The Effect Of Blueberry Anthocyanin As Nutrition Supplement On Efficiency Of Dacarbazine For Melanoma

Melanoma is a kind of malignant tumor originated from neural crest melanocytes.It is highly malignant,strongly aggressive,easily metastasized and poorly prognosis,which seriously threatens human health.In clinic,dacarbazine(DTIC)and other chemotherapy are often used in the middle and late stage of melanoma.However,the effect is not good,which is far from people’s expectation.It is of great significance to introduce tumor nutrition therapy for improving its curative effect.Blueberry is rich in nutrients and anthocyanin(BA),known as the"king of fruits".Research shows that BA has a wide range of pharmacological effects,and BA products are often used as high-grade nutrition.According to the theory of"medicine food homology"in traditional Chinese medicine,nutrients and drugs can not be randomly matched.Therefore,it is of certain significance to explore whether BA is suitable for the adjuvant treatment of melanoma patients using DTIC.Based on the view,the work was done contianing below:(1)Determination of dacarbazine in the urine of melanoma mice by HPLCIn this chapter,a HPLC method with mixed organic solvent and phosphate buffer system as mobile phase was developed.The results showed DTIC had a good linear relationship(r~2=0.999)in the concentration range of 0.25μg/m L～1000μg/m L,and the detection limit and quantitative limit were 0.12μg/m L and 0.25μg/m L,respectively(based on S/N=3 and S/N=10).The recoveries were 98.9%,102%and 99.1%for the three concentrations(50.0μg/m L,375μg/m L and 500μg/m L)with relative standard deviations(RSDs)of 3.2%,1.3%and 1.2%,respectively(n=5).It provided one fast method to detect the content of DTIC in urine for melanoma C57BL/6 mice and evaluate its utilization rate.(2)Mechanism of BA on B16-F10 cellsB16-F10 cells were treated with different concentrations of BA at different times.And then MTT method was used to assess the effect of BA on the proliferation of B16-F10 cells was detected by MTT method.The results showed BA significantly inhibit the growth of B16-F10 cells in vitro.Scratch test;Transwell test and gelatin zymogram showed BA inhibit the migration and invasion of B16-F10 cells.The results of AO/EB and flow cytometry showed BA induce apoptosis of B16-F10 cells.The apoptosis mechanism of B16-F10 cells was studied by Western blotting.It showed that BA induced apoptosis of B16-F10 cells was associated with mitochondrial pathway.Above results provided the basis for the further study of BA as a nutritional supplement in the treatment of melanoma with DTIC.(3)Study on the pharmacological effect of BA as a nutritional supplement to DTIC in the treatment of melanomaIn this section,it was investigated that how BA as a nutrient influenced the therapeutic effect of DTIC treating melanoma C57BL/6 mice.According to the growth rate and tumor weight of melanoma in different doses of BA+DTIC,it can be found growth rate of melanoma in supplemented with nutrition BA is faster than DTIC group.At the same time,the HPLC method was used to monitor the content of DTIC in the urine of C57BL/6melanoma mice.The results showed the content of DTIC in BA+DTIC group was significantly different from DTIC group in the urine of melanoma mice.(4)Study on the mechanism of melanoma treatment with DTIC assisted by BAHE staining was used to observe the morphology of melanoma using BA+DTIC.The results of tumor tissue section staining showed BA+DTIC group was more neutrophils infiltration in the necrotic area,and tumor nuclei in the necrotic area were pyknosis,fragmentation and even dissolution.Effect of BA+DTIC on the proliferation and apoptosis of melanoma was detected by Ki-67 immunohistochemistry and TUNEL staining.The results showed the expression of Ki-67 in BA+DTIC group was higher than that in DTIC group,and apoptosis rate in BA+DTIC group was lower than that in DTIC group.Furthermore,Western blotting was used to detect related proteins to study the possible mechanism of DTIC weakening after BA treatment.The results showed BA could weaken the therapeutic effect of DTIC on melanoma through PI3K/AKT pathway.The above experimental results showed that BA alone can inhibit melanoma to a certain extent,but the efficacy of it was much lower than that of DTIC,a common chemotherapy drug for melanom.Moreover,it was found that BA used as a nutrient can decrease the efficacy of DTIC towards melanoma.Therefore,it is suggested that patients with melanoma treated with DTIC should carefully consider BA and products containing BA using nutritional adjuvant therapy.