The Interaction Between Five Bisbenzylisoquinoline Alkaloids And BCRP And Their Bioactivation Mediated By CYP3A5

Bisbenzylisoquinoline alkaloids are a large and complicated family of phytochemicals, and they are found in hundreds of plant species around world. Many herbs containing alkaloids have been used as traditional medcines in China and other countries. Usually, these chemicals contain two benzylisoquinoline units linked by one or more carbon-carbon bridges or ether bridges. And the substituent groups on the para position of aromatic rings are commonly hydroxyl or methoxyl moieties.Neferine, liensinine, isoliensinine, dauricine and tetrandrine are all bisbenzylisoquinoline alkaloids and their chemical structures are very similar. These five alkaloids have been reported to show multiple pharmacological activities. Because of the promising pharmacological effects, these five alkaloids have received more and more attention for investigating their potential of clinic use, therefore understanding of their intracorporal process and potential toxicity will have significant meanings in guiding clinical application.Transporter proteins play important roles in drug absorption, distribution and elimination. In this study, a transgenic cell model LLC-PK1/BCRP was established using LLC-PK1as parent cell to evaluate the permeability of these alkaloids and to study the interaction between BCRP and them. The results will provide a clue of the potential role that BCRP may play in the absorption and elimination of these alkaloids. On the other hand, to reveal the role that CYP3A5may act in the bioactivation of these five alkaloids and to further confirm if the pulmonary toxicity caused by dauricine and tetrandrine are responsible for the bioactivation produced by CYP3A5, a WI-38/3A5cell line was established. The compare has been made between WI-38/3A5cell line and the control cell line (WI-38/Vector) to find whether the elevation of CYP3A5expression level lead to more severe cytotoxicity to WI-38cell induceded by these alkaloids.1. Establishment of BCRP expressed pig kidney cell model LLC-PK1/BCRP and its biological profileTo establish a pig kidney cell model LLC-PK1/BCRP in which human breast cancer resistance protein was highly expressed, the expression vector pcDNA3.1(+)-BCRP which contained BCRP gene was constructed and transfected into LLC-PK1cells via liposomes. After selecting with G418, population doubling time, flow cytometry and western blot analysis were used to evaluate the cell model. MTT assays were employed to determine the drug resistance index of mitoxantrone and doxorubicin. Invert fluorescent microscope was used to observe the efflux of fluorescence dye Hoechst33342by BCRP, further more, the BCRP’s inhibitor GF120918was applied to reverse the efflux of Hoechst33342. The experiment results showed that the expression of BCRP protein increased in LLC-PK1/BCRP cell. The population doubling time of LLC-PK1/BCRP cell was a little longer than the parental cell LLC-PK1. The resistance index to mitoxantrone and doxorubicin were51.92and6.09times higher than LLC-PK1cell respectively. The efflux of Hoechst33342was significantly enhanced and could be reversed by GF120918. So we established a LLC-PK1/BCRP cell model successfully. This cell model could be a valuable tool to further investigate the biological profile of BCRP and select the substrate and inhibitor of BCRP.2. The interaction between Human BCRP and Five Bisbenzylisoquinoline AlkaloidsBCRP is one of the key factors to drug absorption, distribution and elimination. Bisbenzylisoquinoline alkaloids are a large family of natural phytochemicals with great potential for clinical use. In this study, the interaction between BCRP and five bisbenzylisoquinoline alkaloids (neferine, isoliensinine, liensinine, dauricine and tetrandrine) were evaluated using LLC-PK1/BCRP cell model.The intracellular accumulation and bi-directional transport studies were conducted, and then molecular docking analysis was carried out employing a homology model of BCRP. Our study revealed that the permeability of these five alkaloids was not high, the Papp values were not great. Liensinine and dauricine were substrates of BCRP, at lower concentration (10μM), the net efflux ratios were2.87and1.64respectively. And their cellular accumulation was lower in LLC-PK1/BCRP cells than in LLC-PK1cells. On the other hand, tetrandrine, isoliensinine and neferine were not substrates of BCRP. On the basis of docking studies, a direct hydrogen bond was formed between liensinine and arginine482which is a hot spot of BCRP for substrate specificity; and dauricine had hydrophobic interaction with BCRP. In conclusion, our study indicated that BCRP could mediate the excretion of liensinine and dauricine, thus influence their pharmacological activity and disposition.3. The potential role that CYP3A5played in the bioactivation of five bisbenzylisoquinoline alkaloidsThe early studies indicated that neferine, liensinine, isoliensinine, dauricine and tetrandrine had relative high distribution in animal’s lung; and dauricine and tetrandrine can cause serious pulmonary toxicity after one single toxic dose intraperitoneally. Although the pulmonary toxicity caused by neferin, liensinine and isoliensinine has not been reported, because the dosages used in these study were low. It was reported that CYP3A5was responsible for the bioactivation of dauricine and tetrandrine resulting severe pulmonary toxicity, to further confirm this hypothesis and to explore the potential role that CYP3A5may play in the bioactivation of neferine, liensinine and isoliensinine whose chemical structures are very similar to dauricine and tetrandrine’s, a transgenic cell model WI-38/3A5was established. Through comparing the cytotoxicity produced by thses five alkaloids, we found the increasing of CYP3A5’s expression level resulting a lower cell survival at the same concentration detected by MTT or MTS assays. Because tetrandrine was the most toxic compound among these five alkaloids, we focused on it and conducted further test. The result indicated that WI-38/3A5cell had a higher intracellular ROS level and a lower GSH level compared with WI-38/Vector cell after treating with tetrandrine of the same concentration, and WI-38/3A5cell had more severe cell apoptosis. All the facts indicated that the bioactivation of tetrandrine was truly mediated by CYP3A5, and CYP3A5was responsible for the pulmanory toxicity induced by tetrandrine. Because neferine, liensinine and isoliensinine contained the same chemical group as dauricine and tetrandrine, we assumed that CYP3A5could mediated the bioactivation of these three alkaloids too.