Knipholone anthrone from Kniphofia foliosa induces a rapid onset of necrotic cell death in cancer cells

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1 Knipholone anthrone from Kniphofia foliosa induces a rapid onset of necrotic cell death in cancer cells Fitoterapia, 11/18/2010 Habtemariam S The present study examines the comparative cytotoxicity of knipholone and knipholone anthrone in leukaemic and melonocyte cancer cell lines. It was found that KA induces a rapid onset of cytotoxicity with IC50 values ranging from 0.5 to 3.3 micro M. In comparison to KA, KP was times less toxic to cancer cells. Morphological and biochemical studies revealed that the cytotoxicity of KA was coupled with a quick loss of membrane integrity leading to necrotic cell death. The study identified KA as a new class of natural potential anticancer agent with a wide range of toxicological and pharmacological implications. Knipholone anthrone from Kniphofia foliosa induces a rapid onset of necrotic cell death in cancer cells Solomon Habtemariam, a, a Pharmacognosy Research Laboratories, Medway School of Science, University of Greenwich, Chatham-Maritime, Kent ME4 4TB, UK Received 12 May 2010; revised 9 June Available online 1 July Abstract The present study examines the comparative cytotoxicity of knipholone (KP) and knipholone anthrone (KA) in leukaemic and melonocyte cancer cell lines. It was found that KA induces a rapid onset of cytotoxicity with IC50 values ranging from 0.5 to 3.3 μm. In comparison to KA, KP was times less toxic to cancer cells. Morphological and biochemical studies revealed that the cytotoxicity of KA was coupled with a quick loss of membrane integrity leading to necrotic cell death. The study identified KA as a new class of natural potential anticancer agent with a wide range of toxicological and pharmacological implications. Graphical abstract Knipholone anthrone but not its close structural analogue, knipholone, induces a rapid onset of necrotic cell death in cancer cells.

2 Keywords: Kniphofia foliosa (Asphodelaceae); Knipholone; Knipholone anthrone; Cytotoxicity; Apoptosis; Necrosis Abbreviations: AZ, 6-azauridine; KA, knipholone anthrone; KP, knipholone; LDH, lactate dehydrogenase; PI, propidium iodide; H 2 O 2, hydrogen peroxide Article Outline 1. Introduction 2. Materials and methods 2.1. Materials 2.2. Cell lines and culture conditions 2.3. Cytotoxicity assay 2.4. Detection and quantification of apoptosis/necrosis by fluorescence microscope 2.5. DNA extraction and agarose gel electrophoresis 2.6. Statistical analysis 3. Results 3.1. Comparative cytotoxicity of KA and KP in cancer cells

3 3.2. Effects of KA on cell membrane integrity 3.3. Measurement of apoptotic and necrotic events following KA treatment 3.4. Effect of KA on interneuclosomal DNA fragmentation 4. Discussion Conflict of interest statement Acknowledgements References Fig. 1. Structures of knipholone and knipholone anthrone from K. foliosa with P absolute configuration. Fig. 2. Effects of KA and KP on RAW cells viability. Cell viability was assessed by Alamar Blue assay after 24 h of treatment with KA, KP or 6-azauridine (AZ). Data are mean and SEM values obtained from six replicates of a representative experiment. Percent cell viability effects on four representative cancer cell lines are shown: (A) B16, (B) THP-1, (C) RAW264.7 and (D) U937 cells are shown.

4 Fig. 3. Effects of KA on RAW membrane integrity. The time- and concentration-dependent release of LDH following treatment with various KA concentrations are shown. Data are mean and SEM values (n = 4) from three independent experiments. Fig. 4. Fluorescence microscopic analysis of KA mediated apoptosis/necrosis. RAW and B16 cells were treated with KA or H 2 O 2 for 3 h and stained with annexin V and PI. Mean and SEM (n = 4) percentage of PI-stained cells are shown: *P < 0.01 vs control; ** and *** are P < 0.05 and P < vs H 2 O 2 treatment. An increase in Annexin stained cells were not observed in all KA treatments as evidenced by four independent experiments. Fig. 5.

5 Morphological analysis of KA-treated cells. Cells were incubated for 24 h in the presence or absence of KA concentrations and stained with Hoechst as described in the materials and methods. Photographs were taken at a final magnification of 200 : phase contrast images (right panel) and fluorescent DNA image (left panel) are shown. Photographs numbers indicate: 1 is control cells and 2 4 are treatments with 3, 6 and 12 μm KA respectively. Similar results were obtained when B16 cells were treated with KA concentrations for 24 h. Fig. 6. DNA fragmentation analysis of macrophages. RAW cells (2 106/2ml/well in 6-well plates) were treated with increasing concentrations of KA for 24 h. Genomic DNA was isolated and electrophoresis was carried out using Tris Borate EDTA buffer as described in the materials and methods. Lane 1 is a marker λ DNA hind III, Lane 2 is DNA from untreated control cells, Lane 3 5 are DNA from cells treated with 3, 6 and 12 μm KA respectively. Similar results were obtained when cells were treated for 8 h. Table 1. Effect of KA and KP on cancer cell viability. a Data are mean IC50 values and SEM in μm obtained from 4 to 10 independent experiments. b Data are mean IC25 (25% inhibitory concentration) values and SEM in μm obtained from 4 or 5 independent experiments. Corresponding author. Tel.: ; fax: Fitoterapia Volume 81, Issue 8, December 2010, Pages

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