Background Calotropis gigantea (CG) is a tall and waxy flower that is used as a traditional remedy for fever, indigestion, rheumatism, leprosy, and leukoderma. However, the precise mechanisms of its anticancer effects have not yet been examined in human non-small cell lung cancer (NSCLC) cells. In this study, we investigated whether CG extract exerted an apoptotic effect in A549 and NCI-H1299 NSCLC cells. Methods The ethanol extract of CG was prepared, and its apoptotic effects on A549 and NCI-H1299 NSCLC cells were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining, cell cycle analysis, real-time polymerase chain reaction (RT-PCR), western blotting, JC-1 staining, and ROS detection assay. Results The CG extract induced apoptosis through the stimulation of intrinsic and extrinsic signaling pathways in A549 and NCI-H1299 lung cancer cells. Cell cycle arrest was induced by the CG extract in both cell lines. Reactive oxygen species (ROS), which can induce cell death, were also generated in the CG-treated A549 and NCI-H1299 cells. Conclusions These data confirmed that CG caused apoptosis through the activation of extrinsic and intrinsic pathways, cell cycle arrest, and ROS generation in A549 and NCI-H1299 lung cancer cells. Thus, CG can be suggested as a potential agent for lung cancer therapy. Electronic supplementary material The online version of this article (10.1186/s12906-019-2561-1) contains supplementary material, which is available to authorized users.

Context: Methyl lucidone (ML) from the dried fruit of Lindera erythrocarpa Makino (Lauraceae) exhibits cytotoxic effects in various cancer cell lines. However, its effects on ovarian cancer cells remain unknown. Objective: This study evaluates the mechanism of ML-induced apoptosis, cell cycle distribution in ovarian cells. Materials and methods: The cytotoxic effect of ML (2.5-80 mM) on OVCAR-8 and SKOV-3 cells was evaluated by MTS assay for 24 and 48 h. Apoptosis and cell cycle arrest were analysed by flow cytometry. PCR, western blot analyses were performed to examine the related signalling pathways. Results: ML induced significant cellular morphological changes and apoptosis in ovarian cancer cells, leading to an antiproliferative effect (IC 50 ¼ 33.3-54.7 mM for OVCAR-8 and 48.8-60.7 mM for SKOV-3 cells). Treatment with ML induced cleavage of caspase-3/9 and PARP and release of cytochrome c from the mitochondria. Moreover, ML downregulated the expression of Bcl-2 and Bcl-xL and induced cell cycle arrest in the G 2 /M phase. Additionally, ML suppressed the expression of cyclin-A/B and promoted that of the cyclin-dependent kinase inhibitors p21 and p27. The expression of death receptors was not altered. Interestingly, ML also inhibited the activity of PI3K/Akt and NF-jB. Discussion and conclusions: ML caused G 2 /M phase arrest and apoptosis in ovarian cancer cells by activating intrinsic apoptotic pathways and suppressing the PI3K/Akt survival pathway. ML may be a potential anticancer agent to suppress ovarian cancer proliferation; thus, to improve the survival rate of cancer patients.

The compound (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI) is known as an inhibitor of dual specific phosphatase 1/6 and mitogen-activated protein kinase. However, its precise anti-lung cancer mechanism remains unknown. In this study, the effects of BCI on the viability of non-small cell lung cancer cell lines NCI-H1299, A549, and NCI-H460 were evaluated. We confirmed that BCI significantly inhibited the viability of p53(-) NCI-H1299 cells as compared to NCI-H460 and A549 cells, which express wild-type p53. Furthermore, BCI treatment increased the level of cellular reactive oxygen species and pre-treatment of cells with N-acetylcysteine markedly attenuated BCI-mediated apoptosis of NCI-H1299 cells. BCI induced cellular morphological changes, inhibited viability, and produced reactive oxygen species in NCI-H1299 cells in a dose-dependent manner. BCI induced processing of caspase-9, caspase-3, and poly ADP-ribose polymerase as well as the release of cytochrome c from the mitochondria into the cytosol. In addition, BCI downregulated Bcl-2 expression and enhanced Bax expression in a dose-dependent manner in NCI-H1299 cells. However, BCI failed to modulate the expression of the death receptor and extrinsic factor caspase-8 and Bid, a linker between the intrinsic and extrinsic apoptotic pathways in NCI-H1299 cells. Thus, BCI induces apoptosis via generation of reactive oxygen species and activation of the intrinsic pathway in NCI-H1299 cells.

Cigarette smoke (CS) is a major risk factor for emphysema, which causes cell death in structural cells of the lung by mechanisms that are still not completely understood. We demonstrated previously that CS extract (CSE) induces caspase activation in MRC-5 human lung fibroblasts, activated protein kinase C-η (PKC-η), and translocated PKC-η from the cytosol to the membrane. The objective of this study was to investigate the involvement of PKC-η activation in a CSE-induced extrinsic apoptotic pathway. We determined that CSE increases expression of caspase 3 and 8 cleavage in MRC-5 cells and overexpression of PKC-η significantly increased expression of caspase 3 and 8 cleavage compared with control LacZ-infected cells. In contrast, dominant negative (dn) PKC-η inhibited apoptosis in MRC-5 cells exposed to CSE and decreased expression of caspase 3 and 8 compared with control cells. Exposure to 10% CSE for >8 h significantly increased lactate dehydrogenase release in PKC-η-infected cells compared with LacZ-infected cells. Additionally, PKC-η-infected cells had an increased number of Hoechst 33342 stained nuclei compared with LacZ-infected cells, while dn PKC-η-infected cells exhibited fewer morphological changes than LacZ-infected cells under phase-contrast microscopy. In conclusion, PKC-η activation plays a pro-apoptotic role in CSE-induced extrinsic apoptotic pathway in MRC-5 cells. These results suggest that modulation of PKC-η may be a useful tool for regulating the extrinsic apoptosis of MRC-5 cells by CSE and may have therapeutic potential in the treatment of CS-induced lung injury.

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-