Inhibitory Effect Of VEGF Antisense Oligonucleotide On Human Colorectal Cancer Cells Under Hypoxia In Vitro

BackgroundColorectal carcinoma,whose mortality rate is the fourth in all deaths because of cancers,is the third most common cancer in the world.And its incidence rate is also rising at the increase speed of 4%,which is two times as the average level of the world,in our country every year.Although the chemotherapy cooperating with the surgery,the core means to treat colorectal cancers today,can prolong the survival time of patients in a certain extent,it has the defects such as poor drug targeting, drug side effects,et al.Therefore,it is particularly necessary to research and develop new types of anticancer drugs with the guide of target therapy strategy.As the biotechnology was rapidly developed in the field of medicine and the pathogenesis of the tumor was deeply understood from the cellular and molecular level,biotherapy of tumor has entered a new era.Cancer molecular targeted therapy was in the method of using a certain carrier of specificity to deliver drug or other anti-tumor substances to the tumor site.The therapeutic effect or drug itself was restricted to target cells,tissues or organs without affecting normal ones,so the curative effect was enhanced and the side effects were reduced.Target therapy includes three levels,the first one is organ-specific,it only has effect on a certain organ;the second is called cell-targeted,it only affects certain types of tumor cells which are apoptosis when combined with the drugs selectively; the third is molecular-targeted,the drug is combined with a certain kind of protein or a fragment of the nucleotide or a gene product to exert biological effects.Molecular targeted therapy is very important to the treatment of tumor,and it has many advantages such as specificity and effectiveness.Now it is being studied hotly both at home and abroad.Vascular endothelial growth factor(VEGF)is the central focus in areas of tumor angiogenesis,it not only plays an important role in tumor growth and metastasis,but also has some influence on proliferation and apoptosis of the tumor cell itself.Now there are at least five kinds of VEGF hypotypes have been found,namely,VEGF121, VEGF165,VEGF 189,VEGF206 and VEGF 145,which come from the same gene through different mRNA splicing ways.VEGF121 and VEGF 165 are two of the most important isomers in this family.VEGF receptors include VEGFR-1/flt-1, VEGFR-2/KDR/flk and VEGFR-3/flt-4.However,only the first two can specifically bind with VEGF,VEGFR-1 and VEGFR-2 mainly expressed on vascular endothelial cells,but have now also been found on the tumor cell surface.Recent research show that VEGF over-expressed in many kinds of cancer tissue.VEGF and its receptors play a key role in tumor growth and metastasis and VEGF/VEGFR targeted drugs have great advantages compared with the traditional anticancaer drugs,and many of them have entered clinical trials stage or have been applied to clinical.For VEGF/VEGFR targeted therapy,in addition to inhibiting VEGF and VEGFR themselves,we can also block the signal transduction pathway after VEGF and VEGFR have bound together.Small molecule VEGF receptor tyrosine inhibitor (Receptor tyrosine kinase inhibitors,RTKIs) including Sunitinib,Sorafenib, PTK-787,Zactima,AG-013736 and so on.In the deterioration of solid tumors,the extracellular matrix microenvironment plays an important role.Oxygen partial pressure of tumor tissue[p(O2)]is 0-20 mmHg(1 mmHg=0.1133 kPa),but in normal tissue it is higher than 40 mmHg.Hypoxia is a basic characteristic of the tumor micro-environment,one hand, hypoxia induces Na-K pump dysfunction,lysosome rupture,and endonuclease activation,triggers cell necrosis and apoptosis.On the other hand,hypoxia is the strongest stimulus of VEGF expression,it can indruce the angiogenesis and promote tumor cell growth by increasing the expression of VEGF.Antisense technology can downregulate protein expression by inhibiting the transcription process from DNA to mRNA or from mRNA to protein.According to the different binding sites,antisense technology can be divided into anti-sense DNA, anti-sense mRNA and the self-catalytic ribozyme,among which Antisense oligodeoxynucleotides(ASODN) is the most commonly used,it has the characteristics of high degree of target specificity,easy design,variety and easy synthesis,which are unparalleled to those of conventional drugs,so they have enormous research value.In addition,it has nothing to do with cell cycle when antisense nucleic acid is transfected into cells,no matter proliferating cells or non-proliferating cells.It does not contain viral sequences,does not produce an immune response and would not integrate into the host chromosome.Because of these advantages,antisense technology has great development both in basic research and clinical application of malignant turners.Based on the research both home and abroad,the synthetic VEGF antisense oligonucleotide was transfected into HT-29 cells with Lipofectamine under hypoxia conditions,reverse transcription-PCR and Western blot method were used to detect the expression of VEGF and its receptor,proliferation and apoptosis were studied by MTT and flow cytometry.ObjectivesTo simulate hypoxic microenviroment in vitro and learn the impact of synthetic antisense oligodeoxynucleotides on the expression of VEGF and its receptor under hypoxic conditions in vitro,as well as those on cells,thus provide a theoretical and experimental basis for gene therapy for colorectal cancer.MethodsCoCl₂ was used to build a hypoxic environment for HT-29 cells after 24 hours cultivation under normal environment in vitro,and the final concentration of CoCl₂ was150μmol/L.RT-PCR was used to detect the mRNA expression of VEGF and KDR after 48 hours cultivation under hypoxic environment.Protein expression was detected by Western blot method 72 hours later.VEGF ASODN and SODN were transfected into HT-29 cells by LipofectamineTM2000 after 12 hours hypoxic cultivation,mRNA expression in each group was detected by semi-quantitative RT-PCR 48 hours after transfection,the results were compared with that of the control group.Western blot was applied to measure the protein expression 48h and 72h after tranfection.Cell proliferation was detected by MTT,there are 4 concentrations in ASODN group:400nmol/L,600 nmol/L,800 nmol/L and 1000 nmol/L,the concentration of the SODN group was 1000 nmol/L and the cells in control group were transfected with liposome only,each group mentioned above has 4 time point:24h,48h,72h and 96h,the results were analyzed through the method of analysis of variance.The cell apoptosis was detected by flow cytometry.Results(1)The mRNA and protein expressions of VEGF were significantly increased under hypoxic conditions compared with those of the control groups(0.914±0.015 VS 0.442±0.026,P＜0.01)and(0.919±0.004 VS 0.420±0.011,P＜0.01).(2) There is significant difference of VEGF mRNA expression among different groups(F= 2299.046,P＜0.01).The VEGF mRNA expression in ASOND group(0.455±0.022) is lower than those in SODN group(0.915±0.005) and liposome group (0.934±0.018)(P＜0.01).There is also significant difference of KDR mRNA expression among different groups(F=671.060,P＜0.01).The KDR mRNA expression in ASOND group(0.374±0.020) is lower than those in SODN group (0.801±0.032) and liposome group(0.797±0.032)(P＜0.01).(3) Western blot confirmed the specific bands which in line with the molecular weight of VEGF and KDR,there is significant difference of VEGF protein expression among different groups(F=14706.116,P＜0.01),and the bands in both ASODN 48h(0.622±0.004) and 72h(0.486±0.005) groups were significantly weaker than those in the SODN(0.898±0.004) and liposome(0.896±0.007) groups,and the band in 48h group is weaker than that in 72 group(P＜0.01).There is also significant difference of KDR protein expression among different groups(F=8593.048,P＜0.01),and the bands in both ASODN 48h(0.527±0.004) and 72h(0.417±0.003) groups were significantly weaker than those in the SODN(0.788±0.010) and liposome(0.782±0.003) groups,and the band in 48h group is weaker than that in 72 group(P＜0.01).(4) MTT revealed that there are statistical differences of the cell proliferation among different time points(Ftime=4321.096 P＜0.001) and different groups(Fgroup=1988.745 P＜0.001),and there is interaction effect between time and group(FIE=503.131 P＜0.001).There is statistical difference between two random time points(P＜0.05) and groups(P＜0.001) except that between SODN group and liposome group(P＞0.05).There are significant differences between different time points in each ASODN concentration(P＜0.05),and it is not happen in SODN group and liposome group(P＞0.05),(4) Apoptotic index of HT-29 cells in ASODN group was obviously higher than that in the control group 72 hours after transfection. [(25.31±4.62)%VS(3.06±0.64)%,P＜0.05].ConclusionHypoxia can promote VEGF expression,and increase KDR expression through positive feedback mechanism,LipofectamineTM2000-mediated VEGF ASODN inhibited the HT-29 proliferation by descending the VEGF gene expression in vitro under hypoxic conditions.