The Research On The Activation Of MAPK In Non-small Cell Cancer And The Effect Of MAPK Pathway In The Antiproliferative Response Of A549 Human Lung Adenocarcinoma Cells To Resveratrol

The p38 mitogen-activated protein kinase (p38 MAPK), c-jun N-terminal kinases/stress-activated protein kinase (JNK/SAPK) and extracellular signal-regulated kinase (ERK) are three main members of MAPK family. Upon activation, MAPK transmits signals from a variety of stimuli from the cell membrane to the nucleus, where it regulates gene transcription through affection chromatin structure and modifying the activity of transcription. Previous studies have revealed that MAPK could play key roles in the activation of inflammatory factor and development, differentiation and apoptosis in cancer cells.Resveratrol (trans-3, 4', 5-trihydroxystilbene) is well-known as an anti-inflammatory, antioxidant and cardioprotective compound. Recently, some studies have also demonstrated the potent cancer chemopreventive effects of resveratrol on a wide variety of tumor cells. However, there has little reports regarding resveratrol's biological effect on the lung cancer cells. Moreover, the precise mechanisms of resveratrol-induced apoptosis in cancer cells remain unclear.To study the effect of MAPK pathway on lung cancer and provide some theory evidence and guide to the clinic treatment scheme of cancer, this subject was dividedinto two parts: the activation of p38MAPK, JNK, and ERK on non-small cell lung cancer were examined by Western-blot and the effect of MAPK in the antiproliferative response of A549 human lung adenocarcinoma cells to resveratrol were analyzed by MTT, Western-bolt, and flow cytometry.Section One expression of phospho-MAPK in human non-small cell cancerMETHODS: Forty-three primary non-small cell lung tumors and paired normal lung tissue from the same patients were collected from the Department of Chest Surgery, First Affiliated Hospital of Zhejiang University School of Medicine in 2006. We investigated the expression of phospho-p38MAPK, phospho-ERK and phospho-JNK in these samples with Western-blot analysis. Then we studied the relationship between clinicopathological parameters and levels of p38MAPK and ERK activation. These clinicopathological parameters included sex, age, tumor size, lymph node metastasis, differentiation, histology type, and histopathologic grading.RESULTS: We found that phospho-p38MAPK and phospho-ERK were obviously activated in the tumor tissues compared with the matched normal lung tissue, while the phospho-JNK was not. In addition, activation of p38MAPK was associated with Tumor size.Section TwoThe effect of MAPK pathway in the antiproliferative response ofA549 human lung adenocarcinoma cells to resveratrolMETHODS: A549 cells were incubated with increasing concentrations (0-100μmol/L) of resveratrol for 6-72 hours. The proliferative response was evaluatedusing MTT assay. After A549 cells were treated with 50μmol/L of resveratrol for 24 hours, we analyzed the cell cycle and the ratio of apoptotic cells in all cancer cells. A549 cells were exposed to increasing concentrations of resveratrol (0-100μmol/L) for 24 h. The protein levels of MAPK expression in resveratrol-treated A549 cells were assessed by Western-blot analysis. The protein levels of caspase-3, cleaved caspase-9, cleaved poly(ADP-ribose) polymerase (PARP),p53 and Bcl-2 family were also quantitated by Western-blot analysis.RESULTS: Resveratrol inhibited proliferation of A549 human lung adenocarcinoma cells in a dose- and time-dependent manner. Resveratrol induced S phase cell cycle arrest and subsequent apoptosis. Western-blot analysis indicated that treatment of A549 cells with resveratrol for 24 h increased the level of cleaved caspase-9 and cleaved PARP fragment and decreased the level of pro-caspase-3. We also found that treatment of A549 cells with resveratrol for 24 h increased the level of phospho-p38MAPK and decreased the level of phospho-ERK, while the level of phosphor-JNK was influenced obviously. In addition, resveratrol treatment results in modulation of the levels of p53 and Bcl-2 family proteins in a manner that promotes apoptosis in A549 cells.CONCLUSIONS1. ERK and p38MAPK were activated in tumor sample compared with normal lung tissue. Activation of p38MAPK was associated with Tumor size and Age of patients. These results suggested that p38MAPK and ERK may play important roles in lung carcinoma.2. Resveratrol inhibited proliferation of A549 human lung adenocarcinoma cells in a dose- and time-dependent manner. Resveratrol induced S phase cell cycle arrest and subsequent apoptosis. The results of our study suggest that resveratrol may be an effective chemopreventive agent for human lung cancer.3. Resveratrol induced death signaling is mediated through thecaspase-9/caspase-3/PARP pathway. It suggested us mitochondria pathway maybeinvolved with resveratrol-induced apoptosis. 4. Resveratrol promotes apoptosis in A549 cells via modulation of thep38MAPK/p53 pathway and Bcl-2 family proteins. It suggested that p38MAPKmight be a convergence point in cancer therapy.