Antileukemic Efficacy of Anthocyanins In-Vitro and In-Vivo and In Combination with a DNA Vaccine Against Ph⁺ Acute Lymphoblastic Leukemia

Abstract 3267

Polychemotherapy has dramatically improved the outcome of patients with acute lymphoblastic leukemia (ALL) and leads to long term remission in 70–80 % of children and 40–50% of adults. However, it is likely that after remission the immune system plays an essential part in the eradication of minimal residual disease. Since chemotherapy compromises the immune system, compounds without immunosuppressant activity should be developed for maintenance therapy in order to reduce the relapse rate. Anthocyanins are a group of naturally occurring phenolic compounds widely available in fruits and vegetables in human diets. They have broad biological activities including antimutagenesis and anticarcinogenesis, which are generally attributed to their antioxidant activities.

We studied the effects and the mechanisms of anthocyanin-rich berry extract and the most common types of anthocyanins, cyanidin-3-rutinoside and dephinidin, in several leukemia cell lines. Proliferation was significantly reduced in the murine Ph⁺ ALL cell line BM185 by berry extract as well as by cyanidin-3-rutinoside and dephinidin which was comparable to the effect seen with imatinib or 6-mercaptourine and methotrexate. In the leukemia cell lines HL-60, Sup-B15 and K562 the anti-leukemia efficacy of berry extract was superior compared to cyanidin-3-rutinoside or dephinidin. Apoptosis was induced by upregulation of caspases 3 and 9.

We then evaluated the therapeutic effect of berry extract and cyanidin-3-rutinoside with or without a DNA-based vaccine in a syngeneic Balb/c mouse model. Non-viral minimalistic immunogenically defined gene expression vectors (MIDGE) encoding a BCR-ABL^p185 fusion specific peptide, GM-CSF and IL12 were used for in vivo transfection of murine skin. In addition, we used natural DNA-based double stem-loop immunomodulators (dSLIM), containing 6 CpG-motifs and exhibiting an unique structure, as non-specific immune adjuvant. One day after the application of a lethal challenge dose of the syngeneic BCR-ABL^p185 expressing leukemia cell line BM185, mice received either i) daily oral applications of berry extract (500mg/kg/d) or ii) intraperitoneal applications of cyanidin-3-rutinoside (50 mg/kg/d) or iii) were immunized twice with the DNA vaccine BCR-ABL/GM-CSF/IL-12/dSLIM intracutaneously. The fourth group received both, cyanidin-3-rutinoside and the DNA-vaccine and the fifth group was treated with berry extract and the DNA-vaccine. While the treatment with berry extracts or cyanidin-3-rutinoside alone had no benefit compared to the control, mice which received the DNA-vaccine showed a significant longer tumor-free and overall survival compared to the control and a survival rate of 56%. Interestingly, the combined treatment with DNA-vaccine and berry extract but not with DNA-vaccine and cyanidin-3-rutinoside further improved the outcome and lead to a significant longer tumor-free and overall survival compared to the DNA-vaccine alone and a survival rate of 90%. In conclusion, we provide data that anthocyanin-rich berry extract combined with a leukemia-specific DNA-vaccine acts synergistically in the treatment of Ph⁺ ALL. This approach may have implications to optimize maintenance therapy in patients with Ph⁺ ALL.