and CD13 (myelomonocytic cells), glycophorin A (erythroid), CD61 (megakaryocyte). Total Erk1-2, Akt, p38 and P-Erk1-2 (thr202/tyr204), P-Akt (Ser473) and P-p38 (thr180/tyr182) were assessed by Western blot. Synergism was studied at sublethal levels of tangeretin (0-50 μM) and imatinib (0-1 μM) and scored by the multiplicative model of Valeriote and Lin. P-glycoprotein expression and function was studied by flow cytometry (4E3, Daco MRK16 Kamiya Chemicals Abs and DiOC6). Bcr-abl mRNA transcription was assayed by RT-PCR.

Results

Tangeretin did not affect total Erk1-2, Akt and p38 levels, nor the phosphorylation of Akt and p38 but inhibited constitutive Erk1, 2 phosphorylation in K562. Tangeretin inhibited growth with a LD50 of 50-70 μM and induced cell death in the 100 μM dose range. Cytological signs of apoptosis and extensive vacuolization were observed from 10 μM on at day 5 of treatment. PARP cleavage was demonstrated at 100 μM after 24hrs. No signs of differentiation were observed by cytology or flow cytometry in contrast to previous reports on other Erk-inhibitors with K562. Coculture in tangeretin (0-50 μM) and imatinib (0-5 μM) demonstrated synergism in growth inhibition. To account for imatinib potentiation by P-glycoprotein inhibition reported with tangeretin, P-glycoprotein expression and function were measured and found to be negative. To evaluate the influence of tangeretin on the bcr-abl transcription of imatinib treated K562 cells, RT-PCR was performed, and but coculture did not effect bcl-abl mRNA levels over consecutive 6 days.

Conclusion

The citrus flavonoid tangeretin is capable of inducing apoptosis, growth arrest in bcr-abl positive K562 cells in part through inhibition of Erk-phosphorylation. Tangeretin synergizes with imatinib in inducing growth inhibition in a P-glycoprotein independent way.