Calotropis gigantea extract induces apoptosis through extrinsic/intrinsic pathways and reactive oxygen species generation in A549 and NCI-H1299 non-small cell lung cancer cells

CG was dissolved in 0.05% dimethyl sulfoxide (DMSO) and used for biological assays. CellTiter 96® AQueous One Solution Cell Proliferation Assay Reagent [MTS; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] was purchased from Promega (Madison, WI, USA), and propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies specific to PARP, caspase-3, caspase-8, caspase-9, Bcl-2, Bcl-xL, Bax, Bid, and cytochrome c were sourced from Cell Signaling Technology (Beverly, MA, USA). Anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody and anti-mouse IgG HRP-conjugated secondary antibody were obtained from Millipore (Billerica, MA, USA). Antibodies specific to p21, p27, cyclin D1, cyclin E, cyclin A, SOD-2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazoly carbocyanine chloride) was obtained from Enzo (New York, USA), FITC-annexin V apoptosis detection kit I was obtained from BD Biosciences (San Diego, CA, USA), and 2′,7′-dichlorofluorescin diacetate (DCF-DA) was procured from Abcam (Cambridge, UK).

The ethanol extract of the whole plant of C. gigantea (L.) W.T. Aiton (Asclepiadaceae) was supplied by Foreign Plant Extract Bank (No. FBM085–042; Daejeon, Korea). The plant was collected in Yunnan Province of China in 2008 and authenticated by Jin Hang, the Chief of the Medicinal Plants Research Institute, Yunnan Academy of Agricultural Sciences (YAAS) (Yunnan, China). A voucher specimen (YASS3533–2) was deposited at the herbarium of YAAS. To prepare the material, the air-dried whole plant of the C. gigantea sample (100.0 g) was mixed in 95% ethanol (800 mL × 2), and the mixture was shaken at room temperature for 2 h. The extracts were combined and concentrated in vacuo at 40 °C to produce a dried extract, which then was used for phytochemical analysis and biological assays.

Tentative identification of compounds from C. gigantea extracts were carried out using an ACQUITY UPLC (Waters Corporation, Milford, MA) system connected to a Micromass QTof Premier™ mass spectrometer (Waters Corporation, Milford, MA) with an electrospray ionization device. The operation parameters used in the negative ion mode were: capillary voltage, 2300 V; cone voltage, 50 V; ion source temperature, 110 °C; desolvation temperature, 350 °C; flow rate of desolvation gas (N₂), 500 L/h; mass scan range, 100–1500 Da; and scan time, 0.25 s. Leucine enkephalin was used as the reference compound (m/z 554.2615 in negative ion mode). The gradient elution program comprised: 0 min, 10% B; 0–1.0 min, 10% B; 1.0–12.0 min, 10–100% B; wash for 13.4 min with 100% B; and a 1.6 min recycle time. The injection volume was 2.0 mL, and the flow rate was 0.4 mL/min.

A549 and NCI-H1299 cells were purchased from the American Type Culture Collection (ATCC: Manassas, VA, USA). The human keratinocytes HaCaT cells (ATCC) were used as the control cells. The cells were cultured in RPMI 1640 medium (Welgene, Gyeongsan, South Korea) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT, USA) and maintained in an incubator at 37 °C in an atmosphere of 5% CO₂/95% air with saturated humidity.

Cell viability was examined by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The cells were seeded in 100 μL medium/well in 96-well plates (A549 cells: 0.7 × 10⁴ cells/well; NCI-H1299 cells: 0.9 × 10⁴ cells/well) and allowed to grow overnight. After 24 h, different concentrations of CG extract were added, and the cells were returned to the incubator for a further 24 or 48 h. Subsequently, the medium (100 μL) was removed and incubated with 100 μL MTS with PMS mix solution for 40 min to 1 h at 37 °C. The optical density at 492 nm was measured for each well by using an ELISA reader Apollo LB 9110 (Berthold Technologies GmbH, Zug, Switzerland).

A549 cells (1.5 × 10⁵ cells) and NCI-H1299 cells (2.0 × 10⁵ cells) were seeded in 1.5 mL medium/well in 6-well plates overnight. The cells were treated with various concentrations of CG extract for 48 h, harvested using trypsin, and washed with PBS. Annexin V and PI staining were performed by using FITC-Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA) in accordance with the manufacturer’s instructions. The staining was analyzed by flow cytometry using a FACSCalibur instrument and CellQuest software (BD Biosciences, San Jose, CA, USA).

The cell cycle distribution was analyzed by PI (propidium iodide) staining and flow cytometry. A549 (1.5 × 10⁵ cells) and NCI-H1299 cells (2 × 10⁵ cells) were seeded in 1.5 mL medium/well in 6-well plates for overnight growth and treated with various concentrations of CG extract. After 48 h, the cells were harvested with trypsin and fixed with 80% ethanol for > 1 h. Subsequently, the cells were washed twice with cold phosphate-buffered solution (PBS) and centrifuged. The supernatant was removed, and the pellet was re-suspended and stained in PBS containing 50 μg/mL PI and 100 μg/mL RNase A for 20 min in the dark. The staining was analyzed by flow cytometry using a FACSCalibur instrument and CellQuest software (BD Biosciences, San Jose, CA, USA) to calculate the DNA content.

A549 cells were treated with CG for 48 h, harvested, and lysed in 1 mL easy-BLUE™ (iNtRon Biotechnology, SungNam, Korea). The RNA was isolated in accordance with the manufacturer’s instructions, and cDNA was obtained by using M-MuL V reverse transcriptase (New England Biolabs, Beverly, MA, USA). Real-time qPCR was performed using a relative quantification protocol using Rotor-Gene 6000 series software 1.7 (Qiagen, Venlo, Netherlands) and a SensiFAST™ SYBR NO-ROX Kit (BIOLINE, London, UK). The expression of all target genes was normalized to that of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, GAPDH. Each sample contained one of the following primer sets: Fas F: 5′-CGGACCCAGAATACCAAGTG-3′ and R: 5′-GCCACC CCAAGTTAGATCTG-3′; FasL F: 5′-GGGGATGTT TCAGCTCTTCC-3′ and R: 5′-GTGGCCTAT TTG CTT CTCCA-3′; DR5 F: 5′-CACCTTGTACACGATGC TGA-3′ and R: 5′-GCTCAACAA GTGGTCCTCAA-3′; FADD F: 5′-GGGGAAAGATTGGAGAAGGC-3′ and R: 5′-CAGTTCTCAGTGACTCCCG-3′; SOD2 F: 5′-TATAGAAAGCCGAGTGTTTCCC-3′ and R: 5′-GGGATGCCTTTCTAGTCC TATTC-3′; Catalase F: 5′-GGGATCTTTTAACGCCATT-3′ and R: 5′-CCAGTTTACCAA CTGGATG-3′; Thioredoxin F: 5′-GAAGCTCTG TTTGGTGCTTTG-3′ and R: 5′-CTCGAT CTGCTTCACCATCTT-3′; GAPDH F: 5′-GGCTG CTTTTAACTCTGGTA-3′ and R: 5′-TGG AAGATGGTGATGGGATT-3′.

A549 and NCI-H1299 cells were treated with CG at various concentrations for 48 h, harvested, washed with PBS, and centrifuged (13,000 rpm, 1 min, 4 °C). The cell pellets were resuspended in lysis buffer containing 50 mM Tris (pH 7.4), 1.5 M sodium chloride, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and a protease inhibitor cocktail. The cell lysates were mixed on a rotator at 4 °C for 1 h and clarified by centrifugation at 13,000 rpm for 30 min at 4 °C. The protein content was estimated by using a Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and UV spectrophotometer. The cell lysates were loaded onto a 10–12% gel, separated by SDS-polyacrylamide gel electrophoresis (PAGE), and the protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Next, the membranes were blocked with Tris-buffered saline containing Tween-20 (TBST) (2.7 M NaCl, 1 M Tris-HCl, 53.65 mM KCl, and 0.1% Tween-20, pH 7.4) and 5% skim milk for 30 min at room temperature. The membranes were incubated overnight at 4 °C with primary antibodies targeting specific proteins. After three washes with TBST for 10 min each, the membranes were incubated with a secondary antibody (HRP-conjugated anti-rabbit or anti-mouse IgG) for 2 h at room temperature. After three washes with TBST, the blots were analyzed by using a chemiluminescence detection kit (Advanstar, Cleveland, OH, USA). Western blotting bands were quantified by using ImageJ software version 1.5 [22]. The respective band intensities were normalized to GAPDH.

A549 and NCI-H1299 cells treated with CG were collected and fractionated by using the Mitochondria/cytosol fractionation kit (BioVision Inc., San Francisco, CA, USA) in accordance with the manufacturer’s instructions. The treated cells were harvested with trypsin-EDTA and centrifuged at 600×g for 5 min at 4 °C. The cell pellets were suspended in 1 mL cytosol extraction reagent. The suspensions were incubated on ice for 10 min, homogenized in a sonicator, and centrifuged at 16,000×g for 10 min at 4 °C. The supernatant was isolated and centrifuged again at 10,000×g for 30 min at 4 °C; the resulting supernatant, constituting the cytosolic fraction, was transferred to a pre-chilled tube. The resulting pellet, constituting the mitochondrial fraction, was used in subsequent experiments.

We evaluated MMP (Δψm) by JC-1 staining and flow cytometry. A549 (3.8 × 10⁵ cells) and NCI-H1299 (4.3 × 10⁵ cells) cells were seeded into 3 mL medium in a 60-mm culture dish and treated with various concentrations of CG. The cells were harvested with trypsin-EDTA and transferred into 1.5 mL tubes. JC-1 (5 μg/mL) was added to the cells and mixed until it was completely dissolved. Subsequently, the cells were incubated in the dark for 10 min at 37 °C, centrifuged (300×g, 5 min, 4 °C), washed twice with PBS, and resuspended in 200 μL PBS. The solutions were protected from light and analyzed by using a FACSCalibur instrument and CellQuest software (BD Biosciences, San Jose, CA, USA).

We used a DCF-DA cellular ROS detection assay kit (Abcam, UK) to detect the accumulation of intracellular ROS in A549 and NCI-H1299 cells. A549 (0.7 × 10⁴ cells) and NCI-H1299 (0.9 × 10⁴ cells) cells were seeded into 96-well plates and incubated for 24 h in the dark. The cells were then stained with 25 μM DCF-DA for 45 min and treated with various concentrations of CG (0, 3.75, 7.5, and 15 μg/mL) for 48 h. The mean fluorescence intensity (MFI) of each well was quantified by using a fluorescence microplate reader (Gemini EM, Molecular Devices, USA) at the excitation and emission wavelengths of 485 and 538 nm, respectively.

UPLC-PDA-QTof-MS analyses were performed by using a C18 column with a linear gradient of acetonitrile/water. All peaks were characterized by using mass (Fig. 1). Presented in Table 1 are the retention times, UV-Vis absorption maxima, and mass spectral data of the molecular ions of compounds in the CG extract: quercetin 3-rutinoside, kaempferol-4′-O-rutinoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, deglucoerycordin, 15ß-hydroxycalo tropin, frugoside, and trihydroxyoctadecenoic acid. Various rutinosides were found in CG extract and isorhamnetin-3-O-rutinoside, one of the phytochemicals found in this experiment, has been reported to have anti-cancer effects [23].

The cytotoxic effect of CG on HaCaT, A549, and NCI-H1299 cells was determined by using an MTS assay. Three cell lines were treated with different concentrations of CG for different time periods (up to 15 μg/mL for 24 and 48 h). The viability of A549 and NCI-H1299 cells decreased in a dose-dependent manner after CG treatment (Fig. 2b and c), but that of HaCaT human normal keratinocytes was not affected by CG (Fig. 2a), confirming that CG extract exerted cytotoxic effects in A549 and NCI-H1299 human non-small cell lung cancer (NSCLC) cells only. For the positive control sample, A549 and NCI-H1299 cells were treated with doxorubicin, a chemotherapy drug. Similarly, doxorubicin decreased the viability of A549 and NCI-H1299 cells in a dose-dependent manner (Additional file 1. Figure S2). Thus, we focused our subsequent experiments to verify the mechanism through which the CG-induced apoptosis occurred in A549 and NCI-H1299 cells.

As the viabilities of A549 and NCI-H1299 cells for 48 h treated with CG were decreased in a dose-dependent manner, the changes in cell morphology and cell death were observed by using phase-contrast microscopy. Cell morphologies became more rounded and interacted less with surrounding cells after treatment with high concentrations of CG in A549 (Fig. 3a) and NCI-H1299 (Fig. 3b) cells than in the untreated A549 and NCI-H1299 cells. This indicated that CG could alter cell morphology and subsequently induce cell death [24]. For further evidences of the effects of CG, CG treated A549 and NCI-H1299 cells were stained with Annexin V and PI [25]. When apoptosis occurs in the cells, lipid phosphatidylserine (PS) is translocated from the inner to the outer membrane of cells, a so-called “flip-flop” movement, which allows PS to be stained with Annexin V [25]. Furthermore, pores appear in the cell membranes during necrosis or late apoptosis and mediate PI binding to DNA. Annexin V-FITC/PI staining indicated the occurrence of apoptosis in A549 (Fig. 3c) and NCI-H1299 (Fig. 3d) cells after treatment with CG. When both cell types were treated with CG for 48 h, the numbers of early and late apoptotic cells were dramatically increased, and the number of live cells decreased. These results indicated that the death of A549 and NCI-H1299 cells induced by CG was mediated by apoptosis.

p53 is well known as a tumor suppressor protein [26] and it stimulates its downstream factor, p27 [27]. The cyclin-dependent kinase inhibitor p27 has the ability to control the cell cycle, which regulates cyclin D [28]. Proteins in the cyclin family, such as cyclins D1, E, and A, are each involved in specific phases of the cell cycle. The expression of p53 in A549 cells was increased as CG concentration increased (Fig. 4a). In addition, phosphorylated p53 (pp53; the activated form of p53), and p27 were upregulated by CG, whereas p21 was not altered (Fig. 4a). This suggested that p53 and p27 were stimulated by CG and induced the death of A549 cells through inhibiting of the cell cycle. However, in p53-null NCI-H1299 cells (Fig. 4b), p27 and p21 were not affected by CG treatment as expected. The cell cycle of CG-treated A549 (Fig. 4c) and NCI-H1299 (Fig. 4d) cells was analyzed by using flow cytometry. In the sub-G1 phase, apoptotic cells could be distinguished from fragmented DNA, which is a marker of apoptosis [29, 30]. In our study, the cell cycle analysis showed that A549 (Fig. 4e) and NCI-H1299 (Fig. 4f) cells in the sub-G1 phase increased in a dose-dependent manner by CG treatment. Furthermore, cyclin D1, especially related to the sub-G1 phase, and cyclin A were downregulated by CG treatment in A549 (Fig. 4g) and NCI-H1299 (Fig. 4h) cells, although cyclin E was not altered. These results indicated that CG extract inhibited the cell cycle of A549 and NCI-H1299 cells, by inducing the restrictions against unlimited cell growth.

The extrinsic apoptosis pathway is one of the main factors to leading cell death [31]. The interactions between death ligands and death receptors promote the formation of death-inducing signaling complex (DISC), which activates caspase-8 [32]. To confirm the mRNA expression of factors of the extrinsic pathway, real-time qPCR was performed. The mRNA expression of death receptor 5 (DR5), Fas-associated protein with death domain (FADD), Fas, and Fas ligand (FasL) were increased in CG-treated A549 (Fig. 5a) and NCI-H1299 (Fig. 5b) cells. Furthermore, the pro-forms of caspase-8 expression were decreased by CG in a dose-dependent manner, and the cleaved forms appeared after treatment with high concentrations of CG in A549 (Fig. 5c) and NCI-H1299 (Fig. 5d) cells. These results demonstrated that CG effectively induced cell death through the extrinsic apoptosis pathway in A549 and NCI-H1299 cells.

The extrinsic and intrinsic apoptotic pathways intersect in the mitochondria [33]. Activated caspase-8 cleaves the protein Bid. Cleaved Bid induces Bax-dependent outer mitochondrial membrane permeabilization and the release of cytochrome c [9]. In this study, Bid expression level was decreased, whereas Bax was enhanced in A549 cells after treatment with CG (Fig. 6a). Bcl-2, an inhibitory factor in the intrinsic apoptosis pathway, also decreased, whereas the levels of Bcl-xL were unaltered. These levels were altered in a similar manner in NCI-H1299 cells (Fig. 6b). Thus, these results suggested that MMP was decreased owing to mitochondrial dysfunction. The fluorescence of JC-1-stained cells changes from orange to green during the process of apoptosis and during a decrease in MMP. The orange fluorescence of A549 (Fig. 6c) and NCI-H1299 (Fig. 6d) cells exhibited a dose-dependent leftward shift after treatment with CG. Moreover, cytochrome c from the mitochondrial membrane appeared at high concentrations in the cytosol of CG-treated A549 (Fig. 6e) and NCI-H1299 (Fig. 6f) cells, as shown by western blotting. Mitochondrial dysfunction is a very important signal in the intrinsic pathway of apoptosis [33], and collapse of the mitochondrial membrane causes the release of caspase-9. This study confirmed these factors, such as caspase-9 and caspase-3, which are controlled by Bcl-2, were cleaved to induce apoptosis in a dose-dependent manner after CG treatment in A549 (Fig. 6g) and NCI-H1299 (Fig. 6h) cells, as determined by western blotting. The cleaved forms of caspase-9 and caspase-3 were found after treatment with the highest concentration of CG in both cells, and finally, PARP, the key element of DNA repair, was cleaved and inactivated (Fig. 6g and h). In addition, in cells treated with doxorubicin (a positive control group), PARP was cleaved to induce apoptosis (Additional file 1. Figure S3). These results indicated that CG induced apoptosis through the mitochondrial intrinsic signaling pathway in A549 and NCI-H1299 cells.

There are many studies on the relationship between ROS and apoptosis [29, 34]. We examined the generation of ROS, which is another important cause of cell death. ROS levels can be increased dramatically by environmental stress and result in significant damage, termed oxidative stress [5]. Therefore, we investigated whether CG increased ROS levels in A549 and NCI-H1299 cells. CG-treated A549 and NCI-H1299 cells produced ROS in a dose-dependent manner (Fig. 7a and b). Furthermore, the mRNA expression of the ROS scavenger, SOD2, which has an anti-apoptotic role, was decreased in a dose-dependent manner by CG treatment in A549 (Fig. 7c) and NCI-H1299 (Fig. 7d) cells and protein expression of it had a same result in both cells (Fig. 7e and f). In addition, there was a decrease in the expression of catalase, but the expression of thioredoxin (TXN) was not altered (Additional file 1. Figure S4). This study suggested that the generation of ROS mediated CG-induced apoptosis in A549 and NCI-H1299 cells.

To confirm that CG extract induced apoptosis mediated by ROS generation, we used the ROS scavenger NAC [29, 35] to examine cell viability and ROS generation. In the CG/NAC-treated groups, cell viabilities were dramatically recovered to almost 100%, compared with the viability in A549 (Fig. 8a) and NCI-H1299 (Fig. 8b) cells treated with CG only. ROS levels were also decreased both in A549 and NCI-H1299 cells treated with CG and NAC (Additional file 1. Figure S5), compared with the expression in cells treated with CG only. Moreover, NAC restored the decrease in Bcl-2 and Bax after CG treatment in A549 (Fig. 8c) and NCI-H1299 (Fig. 8d) cells. Collectively, these results indicated that CG exerted anti-lung cancer effects through ROS-mediated apoptosis and that the inhibition of ROS generation by ROS scavenger NAC sufficiently blocked CG-induced apoptosis.