| Mammary gland tumors are the most common tumors in the female dog. Approximately half of canine mammary gland tumors are malignant. It is very crucial to find appropriate biomarkers to contribute to tumor diagnosis, predict outcomes of the disease. Recently, CHIP (Carboxy terminus of Hsc70interacting protein)expression has also been reported as decreased in human mammary and gastric cancere and was believed to be a tumor suppressor protein.In our paper we discussed the significance of CHIP expression in canine mammary tumor. In tumor chemotherapy, bad-side effects and drug resistance are the main cause of failure. The combination of chemotherapy drugs maybe overcome the problems above.5-Fluorouracil (5-FU) is the first-line chemotherapeutic agent in colon cancer and its side effects are relatively high. NVP-BEZ235is the dual inhibitor of PI3K/AKT pathway and has low side effects.Therefore, we investigated the mechanism of the combinational therapy of5-FU and NVP-BEZ235in colon cancer.1. By immunohistochemistry and fluorescence quantitative analysis, we investigated CHIP expression and gene transcription levels in canine mammary tumors. We found that CHIP expression was low in68%of canine malignant mammary tumors while higher expression in87%of canine benign tumors. The immunohistochemical tests of CHIP demonstrated a significant correlation between the CHIP expression and histopathological diagnosis(P=0.007). And the relationship between outcome and immunostaining was statistically different (P=0.034). The transcriptional level of CHIP in normal mammary gland tissues differed significantly from those in malignant tumors (P=0.001). The transcriptional level of CHIP between benign and malignant tumors was significantly different (P=0.009).CHIP expression was a significant prognostic factor for overall survival (P=0.050).2.Using CCK8, flow cytometry and Hochest staining techniques we analyzed cell viability and apoptosis after treatment on HCT-116with the single agent or combination of5-FU and NVP-BEZ235.The results showed that the combination of5-FU and NVP-BEZ235compared with single agent more significantly decreased the viability of cancer cells and induced cancer cell apoptosis.3. In order to further study the mechanism that5-FU and NVP-BEZ235induced apoptosis of HCT-116cells, we studied the changes of p53/PUMA and PI3K/AKT/PUMA signal pathways after5-FU and NVP-BEZ235alone or combined treating on wt-HCT-116, RHO, HCT-116(p53-/-), HCT-116(PUMA-/-) and HCT-116(Bax-/-) cells by the Western blot and RNAi technology. The results showed that5-FU induced cancer cell apoptosis through p53/PUMA pathway with the activation of AKT; NVP-BEZ235induced cancer cell apoptosis by activating PI3K/AKT/PUMA signal pathway and it was p53-independent; the combination induced cancer cell apoptosis by the activation of the both p53/PUMA and P13K/AKT/PUMA pathway, and compared with the single agents more significantly induced cancer cell apoptosis.4In the animal experiment, we confirmed the effect of the treatment on null mice bearing tumors with the single agent or combination of5-FU and NVP-BEZ235by the tumor volume and weight of tumor. The results showed that that the single agent or the combination could significantly inhibit the growth of tumor and induce cell apoptosis and the effect of the combination was better.Our results suggest that CHIP may be a prognostic protein in canine mammary cancer. In addition, we confirmed that the combination of5-FU and NVP-235effectively inhibited the growth of cancer cells, induced the cancer cell apoptosis and inhibited the tumor growth. The results also showed that apoptosis was induced by activating the both p53/PUMA pathway and P13K/AKT/PUMA pathway. In conclusion, the combination of5-FU and NVP-BEZ235maybe provide a more effective method for the cancer treatment. |
