Pristimerin inhibits thioredoxin reductase in the treatment of non-small cell lung cancer

Abstract

Elevated cellular oxidative stress is a common marker of cancer cell dysregulation caused by malignant transformation. Thioredoxin reductase (TrxR, encoded by TXNRD) is a crucial enzyme that regulates cellular oxidative stress and the survival of many types of cancer cells. Therefore, targeting TrxR may lead to selective cell death in cancer cells. Pristimerin, a plant triterpenoid, increases the accumulation of reactive oxygen species (ROS) in cells, but its specific regulatory mechanism is unclear. Herein, we found that pristimerin selectively targets TrxR and subsequently induces apoptosis in human non-small cell lung cancer cells, and inhibits tumor growth in vivo with low toxicity to normal cells. Pristimerin was found to inhibit cancer cell growth primarily by inhibiting cellular TrxR, thereby compromising TrxR’s antioxidant function in cells and resulting in the accumulation of oxidized Trx. Furthermore, excessive ROS accumulation stimulated by pristimerin triggered tumor-specific amplification of oxidative stress in cancer cells and ultimately led to targeted destruction of cancer cells. Our data may support the development of potential therapeutic molecules as selective anticancer agents targeting highly enriched TrxR in cancer cells.

Main article text

1. INTRODUCTION

Natural products and their analogs have played major roles in drug discovery and design, particularly in the treatment of cancer and infectious diseases [1, 2]. The diversity of natural products, along with their complex structural scaffolds, provide unique advantages in the drug discovery process [1, 3]. Notably, the identification of biological activity in natural products can provide valuable insights into the efficacy and safety of traditional medicines for treatment purposes [4]. Consequently, interest in candidate drug libraries rich in biological activity has grown in pharmaceutical research, owing to their ability to cover a wider chemical space [5, 6].

Pristimerin is a natural product that was initially discovered as an antibacterial molecule in Pristimerae indica and Pristimerae grahami (Celastraceae) [7, 8]. Numerous studies have revealed this compound’s diverse biological activities and pharmacological effects, including anti-tumor [9, 10], anti-inflammatory [11], anti-malarial [12], and anti-infectious [13, 14] activity. Pristimerin also induces various forms of cell death, such as apoptosis [10, 15, 16], autophagy [17, 18], and necrosis [19]. Its roles have been extensively studied, including its inhibitory effects on the AIM2 inflammasome [17], NF-κB signaling [20], MAGL [21], and the Wnt/β-catenin pathway; its activation of JNK, the reactive oxygen species (ROS)/ASK1/JNK pathway, and the ROS-dependent ER stress/Noxa pathway [22]; and its modulation of AIM2-PYCARD/ASC stability [17] and MAPK signaling pathways [9]. Moreover, pristimerin has been found to preventing sperm hyperactivation through improvement of PPARα pathway [23] and induction of a significant increase in Ca²⁺ levels [24, 25]. Notably, its inhibitory activity and mechanism in the proliferation of various cancers have garnered considerable attention. Pristimerin enhances the accumulation of ROS in cells [18, 22, 26, 27], although the specific molecular regulation mechanism remains unclear.

The thioredoxin system is a crucial mammalian antioxidant system comprising thioredoxin (Trx, encoded by TXN), thioredoxin reductase (TrxR, encoded by TXNRD), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) [28–30]. This system begins with the electron donor NADPH; TrxR subsequently transfers electrons to Trx [31], which in turn transfers electrons and results in ROS clearance [32, 33]. The thioredoxin system plays crucial roles in redox homeostasis and DNA protection against oxidative-stress-related DNA damage [34, 35]. The thioredoxin-specific enzyme TrxR, which is a selenase and NADPH-dependent flavoprotein, plays a crucial role in facilitating the interaction between NADPH and Trx in the system [31, 36]. Activation of the thioredoxin system is essential in cancer [29]; Trx and TrxR are widely expressed in non-small cell lung cancer (NSCLC) and are involved in the activation of transcription factors and the regulation of apoptosis [37]. In clinical samples, TrxR is considered a prognostic marker for human NSCLC: elevated expression of TrxR is positively correlated with advanced clinical stages and poor patient survival rates [37–39]. In practice, auranofin (AF), an inhibitor of TrxR, is an effective anticancer agent in the treatment of NSCLC [40], chronic lymphocytic leukemia [41], epithelial ovarian cancer [42], and peritoneal cancer and fallopian tube cancer [43–45]. In addition, many TrxR inhibitors with antitumor activity obtained from natural products have further demonstrated TrxR’s promise as an anticancer target [46–52]. Therefore, the development of novel TrxR inhibitors from natural products for NSCLC treatment has great potential. Herein, we found that plant triterpenoid pristimerin has effective anticancer effects on NSCLC by inhibiting TrxR, thus providing an experimental basis for pristimerin to further become an anti-cancer agent.

2. MATERIALS AND METHODS

2.1 Materials and reagents

Pristimerin (purity ≥98.0%) was obtained from TargetMol Chemicals Inc. (Boston, Massachusetts, USA). Cisplatin (purity ≥99.0%) and bovine serum albumin were purchased from Beyotime (Nantong, China). The HeLa-shTrxR1 cells and HeLa-shNT cells were generated as described in previous articles from our research group [53, 54]. Prof. Jianqiang Xu (Dalian University of Technology, China) provided recombinant rat TrxR1 [55]. U498C TrxR1 mutant (Sec→Cys) and recombinant E. coli Trx were prepared as previously described [56]. RPMI 1640, DMEM, and antibiotics were supplied by Thermo Fisher Tech Co., Ltd-CN (Shanghai, China). NADPH was purchased from Yuanye Bio-Tech Co., Ltd. (Shanghai, China). Glutathione (GSH), yeast glutathione reductase, oxidized glutathione (GSSG), bovine insulin, L-buthionine-sulfoximine (BSO), N-acetyl-L-cysteine (NAC), Hoechst 33342, DTNB, MTT, DMSO, DCFH-DA, DHE, and 2,3-dimercapto-1-propanesulfonic acid (DMPS) were purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS) was obtained from HyClone (Logan, USA). Primary antibodies to Trx1 (anti-Trx1, sc-28321), TrxR (anti-TrxR, sc-28321), GAPDH (anti-GAPDH, sc-28321), Actin (anti-Actin, sc-28321), caspase-3 (anti-caspase-3, sc-28321), Bax (anti-Bax, sc-28321), and Bcl-2 (anti-Bcl-2, sc-28321) were from Abcam (Cambridge, UK). The TRFS-green dye was purchased from MedChemExpress (www.medchemexpress.com). The Annexin V-FITC/PI Apoptosis Detection Kit was from Vazyme Bio-Tech (Nanjing, China).

2.2 Cell culture

A549, HeLa, HepG2, and L02 cells were purchased from the Shanghai Institute of Biochemistry and Cell Biology. A549 cells were maintained in RPMI 1640, and HepG2, HeLa, and L02 cells were maintained in DMEM. All cell lines were incubated in DMEM with 10% FBS, and 100 units·mL⁻¹ penicillin and streptomycin, at a temperature of 37°C in a 5% CO₂ incubator. HeLa-shTrxR1 and HeLa-shNT cells were grown under standard culture conditions with an additional 1 μg·mL⁻¹ puromycin.

2.3 Cell viability assays

The cytotoxic activity of pristimerin was evaluated with MTT assays. In this method, a specific number of cells were incubated with various concentrations of different compounds for predetermined times. The control group was treated with the maximum amount of DMSO, whereas the blank group consisted of cells treated with only the culture medium. After the specified incubation period, a 100% DMEM solution containing 0.5 mg/mL MTT was added to each well and incubated for 4 h. Afterward, 100 μL triple solution was added to each well and incubated overnight. Finally, the absorbance per well (OD₅₇₀ nm) at 570 nm was measured with a full-wavelength microplate reader. In the NAC and BSO experimental groups, cells were initially treated with 50 μM BSO for 24 h, or 5 or10 mM NAC for 24 h, then exposed to various concentrations of pristimerin. The cell survival rate was obtained with the formula [(OD_{experimental group}−OD_blank)/(OD_{control group}−OD_blank)] × 100.

2.4 Colony formation assays

In a six-well plate, 1 × 10³ A549 cells were incubated with various concentrations of pristimerin for 24 h under standard culture conditions. The culture medium was replaced every 2 days after the removal of pristimerin. On the 10th day of incubation, the culture medium was discarded, and the cells were fixed (in 4% paraformaldehyde and 0.5% crystal violet) for 30 min. Subsequently, the six-well plate was photographed, and the colony count was determined.

2.5 In vitro TrxR activity assays

In 96-well plates, TrxR pre-reduced by NADPH (final concentration of 80 nM) or U498C TrxR (final concentration of 300 nM) was incubated with various concentrations of pristimerin in buffer (50 mM Tris HCl, 1 mM EDTA, pH 7.4) at 37°C for 2 h. The control group was treated with the maximum amount of DMSO. A 50 μL solution containing 200 μM NADPH and 2.5 mM DTNB was subsequently added, and the linear change in absorbance at 412 nm in the first 3 min was read in buffer with a microplate reader.

2.6 Molecular docking

Molecular covalent docking was simulated with Schrödinger molecular modeling software (Schrödinger, LLC, New York, NY, USA, Release 2015). The ligands were prepared with the Ligprep 3.3 module from Schrödinger 2015. The Protein Preparation Wizard module in Schrödinger was used to prepare rat TrxR1 protein (PDB code 3EAN). All heteroatoms and water molecules of TrxR1 were removed, and all hydrogen atoms were added. The Sec498 residue in the TrxR1 chain a was set as the center of the docking box, and the Michael addition reaction was simulated for molecular covalent docking under default parameters.

2.7 Cellular TrxR activity assays

The activity of TrxR in cells was determined with insulin endpoint assays. A549 cells were treated with various concentrations of pristimerin for 24 h in 100 mm dishes. After the cells were collected, total protein was extracted and quantified, and 20 μL lysate containing 0.33 mM insulin, 15 μM E. coli Trx, as well as a buffer containing 0.66 mM NADPH were incubated at 37°C for 30 min (50 μL final volume). A 200 μL solution containing 6 M guanidine hydrochloride and 1 mM DTNB (pH 8.0) was added to each well and incubated at room temperature for 5 min, and the absorbance was read at 412 nm with a microplate reader.

2.8 Live cell imaging with the TrxR probe TRFS-green

In 12-well plates, A549 cells were treated with pristimerin at final concentrations of 0, 0.5, 1, or 2 μM for 24 h. The medium was then replaced with fresh FBS-free medium containing 10 μM TRFS green for another 4 h. After the medium was removed, the cells were washed three times with PBS, then imaged in the green channel with a fluorescence microscope (Floid Cell Imaging Station).

2.9 Cellular ROS measurement

In 12-well plates, cells were treated with pristimerin at final concentrations of 0, 0.5, 1, or 2 μM for 24 h. The medium was then replaced with fresh FBS-free medium containing 10 μM DCFH-DA or 10 μM DHE, and incubated for 30 min in the dark. After the medium was discarded, the cells were washed with PBS three times, then imaged under a fluorescence microscope (Floid Cell Imaging Station) in the green or red channels.

2.10 Total glutathione and GSSG assays

In 100 mm dishes, A549 cells were incubated with 0, 0.25, or 1 μM pristimerin for 24 h. The cells were counted and collected, resuspended in 1 mL PBS, and then divided into a total GSH group (300 μL) and a GSSG group (600 μL). In the total GSH group, NEM was added to a final concentration of 1 mM, and cells were incubated in the dark for 10 min. The cells were centrifuged, the supernatant was discarded, and the cells were washed with PBS and centrifuged three times. The GSSG group was not treated. The supernatant was discarded after centrifugation, and the cell pellet was collected. The cells were sonicated in protein extraction solution (0.1 M potassium phosphate buffer, 5 mM EDTA, pH 7.4) containing 1.0% sulfosalicylic acid and 0.1% Triton X-100. After centrifugation at 3000 g at 4°C for 10 min, all supernatants were transferred immediately to a new centrifuge tube and diluted with KPE buffer without sulfosalicylic acid to a sulfosalicylic acid concentration of 0.5%. In a 96-well plate, 20 μL of total GSH group sample diluted ten-fold or 20 μL of GSSG group sample was added. Another 60 μL KPE buffer, followed by 60 μL of KPE buffer containing 1.68 mM DTNB, 0.8 mM NADPH, and 3.33 U/mL glutathione reductase, was added to each well. The GSH and GSSG standard curves were prepared with KPE buffer containing 0.5% sulfosalicylic acid in the same manner for the total GSH group and GSSG group. The linear change in absorbance at 412 nm in the first 3 min was immediately read with a microplate reader, and the content of total GSH and GSSG was calculated with a standard curve.

2.11 Cell-free thiols assays

A549 cells were incubated with various doses of pristimerin for 24 h in 100 mm dishes. Total protein was subsequently extracted from the cells with RIPA buffer and quantified. Twenty microliters of lysate containing 30 μg total protein or 20 μL standard Cys working concentration was added to 96-well plates. The mixture was then treated with 80 μL of 6 M guanidine hydrochloride solution (pH 8.0) containing 1 mM DTNB for 5 min at room temperature, and the absorbance at 412 nm was measured with a microplate reader.

2.12 Trx redox state determination

A549 cells were seeded in 100 mm dishes for attachment culture and incubated with various concentrations of pristimerin for 24 h at 37°C. The cells were then collected, washed with PBS, and lysed in RIPA buffer. Total cellular protein was quantified, and the lysates were divided into two groups: a total Trx group and a treatment group. In the total Trx group, no follow-up treatment was performed, and the total Trx was directly denatured at 100°C for 5 min after addition of loading buffer. The samples were then subjected to immunoblot analysis. In the treatment group, samples were added into PAO-Sepharose, incubated at room temperature for 30 min, and vortexed every 5 min. The supernatant was then separated to obtain oxidized Trx. After the beads were washed three times with buffer, the bound reduced Trx was eluted with 20 mM DMPS every 5 min six times. Trx content in all samples was determined by western blot analysis.

2.13 Western blotting

A549 cells were treated with various concentrations of pristimerin for 24 h, and the total cellular protein was extracted with RIPA buffer. Samples with equal amounts of total cellular protein were separated by gel electrophoresis (SDS-PAGE). The proteins were then transferred to PVDF membranes, blocked with 5% nonfat milk for 1 h, and incubated with primary antibodies to caspase 3, Actin, Bax, or Bcl-2 (1:1000 dilution) at 4°C overnight. After three washes with TBST, the PVDF membrane was further incubated with a secondary antibody with HRP (1:4000 dilution) for 1 h. After the secondary antibody was washed away three times with TBST, a chemiluminescence kit was used to detect the protein bands.

2.14 Hoechst 33342 probe for live-cell nuclear imaging

A549 cells were incubated with pristimerin (final concentrations of 0, 0.25, 0.5, or 1 μM) in 12-well plates for 24 h. The medium was then replaced with fresh FBS-free medium containing 5 μg/mL Hoechst 33342 and incubated in the dark for another 30 min. After the medium was removed, the cells were washed three times with PBS, then imaged in the blue channel with a fluorescence microscope.

2.15 Caspase 3 activity assays

A549 cells were incubated with various concentrations of pristimerin in 100 mm dishes for 24 h. After the cells were collected, the proteins were extracted with RIPA buffer, and the total cellular proteins were quantified. In 96-well plates, lysate of 30 μg protein were incubated with 50 mM HEPES containing 10 mM DTT, 5% glycerol, 0.2 mM Ac-DEVD-pNA, 2 mM EDTA, and 0.1% CHAPS, pH 7.4, for 3 h at 37°C. The absorbance at 405 nm was read with a microplate reader.

2.16 Annexin V-FITC/PI staining

A549 cells were treated with various concentrations of pristimerin in six-well plates for 48 h, and the cells were subsequently washed with PBS and collected. The cells were stained with an Annexin V-FITC/PI double staining apoptosis detection kit. Viable cells, apoptotic cells, and necrotic cells were detected by flow cytometry. The data were analyzed in Cell Quest software.

2.17 Animals and xenograft assays

Approximately 4-week-old female BALB/c mice were obtained from Charles River Laboratories (Beijing, China). All mice were housed at the GLP experimental center of Lanzhou University, and all animal experiments were performed in accordance with the relevant guidelines and regulations, and approved by the institutional animal care and use committee. This study was approved by the Ethics Committee of Lanzhou University School of Pharmacy (ethics approval number: 20200323). Six-week-old (∼18 g) female Balb/c nude mice were injected with 5 × 10⁶ A549 cells and divided into three groups. When the tumor size reached a certain volume, the mice were administered intraperitoneally with a control containing 2% DMSO, 40% PEG400, and 5% Tween-80 in normal saline, pristimerin (2 mg/kg) [20], and CDDP (2 mg/kg), once every 3 days for 21 days. Tumor length, width, and weight were recorded every 3 days, and the tumor volume was calculated. After the experiment, the mice were dissected, and the tumor tissues and various organs were collected to calculate the average tumor weight and organ index [57].

2.18 Statistical analysis

All experiments were conducted at least three times, and the data are presented as mean ± SE. Statistical differences (p < 0.05) between groups were analyzed with Student’s t-test. The densities of targeted bands or positive cells were analyzed in ImageJ software.

3. RESULTS

3.1. Inhibition of TrxR by pristimerin in vitro

The naturally occurring quinonoid triterpene pristimerin is recognized for its α-methylene-γ-methylene moiety [58] ( Figure 1A ), similar to that in known TrxR inhibitors, such as micheliolide [59] and securinine [60]. Pristimerin has also been suggested to be a TrxR inhibitor. To investigate this possibility, we evaluated its inhibitory effect on pure TrxR in vitro. Preincubation of pristimerin with reduced recombinant rat TrxR1 (WT TrxR1) resulted in dose-dependent inhibition of the enzyme, with an IC₅₀ value of 4.26 μM ( Figure 1B ). Similarly, we examined the inhibitory efficacy of pristimerin on U498C TrxR1, a WT TrxR1 mutant with Sec498 mutated to Cys, which exhibited almost no inhibitory effect on the U498C TrxR1 enzyme ( Figure 1B ). The selective inhibition of WT TrxR1 but not U498C TrxR1 by pristimerin suggested that the Sec residue is specifically targeted by pristimerin. This crucial finding was further confirmed through molecular docking analysis of the probable binding site of pristimerin in TrxR1 protein. Pristimerin inserts into TrxR1, and the α-methylene-γ-methylene moiety from pristimerin ( Figure 1 highlights); i.e., the carbon at position 6 of pristimerin forms a covalent bond with the Sec498 residue in the redox center of the C-terminal active site of TrxR1 ( Figure 1C and 1D ). The specific targeting of Sec498 by pristimerin may ablate the redox activity of TrxR in cancer cells.

Figure 1 |

Pristimerin selectively inhibits TrxR in vitro.

(A) The structure of pristimerin. (B) Purified WT TrxR1 and U498C TrxR1 were inhibited by pristimerin. NADPH-reduced WT TrxR1 and U498C TrxR1 were treated with various concentrations of pristimerin for 2 h, and activities were measured with DTNB assays. (C) Molecular docking analysis, used to simulate and examine the interaction between pristimerin and the receptor protein, revealed that the carbon at position 6 of pristimerin covalently binds to Sec498 of TrxR. (D) The 2D binding mode of pristimerin and TrxR’s Sec498 binding. Pristimerin assumed an optimized conformation, yielding a docking score of −4.233 kcal/mol, with a covalent bond length of 2.0 Å. **, p < 0.01 vs the control group.

3.2 Pristimerin inhibits cancer cell proliferation

To investigate the biological effects of the specific targeted inactivation of TrxR by pristimerin, we first tested the cytotoxicity of pristimerin toward various cancer cells. Pristimerin inhibited the malignant proliferation of A549, HeLa, and HepG2 cells in a dose-dependent manner, and the cytotoxic IC₅₀ values obtained after 48 h incubation were 0.81, 1.25, and 1.46 μM, respectively ( Figure 2A ). We further compared the cytotoxicity of pristimerin against L02 cells, an undifferentiated lung epithelial cell line, and A549 cells. Interestingly, we observed a significant difference in the cytotoxicity of pristimerin toward L02 cells and A549 cells after 48 h incubation, with IC₅₀ values of 1.78 and 0.81 μM, respectively, indicating a difference greater than two-fold ( Figure 2B ). Importantly, the inhibition of the proliferation of A549 cells by pristimerin was time dependent ( Figure 2C ). For visualization, we treated A549 cells with 0.5 and 1 μM of pristimerin, then performed crystal violet staining. Pristimerin inhibited the formation of A549 cell colonies in a dose-dependent manner ( Figure 2D and 2E ). These results together demonstrated that pristimerin effectively inhibits the malignant proliferation of A549 cells.

Figure 2 |

Pristimerin has cytotoxic and proliferation-inhibiting effects on tumor cells.

(A) MTT assays were used to assess pristimerin’s cytotoxicity toward A549, HeLa, and HepG2 cells after 48 h incubation. (B) MTT assays were used to assess pristimerin’s cytotoxic effects on L02 and A549 cells. (C) The time-dependent cytotoxicity of pristimerin toward A549 cells was assessed with MTT assays after 24 and 48 h of incubation. (D) Pristimerin dose-dependently inhibits the formation of A549 cell colonies. A549 cells were treated with pristimerin for 24 h and allowed to grow for 10 days before staining with crystal violet. (E) Counts of cell colonies. Data are shown as mean ± SD. ** and ^^, p < 0.01 vs the control group.

3.3 Intracellular TrxR is involved in pristimerin cytotoxicity

Because pristimerin effectively interacts with pure TrxR, we reasoned that the performance of intracellular pristimerin in inhibiting the malignant proliferation of A549 cells might be associated with the inhibition of intracellular TrxR activity by pristimerin. Therefore, we determined the inhibitory effect of pristimerin on intracellular TrxR. First, we performed a classical Trx-mediated insulin reduction assay to confirm the inhibitory effect of pristimerin on TrxR [61]. Pristimerin impaired TrxR activity in a dose-dependent manner; AF, a classical inhibitor of TrxR, served as a positive control ( Figure 3A ). Notably, the IC₅₀ value for pristimerin in inhibiting TrxR in A549 cells was 1.65 μM; this value strongly correlated with inhibition of cell proliferation in the same cell line. Second, we developed a TRFS-green probe to evaluate the activity of intracellular TrxR after pristimerin treatment [62]. The fluorescence intensity in A549 cells treated with pristimerin significantly decreased in a dose-dependent manner, thereby indicating that pristimerin inhibited TrxR activity in cells ( Figure 3B ). To further explore whether the cytotoxicity of pristimerin might be associated with an interaction with TrxR, we transfected an shRNA plasmid specifically targeting TrxR1 to generate HeLa cells with stable knockdown of TrxR1 expression (HeLa-shTrxR1). Non-targeting shRNA plasmids were transfected to generate control cells (HeLa-shNT) [53, 54]. We evaluated the differences in intracellular TrxR activity and expression between groups ( Figure 3D and 3E ). Interestingly, pristimerin showed a synergistic effect on HeLa-shTrxR1 cells, thus confirming that intracellular TrxR is indeed involved in the cytotoxic effect of pristimerin ( Figure 3F ), in agreement with findings that TrxR knockdown leads to drug-specific changes in the cytotoxicity of therapeutic small molecules [63]. Collectively, pristimerin’s ability to inhibit tumor cell proliferation is due primarily to its inhibition of intracellular TrxR.

Figure 3 |

Pristimerin selectively inhibits TrxR in A549 cells.

(A) Intracellular TrxR activity in A549 cells is inhibited by pristimerin and the positive drug AF. Pristimerin and 1 μM AF were used to treat A549 cells for 24 h, and the endpoint trypsin assay was used to determine intracellular TrxR activity. (B) The TRFS-green probe was used to image intracellular TrxR in A549 cells, which were grown in 12-well plates and incubated with pristimerin for 24 h before staining for intracellular TrxR activity with the TrxR probe TRFS-green. Scale bar: 20 μm. (C) ImageJ software was used to measure positive cells in (B). (D) Insulin endpoint assays were used to assess the knockdown efficiency of TrxR1 in HeLa-shNT and HeLa-shTrxR1 cells. (E) Expression of TrxR in HeLa-shNT and HeLa-shTrxR1 cells. (F) MTT assays were used to assess the effect of TrxR1 knockdown in A549 cells on the cytotoxicity of pristimerin. Data are shown as means ± SD. *, p < 0.05, **, p < 0.01 vs the control group.

3.4 Pristimerin induces intracellular accumulation of oxidized Trx

The catalytic role of TrxR in the NADPH-dependent reduction of disulfide bonds in oxidized Trx is crucial for maintaining intracellular redox levels [64]. The inhibition of intracellular TrxR by pristimerin might lead to the accumulation of intracellular oxidized Trx; therefore, we assessed the ratio of reduced Trx to oxidized Trx in A549 cells after pristimerin treatment. To do so, we used PAO-Sepharose to retrieve reduced Trx while retaining oxidized Trx in solution [54, 65]. We subsequently examined the protein expression of the treated samples. Intracellular oxidized Trx increased with increasing pristimerin concentrations in A549 cells ( Figure 4A ), thereby suggesting that pristimerin stimulates the accumulation of oxidized Trx. The ratio index of reduced Trx to oxidized Trx decreased significantly after pristimerin treatment ( Figure 4B ), thus indicating that the inhibition of intracellular TrxR by pristimerin blocked the reduction of Trx and resulted in a state of oxidative stress.

Figure 4 |

The redox state of Trx in A549 cells treated with pristimerin.

(A) With a PAO-Sepharose pull-down assay, oxidized Trx (O) and reduced Trx (R) in protein extracts from A549 cells were separated and assessed with western blotting to determine intracellular Trx redox status after pristimerin treatment. (B) ImageJ software was used to analyze the decrease in the ratio of reduced Trx to oxidized Trx. Data are shown as means ± SD. **, p < 0.01 vs the control group.

3.5 Pristimerin induces intracellular ROS accumulation

The activity of TrxR is crucial for maintaining intracellular redox levels in the thioredoxin system [66]. TrxR not only directly scavenges ROS through Trx but also supports the activity of various antioxidant enzymes [66, 67]. Consequently, we reasoned that inhibiting TrxR with pristimerin might exacerbate oxidative stress by further converting the enzyme into a ROS generator. To confirm this possibility, we assessed ROS levels in A549 cells after pristimerin treatment. DCFH-DA, a universal ROS probe, was used to stain the treated cells [68]. A burst of ROS was observed after stimulation with pristimerin ( Figure 5A and 5B ). We further used DHE, a dye that easily penetrates the plasma membrane and intercepts ROS, particularly superoxide, and forms products emitting red fluorescence [68]. Pristimerin-treated cells exhibited red fluorescence ( Figure 5C and 5D ), thus further confirming ROS accumulation. Together, our results indicated that pristimerin induces ROS accumulation in A549 cells.

Figure 5 |

Pristimerin affects ROS levels in A549 cells.

ROS levels in A549 cells were measured with DCFH-DA (A) and DHE (C). A549 cells were incubated with pristimerin for 6 h, then stained with DCFH-DA (10 μM) or DHE (10 μM) for 30 min (B and D). ImageJ software was used to measure the positive cells in (A) and (C). Scale bar: 20 μm. Data are shown as means ± SD. **, p < 0.01 vs the control group.

3.6 Pristimerin induces oxidative stress in A549 cells

Because pristimerin selectively inhibited TrxR and resulted in the accumulation of intracellular ROS, we tested other oxidative stress-related parameters to confirm that pristimerin-treated A549 cells were under oxidative stress. We initially assessed intracellular GSH levels. The treatment of cells with pristimerin increased total intracellular GSH levels ( Figure 6A ), possibly as a compensatory effect after inhibition of the thioredoxin system [69]. Moreover, intracellular levels of GSSG in pristimerin-treated cells were markedly greater than those observed in the control group ( Figure 6B ), and the GSH to GSSG ratio was significantly diminished ( Figure 6C ), thus further confirming that pristimerin-treated A549 cells were under oxidative stress. Additionally, the decrease in total thiol levels observed in cells treated with pristimerin might have been a response to the aforementioned accumulation of ROS ( Figure 6D ). Moreover, pretreatment of A549 cells with BSO increased the cytotoxicity of pristimerin by depleting cellular GSH ( Figure 6E and 6F ). However, pretreatment with NAC impaired the cytotoxicity of pristimerin ( Figure 6F ), thereby suggesting that high concentrations of NAC might protect against the cytotoxic effects of pristimerin. GSH is a key component of the glutathione system, another redox-regulatory network in cells in addition to the thioredoxin system [70]. The depletion of cellular GSH suggested that the thioredoxin system is involved in the cellular action of pristimerin and sensitizes cells to this treatment. In conclusion, the accumulation of GSSG, and the findings that NAC protected against cell death, whereas GSH depletion promoted cell death, further indicated the oxidative stress state of pristimerin-treated cells.

Figure 6 |

Pristimerin induces oxidative stress in A549 cells.

(A), (B), and (C) show the changes in intracellular total GSH, GSSG, and the GSH/GSSG ratio after pristimerin treatment, respectively. Enzymatic cycling was used to determine the changes in intracellular total GSH and GSSG after 24 h pristimerin treatment of A549 cells. (D) Intracellular thiols in A549 cells were measured by DTNB titration to determine changes in intracellular total thiol levels after pristimerin treatment for 24 h. (E) Enzymatic cycling was used to determine changes in intracellular total GSH after 50 μM BSO treatment for 24 h. (F) MTT assays were used to assess the effects of NAC and BSO on pristimerin cytotoxicity. A549 cells were pre-incubated with NAC for 24 h before assessment of cell viability. Data are shown as mean ± SD. *, p < 0.05, **, ^##, and ^^, p < 0.01 vs the control group.

3.7 Pristimerin induces apoptosis in A549 cells

Our findings demonstrated that the cytotoxicity of pristimerin-targeted TrxR on A549 cells occurred primarily through the induction of apoptosis. We conducted multiple assays to confirm pristimerin-induced apoptosis in A549 cells. To demonstrate the morphological changes in cell nuclei, we performed Hoechst staining. Hoechst 33342 dye specifically binds double-stranded DNA and produces blue fluorescence after penetrating the cell membrane. As shown in Figure 7A , control cells showed normal round nuclei with weak and uniform fluorescence. After treatment with pristimerin, A549 cells exhibited condensed hyperfluorescent nuclei, a morphology characteristic of apoptosis. The activation of caspase 3, a crucial component of the apoptotic mechanism, was also determined after pristimerin treatment. Pristimerin activated caspase 3 in a concentration-dependent manner ( Figure 7B ). Additionally, the apoptotic population was quantified with membrane Annexin V-FITC/PI double staining and analyzed through flow cytometry (scatter plot in Figure 7C ; quantification in Figure 7D ). Approximately 35% of A549 cells underwent apoptosis after 48 h treatment with 1 μM pristimerin. Finally, we analyzed the expression levels of proteins associated with apoptosis. Pristimerin increased the levels of cleaved caspase 3, an activated form of caspase 3, in a dose-dependent manner ( Figure 7E and 7F ). Moreover, the levels of inactivated zymogen procaspase-3 gradually decreased ( Figure 7E and 7F ). Our findings suggested that pristimerin treatment induced apoptosis in cancer cells. Specifically, pristimerin treatment decreased the expression of the apoptosis inhibitor protein Bcl-2 and increased the expression of the pro-apoptotic protein Bax ( Figure 7E and 7G ). Hence, we concluded that pristimerin is cytotoxic to A549 cells by inducing apoptosis.

Figure 7 |

Pristimerin induces apoptosis in A549 cells.

(A) Hoechst 33342 dye was added to stain the nuclei of A549 cells cultured with pristimerin for 24 h, and nuclear morphological changes were observed. Scale bar: 20 μm. (B) Pristimerin treatment increased caspase-3 activity in A549 cells, as assessed with a colorimetric assay. (C) Annexin V-FITC/PI double staining assays were used to analyze apoptosis in A549 cells treated with pristimerin for 48 h in six-well plates. Cells were analyzed by flow cytometry (Q3, live cells; Q2 and Q4, apoptotic cells; Q1, necrotic cells). (D) Analysis of number of cells in (C). (E) Western blots assessing protein extracts from A549 cells treated with pristimerin for 24 h for the expression levels of apoptosis-associated proteins. (D) Analysis of number of cells in (C). (E) Expression levels of apoptosis-associated proteins were determined with western blots of protein extracts from A549 cells treated with pristimerin for 24 h. (F) and (G) Quantification of procaspase-3/cleaved-caspase-3 and Bax/Bcl-2 in (E). The results are presented as mean ± SD of three samples. **, ^## and ^^, p < 0.01 compared with controls in (B), (D), (F), and (G).

3.8 Curbing the progression of lung tumor growth in vivo

We further investigated the inhibitory effect of pristimerin on the growth of A549 cells in an in vivo model. A549 cells were subcutaneously implanted into the ventral side in nude mice, and pristimerin administration commenced 7 days after implantation. Substantial inhibition of tumor growth was observed with this treatment compared with vehicle control ( Figure 8A and 8C ). No noticeable differences in body weight were observed in mice treated with pristimerin compared with the untreated group ( Figure 8B ). Treatment with 2 mg/kg of pristimerin inhibited tumor growth in nude mice from day 15 onward ( Figure 8D ). Specifically, tumor volume showed inhibitory effects from days 15 to 21 ( Figure 8D ). The tumor weights were significantly lower in nude mice treated with pristimerin than in the control group ( Figure 8E ). Organ indexes were used to assess the toxicity of antitumor drugs on organs. The statistical results indicated no significant difference in organ indices between the treatment and carrier groups ( Figure 8E ). Therefore, pristimerin did not have significant toxic effects on vital organs in mice. These results demonstrated the anti-cancer effects and strong therapeutic potential of pristimerin in vivo.

Figure 8 |

The progression of lung tumor growth is repressed with pristimerin in an in vivo xenograft model.

(A) Pristimerin experimental design for A549 subcutaneous tumors. (B) Representative tumor images of tumor-bearing mice after 21 days of treatment with pristimerin, CDDP, and vehicle. (C) Trends in weight changes in mice. (D) Trends in tumor volume were calculated every 3 days after treatment with pristimerin, CDDP, and vehicle. (E) Tumor weights after the treatment ended in the treatment group and the vehicle group and organ (heart, spleen, lung, kidney, and liver) indexes after treatment with pristimerin, CDDP, and vehicle. The results are expressed as mean ± SD, and **, p < 0.01, ***, p < 0.001 versus the vehicle groups in (D) and (E).

4. DISCUSSION

The inhibition of TrxR has emerged as a research focus because of its high expression in tumor cells, and its association with tumor cell proliferation and treatment resistance [30]. Researchers are currently investigating TrxR inhibitors with high selectivity and efficacy, to study their anti-tumor activity [71–73]. In this regard, we found that pristimerin selectively inhibited TrxR while having almost no effect on U498C TrxR ( Figure 1 ). This selectivity enabled pristimerin to effectively target the overexpressed TrxR in tumor cells and inhibit their proliferation. Furthermore, pristimerin demonstrated lower cytotoxicity toward normal cells than tumor cells ( Figure 2 ). Although the significance of this selectivity in transformation might not be readily apparent, notably, pristimerin, a naturally occurring substance, exhibited initial selectivity. Thus, pristimerin has promise as a lead molecule in this context.

Cancer cells often exhibit elevated oxidative stress because of abnormal activation of cellular signaling pathways and metabolism [74]. This imbalance between ROS production and clearance results in cell cycle arrest and apoptosis [75]. TrxR is widely expressed in NSCLC and is involved in the activation of transcription factors and regulation of cell apoptosis in this type of cancer [37]. Inhibition of TrxR activity with pristimerin impaired the ability of the thioredoxin system to clear ROS ( Figure 3 ), thus leading to substantial ROS accumulation and the induction of NSCLC death. Additionally, we focused on the stimulation of ROS accumulation with various concentrations of pristimerin. We used two distinct ROS probes, DCFH-DA and DHE, to label cells treated with pristimerin. Subsequently, we used ImageJ software to measure fluorescence intensity, thus enabling the preliminary quantification of ROS accumulation in cells after exposure to various pristimerin concentrations ( Figure 5 ). Furthermore, we conducted extensive examinations of downstream signaling reactions prompted by ROS accumulation. Our assessments included indicators such as total thiol, GSH, GSSG, and Trx redox state ( Figures 4 and 6 ). Collectively, these findings provide evidence of intracellular oxidative stress elicited by ROS accumulation induced by pristimerin.

Pristimerin has been shown to induce tumor cell apoptosis and autophagy in various types of cancer [17, 76], including human breast cancer, through activation of the ROS/ASK1/JNK pathway [26]. The inhibition of TrxR by pristimerin hinders Trx reduction and Trx-ASK1 binding, thus activating apoptosis ( Figure 7 ). These signaling pathways enabled pristimerin to inhibit the proliferation of tumor cells and decrease tumor growth in an in vivo model, with almost no effect on the weights and organs of nude mice ( Figure 8 ). The targeting of TrxR by pristimerin opens new possibilities for the development of small-molecule inhibitors as potential cancer chemotherapy drugs.

5. CONCLUSIONS

This study demonstrated that pristimerin is a novel inhibitor of TrxR, which induces tumor cell apoptosis through a previously unrecognized mechanism. The interaction between pristimerin and TrxR in cells may contribute to understanding of the mechanism of action of this quinolomethide triterpenoid in vivo. The unique targeting mechanism of pristimerin on TrxR highlights its potential as a candidate anti-tumor therapy.

DECLARATION OF COMPETING INTERESTS

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported herein.

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Graphical abstract

Highlights

• Pristimerin inhibits thioredoxin reductase.

• Pristimerin inhibits non-small cell lung cancer growth in vitro and in vivo.

• Pristimerin works by inhibiting TrxR-triggered ROS-mediated oxidative stress.

• The TrxR/Trx/ROS/oxidative stress axis mediates pristimerin-induced cell apoptosis.

• Pristimerin has excellent potential to be developed as an anti-cancer agent.

In brief

This research elucidates a novel target of pristimerin for the treatment of non-small cell lung cancer. Specifically, pristimerin demonstrates a selective anti-cancer effect by modulating thioredoxin reductase, leading to the induction of oxidative stress-mediated apoptosis.