Jenny Ling-Yu Chen1,2,3, Chun-Kai Pan1,4, Yu-Li Lin5, Ching-Yi Tsai4,6, Yu-Sen Huang1,7, Wen-Chi Yang2,3, Feng-Ming Hsu2,3, Sung-Hsin Kuo2,3, Ming-Jium Shieh8. 1. Department of Radiology, National Taiwan University College of Medicine, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan. 2. Division of Radiation Oncology, Department of Oncology, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan. 3. Cancer Center, College of Medicine, National Taiwan University, No. 57, Lane 155, Section 3, Keelung Road, Taipei, Taiwan. 4. Department of Medical Research, National Taiwan University Hospital, No. 7, Chung-Shan South Road, 100, Taipei, Taiwan. 5. Department of Medical Research, National Taiwan University Hospital, No. 7, Chung-Shan South Road, 100, Taipei, Taiwan. linyuli888@gmail.com. 6. Institute of Toxicology, National Taiwan University College of Medicine, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan. 7. Department of Medical Imaging, National Taiwan University Hospital, No. 7, Chung-Shan South Road, Taipei, Taiwan. 8. Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan.
Abstract
PURPOSE: Development of a safe and effective systemic chemotherapeutic agent for concurrent administration with definitive thoracic radiotherapy remains a major 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. METHODS: In vitro radiosensitization of A549 and LLC cell lines was evaluated by colony formation assay, γH2AX fluorescent staining and western blot assay, and annexin V staining. A radiosensitization 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. RESULTS: Treatment with PEGylated liposomal doxorubicin decreased A549 and LLC cell proliferation in a dose-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 a radiosensitizer than free doxorubicin in vivo. Pharmacokinetics evaluation showed a longer 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. CONCLUSIONS: Our study provides preclinical in vitro and in vivo evidence of the effectiveness of PEGylated liposomal doxorubicin as a radiosensitizer, supporting its potential clinical development as a component of chemoradiotherapy.
PURPOSE: Development of a safe and effective systemic chemotherapeutic agent for concurrent administration with definitive thoracic radiotherapy remains a major 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. METHODS: In vitro radiosensitization of A549 and LLC cell lines was evaluated by colony formation assay, γH2AX fluorescent staining and western blot assay, and annexin V staining. A radiosensitization 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. RESULTS: Treatment with PEGylated liposomal doxorubicin decreased A549 and LLC cell proliferation in a dose-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 a radiosensitizer than free doxorubicin in vivo. Pharmacokinetics evaluation showed a longer 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. CONCLUSIONS: Our study provides preclinical in vitro and in vivo evidence of the effectiveness of PEGylated liposomal doxorubicin as a radiosensitizer, supporting its potential clinical development as a component of chemoradiotherapy.
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