Cytotoxic activity of doxorubicin “loaded” neutrophils against human mammary carcinoma (HTB-19)

Abstract

Neutrophils were intra-cellularly “loaded” with the chemotherapeutic agent, doxorubicin applying a variety of incubation conditions in order to identify parameters which maximize chemotherapeutic incorporation, while simultaneously preserving optimal viability and chemotactic responsiveness. Doxorubicin “loaded” neutrophils (DLN) were produced in triplicate at different combinations of incubation conditions such as temperature (4° C, 37° C); duration (0, 1, 2 hours); and doxorubicin concentration (20, 40, 60µg/ml). Chemotactic responsiveness of rinsed DLN preparations was subsequently assessed against the neutrophil peptide chemotactic agent, formyl methionyl leucyl phenylalanine (fMLP, 10⁻⁶ M) utilizing a modified 96-well Boyden chemotactic chamber apparatus. Viable, fMLP-responsive DLN preparations were subsequently detected with MTT vitality staining reagent. At sub-physiological incubation temperatures (4° C), profound declines in the viability of DLN preparations were detected when simultaneously incubated with doxorubicin formulated at concentrations greater than 10µg/ml. In contrast, DLN preparations incubated at 37° C displayed diminished viability only when incubated with doxorubicin formulated at a concentration of 60µg/ml. Viable DLN populations were subsequently evaluated to determine their ability to exertin vitro cytotoxic activity against monolayer populations of human mammary carcinoma (HTB-19) propagated in a tissue culture environment. The lethal effect which DLN preparations inflicted towards HTB-19 populations was substantially greater than was observed with an equivalent population of untreated neutrophils. Maximalin vitro cytotoxic activity was detected with DLN preparations produced at 37° C in the presence of doxorubicin formulated at a concentration of 40µg/ml. In contrast, DLN preparations produced at an incubation temperature of 37° C, and a doxorubicin concentration of 20µg/ml displayed relatively lower levels ofin vitro cytotoxic activity against HTB-19 monolayer populations.

The degree ofin vitro cytotoxic activity exerted against HTB-19 monolayer populations by DLN preparations was directly influenced by the duration of the challenge period. Maximalin vitro cytotoxic activity was observed when HTB-19 monolayer populations were challenged with DLN preparations for a period of 96-hours duration at 37° C. Challenge periods of 48-hours duration produced levels ofin vitro cytotoxic activity which were substantially lower than those observed for challenge periods of 96-hours duration. Optimalin vitro cytotoxic activity was recognized when DLN preparations were allowed to establish direct contact with HTB-19 monolayer populations at an estimated DLN:HTB-19 cellular ratio of approximately 5:1 (37° C, CO₂, 6%). Significantly lessin vitro cytotoxic activity was recognized when DLN preparations were only permitted indirect cellular contact with HTB-19 monolayer populations which was achieved through the application of a semi-permeable 3µM pore membrane partition.

In vitro cytotoxic activity of DLN populations was not inhibited by the anti-oxidant agent, dimethyl sulfoxide (DMSO), but was inhibited in the presence of glutathione (GSH), superoxide dismutase (SOD), and vitamin E (α-tocopherol). Similarly,in vitro cytotoxic activity of DLN populations was also inhibited in the presence of sodium heparin (serine esterase inhibitor), and dexamethasone (inhibitor of neutrophil activation-degranulation phenomenon). Experimental results observed in these investigations collectively imply that thein vitro cytotoxic activity exerted by DLN preparations against HTB-19 populations is in part attributable to neutrophil-mediated cytotoxic immunity. This innate property of neutrophil populations involves their capacity to generate highly reactive oxygen “free” radical species (O₂, HO, H₂O₂), and synthesize degradative proteolytic enzyme fractions. These endogenous substances are intimately involved in the processes of neutrophil-mediated cytotoxic immunity when “released” into the extracellular milieu and may potentially act in concert with the anti-neoplastic properties of chemotherapeutic agents such as doxorubicin.