bLAC triggers a lysosomal death pathway in transformed cells

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Abstract:

bLAC (bovine α-LactAlbumin Complex) is a molecular complex consisting of partially unfolded alpha-lactalbumin and a stabilizing co-factor, oleic acid. Originally the human version of bLAC, called HAMLET (human α-Lactalbumin Made Lethal to Tumor cells), was isolated from a fraction of milk, with the ability to kill transformed and immature mammalian cell lines (Håkansson A. et. al. 1995)1. In vivo Hamlet has been shown to have anti-tumoral effects on glioblastoma xenografts in rats (Gustafsson L. et. al. 2005)2, and nonmuscle invasive transitional cell bladder carcinomas in humans (Mossberg AK et. al. 2007)3. In this study we investigate the transformation specificity of bLAC in mouse fibroblast cell line, NIH-3T3, transfected with either an empty retroviral vector or a retroviral vector containing vHa Ras- or cSrcY527F oncogene. Cell viability (survival) was evaluated with MTT assay and cell death (lysis of the outer membrane) with the LDH release assay. Interestingly, our results show, that both oncogenes sensitize the cells to bLAC induced cell death. This finding is of importance with regard to the application of bLAC as a potential new cancer drug. The mechanism whereby bLAC induces cell death is still unclear, and we therefore further sought to investigate this. First we show that bLAC induces caspase-independent cell death, since cell death cannot be inhibited by zVAD-fmk, a caspase inhibitor. Further we performed experiments to elucidate a possible role of the lysosomal compartment in bLAC mediated cell death. We found that Cathepsin L was released from the lysosome into the cytosol in MCF-7 breast carcinoma cell line after treatment with bLAC. A population of bLAC treated cells (those that rounded up) also had Bax activation, however this was always accompanied with translocation of Cathepsin L into the cytosol. Furthermore, expression of Hsp70–2 an inhibitor of lysosomal cell death (Daugaard M. et. al. 2007)4, in MCF-7 cells, protected the cells against bLAC induced cell death. In conclusion our data suggests that bLAC kills transformed cells via a caspase-independent, lysosomal death pathway.

1. Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 8064–8068, August 1995.

2. J. Nutr. 135: 1299–1303, 2005.

3. Int. J. Cancer. 2007 Sep. 15;121(6):1352–9.

4. Cancer Research 67, pp. 2559–2567, March 15, 2007.

Document Type: Abstract

DOI: http://dx.doi.org/10.1111/j.1600-0463.2008.001165_62.x

Affiliations: 1: Apoptoselaboratoriet, Kræftens Bekæmpelse, Strandboulevarden 49, 2100 København Ø 2: NatImmune A/S, Symbion Science Park, Fruebjergvej 3, 2100 København Ø

Publication date: May 1, 2008

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