Effect Of Vitamin B₂ On Anti-colon Tumor Activity Of Pentafluorouracil

Due to the accelerated pace of modern life and changes in dietary habits,the incidence of colon cancer in China has increased year by year and is developing towards a younger age.The mortality rate of colon cancer is second only to common lung cancer and liver cancer.5-fuorouracil（5-FU）is one of the common used therapeutic drugs.However,clinical datas show that 5-FU alone in the treatment of advanced colon cancer patients with a success rate of only10%-15%,accompanied by drug resistance and strong side effects.In order to improve the therapeutic effect of drugs,the combination of drugs and active substances has become a hot research direction in recent years.Vitamins,which are wide range of food sources,as one of the essential substances in human life activities,bear diverse physiological functions,and recent studies have shown that they have potential anticancer properties.Clinically,vitamin dietary supplementation is a common phenomenon in cancer patients.Therefore,this experiment combines 14 common vitamins with 5-FU to explore its effect on 5-FU anti-colon tumor activity,and screens vitamins with the potential to improve the efficacy of 5–FU,and then through in vitro and in vivo experiments to explore whether it can synergize with 5-FU to inhibit colon tumor activity,thereby reducing the amount of 5-FU,alleviating its side effects,providing a theoretical basis for clinically recommending dietary recommendations for colon cancer patients.We mainly get the following results:（1）Comparison of the effects of 14 vitamins on the cytotoxicity of 5-FU in two kinds of colon cancer cellsMTT cell proliferation toxicity test was used to compare the effects of 14 vitamins(VB₁,VB₂,VB₃,VB₅,VB₆,VB₉,VC,DL-αlipoic acid,VE,VD₃,VK₁,GSH,VB₁₂,biotin)at three concentrations alone and combining with 5-FU on the growth and proliferation of two types of colon cancer cells HCT116 and C26.The IC₅₀（half lethal concentration）of 5-FU on C26 and HCT116 cells was 1.13±0.80μmol/L and 15.2±2.28μmol/L,respectively.After comparing the14 vitamins at three concentrations alone and combing with IC₅₀ concentrations of 5-FU,VB₂alone and in combination had the highest inhibition rate in both C26 and HCT116 cytotoxicity tests.Therefore,VB₂ was chosen to combine with 5-FU for subsequent anti-tumor activity experiments in vitro and in vivo.（2）The combined effect of vitamin B₂ and 5-FU on colon tumor activity in vitroThe Chou-Talalay Method was used to quantitatively evaluate the effect of vitamin B₂combined with 5-FU on cell anti-colon tumor activity.The combination index（CI）（CI=1additive effect;CI<1 synergistic effect;CI>1 antagonism）and good concentration ratio of vitamin B₂ and 5-FU in HCT116 and C26 cells were obtained by CompuSyn software and cytotoxicity test results.The results showed that when the inhibition rate of C26 and HCT116cells was 50%,75%and 90%,5-FU combined with VB₂,the corresponding CI values were0.52,0.26,0.13 and 0.91,0.77,0.68,respectively.Because these CI values are all less than 1,the combination of 5-FU and VB₂ had a synergistic effect and the optimal concentration ratios for both C26 and HCT116 cells were 1:20 and 1:2,respectively.（3）The effect of vitamin B₂ combined with 5-FU on its anti-colon tumor activity in vivoThe Balb/c colon cancer tumor-bearing mouse model was used to observe the effect of vitamin B₂ combined with 5-FU on the anti-colon tumor activity of 5-FU in mice.To investigate the effects of drug combination on tumor growth and immune function in mice,the tumor volume,tumor ratio index,tumor inhibition rate and liver and spleen index of mice that were treated with different drugs were detected.Besides,elisa enzyme-linked immunosorbent assay was used to test the changes of tumor necrosis factor（TNF-α）,interleukin-1（IL-1）and interleukin 3（IL-3）inflammatory factors in the blood of mice.The final results showed that the tumor inhibition rate of mice which were treated with VB₂ and 5-FU increased from 51.53±2.32%to 76.18±4.37%;while the tumor body ratio decreased from 0.16±0.03 to 0.07±0.01,and the difference was significant（p<0.001）;spleen index of drug combination group was significantly lower than 5-FU group;liver and kidney index and blood urea nitrogen and plasma creatinine levels were not statistically different;and compared with the 5-FU treatment group,whose plasma uric acid content was 108.50±18.66μmol/L,the uric acid content of the combination group was only 59.75±11.51μmol/L,which was significantly decreased（p<0.001）.After combining with VB₂,the blood inflammatory factor interleukin-1（IL-1）,interleukin 3（IL-3）,tumor necrosis factor（TNF-α）levels are reduced too.（4）Molecular mechanism of vitamin B₂ affecting anti-tumor activity of 5-FUThe effect of vitamin B₂ combined with 5-FU on the cycle of colon cancer cells HCT116and C26 was detected by flow cytometry PI single staining assay.And the influence of the combination of vitamin B₂ with 5-FU on expression of protein that related to cell cycle and apoptosis was detected by western blotting.It was found that the 5-FU or VB₂ alone cleaves the PARP protein,but the cleavage band of the combination group is enhanced significantly.At the same time,the LC3B protein is up-regulated and the Bad protein expression is increased.The phosphorylation of p53 protein induces the downstream cyclin p21 protein up-regulated,which promotes apoptosis in cancer cells.In the cycle experiment,the proportion of G₁ phase of combination group in C26 cells was 75.12±4.90%compared with 64.02±2.54%in 5-FU group,while the proportion of G₁ phase in HCT116 cells increased from 61.09±0.15%to 70.44±0.75%,and the difference was significant（p<0.001）.Therefore,the results of this study indicate that different vitamins have an effect on the anti-colon tumor activity of 5-FU,and VB₂ has a significant effect on the anti-tumor activity of5-FU.VB₂ combined with 5-FU can synergistically reduce the viability of C26 and HCT116colon cancer cells by enhancing 5-FU pro-apoptosis and autophagy to cause G₁ arrest in cells,inhibit tumor growth in Balb/c tumor-bearing mice,reduce uric acid content and mitigate the inflammatory response.These results show that the combination of VB₂ and 5-FU can improve the curative effect and reduce the side effects and therefore provide a new direction and theoretical basis for the future clinical application of colon cancer patients with scientific dietary advice and improving the efficacy of chemotherapy drugs.