The erbstatin analogue methyl 2,5-dihydroxycinnamate cross-links proteins and is cytotoxic to normal and neoplastic epithelial cells by a mechanism independent of tyrosine kinase inhibition

Abstract

Differentiation therapy is an attractive option for the treatment of superficial, localized neoplastic lesions of the skin. Topical application of agents that induce differentiation could selectively inhibit tumor cell growth, inducing a program of cell death with the production of cross-linked protein envelopes as the terminal event of this process at the skin surface, effectively eliminating the neoplastic phenotype. The nonspecific kinase inhibitor staurosporine induces cornified envelope assembly in neoplastic keratinocytes and causes tumor regression (A. A. Dlugosz and S. H. Yuspa, Cancer Res., 51: 4677-4684, 1991). In pursuit of less toxic agents, specific tyrosine kinase inhibitors were tested for the ability to induce differentiation in keratinocyte-derived cells. Of a range of inhibitors tested, only MC was able to induce cross-linked protein and consequent cell death in mouse and human primary normal keratinocytes, 308 neoplastic mouse keratinocytes, HPV-18-infected immortalized human keratinocytes, and human lines SQCC-Y1 (squamous carcinoma) and A431 (epidermoid carcinoma). MC increased cross-linked protein in a dose-dependent manner (0.05-1 mM). To confirm differentiation, MC-treated mouse primary normal keratinocytes were tested for activation of the endogenous cross-linking enzyme transglutaminase, but no association was found between transglutaminase activity and MC-induced protein cross-linking. MC also induced protein cross-linking in the fibroblast cell line NIH3T3 and in B16 melanoma cells, in which cornified envelope assembly is not part of the differentiation process. This cross-linking occurred at 4 degrees C, suggesting a nonphysiological process. Western blot analysis of an in vitro assay with purified EGF receptor showed that MC was able to cross-link the receptor. As in NIH3T3 cells, DTT inhibited cross-linking, suggesting that oxidation of MC or an acceptor group may be required for this effect. Thus, MC does not induce differentiation by a physiological mechanism in epithelial cells but causes chemical protein cross-linking into cornified envelope-like structures at high concentration.