| Objective: The aim of this study was to explore the effects of the diene propyldisulfide (DADS) and LIMK1on the biological characteristics of SW480colorectalcancer cells, to observe the tumor forming and growth of human SW480colon cancercells affected by the DADS and LIMK1in vivo of nude mice, and to explore themolecular mechanisms of the diallyl disulfide (DADS) to suppress SW480cellsproliferation, migration and invasion.Methods Build PIRES2-LIMK1eukaryotic expression vector,the vector containsLIMK1gene CDS sequence that encoding human LIMK1protein. ThePIRES2-LIMK1eukaryotic expression vector was transfected into SW480cells, highexpression of LIMK1protein stably transfected LIMK1/SW480cell screening.RT-PCR, Western blot detection DADS before and after treatment, high expressionof LIMK1on the expression of LIMK1mRNA and protein in SW480cell line. MTTassay, flow cytometry, wound healing assay and Transwell invasion chamber assayDADS before and after treatment,the high expression of LIMK1effect on SW480cell proliferation, cell cycle, migration and invasion. Subcutaneous injection of humancolon cancer cell line SW480, human colon cancer xenograft model was established,the high expression of LIMK1cells, cells in SW480group, LIMK1interference groupcells were in vitro without drug treatment and the45mg. L-1DADS treatment for24 hours, inoculated subcutaneously in nude mice. Every6days, weighing the weight ofnude mice and tumor volume were measured, observing the growth of tumors.32days later, the nude mice were killed, separation and mass, weight, measurement andweighing tumor volume, tumor inhibition rate was calculated, and the transplantedtumor tissue for immunohistochemical analysis of the tissue, the expression oftransplanted tumor in LIMK1, cofilin1, Rac1, PAK1, ROCK1and Destrin.Results1. Successfully to build LIMK1high protein expression stablely transfectsLIMK1/SW480cells.Enzyme digestion and sequencing results confirmed that the PIRES2-LIMK1eukaryotic expression vector is successfully built.PIRES2-LIMK1eukaryoticexpression vector transfects SW480cells, Selected by G418brush after2weeks, thereis green fluorescent in the cytoplasm of the infected cells under the microscope,rt-pcr and Western blotting test showed that the protein expression of LIMK1/SW480cells and LIMK1mRNA is increased (P <0.05), which suggests the stablecell lines LIMK1/SW480of LIMK1high expression is successfully acquired.2. DADS can decrease LIMK1mRNA and protein expression, inhibitsphosphorylation of LIMK1and cofilin1,LIMK1expression can promote thephosphorylation of cofilin1.RT-PCR and Western blot test showed that puts45mg L-1DADS in the LIMK1over expression group,blank vector group and SW480group respectively, after24hours, LIMK1mRNA and protein expression significantly decreased (P <0.05), P-LIMK1and P-cofilin1reduced significantly (P <0.05). Western blotting testshowed that P-cofilin1and LIMK1over expression group was obviously higher thanempty vector and SW480group (P <0.05), Western blot showed that DADS andLIMK1over expression have no statistical significance (P>0.05) of SW480cells andcofilin1expression.3. DADS inhibits the value-added of SW480cell migration, ever LIMK1expression can promote the value of SW480cell migration. Determined by MTT, according to the results of LIMK1high value-added activityexpression group was obviously higher than that of SW480and empty vector group (P<0.05), and DADS after processing, high LIMK1express group SW480cellproliferation activity and empty vector group were suppressed (P <0.05)Scratch the experimental results show that the scratch after24h, healing LIMK1high expression group was obviously faster than SW480and empty vector group (P <0.05), and DADS after processing, high LIMK1express group SW480cells scratchesthe healing rate and empty vector group were suppressed (P <0.05), indicating thatLIMK1expression can promote cell migration ability, DADS inhibits SW480cells’ability to migrateTranswell experimental results show that the Transwell Chambers cultured cellsafter24hours, LIMK1high expression in membrane cells was significantly more thanthe SW480group and empty vector group (P <0.05), and DADS after processing, thecell membrane cell number three group were decreased significantly (P <0.05),indicating that LIMK1expression can enhance the invasive ability of the SW480cells,DADS inhibits the invasion ability of SW480cells.4. DADS can inhibit human SW480colon cancer cells growth which have beentransplantated in nude mice, LIMK1expression can promote the growth ofnude mouse transplantated tumor,interferencing LIMK1protein expression canrestrain the growth of the nude mouse transplantated tumor.The model that human SW480colon cancer cells transplantated into ude mice hasbeen success, by measuring the nude mice transplanted tumors’s volume everygroup,the weight of the nude mouse transplantated tumor and calculating it, theresults show that SW480cells treated by DADS has been significantly inhibited(P <0.05)),LINMK1expressed group of nude mouse transplantation tumor growthsignificantly faster than the group treated by SW480and interferenced group (P <0.05), after the silence LINMK1,the nude mouse transplantated tumor growthsignificantly slower than the group treated by SW480and LIMK1expression. Conclusions1. The overexpression of LIMK1promoting the proliferation,migrationandinvasion of SW480cells,and reducing the inhibiting effection of DADS isrelated to cofilin1phosphorylation.2. DADS inhibiting the proliferation,migration andinvasion of human colon cancerSW480cells is related to downregulating the LIMK1and cofilin1phosphorylation.3. The diallyl disulfide (DADS) can inhibit the growth of SW480cell oftransplanted tumor in nude mice,The overexpression of LIMK1could promote itsgrowth,And silence LIMK1can hinder its growth.4. The diallyl disulfide (DADS) and silenceLIMK1protein can downregulate theexpression of Vimentin, P53, Ki-67, CD34, and upregulate the expression of E-cadherin, while the effects of LIMK1overexpression are opposite. |
PDF Full Text Request