Beyond the Abstract - Treatment of prostate carcinoma with (Galectin-3)-targeted HPMA copolymer-(G3-C12)-5-Fluorouracil conjugates, by Yuan Huang, et al

BERKELEY, CA (UroToday.com) - Prostate carcinoma is one of the leading causes of cancer-related deaths, mainly owing to its metastasis to distant tissues such as the bone, lung and liver.

140776501xChemo- and radiation therapies are largely ineffective against advanced prostate cancer. To achieve a high degree of specificity to the target tissue, the targeted antigen or receptor should be expressed at a sufficiently high density on the surface of the target cells. Increased expression of membrane-bound galectin-3 (Gal-3) by prostate carcinoma has repeatedly been observed to correlate with more poorly differentiated and increased metastatic potential. G3-C12 peptide, identified by using combinatorial phage display techniques, was found to specifically bind to the carbohydrate-recognition domain (CRD) of Gal-3 and distributed preferably in PC-3 tumor than in normal organs. Based on the enhanced permeability and retention (EPR) effect, N-(2-hydroxypropyl) methacrylamide (HPMA) polymeric carriers conjugated targeting moieties were studied for active accumulation of the polymeric system specifically at the tumor site or even cancer cells through receptor-mediated endocytosis to achieve active targeting with concomitant improvement of therapeutic efficacy.

The aim of this study was to develop a G3-C12-mediated drug delivery system based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers targeting to Gal-3-expressed human PC-3 prostate carcinoma cells. 5-Fluorouracil (5-Fu), an anti-tumor agent, was selected as a model drug. G3-C12, a binding peptide, which specifically binds to the CRD of Gal-3, was attached to HPMA copolymers as a targeting moiety. The non-modified (P-Fu, FITC-P-Fu and pHPMA) and G3-C12-modified HPMA copolymer conjugates (P-(G3-C12)-Fu, FITC-P-(G3-C12)-Fu and P-(G3-C12)) were successfully synthesized and characterized. The G3-C12 contents of P-(G3-C12)-Fu, FITC-P-(G3-C12)-Fu and P-(G3-C12) were 3.0, 2.6 and 2.4 mol%, respectively.

Compared with FITC-P-Fu, FITC-P-(G3-C12)-Fu displayed a superior intracellular internalization in prostate carcinoma PC-3 cells over-expressed Gal-3, which could be observed under confocal laser scanning microscopy and analyzed by flow cytometry. The significant localization of FITC-P-(G3-C12)-Fu on the cell surface suggested its internalization mechanism might be a membrane-associated adsorptive endocytosis. Besides, a significant reduction of cellular uptake of FITC-P-(G3-C12)-Fu was found when co-incubating with free G3-C12, which was attributed to the competitive inhibitory. Accordingly, it was demonstrated that the enhanced cellular uptake of FITC-P-(G3-C12)-Fu was due to the G3-C12 attachment by receptor mediated endocytosis, which was in the line with the results of Gal-3 over-expressed CRC tumor cells.

Following with the enhanced intracellular internalization, the MTT assay showed that PC-3 cells were more resistant to 5-Fu (IC50 = 814.1±151.1 μg/ml) but became more sensitive to HPMA copolymer-drug conjugates. P-(G3-C12)-Fu displayed a higher cytotoxicity (IC50 = 33.94±1.00 μg/ml, p<0.05) than P-Fu (IC50 = 54.12±2.43 μg/ml, p<0.05). Otherwise, the morphological analysis with acridine orange / ethidium bromide (AO/EB) staining and quantitative analysis for cell apoptosis with Annexin V-FITC / Propidium iodide (PI) staining further demonstrated a significantly enhanced apoptosis-induction of P-(G3-C12)-Fu, compared with 5-Fu and P-Fu (p<0.01) .

Intravascular cancer cell adhesion plays a significant role in the metastatic process of prostate carcinoma. Therefore, it was important to assess if the conjugates were able to suppress the migration of PC-3 cells. G3-C12 can efficiently inhibit carbohydrate-mediated and metastasis-associated adhesion of PC-3 cells, which was involved in Gal-3 over-expression. In this study, it was observed that G3-C12-modified conjugates had the strongest migration inhibition possibly due to the inhibition of cell growth activity and the decrease of adhesion.

To evaluate the in vivo behavior of non-modified and G3-C12-modified copolymers, the copolymers were radio-labeled with iodine-131 via the tyrosinamide groups. Subsequently, the 131I-labeled copolymers, named pHPMA and P-(G3-C12), were intravenously administrated to mice bearing PC-3 tumor xenografts. In vivo biodistribution studies were performed with radio-labeled conjugates and 2D scintigraphic imaging, which implied that Gal-3-targeted conjugates could be accumulated in tumor effectively. The attachment of G3-C12 could fasten the delivery of 5-Fu to targeting tumor tissues and increase the tumor accumulation of drug, as well as diminish the clearance of conjugates from targeting tumor tissues. Meanwhile, the significant reduction of P-(G3-C12) in other normal organs within 120 h p.i. suggested its potential to fasten the clearance of conjugates from non-targeted organs. Besides, the higher tumor-to-blood and tumor-to-kidney ratio of P-(G3-C12) over pHPMA further demonstrated that G3-C12 attachment elevated the accumulation in tumor, accelerated the drug clearance from the circulation and even the whole body.

Finally, Gal-3-targeted conjugates displayed a much stronger anti-tumor ability in vivo than 5-Fu and non-targeted ones. P-(G3-C12)-Fu exhibited a superior inhibition of tumor growth (71%, p<0.01), compared to 5-Fu (23%, p<0.05) and P-Fu (37%, p<0.05), and processed the cavernous region and systolic depression in tumors which was attributed to the increased mean density of tumor after P-(G3-C12)-Fu treatment.

Consequently, G3-C12, as a targeting moiety, can be utilized to significantly promote the targeted prostate cancer therapy by attaching to the drug delivery system.

Written by:
Yang Yang, Zhou Zhou, Shuang He, Tingting Fan, Yun Jin, Xi Zhu, Chunhui Chen, Zhi-rong Zhang, and Yuan Huang* as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.

Key Laboratory of Drug Targeting and Drug Delivery, Ministry of Education, West China School of Pharmacy, Sichuan University. No. 17, Block 3, Southern Renmin Road, Chengdu 610041, P.R. China
*Corresponding author Email:

Treatment of prostate carcinoma with (Galectin-3)-targeted HPMA copolymer-(G3-C12)-5-Fluorouracil conjugates - Abstract

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