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 [1–4]. 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 IC50 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. 1d–f). 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).

Fig. 1
figure 1

Melittin nano-liposomes induced apoptosis in hepatic carcinoma cells in vitro and vivo and inhibit hepatocellular carcinoma in LM-3 xenograft tumor model. a HepG2 cells were cultured with vehicle, blank liposomes (2 μM), melittin (2 μM), or melittin nano-liposomes (2 μM) for 24 h, stained with annexin V-FITC and PI, and analyzed by flow cytometry. b Western blot analysis of apoptosis-related proteins after HepG2 cells were treated with vehicle, liposomes (2 μM), melittin (2 μM), or melittin nano-liposomes (2 μM) for 24 h. c HepG2 cells were pretreated with or without the caspase inhibitor Z-VAD-FMK for 6 h and then treated with vehicle, liposomes (2 μM), melittin (2 μM), or melittin nano-liposomes (2 μM) for 24 h. Western blot analysis of Bcl-2 and procaspase-3 was carried out subsequently. d TUNEL assay for detecting apoptosis in tumor tissues of the hepatocellular carcinoma Hepa 1-6 orthotopic tumor model. e Western blot analysis of apoptosis-related proteins of Hepa 1-6 tumor tissues. f Live imaging photos and the fluorescence degree of vehicle, blank liposomes (8 mg/kg), melittin (2 mg/kg), melittin nano-liposomes (2, 4, and 8 mg/kg), and sorafenib (30 mg/kg) treated groups in the LM-3 orthotopic implanted tumor model. The data are presented as the mean ± SEM. Statistical significance was calculated using Student’s t test (*p ≤ 0.05)

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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).

Fig. 2
figure 2

Melittin nano-liposomes showed reduced toxicity in vivo. a Photos of the tails of each group at the end point of treatment. These mice were from the LM-3 model. b TUNEL assay for detecting apoptosis in the liver tissue after mice (n = 10) were treated with vehicle, blank liposomes (8 mg/kg), melittin (2 mg/kg), and melittin nano-liposomes (2 mg/kg) for 2 weeks. c Peripheral blood was collected from the ICR mice and analyzed by automated hematology analyzer

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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.