Effects of hyaluronidase on doxorubicin penetration into squamous carcinoma multicellular tumor spheroids and its cell lethality

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Abstract
Doxorubicin is an anticancer agent widely used in the treatment of human cancer. The major limitation of this drug governing the cell-killing effect appears to be its poor penetration into a tumor mass. We have studied the effects of hyaluronidase on the penetration and cell-killing effect of doxorubicin using multicellular tumor spheroids (MTS). MTS approximately 500 μm in diameter were produced by a liquid-overlay culture technique from PC-10 lung and HEp-2 laryngeal squamous carcinoma cell lines. Cells in MTS and monolayer were exposed to hyaluronidase for various lengths of time; this was followed by a 1-h resting interval and a subsequent 1-h exposure to doxorubicin. MTS and monolayer cells were then trypsinized to a single-cell suspension and subjected to clonogenic assay. Hyaluronidase at a concentration of 25 U/ml or 250 U/ml was nontoxic to the monolayer cells. For PC-10 MTS, pretreatment with 25 U/ml hyaluronidase for 24 h and 72 h resulted in approximately 20% increases in Doxorubicin cell killing at the median (IC50) dose as compared to doxorubicin alone. HEp-2 MTS were more sensitive to the hyaluronidase pretreatment. Thus, a 1-h exposure to the enzyme produced a 40% increase in doxorubicin-induced cell death at the IC50 dose. A fluorescence microscopic study revealed that a 1-h exposure of MTS to doxorubicin produced doxorubicin fluorescence only in the one or two outer layers of MTS. When MTS were pretreated with hyaluronidase, there was enhanced penetration of doxorubicin fluorescence into the MTS core. Hyaluronidase-induced enhancement of Doxorubicin penetration and its cell-killing effect is dependent on the exposure time and tumor cell origin. These data suggest that anecdotal reports of hyaluronidase-enhanced activity of preclinical chemotherapy deserve a controlled trial.