Evaluation of the Anti-Liver Cancer Activity of Protein Fractions Isolated from Adenium obesum Leaf Extract
Ashkan Hajinourmohammadi, Jamil Zargan, Hanieh Jafary, Firooz Ebrahimi
Abstract
Introduction: Liver cancer is the third leading cause of cancer-related death. Plantderived therapeutics have played a significant role in preventing and treating many diseases, including cancers. The present study investigated the anticancer properties of protein fractions from the green leaf extract of Adenium obesum (A. obesum) in the laboratory. Methods: Protein fractions of leaf extract were separated using reversed-phase high-performance liquid chromatography (RP-HPLC). The cytotoxicity of protein fractions was studied by MTT and sulforhodamine B assays. The apoptotic cell death was examined using the alkaline comet assay, and redox-related indicators were assessed using the catalase enzyme activity assay, glutathione content, and nitric oxide release. The RBC hemagglutination test investigated the possible presence of ribosome-inactivating proteins (RIPs) in the most toxic protein fraction, and the LD50 of the protein fraction with the highest anticancer effects was determined. The amino acid sequence of fraction proteins was determined by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method. Results: The results showed that protein fraction 8 had the highest toxicity in the HepG2 cell line, with an IC50 of 0.16 μg/mL. This fraction induced hemagglutination in red blood cells at concentrations higher than 65 μg/mL. The apoptosis was induced in the HepG2 cells following treatment with the concentrations of 0.08, 0.16, 0.32, and 0.64 μg/mL. Moreover, the redox potential of the treated cells was changed after treatment. The in vivo cytotoxicity investigation of this fraction in mice showed that it is not toxic for animals in concentrations up to 800 μg/kg, indicating its safety potential for pharmaceutical applications. The protein extract in the aforementioned fraction contained two proteins (22 and 53 kD) as determined by electrophoresis and sequencing methods. Conclusion: The findings of this investigation demonstrated that the protein content of fraction 8 derived from A. obesum leaf extract possesses anticancer activity in the HepG2 cell line. The two isolated proteins from this fraction are novel and have been reported for the first time. Further investigations should be performed to evaluate the treatment potential in in vitro/vivo conditions.