The Effect And Mechanism Of Paeonol On Tumor Malignant Biological Behavior In Inflammation Microenvironment

Background and Purpose:In recent years,’inflammation’ has become an increasingly important driving force to accelerate cancer metastasis, thus attracting people’s great attention to find the molecular regulation mechanism shared between tumor metastasis and inflammation, which becomes the target of varied anti-inflammatory drugs for tumor metastasis treatment in clinic, and a new therapeutic intervention means with certain achievements. Radix Paeonol oniae Alba is a Traditional Chinese Medicine for promoting blood circulation by removing blood stasis. Radix Paeonol oniae Alba and Cortex Moutan are frequently used in classic and proved formulas with anti-cancer effects. Paeonol (Pae), the main active ingredient has a wide range of pharmacological activities, such as antipyretic and analgesic, antibacterial and anti-inflammatory and immune regulation functions, which has already been applied in clinical anti-inflammatory and analgesic, and anti-allergy treatment. Recently, the anti-tumor activity of paeonol has been gradually taken seriously, but its mechanism has not yet been elucidated. This study is to explore the effect and molecular mechanism of paeonol on tumor malignant biological behavior, which will be a useful supplement to Traditional Chinese Medicine for promoting blood circulation by removing blood stasis and its action mechanism of tumor metastasis.Main Methods:The study was divided into three parts. In Part I, MTS and LDH were applied to detect function of paeonol on varied tumor cell growth and its cytotoxic effects; then flow cytometry analysis were used to analyse impact of paeonol on tumor cell apoptosis; further, western blotting analysis was applied for apoptosis protein expression; ELISA detected impact of paeonol on cytokines expression. In Part Ⅱ, the wound-healing assay, the colloidal gold single cell migration assay and transwell invasion assay investigated impact of paeonol on TNF-a and IL-6 induced cell migration and invasion abilities; the western blotting analysis was applied to analyse migration and invasion-related proteins; the mechanism exploration centered on the IL-6-STAT3 and TNF-alpha-NF-kappa B signaling pathway:western blotting analysis for related protein expression; immunofluorescence detection for STAT3 and NF-κB nuclear translocation, and dual-luciferase reporter gene assay for NF-κB promoter transcriptional activity. In Part III, after paeonol in vitro incubation, melanoma experimental model was established to evaluate the impact of paeonol on B16F10 cells in vivo invasive ability changes, with ELISA analysis and fluorescence immunohistochemical approaches detecting inflammation and invasion-related indicators; in vivo administration was applied in melanoma experimental model, pulmonary nodules were counted to evaluate the efficacy, and later with ELISA detecting serum inflammatory expression.Main Results:Part Ⅰ:Larger doses of paeonol can inhibit varied cancer cell proliferation and induce apoptosis, and the mechanism may be related to reducing Bcl-2/Bax ratio and inhibitting cancer cells’ secretion of TNF-alpha, IL-1β, IL-6 and TGF-β inflammatory factor.Part Ⅱ:paeonol can inhibit the TNF-a or IL-6 induced cancer cells migration and invasion, which also involved inhibition of MMPs and influencing EMT-associated protein. In the mechanism study, paeonol can inhibit P-STAT3 protein expression and STAT3 nuclear translocation in IL-6 induced cancer cells. It can also inhibit NF-κB pathway protein expression, NF-κB nuclear translocation and NF-κB promoter transcriptional activity in TNF-α induced cancer cells.Part Ⅲ:In melanoma experimental model after paeonol’s in vitro incubation, paeonol’s effect of inhibiting in vivo invasion changes of B16F10 cells was demonstrated by reducing pulmonary nodules and lung index, and significant correlation between lung tissue inflammation and invasion indicators. The results of paeonol in vivo administration indicated that it did not show markedly improvement in the pulmonary nodules and lung index of B16F10 experimental model; high doses of paeonol can decrease inflammatory cytokine expression in serum.Conclusions and Significance:This study centers on the malignant biological behaviour, proliferation, migration and invasion of tumor cells, conducts in-depth exploration of paeonol, the main active ingredient of Traditional Chinese Medicine for promoting blood circulation by removing blood stasis, and its anti-tumor activity and mechanism. It confirms that paeonol can inhibit proliferation and induce apoptosis, also inhibit tumor migration and metastasis. Its varied mechanisms were related to the anti-metastatic invasion process, which is highly dependent on the inflammatory pathways. Paeonol can affect tumor cell gene expression, such as cytokines (eg. TNF-a or IL-6), and apoptotic proteins (eg. Bcl-2), matrix metalloproteinases (MMPs) and epithelial-mesenchymal transition (EMT) protein (such as E-cadherin) through NF-κB and STAT3, two key transcription factors combining inflammation with cancer, and can affect the malignant biological behaviour through autocrine and paracrine mechanisms.From the aspect of anti-inflammatory activity, paeonol provides us with a new perspective to unlock the anti-tumor effect of Traditional Chinese Medicine for promoting blood circulation by removing blood stasis; moreover, the multilayer collaborative relationship between NF-κB and STAT3 broadens our ideas and methods of combination therapy, and enlightens us to discover deeper space for Traditional Chinese Medicine study, in order to facilitate the development of clinical anti-tumor drugs.