Anti-cancer Properties Of Aromatic Acid Derivatives From "Huoxuehuayu",TCM As Potential COX-2 Inhibitors

Background and Purpose:Driven by the growing wealth of knowledge on how tumor develops,selectively targeting the key molecular steps emerges as one of the leading anticancer therapies.Substantial evidence indicates that over-expressed cyclooxygenase-2(COX-2)orchestrates multiple steps in cancer progression,while non-steroidal anti-inflammatory drugs(NSAIDs)have been highly recommended as chemopreventive agents for cutting the cancer risk based on their COX-2 inhibitory effect.It’s noteworthy that Traditional Chinese Medicine(TCM)for activating blood and resolving stasis or termed "Huoxuehuayu" herb is much appreciated in cancer treatment.However,the relevant mechanism is poorly understood until today.In this study,we identified a group of aromatic acid derivatives widespread in "Huoxuehuayu" TCM,shared common structural skeleton with NSAIDs that contained a COOH group bonded to an aromatic ring,which implied such a perspective that these compounds might have similar inhibitory action on COX activity according to preliminary structure-activity relationship(SAP)analysis.Therefore,we postulated that the potential anti-cancer effect of "Huoxuehuayu" TCM was mediated by sizeable aromatic acid derivatives co-targeting COX-2 in the cancer context.The aim of the study was to exploit the possible role of aromatic acid derivatives from"Huoxuehuayu" TCM in cancer treatment,and the underlying mechanism focused on COX-2.Experimental content:The major part of the research was launched on the non-small-cell lung cancer(NSCLC),a leading systemic malignant disease in both incidence and mortality worldwide.The first chapter determined the COX inhibitory potency of 13 kinds of aromatic acid derivatives from "Huoxuehuayu" TCM by virtual screening for prediction in silico and purified human COX-2 activity assay for validation in vitro.Particularly,the IC50 data and structural parameters of all the drugs were extended to construct a mathematical model by 2D quantitative structure-activity relationships(QSAR)studies.The second chapter was focused on the significance of COX-2 expression in NSCLC.Clinical data were extracted from the publications and sent to meta-analysis to estimate the association between NSCLC and COX-2 expression.We further chose two cell lines of NCSLC that were A549 and NCI-H1650 human lung adenocarcinoma respectively.We also established experimental models in vitro and in vivo to evaluate the differences in the malignant biology of tumor metastasis accompanied by the basal constitutive expression and enzymatic activity level of COX-2.Moreover,COX-2 inducer and inhibitor were introduced to discuss the central role pf COX-2 in NSCLC progression and assess the therapeutic values by inhibiting COX-2 for cancer treatment.The last chapter investigated anti-cancer activity of aromatic acids derivatives from "Huoxuehuayu" TCM and underlying mechanisms based on COX-2.We then used Danshensu(DSS)as a template to dissect the the COX-2 depentdent relationship mediated by aromatic acid derivatives combined with specific enzymatic inhibitor celecoxib to abrogate COX-2 activity,COX-2 substrate arachidonic acid to promote the enzyme-catalyzed reaction and TNF-a to induce COX-2 expression.Key results:(1)The results of the possible interactions between aromatic acid derivatives from"Huoxuehuayu" TCM and COX-2 by molecular docking demonstrated a good binding activity of the test drugs to COX-2.The docking energy was at the range of-60～-120 KJ·mol-1 and the main interaction force involved was hydrogen-bond.By incubating the test drugs with purified human COX-2 and measuring the remaining enzyme activity,and pIC50 were among 3.80～5.5.The quantitative structure-activity relationships(QSAR)studies were also well performed as a prediction model.(2)Meta-analysis collected from case-control studies showed that COX-2 was over-expressed in NSCLC,especially in adenocarcinoma(ADC)and lymph node metastatic patients.Initially,compared to NCI-H1650 cell line,we observed that over-expressed and consequent hyper-activated COX-2 in A549 cell line manifested enhanced capacity of proliferation and migration.COX-2 selective inhibitor celecoxib significantly suppressed the growth of the two cells,and the COX-2 highly expressed A549 NSCLC was more sensitive to celecoxib’s exposure.Additional evidence were observed that TNF-a increased A549 migration,which could be attenuated by Celecoxib.Taken together,COX-2 promotes tumorigenesis and can a potential target for NSCLC.(3)Experiment verification found a strong inhibition of NSCLC growth inhibition by aromatic acids derivatives,which was significantly correlated with their COX-2 enzymatic avrgating activity,some compounds such as rosmarinic acid was also able to inhibit COX-2 protein expression.We took Danshensu as chemical template and found it could retard the growth and movement of NSCLC via inhibiting COX-2 activity but not expression,and inhibition of COX-2 enzyme activity at the cellular level by reducing the arachidonic acid-induced PGE2 production and this role was independent of its physical and chemical properties as potential antioxidants.Signaling pathway anlysis found that Danshensu within 50 μM could deactivate proliferation-related signaling pathways such as ERK and Akt phosphorylation,inhibition of cell migration by abrogating cascade of FAK-Src complex.(4)In A549 NSCLC xenograft nude mice models,DSS(120 mg/kg/day)failed to demonstrate any evidence of reducing the A549 cells growth in vivo.However,DSS prolonged survival time and reduced 50%mortality of A549 bearing-mice compared to the model group,whereas celecoxib at a pharmacological dose(26 mg/kg/day)could significantly retard A549 cells growth in vivo and partly prolong the survival time but the didn’t show any beneficial effect on morality.Conclusion and highlights:Exploration of the substance basis of TCM treating diseases has been one of bottlenecks in the modern research of TCM.To meet this challenge,our study presented a new strategy reversed to the conventional way which was launched from single ingredients to biological activities.By the principle of structure-activity relationship and referring to those clinical chemicals with definite targets and similar actions with TCM,we can excavate groups of potential compounds explicable for TCM functions and underlying mechanisms.Hopefully,such a mode can be served as a bridge to impetus a true understanding of TCM.In a sense,our paper defined a multi-component therapeutic scheme in "Hwoxuehuayu"TCM that might produce an optimal effect to treat related diseases.Additionally,screening of COX-2 inhibitor arguably brings huge economic and social gains in the field of anti-cancer drug discovery,because representative NSAIDs have profound clinical history and it’s not necessary to reboot any human body based security and pharmacokinetics experiments.Therefore,identifying the new COX-2 inhibitors from widely used herbal products with sufficient safety and clinical efficacy deserves much study in the future work.Here,we found the"Huoxuehuayu" TCM such as Salvia miltiorrhiza which has been recognized worldwide and widely applied for treatment of cardiovascular diseases without obvious GI adverse events,is a rich source of potent pharmacological inhibitors of COX-2.The molecular basis of"Huoxuehuayu"TCM in cancer therapy is mediated by the numerous aromatic acids not only via inhibiting COX-2 enzymatic activity but also reducing COX-2 protein expression.All together,the current work might enrich our understanding of the original meaning and theoretical significance of "Huoxuehuayu" TCM and offer valuable opportunities for its clinical anti-cancer practice.