The Studies Of Transport Mechanisms Of Riboflavin Laurate And It’s Effect On Helf Cells Induced By Chemo-or Radio-therapy

Tumor blood vessels provide sufficient oxygen and nutrients for tumor occurrence anddevelopment, targeting tumor angiogenesis can inhibit the tumor growth. Vascularendothelial growth factors (VEGFs) are important regulators of vascular developmentduring angiogenesis, and exert their biological activities by binding to their receptors,such as VEGFR1, VEGFR2, and VEGFR3. VEGFR2is a key receptor for tumorangiogenesis, through affecting vascular permeability, proliferation, survival andmigration of vascular endothelial cells. VEGFR2may be a good therapeutic target, andthe attractiveness of VEGFR2is multifaceted. For instance, VEGFR2is not only highlyexpressed on the surface of tumor-related endothelial cells, also expressed directly ontumor cells. So selective blockade of VEGFR2can inhibit tumor angiogenesis, as wellas accelerate apoptosis of tumor cells, ultimately lead to tumor regression. Importantly,selective inhibitions of VEGFR2do not affect any other RTKs, so they should eliminatetoxicity reactions resulting from off-target receptor inhibitions. BC001, underdevelopment by Shandong Buchang Shenzhou Pharmaceutical Co, Ltd, is a fully humanmonoclone antibody that binds VEGFR2. In this study, we will examine the effect ofBC001on inhibiting VEGF-stimulated endothelial cell proliferation, migration,invasion, tube formation in vitro, and leading to the suppression of angiogenesis andtumor growth in vivo.According to CCK-8assay, we found BC001significantly inhibited VEGF-inducedendothelial cells (ECs) viability, the maximal inhibition ratio was60.75%and theinhibitory effects were linear with concentration-dependent, and the IC50was 13.3296±5.5353μg/ml. The effects of BC001on the HUVECs chemotactic motilitywere measured by wound-healing migration assay and Transwell cell invasion assay,these results indicated BC001could impair the migration and invasion of HUVECs.Because tube formation of endothelial cells is the key process of tumor angiogenesis, socapillary-like structure formation in vitro was examined, and BC001indeed abated tubeformation of endothelial cells. In addition, our study established the model of cancerstem cells (CSCs) transdifferentiation into ECs, and BC001negatively impacted thetransdifferentiation of VEGFR2+CSCs. These data suggested BC001inhibited tumorangiogenesis in vitro.Then, we wanted to illuminate the anti-tumor effect of BC001in vivo, according toproof-of-concept of preclinical studies, so we firstly established the mouse melanomacell line B16F10xenografts model in C57mice to screen the inhibitory properties of theanti-mouse VEGFR2monoclone antibody, and results confirmed that anti-mouseVEGFR2antibody-treated mice had significantly smaller tumors. To investigate theantitumor effect of BC001, we established the human colorectal cancer cell HCT116xenografts in nu/nu mice, the significant antitumor effects were also apparent. Tumortissues were stained with HE or subjected to heat-induced epitope retrieval forsubsequent immunohistochemistry (Ki67, Caspase3, TUNEL, CD31, VEGFR2), therewas no doubt that BC001could lead a shrinkage on tumor angiogenesis and tumorgrowth. Additionally, BC001also reduced BGC823xenografts growth significantly.Taken together, our studies indicate that BC001is a potential inhibitor of tumorangiogenesis and tumor growth. Riboflavin is indispensable and important for normal cellular functions, growth anddevelopment in all aerobic forms of life, participating in a myriad of biochemicalreactions, including carbohydrate, lipid and amino acid metabolism. However, humansare not able to synthesize riboflavin by themselves, so they are vulnerable to developriboflavin deficiency, which may lead to a variety of clinical abnormalities. Riboflavinlaurate (RL) is a new sustained-release and oil preparation of riboflavin, and currentviews merely consider that free riboflavin, is released and transported fromintramuscular injection site to whole body, thereby producing sustained clinical effects,but the definite transport mechanisms of RL are uncertain. And Caco-2cell model hasbeen widely used in vitro model, to study the transport and absorb mechanisms of drugs,so Caco-2cell monolayer was established to demonstrate the transport mechanisms ofthe free RL. According to our results about Papp,%T, EfR, we found RL is able totransport through the Caco-2cell monolayer as the prototype, and the transport is linearwith time and concentration dependent, moreover, it is not only via active transport, alsothrough efflux.RL has been widely used as an army medication for riboflavin deficiency in variousdiseases for decades. Several research groups demonstrated undoubtedly, RL shown thepotential to protect the patients from the oral or gastrointestinal mucositis induced bychemotherapy or radiotherapy in clinic. However, as so little is known about thepreventive effect at the cellular level, we investigated a possible role of RL in reducingchemotherapy or radiotherapy induced toxicities in vitro. We utilized cis-platinum and60Co-γ to establish Chemotherapy, radiotherapy caused cell damage models on Helf cells, illuminating the therapeutic or protective effects of RL. Interestingly, RL indeedameliorated either chemotherapy or radiotherapy-induced toxicities, and the perfecteffect is greater than that of riboflavin.