Dihydroartemisinin enhances gefitinib cytotoxicity against lung adenocarcinoma cells by inducing ROS‐dependent apoptosis and ferroptosis

Abstract

The application of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs), such as gefitinib, has shifted lung cancer treatment from empirical chemotherapy to targeted molecular therapy. However, acquired drug resistance is inevitable in almost all non‐small cell lung cancer (NSCLC) patients treated with gefitinib. Combined treatment with dihydroartemisinin (DHA) and gefitinib produced a better inhibitory effect on lung adenocarcinoma than gefitinib treatment alone; however, the specific mechanism remains unclear. In this study, we aimed to assess the underlying mechanism of this combination treatment. We prepared gefitinib‐resistant A549 cells and investigated whether apoptosis and ferroptosis were involved in the sensitizing effect of DHA. Treatment with 5 μM gefitinib resulted in rupturing and floatation of A549 cells in the medium, while A549‐GR cells were found to be insusceptible to gefitinib. However, treatment with DHA substantially inhibited the proliferation of A549‐GR cells in a dose‐dependent manner accompanied by increased apoptosis and ferroptosis. The accumulated reactive oxygen species (ROS) was crucial for the inhibitory effect of DHA on A549‐GR cells. Moreover, cellular autophagy was significantly upregulated post‐DHA treatment. The combined treatment of DHA and gefitinib resulted in inhibition of proliferation of A549, H1975, and HCC827 cells, and ROS accumulation and a remarkable induction of apoptosis was observed in A549‐GR cells. DHA significantly induced apoptosis and ferroptosis in a dose‐dependent manner and exhibited high cellular toxicity on A549‐GR cells when combined with gefitinib.

Abbreviations

3‐MA

3‐methyladenine

Bcl‐2

B‐cell lymphoma 2

DFO

deferoxamine

DHA

dihydroartemisinin

EGFR

epidermal growth factor receptor

EGFR‐TKIs

epidermal growth factor receptor‐tyrosine kinase inhibitors

FTH

ferritin heavy chain

GPX4

glutathione peroxidase

GSH

glutathione

IRP2

iron regulatory protein

LC3

microtubule‐associated protein 1 light chain 3

LDH

lactate dehydrogenase

NAC

N‐acetyl‐L‐cysteine

NSLC

non‐small cell lung cancer

p62

autophagic protein sequestosome 1

PARP

poly ADP‐ribose polymerase

PVDF

polyvinylidene fluoride

ROS

reactive oxygen species

1. INTRODUCTION

Lung cancer is a malignancy that contributes to almost 1.8 million deaths annually. ¹ , ² Non‐small cell lung cancer (NSCLC) accounts for the vast majority of lung cancer, and most patients lose the opportunity to undergo surgery after the initial diagnosis. Surgery combined with radiotherapy and chemotherapy is currently recognized as the best supportive treatment for lung cancer; however, this treatment strategy has an ideal therapeutic effect only on early‐stage patients. ³ Despite strong evidence demonstrating that standard chemotherapy can prolong the overall survival (OS) and improve quality of life, its efficacy appears to have reached a plateau, especially in advanced NSCLC patients, whose median OS and 5‐year survival rate are only 1 year and 3.5%, respectively. ⁴

Targeted therapy has made great progress in the clinical treatment of lung adenocarcinoma (LUAD) patients. Among these targeted driver genes, the epidermal growth factor receptor (EGFR) mutation is the most widely studied with a high detection rate in LUAD patients in Asia, and the activated EGFR signal can promote cell proliferation, differentiation, and angiogenesis by participating in related pathways. EGFR‐tyrosine kinase inhibitors (EGFR‐TKIs) have been used for advanced LUAD patients with EGFR‐sensitive mutations as first‐line treatment. ⁵ , ⁶ Clinical studies have shown that, compared to chemotherapy, EGFR‐TKIs considerably prolong survival with more precise curative effects, cause less damage to normal tissues, and result in a lower incidence rate of adverse reactions in LUAD patients. ⁷ , ⁸ , ⁹ However, owing to acquired drug resistance, most patients always develop resistance 8–13 months after EGFR‐TKI treatment. ¹⁰ Drug resistance greatly influences the clinical application and therapeutic efficacy of first‐line EGFR‐TKIs such as gefitinib; therefore, improving the sensitivity of tumor cells to EGFR‐TKIs is critical.

Artemisinin compounds extracted from Artemisia annua completely inhibit the growth of Plasmodium and are widely used in the treatment of malaria. ⁹ , ¹¹ Dihydroartemisinin (DHA), the main derivative of artemisinin, exhibits good antitumor activity in regulating apoptosis, cell cycle, metastasis, and the immune system. ¹² , ¹³ , ¹⁴ The combination treatment of DHA and gefitinib enhances the inhibitory effect of the latter; however, no further investigation into the underlying mechanism has been performed. ¹⁵

Therefore, in this study, we aimed to analyze the mechanism underlying the effect of DHA on gefitinib. We treated gefitinib‐resistant A549 cells (A549‐GR) with DHA. Results showed that DHA significantly induced apoptosis and ferroptosis in the A549‐GR cells, which was mediated by the upregulated concentration of reactive oxygen species (ROS), as well as the induction of cell autophagy. The promising results of combining DHA and the EGFR‐TKI, gefitinib, make this a potentially viable therapeutic strategy for drug‐resistant cells.

2. MATERIALS AND METHODS

2.1. Cell culture

The human lung adenocarcinoma cell line, A549, HCC827, and H1975 were acquired from the Shanghai Institute of Country Cell Bank and cultured in a 37°C humidified atmosphere. Cells were grown in Dulbecco's Modified Eagle Medium (Hyclone) containing 10% fetal bovine serum (Gibco).

2.2. Preparation of A549‐GR cells

A549 cells were digested and inoculated in 25 cm² cell culture flasks overnight. The medium was then replaced with fresh medium containing 0.5 μM gefitinib (MCE). After drug treatment for 24 h, the culture medium was discarded and replaced with a drug‐free medium. Cells were digested and subcultured when they reached confluence in the culture flasks. The drug treatment was repeated 6–8 times at a concentration of 0.5 μM until the cells grew stably at this concentration. The drug concentration was increased to 1 μM and the cell culture was continued in a manner similar to that described above. The drug challenge was repeated at concentrations of 0.5, 1, 2, and 2.5 μM until cells were found to be resistant in the medium containing 2.5 μM gefitinib.

2.3. CCK‐8 assay

For drug resistance detection, cells were seeded into 96‐well plates. The cell culture was replaced and treated for 24 h with the indicated concentration of gefitinib. The cell culture was then treated for 2 h in the presence of a Cell Counting Kit‐8 (Beyotime Biotech, China), where the absorbance was examined at 450 nm. To detect cytotoxicity, the A549‐GR cells were treated with DHA; combined treatment of DHA, N‐acetyl‐L‐cysteine (NAC), and glutathione (GSH); DHA combined with DFO or ferrostatin‐1; DHA plus 3‐MA; or DHA plus gefitinib at indicated doses for 24 h; cell viability was examined as above. All reagents were purchased from MedChemExpress (MCE).

2.4. Cell number assay

A549‐GR cells were seeded into 6‐well plates and treated with DHA, combined treatment of DHA and 3‐MA, or DHA plus gefitinib in indicated dose for 24 h. Cells were digested, and the number was calculated using an optical microscope.

2.5. Cellular morphology examination

A549 and A549‐GR cells were treated with the indicated concentration of gefitinib for 24 h. The cellular morphology was imaged using an optical microscope under 400x magnification.

2.6. Flow cytometry analysis for cell apoptosis

A549‐GR cells were treated with DHA, combined treatment of DHA and NAC, or DHA plus gefitinib for 24 h. Cells were then digested, centrifuged, resuspended, and stained with Annexin V (Biolegend) and PI buffer (Beyotime Technology). Cell apoptosis was detected using BD FACS CaliburTM device (Franklin Lakes, NJ).

2.7. GSH assay

Cells were seeded in 6‐well plates and treated with DHA, a combined treatment of DHA and NAC or DHA plus z‐VAD‐FMK (MCE) for 24 h. The supernatant was used to determine the concentration of GSH after the cells were freeze‐thawed and the protein was removed.

2.8. Lactate dehydrogenase assay

A549‐GR cells were treated with DHA, combined treatment of DHA and 3‐MA, or DHA plus gefitinib in the indicated dose for 24 h. The Lactate dehydrogenase (LDH) assay kit (Beyotime Technology) was used and the absorbance was detected at 490 nm.

2.9. Immunofluorescence assay

A549‐GR cells were infected with RFP/GFP‐LC3 adenovirus. After 12 h of transfection, cells were treated with DHA or dimethyl sulfoxide for 24 h. The cells were harvested and fixed using 4% paraformaldehyde (Sangon Biotech, China). Cell nuclei were stained using 4′,6‐diamidino‐2‐phenylindole solution (Beyotime Technology), and the fluorescence was blocked using a DAKO Fluorescence Mounting Medium (Agilent Technologies Inc., USA). The autophagy flux was analyzed using ZEN Imaging Software (Zeiss).

2.10. Lipid ROS detection

A549‐GR cells were treated with DHA for 24 h. The cells were then digested, centrifuged, resuspended in binding buffer, and incubated with a BODIPY C11 probe (5 μM) for 15 min. The flow cytometry was then measured, and the mean fluorescence was analyzed using FlowJo Software.

2.11. Relative ROS detection

Cells were treated with various concentrations of DHA, combined treatment of DHA and NAC, or DHA plus gefitinib at indicated doses for 24 h. Following harvesting, the cells were digested, centrifuged, and then incubated with 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) (5 μM) for 30 min. The relative fluorescence intensity of DCF was detected through flow cytometry.

2.12. Western blotting

Cells were treated with various concentrations of DHA for 24 h. The cells were lysed on ice with western and IP lysis buffer (Sangon Biotech), followed by centrifugation and the collection of the resulting supernatant. The protein concentration was detected using a BCA kit (Beyotime Technology) and equaled, and then the total protein was separated and transferred. After blocking with Quick‐Block Western solution (Beyotime Technology), the PVDF membrane was incubated with antibodies at 4°C overnight. The antibodies were acquired from Cell Signaling Technology (c‐Caspase 3, Caspase 3, PARP, Bcl‐2, β‐actin, IRP2, p62, and Beclin1) and Thermo Fisher Scientific (LC3, FTH, and GPX4). The PVDF strips were subsequently incubated with corresponding secondary HRP‐conjugated antibodies for 2 h. The protein bands were visualized using an ECL kit (Beyotime Biotech), and the relative density of each band was analyzed using ImageJ software.

2.13. Statistical analysis

All data in this study were obtained from three independent experiments and are represented as mean ± standard deviation. The results were analyzed using Statistical Package for Social Sciences software (SPSS 22.0). The differences between the two groups were compared using the Student's t‐test method or the one‐way ANOVA method. The difference was considered statistically significant when p < 0.05.

3. RESULTS

3.1. Preparation of gefitinib‐resistant A549 cells

To determine the drug resistance ability of A549‐GR cells, we analyzed the cell viability with or without gefitinib treatment in A549 and A549‐GR cells. As shown in Figure 1A, there was no difference in the proliferation rate between the two groups of cells without gefitinib treatment, whereas the viability in A549 cells, but not A549‐GR cells, was significantly inhibited when treated with gefitinib (Figure 1B). Moreover, the results of cellular morphology examination were consistent with those of the CCK‐8 assay, showing a marked reduction and distortion of A549 cells when treated with 2.5 μM gefitinib. In the medium containing 5 μM of gefitinib, a mass of cells ruptured and floated, while A549‐GR cells were eumorphism under gefitinib challenge (Figure 1C).

FIGURE 1.

The cytotoxicity of gefitinib on A549 and A549‐GR cells. (A) 2 × 103 A549 normal cells and A549‐GR cells were seeded in 96‐well plates and detected cell viability every 12 h; (B) Cells were treated with the indicated dose of gefitinib for 24 h. The cell viability was examined by CCK8 assays. The data were repeat three times (ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001); (C) 1 × 10⁶ A549 normal cells and A549‐GR cells were treated with 2.5 or 5 μM gefitinib for 24 h and the cellular morphology was photographed at 100x magnification. scale bar = 50 μm.

3.2. DHA suppressed cell viability by inducing apoptosis and ferroptosis

To identify the cytotoxicity of DHA in A549‐GR cells, various concentrations of DHA were treated cells for 24 h, and the cell viability, number, and LDH release rate were assessed. The cell proliferation and number were significantly reduced in a DHA dose‐dependent manner, whereas the LDH release ratio was comparable when cells were treated with 12.5 and 25 μM DHA (Figure 2A–C). After treatment with 12.5, 25, and 50 μM DHA for 24 h, the percentage of early apoptotic cells, as well as late apoptotic cells, increased with the high dose of DHA (Figure 2D). We also analyzed the expression of apoptosis‐related proteins, and found that the cleaved‐Caspase 3 was upregulated, whereas PARP and Bcl‐2 were downregulated after DHA treatment (Figure 2E).

FIGURE 2.

DHA‐induced apoptosis of A549‐GR cells in a concentration‐dependent manner. (A) 2 × 10³ A549‐GR cells were seeded in 96‐well plates and treated with the indicated concentration of DHA foe 24 h. The viability was detected by CCK8; (B) Cell number was calculated post‐DHA treatment for 24 h; (C) 2 × 10³ A549‐GR cells were treated with DHA and the LDH release ratio was examined according manufacturer's protocol; (D) 5 × 10⁵ A549‐GR cells were stained with Annexin V and PI buffer after treated with DHA. The percentage of the apoptotic cell was analyzed with flow cytometry; (E) The apoptotic‐related proteins in A549‐GR cells were detected through western blot post‐DHA treatment. The relative gray value of cleaved‐caspase3, caspase‐3, PARP and Bcl2 was analyzed by Image J software. The data were repeat three times (*p < 0.05, **p < 0.01, ***p < 0.001).

Furthermore, the viability of A549‐GR cells was significantly decreased in a dose‐dependent manner, whereas the cell apoptosis rates were comparable in cells treated with 12.5 and 25 μM DHA. Therefore, we further investigated whether ferroptosis was the mechanism underlying the effect of DHA. The accumulation of lipid peroxidation products is a crucial characteristic of ferroptosis. Lipid ROS are involved in an oxidation–reduction reaction with GSH, thus consuming intracellular GSH. Therefore, we explored the level of relative lipid ROS and GSH. We found that GSH was significantly decreased, whereas lipid ROS levels were increased post‐DHA treatment (Figure 3A,B). The expression of ferroptosis‐related proteins GPX4 and FTH was markedly downregulated, thus contributing to the release of free iron (Figure 3C). In addition, apoptotic cell alteration was not significant, whereas cell viability was reversed when cells were co‐treated with DHA and ferroptosis inhibitor (Figure 3D,E). Cell ferroptosis was neither influenced when cells co‐treated with DHA and z‐VAD‐FMK (a pan‐inhibitor of caspases) (Figure 3F). These results indicate that ferroptosis was independent of apoptosis, whereas they were all closely connected with ROS, and the inhibitory effect of DHA on A549‐GR cells was partly achieved through ferroptosis.

FIGURE 3.

DHA‐induced ferroptosis in A549‐GR cells. (A) 5 × 105 A549‐GR cells were treated with various dose of DHA for 24 h, then cells were stained with 5 μM BODIPY C11 probe for 15 min. The lipid ROS production was detected and mean fluorescence was analyzed by flowJo Software; (B) The GSH% was measured post DHA treatment for 24 h; (C) Cells were treated with 25 μM DHA for 24 h, the ferroptosis‐related proteins were examined and the relative grayscale value was analyzed; (D) Apoptotic cells were detected when cells were treated with DFO plus DHA; (E) 2 × 10³ A549‐GR cells were treated with 25 μM DHA plus 100 μM deferoxamine or 1 μM ferrostatin‐1for 24 h, then cell viability was assessed; (F) A549‐GR cells were treated with Z‐VAD‐FMK (20 μM)and DHA (25 μM) for 24 h and the GSH% was measured. The data were repeat three times (*p < 0.05, **p < 0.01, ***p < 0.001).

3.3. ROS are crucial for DHA in inhibiting the viability of A549‐GR cells

ROS have a crucial antibacterial, anti‐inflammatory, and tumor‐inhibitory role. Under normal circumstances, ROS are constantly eliminated by the antioxidant system to maintain a dynamic balance. Excessive ROS are responsible for triggering lipid peroxidation damage in biofilms and macromolecules. We next examined the relative ROS level post‐DHA treatment and found that the content of ROS was significantly increased when cells were treated with 25 μM DHA (Figure 4A). To further evaluate whether ROS was involved in DHA‐induced apoptosis and ferroptosis, we utilized NAC, a ROS scavenger, for detect any changes in cytotoxicity caused by DHA. As shown in Figure 4B, the proliferation of A549‐GR cells was remarkably suppressed when treated with DHA, while this was reversed when the cells were co‐treated with DHA and NAC or GSH. Intercellular GSH was significantly increased in the DHA and NAC group (Figure 4C). Moreover, the relative ROS levels were reduced post‐DHA and NAC treatment (Figure 4D). Cell apoptosis and ferroptosis were also inhibited by NAC and DHA treatment (Figure 4E,F), indicating that ROS play an important role in the inhibitory effect of DHA on A549‐GR cells.

FIGURE 4.

DHA‐induced ROS generation promoted apoptosis and ferroptosis in A549‐GR cells. (A) 1 × 10⁶ A549‐GR cells were treated with various dose of DHA for 24 h, then cells were labeled with 5 μM DCFH‐DA for 30 min and the DCFH‐DA sensitive ROS was detected; (B) 2 × 10³ A549‐GR cells were treated with 25 μM DHA combined 5 mM NAC or GSH for 24 h, then cell viability was detected; The GSH% (C), DCFH‐DA sensitive ROS (D), apoptotic cells (E) and lipid ROS (F) were assessed post‐A549‐GR cells were treated with DHA and NAC for 24 h. The data were repeat three times (ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

3.4. DHA treatment upregulated cellular autophagy

Accumulating research has demonstrated the relationship between ROS and autophagy. The excessive accumulation of ROS leads to oxidative stress, mitochondrial dysfunction, and autophagy induction. Conversely, autophagy induces phagocytosis and degradation of ROS against oxidative stress. We monitored autophagy flux by using the GFP‐RFP virus and found that cell autophagy was markedly upregulated after DHA treatment (Figure 5A). The expression of autophagy‐related proteins was also examined, and the level of LC3 and Beclin1 was increased, while that of p62 was reduced (Figure 5B). DHA‐induced autophagy was markedly inhibited when cells were co‐treated with NAC (Figure 5C). The cell proliferation and number were significantly decreased when cells were treated with DHA, and this was further decreased when cell autophagy was suppressed (Figure 5D,E). The LDH release ratio, apoptotic cells and related lipid ROS was significantly increased in the DHA plus 3‐MA group (Figure 5F–H).

FIGURE 5.

DHA‐induced autophagy attenuated its induction of apoptosis and ferroptosis in A549‐GR cells. (A) 1 × 10⁵ A549‐GR cells were infected with GFP‐RFP‐LC3 virus. The autophagy flux was assessed post‐cell treated with 25 μM DHA for 24 h. The GFP/RFP‐LC3 puncta was analyzed by Image J software. Scale bar =10 μm. (B) After treated with the indicated dose of DHA, the autophagy‐related proteins were measured; Cells were treated with NAC plus DHA and the autophagy‐related proteins were detected (C); The (D) cell viability, (E) cell number, (F) LDH release assay, (G) relative lipid ROS and (H) cell apoptosis were examined post‐cells treated with 25 μM DHA and 5 mM 3‐MA. The data were repeat three times (ns, not significant, **p < 0.01, ***p < 0.001).

3.5. DHA combined with gefitinib revealed remarkable toxicity in A549‐GR cells

To explore the application of DHA in low doses, a combination of DHA and gefitinib was used to detect the inhibitory effect on HCC827, H975, A549, and A549‐GR cells. As shown in Figure 6A–C, the combination of DHA and gefitinib markedly suppressed the proliferation of lung adenocarcinoma cells and resulted in high LDH content. Cellular ferroptosis and autophagy also increased when A549‐GR cells were co‐treated with gefitinib and DHA (Figure 6D,F). Furthermore, apoptotic cells, especially the percentage of late apoptotic cells, significantly increased in the DHA and gefitinib‐treated group, which was consistent with the levels of relative ROS (Figure 6E,G).

FIGURE 6.

DHA sensitized lung cancer cells to the cytotoxicity of gefitinib by inducing ROS‐dependent apoptosis and ferroptosis. (A) 2 × 10³ HCC827, H1975, A549, and A549‐GR cells treated with 25 μM of DHA and 5 μM gefitinib for 24 h. The viability was detected by CCK8; (B) Cell number was calculated and (C) The LDH release assay was also examined post DHA plus gefitinib treatment for 24 h; A549‐GR cells were treated with DHA plus gefitinib for 24 h, then (D) relative lipid ROS, (E) DCFH‐DA sensitive ROS, (F) autophagy‐related protein level, (G) and cell apoptosis was examined. The data were repeat three times (ns, not significant, **p < 0.01, ***p < 0.001).

4. DISCUSSION

Almost all NSCLC patients treated with EGFR‐TKIs inevitably develop acquired drug resistance, which poses great challenges to follow‐up treatments. The T790M mutation, appearing in 60% of patients, is the leading cause of patients acquiring resistance to EGFR‐TKIs. The amplification of MET is also involved in acquired drug resistance of TKIs, accounting for approximately 5%–22%. ¹⁶ , ¹⁷ , ¹⁸ The amplification of Her2, activation of the bypass signaling pathway, and the transformation of the phenotype or tissue are also reported to be responsible for acquired drug resistance in NSCLC patients. ¹⁹ In the present study, we prepared gefitinib‐resistant A549 cells (Figure 1) and found that DHA significantly promoted the inhibitory effect of gefitinib on A549‐GR cells by inducing the excessive generation of ROS (Figure 6).

ROS are mainly produced by redox reactions and have a dual effect on tumor cells. Low doses of ROS can be used as secondary messengers that mediate multiple signal transduction pathways such as the activation of the MAPK/ERK signaling pathway. However, excessive ROS promotes oxidative stress, damages cells, and even leads to cell death. ²⁰ , ²¹ , ²² , ²³ In this study, DHA treatment resulted in a high concentration of ROS which significantly inhibited A549 cell proliferation. The inhibitory effect of DHA was reversed with decreasing apoptosis and ferroptosis when cells were cotreated with GSH or NAC (Figure 4).

In tumor cells, high concentrations of ROS can lead to oxidative damage of proteins, DNA, lipids, and ultimately, the induction of cell apoptosis and ferroptosis. ²⁴ , ²⁵ In A549‐GR cells, DHA‐induced ROS contributed to dose‐dependent cell apoptosis accompanied by increased Bcl‐2 levels and upregulated cleaved Caspase 3 expression (Figure 2). Bcl2‐family proapoptotic proteins trigger cell apoptosis and further apoptotic execution is mediated by Caspase‐3. ²⁶ , ²⁷ Ferroptosis, which is different from cell apoptosis and necrosis, is characterized by a dependence on iron ions and ROS to induce lipid peroxide accumulation. Cysteine utilization, glutathione biosynthesis, polyunsaturated fatty acid metabolism, and phospholipid regulation are crucial factors in ferroptosis. ²⁸ , ²⁹ , ³⁰ In this study, accumulated lipid ROS post‐DHA treatment contributed to a decrease in GSH, an upregulation of IRP2, and a significant downregulation of GPX4. Cell proliferation was partly preserved when cells were cotreated with DHA and ferroptosis inhibitor, DFO or ferrostatin‐1 (Figure 3). Moreover, the combined treatment of DHA and gefitinib demonstrated remarkable cytotoxicity in lung adenocarcinoma cells by eliciting ferroptosis and apoptosis, as well as reversing the tolerance of A549‐GR cells to gefitinib (Figure 6).

Autophagy is a highly conserved process that maintains cell homeostasis through the degradation of damaged organelles or misfolded proteins. ³¹ In tumor cells, autophagy also plays a dual role in the regulation of cell survival and death. To maintain survival, autophagy is upregulated when tumor cells suffer from starvation, hypoxia, endoplasmic reticulum stress, or radiation. However, prolonged autophagy activation would lead to excessive “self‐extinction” and thus induce apoptosis. ³² , ³³ Growing evidence has demonstrated that ROS are responsible for regulating cell autophagy under various conditions. ³⁴ , ³⁵ , ³⁶ , ³⁷ In our study, we found that in A549‐GR cells treated with DHA, LC3 and Beclin1 accumulated, p62 was degraded, and Bcl‐2 levels decreased, indicating autophagy was upregulated. In addition, the cell viability was further decreased when cellular autophagy was inhibited (Figure 5). Autophagy was further induced when cells were co‐treated with DHA and gefitinib, however, dramatically greater of ROS outdistanced the covering of protective autophagy and results more significant apoptosis (Figure 6). Moreover, growing evidence has demonstrated the correlation between autophagy and ferroptosis. Ferroptosis is triggered when GPX4, the crucial protein, is degraded. ³⁸ , ³⁹ , ⁴⁰ In this study, we found that DHA treatment decreased the levels of GPX4, while autophagy was upregulated, which was consistent with the findings of the previous studies (Figure 3).

In conclusion, DHA treatment induced significant apoptosis and ferroptosis through ROS accumulation and as a result, an increase in the sensitivity of A549‐GR cells to gefitinib was observed. These results provide new insights on the research against EGFR‐TKI drug resistance. Nevertheless, the underlying molecular mechanism still requires further exploration.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

Lai X‐Y, Shi Y‐M, Zhou M‐M. Dihydroartemisinin enhances gefitinib cytotoxicity against lung adenocarcinoma cells by inducing ROS‐dependent apoptosis and ferroptosis. Kaohsiung J Med Sci. 2023;39(7):699–709. 10.1002/kjm2.12684