| ObjectiveHepatocarcinoma is one of the first kiljer malignancy in china. Hence, it would be of significance to find out the genetic factors involving the cell infmi-ting growth and signal controUing. There are lots of evidences showing that cancer is a disease uncontroling in apoptosis, and it is a result of losting local net balance in concern between inhibitors and accelerants of angiogenesis. The most important accelerants are bFGF and VEGF, but the mechanism is still unclear. Some reports show that it is involved of PI3K/PKB signal introduction pathway.bFGF, which is widely distributed in body, is an important member of FGF family. It takes part in many physiology and pathology process such as vascular-ization, injure repair, neural regeneration and tumor growth. Much of the function of bFGF is related with the activity of tyrosine protein kinase receptor and MAPK.PKB, which is also called Akt, is one of Ser/Thr protein kinase. The phosphorylation of two sites, Thr in the 308 and Ser in 473 , is necessary for full activity in body. The PI3K activated by growth factor can phosphorylate different substrate and produce corresponding products; PtdIns-3-P, PtdIns-3, 4-P2 (PIP2) and PtdIns-3,4,5-P3 (PIP3). PIP2 and PIP3 are regulated independently by several kinds of kinase and phosphatase. Some reports show that PIP3 can interact with the SH2 and PH domain of some protein. The phosphorylated PKB transfers to the nuclear from the cytoplasm and stimulus the transcription of gene concerned with proliferation and synthesis of protein, and thus, regulates the proliferation of cells.Apoptosis is a process of active cellular self-destruction according to its own programs and controlled by accelerating factors such as p53, Fas and inhibitorssuch as bcl, CAP family and survivin. The sequence of survivin include three phosphoric sites of PKC, two phosphoric sites of Tyr kinase and one phosphoric site of PKA. The mechanism of function in those site is still unclear, but it is believed that CDECHR box regulates the dependent expression of cell cycle. Survivin act on Caspase, and interrupt the process of apoptosis.To elucidate the mechanism of PKB mediated apoptosis, we investigate whether bFGF regulates survivin expression via PI3K/PKB pathway by using wortmannin. We prove that the expression of survivin dependent on the PI3K/ PKB pathway, and thus, provide a new strategy for tumor therapy.Materials and methodsCell-culture; Bel-7402 cells were maintained in a humidified 5.0% CO2 environment in Dulbecoos modified eagle medium( DMEM ) supplemented with 10% FBS, 80 units/ml gentamycin. Western blot assay detect the PKB activity. The expression of survivin mRNA was assessed by RT-PCR; Cell cycle analysis; the cell cycle distribution of semiconfluent cell cultures was determined by follow cytomytric analysis of the content of propidium iodide-stained DNA.Results1. We found that activity of PKB in Bel-7402 cell treated with bFGF (25ng/ml) reached to the peak at 10 min , and activity of PKB in Bel-7402 cell treated with 10 min reached to the peak when the concentration of bFGF is 25ng/ml. The activity of PKB(25ng/ml bFGF, l0min) is 2. 81 fold higher than that in controlling group (p < 0.05).2. The expression of survivin mRNA was increased and peaking at 16h(7. 86-fold induction by 10 ng/ml bFGF) in Bel-7402 ceU.3. wortmannin efficiently inhibited the expression level of survivin mRNA and the activity of PKB ( p < 0.05 ).4. FCM analysis showed that bFGF induced S-phase entry in Bel-7402 cell,which was inhibited by wortmannin effectively. Wortmannin can induce ap-optosis which is about of 50 percent of controlling groups, and decrease the expression level of survivin mRNA, 36 percent of controlling groups at 16h.Conclusion1. bFGF rapidly stimulated the activity of PKB in cytosol in Bel-7402 cell;2. bFGF induced the expression of survivin mRNA in Bel-7402 cell;3. wortmannin, the inhibitor of PI3K, can block die expression of survivin mRNA in Bel-7402 cell;...
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