Transport And Killing Mechanism Of Camptothecin-20(s)-o-glycine Ester-[n-(3’α,12’α-dihydroxy-24’-Carbonyl-5’β-cholan)] On Hepatocellular Carcinoma SMMC-7721 Cells

We tested the killing ability of camptothecin-deoxycholic acid derivates synthesized by ourselves on hepatocellular carcinoma SMMC-7721,breast carcinoma MCF-7,and colorectal carcinoma HCT-116 cells using MTT assays.Results showed that Caxmptothecin-20(s)-O-glycine ester-[N-(3’a,12’α-dihydroxy-24’-carbonyl-5’β-cholan)](A2)exerted a high killing ability on SMMC-7721 cells,but showed a relatively low killing ability on MCF-7 and HCT-116 cells,especially with regard to MCF-7.10-(3’α,12’α-dihydroxy-5’β-cholan-24’-carboxyl)-(20s)-camptothecin(C2)had low killing ability on all the three kinds of cells,especially with regard to SMMC-7721 cells,but 10-O-(3-O-(3’α,12’α-dihydroxy-24’-carbonyl-5’β-cholan)-propyl)-(20S)-camptothecin(D2)had a high killing ability on all the three kinds of cells.So A2 is the only compound that has selective killing ability specifically on SMMC-7721 cells,suggesting that A2 may have potential to be used for the target therapy of liver cancer.We detected the morphological change of SMMC-7721 cells treated with A2 using a laser scanning confocal microscope after the cells were dyed by acridine orange solute(AO).Results showed that apparent apoptotic morphology occurred in the cells treated with A2.We also found that the number of apoptotic cells increased depends on the increased concentration of A2 by using a flow cytometry detecting cells dyed by Annexin V-FITC/PI.In order to realize the transport mechanism of A2 into SMMC-7721 cells,we performed the qRT-PCR assays to detect the mRNA expression level alteration of organic-anion transporting polypeptides(OATPs)after cells were treated with A2.Results showed that A2 upregulated OATP1B3 selectively,indicating that A2 interacted with OATP1B3 closely and OATP1B3 may be involved in the transport of A2.We then found that A2 transported into SMMC-7721 cells mediated by the cooperation of active transport,simple diffusion and facilitated diffusion by the uptake assays.Subsequent uptake assays indicated that the transport methods of A2 and deoxycholic acids were similar,and confirmed that OATP1B3 involved in the transport of A2.The cell cycle of SMMC-7721 cells was arrested at S phase and G2/M phase by A2 treatment,indicating that A2 prevented the proliferation of SMMC-7721 cells.Western Blot assays showed that A2 increased the expression level of bak significantly.Procaspase9 and procaspase3 were reduced by A2 slightly.The precursor protein of AIF was increased,and the 57KD protein of AIF occurred,indicating AIF was released into cytoplasm.All the expression alteration of the apoptotic factors demonstrated that A2 induced SMMC-7721 cells apoptosis via mitochondria pathway.