Effects And Mechanisms Of Low Molecular Weight Heparin On The Proliferation And Adhesion In Human Liver Cancer Cells

Objective (1) To detect the effects and molecular mechanisms of low molecular weightheparin on proliferation in vitro human liver cancer cells.(2) To detect the effects oflow molecular weight heparin on cell proliferation after combine with anti-cancer drugcisplatin, assay whether low molecular weight heparin can enhance the sensitivity of theliver cancer cells to anticancer drug, and further explore its mechanisms.(3) To detectwhether low molecular weight heparin can influence the adhesion of liver cancer cellsto vascular endothelial cells, and explore its related mechanisms.Methods (1) Human liver cancer cells (SMMC-7721) were treated with low molecularweight heparin for different time, the effect of LMWH on proliferation was detected byMTT assay. Cultured SMMC-7721cells with different concentrations of LMWHincluding control group, low concentration and high concentration groups for36hours,then detected the cell cycle distributions by flow cytometry with PI simple stain. Theexpressions of Skp2、c-Myc mRNA and proteins were detected by RT-PCR and WesternBlot.(2) Human liver cancer cells(SMMC-7721) were divided into control group,LMWH group, cisplatin group and combination group, Firstly, we detected the cellviability of SMMC-7721cells by MTT after36hours; secondly, the apoptosis rate wasdetected by flow cytometry and AO/EB stain; thirdly, we detected the expression ofcleaved-caspase3by Western Blot; Fourthly, we detected the expressions of Fas、Bcl-2and Bax in order to further explore the apoptotic changes, and analysis the possiblemechanisms.(3) Detected the expressions of selectin ligand-SleX in normal liver cellline (L-02), hepatocellular carcinoma cell line (SMMC-7721and HepG2) by WesternBlot. Detected the expression of E-selectin of human umbilical vein endothelial cells byRT-PCR after stimulated with conditioned medium.(4) SleX high expression cell line-HepG2was chosen as a representative of the liver cancer cell line, and dividedHUVECs into control group (stimulated with conditioned medium only) and LMWHgroups (stimulated with conditioned medium and LMWH). Evaluated the number ofHepG2cells adhered to HUVECs in each group, and detected the expression ofE-selectin in HUVECs of each groups.Results (1) Cell proliferation of LMWH treated groups was inhibited in dose-dependentand time-dependent. Compared with negative control group, the FCM results revealedthat the percent of cells in G0/G1phase increased (P<0.05) and cells in S phasedecreased (P<0.05) in LMWH groups. In addition, the expressions of Skp2and c-MycmRNA and proteins were reduced significantly (P<0.05).(2) MTT results showed that,compared with the control group, growth inhibition was not obvious in low doseLMWH group, but the inhibitory of combination group was much stronger than the cisplatin group. The apoptosis rate of combination group was also much stronger thanthe cisplatin group in both FCM results and AO/EB results. The Western Blot resultsshowed that, the expressions of cleaved-caspase3increased in cisplatin group andcombination group compared with control group, and the combination group was muchhigher. The expressions of Fas also increased in cisplatin group and combination group,but the expressions were quantity. The expressions of Bcl-2and Bax both changedobviously in the combination group.(3) There was almost no expression of SleX inL-02cell lines, and lower expression in SMMC-7721cell lines, but high expression inHepG2cell lines. The expression of E-selectin mRNA in HUVECs increasedsignificantly after stimulated with conditioned medium for5hours.(4)The number ofHepG2cell adhered to CM-stimulated HUVECs decreased obviously in LMWH group,and the expression of E-selectin mRNA in HUVECs decreased too.Conclusions (1) Low molecular weight heparin can inhibit the growth of human livercancer cells, the mechanism may be involed in reducing the expressions of Skp2andc-Myc to arrest cell cycle at G0/G1phase.(2) LMWH can enhance the cell proliferationinhibition of DDP on liver cancer cells and its mechanism may be related to the jointapplication of LMWH can regulate the pathways of apoptosis.(3) LMWH can inhibitthe adhesion between HUVECs and HepG2cells by reducing the expression ofE-selectin in HUVECs.