ABSTRACT
Title
Antitumor activity of nanosponge-encapsulated camptotechin in human prostate tumors
Authors
R. Minelli
Doctoral School of Science and High Technology - Pharmacology
Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Italy
Doctoral School of Science and High Technology - Pharmacology
Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Italy
Abstract
Drug delivery technologies are new and powerful approaches to enhance the pharmacological properties of drugs by improving their availability and stability and by reducing their side effects.
Camptothecin (CPT) is a potent DNA Topoisomerase I inhibitor that presents strong antitumoral efficacy in both hematological and epithelial malignancies [1, 2]. However, its use is limited because of its high degradability and weak solubility. Indeed, the structure of camptothecin presents a lactone ring that is subjected to hydrolysis in physiological conditions with consequent inactivation of the topoisomerase binding activity. In the present study a new formulation of CPT embedded in cyclodextrin-based nanosponges (CN-CPT) [3] has been evaluated in vitro on prostate cancer models. CN-CPT manifested a strong antitumoral efficacy revealed by MTT and colony forming assay on hormonal independent PC3 and DU145 cells, significantly higher as compared to the same dose of free CPT. Moreover, cell cycle analysis and annexin V/PI staining showed that CN-CPT treatments induced significant apoptotic cell death at 1 nM (61.7% apoptotic cell death; p<<0.001), where free CTP (4.36% apoptotic cell death) was not effective. In order to evaluate the induction of DNA damage, the phosphorylation status of histone H2A.X (p-H2A.X) has been evaluated. Results indicated that DNA damage was caused by CN-CPT only in both DU145 and PC-3 cells. To evaluate possible antimetastatic activity of CPT, in vitro experiments of tumor cell adhesion to HUVECs as a model of endothelium and cell motility were performed. Results showed that CN-CPT was effective at lower doses compared to CPT in inducing inhibition of adhesion and motility. In order to evaluate whether androgen receptor signaling could affect the responsiveness to either CPT or CN-CPT the AR expressing murine models (MYC-AD, PTEN8 and PTEN CAP8) and human model (LNCaP) of prostate cancer have been used. Results showed that expression of functional AR diminished the efficacy of both CPT and CN-CPT. However the latter still exerted greater effects compared to CN-CPT. Taken together these results support the use of CPT and the development of nanotechnologies for drug delivery in the treatment of castrate refractory prostate cancer.
1) R.P. Hertzberg, M.J. Caranfa and S.M. Hecht, On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme–DNA complex, Biochemistry 28(1989), pp. 4629–4638.
2) C.H. Takimoto, J. Wright and S.G. Arbuck, Clinical applications of the camptothecins, Biochim. Biophys. Acta 1400(1998), pp. 107–119.
3) S. Swaminathan, L. Pastero, L. Serpe, F. Trotta, P. Vavia, D. Aquilano, M. Trotta, G. Zara and R. Cavalli, Cyclodextrin-based nanosponges encapsulating camptothecin: physicochemical characterization, stability and cytotoxicity, Eur J Pharm Biopharm. 74 (2010), pp. 193-201.
Camptothecin (CPT) is a potent DNA Topoisomerase I inhibitor that presents strong antitumoral efficacy in both hematological and epithelial malignancies [1, 2]. However, its use is limited because of its high degradability and weak solubility. Indeed, the structure of camptothecin presents a lactone ring that is subjected to hydrolysis in physiological conditions with consequent inactivation of the topoisomerase binding activity. In the present study a new formulation of CPT embedded in cyclodextrin-based nanosponges (CN-CPT) [3] has been evaluated in vitro on prostate cancer models. CN-CPT manifested a strong antitumoral efficacy revealed by MTT and colony forming assay on hormonal independent PC3 and DU145 cells, significantly higher as compared to the same dose of free CPT. Moreover, cell cycle analysis and annexin V/PI staining showed that CN-CPT treatments induced significant apoptotic cell death at 1 nM (61.7% apoptotic cell death; p<<0.001), where free CTP (4.36% apoptotic cell death) was not effective. In order to evaluate the induction of DNA damage, the phosphorylation status of histone H2A.X (p-H2A.X) has been evaluated. Results indicated that DNA damage was caused by CN-CPT only in both DU145 and PC-3 cells. To evaluate possible antimetastatic activity of CPT, in vitro experiments of tumor cell adhesion to HUVECs as a model of endothelium and cell motility were performed. Results showed that CN-CPT was effective at lower doses compared to CPT in inducing inhibition of adhesion and motility. In order to evaluate whether androgen receptor signaling could affect the responsiveness to either CPT or CN-CPT the AR expressing murine models (MYC-AD, PTEN8 and PTEN CAP8) and human model (LNCaP) of prostate cancer have been used. Results showed that expression of functional AR diminished the efficacy of both CPT and CN-CPT. However the latter still exerted greater effects compared to CN-CPT. Taken together these results support the use of CPT and the development of nanotechnologies for drug delivery in the treatment of castrate refractory prostate cancer.
1) R.P. Hertzberg, M.J. Caranfa and S.M. Hecht, On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme–DNA complex, Biochemistry 28(1989), pp. 4629–4638.
2) C.H. Takimoto, J. Wright and S.G. Arbuck, Clinical applications of the camptothecins, Biochim. Biophys. Acta 1400(1998), pp. 107–119.
3) S. Swaminathan, L. Pastero, L. Serpe, F. Trotta, P. Vavia, D. Aquilano, M. Trotta, G. Zara and R. Cavalli, Cyclodextrin-based nanosponges encapsulating camptothecin: physicochemical characterization, stability and cytotoxicity, Eur J Pharm Biopharm. 74 (2010), pp. 193-201.