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Cell-penetrating peptide conjugates of gambogic acid enhance the antitumor effect on human bladder cancer EJ cells through ROS-mediated apoptosis

Authors Lyu L, Huang LQ, Huang T, Xiang W, Yuan JD, Zhang CH

Received 7 January 2018

Accepted for publication 17 February 2018

Published 5 April 2018 Volume 2018:12 Pages 743—756


Checked for plagiarism Yes

Review by Single-blind

Peer reviewer comments 3

Editor who approved publication: Dr Anastasios Lymperopoulos

Lei Lyu,1,* Lu-qi Huang,2,* Tao Huang,1 Wei Xiang,1 Jing-dong Yuan,1 Chuan-hua Zhang1

1Department of Urology, Wuhan No 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China; 2Department of Neurology, Wuhan No 1 Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China

*These authors contributed equally to this work

Background: Gambogic acid (GA) is the main active ingredient of resin gamboges and possesses anti-cancer activity toward various human cancer cells. However, clinical application of GA has been limited by its poor aqueous solubility and dose-limiting toxicities. Cell-penetrating peptides (CPPs) are widely used to deliver anti-cancer drugs into cancer cells and to enhance the water solubility of drugs.
Purpose: The object of this study was to synthesize peptide-drug conjugates in which the cell-penetrating peptide TAT (trans-activator of transcription) was conjugated to GA and evaluated the anti-cancer activity of this GA-CPP conjugate (GA-TAT) in EJ bladder cancer cells.
Methods: GA is built onto the TAT, and the GA-TAT conjugates are cleaved from the solid support and purified via HPLC. The equilibrium solubility of GA-TAT was measured using the shake-flask method. The effects of GA-TAT on EJ cell viability and proliferation were determined by MTT assay, Edu assay and colony formation assay, respectively. After treated with 1.0 μM GA-TAT for 24 h, the apoptosis rate of EJ cells were detected by Acridine orange/ethidium bromide (AO/EB) assay and flow cytometry assay. The proteins of caspase-3 (processing), caspase-9 (processing), Bcl-2 and Bax were analyzed by Western blotting, and the intracellular reactive oxygen species (ROS) production was evaluated by a reactive oxygen species assay.
Results: In contrast to free GA, the solubility of GA-TAT in water was significantly improved. Meanwhile, GA-TAT significantly increased EJ cellular uptake, toxicity and apoptosis. Mechanistic analysis revealed that GA-TAT enhanced the anti-cancer effect of GA against EJ cells through ROS-mediated apoptosis. The results were demonstrated that GA-TAT increased the ROS level in EJ cells, and N-acetyl-l-cysteine (NAC; a well-known ROS scavenger) inhibited GA-TAT-induced ROS generation and apoptosis. Additionally, GA-TAT activated caspase-3 and caspase-9 and down-regulated the Bcl-2/Bax ratio, but these effects were largely rescued by NAC.
Conclusion: GA-TAT has outstanding potential for promoting tumor apoptosis and exhibits promise for use in bladder cancer therapy.

Keywords: gambogic acid, cell-penetrating peptides, apoptosis, bladder cancer

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