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Apoptosis induction in human breast cancer (MCF-7) cells by a novel venom l-amino acid oxidase (Rusvinoxidase) is independent of its enzymatic activity and is accompanied by caspase-7 activation and reactive oxygen species production

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Abstract

We report the elucidation of a mechanism of apoptosis induction in breast cancer (MCF-7) cells by an l-amino acid oxidase (LAAO), Rusvinoxidase, purified from the venom of Daboia russelii russelii. Peptide mass fingerprinting analysis of Rusvinoxidase, an acidic monomeric glycoprotein with a mass of ~57 kDa, confirmed its identity as snake venom LAAO. The enzymatic activity of Rusvinoxidase was completely abolished after two cycles of freezing and thawing; however, its cytotoxicity toward MCF-7 cells remained unaffected. Dose- and time-dependent induction of apoptosis by Rusvinoxidase on MCF-7 cells was evident from changes in cell morphology, cell membrane integrity, shrinkage of cells and apoptotic body formation accompanied by DNA fragmentation. Rusvinoxidase induced apoptosis in MCF-7 cells by both the extrinsic (death-receptor) and intrinsic (mitochondrial) signaling pathways. The former pathway of apoptosis operated through activation of caspase-8 that subsequently activated caspase-7 but not caspase-3. Rusvinoxidase-induced intrinsic pathway of apoptosis was accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species, followed by a decrease in cellular glutathione content and catalase activity, and down-regulation of expression of anti-apoptotic proteins Bcl-XL and heat-shock proteins (HSP-90 and HSP-70). Rusvinoxidase treatment resulted in increase of the pro-apoptotic protein Bax, subsequently leading to the release of cytochrome c from mitochondria to the cytosol and activating caspase-9, which in turn stimulated effector caspase-7. Rusvinoxidase at a dose of 4 mg/kg was non-toxic in mice, indicating that it may be useful as a model for the development of peptide-based anticancer drugs.

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Acknowledgments

Authors thank Dr. R. Mukhopadhyay, TU for HEK cell cytotoxicity assay. AKM is the recipient of DBT-Crest award from the Department of Biotechnology, Ministry of Science and Technology, Govt. of India, which supported his participation in this study. Support was also provided by a BioScience Discovery award from COEDIT (to SPM).

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Author notes

  1. Anthony J. Saviola

    Present address: Department of Pharmacology, Weill Medical College of Cornell University, New York, NY, 10065, USA

Authors and Affiliations

  1. School of Biological Sciences, University of Northern Colorado, Greeley, CO, 80639-0017, USA

    Ashis K. Mukherjee, Anthony J. Saviola, Patrick D. Burns & Stephen P. Mackessy

  2. Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, 784028, India

    Ashis K. Mukherjee

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Correspondence to Ashis K. Mukherjee.

Electronic supplementary material

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10495_2015_1157_MOESM1_ESM.tif

Supplementary material 1 Fig. S1. Fractionation of crude D. r. russelii venom (200 mg) on size-exclusion BioGel P-100 column (2.8×80 cm). Fractionation was done with 25 mM HEPES buffer containing 100 mM NaCl, 5 mM CaCl2 (pH 6.8) at a flow rate of 6 ml/h. Protein elution was monitored at 280 nm. Elution of Rusvinoxidase from gel-filtration column is indicated with an arrow. [This supplementary figure was originally published as Figure 1A by Mukherjee and Mackessy, 2013 (reference 3) and is reproduced with permission from Biochim. Biophys. Acta] (TIFF 56 kb)

10495_2015_1157_MOESM2_ESM.tif

Supplementary material 2 Fig. S2. FPLC cation exchange fractionation of Bio-Gel peak 1 (supplementary Fig. S1) using a Tricorn Mono Q 5/50 GL column. After eluting the non-specifically bound proteins of Bio-Gel peak 1 with buffer A (20 mM Tris-HCl, pH 8.0), bound proteins were eluted with a linear gradient of 350 mM NaCl in buffer A at a flow rate of 45 ml/h. Elution of proteins was monitored at 280 nm and 0.75 ml fractions were collected. Elution of Rusvinoxidase is indicated by an arrow in the FPLC chromatogram. [This supplementary figure was originally published as Figure 1 by Mukherjee et al, Thrombosis Research 134 (2014), 1150–1156] (TIFF 71 kb)

10495_2015_1157_MOESM3_ESM.tif

Supplementary material 3 A comparison of residual enzymatic activity and cytotoxicity of Rusvinoxidase post storage at 4°C. The cytotoxicity was measured against MCF-7 cell. Data represent mean ± SD of threedeterminations (TIFF 70 kb)

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Mukherjee, A.K., Saviola, A.J., Burns, P.D. et al. Apoptosis induction in human breast cancer (MCF-7) cells by a novel venom l-amino acid oxidase (Rusvinoxidase) is independent of its enzymatic activity and is accompanied by caspase-7 activation and reactive oxygen species production. Apoptosis 20, 1358–1372 (2015). https://doi.org/10.1007/s10495-015-1157-6

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