Molecular Cloning And Expression Of Rana Chensinensis Onconase Gene And Its Anti-tumor Effects On Human Breast Cancer

Breast cancer is the most common cancer in women worldwide. An imbalance between apoptosis and cell proliferation has been implicated in breast cancer development. Through the stimulation of tumorigenic factors, the normal regulation of cell apoptosis could be inhibited at the genetic level, leading to the abnormal proliferation of tumor cells. Meanwhile, the tumor cells can compete for nutrients with normal cells and then cause damages to the normal tissues or organs. The tumor cells are also capable of metastasis, eventually leading to death of patients. With the continuous development of science and technology, the role of anti-cancer drugs increasingly important, therefore, potential therapies for the treatment of patients with breast tumors are required and have been widely investigated. Onconase, which is derived from Rana pipiens, is able to exert cytotoxic effects in various tumor cells, including cervical, breast, colon, pancreatic and prostate cancer cells. Onconase exerts its antiproliferative and cytotoxic effects by interfering with cell-cycle regulation and induction of programmed cell death have been described in many publications. However, the anti-cancer mechanisms of Onconase are not yet fully understood.In the current study, we cloned a novel Rdchonc gene from Rana chensinensis changbaishanensis and expressed in recombinant E.coli BL21(DE3). A significant amount of soluble protein was expressed in the recombinant E.coli. Ribonucleolytic activity assay indicated that the protein has high nuclease activity. To test whether this recombinant Rdchonc have antitumor activity in human breast cancer MCF-7 and MDA-MB-231 cells, MTT and cell invasion assay were performed and confirmed the inhibitory effects on both MCF-7 and MDA-MB-231 cells. To explore the potential mechanisms of anti-tumor effects of Rdchonc treatment on MCF-7 and MDA-MB-231 cells, both cells were treated with 20 μg/ml Rdchonc. Cell cycle analysis revealed that Rdchonc induced G0/G1 cell cycle arrest in MCF-7 and MDA-MB-231 cells at 20 μg/ml, and we found that the treatment induced the cell apoptosis mediated by up-regulation of Bad and down-regulation of Bcl-2 expression.It was reported that MAPK/ERK kinase(MEK)/extracellular signal regulated kinase(ERK) signaling plays a role in oncogenic transformation and ERK is most relevant to human breast cancer. This signaling pathway is activated by cell surface growth factor receptors and induces cellular proliferation, differentiation, and survival. MEK1/2-ERK1/2 signaling pathway has been proposed to be a potential molecular target for cancer therapies. We postulate that ERK may play a key role in this action. We further found that the Rdchonc treated tumor cells with reduced MEK/ERK1/2 phosphorylation. Thus, Rdchonc has an anti-cancer effect that is mediated by Bcl-2 family regulation and MEK/ERK signaling pathway in the human breast cancer.In summary, this study provides support for the comprehensive utilization and in-depth development of the Rana chensinensis. It also provides an insight into the further development and clinical application of the Rana chensinensis Onconase.