Abstract

Several scientific reports documented the potent anti-cancer effect of various natural products  such as curcumin, epigallocatechin gallate (EGCG), resveretrol via the rise in reactive oxygen species (ROS) and dysfunction of anti-apoptotic gene expressions. The present study evaluated the anticancer potential of lotus derived alkaloids, neferine and isoliensinine on colorectal cancer HCT-15 (CRCs) cells. Neferine/Isoliensinine treatments induced cytotoxicity with sequential enhancement of intracellular ROS, intracellular calcium [Ca2+]i  and mitochondrial membrane potential (ΔψM). Furthermore, cell cycle, Annexin FITC and PI uptake were carried out by flow cytometry. Activation of p38 MAPK and apoptotic genes expression was done by western blotting and reverse transcription polymerase chain reaction (RT-PCR) respectively. Results indicate neferine/isoliensinine induced hyper-generation of ROS was responsible for their cytotoxic effect in CRCs consequently, with a significant increase in [Ca2+]i followed by a significant decrease in the ΔψM. The above treatments induced cell cycle arrest at G1 phase whereas apoptosis was indicated by upregulation of p38 MAPK, Bax, caspase 9, caspase 3, cleaved poly (ADP-ribose) polymerase (PARP) and down-regulation of Bcl2. In conclusion, this is the first study of anticancer effect of neferine/isoliensinine via apoptotic mechanism in CRCs and the results suggest that neferine/isoliensinine induced apoptosis through the ROS generation, activation of p38 MAPK which in turn induces mitochondrial mediated apoptosis.

Key words: Neferine, isoliensinine, reactive oxygen species (ROS), HCT-15, apoptosis.

Abbreviation

AKT, Protein kinase B; CCCP, carbonyl cyanide M-cholorophenyl hydrazine; DAB, 3, 3'-diaminobenzidine; DCFH-DA, 2’,7’-dichlorofluorescin diacetate; DioC6, 3, 3′-dihexyloxacarbocyanine Iodide; Fura-2-AM, fura-2-acetoxymethyl ester; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HRP, horseradish peroxidase; JNK, Janus kinase; mTOR,              mammalian target of rapamycin; NCCS,       National Centre for Cell Science; PARP, poly (ADP-ribose) polymerase; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; TGF, tumor growth factor; TNBC, triple negative breast cancer.