The investigation of molecular mechanisms underlying anti-tumour and anti-neuroinflammation induced by Elephantopus scaber in in vitro and in vivo models / Chan Chim Kei

Chan , Chim Kei (2018) The investigation of molecular mechanisms underlying anti-tumour and anti-neuroinflammation induced by Elephantopus scaber in in vitro and in vivo models / Chan Chim Kei. PhD thesis, University of Malaya.

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Abstract

Elephantopus scaber L. is a perennial herb which is traditionally used for the treatment of various diseases including hepatitis, cancer, inflammation and eczema. Enormous attention has been drawn among the researchers to ascertain its multifarious pharmacological benefits especially anticancer. Considerable evidence has revealed that E. scaber exerts anticancer effect against several cancer cells. In this study, the anti-tumour and anti-neuroinflammatory effects of E. scaber leaves and the plausible underlying molecular mechanisms were elucidated using in vitro and in vivo models. The ethanol extract, hexane, ethyl acetate and aqueous fractions of E. scaber exhibited prominent cytotoxicity towards HCT116 and HT-29 colorectal cancer cells. E. scaber ethyl acetate fraction (ESEAF) revealed the most potent effect against HCT116 cells (IC50= 1.42 μg/mL). Induction of apoptosis by ESEAF resulted in the anti-proliferative effect as evidenced by the morphological and biochemical characteristics: nuclear shrinkage, chromatin condensation, DNA fragmentation and phosphatidylserine externalization. ESEAF potentiated the ROS generation and p53 activation leading to mitochondrial membrane potential depolarization. This consequently resulted in caspases cascade (caspase-3/7 and -9) and cleavage of poly (ADP-ribose) polymerase eventually apoptosis. Besides, N-acetyl-L-cysteine (NAC) abrogated the production of ROS and reversed the ESEAF-induced apoptosis implying that ESEAF mediated ROS-dependent intrinsic apoptosis in HCT116 cells. Bioassay-guided approach has led to the isolation of deoxyelephantopin (DET) from ESEAF which remarkably inhibited the growth of HCT116 cells. DET was discovered to induce cell cycle arrest at S phase which was modulated by a concerted action of cell cycle related-proteins including p21, p53, CDK2, CDK4, cyclin D1, A2, E2 and B1. Similar to ESEAF, a ROS inducing agent, DET dysregulated the Bcl-2 family members accompanied by the attenuation of the XIAP and survivin release and the activation of caspase cascades (caspase-8, -10, -9 and -3) implying the involvement of intrinsic and extrinsic pathways. Moreover, mitigation of PI3K/Akt pathway and activation of MAPK pathway were conferred by DET. Furthermore, the event of autophagy occurred upon ESEAF treatment as corroborated by the accumulation of LC3B-II and p62 degradation. In the presence of NAC, HCT116 cells were rescued from apoptosis and autophagy. Importantly, DET prominently suppressed HCT116 tumour growth on the mouse xenograft model which corresponded with the in vitro findings. In addition, the anti-neuroinflammatory effect of E. scaber leaves were investigated in lipopolysaccharide (LPS)-induced microglia cells (BV-2). In acute toxicity assay, oral administration of 2000mg/kg ESEAF did not result in any mortalities and adverse effects which were substantiated by the normal histopathological evidences. ESEAF also attenuated NF-κB translocation leading to the mitigation of the LPS-induced nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2 and prostaglandin E2 production. The production of pro-inflammatory mediators was suppressed via the inhibition of p38. Upon ESEAF treatment, the formation of ROS in LPS-stimulated BV-2 cells was found to decline following the activation of Nrf2 and HO-1 in which it promotes the scavenging activity of antioxidant enzymes and thus ameliorates neuroinflammation. Conclusively, cumulative findings provided the insight for the potential of E. scaber to be developed as nutraceutical for the intervention of cancer and neuroinflammatory disorders.

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