Thioridazine enhances lysosomal accumulation of epidermal growth factor and toxicity of conjugates of epidermal growth factor with Pseudomonas exotoxin

Abstract

Thioridazine, a phenothiazine calmodulin inhibitor, aggravated the cytotoxic effect of a conjugate (EGF-PE) of epidermal growth factor (EGF) coupled with Pseudomonas exotoxin against cultured HeLa cells. Other phenothiazine calmodulin inhibitors, trifluoperazine and chlorpromazine, also intensified the cytotoxic effect of EGF-PE, whereas N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) had no such effect. By using iodinated epidermal growth factor ([ 125I]EGF), the effect of thioridazine on intracellular transport of EGF was examined. The release of radioactivity associated with [ 125I]EGF into medium was slow in the presence of thioridazine. The Percoll gradient centrifugation pattern showed that thioridazine delayed both the appearance of [ 125I]EGF in lysosomes and the disappearance of [ 123I]EGF from the lysosomes. The pH value in lysosomes was 5.28 in thioridazine-treated HeLa cells, while that in untreated cells was 5.15. Thioridazine was found to inhibit lysosomal enzyme activities of cathepsin B and acid phosphatase, but not β-hexosaminidase when cell extracts were treated with the drug. Electron microscopy showed an increased number of electron-dense bodies, possibly autophagosomes/lysosomes in HeLa cells grown for 48 h with 3 μg/ml thioridazine. The potentiating action of EGF-PE by thioridazine is discussed in relation to the altered lysosomal function in treated cells.