Sustained antiproliferative mechanisms by RB24, a targeted precursor of multiple inhibitors of epidermal growth factor receptor and a DNA alkylating agent in the A431 epidermal carcinoma of the vulva cell line

Abstract

Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion. Here we report on the first prototype of this model, RB24, a masked methyltriazene, that in addition to being an inhibitor on its own was designed to degrade to RB14, ZR08, RB10+a DNA alkylating methyldiazonium species. The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion. Thus, we surmise that these species could alkylate the active site of EGFR, thereby irreversibly blocking its action and that DNA damage could be induced by the methyldiazonium. Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene. The pronounced growth inhibitory potency of RB24 was attributed to its ability to simultaneously damage DNA and irreversibly block EGFR TK activity.

Figure 1

Selective inhibition of EGFR autophosphorylation in intact cells by RB24. Serum-starved A431 cells were preincubated for 2 h with the indicated concentrations of RB24 prior to stimulation with EGF for 15 min. An equal amount of cell lysates was analysed by Western blotting using antiphosphotyrosine antibodies. Membranes were stripped of antiphosphotyrosine and reprobed with anti-EGFR antibodies as a loading control. Band intensities were measured using the SynGene GeneTools software package.

Figure 2

Reverse EGFR autophosphorylation in the presence of RB24, RB14, ZR08 and RB10 in A431 cells. (A) Duplicate sets of cells were treated with 30 μM of designated compound to be tested as a reversible EGFR inhibitor for 90 min. One set of cells was then stimulated with EGF for 15 min, and extracts were made as described under the Western blotting procedure. The other set of cells was washed free of the compound with serum-free media, incubated for 2 h, and further washed twice and incubated for 4 h. This set of cells was then stimulated with EGF, and extracts were made similar to the first set. (B) Comparison between the inhibition of autophosphorylation activity induced by RB24, RB14, ZR08 and RB10. The film was scanned and band intensities were quantified using Syngene GeneTools software. Values are percentage of control of phosphotyrosine/EGFR.

Figure 3

Effect of RB24 on Erk1,2 activation in A431 cells. Serum-starved cells were preincubated for 2 h with the indicated concentrations of RB24 prior to stimulation with EGF for 15 min. Protein lysates were obtained and Western blot was performed as described by Tari and Lopez-Berestein (2000).

Figure 4

Effect of RB24 on c-fos gene expression in A431 cells. Serum-starved cells were preincubated for 2 h with the indicated concentrations of RB24 prior to stimulation with EGF for 30 min. Quantitative analysis of c-fos and G3PDH was preformed by RT–PCR as described in Materials and methods.

Figure 5

Quantitation of DNA damage using the alkaline comet assay. Tail moment was used as a parameter for the detection of DNA damage in A431 cells exposed to RB24, RB14 and RB10 for 30 min. Each point represents at least two independent experiments.

Figure 6

Irreversible growth inhibition for RB24, RB14, ZR08, RB10 and TEM in A431 cells. Cells were exposed to (A) RB24, (B) RB14, (C) ZR08, (D) RB10 or (E) TEM for 2, 8, 12, 24 or 48 h following recovery for a total of 96 h. Cell growth was measured using SRB assay. Each point represents at least two independent experiments run in triplicate.

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