The Screening Of Mitosis Inhibitor And The Studies On The Mechanisms Of Active Compounds

Malignant tumor is currently the major disease threatening hunman's health and life.Discovering novel potent antitumor drugs based on new targets accdoring to the features of tumor cells and new screening models are main task in medicinal chemistry.Separase can regulate the separation of sister chromatids at the metaphase to anaphase transition,which is the key enzyme in mitosis. Inhibition the activity of separase will block mitosis and induce cell death.Our aim is to design a separase inhibitor by using CADD method and to develop as a nvoel antitumor drug.At first,we mold the 3D-structure of separase and then the technique of virtual screen was used to obtain 71 compounds as potential separase inhibitors.MTT assay and flowcytometric analysis showed that a compound named 69# could inhititor the cell growth and induce G2/M block in cell cycle significantly but have no effect on the stability of microtubule or spindle at 50?mol·L-1.We found that LJK-11 could inhibit growth and induced apoptosis of many different types of tumor cells.the IC50 was about 35?mol·L-1.The flowcytometric analysis demonstrated that 69# arrest cells at G2/M phase in a time-and dose-dependent manner.The result of transient transfection separase plasmid DNA to HGC-27 showed that overexpression of separase gene in tumor cell weakened the G2/M block induced by 69# which indicated 69# might inhibit separase and induce cell death.. We also found that 69# had synergistic effect with taxol and vinblastine on inhibiting cell growth and blocking cell mitosis which indicated that separase maybe the target of 69#. But we need more details to prove this point in the follow experiments. Tyrosine kinase inhibitors and mitosis inhibitors are powerful anticancer drugs. Research and development of tyrosine kinase inhibitor and mitosis inhibitors have become a hotpot in the research area of anti-anticancer drugs. In this paper, we have identified LJK-11, a synthetic analog of 5,8-disubstituted quinazolines, as a novel mitotic blocker and tyrosine kinase inhibitor.We found that LJK-11 could inhibit growth and induced apoptosis of many different types of tumor cells. LJK-11 inhibited phosphorylation of HER2. To explore the mechanism of the growth inhibition and cell death effects of LJK-11 on tumor cells, we analyzed the expression and phosphorylation of several major cell growth and survival signaling proteins along the tyrosine kinase signaling pathway, including ERK, JNK, and AKT. The results indicated that LJK-11 had no obvious effect on the phosphorylation of JNK, but increased the phosphorylation of ERK 1/2 and decreased the phosphorylation of AKT, suggesting that inhibition of the PI3K/AKT pathway may be one of the mechanisms that responsible for the LJK-11-induced growth arrest and/or cell death.The immediate cause inducing of cell death was attributed to LJK-11's effect on the cell cycle. It prevented mitotic spindle formation and arrested cells at G2/M phase. In the presence of LJK-11, the chromosomes were unable to achieve bipolar attachment to the spindle and congress to the metaphase plate. The cells therefore remain blocked at prometaphase/metaphase of mitosis and eventually undergoes apoptosis. Detailed in vitro analysis demonstrated that LJK-11 inhibited microtubule polymerization. The mode of action of LJK-11 on microtubule polymerization resembles most closely that of colchicine.LJK-11 also had synergistic effect with colchicine on blocking cell mitosis. It however had neither synergistic nor additive effect with two other anti-microtubule agents, nocodazole or vinblastine. The effect of colchicine on cell mitosis was irreversible, but the effect of LJK-11 was reversible. These data suggest that LJK-11 was different from colchicine.In conclusion, LJK-11 represents a novel anti-microtubule and tyrosine kinase-inhibited agent with therapeutic potential in treating cancer.