| BackgroundColon cancer is one of the prevalent gastrointestinal malignant tumors, it has a high incidence and mortality. Chemotherapy is also a commonly used for colon cancer other than surgery.5-fluorouracil (5-fu) belongs to Pyrimidine analogues and has a similar structure with thymine as a cell cycle specific drug which plays a major role in s phase.5-fu as a widely used chemotherapy drug is often taken to treat colon cancer, despite it does not have a good result, only10%-30%of5-fu alone is utilized, so it is so meaningful to find an efficient Chemotherapy sensitizers to enhance the chemotherapy effect of5-fu. Dihydropyrimidine dehydrogenase (DPYD) is the initial and limited enzyme for5-fu catabolism, and about more than80%of5-fu are degraded into inactivated FUH2. The up-regulated DPYD in tumor tissues accelerate5-fu degradation and impaired anti-cancer effect of5-fu. Therefore, effectively restrain the activity of DPYD may enhance the sensitivity of tumor cells to5-fu, so as to improve the effect of chemotherapy.Peroxisome proliferator-activated recepter (PPAR) is a member of nuclear receptor superfamily. It plays very important roles in regulation of adipocyte differentiation, metabolism of carbohydrates and lipids, inflammation response, cell proliferation and cell apoptosis. PPAR family has three subtypes:PPARα、PPARγ and PPARδ. Among them, PPARγ is well studied. PPARγ’s ligands can be divided into natural and synthetic ligands. The synthetic ligands include TZD compounds and nonsteroidal anti-inflammatory drugs. As insulin sensitizers, TZD compounds in clinically used to treat type2diabetes, therefore improving insulin resistance and preventing diabetes cardiovascular complications. As a PPAR γ agonist, rosiglitazone was found to inhibit the proliferation and promote apoptosis of cells, and can enhance the sensitivity of tumor cells to5-fu chemotherapy.Objective To explore the effect of combination of PPARγ and5-fu on the growth of transplant colon tumor in nude mice, and whether rosiglitazone can enhance sensitivity of colon cancer to5-fu in vitro.Methods1. In vivo experiments:The human colon cancer Lovo cells were transplanted into nude mouse. Two weeks aftertransplantation, nude mice were divided into4groups by tumor size and mass:control,5-fu, rosiglitazone and combination group. Each kind of drugs was taken by intragastric administration to nude mice every day for2weeks. The longest and shortest diameter of tumor were measured every2days and tumor volume and inhibition rate were calculated.2. In vitro experiments:MTT assay was used to detect growth inhibition of5-fu, rosiglitazone and combination ofthese two compoundson human colon cancer Lovo cells; Optical microscope was used to observe the morphological changes of colon cancer cells in each group after treated with10μmol/L rosiglitazone,10μmol/L5-fu and combination of these two compounds and control group treated with DMSO. Fluorescence microscope was used to observe the changes of cell nuclei after stained with Hoechst33342. Flow cytometry was used to detect cell cycle after stained with PI; The protein levels of cyclin A1, cyclin E1and cdk2was explored via western blotting; Detection of mRNA and protein levels of DPYD was conducted via RT-PCR and western blotting.Results1. In vivo experiment:the growth inhibition rate of transplant tumor of nude mice after treated with5-fu, rosiglitazone and combination of these two compounds was74.9%,67.97%, and80.49%, respectively. Rosiglitazone can enhance the inhibitory effect of5-fu on tumor growth. 2. MTT assay results:rosiglitazone can enhance the inhibitory effect of5-fu on cell growth in time and dose dependent manner.3. Optical microscope results:colon cancer cells had much bigger size and appear more apoptosis phenomenon in5-fu group; combination group appear much more obvious apoptotic phenomenon compared with5-fu group.4. After stained with Hoechst33342, more and more apoptotic cells were found in combination group compared with the other groups.5. Flow cytometry assay stained with PI:combination of5-fu and rosiglitazone showed much more S phase cells.6. Protein levels of cyclin A1, cyclin E1and cdk2:5-fu andcombination group can significantly decreased protein level of cyclinA1compared with control group. Combination group also dramatically decreased protein level of cyclinA1compared with5-fu group; Control group had much more protein level of cylinE1, cdk2than5-fu and combination group, but combination group had further decreased protein level of cyclin E1than5-fu group.7. mRNA level of DPYD:5-fu increased mRNA level of DPYD and combination group decreased its mRNA level compared with control group. Protein level of DPYD:Western blotting result showed that5-fu increased protein level of DPYD and combination group decreased the protein level compared with control group.ConclusionRosiglitazone enhances5-Fu-induced LOVO cell growth inhibition through S cycle arrest via reducing DPYD activity and enhancing5-fu utilization. |
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