Combinational Treatment of Imatinib and Allicin Synergistically Inhibits the Growth of BCR-ABL-Positive Leukemia and the Emergence of Drug-Resistant Cells

Imatinib mesylate (Gleevec, Glivec, STI571) is used as a first-line therapy for chronic myeloid leukemia (CML) to inhibit the tyrosine kinase activity of BCR-ABL. Although imatinib has greatly improved CML patient prognosis, imatinib-resistant cells often develop in about 30-40% of patients after treatment, causing major therapeutic failure. Several second and third generation tyrosine kinase inhibitors, such as dasatinib, nilotinib and ponatinib, have been developed, but none of them are able to completely block the development of drug-resistance. Therefore, new therapeutic approaches are in need to prevent or reduce the emergence of drug-resistant cells in the early stages of imatinib therapy.

In this study, we show that the combinational treatment of allicin, an active component in garlic, and imatinib effectively blocks imatinib-resistance in K562 BCR-ABL-positive leukemia cells. Both imatinib and allicin had dose-dependent effects on cell viability during the first 3 days of treatment. However, the growth inhibitory effect did not last and resulted in re-growth of cells when treated with allicin alone (2 microgram/ml) or 200 nM of imatinib, which is close to the IC50 value in our K562 cells determined on day 3. In contrast, the combination of allicin with imatinib still inhibited cell growth by day 6. Wright-Giemsa stainings showed that the combinational treatment of imatinib and allicin also caused erythroid differentiation.

To identify the molecular mechanisms of imatinib and allicin's actions, we analyzed RNA expression after single and combinational treatments with imatinib and allicin. The results demonstrated that both imatinib and allicin increased the expression of alpha -globin and gamma -globin. The expression of glycophorin A (GYPA), a marker of erythroid differentiation, was synergistically increased by the combinational treatment of imatinib and allicin. One of the notable changes we observed was a decrease of p21 (cyclin-dependent kinase inhibitor 1A; CDKN1A) expression when cells were treated with the IC50 dosage of imatinib. Surprisingly, when allicin was treated together with imatinib, the p21 decrease by imatinib was completely inhibited. Allicin alone did not increase p21, suggesting that allicin does not control p21 expression directly, but instead antagonizes the action of imatinib downregulating p21.

More importantly, a long-term treatment of K562 cells with imatinib and allicin showed that allicin was able to completely block the emergence of imatinib-resistant cells in vitro. Viable cells started to grow again within 12 days when treated with imatinib or allicin alone. However, no resistant cells were observed after up to 40 days when cells were cultured with the combinational treatment.

Taken together, the combinational treatment of imatinib and allicin has a synergistic effect on blocking leukemia cell expansion as they enhance erythroid differentiation and prevent cell cycle progression of residual resistant cells by attenuating the negative effects of imatinib on p21 expression. Our findings suggest that combining imatinib and allicin could be a potential therapeutic to prevent drug-resistance in BCR-ABL-positive leukemia.