The Relationship Between The Expression Of Phosphorylation Of P70 S6 Kinase (P-S6K1) And The Sensitivity Of Rapamycin Therapy In Lung Cancer

Background:Tumor cell growth was regulated by complicated, subtile network. mTOR was especially important in the regulator system, which can integrate the signaling pathway stimulated by amino acide, energy and growth factors. mTOR was involved in multitude cell function, such as genetic transcription,protein translation, ribosomal biosynthesis, cell apoptosis and so on. It has been confirmed presently that mTOR signaling pathway was regulated abnormally in many tumors. The growth factors activated PI3K through transmembrane signaling transduction, then generated PtdIn (3, 4) P2, PtdIn (3, 4, 5) P3, which combined with PDK and AKT by PH domain and activated PDK-1. Activated PDK-1 phosphorylated AKT and activated it, then AKT phosphorylated mTOR directly, activated mTOR affected the downstream subsets. Activated mTOR can control two different subsets at least: p70 S6 kinase (S6K1) and eIF4E-binding protein 1 (4E-BP1), which were critical regulatory factors in protein translation. Rapamycin was a macrolide antibiotic, which can inhibit mTOR specifically and block the phosphorylation of S6K1 and 4E-BP1. Now it was proved that Rapmycin and its derivatives (CCI-779, RAD001, AP23573 and so on) had targeted anticancer role. They can block cancer cell to G1 phase, induce cell apoptosis, inhibit neovascularization, restrain tumor invasion and metastasis. However, National Cancer Institute (NCI) studied Rapamycin had lethal effect on 60 different tumor cell lines, discovered Rapamycin only can inhibit parts of tumor cell lines, especially inhibited breast cancer, ovarian cancer, leukemia, lung cancer, renal cancer and so on. It was proved again by Noh Woo-Chul and Boffa DJ. Noh Woo-Chul firstly discovered Rapamycin sensitivity of various cell lines was different in the same tumor (12/15 breast cancer cell lines were sensitive to Rapamycin), and indicated overexpression of S6K1, expression of phosphorylated AKT and changes in cyclin D1 have concerned with Rapamycin sensitivity of breast cancer cell lines. But there were less reports on Rapamycin sensitivity of lung cancer cell lines, the relationship between P-S6K1 and Rapamycin sensitivity of lung cancer cell lines, the expression of P-S6K1 in lung cancer tissues. In the study , by surveying Rapamycin sensitivity of various lung cancer cell lines and detecting the expression of differential protein in various lung cancer cell lines, the relationship between differential protein (P-S6K1) and Rapamycin sensitivity of lung cancer cell lines was explored, the theory foundation was established for searching for the sensitive marker of mTOR inhibitor-Rapamycin targeted therapy in lung cancer. By detecting the expression of P-S6K1 in lung cancer tissues and normal lung tissues, analyzing the relationship between the expression of P-S6K1 and their clinical pathological character, the relationship between the expression of P-S6K1 and their clinical pathological character and its clinical significance was explored. The study was trying to provide theory foundation for using alternatively mTOR inhibitors (Rapamycin and its derivatives) in lung cancer therapy clinically.Objectives: 1. Human lung cancer cell lines (HLAMP, H1299, A549 and PAa) were treated by mTOR targeted inhibitor-Rapamycin and the inhibition role of Rapamycin on lung cancer cell was investigated. Lung cancer cell lines were divided into Rapamycin sensitive group and non-sensitive group according to inhibite rate.2. The difference of mTOR signaling pathway-associated protein (AKT, S6K1, P-S6K1, eIF4E, 4E-BP1 and so on) in Rapamycin sensitive and non-sensitive lung cancer cell was investigated, the change of the differential protein (P-S6K1) before and after Rapamycin was detected, the relationship between P-S6K1 and Rapamycin sensitivity in lung cancer was analyzed.3. The expression of P-S6K1 protein in lung cancer tissues and normal lung tissues were investigated, the relationship between P-S6K1 expression and clinical pathological character was analyzed. Theory foundation was provided for using alternatively mTOR inhibitors (Rapamycin and its derivatives) in lung cancer patient.Methods: 1. Lung cancer cell lines (HLAMP, H1299, A549 and PAa) were treated by different concentration of Rapamycin, the inhibitory effect on human lung cancer cell lines was determined with MTT method. The lung cancer cell lines were divided into Rapamycin sensitive group and non-sensitive group according to experiment results.2. The expression of mTOR signaling pathway-associated protein (AKT, S6K1, P-S6K1, eIF4E, 4E-BP1 and so on) in two groups of lung cancer cell lines was detected with Western Blot. Differential protein (P-S6K1) was searched. The change of P-S6K1 protein expression before and after Rapamycin treatment was detected with Western Blot, the relationship between P-S6K1 protein expression and Rapamycin sensitivity was explored in lung cancer cell lines.3. The expression of P-S6K1 protein in cancer tissues from 100 lung cancer patients and normal lung tissue from 20 lung nonmalignant disease patients was detected with immunohistochemical method. According to the clinical data, the relationship between P-S6K1 and lung cancer clinical pathological character and the significance was analyzed statistically.Result: 1. With the exception of H1299, HLAMP, A549 and PAa 3 human lung cancer cell lines were inhibited by varied dose of Rapamycin (0.1, 1, 10, 100nmol/L). The inhibition rate was enhanced with the increased drug concentrations (P<0.05), the inhibition rate was largest when the concentration was 100nmol/L. HLAMP, A549 and PAa were sensitive to Rapamycin (inhibition rate≥30%), H1299 was non-sensitive to Rapamycin (inhibition rate<30%). OD of Rapamycin sensitive group (HLAMP, A549 and PAa) between the two consecutive sets were compared, the difference was significant. OD of Rapamycin non-sensitive group (H1299) between the two consecutive sets were compared, the difference was no significant (P>0.05).2. mTOR signaling pathway-associated proteins, including AKT, S6K1, 4E-BP1, eIF4E, were expressed in all four human lung cancer cell lines. P-S6K1 protein was high-level expression in sensitive cell lines, not detected in non-sensitive cell line. And the expression of P-S6K1 was lower after rapamycin (6h, 12h, 18h and 24h) than before rapamycin in HLAMP and A549 cell lines.3. The P-S6K1 positive-expression rate was 15% (3/21) in normal lung tissue, was 62% (62/100) in lung cancer tissue, and in small cell lung cancer tissue (81%, 17/21) was higher than in non-small cell lung cancer tissue (59%, 45/79). The expression of P-S6K1 protein was closely correlated with lung cancer pathological grouping (P<0.05), but not significantly correlated with gender of patient, lymph note metastasis, anatomic site, tumor size, degree of histological differentiation and TNM stage (P>0.05).Conclusion: 1. Rapamycin can inhibit human lung cancer cell growth, but Rapamycin non-sensitive cell lines were exist.2. The expression of P-S6K1 in human lung cancer cell lines may reflect sensitivity to Rapamycin.3. The expression of P-S6K1 was high in lung cancer, was low in lung nonlignant disease. P-S6K1 may be a new index in identification of lung cancer and lung nonmalignant disease.4. The expression of P-S6K1 in small cell lung cancer was much higher than non-small cell lung cancer, the result suggested clinical effect of mTOR inhibitors-Rapamycin and its derivatives may be much better in small cell undifferentiated lung cancer.