The Anti-tumor Effects And Molecular Mechanisms Of Cinobufacini On Breast Cancer

Background & Objective: Cinobufacini, a yellow water-soluble extract, is a traditional Chinese medicine prepared from the dried toad skin. The main pharmacological function includes anti-tumor effects, regulation of immune function and anti-viral effects. It has been widely used in clinical therapy for various types of cancers. The anti-tumor effects of cinobufacini on breast cancer were reported rarely, and its mechanisms are also unclear. We observed the effects of cinobufacini on tumor growth and lung metastasis in mouse breast cancer model and breast cancer lung metastasis model in this study. Meanwhile, in vitro, we investigated the inhibition effects of cinobufacini on proliferation and migration of human breast cancer cells MDA-MB-468 and BT549. The experimental results provide a scientific basis for clinical application of cinobufacini.Methods: In order to explore the effects of cinobufacini on the mammary tumor growth, we constructed the mouse breast cancer model by orthotopic implantation. To test the effects of cinobufacini on lung metastasis, we built the breast cancer lung metastasis model by tail vein injection of breast cancer cells RCT-D419. In vitro, MDA-MB-468 and BT549 cells were used to assay the effects of cinobufacini on human breast cancer cells. Cell viability of human breast cancer cells MDA-MB-468 and BT549 was assessed using CCK-8 assay; Cell-cycle status of MDA-MB-468 and BT549 cells was assessed by Propidium Iodide(PI) staining; Cell migration was assessed by scratch assay; Apoptosis-inducing effects in human breast cancer cells MDA-MB-468 and BT549 cells were evaluated by Hoechst33258 staining and Annexin-PI staining; And the expression of cleaved-PARP, p Akt(ser473), p S6 RP and Vinculin proteins were evaluated by Western blot, respectively. The results were analyzed using SPSS v17.0 software.Results: Cinobufacini inhibited the proliferation of human breast cancer cells MDA-MB-468 and BT549. The effect of growth inhibition was elevated with increased dose and extended time. It demonstrated a significant dose- and time-dependent growth inhibitory effect of cinobufacini on MDA-MB-468 and BT549 cells. The induction of G2/M cell-cycle arrest was also seen in the human breast cancer cells MDA-MB-468 and BT549 treated by cinobufacini for 24 h. Cinobufacini suppressed the migration of BT549 cells markedly and suppressed the migration of MDA-MB-468 cells slightly. In FVB mouse breast cancer model and breast cancer lung metastasis model, cinobufacini not only shrank the tumor volume but also inhibited the breast cancer lung metastasis significantly in the treatment of 14 days compared with control group. The apoptosis, induced by cinobufacini, was confirmed by Hoechst33258 staining, Annexin V-PI staining and increased expression of the cleaved-PARP protein in human breast cancer cells MDA-MB-468 and BT549. In Hoechst33258 staining, MDA-MB-468 and BT549 cells, which were treated with cinobufacini for 24 h, showed a typical apoptotic morphology characterized by volume reduction and appearance of apoptotic bodies in nuclei. In Annexin V-PI staining, the apoptotic rate of MDA-MB-468 and BT549 cells was elevated after treated with cinobufacini for 24 h. The results of western blot showed that the expression of cleaved-PARP, the important regulator of apoptosis, increased significantly in MDA-MB-468 cells and BT549 cells. In human breast cancer cells MDA-MB-468, PI3K/Akt signaling pathway was inhibited by cinobufacini. The expression level of p Akt(ser473) and p S6 RP proteins in MDA-MB-468 cells decreased. But the PI3K/Akt signaling pathway was activated in human breast cancer cells BT549, the expression level of p Akt(ser473) and p S6 RP proteins increased after treated with cinobufacini for 24 hours. Combination of cinobufacini and GDC-0941(PI3Kα/δinhibitor) did not induced apoptosis significantly compared with either cinobufacini or GDC-0941 alone, though inhibited the activity of PI3K/Akt signaling pathway and the expression of p Akt(ser473) and p S6 RP proteins in human breast cancer cells BT549.Conclusion: Our data showed that cinobufacini suppressed proliferation, induced G2/M cell cycle arrest and inhibited the activity of migration in human breast cancer cells MDA-MB-468 and BT549. In vivo, we further confirmed that cinobufacini shrank the breast cancer volume and suppressed breast cancer lung metastasis in mouse breast cancer model and breast cancer lung metastasis model. The results in human breast cancer cells MDA-MB-468 and BT549 and mouse breast cancer model indicated that cinobufacini could be a potential agent for treating breast cancer in clinical therapy. We revealed that cytotoxicity of cinobufacini was caused through cell apoptosis by Hoechst33258 staining and Annexin V-PI staining in human breast cancer cells MDA-MB-468 and BT549. Moreover, we found that cinobufacini induced apoptosis of MDA-MB-468 cells via down-regulation of PI3K/Akt signaling pathway. However, BT549 cells exhibited up-regulation of PI3K/Akt signaling pathway in spite of elevated cinobufacini-induced apoptosis. Combination of cinobufacini and GDC-0941 did not induced apoptosis significantly compared with either cinobufacini or GDC-0941 alone, though inhibiting the activity of PI3K/Akt signaling pathway in BT549 cells. Our results suggested cinobufacini-induced apoptosis of BT549 cells through potential PI3K/Akt independent mechanisms that determine dominance of controlling apoptosis over that of PI3K/Akt itself. The specific mechanism should be investigated in future research.