| Malignant tumors are a serious threat to human health and have been a major public medical problem.Exploring effective methods of treating malignant tumors has long been the focus of medical research in the world.In recent years,with the development of our society,people’s lifestyle and the ecological environment changes,the incidence of malignant tumors resulting in increasing social burden is extremely serious.Breast cancer is a common malignancy among women.In recent years,epidemiological survey shows that the incidence and mortality of breast cancer have been increased for years.Aspirin,one of classic non-steroidal anti-inflammatory drugs,is widely used in clinical fields.In recent years,with the deepening of the pharmacological effects of Aspirin,more and more evidence showed that it has anti-tumor effect.Aspirin reduces the risk of cancer,effectively prolongs the survival of cancer patients and reduces the recurrence of cancer.At present,a large number of experimental results show that Aspirin inhibits the proliferation of a variety of tumor cells in vitro and in vivo.However,the anti-tumor mechanism of Aspirin has not been fully revealed because of the complex pharmacological.In addition,the molecular mechanism of antitumor effect of Aspirin is different.Anti-tumor effect of Aspirin is lack of systematic and improved experiments in vivo,which also limits the Aspirin apply in the clinical application of the tumor.Compared with colorectal cancer and other gastrointestinal malignancies,Aspirin for breast has been seldom researched.For its anti-cancer mechanism of the study is also a lack of systematic exposition,and there is still obvious controversy in some issues.Canine mammary gland tumors for veterinary clinical are common.Its conventional treatment in the current clinical is surgical resection similar to human breast cancer,for the malignant and associated with the metastasis of canine mammary gland tumors,the simple surgical treatment is not ideal.Surgery with the system of adjuvant therapy is particularly necessary.Because dogs and human living environment are closely related,it has been the ideal experimental animal model for medical research.In addition,canine mammary gland tumors and human breast cancer are highly similar in the pathogenesis and course of disease development and so on.The study of canine mammary gland tumor can be not only for veterinary clinical exploration effective treatment programs,but also for clinical data to provide an important reference.Based on the preliminary study of Aspirin and the importance of dog as a model of breast cancer research,and there is no report about Aspirin on canine mammary gland tumor.This study was to investigate the inhibitory effect of Aspirin on mammary gland tumor cells and its molecular mechanism,which provide a theoretical basis for the next step in evaluating the antitumor effect of Aspirin in the model of breast cancer and lay a foundation for the early application of aspirin to the clinical treatment of breast cancer.In this study,CCK-8 and clone formation experiment were used to detect the effect of aspirin on the proliferation of breast cancer cells.The tumor model of breast cancer was established in nude mice,and the antitumor effect of aspirin was observed.The effect of aspirin on the apoptosis of breast cancer cells was detected by Annexin V-FITC staining.The expression of PARP,Bcl-2 and Bax protein in breast cancer cells was detected by western blot.The expression of Cyclin D1,CDK4,E2F1 and p21 protein was detected by western blot in order to reveal the mechanism of aspirin blocking cell cycle progression.Western blot was used to detect the expression of PI3 K / Akt and Wnt / β-catenin signaling pathway in aspirin-treated human breast cancer cells.The expression of β-catenin protein in the nucleus of breast cancer was further detected by aspirin.The effects of aspirin on the localization and expression of p-GSK3βand β-catenin in breast cancer cells were detected by immunofluorescence and immunohistochemical staining.The effects of aspirin on the proliferation of mammary gland tumor cells were investigated.Adjustment mechanism.The effect of aspirin on the migration and invasiveness of mammary gland tumor cells was detected by Transwell chamber test.The expression of VEGF and E-cadherin in the breast tumor of Aspirin was detected by western blot.Effect of metastasis ability of breast cancer cells.To evaluate the combined effect of aspirin with cisplatin or tamoxifen to lay the theoretical basis for the combined use of aspirin.The results showed:(1)Aspirin inhibits the proliferative activity of CHMp and CHMm in canine mammary gland tumor cell lines in vitro and inhibits the clonal formation of CHMp and CHMm in a dose-and time-dependent manner.In vivo experiments,Aspirin inhibits the growth of canine mammary gland tumor transplanted tumor in nude mice.Aspirin had the inhibitory effect on proliferation of canine mammary gland tumor cells in vitro and in vivo.(2)Aspirin could not induce apoptosis on CHMp.The expression of PARP,Bcl-2 and Bax protein in CHMp cells was not affected by Aspirin.But the number of late apoptotic and necrosis cells of CHMm induced by Aspirin was significantly increased in 10 m M group comparing with other groups.In addition,western blot analysis showed that within the group of 10 m M,there was a significant increase in the cleaved-PARP protein level of CHMm cells.A significant decline was observed in Bcl/Bax ratio compared to Aspirin treated CHMm cells and control.Aspirin Induces canine mammary gland tumor cells blocking in G0/G1 phase.The expression of Cyclin D1,CDK4 and E2F1 proteins in the cells was down-regulated,and the expression of p21 protein was up-regulated by Aspirin,which indicated that Aspirin could induce the cell cycle arrest.(3)By detecting the related protein in PI3 K / Akt signaling pathway,Aspirin inhibited PI3 K /Akt signaling pathway by down-regulating the expression of PI3 K and p-Akt in canine mammary gland tumor cells.(4)Aspirin inhibits Wnt/β-catenin signaling pathway,down-regulates the expression of pGSK3β and β-catenin protein,inhibits the expression of β-catenin protein in the nucleus and inhibits its downstream regulatory molecules c-myc,PPAR-δ.Aspirin reduced the expression of VEGF and increased the the expression of E-cadherin in canine mammary gland tumor cells,prevented the migration and invasion of canine mammary gland tumor cells,and thus inhibited the ability of metastasis.(5)Aspirin and cisplatin,tamoxifen respectively showed additive effect. |
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