The Effects And Mechanisms Of RNA Interference Targeting Focal Adhesion Kinase Enhances Colon Carcinoma 5-fluorouracil Chemosensitivity

Background and ObjectivesColorectal cancer is the second leading cause of cancer deaths after lung cancer in Western countries. In China, colon cancer is also one of the most common cancers. Preexistent or acquired resistance of cancer cells against genotoxic injury caused by ionizing radiation or cytotoxic drugs is a prevalent and clinically highly relevant phenomenon .In recent years, researchers increasingly investigated the modulating effects of cell–matrix interactions on the cytotoxic power of genotoxic drugs and ionizing radiation. Intriguingly, the majority of clinically used chemotherapeutic agents as well as irradiation displayed significantly less cytotoxic in matrix adhered cells in vitro as compared to control cells on nonspecific substrates.the growth in close cell–cell contact could be a key event in the observed resistance.Compared with monolayer culture, tumour cells cultured as multicellular spheroids exhibit much higher levels of resistance to chemotherapeutic agents, a phenomenon known as multicellular resistance (MCR).Normal cells undergo apoptosis or'anoikis'when they lose adhesion to extracellular matrix (ECM) .In contrast, cancer cells are more resistant to anoikis and can grow anchorage independently. Apoptosis is arguably the most potent defense against cancer because it is the mechanism used by metazoans to eliminate deleterious cells. Many chemopreventive agents appear to target signaling intermediates in apoptosis-inducing pathways.One of the proteins implicated in the mechanism of anoikis is focal adhesion kinase (FAK), a nonreceptor tyrosine kinase that is involved in the control of cell-extracellular interactions such as spreading, migration, motility, and survival.FAK is overexpressed by a variety of human tumors including colon cancers, making FAK a rational target for therapeutic intervention. Recently, Kornberg and Fleigel demonstrated a potential role for FAK in chemoresistance.RNA interference using siRNA is a powerful technique that allows highly specific suppression of individual gene expression. Recent reports have indicated that siRNA has advantages over antisense oligonucleotides in vitro and in vivo, thought to be due in part to the greater resistance of siRNA to nuclease degradation.Compared to monolayers, multicellular spheroids are known to reproduce some features of solid tumors such as the presence of a subpopulation of cells with a restricted oxygen supply, responsible for a decrease in cell proliferation and development of necrotic regions.Here we used multicellular spheroid cultures to study the resistance to chemotherapy were investigated. Here we show, for the first time, that suppression of FAK expression by shRNA interference increases the susceptibility of colon carcinoma cells to 5-FU in vitro. We also demonstrate that FAK shRNA interference can suppress FAK expression in vivo and that this treatment enhances the therapeutic efficacy of 5-FU in a nude mouse xenograft model of colon carcinoma. These findings identify FAK as not only a determinant of colon carcinoma chemoresistance, but also a promising therapeutic target.Methods1. Immunohistochemistry and Western blotting were used to examine the expression of FAK in four colon cancer lines (LoVo, HCT116, HT-29 and SW480), and Immunohistochemistry was used to examine the expression of FAK , FAK pY397,AKT,NF-κB,Bcl-2 and Caspase-3 in colorectal cancer samples.2. To inhibit the expression of FAK in colon carcinoma HT-29 cells by generation of RNA interference(RNAi) construct targeting FAK, and the effect of FAK silence were detected by RT-PCR ,Real-time PCR,Immunohistochemistry and Western blotting.3. Three dimensional multicellular spheroid cultures were used for experiments.Spheroids were trypsinized for cell counting using a hemocytometer.MTT and CCK-8 assay were employed to detect the effect of FAK silence on the sensitivity of multicellular spheroids to 5-FU. Flow cytometry were exploited to detect 5-FU-induced apoptosis of multicellular spheroids.4. The expression of FAK,FAK pY397,AKT, NF-κB ,Caspase-3 and Bcl-2 in colon cancer multicellular spheroid before and after RNAi were detected by Western blotting., then to explore the molecule mechanisms of suppressing expression of FAK enhances colon carcinoma 5-fluorouracil chemosensitivity.5. The effects of RNA interference targeting FAK on tumor growth inhibition were detected by using the nude mouse xenograft model.6. The expression of FAK,FAK pY397,AKT, NF-κB ,Caspase-3 and Bcl-2 in nude mouse xenograft were detected by Western blotting. To explore the molecule mechanisms of suppressing expression of FAK can inhibit tumor growth in the nude mouse xenograft.Results1. FAK protein expression level in the four colon carcinoma cells are high,of which HT-29 was highest. Positive expression of FAK,FAKpY397,AKT, NF-κB ,Caspase-3 and Bcl-2 were found in 82.43%,45.95%,52.70%,59.46%,48.65%,70.27% of cases of colorectal cancer respectively, which were much different than that in normal samples.The positive expression of FAK correlated with the degree of differentiation of cancer cells and lymph node metastasis(P<0.05), but not related to either patient's gender, age, tumor location, tumor size or Dukes'stage(P>0.05). Positive expression of AKT significantly related to cellular differentiation (P<0.05). Positive expression of NF-κB significantly related to lymph node metastasis(P<0.05). The positive expression of FAK correlated with the expression of AKT and NF-κB(P<0.05), not with FAKpY397,Caspase-3 and Bcl-2(P>0.05).2. The recombinant plasmids were completely coincided with the designs and were stably transfected into HT-29 cell lines.After RNA interference targeting FAK, the protein expression of FAK was reduced significantly as detected by immunocytochemistry and Western blotting, FAK mRNA expression was decreased markedly as detected by RT-PCR and Real-time PCR. Compared with untransfected group,FAK mRNA and protein expression of transfected pGenesil-1-FAK vector group decreased respectively 79.2%±2.97% and 79.9%±5.44%, but not decreased significantly in transfected negative control vector group(P>0.05).3. The results of spheroid growth: the growth rate of all cell groups had a doubling time of 6 days. Compared with untransfected group,each point growth rate of both transfected negative control vector group and transfected pGenesil-1-FAK vector group were not different markedly(P>0.05). The results assayed by MTT:Compared with untransfected group,the sensitivity to 5-FU(IC50)of transfected negative control vector cells didn't chang markedly(P>0.05), But the sensitivity to 5-FU(IC50)of transfected pGenesil-1-FAK vector group decreased evidently(P<0.01).The results of CCK-8 assay were so as MTT results. Apoptosis rates were detected by flow cytometer in multicellular tumor spheroids after 5-FU challenging. Compared with the rates of apoptosis of untransfected group(8.87%±0.57%), the rates of apoptosis of transfected negative control vector group(8.99%±0.55%) didn't chang markedly(P>0.05),but apoptosis rates of transfected pGenesil-1-FAK vector group both with 5-FU-induced (21.50%±4.62%) and with no drug-induced (16.48%±1.18%)increased significantly(P <0.01), futhermore,5-FU-induced apoptosis rates were higher than non-drug-induced apoptosis rates significantly(P <0.01).4. The protein expression change of FAK,FAK pY397,AKT, NF-κB ,Caspase-3 and Bcl-2 in multicellular tumor spheroids cells were detected by Western blotting. Compared with untransfected group, the protein expression change of transfected negative control vector group was not significantly(P>0.05),but the protein expression of FAK,FAK pY397,AKT, NF-κB and Bcl-2 in transfected pGenesil-1-FAK vector group decreased , and apoptosis-inducing molecule caspase-3 expression rised.5. FAK shRNA interference promotes 5-FU cytotoxicity were detected in the nude mouse xenograft model. Mice in group A and B had a mean tumor mass of 4.204 g±0.658g and 4.013 g±0.540 g respectively, the different between them was not significant (P>0.05). group C,Dand E had a mean tumor mass of 2.427 g±0.394 g, 2.833 g±0.331 g, 2.741 g±0.251 g respectively, the different among them was not significant too(P>0.05).But group F which vaccinate transfected pGenesil-1-FAK vector cell and treated with 5-FU had a mean tumor mass of 1.105 g±0.159g, this equated to a tumor growth inhibition of 73.72% , the different was significant compared to the other groups(P < 0.05).6. The protein expression of FAK,FAK pY397,AKT, NF-κB ,Caspase-3 and Bcl-2 in nude mouse xenograft were detected by Western blotting. Compared to group A, all of the protein expression in group B,D and F were not large different respectively. But in group C and group F which vaccinate transfected pGenesil-1-FAK vector cell, the protein expression of FAK, FAK pY397, NF-κB ,AKT and Bcl-2 were lower than the other groups,and Caspase-3 protein expression was higher than the other groups, the protein expression between group C and group F was not large different respectively.Conclusions1. Of the four colon carcinoma cells, FAK protein expression level in HT-29 was highest. In colorectal cancer samples, FAK,AKT and NF-κB is overexpressed, moreover AKT and NF-κB are related with FAK,and caspase-3 expression is lower than the normal, which imply that they may play an important role in colorectal cancer progression.2. The RNA interference vectors targeting FAK were constructed and transfected successfully, the expression of targeting gene FAK was inhibited effectively.3. The suppressing expression of FAK can not affect spheroids growth, but can stimulate spheroids cells apoptosis and increase spheroids apoptosis-inducing-5-FU markedly, and enhance the sensitivity of HT-29 spheroids cells to 5-FU significantly.4. The suppressing expression of FAK can inhibit tumor growth in the nude mouse xenograft, increase the sensitivity of tumor to 5-FU and reverse multicellular resistance efficiently.5. The suppressing expression of FAK can down-regulated expression of Akt and NF-κB, then promotes cell apoptosis, increases the sensitivity of tumor to 5-FU and reverses multicellular resistance efficiently.6. Signaling pathways FAK-- Akt--NF--κB is one of mechanisms of FAK mediating multicellular resistance.