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Many oncogenes encode tyrosine kinases and the identification of appropriate inhibitors is an appealing proposition for the development of selective anticancer agents. These studies outline some screening models which we are currently developing in which we have evaluated a variety of compounds, reported to have tyrosine kinase inhibitory activity, as antitumor agents. In an enzyme based assay, using isolated pp60c-src, a variety of indolocabazoles, flavonoids, tyrophostins, herbimycin A and natural product cytotoxic agents were tested. Genistein, quercetin, staurosporine and herbimycin A were selected for comparison in whole cell studies. Soft agar colony formation of an activated c-src transformed NIH3T3 line was inhibited (IC50) by genistein (4.5 mug/ml) and quercetin (6.5 mug/ml) at similar concentrations and by herbimycin A (7 ng/ml) and staurosporine (1.3 ng/ml) at much lower concentrations. However, these agents inhibited colony formation of v-raf, H-ras and polyoma transformed NIH3T3 lines at comparable concentrations suggesting no selective inhibition of the src mediated transformation was being produced. Furthermore, quercetin failed to inhibit tyrosine phosphorylation of cellular proteins at concentrations shown to inhibit colony formation. Nevertheless. daily quercetin treatment (ip) for seven days at 1000 and 500 mg/kg/day did produce a meaningful increase in median survival time of athymic mice inoculated ip with 106 c-src transformed NIH3T3 cells. The screening models described are now being used to identify and develop new tyrosine kinase inhibitors from natural products and assess their potential as antitumor agents.

The A431 tumor Xenograft was characterized as a model to test EGF-receptor antagonists. Removal of the EGF rich submandibular gland from athymic mice, or stimulation of EGF production in the submandibular gland by testosterone administration both failed to influence tumor growth. Infusion of EGF from alzet minipumps raised circulating EGF levels, however, A431 tumor growth was not significantly changed. Treatment on the other hand (IP every 4 days) with a monoclonal to the human EGF receptor (225 antibody) produced a dose (1-0.25 mg/inj) related inhibition of Subcutaneous (s.c.) implanted A431 tumor growth. Long term inhibition (> 114 days) of tumor growth was related to the number of doses of antibody administrated with 9/10 tumors not re-growing after 9 doses of 1 mg/mouse/inj given 4 days apart. A dose related inhibition of A431 tumor growth when implanted under the subrenal capsule (src) was also established for the EGF-receptor antibody and tumor regression was frequently observed with doses greater-than-or-equal-to 0.5mg/mouse/inj. These studies indicate that A431 tumor growth is insensitive to modulation of host EGF but can be inhibited by agents that block the EGF-receptor. The s.c. and src implanted A431 tumor models that have been established will now be used to evaluate the antitumor potential of EGF inhibitors identified from our screening programs.