Effects Of PCNA Antisense Oligodeoxy-nucleotides Mediated By Galactose-receptor On Hepatocellular Carcinoma Bel-7402 Cells

Objective:To study the targeting and effectively inhibitory effects of PCNA antisense oligodeoxynucleotides mediated by galactose-receptor on hepatocellular carcinoma Bel-7402 cells.Methods:1. Antisense oligodeoxynucleotides(ASODN) were mixed with galactose-polyethyleneimine (Gal-PEI) to form Gal-PEI-ASODN complexes. The uptake rates and mean fluorescence intensity of FAM labeled Gal-PEI-ASODN or ASODN on Bel-7402, GLC-82 and K562 cell lines were tested by flow cytometry, morphologies of FAM labeled Gal-PEI-ASODN or ASODN entering these cells were observed under phase-contrast fluorescence microscope; the inhibitory effects of Gal-PEI-ASODN on the proliferation of the three kinds of cells: Bel-7402, GLC-82, K562, were tested by trypan blue dye at various concentrations of ASODN or Gal-PEI-ASODN.2. The inhibitory effects on the proliferation of Bel-7402 cells were tested by trypan blue dye at various concentrations of antisense oligodeoxynucleotides or at different time points with the same concentration of antisense oligodeoxynucleotides.Incubated with ASODN or Gal-PEI-ASODN(0.125μmol/L) for 48 h: hypodiploid percentage of Bel-7402 cells was analyzed by flow cytometry; DNA ladder was tested by DNA electrophoresis; stained with Hochest 33258, the morphologies of cell apoptosiswere observed under fluorescence microscope.3. 5-fluorouracil(5-Fu), cis-Diamrninedichloroplatinurn(DDP), hydroxy -icamptothecine (HCPT) were added into Bel-7402 cells respectively, then ASODN or Gal-PEI-ASODN was transfected into the cells; chemosensitivity of Bel-7402 cells was evaluated by improved MTT test(CCK-8 agent), and IC50 was calculated.Results:1. Incubated with FAM labeled Gal-PEI-ASODN or ASODN from 0.5 h to 2 h, the uptake rates and the mean fluorescence intensity of Gal-PEI-ASODN-Bel-7402 cell group were significantly higher than that's of ASODN-Bel-7402 cell group, Gal-PEI-ASODN-GLC-82 and Gal-PEI-ASODN-K562 cell group respectively at every time point. Only in the Gal-PEI-ASODN-Bel-7402 cell group, FAM labeled Gal-PEI-ASODN could be uptaken by Bel-7402 cells effectively, while in the other three groups FAM labeled Gal-PEI-ASODN or ASODN could not enter Bel-7402 cells effectively at the same ASODN concentration observed under phase-contrast fluorescence microscope.When the three kinds of cells were treated with Gal-PEI-ASODN complexes respectively at the same various of concentrations for 48 h, Bel-7402 cells' proliferation was significantly inhibited only.2. Treated with ASODN (from 0.05 umol/L to 0.25 umol/L)for 48h, Bel-7402 cells' proliferation was not significantly inhibited;while the concentration of ASODN was much higher (from 10 umol/L to 40 umol/L), the inhibitory effects on the proliferation of Bel-7402 cells were observed, the IC50 of ASODN on Bel-7402 cells' proliferation is 29.3 umol/L. When incubated with Gal-PEI-ASODN complexes (from 0.05 umol/L to 0.25 umol/L)for 48 h, Bel-7402 cells' proliferation was significantly inhibited at different concentrations, the IC50 of Gal-PEI-ASODN is 0.06 umol/L.Treated with ASODN(0.10 umol/L) or Gal-PEI, the proliferation of Bel-7402 cells was not obviously inhibited at different time point; but with Gal-PEI-ASODN(0.10 umol/L),the cells'growth was effectively inhibited.Incubated with very low concentration^. 125 umol/L) of PCNA antisenseoligodeoxynucleotides mediated by galactose receptor, hypodiploid percentage of Bel-7402 cells increases, and cell shrinkage, chromatin condensation, apoptic bodies, formation of DNA ladder can be observed.3. Combined with ASODN(0.03 umol/L), the IC50 of 5-Fu, DDP, HCPT reduced from 16.15 ug/mL, 1.88 ug/mL and 0.31 ug/mL to 12.59 ug/mL, 1.71 ug/mL and 0.27 ug/mL respectively; combined with Gal-PEI-ASODN(ASODN 0.03 umol/L), the IC50 of 5-Fu, DDP, HCPT reduced further to 0.51 ug/mL, 0.90 ug/mL, and 0.10 ug/mL respectively.Conclusion:1. PCNA antisense oligonucleotides mediated by galactose-receptor have good targeting effect on Bel-7402 cells.2. PCNA antisense oligonucleotides mediated by galactose-receptor can be effectively uptaken by Bel-7402 cells, proliferation of the cells is signifantly inhibited, and the dosage of antisense oligodeoxynucleotides is greatly reduced. With very low concentration(0.125 umol/L) of PCNA antisense oligodeoxynucleotides mediated by galactose-receptor, apoptosis of Bel-7402 cells can be induced.3. When a small quantity of PCNA antisense oligonucleotides(0.03 umol/L) mediated by galactose-receptor is combined with chemotherapeutic drugs for hepatocellular carcinoma, dosage of these drugs is reduced ,and side effect or toxic reaction of them may be reduced too, but the inhibitory effects are enhanced. In a word, the chemosensitivity of Bel-7402 cells can be enhanced by PCNA antisense oligonucleotides mediated by galactose-receptor.