Purification of syncytia -producing peptide as a potential therapeutic agent for cancer and viral infection


 Virus-induced syncytium formation, or cell fusion, has been investigated as a potential therapeutic approach for cancer. HVJ virus infection or transfection of membrane glycoproteins of virions have been known to cause syncytium formation in affected cells. Also, it was known that other enveloped viruses such as Herpes viruses, leukaemia viruses induce cell fusion. However, substances or molecules that directly cause syncytium formation have not been identified to date. Here, we identify a peptide that efficiently induces syncytia and report its structure, as an actionable therapeutic agent for cancer and viral infection. We purified and identified the fusion factor from the exosomes of the cells infected with murine leukaemia virus but not producing viruses by column chromatography, mass spectrometry, and amino acid analyses. We confirmed the peptide purified from the cell culture media and synthesized peptides induce syncytia as well as the murine leukaemia viruses, or the membranes or exosomes of MuLV infected cell lines in RFL cells and several cancer cell lines leading to apoptosis. And this peptide suppresses in vivo growth of cancer cells significantly. Furthermore, we found the synthesized peptide can cause fusion of enveloped virions as well as virus infected cells or cancer cell lines. These results nominate the use of this peptide as a potentially promising therapeutic approach for cancer and viral infection through efficient induction of syncytium formation followed apoptosis.

 Kogyo Hospital. LLC, CW2, B1203L, and A549 cell lines were obtained from Cell Bank, RIKEN BioResource Research Centre. (Tokyo, Japan). RM4 cells induced syncytia with RFL cells, but don't fuse each other, the RM4 cells resistant against fusion factor of themselves.

Treatment with RM4 cell's conditioned media and the RM4 plasma membrane.
RFL cells were induced syncytium by cocultivation with the RM4 cells and cell membranes of RM4 cells and exosomes in the culture medium of RM4 cells. We tried to purify the fusion factor from the exosomes produced by RM4 cells 12 .
Purification of a syncytium-inducing peptide and determination of amino acids arrangement from the exosomes produced RM4 cells.
Exosomes derived from RM4 cells were collected by filtration of the RM4-conditioned media through Amicon Ultra-15 filters (Merk Millipore LTD.). The collected exosomes were treated with 70% acetone at room temperature and the supernatant which showed the activity of syncytium formation was concentrated by evaporation. The supernatant was applied on QAE  Sephadex C-25 anion exchanger and SP Sephadex C-25 cation exchanger (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Then the solubilized fraction which has the activity of syncytium formation was applied on Hitachi LaChrom Elite HPLC system Pump L-2130(Hitachi, Co. Ltd, Tokyo, Japan) using X Bridge 18 column (Waters, Ireland) and eluted by 10% Acetonitrile. The fraction which has the activity of syncytia formation was further analysed by mass spectrometry (Shimadzu LC-10A). The LC-MS data were acquired on a liquid chromatography system (Agilent HP1200, Agilent Technologies, Palo Alto, CA) equipped with a C18 column (CAPCELL PAK C18 IF, 2 μm, 50 mm × 2.0 mm ID, Shiseido, Tokyo, Japan) coupled with an electrospray ionization quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent 6520, Agilent Technologies, Palo Alto, CA, USA)31. Solvent A was 5 mM ammonium acetate in water, and solvent B was acetonitrile. The target peptides were eluted at a flow rate of 0.2 mL/min at 40°C with a following gradient program: 0 to 10 min, 10% to 100% solvent B; and 5 min hold at 100% solvent B. The mass spectrometer was operated in both positive and negative mode with the capillary voltage of 3500 V. The nebulizing gas pressure was 30 psi and the dry gas flow rate was 8 L/min at 350°C.
The injection volume was 10 μL. MS/MS spectra to identify the target peptides which were also obtained with QTOF mass spectrometer. Detected range was set from m/z 70 to 3000. Liquid chromatography (Fig.1a) and mass spectrometry (Fig.1b) of the concentrated supernatant of the  solubilized RM4-derived exosomes revealed that certain peptides were enriched in the samples .
We analysed the amino acid sequence of the enriched peptides and identified the peptide sequence as CH3-Pro-Ile-Val-Ser-Gln-Thr-Thr-Ala-Ile-Ala.
The synthetic peptide induces syncytia as well as purified peptide from culture media.
To validate this purified peptide is responsible for syncytium formation, we synthesized the peptide of the same sequence. The synthetic peptide as well as the plasma membrane or the exosomes derived from RM4 cells induced syncytia formation in RFL cells (Fig. 2a). RM4 cells and murine Lewis lung carcinoma (LLC) cells formed syncytia after treatment with this synthesized peptide (Fig. 2b). These results were complicated, because RM4 cells did not form syncytia with RM4 cells itself, cell membranes or exosomes derived from RM4cells. The reason why only purified peptide or, synthesized peptide induced syncytia in RM4 cells and several cancer cells is unknown. There may be exist protective mechanism in the membrane to fuse each other or by exosomes of themselves.

Syncytium formation assay
For the syncytium formation assay, 8x10 4 of RFL or LLC cells per 0.25 ml of the media were plated in each well of 24-well plate (Iwaki 2820-024). After 30min, the media was replaced by  50 µL of the media containing the peptide (1µg/ml). After 16 to 24 hours, the cells were fixed with methanol (Wako, Japan) and Giemsa staining (Muto Chemistry, 15003) was performed according to the manufacturer's instruction.

The peptide induces apoptosis after syncytium formation
It has been shown that viral-induced syncytium formation lead to apoptosis 22 . Thus we investigated if the treatment with the synthetic peptide lead to apoptosis of the treated cells by measuring caspases 23 and annexin V 24 . Caspase 3/4 and annexin V were increased in RFL cells after treatment with the synthetic peptide ( Fig. 2c-e). Induction of apoptosis by the peptides in dose dependent manner were confirmed in RFL cells and RM4 cells (Fig. 2f, g), as well as several cancer cells lines, such as LLC which is a mouse lung cancer cell, CW2 which is a human colon adenocarcinoma cell line, and B1203L which is a human lung squamous cell carcinoma cell line (Fig. 2h-j). The sensitivity of apoptosis followed by syncytium formation by the synthetic peptides were higher in cancer cell lines and RM4, which are viral infected cells, as compared with RFL cells, which are non-viral infected, non-cancer cells (Fig. 2f-j), suggesting existence of therapeutic windows of these synthetic peptides against cancer and viral infection.

The peptide induced fusion in viruses as well as cells
 HVJ viruses, obtained Ishihara Sangyo Kaisya LTD, and RFL cells were observed with electron microscope (JEM-1400Plus; JEOL Ltd, Tokyo, Japan) after treatment by synthesized peptides. Fig.4a shows that HVJ viruses fused each other after treatment with the peptide. In Fig 4b shows the syncytia of RFL cells after treatment of the peptide.

The peptide inhibits tumour growth in vivo
To further evaluate in vivo anti-tumour efficacy of the synthetic peptides, we treated nude mice harbouring subcutaneous tumours of B1203L, a human lung cancer cell line. Tumour growth  was significantly suppressed in the peptide-treated group compared with the vehicle-treated group (Fig. 3a, b) without affecting the body weight of the animals (Fig. 3c). Histological examination of the tumours presented highly cellular tumours in the vehicle-treated group (Fig.   3d) and prominent necrosis in the peptide-treated group (Fig. 3e), suggesting in vivo anti-tumour efficacy of this synthetic peptide.

Treatment of subcutaneous tumour growth by synthesized peptide
The antitumor experiment in mice were performed by HAMRI Co. LTD (Tsukuba, Japan). The that the peptide has not toxicity. The peptide-treated group exhibits necrosis, but vehicle-treated  group does not exhibit necrosis., and there are many live cancer cells. The cancer growth in the nude mice was significantly suppressed in the peptide-treated group.

Discussion
In this study we have identified the substance that induce syncytium formation in several cell