Preclinical evaluation of PEGylated liposomal doxorubicin as an effective radiosensitizer in chemoradiotherapy for lung cancer.

Abstract

Development of asafe and effective systemic chemotherapeutic agent for concurrent administration with definitive thoracic radiotherapy remains amajor goal of lung cancer management. The synergistic effect of PEGylated liposomal doxorubicin and irradiation was evaluated in lung cancer cell lines both in vitro and in vivo. In vitro radiosensitization of A549 and LLC cell lines was evaluated by colony formation assay, γH2AX fluorescent staining and western blot assay, and annexinV staining. Aradiosensitization study with healthy human lung-derived cell line BEAS-2B was performed for comparative purposes. In vivo radiosensitization was evaluated by tumor ectopic growth, cell survival, pharmacokinetics, and biodistribution analyses. Cleaved caspase‑3, the marker for apoptosis, was assessed immunohistochemically in A549 xenograft tumors. Treatment with PEGylated liposomal doxorubicin decreased A549 and LLC cell proliferation in adose-dependent manner. In vitro studies revealed comparable radiosensitizer advantages of PEGylated liposomal doxorubicin and free doxorubicin, showing equivalent DNA double-strand breaks according to γH2AX fluorescent staining and western blot assays, similar numbers of apoptotic cells in the annexin‑V staining assay, and moderately decreased clonogenic survival. In vivo studies demonstrated markedly slow ectopic tumor growth with prolonged survival following treatment with PEGylated liposomal doxorubicin plus irradiation in both A549 and LLC mouse models, suggesting that PEGylated liposomal doxorubicin is more effective as aradiosensitizer than free doxorubicin in vivo. Pharmacokinetics evaluation showed alonger half-life of approximately 40 h for PEGylated liposomal doxorubicin, confirming that the liposomal carrier achieved controlled release. Biodistribution evaluation of PEGylated liposomal doxorubicin confirmed high accumulation of doxorubicin in tumors, indicating the promising drug delivery attributes of PEGylated liposomal doxorubicin. Although free doxorubicin caused histopathologic myocarditis with the cardiac muscle fibers showing varying degrees of damage, PEGylated liposomal doxorubicin caused no such effects. The immunohistochemical expression of cleaved caspase-3-positive cells was greatest expressed in the irradiation and PEGylated liposomal doxorubicin combined treatment group, indicating prolonged tumoricidal effects. Our study provides preclinical in vitro and in vivo evidence of the effectiveness of PEGylated liposomal doxorubicin as aradiosensitizer, supporting its potential clinical development as acomponent of chemoradiotherapy.