Helleborus niger as new cytostatic compound against lymphoma and leukemia in childhood

Abstract

Helleborus niger , also known as Christmas Rose, belongs to the family of Ranunculaceae, a family of flowering plants with about 2500 different species. In anthroposophic medicine H. niger is used as adjuvant drug in the treatment of brain tumors, leukemia, lymphoma and prostate cancer. Although it is widely applied in these malignancies, there are no clinical or preclinical data regarding the effects of H. niger in vivo , ex vivo or in vitro . For this purpose, we investigated the cytotoxic effects of four different standardized aqueous H. niger extracts on various cancer cell lines. We used one whole plant extract, one root extract, one leave extract and one containing only the blossom of H. niger . After 4 h no significant LDH release was measured, thus excluding an unspecific, necrotic damage to the cell membrane. After 24 h a dose-dependent inhibition of proliferation was determined and after 48 h a distinction into early and late apoptotic cells was detected via annexin/PI staining. The cell cycle analysis revealed characteristic hypodiploid DNA after 72 h, once more identifying apoptosis as a cause of the cell death. Apoptotic cell death was detected in the Burkitt-like lymphoma cell line BJAB, the leukemia cell lines NALM-6, Sup-B-15 and REH and the melanoma cell line MEL-HO. Hereby, the whole plant and the root extract revealed the strongest apoptosis induction. In western blot analysis an activation of Caspase-3, the main executioner caspase of apoptosis, could be found after 36-h incubation with the extract. A breakdown of the mitochondrial membrane potential and dose-dependent mitochondrial permeability transition were determined after 48 h, revealing that apoptosis is executed via the mitochondrial pathway. Furthermore, we could find a decreased apoptosis induction in BCL-2 overexpressing melanoma cells. The dependency on Bcl-2 expression is another sign of apoptosis via the mitochondrial pathway. Finally, we evaluated the effect on primary cells ex vivo . Interestingly, we could show a significant apoptosis induction in primary leukemia cells from patients with ALL or AML in childhood, which were partly poor responding to the anthracyclines doxorubicin and daunorubicin. In Nalm6 cells resistant to vincristine and paclitaxel, apoptosis induction by H. niger was as high in the control than in the resistant cell line, so that drug resistance against these two compounds is overcome in vitro . For the first time, we demonstrate in vitro efficacy of H. niger extract in cells of haematological malignancies and thus present an interesting baseline for further in vivo experiments.