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

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


46
Lung cancer, also known as lung carcinoma, is one of the most common diseases in the world [1]. However,

47
owing to very few therapies being available, diverse studies on lung cancer are needed. Lung cancer is classified 48 into non-small cell lung cancers (NSCLCs) and small cell lung cancers (SCLCs) [2,3]. SCLC is a neuroendocrine 49 tumor type, and the size of cells in these cancers is smaller than those in NSCLC. In contrast, NSCLC includes 50 squamous cell carcinomas, large cell carcinomas, and adenocarcinomas. Among these NSCLCs, A549 lung cancer 51 cells (wild-type p53) [4] and p53 null NCI-H1299 lung cancer cells are human non-small cell lung carcinoma cell 52 lines.

53
Lung cancer is caused by uncontrolled cell growth in lung tissues because of defects in cancer suppressor genes

54
[5], which results in the failure of apoptotic signaling and thus cell proliferation. In other words, inducing 55 programmed cell death significantly reduces cancer cell numbers [6]. Apoptosis is a process of programmed cell 56 death that controls cell division. If cells are old, our body causes them to commit suicide by activating an 57 intracellular death process. Cells die by two apoptotic pathways: the intrinsic pathway and extrinsic pathway.

58
First, the intrinsic pathway of apoptosis starts when the mitochondria outer membrane becomes permeable in 59 response to intracellular stressors such as DNA damage, growth factor impairment, or oncogene activation.

60
Cytochrome c and pro-apoptosis proteins are released from permeable mitochondria in a Bax/Bak-dependent 61 manner [7]. These releases are regulated by Bcl-2 family members such as Bcl-2 and Bcl-xL and BH-3 family -6-116

117
Tentative identification of compounds from C. gigantea extracts were carried out using ACQUITY UPLC (Waters

119
Corporation, Milford, MA) with an electrospray ionization device, operating in the negative ion mode, using the 120 following operation parameters: The operation parameters in the negative ion mode were capillary voltage, 2,300

230
UPLC-PDA-QTof-MS analyses were carried out using a C18 column with a linear gradient of acetonitrile/water 231 (Fig. 1). All peaks were characterized using Mass. Table 1 shows the retention times, UV-Vis absorption maxima, 232 mass spectral data of molecular ions of these compounds in the CG extract, namely quercetin 3-rutinoside,

247
The MTS assay showed that the cell viability rate declined in a dose-dependent manner by CG ( Fig. 2A and B).

248
The viabilities of A549 and NCI-H1299 cells treated with the highest (up to 15 µg/ml) CG concentrations at 48 h 249 were both less than 50% that in DMSO (0.01%) treated control cells; the viabilities of A549 and NCI-H1299 cells 250 treated with CG decreased significantly after 48 h. Therefore, CG extract was found to exert cytotoxic effects on 251 lung cancer cells. Thus, our next study was focused on verifying the mechanism underlying NSCLC apoptosis 252 following up to 15 µg/ml of CG for 48 h on NSCLC cells.

H1299 cells 287
Cell cycle disruption is a major cause of apoptosis in cancer calls [21], and many factors such as p53, p27, p21,

288
and cyclins control cell cycle phases. p53 is known as a tumor suppressor gene [22]. When DNA has been 289 damaged, it is activated to induce apoptosis. In addition, phosphorylated p53, p27 and p21, downstream factors 290 of p53, regulate cyclin dependent kinase. The levels of p53 in A549 cells following treatment with 7.5 μg/mL and 291 15 μg/mL of CG extract were increased compared to that of the control, and 3.75 (μg/mL) concentrations and p27 292 was upregulated by CG as same as p53, but p21 was not (Fig. 4A). This suggests that p53 and p27 were stimulated 293 by CG and induced the death of A549 cells by terminating the cell cycle. However, in NCI-H1299 p53-knockout 294 cells, the cell cycle regulatory factors p27 and p21 were not affected by CG treatment (Fig. 4B). The cell cycle 295 analyses of CG treated A549 (Fig. 4C) and NCI-H1299 cells (Fig. 4D) were performed by flow cytometry. Cells 296 in the sub-G1 phase showed fragmented DNA, which is a marker of apoptosis [23,24]. In our study, the cell cycle 297 analysis showed that cells in the sub-G1 phase and S phase increased in a dose-dependent manner with CG 298 treatment for A549 cells (Fig. 4E) and NCI-H1299 cells (Fig. 4F), whereas cells in the G0/G1 and G2/M phases 299 decreased for both cell line. Furthermore, the cell cycle related factors cyclin D1 related to the sub-G1 phase and 300 cyclin A related to the S phase, were downregulated as expected, but cyclin E in A549 cells was not altered by 301 CG treatment (Fig. 4G). However, the levels of cyclin D1, E, and A decreased with treatment of NCI-H1299 cells 302 (Fig. 4H). These results indicated that CG extract had negative effects on the A549 and NCI-H1299 cell cycles,

303
reducing the restrictions against unlimited cell growth. increased in CG-treated A549 (Fig. 5A) and NCI-H1299 cells (Fig. 5C). Furthermore, the pro-forms of caspase-321 8 expression levels decreased in a CG dose-dependent manner, and cleaved forms appeared following treatment 322 with high concentrations of CG in A549 cells (Fig. 5B) and NCI-H1299 cells (Fig. 5D). These results 323 demonstrated that CG was effective in inducing cell death through the process of apoptosis in A549 and NCI-

337
Bid expression level was decreased, while Bax was enhanced in A549 cells following treatment with CG ( Fig.   338  6A). Bcl-2, which is an inhibitory factor in the intrinsic pathway of apoptosis, also decreased, but the levels of

339
Bcl-xL were not altered. These levels were similar in NCI-H1299 and A549 cells (Fig. 6E). Thus, Bax stimulated 340 by Bid and Bcl-2 controlled MMP. The fluorescence of cells stained with JC-1 change from orange to green when 341 apoptosis is in progress and MMP is decreasing. The orange fluorescence of A549 cells (Fig. 6B) and NCI-H1299 342 cells (Fig. 6F) exhibited a dose-dependent leftward shift following treatment with CG. Moreover, cytochrome c 343 from mitochondrial membranes appeared in the cytosol in CG-treated A549 cells (Fig. 6C) and NCI-H1299 cells 344 (Fig. 6G) as determined by western blot. Mitochondrion dysfunction is a very important signal in the intrinsic 345 pathway of apoptosis [27], and mitochondria membrane collapse caused caspase-9 to be released. This study

346
confirmed that factors such as caspase-9 and caspase-3, which are controlled by members of the Bcl-2 family 347 (e.g., Bcl-2 and Bcl-xL) [29] decreased and were cleaved to induce apoptosis in a dose-dependent manner 348 following CG treatment of A549 cells (Fig. 6D) and NCI-H1299 cells (Fig. 6H) as determined by western blot 349 assay. The cleaved forms of these proteins were found following treatment at the highest concentration of CG in 350 both cell lines. This suggests that these cleaved forms activated apoptosis. Finally, the key factor in programmed 351 cell death and DNA repair, PARP, was cleaved by CG in the two types of cells ( Fig. 6D and H). These results

352
indicated CG-induced apoptosis following signaling in the intrinsic pathway.   pre-treated with NAC, and cell viability was confirmed. Cell viabilities dramatically recovered to over 100% in 386 CG and NAC treatment groups, compared with the viabilities in the CG-treated groups of A549 cells (Fig. 8A) 387 and NCI-H1299 cells (Fig. 8B). ROS generation levels also decreased with CG and NAC treatment of A549 (Fig.   388 8C) and NCI-H1299 cells (Fig. 8D), compared to the levels in CG-treated cells. Collectively, these results

389
indicated that CG exerted anti-lung cancer effects through ROS-mediated apoptosis and the inhibition of ROS