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01.12.2017 | Letter to the Editor | Ausgabe 1/2017 Open Access

Journal of Hematology & Oncology 1/2017

A novel melittin nano-liposome exerted excellent anti-hepatocellular carcinoma efficacy with better biological safety

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2017
Autoren:
Jie Mao, Shujun Liu, Min Ai, Zhuo Wang, Duowei Wang, Xianjing Li, Kaiyong Hu, Xinghua Gao, Yong Yang
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-017-0442-y) contains supplementary material, which is available to authorized users.
Abbreviations
HCC
Hepatocellular carcinoma
PD
Progressive disease
PR
Partial remission
SD
Stable disease

Letter to the editor

Melittin is the main effective component of bee venom and has extensive biological functions in vivo, including anti-cancer property. To evaluate the anti-cancer activity of melittin on hepatocellular carcinoma (HCC), a clinical trial containing 40 HCC patients was conducted in Yancheng Second People’s Hospital (Yancheng, China). Patients with melittin treatment showed partial remission (PR) ( n = 4, 10%), stable disease (SD) ( n = 24, 60%), and progressive disease (PD) ( n = 12, 30%), and the disease control rate (CR + PR + SD) was 70%. Toxicity was also assessable in the 40 patients. The most common adverse events were pain at the administration site and skin itch, which disappeared after melittin withdrawal (32 grade 0, 6 grade I, and 2 grade II). This results together with other preclinical studies of melittin indicated that it exerted a significant anti-HCC activity, while serious side effects have restricted the clinical application of melittin in cancer therapy [ 14]. To resolve these problems, melittin was modified with 2% poloxamer 188 and melittin nano-liposomes were prepared (Patent number: CN 101391098 A).
The anti-tumor activity of melittin nano-liposomes was investigated both in vitro and in vivo models (Additional file 1: Materials and methods). In the current study, we found that five hepatic carcinoma cell lines used (Bel-7402, BMMC-7721, HepG2, LM-3, and Hepa 1-6 cells) were sensitive to melittin nano-liposomes, and the IC 50 value was close to melittin, ranging from 1.44 to 2.1 μM (Additional file 2: Table S1). Melittin and melittin nano-liposomes could remarkably induce cell apoptosis compared with vehicle or blank liposomes (Fig.  1a and Additional file 3: Fig. S1). Western blot analysis showed that melittin nano-liposomes (2 μM) increased the expression level of pro-apoptotic proteins, such as Bax and cleaved caspase-3, and decreased anti-apoptotic proteins, including Bcl-2 and PARP, in HepG2 cells compared with the vehicle or blank liposome group (Fig.  1b). The apoptosis process could be partly reversed by the caspase inhibitor Z-VAD-FMK (Fig.  1c). To assess the apoptosis induced by melittin nano-liposomes in vivo, a liver orthotopic xenograft tumor model of Hepa 1-6 cell was established. TUNEL assay revealed that tumor tissues of the melittin nano-liposome (2 mg/kg) group owned a larger proportion of apoptotic cells than the control or blank liposome groups, and the expression levels of apoptosis-regulated proteins in tumor tissue were also detected (Fig.  1df). Melittin nano-liposomes also showed significant inhibition of hepatocellular carcinoma growth in two nude mouse models, including the HepG2 cell subcutaneous xenograft model and LM-3-GFP cell orthotopic xenograft model (Fig.  1g and Additional file 4: Fig. S2).
To evaluate the toxicity of melittin and melittin nano-liposomes, we primarily compared the injury severity of the mouse tails in the LM-3 model where the drugs were administered. The tails of the melittin group showed severe tissue swelling and necrosis while the tails of the melittin nano-liposome group were injured less, even at a high dose of 8 mg/kg (Fig.  2a). To further compare the biological safety of melittin and melittin nano-liposomes, the drugs were intraperitoneally administered to ICR mice for 2 weeks. TUNEL assay revealed that melittin caused slight apoptosis in hepatocytes while melittin nano-liposomes showed lower toxicity to the liver tissue (Fig.  2b). Meanwhile, melittin caused a decrease of lymphocyte percentage and an increase of neutrophils in both peripheral blood and spleen, suggesting that melittin induced an inflammatory response in vivo; however, melittin nano-liposomes showed similar lymphocyte and neutrophil percentages as the control groups. Melittin induced an allergic reaction with significantly increased eosinophils and eosinophil percentage in blood, while melittin nano-liposomes effectively prevented anaphylaxis in the mice. Liposomes and melittin nano-liposomes also caused an increase of splenic B lymphocytes. (Fig.  2c and Additional file 6: Fig. S3).
In summary, our results revealed that melittin significantly delayed HCC development with certain side effects in clinic trial. Novel melittin nano-liposomes showed outstanding anti-HCC potency in vitro and in vivo with a decreased toxicity. The results potentially have clinical implications for melittin nano-liposomes as a promising new drug for HCC therapy.

Acknowledgements

Not applicable.

Fundings

This work was supported by the National Science Foundation of China (Nos. 81673468, 91529304, 81473230, and 81403020), “Major Drug Discovery” science and technology major projects of China (No. 2011ZX09102-001-20), the 111 Project (No. 111-2-07), and Foundation of Nanjing University of Chinese Medicine (13XZR19).

Availability of data and materials

Due to our internal policy, raw data cannot be shared.

Authors’ contributions

LSJ, MJ, and AM initiated and designed the in vitro and in vivo studies. WDW and HKY performed the preclinical animal studies. LXJ performed the in vitro experiments. FJH, GXH, and YY conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

All animals were obtained from SLRC Laboratory Animal Co. Ltd. (Shanghai, China). Mice were bred, treated, and maintained under specific pathogen-free conditions at 24 ± 1 °C and 55 ± 5% humidity in a barrier facility with 12-h light-dark cycles. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of center for new drug evaluation and research, China Pharmaceutical University (Nanjing, China).

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