Apoptosis Induced by Extracts of Helleborus Niger in Different Lymphoma and Leukemia Cell Lines and Primary Lymphoblasts of Children with ALL Is Independent of Smac-Overexpression and Executed Via the Mitochondrial Pathway.
Authors: Jesse Patrick , Mottke Gritt , Seifert Georg , Fulda Simone , Henze Guenter , Prokop Aram 
Published: October 2019

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 complementary medicine Helleborus niger is used as adjuvant drug in the treatment of non-metastasised and metastasised forms of bronchial cancer, abdominal tumours and prostate cancer. It is also applied in myeloproliferative diseases like Hodgkin and Non-Hodgkin lymphoma, leukaemic disorders and AIDS- related diseases like the Kaposi sarcoma. Until now, there is no clinical or preclinical data regarding the effects of Helleborus niger in vivo, ex vivo or in vitro. For this purpose, we investigated the cytotoxic effects of four different standardized aqueous Helleborus niger extracts from the companies Hiscia and Helixor on various cancer cell lines. We used one whole plant extract, one root extract, one leave extract and one containing only the blossom of Helleborus niger. After 4h of treatment with the extracts no significant LDH release was measured, thus excluding an unspecific, necrotic damage of the cell membrane. After 24h a dose dependent inhibition of proliferation up to 69% could be found and after 48h a distinction into early (45,2%) and late apoptotic (45,5%) cells was detected via Annexin/PI staining. The cell cycle analysis revealed characteristic hypodiploid DNA fragments after 72h, once more identifying apoptosis as cause of the cell death. In the Western Blot analysis a processing of Caspase-3 could be found after 36 h incubation with the extract. Apoptotic cell death was detected in the Burkitt-like lymphoma cell line BJAB, the three human acute lymphoblastic leukemia cell lines NALM-6, Sup-B-15 and REH and the melanoma cell line MEL-HO. The apoptosis induction caused by the root extract was higher than the apoptotic cell death in the other extracts. There are two major pathways of apoptosis, the extrinsic pathway via death receptors like FADD and the intrinsic pathway via the mitochondria. In BJAB cells a breakdown of the mitochondrial membrane potential and dose-dependent mitochondrial permeability transition was detected after 48h, revealing that apoptosis is executed via the mitochondrial pathway. Furthermore, we found a decreased apoptosis induction in BCL-2 overexpressing melanoma cells. The dependency of Bcl-2 expression is another sign of apoptosis via the mitochondrial pathway. In contrast, apoptosis induction by Helleborus niger seems to be independent of Smac overexpression, which could be shown in Jurkat cells. In combination with the vinca alkaloid vincristine, which is used in the treatment of ALL, a synergistic effect could be detected. The apoptosis induction was up to 16% higher in combination than in the single treatment. Finally, we evaluated the effect on primary leukemia cells ex vivo. Interestingly, we could show a significant apoptosis induction in primary leukemia cells from 2 patients with ALL or AML in childhood, which were resistant to the treatment with the anthracycline doxorubicin. For the first time, we were able to show that extracts of Helleborus niger induce apoptosis in different cancer cell lines and primary leukemia cells. Apoptosis is executed via the intrinsic pathway and is independent of Smac overexpression. Thus, we present an interesting baseline for the design of upcoming in vivo experiments or clinical trials.