The Regulation Effect Of LIM Kinase1in Osteosarcoma Multidrug Cell

Osteosarcoma is the clinical common malignant tumor，which occurred in themetaphysis of long bones,especially in adolescents and adults. It has character of highmalignancy, recurrence and metastasis. The current clinical treatment of osteosarcomaare surgical operation and neoadjuvant chemotherapy, and the5-year survival rate hasreached60%. Although we has made a great progress in the treatment ofosteosarcoma recently, the treatment effecst of osteosarcoma still needs to beimproved because of multidrug resistance. Therefore, the study on the mechanism ofmultidrug resistance in osteosarcoma has become the important field of molecularbiology.The main mechanism of osteosarcoma mutidrug resistance is by P-glycoproteinmediated. P-gp is a transmembrane protein encoded by MDR1, relative molecularmass170KD.,consisting of1280amino acids. In many normal tissues, such asintestinal epithelial cells, renal tubular cells, bile duct cells, adrenal cortex and bloodbrain barrier has a high P-gp expression. There are higher expressed in resistant tumorcells. Many anticancer drugs were identified by P-gp once they enter into cells bydiffusion. P-gp has conformational change, obtain energy by consuming ATP, and thenefflux the drug into the extracellular. Takada T found BCRP protein is regulated byPI3K/AKT signaling pathway in breast cancer cells. Mutoh found estrogen canactivate PI3K/AKT signaling pathway leading to P-gp, BCRP protein expressionchanges for those estrogen receptor expressed breast cancer cells. Meihong foundestrogen stimulation factor can activate PI3K/AKT/SSH1L signaling pathway.Kazuhiro Katayama found blocking the MAPK signaling pathway coulddown-regulate P-gp protein expression.LIMK1, is a70KD serine/threonine protein kinase. It mainly exists in cytoplasmand be free to shuttle between the cytoplasm and nucleus. Its main physiologicalfunction are Involved in the reorganization of the cytoskeleton protein by inactivatedCofilin. It is important to connect extracellular stimulation and the stability of cellskeleton protein. In recent years, the significance of LIMK1in carcinogenesis causedwidespread concern. The study found LIMK1and high concentration of phosphorylated Cofilin is overexpressed in prostate cancer cells, Inhibition of LIMK1can change the cell proliferation in G/M phase state. Another report showed LIMK1plays an important role in the growth, angiogenesis and invasion in breast cancer. Ournew research suggests inhibition of LIMK1decreased migration and invasion ofosteosarcoma cells. In this study, we put LIMK1as the target to further study theproliferation, migration and tumor resistance effect of osteosarcoma cell so as toprovide theoretical and experimental basis for the study of a new theory.In our previous study,we successfully established a multidrug resistance cellmultidrug osteosarcoma lines, and specified the changes of biological activity ofresistant cells. This study selected cultured human osteosarcoma cell line MG63andMG63/VCR(MG63vincristine resistant cells) as the main object of study. We applythe colony formation assay and cell scratch assay to detect LIMK1in proliferation,migration of MG63cell in vitro,Colony formation assay showed that theproliferation ability of the resistant cell was stronger than the parental cells. However,silencing of LIMK1, the the proliferation ability of the resistant cells were weakerthan the parental cells. Cell scratch assay showed that LIMK1could significantlypromote the drug-resistant cell migration.We detect the different expression of LIMK1and MDR1of the parental cells andresistant cells by many experimental technologies at mRNA and protein level. Theexperiment found that gene and protein expression was higher than that of parent cellin resistant cells. After using gene silencing technology inhibition of LIMK1expression,IC50and P-gp protein were decreased in MG63/VCR cell. We stimulatedMG63\/VCR cells using the Western-blot technology by adding differentconcentrations of VCR to detect AKT activity which may be associated with LIMK1.Results showed that VCR can stimulate MG63/VCR to decrease phosphorylated AKTexpression, which lead to the inhibition of cell proliferation and promote cellsapoptosis.