| Canine mammary tumor(CMT)is a malignant tumor originated from the mammary gland combined with glandular epithelial tissues,including solid,ductal and papillary carcinomas,which accounts for 13.4% of all cancers of canine.Clinically,western medicine treatment modalities are mainly mastectomy combined with chemotherapy,but postoperative recurrence and metastasis of the tumors are commonly seen.Chemotherapy has many disadvantages,such as highly toxic side effects,drug resistance,and high treatment costs.So it is important to develop novel canine breast cancer therapeutic drugs.Chinese herbal therapy is natural,mild and low-toxic,and has been used in human medicine since ancient times.Clinically,the use of Chinese herbal medicine in combination with western medicine therapy to increase efficacy,reduce toxic side effects,improve the patient’s quality of life,enhance the body’s immunity and prolong survival has become one of the current hot spots in tumor treatment research.Toosendanin(TSN)is an active ingredient extracted from the bark of Melia toosendan Seib et Zucc,which has been used as worm repellent in tradition.Recent studies have revealed that TSN has broad-spectrum antitumor activity,exhibiting anticancer effects against human liver,stomach,colon,and breast cancers,but it has not been reported in treatment researches on canine tumors.In this study,we investigated the effects of TSN on the proliferation,migration,invasion and apoptosis of canine breast cancer U27 cells by in vitro cellular assays and constructed an in vivo BALB/c nude mouse transplantation tumor model to investigate the antitumor effects of TSN on canine breast cancer U27 cells and its possible mechanisms,aiming to provide a theoretical basis for the development of targeted drugs for canine breast cancer.(1)Effect of TSN on the proliferation of canine breast cancer U27 cells.the CCK-8method was used to detect the viability of CMT-U27 cells.The morphological changes of CMT-U27 cells were observed by microscopy,the growth of CMT-U27 cells was detected by cell cloning assay,and the DNA synthesis and proliferation of CMT-U27 cells were detected by Ed U incorporation assay.The results showed that TSN treatment with different concentrations(0,1.25,2.5,5,10,and 20 μM)significantly inhibited CMT-U27 cell viability in a time-dose dependent manner(p <0.05),with an IC50 of 19.37 ± 0.79 μM at 48 h,which resulted in concentrations of TSN used in subsequent experiments(5 μM in the low concentration group,10 μM in the medium concentration group,and 20 μM in the high concentration group).After TSN treatment,the cell morphology was observed,and it was found that the cells became rounded and clustered,and the cell gap became wider,and only a few cells survived in the high concentration group.The number of positive cells in the TSN group was significantly lower compared with the control group(p <0.05)in Ed U incorporation assay,and cell proliferation was inhibited.(2)Effect of TSN on the migration and invasion of canine breast cancer U27 cells.The cell migration ability was detected by cell scratch assay,and the cell migration and invasion ability were detected by Transwell assay.The results showed that TSN dose-dependently inhibited the healing of CMT-U27 cell scratches significantly(p <0.05),and the migration rate was 19.01% in the low concentration group,11.92% in the medium concentration group and 7.91% in the high concentration group.The number of migrating cells and invading cells decreased significantly(p <0.05)after TSN treatment in Transwell assay.TSN inhibited the migration and invasion ability of CMT-U27 cells by inhibiting cell proliferation and reducing the number of cells.(3)Apoptosis induction effect of TSN in canine breast cancer U27 cells.Hoechst 33342 fluorescence microscopy was used to detect nuclear morphology,Annexin V/PI flow cytometry to detect apoptosis rate,Western-blot to detect apoptosis-related protein expression,and q RT-PCR to detect apoptosis-related gene expression.The results showed that CMT-U27 cells formed apoptotic vesicles after TSN treatment,and only a small proportion of cells in the high concentration group survived,and the nuclei shrank to a smaller size.Flow cytometry showed that TSN induced early apoptosis in CMT-U27 cells in a dose-dependent manner(p <0.05),with the total apoptosis rate of 24.78% ± 0.59% in the low concentration group,31.37% ± 0.57% in the medium concentration group,and 56.63% ± 0.85% in the high concentration group.Western-blot results showed that increased concentration of TSN upregulated the expression of Bax,cleaved-caspase3,cleaved-caspase9,p53,and cytoplasmic cytcs protein(p <0.01),downregulated the expression of Bcl-2 and mitochondrial cytcs protein(p <0.001).No significant difference of the expression of caspase3 and caspase9(p >0.05)was detected,and the Bax/Bcl-2 ratio was increased(p <0.01).q RT-PCR results showed that increased TSN concentration upregulated the m RNA expression of Bax,p53,and cytcs(p <0.05)and downregulated the expression of Bcl-2 m RNA(p <0.001).mi R-23 a expression was upregulated in CMT-U27 cells(p <0.05),mi R-34 a expression was downregulated(p <0.05),and mi R-374 expression was downregulated(p <0.01).TSN promoted apoptotic response in CMT-U27 cells by regulating apoptosis-related genes and thus affecting related protein expression.(4)In vivo anti-cancer activity of TSN against canine breast cancer.BALB/c nude mouse tumor transplantation model was constructed and used to monitor anti-cancer activity of TSN via body weight,tumor volume and tumor mass of model mice after TSN treatment.Westernblot and q RT-PCR were performed to detect apoptosis-related genes and protein expression in tumor tissue.The results showed that there was no significant difference in body weight(p >0.05),decreased tumor volume(p <0.05)and tumor mass(p <0.05)in the TSN group compared with the control group.(p <0.01).Western-blot results showed that Bcl-2 and intra-mitochondrial cytcs protein expression was down-regulated(p <0.01),and Bax/Bcl-2ratio was increased(p <0.001)in TSN group.q RT-PCR results showed that Bax,p53 and cytcs expression were up-regulated(p <0.01)and Bcl-2 expression was down-regulated(p<0.05)in the TSN group.The expression of mi R-23 a was upregulated in tumor tissues(p<0.05),and the expression of mi R-34 a and mi R-374 was downregulated(p <0.05).TSN affected the expression of related proteins in tumor tissue through regulating apoptosis-related genes and inhibited the tumorigenic ability of CMT-U27 cells in vivo.Conclusion: TSN has effects to inhibit the proliferation,migration and invasion of canine mammary carcinoma U27 cells and to induce apoptosis in vivo;It induces apoptosis of canine mammary carcinoma cells and exerts anti-tumor effects,and the mechanism of it’s apoptosis induction may be achieved through the caspase-cascade reaction in the mitochondrial apoptosis pathway. |
