Chemicals

(+)-ABX was prepared from the fermentation product of Streptomyces FJS31-2 in our laboratory. Chloroquine (CQ) was obtained from Solarbio (Beijing, China), while LY294002 and N-acetyl-l-cysteine (NAC) were acquired from MedChemExpress (Shanghai, China).

Cell lines and cell culture

The human non-small cell lung cancer cell lines A549 (cat. no. CL-0016), NCI-H1299 (cat. no. CL-0165), and the human squamous lung cancer cell line NCI-H226 (cat. no. CL-0396) were sourced from the Scientific Research Center of the First People’s Hospital of Zunyi (China). These cell lines were cultivated in RPMI-1640 medium (Gibco, Australia) supplemented with 10% fetal bovine serum (FBS, Gibco, Australia) under a 5% CO2 atmosphere at 37 °C. The cells were passaged three times to reach a stable growth state before being used for the experiments.

Cell viability assay

The cytotoxicity of (+)-ABX was assessed against A549, H1299, and H226 cells using the CCK-8 method. Briefly, a 100 μL cell suspension (5 × 104 cells/mL) was seeded into 96-well microtiter plates with six replicate wells per group. After 24 h of incubation at 37 °C, 5% CO2, and saturated humidity, the compound was introduced. Different concentrations of (+)-ABX (ranging from 0 to 256 μM) were then added to the plates, followed by a 24-h incubation period. After discarding the liquid from the 96-well plate, each well was rinsed with phosphate-buffered saline (PBS). Next, 90 μL of culture medium and 10 μL of CCK-8 solution (Solarbio, Beijing, China) were added to each well, and the plate was further incubated for an additional 4 h. The absorbance at 490 nm (A) was measured using a microplate reader, and the percentage of inhibition was calculated. The formula for calculating percent cell viability is as follows:

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Colony formation assay

Initially, A549, H1299, and H226 cells were exposed to varying concentrations of (+)-ABX (0, 10, 20, and 40 μM) for 24 h. Following this, the medium was aspirated, and the cells underwent two washes with PBS. The cells were then harvested, reseeded in 6-well plates at a density of 2000 cells per well, and the culture medium was renewed every 2 days. After a 14-day incubation period, the resulting colonies were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 15 min, followed by staining with 0.1% crystal violet (Solarbio, Beijing, China) for 20 min. Colonies containing more than ten cells were quantified under a microscope (Olympus). The presented data represent the average of three independent experiments.

Wound healing assay

The wound healing assay was conducted as previously outlined [31]. A549, H1299, and H226 cells were seeded into 6-well plates at a density of 10 × 105 cells per well. Once a monolayer of confluent cells was established, a wound of approximately 0.5 mm width was generated using a sterile micropipette tip (200 μL), followed by gentle rinsing with PBS twice. Subsequently, the cells were exposed to various concentrations of (+)-ABX (0, 10, 20, and 40 μM) for 24 h. Images documenting the progression of wound healing were captured at 12-h intervals over a 48-h period using an inverted microscope (Olympus). The width of the wound, defined by the gap between the edges of the cell-free area, was quantified using Image-J software (NIH, MD).

Transwell migration and invasion assay

To assess cell migration, A549, H1299, and H226 cells in logarithmic growth phase were seeded into a 6-well plate. Different concentrations of (+)-ABX (0, 10, 20, and 40 μM) were then added for a 24-h incubation period. Subsequently, the cells were digested and adjusted to a concentration of 1 × 106 cells/mL in RPMI-1640 medium supplemented with 0.1% FBS. For the upper chamber of the transwell, 200 μL of the cell suspension was added, while the bottom chamber contained 500 μL of RPMI-1640 medium supplemented with 20% FBS. The transwell chambers were placed in a 37 °C, 5% CO2 incubator (Thermo) for 24 h. Following incubation, the culture medium was aspirated, the chambers were washed twice with PBS, and then the cells were fixed with methanol for 20 min. Subsequently, they were stained with 0.1% crystal violet for 20 min, rinsed with water several times, and imaged under a microscope. The migrated cells were counted after gently removing the upper unimmigrated cells using a cotton swab. For the evaluation of cell invasion, the aforementioned cell suspension was added to the transwell upper chamber that had been precoated with Matrigel. The cells were incubated at 37 °C for 5 h. Following incubation, the residual liquid in the chamber was removed, and each well was supplemented with 70 μL of serum-free medium. An additional 30-min incubation at 37 °C was conducted prior to the experiment. The subsequent steps of the operation were identical to those of the cell migration assay.

Flow cytometry

A549, H1299, and H226 cells were individually seeded into 6-well plates (1 × 105 cells per well) and exposed to (+)-ABX (0, 10, 20, and 40 μM) for 24 h. For apoptosis detection, all cells were collected and processed using the FITC Annexin V Apoptosis Detection Kit (BD) according to the manufacturer’s instructions. The stained cells were washed twice with PBS and analyzed using a flow cytometer (AccuriTM C6 Plus, BD). To analyze the cell cycle, the cells were collected, fixed in 70% ethanol overnight at 4 °C, and then stained with propidium iodide (PI) for 30 min at 37 °C in the dark. Subsequent analysis was performed using a flow cytometer.

Cell apoptosis assay

Apoptosis was assessed using Hoechst 33,342 staining. A549, H1299, and H226 cell densities were adjusted to 1 × 105 cells/mL, and 100 μL of cell suspension was seeded into each well of a 96-well plate. The cells were cultured in a 37 °C, 5% CO2 environment for 24 h. Subsequently, different concentrations of (+)-ABX (0, 10, 20, and 40 μM) were added after the initial 24-h incubation. The medium was discarded, and the cells were washed twice with PBS. Following this, the cells were fixed with 4% paraformaldehyde for 15 min, stained with 100 μL of Hoechst 33,342 solution (10 μg/mL) (Solarbio, Beijing, China), and incubated at 37 °C in a 5% CO2 incubator for 30 min. The staining solution was removed, and the cells were washed twice with PBS. Apoptosis was observed under a fluorescence microscope (Olympus), and the images were captured.

Measurement of mitochondrial membrane potential (MMP)

The cells were exposed to various concentrations of (+)-ABX (0, 10, 20, and 40 μM) for a duration of 24 h. Following this exposure, the culture medium was aspirated, and the cells were subjected to two washes with PBS. Subsequently, 1 mL of JC-1 staining working solution (Solarbio, Beijing, China) was added to each well, and the cells were incubated in a cell culture incubator at 37 °C for 20 min. The culture medium was then discarded, and the cells were washed twice with precooled JC-1 staining buffer. Afterward, 1 mL of PBS was added to each well to resuspend the cells, allowing for observation and image collection using a fluorescent microscope.

Quantitative real-time polymerase chain reaction

Total RNA was extracted from A549, H1299, and H226 cells treated with various concentrations of (+)-ABX (0, 10, 20, and 40 μM) for 24 h using the RNAiso Plus reagent (Takara, Beijing, China). Subsequently, the extracted total RNA was reverse transcribed into first-strand cDNA using the PrimeScript™ RT Master Mix (Takara, Beijing, China). Real-time PCR amplification was carried out utilizing the Real-Time PCR Detection System (CFX96, Bio-Rad, USA) and the SYBR Premix Ex Taq™ (Takara, Beijing, China). The PCR primers (Additional file 1: Table S1) were designed and synthesized by Sangon Biotech (Shanghai, China). The relative mRNA expression levels were determined using the relative standard curve method with β-actin as a reference.

Western blot assay

The cells were seeded in 6-well plates and cultured for 24 h prior to treatment with (+)-ABX (0, 10, 20, 40 µM) or inhibitors (CQ, LY294002, and NAC) for an additional 24 h. Following treatment, cells were lysed using RIPA lysis buffer (Solarbio, Beijing, China), and protein concentrations were determined using the BCA assay. Proteins (20 µg per lane) were separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gel (Solarbio, Beijing, China). Subsequently, the gels were transferred onto polyvinylidene fluoride membranes (Millipore, MA), blocked with 5% BSA blocking buffer (Solarbio, Beijing, China) for 2 h, and incubated overnight at 4 °C with primary antibodies. Antibodies against Beclin1, LC3B, SQSTM1/p62, cleaved-caspase 3, mTOR, phospho-mTOR, phospho-PI3K, phospho-Akt, Akt, Bcl2, p21, cyclin B1, and CDK1 were procured from Zenbio (Chengdu, China). Antibodies against BAX, Bcl2, and β-actin were sourced from Solarbio (Beijing, China), while cleaved-caspase 9 was obtained from Affinity Biosciences (Changzhou, China). PI3K and HRP Goat anti-Rabbit IgG secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Subsequent to incubation with corresponding secondary antibodies at room temperature for 2 h, the membranes were treated with Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA) and imaged using the BIO-RAD ChemiDoc Imaging System (Hercules, CA).

Transmission electron microscopy (TEM)

A549 and H1299 cells treated with (+)-ABX at 20 μM for 24 h were collected and subjected to pre-embedding in 1% agarose solution. Subsequently, the samples were fixed in 1% osmium tetroxide at room temperature in the dark for 2 h. After three washes with PBS buffer, each lasting 15 min, a series of increasing ethanol concentrations were employed for dehydration. The samples were then subjected to infiltration, polymerization, and slicing processes to obtain ultrathin sections measuring 60–80 nm. These sections were stained with 2% uranyl acetate saturated alcoholic solution in the absence of light, followed by staining with 2.6% lead citrate solution to avoid exposure to carbon dioxide. Subsequently, the samples were observed for cellular morphology, and the images were captured using a transmission electron microscope (HT7800, Hitachi, Japan).

RNA-seq and transcriptomic analysis

The H1299 cells were cultured to the logarithmic growth phase. For the (+)-ABX treated groups, the cells were treated with 20 μM and 30 μM of (+)-ABX, respectively, while the control group received an equivalent volume of solvent. Each group had three replicates, and after 24 h, the cells were collected and treated with 1 mL of Trizol reagent individually. The library preparation and transcriptome sequencing experiments were conducted by Beijing Qinge Biotechnology Company (Beijing, China) using the Illumina HiSeq platform. During the analysis of differentially expressed genes, a threshold of fold change ≥ 2 and P-value < 0.05 was utilized for screening. Pathway enrichment analysis of differentially expressed genes was carried out using the KEGG database (www.genome.jp/kegg).

Molecular docking

The three-dimensional conformation of (+)-ABX was optimized using Chem3D 19.0 (CambridgeSoft, USA). The three-dimensional structures of PI3K (1H9O), AKT (2W1C), and mTOR (4DRI) were downloaded from the RSC Protein Data Bank (https://www.rcsb.org) and processed using PyMol (Version 2.6.0a0 Open-Source). Molecular docking studies were conducted using AutoDock Vina (Version 1.5.7) [32], and the docking results were visualized using Discovery Studio 2019 (BIOVIA, USA).

Measurement of ROS levels

The cells were adjusted to a density of 1 × 106 cells/mL. Subsequently, 500 μL of the cell suspension was inoculated into a 24-well plate and cultured for 24 h. Different concentrations of (+)-ABX (0, 10, 20, and 40 μM) were then added for an additional 24 h. Following the removal of the culture medium, 1 mL of preprepared DCFH-DA fluorescent dye (Solarbio, Beijing, China) was added to each well. The cells were incubated in the incubator for 30 min, after which any excess dye that had not entered the cells was washed away with PBS. Finally, 1 mL of PBS was added to each well for resuspension. The cells were excited with blue light using an inverted fluorescence microscope, and the fluorescence intensity was observed.

Tumor xenograft models

The animal experiment protocol was approved by the Laboratory Animal Welfare and Ethical Committee of Zunyi Medical University (ZMU21-2303–105). SPF-grade male BALB/c-nu nude mice (4–5 weeks old) were obtained from Beijing HFK Bioscience CO., Ltd (SCXK J2019-0008). A volume of 100 μL of A549 cell suspension (1 × 106 cells/mL) was subcutaneously inoculated on the back of nude mice. The mental state of the mice was observed every two days, and body weight and tumor volume (0.5 × L × W2, where L is length and W is width) were recorded. After 10 days, when the tumor volume reached approximately 60–80 mm3, the nude mice were randomly divided into two groups: a control group and a (+)-ABX treatment group (2 mg/kg), each containing six mice. The treatment group received intraperitoneal administration of 200 μL of (+)-ABX (2 mg/kg) every other day for 28 days, while the control group received intraperitoneal injections of an equal volume of normal saline. The experiment was terminated after 30 days, at which point the nude mice were euthanized by cervical dislocation. The tumors and major organs (liver, lung, and kidney) were removed, weighed, recorded, and subsequently fixed with 10% formalin for histopathology and immunology evaluation.

Histopathology and immunohistochemistry

After the in vivo experiment, subcutaneously transplanted tumors, liver, kidney, and lung samples from each group of nude mice were subjected to hematoxylin and eosin (H&E) staining to assess the potential organ toxicity of (+)-ABX. The procedure was carried out as outlined below. The tissues were fixed in 10% formalin, dehydrated using varying concentrations of ethanol, paraffin-embedded, and subsequently sectioned. The sections were dewaxed, hydrated, stained with hematoxylin for 20 min, water-rinsed, differentiated with differentiation medium for 30 s, water-soaked for 15 min, stained with eosin for 2 min, water-soaked for 5 min, dehydrated, and sealed with neutral balsam. The images were acquired under a microscope. For immunohistochemical staining (IHC), the slides were incubated with antibodies (Ki-67, cleaved-caspase 3, cleaved-caspase 9, BAX, AKT, LC3A, and Beclin1) at 4 °C overnight, followed by incubation with secondary antibodies at room temperature for 2 h. Subsequently, the slides were mounted using neutral balsam, and optical microscope images were captured. Antibodies Ki67 was sourced from ABclonal (Wuhan, China), LC3A from Zenbio (Chengdu, China), and BAX and cleaved-caspase 3 from Proteintech (Wuhan, China). The HRP-Polymer anti-Mouse/Rabbit IHC Kit was acquired from MXB (Fuzhou, China), and the remaining antibodies corresponded to those used in the western blot assay.

Statistical analysis

All experiments were conducted independently in triplicate, and the outcomes are expressed as mean ± standard deviation (SD). Statistical evaluations were performed using SPSS 29.0 (IBM Corp., Armonk, NY) and GraphPad Prism 8.0 statistical software (GraphPad, La Jolla, CA). Group comparisons were assessed utilizing one-way analysis of variance (ANOVA) and Student’s t-test. A significance threshold of P < 0.05 denoted statistical significance and is denoted by * or △; ** or △△P < 0.01, and *** or △△△P < 0.001. A P-value exceeding 0.05 indicated nonsignificance (ns).