Doxorubicin Hydrochloride Induces Apoptosis In Canine Breast Cancer Cells

In addition to cutaneous carcinoma, canine mammary cancer is also a common tumor in dogs. To date, surgery combined with chemical drugs is the most effective treatment for canine mammary cancer. Based on the facts that there is no effective chemotherapeutic drugs for canine mammary cancer in clinical veterinary, no theory and basis of applying chemical drugs for treating human cancer in curing canine mammary cancer and the long time and the low success rates when developing new drugs,doxorubicin hydrochloride, which has good performance in treating human drug-resistant tumor in vitro, is chosen to study its effect in treating canine mammary cancer. In our study,the mechanism of apoptosis induced by doxorubicin hydrochloride is stuied, with a view to uncover its effecting targets and mechanism, provide a theoretical basis and address the lack of chemotherapy drugs in clinically treating pet disease.Doxorubicin hydrochloride was selected as the inducing agent and the primary and metastatic canine breast tumor cell lines CHMp and CHMm strains was chosen to be the studied cells. After treated with drugs of different amount, the inhibition of doxorubicin hydrochloride on the replication of the two cancer cells was investigated by CCK-8method and the cell morphology was observed. Then by using three different concentrations of drugs treated cells, morphology were observed after 24 h staining;Whether doxorubicin hydrochloride induces apoptosis in the two cell lines is determined by AO/EB; The effect of doxorubicin hydrochlorid on the activity of Caspase-3, 6, 8, 9 was determined by spectrophotometry; The change of Bcl-2 m RNA, Bax m RNA and p53 m RNA were detected by realtime PCR; Finally, cytoplasmic Bax protein expression level was elevated by Western Blotting.The experimental results showed that doxorubicin hydrochloride with different concentrations could effectively induced apoptosis in canine mammary tumor cells. The drug with higher concentration could have the stronger inhibition ability. It is also observed that after treated with doxorubicin hydrochloride, the expression level of Caspase-3,6,8,9in CHMp cell line was significantly up-regulated(P?0.05); The expression level of Caspase-3(P?0.001) and Caspase-6,8,9 in CHMm cell line was also significantly up-regulated(P?0.05). In addition, drugs with different concentrations could significantly up-regulated the expression level of the p53 and Bax m RNA in CHMp cell line(P?0.001),the p53 m RNA(P?0.05), the Bax m RNA(P?0.001) in CHMm cell line, with no influence or significantly down-regulated the expression level of the Bcl-2 m RNA(P?0.001). The Western Blotting result showed that the drug could improved Bax protein expression in both cancer cells and the protein expression level was in accord with the drug concentration. These experimental result show that doxorubicin hydrochloride could increase cell cycle-associated factor p53, apoptosis factor Bax, death receptor pathway factors of Caspase family, activate apoptosis program and induce cell apotosis.This experimental preliminary uncovers the mechanisms of doxorubicin hydrochloride induced canine mammary tumor cell apoptosis, lays the foundation of scientific theory for clinical application of the drug and provides reference for the clinical application and screening pet clinical anti-tumor drugs.