Study On Screening Of Novel Camptothecins Based On Small Intestinal Oligopeptide Transporter PepT1 And In Vitro Antitumor Activity

Camptothecin(CPT)is a terpene indole alkaloid with broad-spectrum ani-tumor activity extracted from a special Chinese tree Camptotheca acuminate.However,the poor water solubility and low stability of camptothecines limit their clinical application.Small intestinal oligopeptide transporter 1(PepTl)is an active transporter widely expressed in human small intestinal epithelial cells,which mediates the small intestinal absorption of dipeptides,tripeptides,and peptidomimetic compounds.In addition,PepTl is less expressed in normal cells and tissues,but is over expressed in some tumor cells.Therefore,the researchers have modified some poorly absorbed drugs into dipeptide or tripeptide-like structures.Mediated by PepTl,to enhance oral absorption and tumor targeting of parent drugs.In this study,we screened a variety of camptothecin compounds based on PepTl and investigated the in vitro antitumor activity of camptothecin analogues against cell lines with high/low expression of PepT1.The main findings were as follows.1.Establish a HEK-293T cell model overexpressing hPepT1:The recombinant plasmid pcMV3-SLC15Al was constructed,and HEK-293T cells were transiently transfected with different concentrations of plasmids selected.The protein expression of hPepTl was observed by Western Blot,and the transport function of hPepTl was investigated by cellular uptake of Gly-Sar,a typical substrate of PepT1,and the optimal transfection concentration of the plasmid was finally determined to be 0.5 μg/well.2.Screening of Camptothecin Compounds Based on PepTl:Using the HEK-293T-hPepTl cell model constructed above,the camptothecin-like compounds were co-incubated with Gly-Sar,and the concentration of Gly-Sar in the cell lysate was detected by LC-MS/MS method,and the compounds with stronger inhibitory effects were screened:HCPT,CPT-Phe CPT-Gly-Val,N-acetyl-CPT-Gly-Val,CPT-Gly-Gly,N-acetyl-CPT-Gly-Gly,t-boc-CPT-Gly-Asp,and the inhibition of Gly-Sar uptake was stronger for the camptothecin compounds without protective groups.3.Screening of PepTl high/low expression cell lines:The expression levels of PepTl in various cell lines were investigated by applying immunofluorescence and Western Blot.The results showed that the expression levels of PepTl were higher in Caco-2,HepG2,and BxPC3,while lower in HL-7702,Hep3B,and MPC2.4.In vitro antitumor activity study of camptothecin compounds:Cell lines with high/low expression of PepTl were selected,and the in vitro effects of seven derivatives HCPT,CPT-Phe,CPT-Gly-Val,N-acetyl-CPT-Gly-Val,CPT-Gly-Gly,N-acetyl-CPT-Gly-Gly,t-boc-CPT-Gly-Asp were investigated by MTT assay using CPT as control.All these compounds showed good antitumor effects.And in HepG2 with high expression of PepTl,the anti-tumor activity in vitro was significantly higher than that of Hep3B with low expression of PepT1.For the camptothecin dipeptide derivatives,the anti-tumor effect was better without the protective groups than that of derivatives containing protective groups.