Aloin Induces Apoptosis Of Canine Breast Tumor Through MAPKs Signaling Pathway

Canine breast tumor is one of the common tumor diseases in veterinary clinic,among which the malignant rate of canine breast tumor is about 50%.This disease seriously threatens the health of pet dogs and reduces their quality of life.The study of canine breast tumor disease has certain practical value.Aloin contains a variety of amino acids and vitamins and other nutrients.Modern pharmacological studies show that aloin has anti-inflammatory,antioxidant and immunity enhancement effects.In addition,aloin has been shown to reduce cell proliferation and induce apoptosis in different human tumor cells.Therefore,these results suggest that aloin is a potential drug for cancer treatment.However,the mechanism of aloin action on canine breast cancer cells is less well studied.Apoptosis,as the key to type I programmed cell death,is closely related to the development of tumor and can be regarded as a potentially beneficial strategy for anti-tumor therapy.The MAPKs signaling pathway sre closely related to the occurrence and development of tumors.Abnormalities in a certain link of the MAPKs signaling axis will cause the uncontrolled growth of cells,leading to the occurrence of tumors and other diseases,suggesting that key proteins or regulatory factors in this signaling pathway may be potential therapeutic targets for tumors.Methods1.In vivo experiment: Mouse mammary cancer 4T1 cells were injected subcutaneously under the fourth breast pad of BALB/ C mice to establish a transplanted tumor mouse model,then the mice were randomly divided into 5 groups(Aloin 0,10,20,30,40mg/kg).Aloin was intraperitoneally injected for 28 days,the weight and tumor volume of the mice were measured every two days.The grafted tumor and adjacent tissue were separated,one part of the tumor tissue was fixed and H&E staining was used to observe pathological changes,the other examined levels of apoptosis-related proteins by Western blot.2.In vitro experiments: Experiments with CMT 1211 and CMT 7364,After growing to logarithmic phase,the cells were cultured on 6-well plates and labeled(Aloin 0,10,20,40,80,160μM),respectively.Cell proliferation activity was detected by CCK-8,and the effect of aloin on cell migration was detected by cell scratch assay.The levels of apoptotic genes Bax,Bcl2 and XIAP were detected by Q-PCR.Western blotting was used to detect migration proteins MMP-2,MMP-9,apoptosis proteins Bax,Bcl2,Cleaved caspase-3,Cleaved PARP,ERK1/2,P-ERK1/2,P38,P-P38,JNK,and P-JNK levels.Cell cycle distribution and apoptosis were detected by flow cytometry.The positions of p-ERK1/2,P-P38 and p-JNK in cells were determined by immunofluorescence assay.Results1.After 7 days of inoculation with 4T1 cells in mice,the tumor formation rate of mice reached 100%.After 28 days of aloin treatment,the volume of transplanted tumors in the treated group was significantly lower than that in the control group in saline,and the volume of transplanted tumors in mice decreased significantly with increasing concentration of aloin treatment.Compared with normal saline control group,aloin 40mg/kg treatment group showed significant increase of necrotizing lymphocytes and obvious nuclear concentration.Western blot of protein extracts from tumor tissues showed that the expression of Bcl-2,an apoptosis inhibitor,was significantly reduced,while the expression of Cleaved Caspase 3,Bax and Cleaved PARP,a pro-apoptosis protein,showed the opposite trend.2.CCK-8 results showed that the viability of mouse mammary tumour cells decreased significantly with increasing concentrations of aloin and increasing duration of action.At the same time,flow cytometry analysis of the cell cycle revealed a significant increase in cells in s-phase after treatment with medium concentrations of aloin.Cellular immunofluorescence detection of p-ERK1/2 expression revealed a significant reduction in the level of intracellular activated ERK1/2 expression in both the aloin medium concentration treatment group and the ERK1/2 inhibitor Ravoxertimib group.Cell scratch assays showed that the migration capacity decreased with the increase of aloin concentration.Q-PCR results showed a significant decrease in the expression levels of the apoptosis inhibiting factor Bcl-2 and a significant increase in the expression levels of the pro-apoptotic factor Bax and a series of apoptotic protein factors XIAP in the aloin treated group.Western blot detection of migration proteins showed a significant decrease in the expression of MMP-2 and a non-significant change in the expression of MMP-9 with the increase in the concentration of aloin treatment.Flow cytometry detection of apoptosis revealed that the apoptosis rate increased significantly with increasing concentration of aloin in the treatment group.Western blot detection of apoptotic proteins revealed that the expression of phosphorylated JNK and p38 decreased significantly in line with the trend of tissue protein expression,and immunofluorescence experiments also showed that aloin inhibited the phosphorylation of JNK and p38.ConclusionAloin inhibited the activation of ERK1/2 in the MAPKs signaling pathway,blocked cells in s-phase and inhibited cell proliferation.By inhibited the phosphorylation of p38 and JNK,it affected the expression of downstream apoptotic and pro-apoptotic factors,induced the accumulation of Bax,and activated the expression of apoptotic proteins such as Cleaved Caspase-3 to induce apoptosis in breast cancer cells,and exerted anti-cancer effects.Therefore,aloin is expected to be a novel herbal medicine against canine mammary tumor.