Targeting And Enhancing Effects Of C-myc Antisense Oligodeoxynucleotides Mediated By Galactose Receptor On Hepatocarcinoma Cells

ObjectiveTo investigate the targeting and enhanced proliferative inhibition effects of c-myc antisense oligodeoxynucleotides mediated by galactose receptor on hepatocarcinoma cells.Methods1 Cell culture: hepatocarcinoma Bel-7402 cell lines, Raji and U937 lympha cell lines werecultured in RPMI-1640 culture medium containing 10 % inactive newborn calf serum, 100 u/mL Penicillin, 100μg/mL Streptomycin, incubated at 37℃, 5 % CO₂ incubator.2 Synthesis of c-myc ASODN, preparation of Gal-PEI-ASODN complex, targeting testsin vitro(1) C-myc ASODN was synthesized by Shanghai Bioengineering Company and modified with phosphorothioate, with or without carboxyfluorescein labeled at 3' terminal, sequence as: 5'-CTT CTC GAG GCA GGA GGG-3'.(2) Preparation of Gal-PEI-ASODN complex: Gal-PEI reagent and c-myc ASODN were dissolved in serum-free RPMI-1640 respectively, mixed in 2:1(V/V), and N:P was 5.0 to obtain Gal-PEI-ASODN complex.(3) Targeting tests in vitro: Bel-7402 cells, U937 cells or Raji cells were incubated with Fluorescence(FAM)-labeled ASODN and Gal-PEI-ASODN for different time. Cell fluorescence uptake rates and mean fluorescence intensity were determined by flow cytometry. The morphology of Gal-PEI-ASODN entering Bel-7402, U937, Raji cells was detected by using phase-contrast fluorescence microscope and cell proliferative inhibitions were determined with trypan blue dye test.3 Detection of cell proliferation, cell cycle, cell necrosis by trypan blue dye test, cell clone test and flow cytometryBel-7402 cells were incubated with Gal-PEI, ASODN, Gal-PEI-ASODN for different time, inhibition of cell proliferative was measured by trypan blue dye test and IC50 wascalculated, cell clones were counted by inverted microscope and the clone forming rates were calculated. The morphology of fluorescence-labeled Gal-PEI-ASODN entering Bel-7402 cells was observed under fluorescence microscope. The change of cell cycle was analyzed with flow cytometry (propidium iodide staining). The percentage of cell apoptosis and necrosis was determined with flow cytometry (Annexin v -FITC and propidium iodide double staining).4 Detection of drug-sensitivity of Bel-7402 cells by improved MTT testBel-7402 cells were incubated for 48 h with different concentrations of drugs in three groups: chemotherapeutic drug group, chemotherapeutic drug combined with Gal-PEI-ASODN group, chemotherapeutic drug combined with ASODN group. Cell proliferation was determined by cell-counting kit, IC50 and the drug-sensitivity were calculated. The combined therapeutic effects (antagonizing, addition and cooperation) were analyzed. These chemotherapeutic drugs were arsenic trioxide, 5-Fu, Pharmorbincin(PHA) respectively.5 Animal experiments(1) Setting up mice transplanted hepatocarcinoma model of Hep A cells:Transplanted hepatocarcinoma mice were killed, the carcinoma cells were collected and adjusted to 1 x 107 cells/mL. 0.2 mL cell suspension was injected into each of the experimental mice intraperitoneally or subcutaneously in the right arm of experimental mice. (2) Treatment of mice transplanted hepatocarcinoma with Gal-PEI-ASODN:Kunming mice were divided into 4 groups: the control group, Gal-PEI group, ASODN group and Gal-PEI-ASODN group and transplanted with hepatocellular carcinoma subcutaneously in the right arm and injected drugs every 48 hours inside the tumor tissues on the 1st, 3rd, 5th, 7th, 9th days after transplantation. On the 13th day, the mice were killed, the weights of subcutaneous tumors were measured, tumor inhibition rates were calculated, and c-MYC protein expression level was examined by SP immunohistochemical method.Results1 Targeting effects of Gal-PEI-ASODN complex on Bel-7402 cell lines(1) Differences of Bel-7402 cell uptake of Gal-PEI-ASODN or ASODN: The uptake rates (88.25 %⁹6.54 %) and the mean fluorescence intensity (38.64 -60.37) of theGal-PEI-ASODN-Bel-7402 cell group were significantly higher than those of ASODN-Bel-7402 cell groups (33.26 %⁵8.33 % and 24.95-38.53 respectively, P <0.01) in the period from 10 min to 60 min.(2) Differences between Bel-7402 cells and U937 cells in Gal-PEI-ASODN uptake: The uptake rates (88.25 %⁹8.66 %) and the mean fluorescence intensity (38.61-111.9) of Gal-PEI-ASODN-Bel-7402 cell group were significantly higher than those of Gal-PEI-ASODN-U937 cell group (11.16 %⁶9.99 % and 8.24-32.62 respectively, P <0.01) in the period from 10 rrin to 4 h.(3) After cells were incubated with fluorescence-labeled Gal-PEI-ASODN and ASODN for 1 h, only a small number of cells in the Gal-PEI-ASODN-raji group, Gal-PEI-ASODN-U937 group and ASODN-Bel-7402 group had assimilated fluorescence-labeled ASODN, while most cells in the Gal-PEI-ASODN-Bel-7402 group had imbibed fluorescence-labeled ASODN observed under phase-contrast fluorescence microscope.(4) After treated with Gal-PEI-ASODN at different concentrations for 48 h, proliferation of Bel-7402 cells was significantly inhibited (PO.01), but no significant inhibition was detected on U937 and Raji cells treated with Gal-PEI-ASODN complexes at the same concentrations (F>0.05).2 Inhibitory effects of Gal-PEI-ASODN complex on proliferation of Bel-7402 cell lines (1) Time effects on cell proliferative inhibition: there were no significant inhibitory effects of Gal-PEI group on Bel-7402 cell proliferation from 0 h to 96 h (P>0.05) compared with the control group, and in ASODN group (20 umol/L), the inhibitory effect could be detected only at 72 h time point (PO.05). However, significant inhibitory effects of Gal-PEI-ASODN group (0.75 umol/L) were detected in every time point (PO.01).(2) Concentration effects on cell proliferative inhibition: the IC5o of ASODN on Bel-7402 cells was determined as 20.9 umol/L, and the IC50 of Gal-PEI-ASODN on Bel-7402 cells was 0.29 umol/L, the inhibitory effect enhanced 72 times. However, no inhibitory effect was detected in Gal-PEI groups at different concentrations (P>0.05).(3) Incubated with Bel-7402 cells for 7 days, Gal-PEI-ASODN inhibited cell clone formation significantly at different concentrations. No significant inhibition was detected in ASODN group and Gal-PEI group compared with the control group (P>0.05).(4) When Bel-7402 cells were incubated with fluorescence-labeled ASODN or Gal-PEI-ASODN for 1 h, only a small number of cells had imbibed ASODN, but a large number of cells had absorbed Gal-PEI-ASODN, and fluorescence-labeled Gal-PEI-ASODN particles distributed on cell membrane surface, or in cytoplasm andnucleus.(5) Cell cycle determination by flow cytometry. In the control group, Gal-PEI group and ASODN group, cell proliferative index was found to be 35.04 %, 33.95 %, 32.90 %, respectively. However, cell proliferative index for Gal-PEI-ASODN group was found to be 23.65 %, decreased by 11.39 % compared to the control group.(6) Necrotic cell percentage determination using AnnexinV-FITC/ PI double staining test. The results showed cell apoptotic rate of 2.77 %, cell necrotic rate of 3.06 % and the normal cell percentage was detected as 94.13 % in the control group; cell apoptotic rate below 4 %, cell necrotic rate below 6.8 % were detected in both ASODN group and Gal-PEI group; cell apoptotic rate of 6.5 %, cell necrotic rate of 15.9 % and the normal cell percentage of 76 % were determined in Gal-PEI-ASODN group.3 Gal-PEI-ASODN enhancing the drug-sensitivity of Bel-7402 cells(1) IC50 was calculated as: 11.09 umol/L for arsenic trioxide group, for the arsenic trioxidecombining Gal-PEI-ASODN group, 3.57 umol/L; for arsenic trioxide combining ASODN group, 10.89 umol/L.(2) IC50 was calculated as: 27.16 ug/mL for the 5-Fu group, for the 5-Fu combining Gal-PEI-ASODN group, 17.15 ug/mL, and for the 5-Fu combining ASODN group, 25.20 ug/mL.(3) IC50 was calculated as: for the PHA group, 3.60 ug/mL; PHA combining Gal-PEI-ASODN group, 1.84 ug/mL; PHA combining ASODN group, 3.57 ug/mL. The combination of As2O3, 5-Fu, PHA with Gal-PEI-ASODN enhanced the chemotherapeutic effect of them on Bel-7402 cells, which was 3.11, 1.58 and 1.96 times as compared with the origins, respectively.4 Inhibitory effects of Gal-PEI-ASODN on the growth of mice hepatocarcinoma(1) Tumor weights (0.478 g ±0.283g) of the Gal-PEI-ASODN group were much lower than that of the control groups (0.800 g ±0.294 g), and the tumor inhibition rate was 40.25 % (P <0.05). The tumor weights (0.631 g ±0.228 g) of the ASODN group were smaller than that of the control group (0.800 g ±0.294 g, ,P>0.05). No difference was tested in tumor weight between the Gal-PEI group and the control group (/*>0.05).(2) In the control group, c-MYC protein was located in cytoplasm and nucleus of hepatocarcinoma cells. The positively stained cells were much more in numbers, stained more intensively, and showed more heterogeneity. In Gal-PEI and ASODN group, the location and intensity of c-MYC expression were the same as the control group. While in the Gal-PEI-ASODN group, c-MYC expression was localized as the same as the above 3groups, but the intensity was weaker, and the positively stained cells were fewer.Conclusions1 There is a targeting effect of Gal-PEI-ASODN on Bel-7402 cells, but no such effects on U937, Raji cells lines are detected.2 There is high inhibitory effect of Gal-PEI-ASODN on Bel-7402 cells' proliferation. Theinhibitory effect of c-myc ASODN on Bel-7402 cell lines increases by 72 folds mediated by galactose receptor. Gal-PEI-ASODN inhibits cell clone formation significantly compared with the control group. While incubated with fluorescence-labeled Gal-PEI-ASODN, fluorescence-labeled particles distribute on cell membrane surface, or in cytoplasm and nucleus. The mechanism by which Gal-PEI-ASODN inhibits cell proliferation maybe that it can restrain the change from Go/Gi period to S period in cell cycle and induce cell necrosis.3 0.25 umol/L Gal-PEI-ASODN can enhance chemotherapeutic drug-sensitivity of Bel-7402cells to AS2O3,5-Fu, PHA. The drug-sensitivity is found to be increased by 3.11, 1.58 and 1.96 times, respectively.4 Gal-PEI-ASODN can inhibit the growth of mice hepatocarcinoma and inhibit the expression of c-MYC protein in hepatocarcinoma cells.