A molecular complex of bovine milk protein and oleic acid selectively kills cancer cells in vitro and inhibits tumour growth in an orthotopic rat bladder tumour model

Abstract

What's known on the subject? and What does the study add?• Novel intravesical therapies are needed for superficial bladder cancer that reduce the risk of infection associated with Bacillus Calmette-Guérin (BCG) and further destabilization of the urothelium associated with cytotoxic chemotherapy. Experimental therapies to date have included photodynamic therapy, oncolytic viruses, gene therapy (antisense oligonucleotides and silencing RNA), cytokine therapy, death receptor agonists (tumour-necrosis-factor-related apoptosis-inducing ligand and anti-DR5 monoclonal antibody), naturally occurring substances (curcumin and deguelin) and human a-lactalbumin made lethal to tumour cells (HAMLET). HAMLET, a natural occurring product in milk, induces apoptosis in urothelial cancer cells but has limitations in clinical application because of its human source. A previous study in patients with bladder cancer has demonstrated that intravesical HAMLET (daily for 5 days before tumour resection) caused selective apoptotic tumour cell death. BAMLET, the bovine equivalent of HAMLET, is a complex of bovine a-lactalbumin and oleic acid (bLAC) that has been shown in vitro to accumulate in the endolysosomal compartment of tumour cells and induce leakage of lysosomal cathepsins into the cytosol followed by activation of the pro-apoptotic protein Bax.• This is the first in vivo study to show that BAMLET (bLAC) induces apoptosis in urothelial cancer cells and controls the growth of high risk urothelial cancer in a syngeneic rat orthotopic model. This same bladder cancer model system has been used to test other novel therapies, including BCG, and therefore provides a relative comparison of its effectiveness with other intravesical therapies. Objective• To investigate the efficacy of a complex of bovine a-lactalbumin and oleic acid (bLAC) to kill urothelial cancer cells in vitro and inhibit tumour growth and progression in a high risk bladder tumour model. Materials and Methods• The cytotoxicity of bLAC to a large panel of urothelial cell cancer (UCC) cells was tested by the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay, using bLA, the folded a-lactalbumin without oleic acid, as a control.• The mechanism of bLAC-inducing cell death was evaluated by annexin V staining, TUNEL (terminal deoxynucleotidyl transferase mediated nick end labelling) assay and sub-G1 DNA analysis.• The selective bLAC cytotoxicity was examined using multicellular spheroids consisting of UCC and non-transformed fibroblasts.• Rats bearing orthotopic tumour received intravesical instillations (twice weekly, for 3 weeks) of bLAC, bLA, BCG or saline, starting 6 days after UCC (AY-27) cell inoculation. Animals were monitored for survival, toxicity and tumour growth control. Results• A dose-dependent bLAC-inducing apoptotic-like cell death was shown in UCC cells tested, including cells refractory to classic apoptosis-inducing agents, whereas bLA showed little cytotoxicity.• bLAC selectively destroyed cancer cells in spheroids.• Intravesical bLA...

Structure and Potential Cellular Targets of HAMLET-like Anti-Cancer Compounds made from Milk Components

“…However, although HAMLET-like compounds have been assayed on 50 different cancer cell lines, the literature contains only 11 types of primary cell cultures, and thus it remains to be seen whether these trends will hold when more types of primary cells are investigated. The 6 immortalised non-cancer cells lines investigated have sensitivities to HAMLET that are in the same range as the cancer cell lines (7,14,16,22). It is not yet clear whether this is due to a general toxicity of HAMLET to healthy cells, or whether HAMLET is targeting a fundamental cancer-like characteristic that is possessed by immortalised non-cancer cell lines.…”

“…In vivo cytotoxicity and efficacy HAMLET and BAMLET reduced bladder cancer tumours without toxicity to healthy tissue in humans (23), rats (14), and mice (24). Nine human patients awaiting transurethral surgery for bladder cancer received HAMLET instillations via urinary catheter for 5 consecutive days (23).…”

“…1) In vitro, HAMLET and BAMLET compounds show cytotoxicity to over 50 cancer cell lines (7,(13)(14)(15)(16)(17)(18)(19)(20) including cancer cells resistant to cell death (21) while most primary cell cultures investigated, with the exception of blood cells, show resistance to toxicity from HAMLET-like compounds (7,13,14). Embryonic cells (7) and immortalised non-cancer cells (7,14,16,22) are sensitive to HAMLET.…”

“…Embryonic cells (7) and immortalised non-cancer cells (7,14,16,22) are sensitive to HAMLET. A summary of results of HAMLET-like compounds on cell lines and primary cells is provided in Figure 2 and Supplementary Table 1, and is informative for the analyses of the mechanistic studies into HAMLET's modes of action and potential targets.…”

“…HAMLET treatment resulted in a reduction of warts and no signs of toxicity in both immunocompetent and immunosuppressed patients (27). An independent group of researchers showed that BAMLET administered by catheter instillation reduced orthotopic bladder tumours without toxicity in rats (14).…”