Calu-1 and A549 cancer cells were obtained from the National Collection of Authenticated Cell Cultures (NCACC) and cultured in DMED medium supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% antibiotic in a humidified incubator at 37˚C and 5% CO₂. Cells with a density of 70% to 80% were deemed ready to adopt measures for subsequent detection. To enhance the effect of the inhibitors used in this study, they were intentionally added to cells 2 h before SS.

The reagents included solasonine (HY-N0070, MCE), Z-VAD-FMK (S7023, Selleck), DCFH-DA (S0033S, Beyotime), Cell Counting Kit-8 (40203ES76, YEASEN), SYTOX green (S7020, Invitrogen), Trolox (C3183, APExBIO), DFO (HY-B0988, MCE), Mito-TEMPO (HY-112879, MCE), Necrostatin-1(HY-15760, MCE), Hoechst 3342 (23491-52-3, Solarbio), Ferrostatin-1 (HY-100579, MCE), RSL3 (HY-100218A, MCE), Bafilomycin A1 (HY-100558, MCE), C11-BODIPY581/591 (D3861, Invitrogen), Erastin (HY-15763,MCE), GSH assay kit (A006-2-1, Nanjing Jiancheng), Cysteine assay kit (BC185, Solarbio), FerroOrange probes (M489, Dojindo), MitoPeDPP (M466-5,Dojindo), and JC-1 (MT09-1, Dojindo).The following antibody purchased from Cell Signaling Technology was GAPDH (5174, CST), the others purchased from BOSTER included GPX4 (BM5231, BOSTER) and SLCA711 (BM5318, BOSTER). According to the manufacturer’s instructions, the drugs were initially dissolved in dimethylsulfoxide (DMSO) at a stock solution.

Approximately 5000 Calu-1 cells and 10,000 A549 cells were seeded into the 96-well plates in triplicate and cultured in the appropriate conditions for 24 h. Incidentally, the objective drugs and reagents involving various inducers and inhibitors were applied based on the manufacturer’s protocol. Specifically, the inhibitors including Z-VAD-FMK (Z-V), Necrostatin-1 (Nec), Ferrostatin-1 (Fer-1), Bafilomycin A1 (Baf-A1), Deferoxamine mesylate (DFO), Mito TEMPO, Trolox, and RSL3 were added to the cells for 2 h prior to SS for pretreatment. The medium was then supplemented with the Cell Counting Kit 8 (CCK8) reagent, and the cells were incubated for 2 h. Finally, the absorbance at 450 nm was measured using a microplate reader (Bio Tek, United States).

The SYTOX Green staining solution was used to visualize cancer cell death. Calu-1 and A549 cells were seeded into 24-well plates at a density of approximately 5*104 cells/well and 1*105 cells/well, respectively, and cultured at 37°C in an incubator. Calu-1 cells were exposed to SS (10 μM, 15 μM, 20 μM) after 24 h, while A549 cells were exposed to SS (20 μM, 25 μM, 30 μM). Meanwhile, the cells were stained with 100 nM SYTOX Green and cultured in the dark before being observed under a fluorescence microscope (Leica, Germany).

The levels of intracellular Fe²⁺ were assessed using FerroOrange probes (Dojindo) according to the manufacturer’s protocol. Cells were drug-treated for the time indicated before being stained with a final concentration of 1 mol/L of FerroOrange for 30 min at 37°C. The signal from the samples was collected using a flow cytometer and analyzed using the GraphPad Prism software.

In the study, DCFH-DA, a cell-permeable probe, was used to measure the levels of cytosolic ROS using flow cytometry and a fluorescence microscope. In brief, cells were drug-treated before being cultured with DCFH-DA for 30 min at 37°C. The level of ROS was measured using the flow cytometer and fluorescence microscope mentioned above after the samples were washed twice with PBS.

C11-BODIPY581/591 was performed on cells by incubating them in PBS containing 5% FBS and 10 μM C11-BODIPY581/591 for 1 h at 37°C in the dark after being treated as indicated. The cells were then rinsed twice with PBS and examined under a fluorescence microscope. The labeled cells were distinguished by a shift in the fluorescence emission peak from 590 nm to -510 nm, which was proportional to lipid ROS generation and would be measured using a flow cytometer.

The glutathione (GSH) concentration was determined using a glutathione assay kit purchased from Jiancheng Bioengineering institute and carried out according to the manufacturer s instructions. The treated cells were harvested and homogenized in PBS on ice, the detected result was read by a microplate reader, and the glutathione in the cell lysate was calculated using the formula provided by the GSH assay kit’s product protocol.

Intracellular cysteine (Cys) levels were determined in the lysates of cells exposed to drugs using a Cys assay kit (BC185, Solarbio) according to the manufacturer’s instructions. The final data obtained from a microplate reader at an absorbance of 600 nm was calculated using the formula provided by the Cys assay kit’s product protocol.

The change in mitochondrial membrane potential was measured using JC-1 (Dojindo) staining as directed by the manufacturer. The ratio of red fluorescence to green fluorescence was used to describe each group.

The MitoPeDPP (Dojindo), a cell-membrane-permeable probe that specifically localizes in mitochondria due to the triphenylphosphonium moiety, will be used to assess lipid peroxidation in mitochondria. Cells were treated as indicated, then washed twice with PBS and stained with 1 μM MitoPeDPP and 10 μM Hoechst nuclear stain in certain experiments. Following that, images of cells were captured at random by a fluorescence microscope with 100 m scale bars.

Total proteins were isolated using a cell lysis buffer supplemented with protease and phosphatase inhibitors (Beyotime, Shanghai, China). Protein concentrations were measured using a BCA protein assay kit (Beyotime). After boiling in the 1% SDS loading buffer, the protein samples were separated by SDS/PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The samples were then incubated with the primary antibodies (GAPDH, SLC7A11, GPX4) overnight before being incubated with the secondary antibody anti-rabbit IgG (925-32211, LICOR) with the membranes. Finally, protein brand images were obtained using an infrared laser two-color image analysis system (Odyssey LICOR, United States). Image J was used to calculate and normalize the objective protein expression level.

In the study, all results obtained from experiments were independently repeated three times and tested for normality. The continuous variables of normal distribution or nearly normal distribution were displayed as average value ± standard deviation. After homogeneity of variance test, one-way ANOVA and LSD post hoc comparisons were used for those meeting data requirements in SPSS 25 or GraphPad Prism 8.0. P < 0.05 was considered significantly different.

Previous studies have demonstrated that SS has anti-proliferative properties. The Calu-1 and A549 cells, which belong to the LUAD cell lines, were used in this study for cytotoxicity experiments and subsequent examinations. The cells were subjected to the treatments described above, and the IC50 values of SS for Calu-1 or A549 cells were determined to be 15.08 or 21.59 μM, respectively ( Figure 1A ). Based on the findings, Calu-1 cells were found to be more sensitive to SS than A549 cells, so we tailored the appropriate drug concentrations for follow-up work. In addition, the cell morphology changed, with the cells becoming shriveled and even broken as the administration concentration increased ( Figure 1B ). Likewise, the fluorescent images indicated by the SYTOX Green confirmed that SS elicited cell death in a dose-dependent manner ( Figure 1C ).

What types of cell death contributed to the solasonine-induced growth inhibition in LUAD cells? This question prompted us to conduct additional research into the work. In the CCK8 assay, the descent was rescued by Z-V, Nec, Fer-1, and Baf-A1, indicating that apoptosis, necroptosis, ferroptosis, and autophagy may play a part in solasonine-induced cell death ( Figure 2A ). Several ferroptosis-related reagents, including inhibitor DFO and inducer RSL3, were used for extra validation to assess the involvement of ferroptosis. Fer-1, a lipid peroxidation inhibitor, and DFO, an iron chelator, are both commonly used ferroptosis inhibitors and are widely used in ferroptosis reports (13). In addition, since their discovery, RSL3 and erastin have been well known for their ferroptosis-induced property (14). The data indicated that Fer-1 and DFO protected both cells from solasonine-induced destruction, however, the toxicity was amplified by the combination of RSL3 and SS ( Figure 2B ). The fluorescent indicator C11-BODIPY581/591 was used to directly observe lipid peroxidation, which is located in the membrane and shifts the fluorescence from red to green when oxidization occurred (15). As expected, solasonine-treated Calu-1 and A549 cells exhibited high fluorescence in green and low fluorescence in red, referring to the accumulation of lipid peroxidation, whereas the Fer-1 restricted this process ( Figures 2C, D ).

Since ferroptosis has been identified, the critical events of the process, which include Fe2+overload and intracellular ROS accumulation, will be investigated and validated in the next step. It should be noted that the occurrence of ferroptosis is dependent on the level of iron content rather than any other metallic element, so measuring the content of Fe2+ is required. Fortunately, Fe2+ was discovered to be required for SS action, as evidenced by the experimental results shown in Figure 3A . The levels of Fe2+ increased as drug concentrations increased. However, a high dose of SS had a remarkable effect, which was completely reversed by DFO. On the other hand, intracellular ROS, while not as specific as lipid ROS in ferroptosis, is a major character that runs throughout the entire process. The images demonstrated that the intracellular ROS marked with DCFH-DA probes was excessively generated when SS was administered ( Figure 3B ). Besides the fluorescent images, the detailed information about the integral fluorescent intensity was presented in the histogram ( Figure 3C ).

When discussing ferroptosis, the glutathione redox system is a popular point of penetration. Intracellular antioxidant enzyme glutathione peroxidase 4 (GPX4), cystine/glutamate transporter SLC7A11, substrate GSH, and Cys all contribute to the antioxidant machinery’s response to redox imbalance. The statistical graph displayed that the GSH and Cys decreased significantly when compared to the control group ( Figures 4A, B ).

Furthermore, the Western blot results revealed that the protein expression of GPX4 and SLC7A11 was altered on the sensitive cell line Calu-1 between SS treated cells and the control ( Figure 4C ), and the gray value was transformed into quantitative data displayed by the bar graph ( Figure 4D ). It is suggested that the inhibition of the expression of protein GPX4 and SLC7A11 by SS had parallels with the effect of the positive control group ( Figures 4E, F ). Incidentally, Fer-1 successfully counteracted the effect of SS on these four redox components to some extent. All the evidence pointed to SS paralyzing the antioxidant defense, collapsing the redox balance, and causing ferroptosis.

It is well known that the mitochondria are the cell’s energy centers, responding to various stimuli and conducting oxidative metabolism in some biological activities. Thus far, studies have shown that mitochondria, a major site for ROS production, play a critical role in ferroptosis (16). Therefore, the relationship between mitochondria and solasonine-induced ferroptosis on Calu-1 cells needs to be revealed.

To begin with, the viability of the cells was determined using the antioxidants Trolox and MitoTEMPO. Trolox, in addition to protecting cells from oxidation, prevents membrane damage for cell integrity, and MitoTEMPO targets superoxide and alkyl radicals found in mitochondria. With the assistance of Trolox and MitoTEMPO, the solasonine-treated cells rejuvenated to a great extent ( Figure 5A ).

According to these findings, mitochondria were found to play a role in solasonine-induced ferroptosis. To gain more insights, mitochondrial functional assessments such as mitochondrial injury and mitochondrial lipid peroxidation assays were launched. According to the diagram, the solasonine-treated group with a low fluorescent ratio showed high mitochondrial membrane potential depolarization paralleled to the positive control. However, this alternation could be reversed after using Fer-1, Trolox, and MitoTEMPO ( Figures 5D, E ). Moreover, the level of mitochondrial ROS production was evaluated using MitoPeDPP dye, a selective probe that targets peroxide in the mitochondrial inner membrane (17). The enhancement of a green, fluorescent signal indicated that erastin or SS action increased mitochondrial ROS production. In addition to Fer-1, Trolox, and MitoTEMPO, they were also capable of reversing excessive mitochondrial ROS production ( Figure 5F ). The data showed that the SS may obstruct mitochondrial function directly and exacerbate the redox imbalance when ferroptosis occurs on LUAC cells.