RESUMEN
BACKGROUND: Trichobakin (TBK), a member of type I ribosome-inactivating proteins (RIPs), was first successfully cloned from Trichosanthes sp Bac Kan 8-98 in Vietnam. Previous study has shown that TBK acts as a potential protein synthesis inhibitor; however, the inhibition efficiency and specificity of TBK on cancer cells remain to be fully elucidated. METHODS AND RESULTS: In this work, we employed TBK and TBK conjugated with a part of the amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA), which contains the Ω-loop that primarily interacts with urokinase-type plasminogen activator receptor, and can be a powerful carrier in the drug delivery to cancer cells. Four different human tumor cell lines and BALB/c mice bearing Lewis lung carcinoma cells (LLC) were used to evaluate the role of TBK and ATF-TBK in the inhibition of tumor growth. Here we showed that the obtained ligand fused RIP (ATF-TBK) reduced the growth of four human cancer cell lines in vitro in the uPA receptor level-dependent manner, including the breast adenocarcinoma MDA-MB 231 cells and MCF7 cells, the prostate carcinoma LNCaP cells and the hepatocellular carcinoma HepG2 cells. Furthermore, the conjugate showed anti-tumor activity and prolonged the survival time of tumor-bearing mice. The ATF-TBK also did not cause the death of mice with doses up to 48 mg/kg, and they were not significantly distinct on parameters of hematology and serum biochemistry between the control and experiment groups. CONCLUSIONS: In conclusion, ATF-TBK reduced the growth of four different human tumor cell lines and inhibited lung tumor growth in a mouse model with little side effects. Hence, the ATF-TBK may be a target to consider as an anti-cancer agent for clinical trials.
Asunto(s)
Neoplasias Pulmonares , Neoplasias de la Próstata , Humanos , Masculino , Animales , Ratones , Activador de Plasminógeno de Tipo Uroquinasa , Sistemas de Liberación de Medicamentos , Línea Celular TumoralRESUMEN
Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of rabbit reticulocyte lysate (IC50: 3.5 pM). In this respect, TBK surpasses the well-studied highly homologous RIP-I trichosanthin (IC50: 20-27 pM), therefore creation of recombinant toxins based on it is of great interest. TBK needs to penetrate into cytosol through the cell membrane and specifically bind to α-sarcin/ricin loop of 28S ribosome RNA to perform the function of specific RNA depurination. At the moment, there is no detailed structural-dynamic information in solution about diverse states RIP-I can adopt at different stages on the way to protein synthesis inhibition. In this work, we report a near-complete assignment of 1H, 13C, and 15N TBK (27.3 kDa) resonances and analysis of the secondary structure based on the experimental chemical shifts data. This work will serve as a basis for further investigations of the structure, dynamics and interactions of the TBK with its molecular partners using NMR techniques.
Asunto(s)
N-Glicosil Hidrolasas/química , Resonancia Magnética Nuclear Biomolecular , Proteínas de Plantas/química , Ribosomas/metabolismo , Espectroscopía de Resonancia Magnética con Carbono-13 , Estructura Secundaria de Proteína , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
Recombinant trichobakin (TBK) has been subjected to study its capacity in inhibiting the growth of three lines of human cancer cell - epidermoid carcinoma of the mouth (KB), rabdomyo sarcoma (RD-26) and myelo sarcoma (2/0-Ag-14), it has been shown that TBK has inhibited the cancer cells. Myeloma sarcoma cells were inhibited by TBK at the lowest dose (ID50 2.55 microgram/ml). For RD-26 and KB cell lines, ID50 = 7.27microgram and 10.5 microgram/ml respectively. After 2 year preservation, protein recombinant contains almost the same inhibition activity from its original one