
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00301-X
10.1016/j.redox.2024.103323
103323
Research Paper
Tryptanthrin targets GSTP1 to induce senescence and increases the susceptibility to apoptosis by senolytics in liver cancer cells
Zhang Yuxuan a
Xiao Biying a
Yuan Shuying a
Ding Lele a
Pan Yongfu a
Jiang Yanyu a
Sun Shenghao a
Ke Xisong b
Cai Lili echo_cll_0806@126.com
a⁎⁎
Jia Lijun ljjia@shutcm.edu.cn
a⁎
a Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, PR China
b Center for Chemical Biology, Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, PR China
⁎ Corresponding author. ljjia@shutcm.edu.cn
⁎⁎ Corresponding author. echo_cll_0806@126.com
20 8 2024
10 2024
20 8 2024
76 10332313 7 2024
5 8 2024
18 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Targeting senescence has emerged as a promising strategy for liver cancer treatment. However, the lack of a safe agent capable of inducing complete senescence and being combined with senolytics poses a limitation. Here, we screened a natural product library and identified tryptanthrin (TRYP) as a potent inducer of cellular senescence in liver cancer cells both in vitro and in vivo. Mechanistically, Glutathione S-transferase P1 (GSTP1), a key regulator for redox homeostasis, was identified as a target protein for TRYP-induced senescence. TRYP directly bound to GSTP1 and inhibited its enzymatic activity, mediating reactive oxygen species (ROS) accumulation, followed by DNA damage response (DDR), consequently contributing to initiating primary senescence. Furthermore, TRYP triggered DNA damage-dependent activation of NF-κB pathway, which evoked senescence-associated secretory phenotype (SASP), thereby leading to senescence reinforcement. Importantly, TRYP exposed the vulnerability of tumor cells and sensitized senescent cells to apoptosis induced by senolytic agent ABT263, a Bcl2 inhibitor. Taken together, our findings reveal that TRYP induces cellular senescence via GSTP1/ROS/DDR/NF-κB/SASP axis, providing a novel potential application in synergizing with senolytic therapy in liver cancer.

Graphical abstract

Image 1

Highlights

• TRYP, as a novel pro-senescence agent, induces obvious cellular senescence in vitro and in vivo with a high safety profile.

• GSTP1 is a direct pharmacological target of TRYP triggering oxidative stress and cellular senescence.

• TRYP-induced senescent cells establish a pro-inflammatory environment to spread and reinforce the senescence.

• TRYP-induced senescence exposes the vulnerability of tumor cells and enhances their sensitivity to senolytic therapy.

Keywords

Tryptanthrin
Cellular senescence
Liver cancer
GSTP1
SASP
ABT263
Abbreviations

TRYP tryptanthrin

SASP senescence-associated secretory phenotype

ROS reactive oxygen species

NF-κB Nuclear factor-kappa B

SA-β-gal Senescence-associated β-galactosidase

GSEA Gene set enrichment analysis

NAC N-acetylcysteine

DA danirixin

RE reparixin

ATM ataxia telangiectasia mutated

DDR DNA damage response

GSTP1 glutathione S-transferase P1

GST glutathione s-transferase

GSH glutathione

DARTS drug affinity responsive target stability

RMSD root-mean-square deviation

MST microscale thermophoresis

BLI biolayer interferometry
==== Body
pmc1 Introduction

Liver cancer ranked as the sixth most common cancer worldwide [1]. Due to its insidious onset, less than 30 % of liver cancer patients are suitable for radical treatment at first diagnosis [2]. Targeted therapies are currently achieving significant success in a broad spectrum of tumors. Unfortunately, liver cancer is among the solid tumors with the fewest somatic mutations and lacks effective targeted therapeutic agents [3]. Strikingly, the strategy of inducing and exploiting the vulnerability of tumor cells by targeting senescence holds great promise in liver cancer treatment [4,5]. Through the induction of cellular senescence, the senescent tumor cells expose the vulnerability to be subsequently targeted for elimination. This approach no longer relies on a specific gene mutation, thereby substantially expanding drug applicability.

Cellular senescence serves as a state of irreversible growth arrest that plays a pivotal role in tumor suppression [6,7]. Various cellular stressors, such as genomic damage, epigenomic perturbations, or oxidative stress, can initiate the process of senescence. Senescent cells exhibit distinct phenotypic changes, including flattened and irregular cell morphology in vitro, loss of Lamin B1 protein, increased β-galactosidase activity, heterochromatic alterations, heightened metabolic activity, and the activation of the senescence-associated secretory phenotype (SASP) [[8], [9], [10]]. The SASP encompasses a group of secreted proteins, including pro-inflammatory cytokines, growth factors, and matrix metalloproteases [11]. Although many chemotherapeutic agents have the potential to induce senescence, apoptosis remains the predominant phenotype [12]. Inducing complete senescence in tumor cells is a crucial prerequisite for utilizing cellular senescence as a therapeutic target. Consequently, an increasing number of studies are focusing on screening senescence-inducing drugs [5,13,14]. Natural products, characterized by their structural diversity and potential in drug development, represent a valuable resource for identifying effective senescence inducers. Therefore, our study aims to screen effective senescence inducers from natural products.

It is widely recognized that various natural phytochemicals possess anti-cancer properties. Indigo naturalis, a traditional Chinese medicine with a history of over 1400 years in treating acute infectious diseases [15], has been shown effective in treating hematologic tumors based on numerous clinical trials [16]. One of the active ingredients identified in Indigo naturalis is tryptanthrin (TRYP) [17]. Studies have emphasized the therapeutic potential and superior safety profile of TRYP, as it belongs to the class of alkaloids [18]. TRYP and its derivatives have demonstrated many biological effects, particularly anti-tumor activity in vitro [[19], [20], [21], [22]]. TRYP is considered a highly promising small molecule compound derived from plants with anti-tumor properties [23,24]. However, it remains unknown whether TRYP can inhibit liver cancer in vivo and induce cellular senescence, despite its primary distribution in the liver [25].

In the present study, TRYP was identified as a potent senescence inducer through screening a library of natural products and significantly impeded tumor progression in liver cancer. Mechanistically, TRYP targeted and inhibited the enzymatic activity of GSTP1, leading to excessive accumulation of reactive oxygen species (ROS), which in turn triggered DNA damage and ultimately induced cellular senescence. Additionally, the DNA damage response (DDR) upon TRYP treatment activated NF-κB pathway, leading to SASP secretion and senescence reinforcement. Importantly, we revealed that TRYP was an excellent pro-senescence agent and proposed the synergistic combination of TRYP with senolytic therapy as an innovative and promising approach for liver cancer therapy.

2 Materials and methods

2.1 Cell culture

Huh7 and HepG2 cell lines were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China), Huh6 was purchased from Procell Life Science&Technology Co.,Ltd (Hubei, China), Hep3B and HCCLM3 were obtained from FuHeng Cell Center (Shanghai, China). All cell lines were cultured in DMEM or MEM (BasalMedia, Shanghai, China) supplemented with 10 % FBS (Excell Bio, China) and 100 μg/mL penicillin-streptomycin (BasalMedia, Shanghai, China). The cells were maintained at 37 °C in a humidified atmosphere with 5 % CO2.

2.2 Antibodies and reagents

The antibodies utilized in this study were as follows: p53 (Santa Cruz, Cat# sc-126), H2AX (Santa Cruz, Cat# sc-517336), γ-H2AX (CST, Cat# 9718), PARP (CST, Cat# 9542), c-PARP (CST, Cat# 5625), p21 (CST, Cat# 2947), p27 (CST, Cat# 2552), p-ATM (CST, Cat# 1981), Lamin B1 (Proteintech, Cat# 112987-1-AP), p16 (Proteintech, Cat# 10883-1-AP), GSTP1 (Abclonal, Cat# A5691), β-actin (HuaBio, Cat# 68190504).

The small molecule compounds used in this study: TRYP (PubChem CID: 73549, 99.94 % purity, Selleck, Cat# S5686), danirixin (DA) (Selleck, Cat# S6620), reparixin (RE) (Selleck, Cat# S8640), KU-55933 (Selleck, Cat# S1092), BAY 11–7082 (Selleck, Cat# S2913), Etoposide (Selleck, Cat# S1225), N-Acetylcysteine (NAC) (MedChem Express, Cat# HY-B0215), TLK199 (MedChem Express, Cat# HY-13634A), ABT263 (Selleck, Cat# S1001). For in vitro studies, a stock solution of TRYP (10 mM) was fully dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, Germany) and stored at −80 °C as small aliquots before use. For in vivo studies, TRYP was freshly prepared and dissolved in saline containing 10 % DMSO with ultrasonic.

2.3 Tumor xenograft model

Male BALB/c nude mice (5 weeks of age) were purchased from SLAC ANIMAL (Shanghai, China) and maintained and treated by established guidelines. The experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM2311130005). The maximum allowable tumor volume was set at 2000 mm3. Subcutaneous injection of 2 × 106 Huh7 cells was performed on the flank of each mouse. Mice were randomized into two groups: a control group receiving daily intraperitoneal injections of vehicle, and a treatment group administered 100 mg/kg of TRYP (The dosage was chosen based on the pharmacokinetic profile of TRYP [26] and the effective dosages utilized in a previous study [27]). Every other day, the mice were weighed, and the length and width of the tumors were measured. Tumor volumes were calculated with the formula V = L × S2/2 (where L is the longest diameter and S is the shortest diameter) by a digital caliper. At the end of the experiment, mice were euthanized, and tumors were excised, photographed, and weighed. To evaluate liver and kidney function, the livers and kidneys of the mice were fixed in 4 % paraformaldehyde and subjected to H&E staining. Additionally, serum samples were collected to assess liver and kidney function.

For the in vivo combination experiment of TRYP and ABT263, mice were randomized into 4 groups: Control (Ctrl), TRYP (100 mg/kg, i.p., q.d.), ABT263 (25 mg/kg, i.g., q.3d.), and TRYP + ABT263 (TRYP combined with ABT263). Other operations are consistent with the above.

2.4 SA-β-gal staining

Senescence-associated β-galactosidase (SA-β-gal) staining was conducted according to the manufacturer's instructions (C0602, Beyotime, Shanghai, China). Briefly, cells were seeded in 12-well plates and treated with TRYP for 72 h. Afterward, the cultured cells were fixed by β-galactosidase staining fixative at room temperature for 20 min, followed by incubation in the staining working solution for approximately 12–24 h at 37 °C. The stained cells were then observed and photographed under a light microscope. The quantification of SA-β-gal staining-positive cells was based on the analysis of five random images. For mouse subcutaneous tumor tissues, frozen sections were prepared, fixed, and stained following the same procedure as mentioned above.

2.5 Immunofluorescence staining

Cells were seeded in 35 mm confocal dishes at a density of 2 × 104 cells/dish. After 72 h of TRYP treatment, the cells were fixed and permeabilized with 0.1 % Triton X-100. Next, cells were blocked with 2 % BSA for 1 h and incubated with the γ-H2AX antibody overnight at 4 °C. The following day, the secondary antibody was incubated in a dark room for 1 h. Nuclei were stained blue with DAPI. Images were acquired using a fluorescent microscope (Keyence, Japan). The percentage of γ-H2AX foci cells was calculated using the ImageJ software.

2.6 Measurement of ROS generation

Cells were collected and stained with CellROX Orange Reagent (Yeasen Biotechnology, Shanghai, China). After incubating at 37 °C for 30 min, the level of ROS was detected by flowmetry. Data were analyzed by FlowJo 10 software.

2.7 RNA sequencing

Huh7 cells were treated with 1 ‰ DMSO or TRYP (5 μM) for 72 h, and total RNA was isolated using Trizol. After transcriptome sequencing library construction, the library preparations were sequenced on an Illumina sequencing platform and 150bp paired-end reads were generated. Reads were aligned to the GRCh38 human reference genome. RNA-seq raw count data was filtered with the criteria of the gene counts >10 at least in one experiment. The differentially expressed genes were analyzed by DESeq2 filtering with the criteria of FC ≥ 2 or ≤0.5, and p < 0.05. KEGG pathway enrichment analysis was performed using the R package clusterProfiler (version 4.2.2), and the enrichment analysis results were visualized in a dot plot by R package enrichplot (version 1.14.2).

Gene set enrichment analysis (GSEA) was performed by the R package clusterProfiler (version 4.2.2). The “SenMayo” gene set [28] was used to assess the enrichment of senescence-associated genes in TRYP-treated versus control cells. Gene sets from Reactome pathways related to “cell cycle checkpoints” (R-HSA-69620) and “DNA double-strand break repair” (R-HSA-5693532) were used to evaluate the enrichment of genes associated with cell cycle arrest and DNA damage repair in TRYP-treated versus control cells. The “HALLMARK_REACTIVE_OXYGEN_SPECIES_PATHWAY” (M5938) gene set was used to assess the enrichment of genes up-regulated by ROS in TRYP-treated versus control cells. The “HALLMARK_INFLAMMATORY_RESPONSE” (M5932) and “HALLMARK_TNFA_SIGNALING_VIA_NFKB” (M5890) gene sets were used to evaluate the enrichment of genes associated with inflammatory response and NF-κB signaling pathway in TRYP-treated versus control cells. Normalized enrichment scores and adjusted P values were shown in the figures.

2.8 DARTS assay

The DARTS assay was performed according to established procedures [29]. In summary, Huh7 cells were lysed using M-PER buffer (Pierce, 78501) containing protease inhibitors, and then incubated with DMSO or 250 μM TRYP for 1 h on a rotator at 4 °C. Subsequently, the cell lysis was added with pronase and incubated at room temperature for 30 min. To prepare the DARTS samples for mass spectrometry analysis, protease inhibitors (11836153001, Roche) were added to the mixture on ice to halt the digestion process.

2.9 Molecular docking simulation

The crystal structure of GSTP1 protein (PDB ID: 7BIA) was obtained from Protein Data Bank (https://www.rcsb.org/), while the 3D structure of TRYP (CAS: 13220-57-0) was retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). Both the GSTP1 and TRYP structures were preprocessed using AutoDock Tools 1.5.6. Subsequently, molecular docking was conducted using AutoDock Vina 1.2.3. Reasonable structures with lower energies were selected, and the resulting visualization views were generated by Pymol 2.5.0. The protein hydrophilicity surface of GSTP1 was mapped by ChimeraX software, with different colors representing the hydrophilicity of amino acids.

2.10 Molecular dynamic simulation

The complexes obtained from docking were utilized as initial structures for molecular dynamics simulations using Gromacs 2023.2. The GAFF small molecule force field and Amber14sb_parmbsc1 protein force field were employed to characterize small molecules and proteins, respectively. The TIP3P water model was used to fill the system with water molecules, and Na+/Cl− was added to ensure electrical neutrality. The simulation time was set to 100 ns at a constant temperature of 298.15 K. Trajectories were obtained by Pymol 2.5.0, while the Root Mean Square Deviation (RMSD) was calculated using Gromacs.

2.11 Microscale thermophoresis

The coding sequence of GSTP1 was inserted into the pET-28a vector (Novagen). His-GSTP1 was expressed in Escherichia coli BL21 (DE3). To detect direct binding, MST was conducted according to the manufacturer's instructions (MO-L018, NanoTemper, German). Briefly, recombinant His-GSTP1 proteins were labeled with MST fluorescence dye. Subsequently, 10 μL TRYP samples at different concentrations were obtained by multiplicative dilution, followed by the addition of 10 μL labeled proteins, and mixed thoroughly. The fluorescence signal was detected by the Monolith NT.115 instrument (NanoTemper Technologies) at 25 °C. The Kd values were calculated by fitting a standard binding curve to the series of diluted ligands.

2.12 Biolayer interferometry

The binding affinity between TRYP and GSTP1 protein was assessed using the BLI technique. Biotinylated GSTP1 was prepared following the manufacturer's instructions (1828 M, Genemore, Jiangsu, China). After pre-wetting the SSA biosensors with PBST (containing 0.02 % Tween 20), the biotinylated protein was directly immobilized on the biosensors in 96-well black plates (655209, Greiner, Germany). TRYP was then diluted to the appropriate concentration with PBST in a final volume of 200 μL per well, while an equal volume of PBST was added to the control wells. The process involved three main steps: a 30-s baseline, followed by a 60-s association, and finally a 60-s dissociation, which was repeated cyclically. Data acquisition and analysis were conducted using the ForteBio Octet Data Acquisition and Data Analysis software (Port Washington, NY, USA).

2.13 In vitro kinetic assay of GSTP1

A GSTP1 kinetic assay was carried out according to the manufacturer's guidelines (Solarbio, Cat# BC0355). The principle underlying the GSTP1 enzyme activity assay involves the GST-catalyzed conjugation of glutathione (GSH) to 1-chloro-2,4-dinitrobenzene (CDNB), which generates the product GS-DNB. This product is characterized by a peak absorbance at 340 nm. In brief, 2 μg of GSTP1 protein was incubated with DMSO or TRYP (3, 10, 30 μM) on ice for 1 h. Following this, the reaction buffer was added to a 96-well UV plate (Corning, Cat# 3635), and mixed with GSH and 1-chloro-2,4-xylene (CDNB). The enzyme activity kinetics assay was conducted by recording the absorbance at 340 nm every 10 s for 60 min. Finally, the enzyme activity of GSTP1 was calculated according to the manufacturer's instructions.

2.14 Senescence conditional medium-related experiments

Huh7 cells were seeded in 6 cm dishes at a density of 1.5 × 105 cells/dish and treated with DMSO or 10 μM TRYP for 96 h. The medium was then removed, cells were washed three times with PBS, and a fresh medium was added. After 24 h, the medium was collected and mixed 1:1 with fresh medium to obtain a conditioned medium. The conditioned medium was added to 12-well plates with 1.5 × 104 Huh7 cells per well, and incubated for an additional 96 h. Finally, SA-β-gal staining assay was performed on the cells. The flowchart was drawn by Figdraw (www.figdraw.com).

2.15 Quantitative and statistical analysis

Data was analyzed using GraphPad Prism software. The data are presented as mean ± standard error of the mean. For normally distributed data, the differences between two groups were tested for statistical significance using independent-sample two-tailed t-tests (unpaired). Four levels of significance were used for all tests (*P < 0.05, **P < 0.01, ***P < 0.001, ns = no significance).

3 Results

3.1 TRYP induces cellular senescence and inhibits proliferation of liver cancer cells

Acute generation of senescent cells facilitates inhibition of tumorigenesis, recruitment of immune cells, and activation of anti-tumor immunity [30]. In this study, we aimed to identify small molecules from natural products that induce senescence in liver cancer cells. Through screening a library of 1445 small compounds, TRYP (Fig. 1A) was identified to strongly induce the senescence phenotype, piquing our interest. TRYP, a compound found in Indigo, is characterized by a simple structure and ease of modification. Previous studies reported a variety of biological activities associated with TRYP and its derivatives, such as anti-bacterial, anti-parasitic and anti-tumor [31], indicating its potential as a highly promising antitumor agent. The IC50 values of TRYP in three liver cancer cell lines (Huh7, HepG2, and Hep3B) were determined to be 11.060, 8.703, and 7.273 μM, respectively (Fig. 1B). Notably, TRYP was found to induce a senescence phenotype across a broad spectrum of liver cancer cell lines, as evidenced by positive staining for senescence-associated β-galactosidase (SA-β-gal) (Fig. 1C–D, Figs. S1A–B). Given that different senescence-inducing stimuli often lead to the loss of Lamin B1, along with frequent upregulation of p16 and p21 [[32], [33], [34], [35]], we further examined senescence-related markers and observed an increase in p21, p27, and p16, as well as a decrease in Lamin B1 upon TRYP treatment (Fig. 1E–F, Figs. S1C–D). Additionally, upregulation of p21 and p16 at the mRNA level was observed (Fig. 1G). DNA damage is a known marker of cellular senescence, we indeed found that TRYP treatment induced DNA damage, as evidenced by γ-H2AX foci formation and increased γ-H2AX protein expression (Fig. 1H–I, Fig. S1E-F). Furthermore, cell cycle analysis showed that TRYP induced G2/M-phase cell-cycle arrest (Fig. S1G), which may lead to the initiation of cellular senescence.Fig. 1 TRYP is a novel pro-senescence agent in liver cancer cells

(A) Identification of TRYP as a potential hit through screening a natural product library.

(B) The half-maximal inhibitory concentrations (IC50) of TRYP in Huh7, HepG2 and Hep3B were shown.

(C-D) Huh7 and HepG2 cells were treated with 0, 1.25, 2.5, 5, and 10 μM TRYP for 72 h respectively, followed by SA-β-gal staining (scale bar = 50 μm) (C). The proportion of staining-positive cells to the overall cells was shown in the bar plot (D).

(E–F) The protein expressions of senescence markers (p21, p16, p27 and Lamin B1) in Huh7 cells treated with TRYP were detected by western blot, with β-actin as a loading control (E). The corresponding statistical analyses of protein expressions were shown (F).

(G) Cells were treated with different doses of TRYP for 72h, and the mRNA expression levels of p21 and p16 were detected by qPCR.

(H) Huh7 cells were treated with 1 ‰ DMSO or TRYP (5, 10 μM) for 72 h, then γ-H2AX foci was determined by immunofluorescence staining (green). DAPI was used to stain the nuclei (blue) (scale bar = 20 μm). The ratio of γ-H2AX foci to total cells was presented in a bar plot (right panel).

(I) Huh7 cells were treated with 1 ‰ DMSO or TRYP (5 μM, 10 μM) for 72 h. Protein expression of γ-H2AX and H2AX were detected by western blot, with β-actin as a loading control (left panel). The corresponding statistical analyses of the western blot bands were shown (right panel).

(J) Huh7 and HepG2 cells were cultured with different concentrations of TRYP for 20 days and subjected to colony formation assay (left panel). A statistical graph of colonies was shown (right panel). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1

Cellular senescence is one of major outcomes of tumor therapy [36]. As shown, TRYP exerted a substantial inhibitory effect on the growth and colony-forming capacity of liver cancer cells in a dose- and time-dependent manner (Fig. 1J, Fig. S1H). To further elucidate the specificity of the senescence phenotype induced by TRYP, we explored whether it triggers apoptosis alongside senescence. The dynamic analysis of cell growth and death revealed that TRYP-treated cells did not exhibit a notable increase in population over time, nor was there a significant accumulation of cell death, even when senescence was particularly pronounced after 72 h of treatment. (Figs. S2A–B). Furthermore, despite the use of high concentrations, TRYP induced apoptosis in only a minimal fraction of cells, which was not proportional to the considerable inhibition of liver cancer cell growth (Fig. S2C). Accordingly, no detectable levels of cleaved PARP (c-PARP) and cleaved Caspase 3 (c-Caspase 3) were observed following TRYP treatment, even when subjected to extended exposure periods (Figs. S2D–E). In contrast, the positive control Etoposide exhibited a pronounced time-dependent escalation in cell mortality, which is a consequence of its established role in triggering apoptosis (Figs. S2A–D). Collectively, these findings confirm that TRYP predominantly triggers cellular senescence in liver cancer cells, leading to a marked inhibition of cell growth.

3.2 TRYP initiates cellular senescence by inducing ROS accumulation

The aforementioned results implied that TRYP induced DDR and cell cycle arrest, hallmarks of cellular senescence. To further elucidate the underlying mechanisms driving TRYP-mediated senescence, an RNA sequencing (RNA-seq) analysis was performed on Huh7 cells following TRYP treatment. 1459 genes were found to be down-regulated, while 1200 genes were up-regulated (Fold change ≥2, Fig. 2A). Gene Set Enrichment Analysis (GSEA) unveiled the enrichment of the SenMayo senescence signature [28] upon TRYP treatment, providing further insight into the modulation of senescence-associated genes by TRYP, which might play pivotal roles in cellular senescence regulation (Fig. 2B). It is well-established that oxidative stress is a pivotal mediator in the induction of cellular senescence [37]. Of particular note is that GSEA analysis revealed a positive enrichment of the HALLMARK_REACTIVE_OXYGEN_SPECIES_PATHWAY (M5938) in TRYP-treated Huh7 cells (Fig. 2C), indicating that oxidative stress may serve as a contributor to the senescence phenotype by TRYP exposure. Thus, we investigated whether TRYP induced ROS production to trigger senescence, and observed a dose-dependent increase in ROS accumulation upon TRYP treatment (Fig. 2D). Treatment with N-acetylcysteine (NAC), a classical ROS scavenger [38], significantly restrained TRYP-induced ROS accumulation (Fig. 2E) and cellular senescence (Fig. 2F). Moreover, NAC treatment effectively alleviated the accumulation of γ-H2AX induced by TRYP (Fig. 2G–H), suggesting that ROS indeed contributes, at least in part, to the DDR triggered by TRYP. Collectively, these findings suggest that TRYP initiates cellular senescence by inducing ROS accumulation accompanied by DDR and cell cycle arrest.Fig. 2 TRYP induces cellular senescence through ROS/DDR axis

(A) Volcano plot of the differentially expressed genes in TRYP treated vs. control group.

(B–C) The GSEA plots for TRYP vs. DMSO display an enrichment for the “SenMayo” and “HALLMARK_REACTIVE_OXYGEN_SPECIES_PATHWAY” gene sets.

(D) TRYP induced ROS accumulation in Huh7 and HepG2 cells. Cells were treated with indicated concentrations of TRYP for 72 h. ROS generation was determined through FACS analysis. The fold change in ROS was displayed (right panel).

(E) After pretreatment with 10 mM NAC for 2h, Huh7 and HepG2 were added with 2.5 μM TRYP. ROS generation was determined (left panel), and statistical analysis was performed (right panel).

(F) The treatment was the same as (E). Cells were subjected to SA-β-gal staining analysis (scale bar = 50 μm). The percentage of positive staining was shown (bottom panel).

(G–H) After pretreatment with 10 mM NAC for 2h or not, 1 ‰ DMSO or 2.5 μM TRYP was added in Huh7 and HepG2 cells and treated for another 72 h, cell lysates were detected by western blot with antibodies against γ-H2AX, H2AX and β-actin (G). The corresponding statistical analyses of the western blot bands were shown (H).

Fig. 2

3.3 TRYP triggers the DDR-dependent NF-κB pathway activation and SASP-related senescence reinforcement

In addition to oxidative stress, we investigated other underlying mechanisms of TRYP-induced cellular senescence in liver cancer cells. Remarkably, GSEA analysis unveiled a pronounced enrichment of the “HALLMARK_INFLAMMATORY_RESPONSE” (M5932) gene set upon TRYP treatment (Fig. 3A), suggesting that TRYP induces cellular senescence along with creating a pro-inflammatory environment. Senescent cells are known to secrete various SASP factors, including growth modulators, matrix metalloproteinases, and particularly numerous pro-inflammatory cytokines and chemokines [8,11]. Consistent with this, RNA sequencing analysis showed up-regulation of multiple cytokines and inflammatory factors upon TRYP treatment (Fig. S3A). Moreover, a set of SASP factors was confirmed to exhibit a concentration-dependent increase following TRYP treatment (Fig. 3B). Based on these observations, we postulated that these secreted cytokines might be responsible for TRYP-induced cellular senescence. To test this hypothesis, proliferating Huh7 cells were cultured with conditioned medium from TRYP-treated senescent Huh7 cells for 96 h (Fig. 3C). These cells displayed an enlarged and flattened morphology, as well as increased SA-β-gal-positive staining (Fig. 3D), indicating the occurrence of paracrine senescence.Fig. 3 TRYP triggers SASP production via p-ATM/NF-κB pathway, further amplifying cellular senescence(A) The GSEA plot for TRYP vs. DMSO displays an enrichment for the “HALLMARK_INFLAMMATORY_RESPONSE” gene set.

(B) Huh7 cells were treated with DMSO or TRYP (5,10 μM) for 72 h. The mRNA levels of CXCL1, CXCL8, CXCL10, IL6, IL15, and TNFα were detected by qPCR.

(C) The flow chart shows the experiment of senescence induction with conditioned medium. Briefly, Huh7 cells were treated with DMSO or 10 μM TRYP for 72 h. The culture was exchanged with fresh medium and continued growing for another 24 h. The supernatant was retained and mixed 1:1 with fresh medium to obtain conditioned medium (CM). Proliferating Huh7 cells were cultured with CM for 96 h and subjected to SA-β-gal staining.

(D) Huh7 cells were treated with control medium or CM in (C), and then subjected to SA-β-gal staining (scale bar = 50 μm). SA-β-gal-positive cells were counted (right panel).

(E) The GSEA plot for TRYP vs. DMSO displays an enrichment for the “HALLMARK_TNFA_SIGNALING_VIA_NFKB” gene set.

(F-G) Cells were treated with 10 μM BAY 11–7082 or vehicle. Two hours later, 2.5 μM TRYP or an equivalent volume of vehicle was added. After 72 h treatment, the expression of CXCL8 was analyzed via qPCR (F). The senescent cells were detected by SA-β-gal staining (G, left panel) (scale bar = 50 μm). Statistical analysis was performed (G, right panel).

(H) Huh7 and HepG2 cells were treated with the indicated concentrations of TRYP for 72 h, cell lysates were subjected to western blot with antibodies against p-ATM (S1981) and β-actin. The corresponding statistical analyses of the western blot bands were shown in the right panel.

(I-J) After pretreatment with DMSO or KU-55933 (1 μM) for 2 h, Huh7 and HepG2 cells were treated with or without 2.5 μM TRYP for 72 h. The mRNA level of CXCL8 was detected by qPCR (I). The senescent cells were detected by SA-β-gal staining (J, left panel) (scale bar = 50 μm). Statistical analysis was performed (J, right panel).

(K) After pretreatment with 10 mM NAC for 2 h, Huh7 and HepG2 cells were cultured with 2.5 μM TRYP for 72h. The mRNA level of CXCL8 was detected by qPCR.

Fig. 3

Since CXCL8 is one of the most significantly upregulated inflammatory factors in TRYP-exposed senescent Huh7 cells (Fig. 3B), leading us to hypothesize that CXCL8 might be a contributing factor in conditioned medium to reinforce senescence. As a member of the CXC chemokine family, CXCL8 is secreted in response to inflammatory stimuli, acting as a multifunctional cytokine with a broad spectrum of functions [39]. Its function primarily depends on its interaction with specific cell surface receptors, CXCR1 and CXCR2 [40]. To further explore the role of CXCL8 in TRYP-induced senescence, we utilized antagonists of CXCR1/CXCR2, danirixin (DA) and reparixin (RE), to disrupt the function of CXCL8. As shown, treatment with DA or RE partially rescued the TRYP-induced SA-β-gal staining-positive cells in Huh7 and HepG2 cells (Fig. S3B). Together, these findings imply that TRYP treatment not only undergoes ROS/DDR-induced cellular senescence but also modulates the microenvironment by secreting SASP, thereby triggering senescence reinforcement.

The regulation of SASP involves several pathways, including the nuclear factor κB (NF-κB), CCAAT-enhancer-binding protein β (C/EBPβ) and p38MAPK, particularly NF-κB signaling pathway [41]. Indeed, GSEA analysis demonstrated that the “HALLMARK_TNFA_SIGNALING_VIA_NFKB” (M5890) gene set was enriched following TRYP treatment, indicating the activation of NF-κB pathway (Fig. 3E). Accordingly, multiple known NF-κB target genes, such as CXCL5, NFKB2, RELB, PIM1, APOE, and SERPINE1, were significantly upregulated upon TRYP treatment (Fig. S3C), indicating that TRYP activates the transcriptional activity of NF-κB, thereby evoking downstream inflammatory responses. To elucidate the role of the NF-κB signaling pathway in inducing SASP factors, we first asked whether inhibition of NF-κB signaling could block CXCL8 production and TRYP-induced senescence. Indeed, inhibition of IκB kinases with BAY 11–7082 effectively blunted the upregulation of CXCL8 (Fig. 3F), the increase of SA-β-gal staining-positive cells (Fig. 3G), and the elevation of the senescence marker p21 (Figs. S3D and F). These data indicate that TRYP treatment stimulates NF-κB pathway activation and further evokes SASP-dependent cellular senescence.

Ataxia telangiectasia mutated (ATM) stands as a critical sentinel of the genome, rapidly mobilizing in response to DNA damage, and its activation is a cornerstone in initiating DDR and cell cycle checkpoints [42,43]. Persistent DDR signaling drives ATM-dependent SASP secretion, including the major inflammatory cytokines [44]. Considering that ATM inhibition dampens NF-κB activation induced by DNA damage [45], we investigated whether TRYP-induced DNA damage could activate the p-ATM/NF-κB signaling pathway, thereby enhancing SASP secretion. As depicted in Fig. 3H, TRYP treatment considerably increased the expression of p-ATM in liver cancer cells (Fig. 3H). Meanwhile, treatment with an ATM kinase inhibitor (KU-55933) also alleviated the upregulation of CXCL8 (Fig. 3I), the increase of SA-β-gal staining-positive cells (Fig. 3J), and the elevation of p21 (Figs. S3E and G), indicating that TRYP-induced ATM-dependent NF-κB activation is associated with SASP-related senescence. Furthermore, treatment with NAC also significantly attenuated TRYP-induced upregulation of CXCL8 (Fig. 3K), indicating that TRYP-induced ROS generation is partially responsible for NF-κB activation and SASP production. Collectively, these results elucidate that TRYP triggers and further augments cellular senescence via ROS/DDR/NF-κB/SASP axis.

3.4 TRYP-induced cellular senescence depends on targeting GSTP1 and inhibiting its enzymatic activity

To further identify the proteins directly binding to TRYP, we conducted a DARTS (drug affinity-responsive target stability) assay, in which the stability of potential targets is protected or is more sensitive to proteolysis upon binding to small molecules [[46], [47], [48]]. After TRYP treatment, glutathione S-transferase P1 (GSTP1) was found to be more sensitive to pronase proteolysis by DARTS combined mass spectrometry (Fig. 4A). GSTP1, recognized for its role as an antioxidant and intracellular detoxifier, facilitates the binding of reduced glutathione to hydrophobic or electrophilic compounds such as ROS, yielding oxidized glutathione [49,50]. Correspondingly, western blot analysis of the DARTS samples confirmed the destabilization of GSTP1 to pronase proteolysis by TRYP (Fig. 4B). Therefore, we hypothesized that TRYP might target GSTP1 directly and inhibit its function, thereby inducing cellular oxidative stress and ultimately driving cellular senescence. Supporting this idea, molecular docking predicted that TRYP bound strongly to GSTP1 with a binding energy of −8.126 kcal/mol. In detail, TRYP and GSTP1 formed three key binding interactions: a π-π interaction with residue F9 and hydrogen bonds with residues Y8 and Y109, respectively (Fig. 4C). Additionally, the hydrophobic surface map showed that TRYP bound in the hydrophobic pocket of GSTP1 as a hydrophobic molecule (Fig. 4D). Moreover, molecular dynamics simulations further demonstrated that the TRYP-GSTP1 complex reached stable conformations after 30 ns, with a root mean square deviation (RMSD) of 1.75 Å (Fig. 4E). To verify whether GSTP1 directly interacts with TRYP, we used recombinant GSTP1 protein to assess its binding affinity with TRYP through microscale thermophoresis (MST) assay. Indeed, TRYP was found to bind to GSTP1 with a dissociation constant (Kd) value of 30.14 μM (Fig. 4F). Furthermore, biolayer interferometry (BLI) analysis also revealed a direct and reversible interaction between TRYP and GSTP1, with an affinity (Kd) of 30.86 ± 2.658 μM, while the association rate constant (Kon) and disassociation rate constant (Koff) were 1.435E3 Ms−1 and 0.044 s−1 respectively (Fig. 4G). Collectively, these findings demonstrate that TRYP directly binds to GSTP1.Fig. 4 TRYP induces cellular senescence by directly binding to GSTP1 and inhibiting its enzymatic activity(A) GSTP1 was identified in DARTS assay as a potential target of TRYP.

(B) DARTS and western blot confirmed that TRYP promoted the hydrolysis of GSTP1 by pronase. The experiment was repeated three times and the statistical analysis was performed (right panel).

(C) Computational docking model between TRYP and GSTP1 protein. Overall structure (left panel) and local binding details (right panel) were shown, with yellow indicating van der Waals forces, red indicating hydrogen bonding, and blue indicating π-π interactions.

(D) Hydrophilicity plot of TRYP and GSTP1 was shown, with hydrophobic regions indicated in yellow, hydrophilic regions in blue, and TRYP represented as a stick model.

(E) The docking complexes were subjected to molecular dynamics simulations for 100 ns, the RMSDs of GSTP1 and GSTP1-TRYP complex were calculated.

(F) The MST assay was used to measure the binding capacity of TRYP to GSTP1.

(G) TRYP binding to GSTP1 was observed by BLI kinetic analysis.

(H) GSTP1 enzymatic activity kinetics was measured in vitro in the presence of DMSO or TRYP. GS-DNB refers to the product of the GSTP1 enzymatic activity assay.

(I) The enzyme activity units of GSTP1 were calculated upon treatment of DMSO or TRYP.

(J) Huh7 cells were treated with 0, 5, 10, and 20 μM TLK199 for 72 h, followed by FACS analysis to assess ROS production.

(K) Following the same treatment as described in (J), senescent cells were identified through SA-β-gal staining (left panel) (scale bar = 50 μm), and statistical analysis was shown (right panel).

(L) Protein expression levels of Lamin B1, p21, γ-H2AX and H2AX were detected by western blot upon treatment of TLK199, with β-actin as a loading control.

(M) Quantitative analysis of gray values was performed on the bands displayed by (L).

(N) Huh7 cells were transfected with siRNA oligos targeting GSTP, then treated with DMSO or TRYP for 72 h, followed by SA-β-gal staining (scale bar = 50 μm).

(O) The percentage of staining-positive cells in (N) was quantified, and the proportion of staining-positive cells relative to the total number of cells in each group is displayed in the bar plot (left panel). The fold change in the percentage of SA-β-gal staining-positive cells between TRYP and DMSO treatments across the three groups is shown in the bar plot (right panel).

(P) The knockdown effect of GSTP1 was detected by western blot, with β-actin as a loading control.

(Q) Quantitative analysis of gray values was performed on the bands displayed by (P). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Intriguingly, TRYP treatment did not significantly alter the protein level of GSTP1 (Fig. S4A). Molecular docking demonstrated TRYP binding within the enzymatic pocket of GSTP1 (Fig. 4C), leading us to hypothesize that TRYP might competitively inhibit GSH binding to GSTP1, potentially suppressing its enzymatic activity. Consistent with this, ligand-free GSTP1 exhibited significant conformational flexibility, whereas TRYP binding helped to maintain the structure of the enzyme active site and promoted the formation of the GSTP1-TRYP complex (Fig. S4B). In support of this notion, TRYP treatment significantly dampened GSTP1 enzyme activity in vitro (Fig. 4H and I), indicating that TRYP directly binds to GSTP1 and inhibits its enzymatic activity by inducing a conformational change. To further substantiate that TRYP induces cellular senescence by inhibiting GSTP1, we next examined whether the known GSTP1 inhibitor TLK199 could also induce cellular senescence. Our results showed that TLK199 treatment enhanced ROS accumulation (Fig. 4J) and induced apparent senescence, as evidenced by SA-β-gal staining analysis (Fig. 4K). Additionally, we observed an increased expression of p21 and γ-H2AX, as well as a decrease of Lamin B1 (Fig. 4L and M). Furthermore, genetic knockdown of GSTP1 also induced cellular senescence, consistent with TRYP or TLK199 treatment (Fig. 4N–Q, Fig. S5). Crucially, knockdown of GSTP1 alleviated the sensitivity of TRYP to induce cellular senescence (Fig. 4N–Q), which further validates the crucial role of GSTP1 in TRYP-induced cellular senescence. Taken together, these findings suggest that TRYP directly targets GSTP1, inhibiting its enzyme activity, which in turn leads to oxidative stress and cellular senescence.

3.5 TRYP induces cellular senescence and suppresses tumor growth of liver cancer cells in vivo

To further validate the anti-tumor efficacy of TRYP in vivo, we established a subcutaneous tumor model with Huh7 in nude mice. Compared to the vehicle group, there were marked decreases in both tumor volume and weight in TRYP-treated group (Fig. 5A–C), elucidating TRYP suppresses tumor growth in vivo. Moreover, immunohistochemical analysis revealed that TRYP treatment significantly inhibited the expression of Ki-67, a marker of cellular proliferation (Fig. 5D). Consistent with the in vitro results, TRYP treatment also increased the SA-β-gal positive area in tumor tissues (Fig. 5E), along with a significant upregulation of p21 expression at both protein and mRNA levels compared to the vehicle group (Fig. 5F and G). Furthermore, TRYP treatment induced the secretion of SASP factors in subcutaneous tumor tissues, as evidenced by the increase in CXCL1, CXCL8, and CXCL10 (Fig. 5H). In addition, throughout the experiment, mice treated with TRYP did not exhibit any general toxic response compared to the control group, as no marked differences in body weight or histological examination of liver and kidney tissues were observed between the TRYP-treated and control groups (Fig. 5I and J). Additionally, liver and kidney function indexes, including ALT, AST, UN, and CREA levels in serum, showed no significant increase compared to the control group (Fig. 5K). These findings collectively demonstrate that TRYP induces cellular senescence and suppresses the growth of liver cancer cells in vivo, without any apparent toxicity or adverse effects.Fig. 5 TRYP induces senescence and suppresses the growth of liver cancer cellsin vivo

(A) Nude mice were sacrificed, and subcutaneous tumors transplanted with Huh7 cells were collected and photographed (n = 6).

(B) The length and width of the subcutaneous tumors were determined every other day by caliper measurement. Tumor volumes were calculated and data were presented in curves.

(C) The tumor weight was measured with an electronic scale after the tumors were harvested.

(D) Representative IHC images of Ki67 staining (left panel). The regions in black boxes were shown at higher magnification (scale bar = 50 μm). Quantification of the percentage of Ki67 positive cells of the control group and TRYP treatment group was shown (right panel). Five fields of view were randomly selected for statistics.

(E) Senescence β-gal staining of tumor tissues was obtained from subcutaneous-implanted nude mice with Huh7 cells (scale bar = 20 μm). The area of positive β-gal staining was statistically analyzed (right panel).

(F) Proteins extracted from xenograft tumors were tested for protein expression of p21 by western blot. The relative gray-scale values of p21 protein expression were shown (right panel).

(G) Total RNA was extracted from xenograft tumors, and the relative p21 mRNA level was assayed by qPCR.

(H) The mRNA levels of CXCL1, CXCL8 and CXCL10 in mice tissues were quantified by qPCR.

(I) Mice were weighed every other day using electronic scales throughout the experiment, and data were presented in curves.

(J) Liver and kidney tissue sections of the mice from two groups were H&E stained and photographed (scale bar = 100 μm).

(K) Serum samples from mice were tested for ALT, AST, UN, and CREA.

Fig. 5

3.6 TRYP synergizes with senolytic therapy in liver cancer treatment

Recently, a novel therapeutic approach known as the “one-two punch” strategy has been proposed for liver cancer treatment [4]. ABT263, a Bcl2 inhibitor, has been reported to induce apoptosis specifically in senescent cells by exploiting their apoptosis-resistant feature [51,52]. To demonstrate the potential application of TRYP-induced senescence in the “one-two punch” therapy strategy, ABT263 was introduced as a senolytic agent to verify its clearance effect on TRYP-induced senescence. As shown, ABT263 substantially enhanced the inhibitory effect of TRYP on colony formation in Huh7 cells (Fig. 6A), and selectively inhibited cell viability in TRYP-treated cells (Fig. 6B). Meanwhile, SA-β-gal staining analysis demonstrated that ABT263 exhibited more selective inhibitory effects on TRYP-induced senescent cells, compared to normally proliferating Huh7 cells (Fig. 6C). As expected, ABT263 induced more pronounced apoptosis in TRYP-induced senescent cells (Fig. 6D). These findings imply that TRYP-induced senescent cells are more sensitive to the senolytic agent.Fig. 6 TRYP increases the sensitivity of senolytic therapyin vitroandin vivo

(A) After pre-exposed to DMSO or 5 μM TRYP for 72 h, Huh7 cells were treated for 24 h with ABT263 (5 μM, 10 μM) or vehicle and subjected to crystal violet staining.

(B) The indicated concentrations of ABT263 were added to TRYP-induced senescent or proliferating Huh7 cells, and cell viability was assayed by ATPlite assay after 72h.

(C-D) After pre-exposed to DMSO or 5 μM TRYP for 72 h, Huh7 cells were treated for 24 h with ABT263 (5 μM, 10 μM) or vehicle. Senescent cells were detected by SA-β-gal staining (scale bar = 100 μm). Senescent cells and total cell numbers were counted (right panel) (C). Apoptotic cells were determined by GreenNuc Caspase-3 assay (scale bar = 100 μm). The proportion of activated Caspase 3-positive cells in the total cells was shown (right panel) (D).

(E) Nude mice were subcutaneously implanted with Huh7 cells, treated with indicated reagents, and tumor tissues were collected and imaged from each of the four groups at the end of the experiment. (n = 6).

(F) The tumor size was measured with a caliper, and the volume was estimated to create a growth curve.

(G) The weight of the tumors in each of the four groups was measured with an electronic scale immediately after the tumor was collected.

(H) Representative IHC images of Ki67 staining (left panel). The regions in black boxes were shown at higher magnification (scale bar = 50 μm). Quantification of the percentage of Ki67 positive cells of the indicated four groups were shown (right panel). Five fields of view were randomly selected for statistics.

(I) Subcutaneous tumor tissues from four groups were subjected to SA-β-gal staining, and the representative images were shown (left panel). The regions in black boxes were shown at higher magnification (scale bar = 100 μm). The β-gal positive area was statistically analyzed (right panel).

(J) Total RNA was extracted from xenograft tumors, and the mRNA level of CXCL8 was detected by qPCR.

(K) Tissue proteins were extracted and detected by western blot using antibodies against c-PARP, PARP, c-Caspase 3, and Caspase 3, with β-actin as a loading control.

(L) The corresponding statistical analyses of the western blot bands were shown. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Next, we further confirmed the anti-tumor efficacy of the “one-two punch” approach by combining TRYP with ABT263 in vivo (Fig. S6A) and found that compared to TRYP or ABT263 treatment alone, the combination of these two agents showed more effective tumor growth inhibition (Fig. 6E–G). Ki-67 staining analysis also revealed significant suppression of the proliferation of liver cancer cells in vivo following treatment with the combination of TRYP and ABT263 (Fig. 6H). Through SA-β-gal staining analysis, we found that the addition of ABT263 significantly reduced the senescent cell proportion (Fig. 6I) and CXCL8 expression (Fig. 6J) upon TRYP treatment, indicating that ABT263 also eliminated TRYP-induced senescent cells in vivo. Moreover, ABT263 treatment also induced marked apoptosis in the combination group (Fig. 6K and L), suggesting that ABT263 eliminates TRYP-induced senescent cells in vivo through the induction of cell apoptosis. Additionally, throughout the experiment, mice treated with TRYP and/or ABT263 did not exhibit any apparent toxic response compared to the control group, with no marked differences in body weight, or histological examination of liver and kidney tissues (Figs. S6B and C). Taken together, these results provide compelling evidence that the combination of TRYP and ABT263 achieves a better therapeutic effect in inhibiting liver cancer tumor growth in vivo.

4 Discussion

Recently, the induction of cellular senescence has emerged as an efficacious strategy for tumor suppression [6]. In the present study, we identified TRYP as a novel senescence-promoting agent that inhibits liver cancer proliferation in vitro and in vivo, and further demonstrated its underlying mechanism to induce senescence. Specifically, TRYP bound to GSTP1 and inhibited its enzymatic activity, leading to ROS accumulation in liver cancer cells. Subsequently, excessive ROS accumulation provoked DDR and initiated cellular senescence. Moreover, TRYP triggered DDR-dependent NF-κB pathway activation and SASP secretion, thus reinforcing the senescence process through feedback mechanisms. Therefore, our results support a model in which TRYP induces liver cancer cell senescence through GSTP1/ROS/DDR/NF-κB/SASP axis, hence rendering the vulnerabilities of tumor cells for elimination by senolytic agents.

It is well-established that cancer cells produce higher levels of ROS compared to normal cells due to their rapid proliferation and metabolic disruption [53]. Consequently, cancer cells may be more sensitive to cell death than normal cells in response to further accumulation of ROS [54], triggering cellular senescence, apoptosis, or ferroptosis [55,56]. Currently, many chemotherapeutic agents are reported to increase ROS levels as part of the mechanism of action [57]. In the present study, we uncovered that TRYP induced excessive ROS accumulation, and triggered cellular senescence by targeting GSTP1, as a potential drug option for liver cancer. GSTP1, a member of the glutathione s-transferase (GST) family, is a major phase II metabolizing enzyme. It contains two active sites: the G-site and the H-site [58]. GSTP1 uses glutathione (GSH) to detoxify reactive compounds (e.g., ROS) generated by oxidative stress [59]. The mechanism involves the binding of a hydrophobic and electrophilic substrate to the H-site and GSH to the G-site. At the G-site, the active residue Y8 abstracts the thiol proton of GSH [60]. We observed that TRYP coincidentally occupied key residues (Y8, F9 and Y109) within the enzymatic pocket of GSTP1. These interactions occur within the enzymatic pocket formed by the G site, suggesting that TRYP may compete with GSH for binding and inhibit the enzyme activity of GSTP1. Molecular dynamics simulations showed significant differences in the structure of enzyme's pocket with and without TRYP. Binding to TRYP stabilized the pocket domain of GSTP1 and maintained the structure of another subunit, potentially facilitating the binding of another small molecule (Fig. S4B). Based on these findings, in vitro enzyme activity assays confirmed that TRYP effectively inhibited the enzymatic activity of GSTP1. Collectively, our results reveal that TRYP directly interacts with GSTP1 and restrains its enzymatic activity, disrupting redox homeostasis and highlighting a pivotal mechanism underlying TRYP-induced cellular senescence.

GSTP1, due to its crucial role in the maintenance of cellular redox homeostasis, is considered a promising anticancer target [61]. Its overexpression is frequently observed in various tumor tissues, correlating with poor prognosis [[62], [63], [64], [65], [66], [67]]. It has been reported that GSTP1 binds to the pro-apoptotic protein c-Jun N-terminal kinase (JNK), thereby inhibiting the apoptotic pathway, which is thought to be associated with resistance to tumor therapy [68]. Therefore, GSTP1 is considered a promising target in cancer therapy, and many studies have focused on finding its inhibitors [69]. The natural product aloe-emodin induces apoptosis in liver cancer cells by inhibiting the binding of GSTP1 to JNK [70]. Curcumin and its analogs interact with a variety of ROS-metabolizing enzymes, including GSTP1, to induce apoptosis and senescence in leukemia cells [71,72]. However, there is no evidence that these drugs bind directly to GSTP1, possibly affecting other targets. Strikingly, TLK199, the most classical inhibitor of GSTP1, is currently in clinical phase II trials and has been shown efficacy in promoting hematopoiesis in myelodysplastic syndromes patients [73]. Indeed, we found that TRYP is a novel enzyme activity inhibitor of GSTP1, indicating its potential as a clinical antitumor agent. Of note, it is reported that inhibition of GSTP1 led to excessive accumulation of ROS and accelerated regulatory T cells (Tregs) aging, leading to uncontrolled inflammation and immune aging [74]. In cancer, Tregs are often exploited to support tumor immune evasion and progression, and high frequencies of intratumoral Tregs have been linked to worse outcomes in patients with various cancers [[75], [76], [77]]. Thus, the function of Tregs in tumors is probably a barrier to effective immunotherapy. Since TRYP has been demonstrated to target GSTP1 and induce cellular senescence, it may synergize with immunotherapy by inducing senescence in Tregs. This suggests the need to evaluate the efficacy of TRYP in an immunocompetent mouse model in the future.

Our findings provide valuable insights into the underlying mechanisms of TRYP-induced cellular senescence and underline the importance of the interplay between senescent cells and their microenvironment. Interestingly, we observed a significant upregulation of CXCL8 upon TRYP treatment, which was also found to be markedly elevated in mice subcutaneous tumors of Huh7 cells treated with TRYP. Moreover, blocking the CXCL8 receptor (CXCR1 and CXCR2) substantially restricted TRYP-induced cellular senescence. This evidence points to a pivotal role of CXCL8 in perpetuating a feedback loop that amplifies paracrine senescence upon TRYP treatment. It has been reported that senescent cells activate a self-amplifying secretory program in which CXCR2 ligands reinforce growth arrest [78,79]. Other SASP factors may also contribute to paracrine senescence and cause tumor suppression [80]. SASP is highly heterogeneous and TRYP-induced SASP tends to be pro-inflammatory, as exemplified by the elevated expression of CXCL8. We speculate that the induction of a pro-inflammatory SASP profile may explain the superior pro-senescence ability of TRYP. Through the secretion of SASP factors, senescent cells establish a pool of pro-senescence inflammatory factors surrounding the tumor cells, constantly reinforcing, maintaining, and disseminating the senescence signaling cascade. In this way, TRYP induces obvious senescence in liver cancer cells at relatively low doses.

As mentioned in our study, TRYP-induced senescent cells tended to generate a pro-inflammatory SASP secretome, which further induced normal proliferating cells to undergo senescence directly. However, it is widely recognized that SASP acts as a double-edged sword [81]. Besides the anti-tumor effect, many studies have highlighted that SASP factors may be more detrimental than beneficial, as they can promote tumorigenesis, progression, metastasis and chemotherapy resistance [7,11]. Therefore, a synergistic and innovative two-step therapeutic paradigm is emerging as a promising anti-tumor strategy. In this approach, tumor cells are first induced to senescence by senescence-inducing agents, followed by a crucial second phase in which these senescent cells are selectively targeted and eliminated by senolytics [[82], [83], [84], [85]]. Senolytic agents, such as Bcl-2 family inhibitors (navitoclax/ABT-263 or ABT-737), have been shown to effectively induce cell death in senescent cells [52]. In our study, we found that ABT-263 effectively and specifically eliminated TRYP-induced senescent cells (Fig. 6), providing a potential “one-two punch” therapy utilizing TRYP for liver cancer. Therefore, we propose that inducing senescence of tumor cells and exploiting the vulnerabilities of senescent cells with senolytics can yield more therapeutic benefits.

Cellular senescence exhibits extremely heterogeneous, depending on various stressors [86]. Our study has revealed that TRYP specifically induced senescence in liver cancer cells without triggering apparent apoptosis. Furthermore, TRYP exhibits several advantages, including a small molecular weight, stable structure, easy modification, low cost and high safety, rendering it an ideal pro-senescence agent. It has been previously reported that TRYP is primarily metabolized in the liver [25], potentially contributing to its favorable therapeutic effects in liver cancer cells. Our current research focuses on liver cancer, where senescence-targeted therapies may hold particular promise due to the paucity of efficacious treatment options available. Prospectively, future endeavors will need to identify the precise subpopulation of cancer cells that must undergo senescence to ensure comprehensive eradication of the tumor. Such insights are pivotal for delineating the precise patient populations who stand to benefit most from this targeted senescence-based therapeutic approach. Moreover, employing this senescence-targeting strategy in a clinically viable treatment necessitates exhaustive investigation, rigorous validation, and practical implementation. Collectively, our findings support strongly the potential of TRYP as an effective senescence-promoting agent, and the combination of TRYP with senolytic therapy offers a promising new treatment option for liver cancer.

CRediT authorship contribution statement

Yuxuan Zhang: Writing – original draft, Project administration, Formal analysis. Biying Xiao: Validation. Shuying Yuan: Validation. Lele Ding: Resources. Yongfu Pan: Methodology. Yanyu Jiang: Methodology. Shenghao Sun: Validation. Xisong Ke: Resources. Lili Cai: Writing – review & editing, Supervision, Data curation. Lijun Jia: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

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

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Data availability

Data will be made available on request.

Acknowledgments

This work was supported by the following funds: Shanghai Frontiers Science Center of Disease and Syndrome Biology of Inflammatory Cancer Transformation (2021KJ03-12 ), National Key R&D Program of China (2022YFC3500200 , 2022YFC3500202 ), National Natural Science Foundation of China (82372984 ). We appreciate Dr. Ni Shuaishuai for the valuable suggestions throughout the entire research.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103323.
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