An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

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Highlights

  • H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication.

  • H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins.

  • Ad-Endo enhanced the cytotoxicity of H101 in NPC cells.

  • Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC.

Abstract

A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

Introduction

Oncolytic adenovirus (Ad) has been demonstrated to lyse selectively cancer cells but not normal cells [1]. E1B55k-deleted Ad dl1520 [2] and H101 [3] are the oncolytic Ad with the most extensive investigation, were firstly applied in clinical trials and H101 has been approved for the clinical application to treat squamous cell carcinoma of the head and neck in China.

The antiangiogenic therapy is another attractive strategy for cancer treatment. Endostatin was previously considered the most potent endogenous angiogenesis inhibitor [4] and was rapidly moved to clinical trials. However, the high instability and shorter half-life made it difficult for clinical application [5]. Promisingly, the antiangiogenic gene therapy can overcome these problems and is likely a potential new approach for the treatment of cancer.

An Ad vector encoding a secreted human endostatin (Ad-Endo) has been confirmed to inhibit tumor growth through antiangiogenesis [6]. The results of preclinical, phase I/II clinical trials suggested that the treatment of solid tumor with Ad-Endo is likely a safe and promising approach [7], [8] (ClinicalTrials.gov identifier, NCT00634595). Even so, it is necessary to find a way for overcoming the problem of limited curative effect due to limited endostatin expression [7], [9], [10].

We presumed that the selective replication of oncolytic Ad would rescue Ad-Endo genome amplification and promote endostatin expression. In this study, we investigated the antitumor effects of the combined treatment of Ad-Endo and H101 on Nasopharyngeal carcinoma (NPC). The results indicate that Ad-Endo and H101 have a synergistic antitumor effect on NPC, which resulted from the promoted antiangiogenic effect of Ad-Endo by H101 and the enhanced oncolysis of H101 by Ad-Endo.

Section snippets

Cells and plasmids

Human NPC CNE-1, CNE-2 cells contain a mutant p53 at codon 280, whereas C666-1 harbors a deletion at codon 249 of p53 and Epstein-Barr virus (EBV) DNA. Human umbilical vein endothelial cells (HUVEC) were cultured in Ham’s F12 nutrient mixture supplemented with endothelial cell growth supplement (BD Biosciences) and 10% FBS. human embryonic kidney 293 cells and CNE-1, CNE-2 cells were cultured in DMEM containing 10% FBS (Invitrogen), and C666-1 cells were cultured in RPMI 1640 with 15% FBS. The

Results and discussion

Ad-Endo has been showed that can inhibit tumor growth through antiangiogenic effects in our previous study [6]. E1B55kD-deficient Ad, such as H101 or Onyx-015, has been confirmed that can selectively lyse cancer cells with abnormal p53 pathway [1], [13]. In addition, late viral RNA export, the cell cycle status of host cells, viral infectivity, and the expression of heat shock proteins may also determine the tumor selectivity of E1B55kD-deficient Ad [13], [14]. In previous studies,

Acknowledgments

This study was supported by National Natural Science Foundation of China (Nos. 81272638, 31170151), National Key Basic Research Program of China (No. 2012CB519003), National High Technology Research and Development Program of China (Nos. 2012AA020803, 2012AA02A204) and Guangzhou Key Program of Science and Technology (No. 2012Y2-00026).

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These authors contributed equally in this paper.

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