Mechanisms Studies Of HMGB1 On The Inflammation Response In Normal Human Bronchial Epithelial Cells And On The Lung Carcinoma Metastasis And Prognosis

Part I. The activation of HMGB1 as a progression factor on inflammationresponse in normal human bronchial epithelial cells throughRAGE/JNK/NF-kB pathwayObjective(s): Non-small cell lung cancer(NSCLC) has been regarded as the leading cause of cancer-related mortality throughout the world. Accumulating evidences showed that inflammation microenvironment on normal human bronchial epithelial(NHBE) cells contributed to the initiation, promotion and development of NSCLC. Extracellular high-mobility group box-1(HMGB-1) has been implicated in the inflammation response leading to the precancerous lesions of non-small cell lung cancer(NSCLC). However, the role of HMGB-1 in the inflammation response in normal human bronchial epithelial(NHBE) cells and its underlying mechanisms were still not fully understood.Methods: In order to screen the optimal stimulation time of HMGB-1, the inflammation-related NF-κB activation and inflammation cytokine IL-10 level at 0, 12, 24, 48 and 72 h was examined after stimulation of 10 μg/ml HMGB-1. To analyze whether HMGB-1 induced NF-κB activation in NHBE cells via RAGE on the cell surface. The specific RAGE-antibody(RAGE-Ab) of 5 μg/ml was used to block the stimulation of HMGB-1. In this experiment, intracellular ROS generation was detected due to the sensitivity of NF-κB activation to ROS overgeneration. To examine whether HMGB-1 induce RAGE activation, immunofluorescence analysis was performed. Inflammation cytokines TNF-α, IL-8, IL-10 and MCP-1 in NHBE cells supernatant were evaluated by ELISA. Western blot analysis was performed to evaluate the activation of HMGB-1 on RAGE, JNK and NF-κB.Results: Our data indicated that HMGB-1 induced inflammation response in NHBE cells through activating RAGE/JNK/NF-κB pathway. The inflammation response in NHBE cells was stimulated by 2.5, 5, and 10 μg/ml HMGB1. However, the receptor for advanced glycation end products(RAGE) blocker RAGEAb(5 μg/ml) or 10 μM c-Jun N-terminal kinases(JNK) inhibitor SP600125 could inhibit HMGB1-induced the release of inflammation cytokines including TNF-α, IL-8, IL-10, and MCP-1 in a dose-dependent manner. Furthermore, HMGB1-induced RAGE protein expression, JNK and NF-κB activation were attenuated by the pretreatment with RAGE-Ab or JNK inhibitor SP600125 in Western blot analysis.Conclusion: Taken together, our data suggested that HMGB1 induced inflammation response in NHBE cells through activating RAGE/JNK/ NF-κB pathway. It might act as a therapeutic target for inflammation leading to the precancerous lesions of NSCLC.Part II. HMGB1 serves as a potential prognostic indicator for lung cellcarcinoma and promotes its invasion and metastasis by MMP-2 viaNF-κB-dependent wayObjective(s): Increasing evidence demonstrated that HMGB1 plays a key role in cancer progression. Our data suggested that HMGB1 induced inflammation response in NHBE cells and it might act as a therapeutic target for inflammation leading to the precancerous lesions of NSCLC. However, HMGB1 expression status and its correlation with the clinicopathological features in lung cancer have not been investigated. In addition, the potential molecular mechanisms underlying the role of HMGB1 in lung cancer are still unknown. This study aimed to detect the clinicopathological and prognostic significance as well as the potential role of HMGB1 in the development and progression of lung cancer.Methods: In order to determine whether HMGB1 expression is changed in human lung cancer, Immunohistochemistry staining was utilized in TMA slides to evaluate the HMGB1 expression in lung cell carcinoma tissues and paired adjacent non-tumor tissues. To further study whether HMGB1 staining in lung cancer patients correlates with a worse prognosis, Kaplan-Meier survival curves were constructed using 5-year overall or disease-specific cumulative survival to compare the patients with high HMGB1 staining to those with low HMGB1 staining. Due to migration and invasion ability is crucial for tumor metastasis, we examined the effects of HMGB1 on migration and invasion of lung cancer cells by transwell assay. To investigate the mechanisms of HMGB1 regulating migration and invasion in lung cancer cells, we performed western blot and gelatin zymography to detect the MMPs protein levels and activities in lung cancer cells. To confirm whether NF-κB activation induced by HMGB1 caused the up-regulation of MMP-2, p65 siRNA and Western blot analysis was used to observe the expression of p65 and MMP-2 in human lung cancer cells.Results: In cohort TMA slide containing 90 cases human lung cancer tissues with paired adjacent non-tumor tissues, we observed that a significantly higher expression of HMGB1 in tumor tissues 51 of 90(56.7%) compared with paired adjacent non-tumor tissues(P < 0.05). HMGB1 expression in the carcinoma tissues of the cohort conspicuously correlated with some clinicopathological features, such as depth of invasion-pT status(P = 0.027), lymph node metastasis-pN status(P = 0.019), and TNM stage(P = 0.012). In the invasion assay, we found that HMGB1 significantly enhanced the ability to migrating through transwell filter inserts. We performed western blot and gelatin zymography to detect the MMP-2 protein levels and activities. Our data showed that the MMP-2 expression and activity were up-regulated by HMGB1 in human lung cancer cells. Moreover, the up-regulated level of MMP-2 protein stimulated by HMGB1 in lung cancer cells could be further blocked by knockdown of p65 expression with a specific siRNA.Conclusion: In summary, our data demonstrated that HMGB1 expression was significantly associated with lung cancer progression. Meanwhile, we demonstrated that HMGB1 promotes lung cancer invasion and metastasis by up-regulating the expression and activity of MMP-2 via NF-κB-dependent ways. These data imply that HMGB1 may be used as a potential prognosis and therapeutic marker for lung cancer.