Article

Molecular mechanisms of mistletoe plant extract-induced apoptosis in acute lymphoblastic leukemia in vivo and in vitro

Department of Pediatric Oncology/Hematology, Otto-Heubner-Center for Pediatric and Adolescent Medicine, Charité, Universitätsmedizin Berlin, Germany.
Cancer Letters (Impact Factor: 5.02). 07/2008; 264(2):218-28. DOI: 10.1016/j.canlet.2008.01.036
Source: PubMed

ABSTRACT Viscum album (Mistletoe) is one of the most widely used alternative cancer therapies. Aqueous mistletoe extracts (MT) contain the three mistletoe lectins I, II and III as one predominant group of biologically active agents. Although MT is widely used, there is a lack of scientifically sound preclinical and clinical data. In this paper, we describe for the first time the in vivo efficacy and mechanism of action of MT in lymphoblastic leukemia. For this purpose, we first investigated both the cytotoxic effect and the mechanism of action of two standardized aqueous MTs (MT obtained from fir trees (MT-A); MT obtained from pine trees (MT-P)) in a human acute lymphoblastic leukemia (ALL) cell line (NALM-6). MT-A, MT-P and ML-I inhibited cell proliferation as determined by Casy Count analysis at very low concentrations with MT-P being the most cytotoxic extract. DNA-fragmentation assays indicated that dose-dependent induction of apoptosis was the main mechanism of cell death. Finally, we evaluated the efficacy of MT-A and MT-P in an in vivo SCID-model of pre-B ALL (NALM-6). Both MTs significantly improved survival (up to 55.4 days) at all tested concentrations in contrast to controls (34.6 days) without side effects.

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Angenommen wird eine endozytotische Aufnahme des Moleküls in die Zelle. Zunächst wurden die ML-I-Liposomen nach verschiedenen Verfahren hergestellt, um jenes mit der höchsten Einschlusseffizienz (EE) des ML-I auszuwählen. Durch Nutzung der Detergensdialyse konnte eine ausreichende EE (8 µg/ml) erreicht werden. Der EC50-Wert auf MOLT4-Zellen (T-Zell-Leukämiezelllinie) des verkapselten ML-I war vergleichbar mit dem des freien ML-I, wobei die Apoptoserate verstärkt ausgelöst wurde und die Nekroserate abnahm. Die apoptotische bzw. nekrotische Wirkung des verkapselten Proteins ist jedoch im Vergleich zum freien ML-I geringer. Eine schnellere und erhöhte Aufnahme von Liposomen in Zellen sollte durch die Funktionalisierung mittels B-Kette des ML-I erreicht werden. Dazu wurde zunächst ein Maleinimidgruppen-spezifischer PEG-Cholesterol-Anker (MPC, PEG = Polyethylenglykol) nach Optimierung einer Anleitung von Pan et al. 2007 synthetisiert. 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Die Kopplung der B-Kette erfolgte mit Hilfe der in der Literatur beschriebenen PIT (Post-insertion Technique) bzw. des Einbaus während der Liposomenpräparation (Conventional Method). Die KE war bei der neuen SPIT vergleichbar zu den herkömmlichen Methoden. Die B-Kette war zudem stabil in der Membran verankert und ließ sich nicht durch Serumproteine desorbieren. Eine nachträgliche Funktionalisierung von Stealth®-Liposomen (PEG-modifizierten Liposomen) mit dem Konjugat war ebenfalls möglich. Zum aktiven Targeting wurden MOLT4-Zellen als Leukämie-Modell mit den B-Ketten-funktionalisierten Liposomen (2 oder 5 mol% PEG-Chol- oder PEG-DSPE-Konjugat) für 2 Stunden inkubiert. Mittels FACS Analyse konnte keine vermehrte Aufnahme festgestellt werden, was zunächst durch ein zu schwaches Endozytosesignal der B-Kette für das Liposom gedeutet wird. Dies wird in Folgearbeiten näher zu untersuchen sein. The main part of the present work is the development of a liposomal drug carrier system with which on the one hand incorporated mistletoe lectin-I (ML-I) is delivered to the target tissue and on the other the B-chain of the protein is used for active targeting of liposomes to tumor cells. Liposomes are convenient vesicles for lipophilic as well as for hydrophilic drugs, which are either incorporated in the hydrophobic bilayer or encapsulated in the aqueous interior for shielding them from the immune system. Misteltoe lectin-I is a RIP type 2 protein which consists of an A- and a B-chain. The A-chain represents the cytotoxic component by inhibiting ribosomal protein synthesis which finally ends in apoptosis. On the contrary the B-chain contacts galactose and α2,6-sialo- neolactoganglioside reidues on cell surfaces. An endocytotic uptake is expected. 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