In vitro cytotoxicity following specific activation of amygdalin by β-glucosidase conjugated to a bladder cancer-associated monoclonal antibody

Abstract

We describe a novel version of antibody‐directed enzyme prodrug therapy (ADEPT), with the use of amygdalin as prodrug. Amygdalin is a naturally occurring cyanogenic glycoside, which can be cleaved by sweet almond β‐glucosidase to yield free cyanide. If amygdalin could be activated specifically at the tumour site, then malignant cells would be killed without the systemic toxicity usually associated with chemotherapy. To this end, we have conjugated β‐glucosidase to a tumour‐associated monoclonal antibody (MAb) (HMFG1) and the conjugate has been tested in vitro for specificity and cytotoxicity subsequent to activation of amygdalin. Amygdalin was cytotoxic to HT1376 bladder cancer cells only at high concentrations, whereas the combination of amygdalin with HMFG1‐β‐glucosidase enhanced the cytotoxic effect of amygdalin by 36‐fold. When 2 concentrations of HMFG1‐β‐glucosidase were compared, the toxic effect was dose dependent. The cytotoxicity of amygdalin was also enhanced by the MAb–enzyme conjugate even when the unbound conjugate was removed from the medium prior to exposure to amygdalin and the cells were washed. In addition to the cytotoxic effect, we also demonstrated specificity, using a MAb–enzyme conjugate that does not recognise the HT1376 bladder cancer cells. Finally, we studied the cytotoxic effect of the conjugate in co‐culture of HMFG1‐positive and ‐negative cell lines (HT1376 and U87MG cells). We demonstrated that the rate of surviving cells corresponds well to the percentage of U87MG (HMFG1‐negative) cells in the flask. Our findings indicate that ADEPT is more effective than non‐directed enzyme activation of a prodrug and can result in a non‐toxic cancer therapy. Int. J. Cancer 78:712–719, 1998. © 1998 Wiley‐Liss, Inc.

Antibody‐guided enzyme therapy of cancer producing cyanide results in necrosis of targeted cells

“…The targeting of cancer cells with an antibody chemically conjugated to a ␤-glucosidase has been reported 11 and was shown to result in growth delay of tumours in vivo. Ideally, a recombinant system needs to be developed, as this can overcome the problem of heterogeneous species that results from chemical conjugation.…”

“…10 The targeting of cancer cells with an antiplacental alkaline phosphatase antibody chemically conjugated to a broad substrate, sweet almond ␤-glucosidase, has been reported with promising results in vivo. 11 Recombinant fusion proteins are worth developing because they generally have advantages over chemically produced molecules in terms of product quality and yield. This is important when considering preclinical or clinical testing.…”

Suppressive Effect of Constituents Isolated from Kernel of Prunus armeniaca on 5 .ALPHA.-Androst-16-en-3-one Generated by Microbial Metabolism

“…Apricot kernels have several bioactive effects and have been prescribed in many traditional Chinese medicines for their antitussive, expectorant, and laxative functions (19)(20)(21). The major effective ingredient of apricot kernels has been reported to be amygdalin and activity related to the generation of hydrogen cyanide follows the decomposition of amygdalin due to the action of b-glucosidase.…”

Enhanced and selective delivery of enzyme therapy to 9L-glioma tumor via magnetic targeting of PEG-modified, ß-glucosidase- conjugated iron oxide nanoparticles

“…The acquisition parameters were set as follows: −128°C for temperature, 9.2 GHz for resonant frequency, and 20 mW for microwave power. The combination of receiver gain (1×10 4 or 1×10 5 ) and modulation amplitude (1 G or 5 G) was dependent on the type of sample as well as the intensity of the observed spectra. A WinEPR workstation (Bruker Corporation) was used to obtain the double integral values of the spectra.…”

“…Magnetic DEPT, which is attracting increasing attention, involves coupling the bioactive prodrug-activating enzyme to magnetic nanoparticles (MNP) 3 that are then selectively delivered to the tumor by applying an external magnetic field. Of all the DEPT strategies, the β-glucosidase/ amygdalin system, 4 in which amygdalin is converted to hydrogen cyanide to kill tumor cells, is the most widely used. The nonspecific toxicity of hydrogen cyanide in normal cells/tissues can be greatly minimized by administering amygdalin with the maximum concentration ratio of β-glucosidase-conjugated MNP in tumor tissue and the blood circulation.…”