Molecular Mechanism Of NKCC1 Promoting Hepatocellular Carcinoma Metastasis And The Exploration Of Its Inhibitor Used As Anticancer Drugs

Cancer is a complex disease. In a particular tissue, the signal response is no longer sufficient for the regulation of cell differentiation, survival, proliferation and death. Cancer cells can proliferate indefinitely, with "eternal life" under suitable conditions, which is not only one of the major biological behaviors of cancer cells, but also the main reason for death. There are six common kinds of cancers worldwide, including lung cancer, liver cancer, stomach cancer, colorectal cancer, breast cancer and esophageal cancer, with increasing rate of incidence and mortality year by year. At present, there are varieties of interventions for cancer therapy, such as surgery, chemotherapy, radiation therapy, and appropriate psychological support. Chemical treatment, also known as drug therapy, has been playing an important role, but its therapeutic effect is influenced by dosage, toxicity and other factors, while small molecule inhibitors with selective killing effects on cancer therapy, such as molecular targets, the active ingredient of small molecule compounds and antibodies targeting tumor cells, can reduce damage to normal tissue, improve treatment effect and reduce side effects, so as to treat cancers or improve the quality of life of patients. In recent years, in order to improve the level of health care, researchers home and abroad have paid a lot of effort for cancer treatment, and achieved remarkable results, but still sorely lack radical means. To understand the source of the molecular mechanisms during cancer development, they are both the most important tasks in the future, therefore, anti-cancer work has a long way to go.Our previous research is concern liver metastasis from in vitro with different metastatic potential of HCC cell lines, tumor-bearing nude mice in vivo model for the study to verify the NKCC1 protein positively correlated with the metastatic potential of HCC, however, by what mechanisms NKCC1 inhibit metastasis of liver cancer cells have not yet been studied. This study focused on selecting the same genetic background, different metastasis of liver cancer cell lines with high metastatic ability MHCC97H cell lines and low metastatic ability MHCC97L cell lines, no metastasis hepatocellular carcinoma cell lines Huh7 and normal liver cells lines LO2 for the study, then they were used to detect the expression of NKCC1 activity, verified WNK1-OSR1/SPAK-NKCC1 pathway key molecules’ total protein and phosphorylation protein expression level involved in among highly metastatic liver cancer cell lines, low metastatic liver cancer cell lines and non-metastatic liver cancer cell lines. The key total protein of WNK1-OSR1/SPAK-NKCC1 pathways involved in and phosphorylated protein was significantly decreased sequentially with its metastatic ability; SBFI detected Na+ concentration in highly metastatic liver cancer cell lines, low metastatic liver cancer cell lines and non-metastatic liver cancer cell lines cells with or without TMZ, NKCC1 activity also showed a decreasing trend in turn. Temozolomide is a kind of chemotherapy drugs, can significantly increase the expression of this pathway in WNK1, OSR1, activity and expression of NKCC1 protein, siRNA interference or OSR1 WNK1, the reduced intracellular sodium ion concentration, and ultimately reduce the NKCC1 activity. Experiments show that, WNK1 and OSR1 by regulating the activity of NKCC1, which led to the transfer of liver cancer cells. NKCC1 inhibitor bumetanide and RNAi interference or OSR1/WNK1 can significantly reduce metastasis of liver cancer cells.Secondly, the use of bumetanide inhibit liver cancer cell proliferation, we expanded the scope of its application, other than the seven selected strains of liver tumor cell lines studied. Combined with the results of previous studies, this paper uses Western Blot verified NKCC1 outside HCC seven kinds of tumor cells, namely lung cancer cell lines (A549), colorectal cancer cell lines (HCT116), chronic myeloid leukemia cell line source (K562), esophageal cancer cell lines (Eca109), cervical carcinoma cell line (Hela), T cell leukemia cell line (Jurkat), breast cancer cell lines (MCF7) in the expression, followed by CCK-8 assay NKCC1 inhibitor bumetanide above seven different tissue sources inhibition rate of tumor cell lines and compared.Bumetanide to lung cancer cell line (A549) and colorectal cancer cell lines (HCT116) inhibition rate is the maximum, ie, the lowest IC50 values in the experiment measured. These laid a strong foundation to improve treatment by a combination method of bumetanide and liver cancer chemotherapy drugs clinically in the future.