Effects Of Integrin Receptor Mediated Bcl-2 Antisense Oligodeoxynucleotides On Colon Carcinoma Cells

Objective:According to the transmenbrance receptor of colon carcinoma, the study was to evaluate targeting delivery and inhibitory proliferation effects of bcl-2 antisense oligodeoxynucleotide (ASODN) mediated by integrin ligand-polyethyleneimine (RGD-PEI) on human colon carcinoma cell line caco-2 . RGD-PEI-ASODN combined with arsenic trioxide (AS₂O₃) or hydroxy icamptothecine(HCPT) to treat caco-2 cell, which increased sensitivity of AS₂O₃ or HCPT for colon carcinoma.Methods:1. Mixing bcl-2 ASODN with RGD-PEI got RGD-PEI-ASODN. Colon carcinoma cell line caco-2 and liver cell line LO-2 were cultured with fluorescence-labeled RGD-PEI-ASODN and ASODN. The mean intracellular fluorescence intensity of cells and uptake rates of bcl-2 antisense oligodeoxynucleotide were measured by flow cytometry, and were observed under phase-contrast fluorescence microscopy.2. Inhibitory effects of RGD-PEI-ASODN of various concentrations and time points on proliferation of caco-2 cell were inspected by trypan blue dye test. Cultured with RGD-PEI-ASODN for 48 hours, hypodiploid percentage of caco-2 cell was determined by flow cytometry and Apoptotic morphology of caco-2 cell was observed under fluorescence microscopy.3. Combined with RGD-PEI-ASODN, arsenic trioxide or hydroxy icamptothecine treated for colon carcinoma cell line caco-2. Inhibitory effects of cell were determined byadvanced MTT method, and IC50 was calculated. Result:1. The uptake rate and intracellular mean fluorescence intensity of RGD-PEI-ASODN cultured cells were higher than those of ASODN cultures cells. Cultured with RGD-PEI-ASODN, the uptake rate and intracellular mean fluorescence intensity of caco-2 cell were significantly higher than those of LO-2 cell. Observed by phase-contrast fluorescence microscope, RGD-PEI-ASODN entered caco-2 cell effectively, but can not entered control group cells significantly.2. Proliferation of caco-2 cell decreased significantly after treatment with RGD-PEI-ASODN compared with control groups (P<0.05) and showed dose-dependent and time-dependent inhibitory effects. After incubation with RGD-PEI-ASODN for 48 hours, hypodiploid percentage of caco-2 cell was higher than that of the control group. Stained with Heochst33258, many caco-2 cells exhibited apoptotic cell morphology such as cell shrinkage, nuclear condensation and nuclear fragmentation under fluorescence microscope.3 The proliferation of caco-2 cell IC50 of AS2O3 was 1.18umol/L. Combined with RGD-PEI-ASODN, the proliferation of caco-2 cell IC50 of As2O3 was 0.114ujnol/L. 0.1 umol/L. 0.25 nmol/L and 0.5 umol/L As2O3and 0.75 umoVL RGD-PEI-ASODN both treated caco-2 cell, which had cooperativity effects. The proliferation of caco-2 cell IC50 of hydroxy icamptothecine was 0.133ug/mL. Combined with RGD-PEI-ASODN, the proliferation of caco-2 cell IC50 of hydroxy icamptothecine was 0.071ug/mL. 0.08 ng/mL ^0 0.1 ug/mL hydroxy icamptothecine and 0.75 uinol/L RGD-PEI-ASODN both treated caco-2 cell, which had cooperativity effects.Conclusion:1. RGD-PEI-bcl-2 ASODN has higher targeting delivery effect on caco-2 cell.2. RGD-PEI-ASODN has significant inhibitory effect on proliferation of caco-2 cell.3. RGD-PEI-ASODN can increase the sensitivity of AS2O3 treatment for colon carcinoma.