| Background:Oral Squamous Cell Carcinoma(OSCC) was one of the common malignant tumors of the head and neck, with the features of fast proliferation and strong local invasion. Therefore, it has a great significance to study of OSCC proliferation, invasion and early predictor in improving the rates of cure and survival of patients. Squamous cell carcinoma of tongue (TSCC) was the most common malignant in OSCC. Abundant blood supply and frequent activities is special feature of the tongue, so that tongue squamous cell carcinoma in addition to have the characteristics of head and neck cancer, It made the cancer easy to transfer to the neck lymph node, these biological characteristics of tongue squamous cell carcinoma, especially easy early metastasis made the Squamous cell carcinoma of tongue easy to recurrence. This has seriously affected on the prognosis of patients, also caused great difficulties to treatment and prevention of oral and maxillofacial tumors.Cells through a complex network formed by variety transduction pathway, cell can control proliferation and apoptosis signal, PI3K/Akt signal transduction pathway is the classic one which has been certified, the phosphatidylinositol3-kinase (phosphatidylinosito-13kinase, PI3K) and protein kinase B (AKT) is critical protein molecule in the signaling pathway, AKT activation by affecting the downstream activation state of a variety of effector molecules in cells plays a key role in inhibition of apoptosis, promoting proliferation, it is closely related to development human Expression imbalance in a variety of human tumor cells, P13K/Akt signaling pathway regulate tumor cell proliferation and apoptosis, and also been closely related with angiogenesis and tumor invasion and metastasis, it affects the patients prognosis.but it provide a new target for the treatment of cancer which is the focus of cancer research.3-phosphoinositide-dependent kinase-1(PDK1) is key activation kinase of Akt upstream, which directly affects the degree of activation of AKT, thus affecting the regulation of AKT downstream proteins, including the regulation of gene expression, cell cycle regulation, cell proliferation and differentiation. Therefore, with the structure and mechanism of action of the protein molecules has been constantly confirmed, the importance and impact of PDK1in PI3K/Akt signaling pathway, It has been pay more and more attention, to target treatment of signaling pathway has broad application prospects in the areas of anti-tumor.Objective:In order to study the effect of PI3K/PDK1/Akt signaling pathway to proliferation and protein expression of tongue squamous cell carcinoma,The experiments selected a certain concentration gradient Wortmannin, a specific inhibitor of PI3K and OSU-03012of PDK1inhibitor to treatment tongue squamous cell carcinoma Tca8113cultured in vitro, they can blocking or combined blocking PI3K/PDKl/Akt signaling pathways, according to after impact of the Tca8113cell proliferation inhibition and detect of SHIP2protein expression level, we can get some theoretical basis for squamous cell carcinoma of tongue targeted therapy.Materials and methods:1. cell lines:Human tongue squamous cell carcinoma cell lines Tca8113kindly provided by Shanghai Jiaotong University Affiliated Ninth People’s Hospital of Oral and Maxillofacial Surgery Laboratory2. Main material:RPMI-1640medium (including double-antibody), fetal bovine serum (Hangzhou Evergreen), of wortmannin (prismis diffuse penicillin) were purchased from Sigma (USA), dissolved in DMSO. PDK1, the inhibitor OSU-03012purchased from American selleckchem. SHIP2rabbit anti-human polyclonal antibody (c-term)(AP8473e) American ABGENT.3.Methods:1Tca8113cells were cultured in RPMI1640medium with10%fetal bovine serum,Tca8113cell lines cultured at37℃,5%CO2saturated humidity incubator, The medium was changed every two days, until the cell growth of approximately70-80%of the bottom of the culture flask, it be digested by trypsin of0.25%and passaged.2To detect the effect of proliferative activity that wortmannin and OSU-03012respectively and Combination apply to the tongue squamous cell carcinoma cells Tca8113by MTT assay3Using RT-PCR to detected the effection of expression of a certain concentration OSU-03012to Tca8113cells SHIP2protein mRNA.4Using the method of SP cell immunocytochemistry and western blot to detect the expression of SHIP2in Tca8113cellResults:MTT assay of OSU03012(OSU) on Tca8113cell proliferation:At the same site of time, the differences were significant different and statistically significant between different concentration groups.(P<0.05);At the same concentration, Tca8113cell proliferation inhibition rate was gradually increased with the prolongation of time.MTT assay of Wortmannin (WM) on Tca8113cell proliferation:when WM concentration was less than500nmol/L, Tca8113cell proliferation inhibition is not obvious; The effects were more obvious at500nmol/L and1μmol/L concentration of WM to the proliferation of Tca8113cell, with the prolongation of time, Tca8113cell proliferation inhibition is not enhanced.The effect of WM combined with OSU03012on Tca8113cell proliferation show that joint WM can enhance the cell proliferation inhibition rate of the TCA8113cell.The RT-PCR assay which detection SHIP2mRNA expression show that the expression of SHIP2mRNA can be expression in the three groups, and the Oμmol/L group mRNA expression is higher than any other group. The expression level of SHIP2mRNA of expression group is lower than control group. But there was no statistically significant difference between OSU5μmol/L and OSU8μmol/L groups.The Immunocytochemistry and western blot detection SHIP2protein expression was show that SHIP2protein expressed in every group, the Oμmol/L group expression was higher than any other group,the OSU8μmol/L group was the lowest expression level, But there was no statistically significant difference between OSU5μmol/L and OSU8μmol/L groups.Conclusion:1. OSU03012is able to inhibit TCA8113cells proliferation, the TCA8113cell proliferation inhibition rate is gradually increased with the concentration increased and the prolongation of time2. The effects are more obvious at500nmol/L and1μmol/L concentration of WM to the proliferation of Tca8113cell. with the prolongation of time, TCA8113cell proliferation inhibition is not enhanced.3. The effect of WM combined with OSU03012on TCA8113cell proliferation can enhance the cell proliferation inhibition rate.4. SHIP2protein expressed in TCA8113cell, OSU03012can reduce SHIP2protein expression level.5. PDK1/PI3K/AKT signaling pathway plays an important role in the proliferation of TCA8113cells, OSU03012can block the PI3K/PDK1/AKT signaling pathway. |
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