The Characteristics Of RLj-RGD3 Toxin On Hep2 Cells Both In Vivo And In Vitro

rLj-RGD3 is a recombinant protein derived from the buccal gland of lampreys.Previous studies have confirmed that rLj-RGD3 has strong activities on anti-angiogenesis,anti-thrombosis and anti-tumor.Furthermore,the purification process of rLj-RGD3 has been established in the previous study.The objective of this study is to investigate the biological characteristics of rLj-RGD3 toxin on human laryngeal squamous carcinoma Hep2 cells both in vivo and in vitro and the molecular mechanisms which account for this process would also be clarified.MTT assay was firstly used to shown the inhibition of rLj-RGD3 on Hep2 cells’ proliferation,and its half maximal concentration was measured as 1.23 μM.Morphological observation,DNA Ladder,flow cytometry,TUNEL and Hochest 33258 staining assays showed that rLj-RGD3 could induce Hep2 cell apoptosis in vitro,and the number of apoptotic cells increased in a dose-dependent manner.Subsequently,the inhibitory effects of rLj-RGD3 on the migration and invasion of Hep2 cells in vitro was due to its abilities on cytoskeleton distraction,confirmed via wound healing,cytoskeleton observation,transwell migration and invasion assyas.Finally,western blot assay showed that the content of activated caspase-9,3and 7,as well as the inactivated PARP was significantly up-regulated in the rLj-RGD3 administration group;while the content of activated caspase-8 and Bcl-2 didn’t show any differences.Furthermore,rLj-RGD3 significantly reduced the levels of p-Akt,p-FAK,p-PLC and paxillin in the rLj-RGD3 treated group,and the level of p-p38 were significantly increased.This meant that rLj-RGD3 might inhibit Hep 2 cells throught endogenous pathways and integrin-related signaling pathways.To figure out the antitumor activity of rLj-RGD3 in vivo,xenograft models of BALB/c nude mice were established in this study.And rLj-RGD3 showed a significant inhibitory effect on Hep2 xenograft tumor without inducing toxicity.In summary,rLj-RGD3 showed a significant anti-tumor activity on Hep2 cells both in vivo and in vitro studies.Therefore,rLj-RGD3 is expected to become a novel anti-tumor drug.Good results would be achieved both in the early treatment and postoperative metastasis prevention.