Hemistepsin A induces apoptosis by modulating the reactive oxygen species-dependent PI3K/Akt signaling pathway in human lung carcinoma A549 cells

Abstract

Hemistepsin A is a sesquiterpene lactone isolated from plants of the family. Recently, this compound was reported to be a bioactive compound that is beneficial for numerous health problems without side effects; however, its effect on lung cancer cells has not yet been studied. Therefore, in this study, we investigated the anticancer activity of hemistepsin A in human lung carcinoma A549 cells. This study showed that treatment with hemistepsin A induces apoptosis by activating caspase cascade and reducing the expression of inhibitors of apoptotic protein family members. Additionally, hemistepsin A disrupted mitochondrial integration by altering the levels of Bcl-2 family proteins to increase the cytoplasmic release of cytochrome c. Moreover, hemistepsin A reduced the activation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, and pretreatment with a PI3K inhibitor markedly augmented the cytotoxic effect of hemistepsin A on A549 cells. Furthermore, hemistepsin A significantly enhanced the production of intracellular and mitochondrial reactive oxygen species (ROS), whereas ROS scavengers restored the reduced viability by attenuating DNA damage and apoptosis by blocking the hemistepsin A-mediated inactivation of the PI3K/Akt pathway. Our findings demonstrate that hemistepsin A induces apoptosis in A549 cells by generating ROS, which subsequently inhibits the PI3K/Akt pathway, suggesting that ROS generation is involved as an early inducer of hemistepsin A-mediated anticancer activity.

INTRODUCTION

Sesquiterpene lactones are a class of natural terpenoid compounds most commonly found in plants of the genus Centaurea [1,2]. They contain a cyclic arrangement with 15 carbon atoms and are characterized by the presence of an α-methylene-γ-lactone moiety [3]. Several studies have reported that these natural terpenoids have multiple physiological properties, such as anti-parasitic, antioxidant, anti-inflammatory, anti-fibrotic, anti-metabolic, cardiovascular, and neurological protective effects, without side effects [4-7]. The anticancer effects of sesquiterpene lactones have been extensively reported over the past few years in various tumor cell lines and in in vivo experimental models. They can block angiogenesis, cell migration, and invasion and improve chemosensitivity to anticancer drugs through multiple mechanisms of action [8-10]. Moreover, many exhibit anticancer activity by mediating intracellular signaling molecules to increase cell cycle arrest, autophagy, and apoptosis [11-13]. In certain cancer cells, including hepatocellular carcinoma, colorectal cancer, leukemia, and lung cancer, sesquiterpene lactones inhibit cell proliferation in a reactive oxygen species (ROS)-dependent manner [14-17]. Numerous studies have shown that excessive production of ROS by oxidative stress acts as a detrimental factor in normal cells and is recognized as a critical regulator of chemotherapeutic agent-mediated apoptosis in many types of cancer cells [18-20].

In addition, the anticancer activity of hemistepsin A, a sesquiterpene lactone extracted from Hemistepta lyrata (Bunge), which belongs to the family Compositae, was recently confirmed in several human cancer cell lines [16,21-25]. Although hemistepsin A blocked oxidative stress-induced cell damage by inhibiting ROS production under conditions that did not cause cell damage in human keratinocytes [26], ROS production was a major factor in the induction of apoptosis in human cancer cell lines. For example, Kim et al. [21] reported that hemistepsin A induces apoptosis in prostate cancer cells, which could be blocked by protective autophagy signaling by activating ROS-mediated AMP-activated protein kinase (AMPK) signaling.

Since the activation of AMPK by ATP depletion is associated with abnormalities in energy metabolic pathways, their results support the finding that various sesquiterpene lactones induce apoptosis through mitochondrial damage [27,28]. Furthermore, in colorectal cancer, hemistepsin A has been shown to induce mitochondrial ROS-mediated apoptosis by enhancing the metabolic conversion from glycolysis to mitochondrial oxidative phosphorylation [23]. Cho et al. [22] suggested that hemistepsin A enhanced the sensitivity of sorafenib-mediated cytotoxicity in hepatocellular carcinoma cells by promoting ROS production and the accumulation of glutathione protein adducts, which correlated with the inhibition of transcriptional activation of signal transducer and activator of transcription 3. However, studies on the anticancer activity and related mechanisms of hemistepsin A are limited and have not yet been conducted in human lung cancer cells. Therefore, the objective of this study was to examine the role of ROS in the anticancer activity of hemistepsin A derived from H. lyrata in human lung carcinoma A549 cells.

METHODS

Cell culture and hemistepsin A treatment

A549 cells (CCL-185; American Type Culture Collection) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and antibiotic mixtures at 37°C in an atmosphere of 5% CO₂ in the air. All materials necessary for cell culture were purchased from WelGENE, Inc. Hemistepsin A isolated from H. lyrata (Bunge). Bunge [29] was kindly provided by Professor Sang Chan Kim (Daegu Haany University, Gyeongsan, Republic of Korea) and solubilized in dimethyl sulfoxide (Thermo Fisher Scientific, Inc.) to prepare a stock solution, which was then diluted to various concentrations with the culture medium before treating the cells.

Cell viability detection

A549 cells were cultured in a medium containing various concentrations of hemistepsin A for 48 h or pretreated with or without 20 µM carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD-fmk), 50 µM necrostatin-1 (Sigma-Aldrich Co.), 10 µM LY294002 (Cell Signaling Technology, Inc.), or 10 mM N-acetyl-L-cysteine (NAC, Thermo Fisher Scientific, Inc.) for 1 h and then treated with or without 20 µM hemistepsin A for 48 h. After treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed as previously described [30]. Cell images were obtained using an inverted phase-contrast microscope (Carl Zeiss).

Quantitative assessment of apoptosis using flow cytometry

To investigate the degree of apoptosis induction in A549 cells cultured under different conditions, an Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection Kit (BD Biosciences) was used. According to the manufacturer’s procedure, harvested cells were suspended in binding buffer, and annexin V-FITC and propidium iodide (PI) buffer were added and allowed to react for 20 min, according to a previously described method [31]. The cell suspension was then analyzed with the Muse Cell Analyzer (Millipore Corporation).

Assessment of apoptosis by nuclear morphology change

To evaluate the effects of hemistepsin A on nuclear morphology, 4′,6′-diamidino-2-phenylindole (DAPI) staining was carried out. Briefly, cells were collected, fixed with 3.7% paraformaldehyde (Sigma-Aldrich Co.) for 10 min, and then stained with 1 μg/ml DAPI (Thermo Fisher Scientific, Inc.) for 10 min [32]. The morphology of the DAPI-stained nuclei was observed using a fluorescence microscope (Carl Zeiss).

Protein extraction and immunoblot analysis

Whole-cell lysates were prepared from control and hemistepsin A-treated cells in the presence or absence of NAC, as previously described [33]. Mitochondrial and cytoplasmic fractions were isolated using a Mitochondrial Fractionation Kit (Active Motif Inc.). Equal amounts of protein were fractionated via electrophoresis using sodium dodecyl sulfate-polyacrylamide gels and transferred to Immun-Blot PVDF membranes (Bio-Rad Laboratories, Inc.). The membranes were hybridized with primary antibodies and incubated with secondary antibodies conjugated to horseradish peroxidase. Immunoreactive proteins were visualized using an enhanced chemiluminescence kit (Sigma-Aldrich) according to the manufacturer’s protocol. Antibodies were purchased from Santa Cruz Biotechnology, Inc., Abcam, Inc., and Cell Signaling Technology, Inc. Cytochrome c oxidase subunit IV (COX IV) and β-actin were used as loading controls for mitochondrial and cytosolic proteins.

Analysis of caspase activity

The activity of each caspase was calculated using Colorimetric Caspase Activity Assay Kit (Abcam, Inc.) based on the hydrolysis of fluorescent substrate peptides by activated caspases. Briefly, after resuspending the cells in a cell lysis buffer, the supernatants were reacted with each caspase substrate according to the manufacturer’s instructions. Finally, the concentration of p-nitroaniline released from the substrates was determined using a microplate reader.

Mitochondrial membrane potential (MMP) assay

To analyze the MMP of A549 cells according to hemistepsin A treatment, 5,5,6,6’‐tetrachloro‐1,1’,3,3’‐tetraethylbenzimi‐dazoylcarbocyanine iodide (JC-1) fluorescent dye (Abcam, Inc.) was used. According to the protocol, cells treated with hemistepsin A were collected and immediately stained with 10 μM JC-1 for 30 min. MMP values were calculated using flow cytometry [34], and cells stained with JC-1 were imaged using a fluorescence microscope [35].

Measurement of ROS production

The levels of intracellular and mitochondrial ROS were measured using 2',7'-dichlorofluorescein diacetate (DCF-DA) and MitoSOX Red (Thermo Fisher Scientific, Inc.), respectively. which could be oxidized into fluorescent DCF by ROS. In brief, hemistepsin A-treated cells with or without NAC were incubated with 10 μM DCF-DA (Cayman Chemical Co.), and then the levels of intracellular ROS were measured using flow cytometry, following the manufacturer’s instructions. In brief, cells were cultured for 1 h in the presence or absence of 10 mM NAC or 2 μM Mito-TEMPO (Sigma-Aldrich Co.), then treated with hemistepsin A for 1 h, and then incubated with 10 μM DCF-DA (Cayman Chemical Co.) or 5 μM MitoSOX at 37°C with 5% CO₂ for 20 min, and then the levels of intracellular ROS were measured using flow cytometry, following the manufacturer’s instructions. The levels of DCF-DA fluorescence in the cells were detected using fluorescence microscopy, following the same procedure as previously described [36]. For mitochondrial ROS labeling, cells were incubated with 0.5 μM MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. The cells were stained with 200 nM Mitotracker Red, a mitochondrial marker (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Nuclei were counterstained with DAPI, and fluorescence images of MitoSOX and Mitotracker were acquired using a fluorescence microscope. Nuclei were counterstained with DAPI, and fluorescence images of MitoSOX were acquired using a fluorescence microscope.

Comet assay (single gel electrophoresis assay)

A comet assay was used to assess DNA damage. Briefly, hemistepsin A-treated cells in the presence or absence of NAC were collected, and the comet assay was performed using the Comet Assay Kit (Trevigen, Inc.), as described by the manufacturer. Randomly selected images were acquired using a fluorescence microscope.

Detection of 8-hydroxy-2'-deoxyguanosine (8-OHdG) for DNA damage analysis

After harvesting and washing, the levels of 8-OHdG, a common oxidized nucleoside in DNA, were determined using an enzyme-linked immunosorbent assay kit (Abcam, Inc.). The cells were mixed with the buffer and standards provided in the kit according to the manufacturer’s instructions and then reacted with the 8-OHdG antibody. The cells were then washed with buffer, and the absorbance was read using an ELISA reader (Dynatech Laboratories) at 405 nm, as previously reported [37].

Statistical analysis

All data were statistically analyzed using GraphPad Prism 5.03 software (GraphPad Software Inc.) using an unpaired two-tailed Student’s t-test and one-way analysis of variance. All results are expressed as the mean ± standard deviation (SD) of at least triplicate independent experiments. p-values lower than 0.05 were considered significant.

RESULTS

Hemistepsin A inhibited cell survival and induced apoptosis

Before conducting this study, we compared the cytotoxicity by treating various concentrations of hemistepsin A for different periods of time, and set the appropriate time for studying the mechanism of cell death induction to 48 h and the highest treatment concentration to 20 µM. As shown by the MTT assay results in Fig. 1A, hemistepsin A suppressed the viability of A549 cells in a concentration-dependent manner. Fig. 1B shows the morphological changes of A549 cells after 20 µM hemistepsin A treatment. Compared to that in the control cells, after incubation with hemistepsin A, cell morphology was severely distorted, resulting in branching and loss of contact with adjacent cells. In addition, microscopic fluorescent examination using DAPI staining showed that hemistepsin A-treated cells exhibited chromosomal fragmentation and condensation and nuclear blebs, which are characteristics of apoptosis-induced cells in the nucleus (Fig. 1C, D). Therefore, flow cytometry was performed using Annexin V/PI double staining to determine the frequency of apoptosis induced by hemistepsin A treatment. As represented in Fig. 1E, with increasing concentrations of hemistepsin A, the proportion of annexin-positive cells, indicating apoptosis, was markedly elevated compared to that in the untreated control cells. As represented in Fig. 1E, when the treatment concentration of hemistepsin A was 5 µM or higher, the proportion of annexin-positive cells, indicating apoptosis, was markedly elevated compared to that in the untreated control cells. These data indicate that hemistepsin A-induced inhibition of A549 cell proliferation was due to the induction of apoptosis. These data indicate that hemistepsin A-induced inhibition of A549 cell proliferation was due to the induction of apoptosis.

Fig. 1. Inhibition of cell viability and induction of apoptosis by hemistepsin A in A549 cells.

(A) Cell viability of A549 cells treated with the indicated concentrations of hemistepsin A for 48 h was analyzed using the MTT assay. (B) Cells were treated with 20 μM hemistepsin A for 48 h and imaged under an inverted phase-contrast microscope (magnification 200×). (C) Morphological changes in DAPI-stained nuclei were captured using a fluorescence microscope (magnification 400×). (D) Frequencies of apoptotic nuclei were expressed as a percentage of the total number of cells. (E) After staining with annexin V-FITC/PI, the degree of apoptosis induction was analyzed using flow cytometry. (E) Frequencies of apoptotic cells were expressed as a percentage of annexin V-positive cells to the total number of cells. **p < 0.01, and ***p < 0.001 vs. control cells. Each number represents the total frequency of cells in the early stage (annexin V positive) of apoptosis and cells in the late stage (annexin V and PI double positive) of apoptosis. **p < 0.01, and ***p < 0.001 vs. control cells. Values are presented as mean ± SD. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4’,6’-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Hemistepsin A activated caspases

To verify the underlying mechanisms of hemistepsin A-mediated apoptosis in A549 cells, we investigated changes in apoptosis-related proteins. As indicated in Fig. 2A, when A549 cells were exposed to hemistepsin A, the levels of the inactive forms of caspase-8, -9, and -3 were greatly reduced, and their activities were significantly increased (Fig. 2B). In addition, hemistepsin A treatment suppressed the expression of apoptosis protein (IAP) inhibitor of apoptosis protein (IAP) family members (Fig. 2C). Moreover, hemistepsin A greatly promoted the expression of the representative pro-apoptotic protein Bax but reduced the expression of Bcl-2, a member of the anti-apoptotic protein family (Fig. 2C). Next, we used z-VAD-fmk, a pan-caspase inhibitor that irreversibly binds to the catalytic site of caspases, to determine whether hemistepsin A-induced apoptosis is caspase-dependent. As shown by the MTT assay results in Fig. 2D, z-VAD-fmk strongly rescued hemistepsin A-induced cytotoxicity. However, necrostatin-1, a necroptosis inhibitor, failed to abrogate the inhibitory effects of hemistepsin A on cell viability (Fig. 2E). These results suggest that hemistepsin A induces caspase-dependent apoptosis but not necroptosis in A549 cells.

Fig. 2. Activation of caspase and reduction of IAP family member expression in hemistepsin A-treated A549 cells.

(A, C) After 48 h of treatment with hemistepsin A at the indicated concentration, the expression change of the indicated proteins was investigated using the total protein and the corresponding antibodies. Equal loading was confirmed with β-actin. (B) Activity of each caspase was expressed as a relative value of the untreated control group. (D, E) Cells were pretreated with 100 μM z-VAD-fmk or 50 μM necrostatin-1 for 1 h and subsequently treated with 20 μM hemistepsin A for 48 h. Cell viability was analyzed using the MTT assay. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control cells; ^##p < 0.01 vs. hemistepsin A-treated cells. Values are presented as mean ± SD. IAP, inhibitor of apoptosis protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; NS, not significant.

Hemistepsin A-induced mitochondrial impairment

To explore the role of mitochondria in hemistepsin A-induced apoptosis, we examined whether hemistepsin A alters mitochondrial permeability. As shown in Fig. 3A, flow cytometry results showed that hemistepsin A treatment disrupted MMP, which was associated with the upregulation of cytochrome c levels in the cytosolic fraction, together with a concomitant downregulation at the mitochondrial level (Fig. 3B). The fluorescence intensity of the JC-1 cells was measured using a fluorescence microscope. As MMP was lost in the cells treated with hemistepsin A, the J-aggregates dissipated into monomers, leading to a shift from red to green fluorescence (Fig. 3C). These data demonstrate that hemistepsin A can destroy mitochondrial integrity by regulating the Bcl-2 family of proteins in A549 cells.

Fig. 3. Induction of mitochondrial dysfunction by hemistepsin A in A549 cells.

(A) To measure MMP changes, cells stained with JC-1 were subjected to flow cytometry. (A) Representative histogram profiles were represented (values at the bottom of the box indicate the percentage of cells with depolarized mitochondrial membranes). (B) Statistical analysis results of cells with loss of MMP after treatment with hemistepsin A were represented. (C) Statistical analysis of the frequency of JC-1 monomers indicating MMP loss is presented. (B) After the separation of mitochondrial (MF) and cytoplasmic fractions (CF), the expression of cytochrome c was detected using immunoblotting. COX IV and β-actin were probed as loading controls for each fraction. (C) JC-1-stained cells were observed under a fluorescence microscope (cells with high MMP show red fluorescence, while cells with low MMP show green fluorescence). Nuclei were counterstained with DAPI (blue). ***p < 0.001 vs. control cells. Values are presented as mean ± SD. MMP, mitochondrial membrane potential; JC-1, 5,5,6,6’‐tetrachloro‐1,1’,3,3’‐tetraethylbenzimi‐dazoylcarbocyanine iodide; COX IV, cytochrome c oxidase subunit IV; DAPI, 4’,6’-diamidino-2-phenylindole.

Hemistepsin A increased the production of ROS

Subsequently, DCF-DA staining was performed to determine whether the mitochondrial dysfunction caused by hemistepsin A was induced through ROS generation. Flow cytometry results indicated that intracellular ROS production increased within 30 min of treatment with hemistepsin A, peaked after 1 h, and then decreased (Fig. 4A). However, in the presence of NAC, an ROS quencher, the hemistepsin A-induced accumulation of intracellular ROS was significantly suppressed at the control level. In addition, ROS generation in cells was confirmed using immunofluorescence staining under the same experimental conditions. Consistent with the flow cytometry data, after 1 h of hemistepsin A treatment, the intensity of green fluorescence, indicating intracellular ROS generation, was much stronger than that in untreated control cells (Fig. 4B). Similar to the flow cytometry results, the hemistepsin A-induced green fluorescence intensity was very low in cells pretreated with the ROS scavenger NAC. To confirm that mitochondria are the major source of ROS generated by hemistepsin A, we used MitoSOX, a specific dye for mitochondrial ROS, and observed that the fluorescence intensity of MitoSOX significantly increased in hemistepsin A-treated cells (Fig. 4C). However, in the presence of Mito-TEMPO, a mitochondrially targeted antioxidant, the fluorescence intensity of hemistepsin A-induced MitoSOX was significantly reduced (Fig. 4C). Furthermore, the cell viability of A549 cells, which was reduced by hemistepsin A treatment, was significantly attenuated by pretreatment with NAC as well as Mito-TEMPO (Fig. 4D), suggesting that mitochondrial ROS generation may underlie mitochondrial dysfunction induced by hemistepsin A.

Fig. 4. Increased ROS accumulation by hemistepsin A in A549 cells.

(A) Cells were incubated at different times in a medium containing 20 μM hemistepsin A or pretreated with 10 mM NAC for 1 h and then treated with 20 μM hemistepsin A for 1 h. After DCF-DA staining, the frequency of DCF-positive cells was analyzed using a flow cytometer. (B) Cells treated with NAC for 1 h or not were exposed to hemistepsin A for 1 h, and then the degree of ROS generation level (green) was confirmed using fluorescence microscopy. Nuclei were identified using DAPI staining (blue). (C) Cells treated with 2 μM Mito-TEMPO for 1 h or not were exposed to hemistepsin A for 1 h. After treatment, cells were stained with MitoSOX (red) to detect mitochondrial ROS, and nuclei were identified using DAPI staining (blue). (D) Cells were pretreated with 10 mM NAC or/and 2 μM Mito-TEMPO for 1 h and then treated with 20 μM hemistepsin A for 48 h. Cell viability was analyzed using the MTT assay. ***p < 0.001 vs. control cells, ^###p < 0.001 vs. hemistepsin A-treated cells. Values are presented as mean ± SD. ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; DCF-DA, 2’,7’-dichlorofluorescein diacetate; DAPI, 4′,6′-diamidino-2-phenylindole.

Hemistepsin A triggered ROS-mediated DNA damage

We further analyzed whether the induction of cytotoxicity by hemistepsin A treatment correlated with DNA damage. Immunoblotting results demonstrated that γH2AX phosphorylation (p-γH2AX), a sensitive marker of DNA damage response, was increased in a concentration-dependent manner after exposure of A549 cells to hemistepsin A (Fig. 5A). This indicated that DNA damage was induced by hemistepsin A and was consistent with the results obtained by the comet assay, which detects single-strand breaks and the generation of 8-OHdG, a marker of oxidative DNA damage (Fig. 5C, D). However, γH2AX phosphorylation was almost completely abolished in hemistepsin A-treated cells in the presence of NAC (Fig. 5B). In addition, DNA-forming tail-like structures and increased 8-OHdG levels were offset in cells in which ROS production was blocked (Fig. 5C, D), suggesting that ROS are essential for hemistepsin A-induced DNA damage in A549 cells.

Fig. 5. Induction of ROS-dependent DNA damage by hemistepsin A in A549 cells.

Cells were incubated for the indicated concentration of hemistepsin A or pretreated with 10 mM NAC for 1 h and then treated with 20 μM hemistepsin A for 48 h. (A, B) Expression level of p-γH2AX was investigated using immunoblotting. (C, D) DNA damage degree was evaluated using comet assay and 8-OHdG level. (C) Representative fluorescence images of the comet assay were shown. (D) Levels of 8-OHdG formation were measured using ELISA. ***p < 0.001 vs. control cells, ^###p < 0.001 vs. hemistepsin A-treated cells. Values are presented as mean ± SD. ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; 8-OhdG, 8-hydroxy-2’-deoxyguanosine.

Hemistepsin A-induced inactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in a ROS-dependent manner

Based on the above results, we investigated the impact on PI3K/Akt signaling to further explore the potential targets of molecular mechanisms. The expression of phosphorylated PI3K (p-PI3K) decreased in a concentration-dependent manner in hemistepsin A-treated cells when compared to that in control cells without changes in the expression of its total protein. Similarly, the phosphorylation levels of Akt (p-Akt) and the mammalian target of rapamycin (mTOR, p-mTOR), which are well-established downstream factors of PI3K, were dramatically suppressed by hemistepsin A (Fig. 6A). To clarify the role of the PI3K/Akt pathway in A549 cells, we evaluated the effects of pretreatment with the PI3K inhibitor LY294002 and NAC on cell viability and apoptosis before hemistepsin A treatment. The results showed that LY294002 significantly enhanced the induction of apoptosis by hemistepsin A and increased the hemistepsin A-induced decrease in cell viability, whereas NAC significantly attenuated this cytotoxic effect and markedly ameliorated the inhibitory effect of hemistepsin A on PI3K and Akt phosphorylation (Fig. 6B–D). These findings suggest that hemistepsin A inhibits proliferation and promotes apoptosis in A549 cells by inactivating the ROS-dependent PI3K/Akt signaling pathway.

Fig. 6. Role of PI3K/Akt signaling and ROS in hemistepsin A-mediated cell viability inhibition and apoptosis in A549 cells.

Cells were cultured in a medium containing the indicated concentration of hemistepsin A for 48 h, treated with 10 mM NAC for 1 h, and treated with 20 μM hemistepsin A for 48 h. (A, B) At the end of the treatment time, the expression changes of the indicated protein were investigated using the corresponding antibodies. Equal loading was confirmed with β-actin. (C) After staining with annexin V-FITC/PI, the degree of apoptosis induction was analyzed using flow cytometry frequencies of apoptotic cells were represented by the percentage of annexin V-positive cells in the total number of cells. (D) Cell viability was analyzed using the MTT assay. ***p < 0.001 vs. control cells, ###p < 0.001 vs. hemistepsin A-treated cells. Values are presented as mean ± SD. PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; ROS, reactive oxygen species; NAC, N-acetyl-L-cysteine; FITC, fluorescein isothiocyanate; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; mTOR, mammalian target of rapamycin.

DISCUSSION

One of the most important characteristics observed during the conversion of normal cells into cancer cells is resistance to apoptosis. Therefore, the identification of therapeutic agents that induce apoptosis in cancer cells may be an appropriate approach for developing anticancer drugs. In this study, we evaluated the effect and underlying mechanism of action of hemistepsin A, a sesquiterpene lactone isolated from H. lyrata, on the induction of apoptosis in human lung cancer A549 cells. The results showed that hemistepsin A significantly inhibited the proliferation of A549 cells by promoting ROS-dependent DNA damage and apoptosis induction, accompanied by inactivation of the PI3K/Akt signaling pathway.

Apoptotic events are largely divided into extrinsic and intrinsic pathways based on initiation signals. Among the various factors involved in the induction of apoptosis, caspases play an important role as executors of the apoptotic signaling pathway [38,39]. Of the two typical pathways, the death receptor (DR)-mediated extrinsic pathway is initiated by an increase in caspase-8 activity upon binding of death ligands to DRs on the cell surface, while the mitochondria-mediated intrinsic pathway is mediated by an increase in caspase-9 activity caused by mitochondrial dysfunction. Activation of caspase-8 and caspase-9 completes apoptosis by activating effector caspases, including caspase-3, caspase-7, caspase-8, and caspase-9, which are recognized as initiator caspases in both pathways [39,40]. In the current study, hemistepsin A-treated A549 cells displayed morphological features of apoptosis, such as cytoplasmic contraction and nuclear condensation. Flow cytometry analysis with annexin V/PI staining showed that hemistepsin A could induce apoptosis. As a result of investigating which caspase was activated for the initiation of apoptosis by hemistepsin A, the levels of the inactive form of caspase-3, as well as initiator caspases such as caspase-8 and caspase-9, gradually decreased as the concentration of hemistepsin A increased, and their activity was significantly increased. IAP family members can directly or indirectly regulate the activity of caspases in the cell [41,42], and an increase in the activity of these enzymes by hemistepsin A is related to the suppression of the expression of IAP family proteins, such as cIAP-1, c-IAP2, and XIAP. Consistent with the results of previous studies [22-24], the cleavage of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme degraded by activated caspase-3 [42,43], was induced. Furthermore, our results showed that the cytotoxic effect of hemistepsin A in A549 cells was mediated through caspase-dependent apoptosis, in which both intrinsic and extrinsic pathways were simultaneously involved, rather than necroptosis.

Bcl-2 family proteins are key regulators of mitochondrial proliferation and can inhibit or promote apoptosis and the process that initiates apoptosis by releasing cytochrome c, which activates the apoptotic caspase cascade to destroy cells [38,39]. Among them, the pro-survival members of the Bcl-2 family, such as Bcl-2, support cell survival by suppressing apoptotic activity, whereas the pro-apoptotic members of this family, including Bax, attack the mitochondria and induce apoptosis by increasing mitochondrial outer membrane permeability [38,39]. In this study, the expression of Bcl-2 was downregulated, whereas the levels of Bax and cytosolic cytochrome c were upregulated by treatment with hemistepsin A, which was associated with the loss of MMP. In contrast, oxidative stress is a key factor in regulating cellular function and proliferation. Although a certain level of ROS is necessary to maintain cellular function under normal physiological conditions, excessive ROS accumulation can lead to cell death and oxidative damage to DNA [44,45]. Growing evidence suggests that the regulation of ROS is an important factor in apoptosis and that oxidative stress caused by intracellular redox imbalance can lead to excessive production of ROS, which promotes apoptosis [44,46]. According to previous data, including our previous studies, certain types of sesquiterpene lactones, including hemistepsin A, can protect cells from oxidative damage by blocking the formation of ROS in various tissue-derived normal cells [13,26,47] but can induce apoptosis through the generation of ROS in cancer cells [16,17,23]. Furthermore, members of the pro-apoptotic Bax family have been shown to reduce ROS production during mitochondrial respiration, whereas pro-survival members promote ROS production, thereby promoting cancer cell activity [38,39]. Therefore, we investigated whether ROS generated by hemistepsin A acts as an important regulator of apoptosis in A549 cells. Using the DCF-DA dye, the amount of ROS production peaked within 1 h after hemistepsin A treatment, which was eliminated in the presence of the ROS scavenger NAC. In addition, mitochondrial superoxide levels significantly increased in hemistepsin A-treated cells, indicating that the major source of ROS was the mitochondria. Moreover, concomitant with the decrease in MMP, the fluorescence intensity of MitoTracker, which fluoresces when oxidized in living cells and is sequestered into the mitochondria according to the membrane potential, was significantly reduced in hemistepsin A-treated cells. Using the DCF-DA dye, the amount of intracellular ROS production peaked within 1 h after hemistepsin A treatment, which was eliminated in the presence of the ROS scavenger NAC. In addition, mitochondrial superoxide levels were markedly increased in hemistepsin A-treated cells, but were largely attenuated in the presence of Mito-TEMPO, a mitochondria-targeted superoxide dismutase mimetic, suggesting that mitochondria may be the main source of ROS induced by hemistepsin A. These findings indicate that ROS generation following hemistepsin A-induced mitochondrial damage/dysfunction is responsible for apoptosis in A549 cells. Furthermore, we confirmed that the blockade of artificial ROS generation by NAC could reverse the DNA damage induced by hemistepsin A through various methods of detecting DNA damage, including p-γH2AX expression, comet assay tail formation, and 8-OHdG generation. These findings show that hemistepsin A induces DNA damage and apoptosis in A549 cells, indicating that oxidative damage plays a key function in hemistepsin A-induced cytotoxicity in A549 cells.

Various intracellular signaling pathways play critical roles in transmitting cellular information, and analyzing their roles is a hot topic in exploring the mechanisms of anticancer drugs. The PI3K/Akt signaling pathway is often constitutively overactivated in many types of cancer, including lung carcinoma, and is known to promote their growth and proliferation, as confirmed in previous studies [48,49]. Therefore, the inhibition of the PI3K/Akt signaling pathway is a promising target for many anticancer drugs. In this study, PI3K/AKT PI3K/Akt signaling was inactivated after treatment of A549 cells with hemistepsin A, as reflected by the downregulation of the phosphorylated levels of PI3K, Akt, and mTOR, which is consistent with the results of previous studies on other types of sesquiterpene lactones [50,51]. Moreover, the levels of phosphorylated PI3K, AKT, and PARP were significantly restored in the presence of NAC. Consistent with these results, pretreatment with NAC to block ROS generation effectively reversed the growth inhibitory effect and apoptosis induced by hemistepsin A. Conversely, PI3K-specific inhibitors further potentiated hemistepsin A cytotoxicity, indicating that hemistepsin A treatment exerted antitumor effects on A549 cells in a PI3K/AKT PI3K/Akt signaling-dependent manner. Our results indicate that elevated ROS levels in hemistepsin A are important events in hemistepsin A-induced apoptosis and act as upstream regulators of the inactivation of the PI3K/Akt signaling pathway (Fig. 7). Although the major intracellular source of ROS is the mitochondria, it is clear that excessive accumulation of ROS is a mediator of mitochondrial dysfunction because ROS are highly reactive towards various macromolecules involved in the electron transport chain [52,53]. However, because ROS generation is associated with various pathways of redox homeostasis and cellular signaling, in addition to the mitochondrial damage associated with aerobic respiration [52,54], further detailed investigation into the origin of ROS generation would be of interest. In addition, since oxidative stress has contradictory aspects of initiating tumorigenesis and supporting cancer cell proliferation or inducing cell death, the focus on the induction of apoptosis by hemistepsin A in cancer cells may be a limitation of this study [55,56]. Therefore, studies that can resolve a comprehensive understanding of the induction of oxidative stress by hemistepsin A from different perspectives are also required. In addition, since oxidative stress has contradictory aspects such as initiating tumorigenesis and supporting cancer cell proliferation or inducing cell death [55,56], studies that can address the induction of oxidative stress by hemistepsin A from various perspectives are also needed to comprehensively understand it. Although our study provides valuable insights into the cytotoxic effects of hemistepsin A in A549 lung cancer cells, it has limitations, particularly in that it did not include experiments using immortalized lung epithelial cell lines, such as human small airway epithelial cells (HSAEC) or human bronchial epithelial cells (BEAS-2B). These cell lines could serve as models for normal lung cells and would be useful for evaluating whether hemistepsin A induces ROS production and cytotoxicity in a non-cancerous context. Due to resource and time constraints, we were unable to incorporate these cell lines into the current study. However, we recognize that investigating the effects of hemistepsin A on ROS production in normal lung epithelial cells would be an important next step in understanding the compound’s selectivity and potential toxicity toward non-cancerous cells. Future studies could address this gap by utilizing HSAEC or BEAS-2B cells to confirm whether the observed effects are specific to cancer cells or extend to normal lung epithelial cells. Additionally, further experiments using primary cultured normal lung cells could offer a more comprehensive understanding of the differential responses to hemistepsin A in cancer versus normal cells. These studies would help to better define the therapeutic window of hemistepsin A and its potential as a targeted anti-cancer agent.

Fig. 7. Mechanism of hemistepsin A-induced cytotoxicity in A549 cells.

The anticancer effect of hemistepsin A is achieved at least by the induction of excessive mitochondrial ROS generation-mediated mitochondrial dysfunction and inactivation of the PI3K/Akt signaling pathway. ROS, reactive oxygen species; PI3K, phosphatidylinositol 3-kinase; Akt, protein kinase B; NAC, N-acetyl-L-cysteine; MMP, mitochondrial membrane potential; IAP, inhibitor of apoptosis protein; PARP, poly(ADP-ribose) polymerase.

In conclusion, the results of this study show that hemistepsin A induces DNA damage by increasing intracellular and mitochondrial ROS production in human lung carcinoma A549 cells, consequently decreasing cell survival and inducing apoptosis. However, the effects of hemistepsin A were markedly abrogated by a ROS scavenger, demonstrating that hemistepsin A possesses ROS-dependent anticancer activity in A549 cells. Furthermore, our data show that hemisthesin hemistepsin A inhibited the ROS-mediated PI3K/Akt pathway, promoting apoptosis and inhibiting cell proliferation. These findings demonstrate that ROS generation in A549 cells acts as an upstream event in the hemistepsin A-induced induction of apoptosis and inactivation of the PI3K/Akt pathway. Therefore, our results suggested that hemistepsin A may have promising prospects as a therapeutic agent for lung cancer. Nonetheless, in-depth studies on hemistepsin A components and anticancer activity in animal tumor models according to cancer cell types are needed.