Calotropis gigantea extract induces apoptosis through extrinsic/intrinsic pathways and upregulation of reactive oxygen species in non-small-cell lung cancer cells

Abstract

21Background: Calotropis gigantea (CG) plant grows in Asia and tropical Africa. However, the precise mechanisms 22 of its anticancer effects have not yet been examined in human non-small cell lung cancer (NSCLC) cells, A54923 and NCI-H1299 cells. 24Purpose: This study was focused on the anti-cancer effects of CG extract on non-small cell lung cancer (NSCLC) 25 cells. 26Methods: The cytotoxic effects of CG extract on NSCLC, A549 and NCI-H1299 cells, were detected by MTS 27 assay, microscope and DAPI staining. Apoptosis was determined by annexin V-FITC/PI staining, cell cycle 28 analysis, western blotting, quantitative polymerase chain reaction, and JC-1 staining. 29Results: First, CG showed significant dose-dependent cytotoxicity in NSCLC, A549, and NCI-H1299 cells. In 30 addition to induction of caspase-8 processing, CG induced apoptosis by upregulating mRNA expression levels of 31 extrinsic pathway molecules such as Fas, Fas ligand (FasL), Fas-associated protein with death domain (FADD) 32and death receptor 5 (DR5). Also, mitochondrial membrane potential (MMP) was collapsed, and intrinsic pathway 33 molecules such as poly (ADP-ribose) polymerase (PARP), caspase-3, and caspase-9 were processed by CG. 34Moreover, reactive oxygen species (ROS) were generated in a CG dose-dependent manner, and inhibition of ROS 35 by NAC, ROS scavenger, recovered A549 and NCI-H1299 cell viability. 36Conclusion: These results indicate that CG causes apoptosis by activating the extrinsic and intrinsic pathways and 37 generating ROS in NSCLC cells. These results suggest that CG can be used as a lung cancer therapeutic agent. 38 39 Keyword: Apoptosis, non-small cell lung cancer, Calotropis gigantea, anti-cancer, intrinsic and extrinsic 40 pathways, ROS 41 42 Abbreviations used: CG, Calotropis gigantea; NSCLC, non-small cell lung cancer; FasL, Fas Ligand; 43 FADD, Fas-associated protein with death domain; DR5, death receptor 5; PARP, poly (ADP-ribose) 44 polymerase; ROS, reactive oxygen species -3-

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine

“…Calotropis gigantea extracts have been shown to have anticancer activity in a range of cancer cells. C. gigantea whole plant extract exhibited cytotoxicity in A549 and NCI-H1299 non-small-cell lung cancer cells 62 . Ethanolic extracts from roots, flowers, and leaves had cytotoxic effects on T47D breast cancer cells 63 .…”

“…Chemical extracts from several plants promoted anticancer activity by inducing the generation of ROS 40 , 80 – 83 . In the non-small-cell lung cancer cell lines A549 and NCI-H1299, ROS generation after treatment with ethanolic extracts from the whole plant of C. gigantea was shown to mediate apoptotic cell death 62 . Coroglaucigenin isolated from the stems and leaves of C. gigantea, inhibited antioxidant molecules, resulting in enhanced ROS generation 21 .…”

Calotropis gigantea stem bark extract induced apoptosis related to ROS and ATP production in colon cancer cells

“…gigantea plant have been reported to exhibit anticancer activity. The ethanolic extracts prepared from the whole plant exhibited cytotoxicity in A549 and NCI-H1299 non-small cell lung cancer cells after 48 h of treatment [ 44 ]. Ethanolic extracts of the roots and leaves of C .…”