Effect And Its Mechanism Of Monomer DMDD,Isolated From The Root Of Averrhoa Carambola L.,on Subcutaneous Transplantation Tumor In Mice With Breast Cancer And Liver Cancer

Objective:In this study,the effect and its mechanism of monomer 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione(DMDD),isolated from the root of Averrhoa Carambola L.,on subcutaneous transplantation tumors in mice with breast cancer and liver cancer were studied.Methods:1.Effect and its mechanism of DMDD on subcutaneous transplantation tumor in mice with breast cancerThe transplanted tumor models were established in 60 Balb/c mice by subcutaneous injection of breast cancer 4T1 cells.50 transplanted tumor mice were randomized into 5 groups named model group,doxorubicin(DOX)group,DMDD low dose(DMDD-L)group,DMDD medium dose(DMDD-M)group and DMDD high dose(DMDD-H)group with 10 mice in each one and 10 normal mice were taken as the normal group.The mice were sacrificed and serum and the transplanted tumor tissues were obtained after 14 days of continuous administration.The contents of interleukin 2(IL-2),interleukin 4(IL-4)and interleukin 10(IL-10)in the serum were detected by enzyme-linked immunosorbent assay(ELISA)kit.The pathological changes of tumor tissue were observed using HE staining and transmission electron microscopy and TUNEL staining was applied to detect the ability of DMDD to induce apoptosis.The m RNA expression levels of RAF1,MEK1/2,ERK1/2,Bax,and Bcl-2were analyzed by RT-q PCR.The protein expression levels of p-RAF1,p-MEK,and p-ERK were assayed by immunohistochemistry.The expression levels of proteins RAF1,MEK,ERK,JNK,P38,p-RAF1,p-MEK,p-ERK,p-JNK,p-P38,Bax and Bcl-2 were examined by Western blotting(WB).2.Effect and its mechanism of DMDD on subcutaneous transplantation tumor in mice with liver cancer45 nude mice were selected and subcutaneously injected with liver cancer Bel-7404 cells to establish the transplanted tumor model.When the tumors grew to 100 mm~3 in size,40 of them were randomly divided into 5 groups including model group,DOX group,DMDD-L group,DMDD-M group and DMDD-H group with 8 mice in each one and 8 unmodeled mice were taken as the normal group.The mice were sacrificed and the blood,liver,spleen and tumor mass of the mice were obtained after 12 days of continuous administration.Tumor volume,tumor weight and tumor inhibition rate were analyzed to evaluate its anticancer effect.spleen index and liver index were calculated.White blood cells(WBC),red blood cells(RBC),hemoglobin(HGB)and platelets(PLT)of the blood were detected.The expression of IL-2,IL-4,IL-10,tumor necrosis factorα(TNF-α),transforming growth factorβ1(TGF-β1)and vascular endothelial growth factor(VEGF)were detected by ELISA.The pathological changes of tumor tissues were observed by HE staining and transmission electron microscopy and the apoptosis-inducing ability of DMDD was detected by TUNEL staining.The m RNA expression level of RAF1,MEK1/2,ERK1/2,Bax and Bcl-2 were detected by RT-q PCR.Semi-quantitative analysis of p-RAF1,p-MEK,p-ERK,Bax and Bcl-2 was conducted by immunohistochemistry.The expression level of proteins RAF1,MEK,ERK,JNK,P38,p-RAF1,p-MEK,p-ERK,p-JNK,p-P38,Bax and Bcl-2 were assessed through WB.Results:1.Effect and its mechanism of DMDD on subcutaneous transplantation tumor in mice with breast cancer(1)Compared with the model group,the tumors in the DMDD group grew slowly.The tumor weight of DMDD group was smaller than that of model group(P<0.05 or P<0.01).The tumor inhibition rates of DOX group,DMDD-L group,DMDD-M group and DMDD-H group were 41.9%,26.59%,37.06%and43.80%,respectively.(2)Compared with the model group,IL-2 expression was increased in the DMDD group,while IL-4 and IL-10 were decreased(P<0.05 or P<0.01).(3)The results of HE staining suggested that in the model group,the cancer cells were closely arranged,large in volume,pleomorphic in nucleus,and the nucleoli were obvious.DOX group and DMDD group:reduced cell number,loose arrangement,and nuclear shrinkage.(4)The results of Transmission electron microscopy were as follows:large nucleus with obvious nucleoli and integrated intra-cellular organelles were observed in the model group.Cell shrinkage,nuclear pyknosis,chromosome aggregation and vacuolar degeneration were noticed in DOX group.cell shrinkage and rupture,nuclear pyknosis,chromosome aggregationand plenty apoptosis body were appeared in DMDD-H group.(5)TUNEL staining indicated that the rate of positive apoptotic cells in the DMDD groups were higher than that in the model group and DOX group(P<0.05or P<0.01).(6)RT-q PCR results showed that compared with the model group,RAF1,MEK1/2,ERK1/2 and Bcl-2 m RNA expressions were decreased and Bax expression was increased in DMDD group(P<0.05 or P<0.01).(7)Immunohistochemical results suggested that compared with the model group,expressions of p-RAF1,p-MEK and p-ERK in DMDD group were down-regulated(P<0.05 or P<0.01).(8)WB results showed that,compared with the model group,the protein expression levels of p-RAF1,p-MEK,p-ERK,p-JNK,p-P38 and Bcl-2 in the DMDD group were down-regulated,while the expression level of Bax protein was up-regulated(P<0.05 or P<0.01).2.Effect and its mechanism of DMDD on subcutaneous transplantation tumor in mice with liver cancer(1)Compared with the model group,the tumors in the DMDD group grew slowly.The tumor weight of DMDD group was lower than that of model group(P<0.01).The tumor inhibition rates of DOX group,DMDD-L group,DMDD-M group and DMDD-H group were 49.38%,47.36%,63.13%and 66.39%,respectively.(2)Compared with the model group,there was no difference in liver index and spleen index in DMDD group(P<0.01).(3)Compared with the model group and DOX group,the content of WBC,HGB and PLT in DMDD group were increased(P<0.05 or P<0.01).(4)Compared with the model group,IL-2,IL-4,IL-10,TNF-α,TGF-β1 and VEGF were decreased in the DMDD group(P<0.05 or P<0.01).(5)The results of HE staining suggested that in the model group,the cancer cells were large and closely arranged and the nucleoli were obvious.DOX group and DMDD group:loose cell arrangement,reduced number,and nuclear condensation.(6)Transmission electron microscopy observations showed that in the model group,the nuclei were large,nucleoli were obvious,and chromatin was evenly distributed.DOX group:nuclear concentration,chromatin condensation and marginalization,a large number of cavitation formation.DMDD-H group:nuclear shrinkage,chromatin agglutination and marginalization,a large number of vacuoles and apoptotic bodies were observed.(7)TUNEL staining indicated that the rate of positive apoptotic cells in the DMDD group was higher than that in the model group(P<0.05 or P<0.01).(8)RT-q PCR results showed that compared with the model group,RAF1,MEK1/2,ERK1/2 and Bcl-2 m RNA expression were decreased and Bax expression was increased in DMDD group(P<0.05 or P<0.01).(9)Immunohistochemical results showed that compared with the model group,expressions of p-RAF1,p-MEK,p-ERK and Bcl-2 in DMDD group were down-regulated,while Bax expression was up-regulated(P<0.05 or P<0.01).(10)WB results indicated that compared with the model group,the protein expression levels of p-RAF1,p-MEK,p-ERK,p-JNK,p-P38 and Bcl-2 in the DMDD group were down-regulated and the expression levels of Bax protein were up-regulated(P<0.05 or P<0.01).Conclusions:DMDD can induce apoptosis of subcutaneous xenograft cells in breast cancer mice,and the mechanism may be related to enhancing immune function and regulation of MAPK pathway in mice.DMDD can induce the apoptosis of subcutaneous xenograft cells in liver cancer mice,and the mechanism may be related to the fact that DMDD can enhance the immune function of mice,improve blood routine indexes,reduce the secretion of cytokines,and regulate the MAPK pathway.