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. 2025 Jul 17;8(8):2710–2724. doi: 10.1021/acsptsci.5c00308

Teniposide Triggers DNA Repair Inhibition by Binding and Ubiquitination of Apurinic/Apyrimidinic Endonuclease 1 to Boost Oxidative DNA Damage for Lung Cancer Destruction

Ning Han †,‡, Jun Hu †, Zhong-Xiong Xie §, Lei Zhang †, Liu-Gen Li †, Xin-Ran Yu †, Cunqing Kong †, Fan Leng †, Cui Hu †, Qiufang Zhang †,*, Mei-Fang Wang ‡,*, Tong-Fei Li †,‡,*
PMCID: PMC12340644  PMID: 40810145

Abstract

Teniposide (Ten/VM-26) is low in toxicity and has proven to be effective in destroying malignant cells at low doses. However, the target and molecular mechanism of Ten/VM-26 are poorly understood, which limits its clinical application against solid malignant cancers. Apurinic/apyrimidinic endonuclease 1 (APEX1) expression is upregulated in lung cancer, which could effectively suppress DNA damage. The present study aims to explore how Ten/VM-26 regulates APEX1 and thereby exploits its antilung cancer effects. Ten/VM-26 possessed powerful antilung cancer efficacy in vitro and organoid models. Furthermore, the findings of in vivo experiments evidenced that Ten/VM-26 could suppress the growth of tumor grafts without impacting the vital organs or body weight of mice. RNA-sequence analysis revealed that Ten/VM-26 treatment led to differentially expressed genes (DEGs), which were enriched in the DNA damage-associated biological process (BP). Reactive oxygen species (ROS) generation mediated by Ten/VM-26 was the major contributor to its anticancer effect. The in-depth investigation identified that APEX1 was efficiently expressed in lung cancer tissues, leading to a poor prognosis. Interestingly, APEX1 was downregulated in the presence of Ten/VM-26, which further abolished the protection of DNA, resulting in robust DNA damage. Further findings discovered that Ten/VM-26 could bind to APEX1 and thereby dampen its function. In contrast, APEX1 recovery attenuated the Ten/VM-26-induced DNA damage and anticancer efficacy. In summary, these data make a strong argument for the notion that Ten/VM-26-mediated inhibition of APEX1 contributes to DNA damage and thereby achieves favorable antilung cancer effects, wherein Ten/VM-26 could down-regulate APEX1 by binding and ubiquitination. The current study presents a critical target and mechanism for Ten/VM-26-mediated antilung cancer therapy.

Keywords: apurinic/apyrimidinic endonuclease 1 (APEX1), lung cancer, Teniposide (Ten/VM-26), DNA damage, reactive oxygen species (ROS)

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The phytomedicine podophyllotoxin is an extract of Podophyllus emodii Wall, a natural plant from Tibet, China, which is synthetically modified to obtain the derivative Teniposide (Ten/VM-26), with a very favorable cytotoxic effect against cancer within a certain concentration range. − Previous studies showed that podophyllotoxin disrupted spindle filament formation by binding to microtubule proteins. , In addition, a few studies suggested that podophyllotoxin could inhibit DNA replication, with the possibility of having a direct target in DNA. , Moreover, Ten/VM-26 demonstrated excellent DNA-damaging capability for cancers resistant to chemotherapeutic agents. , Thus, it is evident that Ten/VM-26 should be considered a multitargeting chemotherapeutic agent, whose intracellular mechanism of anticancer activity needs to be further elucidated.

Apurinic/apyrimidinic endonuclease 1 (APEX1) is a molecule with high expression in a variety of solid tumor tissues, localized predominantly in the nucleus, with a small amount also distributed in the cytoplasm. APEX1 participates in the process of DNA repair by cleaving sugar–phosphate chain deletion sites in DNA. − Moreover, APEX1 remedies apoptosis due to DNA damage triggered by oxidative stress in cancer cells and regulates multiple cell signaling pathways. − Currently, there are fewer studies on whether APEX1 can impair cancer resistance to chemotherapeutic agents. However, APEX1 high expression increases the resistance of malignant cells to damage and may result in the development of resistance to chemotherapeutic agents targeting DNA. To date, several studies have harnessed pharmacological inhibition of APEX1 to achieve antitumor effects. For instance, the team led by Long identified a small molecular blocker of APEX1 and elucidated its antitumor efficacy. Ullah, on the other hand, reported that Cephalomannine could act as an oncostatic agent by inhibiting APEX1. ,, These studies suggested that screening and characterizing phytomedicine that can inhibit APEX1 is significant for tumor chemotherapy.

Based on this idea, should it be demonstrated that Ten/VM-26 has the ability to inhibit APEX1 and damage DNA at the same time, it would suggest that Ten/VM-26 inhibits damage resistance while causing damage to cancer cells, resulting in excellent antitumor efficacy. Therein, the binding and regulation of Ten/VM-26 on APEX1 were highlighted. Additionally, we evaluated whether Ten/VM-26 boosted anticancer efficacy and caused more severe DNA damage to lung cancer cells by inhibiting APEX1 in the lung cancer model, which has the highest incidence and mortality rate among malignant tumors.

Methods

In Vitro Lung Cancer Cell Culture and Organoid Induction

The H1299, Lewis, and A549 lung cancer cell lines were procured from the Cell Bank of Shanghai Institutes for Biological Sciences (Shanghai, China) and utilized as the tumor model in the present work. These cell lines were incubated in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10–15% fetal bovine serum (QmSuero/Tsingmu Biotechnology, China). The cells were cultured in a humidified environment (5% CO2, 37 °C). To construct the 3-dimensional culture model of Lewis cells, the cells were incubated in an agarose-coated plate and then treated with Ten/VM-26 after 10 days. To establish the lung cancer organoid model, cells collected from malignant pleural effusion were obtained after centrifugation and red blood cell lysis, then mixed with matrix gel and cultured in organoid-conditioned medium (356231, Corning, USA). The cells were photographed every 2–3 days, identified, and treated after 10 days. Approval for this study was obtained from the Ethics Committee of Hubei University of Medicine. The concentrations of Ten/VM-26 (29767–20–2, MACKLIN, China, > 98% purity) were screened at 0.25, 0.5, 1, 2.5, 5, and 10 μM with a treatment time of 24 h. Doxorubicin (DOX), the positive drug utilized in the in vitro research, was used at a concentration of 5 μg/mL. The chemical structure of Ten/VM-26 is presented in Figure A,B.

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Ten/VM-26 possessed pronounced antilung cancer ability in vitro. (A, B) Mapping out the chemical structure of Ten/VM-26. (C, D) The cell viability of A549 and Lewis cells treated with Ten/VM-26 was assayed by CCK-8 (n = 3). (E–H) Annexin-V (FITC or APC labeled)/PI staining analysis of the apoptosis rate of Ten/VM-26-treated A549 and Lewis cells. The cells were harvested by flow cytometry, and the Annexin-V positive cells were calculated (n = 3). (I, J) WB detection of the effect of Ten/VM-26 on the expression of apoptosis and proliferation-associated proteins. (K, L) The proliferation of Lewis cells was analyzed using the colony formation experiment. The colony number was calculated. (M) The Lewis cells were carried out the 3-D culture, then labeled with Calcein/PI after Ten/VM-26 treatment, which were photographed using the confocal laser scanning microscopy (Z-scanning). (N) The lung cancer organoid was constructed. The morphology of the organoid was imaged. Statistic values presented were means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). LCO: lung cancer organoid.

In Vivo Animal Experiments

4-week-old (20–22 g), male C57B/L mice were obtained from Hubei University of Medicine Laboratory Animal Center (Shiyan, China). All animal experiments were approved by the Animal Care Committee at the Hubei University of Medicine (Protocol No. 2023105). Isoflurane treatment was employed for all inoculations and administrations. To investigate the efficacy of Ten/VM-26, Lewis lung cancer cells (2 × 106 cells/0.1 mL in PBS) were injected subcutaneously to yield lung cancer cell-bearing mice, which were randomized into 5 groups and administered Ten/VM-26 when the grafts reached 400–500 mm3. The mice were treated with PBS, Ten/VM-26 (5, 10, and 20 mg/kg b.w.), or DOX (5 mg/kg b.w.) (i.p., once a day, 3 days in total). The DOX administration served as the positive control in the in vivo experiment. The concentrations of Ten/VM-26 were set with reference to previous studies for improvement. The mice were sacrificed when the treatment was completed, and the tumor grafts were harvested and used for histological detection or ROS measurement.

Viability Analysis of Lung Cancer Cells

To analyze the effect of Ten/VM-26’s effect on lung malignant cells, the above cells treated with Ten/VM-26 were inoculated in 6-, 24-, or 96-well plates, which were utilized for protein extraction, apoptosis analysis, and the CCK-8 assay. Western blots (WB) were used to detect the expression of apoptosis- and proliferation-associated molecules in the extracted proteins. For the CCK-8 assay, the Lewis and A549 cells were incubated with CCK-8 (C0042, Beyotime, Shanghai, China), and their absorption at 450 nm was measured using a Multifunctional Enzyme Labeler (SpectraMax i3, Molecular Devices). Moreover, the cells labeled by Annexin-V/PI or Annexin-V/7-ADD reagents (CHAMOT BIOTECHNOLOGY CO., Ltd., China) were analyzed using flow cytometry (Cytoflex, Beckman Coulter, USA), wherein the apoptosis rate of lung cancer cells (Annexin-V-positive cells) was measured. Finally, the 3-D cultured cells were labeled with Calcein/PI for the viability assay.

In Vivo Anticancer Efficacy Detection

For investigation of the antilung cancer efficacy in tumor-bearing mice, the volume of the tumor grafts and the weight of the mice were monitored during the treatment period. The size and quality of the tumor grafts were measured after the Ten/VM-26 treatment. The ROS generation in the cancer tissues was detected after DCFH-DA staining. Additionally, the apoptosis of malignant cells in the ultrathin slices of tumor tissues was assayed using the TUNEL staining. The methods of histological detection (HE, TUNEL, and IHC staining) are described as follows.

Network Pharmacology Analysis and RNA-Sequencing

The targets for lung cancer were collected from the GeneCards website (https://www.genecards.org/) and targets for Ten/VM-26 were collected from the PharmMapper, TCMID, and TCMSP databases for the purpose of obtaining cotargets of Ten/VM-26 and lung cancer. The relevant target proteins were then converted to their corresponding genes using the UniProt database, with the shared genes available through the Draw Venn Diagram website (http://bioinformatics.psb.ugent.be/webtools/Venn/). The shared genes were analyzed on the STRING website (https://cn.string-db.org) to acquire protein–protein interaction (PPI) data, which were visualized by the Cytoscape program. For the RNA-sequencing assay, the mRNA extracted from the Lewis lung cancer cells was collected, and its consistency, completeness, and purity were analyzed by the NanoDrop 2000 and RNA Nano 6000 assay kits. Sequencing libraries were created as per the manufacturer’s recommendations using the NEBNext UltraTM RNA Library Preparation Kit for Illumina (NEB, USA), and index codes were appended to the sequence of every sample. Differentially expressed genes (DEGs) were screened using the read count data of the gene expression in individual samples. The screening process was based on the fold change in expression levels among samples. Using the corresponding databases, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Genome Enrichment Analysis (GSEA) were employed to carry out the deep analysis. The raw data can be found in the Bioinformatic Cloud (https://biosys.bgi.com/#/loading/bgi/) online platform.

Bioinformatic and Western Blot (WB) Analysis of APEX1

For the evaluation of APEX1 expression in tissues, spatial distribution, clinical significance, and immune relevance in lung cancer, the Gene Expression Profiling Interactive Analysis (GEPIA), Tumor Immune Estimation Resource (TIMER), and Tumor Immune Single-cell Hub (TISCH) websites (http://gepia2.cancer-pku.cn/, https://cistrome.shinyapps.io/timer/, and http://tisch.comp-genomics.org/) were utilized. − APEX1 normalized gene expression and clinical prognosis were analyzed in the TIMER and GEPIA databases. The spatial expression of APEX1 (e.g., APEX1 expression in immunocytes and malignant cells) was mapped using the TISCH database. The correlation of APEX1 with immunocyte infiltration was analyzed by the GEPIA2 database. Additionally, the detection of APEX1 expression in lung cancer cells was performed by WB.

Molecular Docking, CETSA-WB, MST, DARTS

The structure of APEX1 (PDB code: 6MK3) was determined from the RCSB PDB protein database (https://www.rcsb.org). The molecules were treated with PyMOL, followed by AutoDock measurement of the energetic binding to Ten/VM-26. The chemical structures of Ten/VM-26 were identified and downloaded from the PubChem website (https://pubchem.ncbi.nlm.nih.gov/). Screening of the most favorable free energy combinations was performed based on the principles described by AutoDock Vina (http://vina.scripps.edu) and visualized using PyMOL. To further investigate the affinity of Ten/VM-26 to APEX1, Cellular Thermal Shift-Western blots (CETSA-WB) were carried out as referenced in our published paper. Lewis cells were treated by Ten/VM-26 for 24 h, whose cellular proteins were extracted and divided into 6 equal portions for heating and then cooled for 3 min. Finally, the thermal stability of APEX1 was measured using WB. To more comprehensively measure the binding of Ten/VM-26 to APEX1, the Pronase stability of APEX1 was detected using drug affinity responsive target stability (DARTS), as referenced in our previous work. The proteins were treated with Ten/VM-26 after dividing and further incubated with screened concentration of Pronase (0.2 μg/mL). The expression of APEX1 was then measured using Western blots. To quantitatively calculate the K d value of Ten/VM-26 binding to APEX1, different concentrations of Ten/VM-26 were incubated with fluorescein-labeled purified protein APEX1 (DT10232–1, Detai Bioengineering Co., Ltd.), which was further analyzed using microscale thermophoresis (MST) equipment (Monolith NT.115, NanoTemper).

ROS and DNA Damage Detection

The ROS generation of lung cancer cells labeled by the DCFH-DA dye was detected by flow cytometry, wherein the DCF (ROS) fluorescence represented oxidative stress. N-acetyl cysteine (NAC) was utilized to inhibit oxidative stress. The concentration of NAC used in the present work was 3 mM, and the treatment time was 2–3 h. The DNA damage molecules (e.g., γ-H2A.X and p53) of A549 and Lewis lung cancer cells were assayed by Western blots. For analysis of the DNA damage repair ability of APEX1, 8-xox-dG accumulation within lung cancer cells was observed. Briefly, the cells treated with Ten/VM-26 were fixed and then labeled with an anti-8-xox-dG antibody (ab62623, Abcam, Cambridge, UK) and a fluorescence-labeled secondary antibody, which were further stained with Hoechst 33342 before laser scanning confocal microscopy (LSCM, FV3000RS, Olympus, Japan). Finally, the level of DNA double-strand breakage (DDSB) in lung cancer cells was evaluated using the comet experiment as reported in our previous work. , In brief, a suspension of Ten/VM-26-incubated lung cancer cells in PBS was mixed with low melting point agarose (LMPA). Then, the mixture was dripped slowly onto a glass slide covered with agarose gel. Electrophoresis of the gel was carried out at 20 V, 280 mA for 22 min, followed by lysis in alkaline lysate and neutralization with Tris HCl . Hoechst 33342 was utilized to stain the nuclei for the LSCM.

Immunofluorescence Colocalization

The A549 and Lewis cells were treated by 5-Aminofluorescein (5-FMA)-labeled Ten/VM-26 for 20 h and then were fixed, permeabilized, and blocked before incubation with the APEX1 primary antibody. The primary antibody-incubated cells were further incubated with fluorescence (PE)-labeled secondary antibody for another 1 h at 37 °C. The treated cells were washed and subsequently stained with Hoechst 33342. The location of Ten/VM-26 (green fluorescence) and APEX1 (red fluorescence) was observed using confocal laser scanning microscopy. Additionally, the tumor-bearing mice were administrated with 5-FMA-labeled Ten/VM-26 to investigate drug distribution using the in vivo imaging system.

Overexpression of APEX1

APEX1 overexpression plasmid was constructed, and the effect was validated by transfection, qRT-PCR, and WB. For the transfection of the APEX1 overexpression plasmid, the Lewis cells were transfected by APEX1 plasmid and Lipofectamine 8000 (Lipo 8000) for about 4–8 h before incubating with Ten/VM-26. After transfection, the medium containing Lipo 8000 and APEX1 plasmids was substituted with fresh medium. The efficient overexpression of APEX1 was validated by qRT-PCR and WB measurements. Then, DNA damage and apoptosis of Lewis cells were detected as described before.

Ubiquitination of APEX1 Analysis

The APEX1 within the Lewis cells treated by Ten/VM-26 was pulled down by the APEX1 antibody and agarose beads, respectively, yielding the APEX1 protein complex, to which the sample preparation buffer for the SDS-polyacrylamide gel electrophoresis (PAGE) was added. The gel, after electrophoresis, was transferred to the PVDF membrane, which was further treated with Ubiquitin antibody overnight and then treated with horseradish peroxidase-conjugated secondary antibody before exposure with ECL kits. The bands cannot be cropped when using antibody hybridization. The proteasome inhibitor MG-132 was harnessed as the APEX1 Ubiquitination inhibitor.

Flow Cytometry Assay and Western Blot Experiment

FITC and PI signaling were obtained in the FITC and PE channels. The excitation and emission wavelengths were 561 and 585 nm in the PE channel, and 488 and 525 nm in the FITC channel. The 7-ADD signaling was collected in the PC5.5 channel, with excitation and emission wavelengths of 561 and 690 nm, respectively The APC signaling was collected in the APC channel, with excitation and emission wavelengths of 638 and 660 nm, respectively . Filtration of the cells into flow cytometer-specific tubes was regulated to the appropriate voltage prior to collection of the cells. For each assay, at least 1 × 104 cells were collected per sample. The mean fluorescence intensity (MFI) was quantified by using the geometric mean. Lewis and A549 cells were lysed and treated with a protease inhibitor. Cell lysates were centrifuged, and the supernatant containing protein was collected. Equal amounts of proteins were partitioned by SDS-PAGE before being transferred to a PVDF membrane. The membranes were blocked with 4–5% bovine serum albumin and treated with primary antibodies overnight at 4 °C, including p53 (bs-2090R, Bioss, Beijing, China), γ-H2A.X (bs-3185R, Bioss, Beijing, China), APEX1 (10203–1-AP, Proteintech, Wuhan, China), Caspase-3 (GTX110543, GeneTex, Taiwan, China), Bax (GTX109683, GeneTex, Taiwan, China), PCNA (10205–2-AP, Proteintech, Wuhan, China), Ki67 (bs-23103R, Bioss, Beijing, China), Ubiquitin (10201–2-AP, Proteintech, Wuhan, China), and GAPDH (PMK053C, BioPM, Wuhan, China). The membrane was then incubated with a horseradish peroxidase-conjugated secondary antibody. Blots were visualized using an ECL kit and revealed with a bioimaging system (170–8265, BIO-RAD).

qPCR Measurement and Histopathological Detection

Extracting mRNA from A549 and Lewis cells, reverse transcribing it into cDNA, and then amplifying it with an SYBR Green qPCR Master Mix kit (PC3301, Beijing, Aidlab). The quantitative expression was measured by qRT-PCR (BIO-RAD CFX96 Touch, BIO-RAD). The data analysis was carried out with Bio-RAD CFX Manager. The primer sequences were as follows: Mouse APEX1 Forward: ATGCTCCTGGAATGTGGATGGG. Mouse APEX1 Reverse: GTTTGTTCTCCGAGCACTTGGTC. Mouse GAPDH Forward: AGGTCGGTGTGAACGGATTTG. Mouse GAPDH Reverse: TGTAGACCATGTAGTTGAGGTCA. Alternatively, fixation of tumor tissue and vital organs was followed by sectioning for histopathological testing. The sections were stained with hematoxylin and eosin for 3–5 min, respectively, after dehydration. For detection of the protein expression in tumor grafts, the sections were subjected to immunohistochemistry (IHC) staining. Briefly, the sections were incubated with 3% hydrogen peroxide at room temperature for 10 min after dehydration, blocked with 3–5% BSA for 60 min, and incubated with primary antibody overnight at 4 °C. The sections were further stained with a peroxidase-conjugated secondary antibody for 1–2 h at room temperature. Primary antibodies used in the IHC assay included p53, γ-H2A.X, and APEX1, as mentioned above. To carry out TUNEL staining, the sections were permeabilized with 0.1% Triton-100 prior to staining with FITC-dUTP. Then, the nuclei were dyed with DAPI. Alternativly, to observe the morphology of the organs, the sections were subjected to HE staining. Lastly, the samples were observed using orthogonal microscopy.

Statistical Method

The quantitative data in the current work were displayed as means ± standard deviation (SD). The difference between groups was statistically analyzed by One-way analysis of variance (ANOVA) and followed by the Tukey test. p-values <0.05 were considered statistically significant. Statistical analysis was conducted using GraphPad Prism version 8.0.

Results

Teniposide Exhibited Robust Antilung Cancer Efficacy

To begin with, a comprehensive assessment of Ten/VM-26’s antilung cancer efficacy was presented. The chemical structure of Ten/VM-26, with a purity of 98%, was mapped out as shown in Figure A,B. Significantly, as suggested by the CCK-8 assay in Figure C,D, Ten/VM-26 (0.5 and 1 μM) suppressed the cell viability of A549 and LLC, which indicated potent antilung cancer efficacy with low doses of Ten/VM-26. Consequently, the apoptosis rate of A549 and LLC was elevated dramatically in the presence of Ten/VM-26, with 5 μM doxorubicin (DOX) serving as the positive control (Figure E–H). Furthermore, Ten/VM-26 treatment enhanced the expression of Bax and Cleaved-caspase-3 and dampened the expression of PCNA and Ki67 in A549 and LLC (Figures I,J and S1), indicating increased apoptosis and decreased proliferation of lung cancer cells. Ten/VM-26 treatment also impaired the colony formation of LLC (Figure K,L). Interestingly and excitingly, Ten/VM-26 exhibited a robust tumor-inhibitory effect in the lung cancer cell 3-D culture model (Figure M) and organoid model (Figure N), which provides strong data for the antilung cancer effectiveness of Ten/VM-26. Collectively, the above results demonstrated that Ten/VM-26 possesses a pronounced antilung cancer effect in vitro.

To confirm the in vivo relevance of the in vitro findings, LLC-bearing mice were constructed and treated by Ten/VM-26. The growth of tumor grafts was repressed upon the addition of Ten/VM-26, showing a somewhat dose-dependent effect and demonstrating the best antitumor efficacy at a concentration of 10 mg/kg body weight as displayed in Figure A–C. Actually, a high concentration (20 mg/kg) of Ten/VM-26 may destroy immunocytes in tumor tissue, disrupting the antitumor immune response, which may be the major reason for its slightly poorer efficacy compared to the medium concentration (10 mg/kg). Based on this, the ROS generation within the tumor grafts of mice treated with Ten/VM-26 was increased (Figure D,E), indicating that oxidative damage may have occurred, , which was further validated in the following section as presented in Figure A–D. Indeed, these data evidenced that Ten/VM-26 evokes oxidative stress in malignant cells, which may be the essential contributor to DNA damage. Finally, corresponding with the findings in Figure A–C, the TUNEL fluorescence was enhanced in the tumor grafts of Ten/VM-26-treated mice (Figure G), evidencing increased apoptosis of malignant cells in cancer tissues. More importantly, there was no weight loss in the mice at this dose, proving that the agent is virtually nontoxic to the organism (Figure F). Additionally, Ten/VM-26 exhibited minimal side effects, as characterized by the unchanged morphology of vital organs during the treatment (Figure S2). Altogether, this proof implicated that Ten/VM-26 has favorable in vivo anticancer efficacy. The data presented above provide crucial evidence that Ten/VM-26 was an effective anticancer chemotherapeutic agent.

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Ten/VM-26 inhibited the growth of tumor grafts in lung cancer-bearing mice. (A, B) The size of tumor grafts was photographed and weighted after the Ten/VM-26 treatment (n = 5). (C) During Ten/VM-26 treatment, the volume of tumor grafts was monitored (n = 5). (D, E) The ROS generation within the tumor tissues was assayed by DCFH-DA staining and flow cytometry (n = 3). (F) The body weight of tumor-bearing mice treated by Ten/VM-26 was recorded throughout the treatment (n = 5). (G) Apoptotic cells in tumor grafts were detected by TUNEL staining. Green fluorescence indicatesTUNEL-labeled cells. Statistic values presented were means ± SD (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

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NAC abated Ten/VM-26-mediated DNA damage and improved efficacy in lung cancer cells. The Lewis cells were cotreated with NAC and Ten/VM-26. (A, B) The ROS generation was detected by the DCFH-DA probe. (C) The cell viability was detected by CCK-8. (D, E) Apoptosis rate was analyzed by flow cytometry. (F, G) Colony formation experiments indicated the proliferation of cancer cells, which were counted. (H, I) The comet experiment evaluatedDNA double-strand breakage (DDSB) of Lewis cells. The length of the comet tail was calculated. (J) The 8-oxo-dG accumulation was detected and photographed by confocal laser scanning microscopy. Statistic values presented were means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Teniposide Inactivated APEX1 and Led to Lethal Oxidative DNA Damage

Irreparable oxidative DNA damage can be devastating to tumor cells as it serves as a major mechanism of chemotherapy. − To provide further clarification on whether Ten/VM-26 treatment indeed inflicted irreversible oxidative DNA damage on lung cancer cells, we therefore undertook the following study. As suggested by Figure A–D, Ten/VM-26 treatment led to increased ROS generation. Therefore, elevated expression of p53 and γ-H2A.X (Figure E,F), increased 8-xox-dG accumulation (Figure G,H), and stronger comet tails (Figure I–L) with the treatment of Ten/VM-26 in A549 and LLC cells implicated that vigorous DNA damage was involved. In consequence, the results in Figure S3 provided evidence supporting the conclusion that Ten/VM-26 led to obvious DNA in the cancer grafts of tumor-bearing mice. To further demonstrate that oxidative DNA damage was the crucial reason for Ten/VM-26-mediated antilung cancer efficacy, the oxidative stress inhibitor was employed. As depicted in Figure A,B, N-acetylcysteine (NAC) decreased ROS accumulation, indicating that NAC successfully blocked the oxidative stress triggered by Ten/VM-26. The following findings confirmed that Ten/VM-26-driven oxidative stress was the crucial contributor to DNA damage and anticancer efficacy, as characterized by the dampened comet tails (Figure H,I), 8-xox-dG accumulation (Figure J), increased cell viability (Figure C), decreased apoptosis (Figure D,E), and accelerated colony formation (Figure F,G).

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Ten/VM-26 caused devastating oxidative DNA damage in lung cancer. (A–D) The ROS generation within A549 and Lewis cells was detected by the DCFH-DA probe. (E, F) The biomarkers (p53 and γ-H2A.X) of DNA damage in A549 and Lewis cells were assayed by WB. (G, H) The 8-oxo-dG accumulation, which reflected the oxidative damage of DNA, was detected by the F488 probe in A549 and Lewis cells (green fluorescence). (I–L) The comet experiment evaluated DNA double-strand breakage (DDSB) in A549 and LLC cells, which exhibited prominent tails. The length of the comet tail was calculated. Statistic values presented were means ± SD (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Both network pharmacology and sequencing results indicated that the expression of differentially expressed genes (DEGs) after Ten/VM-26 treatment was observed in association with DNA damage repair. As exhibited in Figure A,B of the network pharmacology analysis, exactly as expected, Ten/VM-26 had coshared targets of DNA damage (TP53 and PARP1) with lung cancer. We therefore conducted RNA sequencing to further analyze the DEGs and their enrichment. The volcano and heat-map analyses showed that Ten/VM-26 treatment facilitated the upregulation of 3183 genes and the downregulation of 3392 genes (Figure C,D). These DEGs were abundant in the cell nucleus and were strongly involved in DNA damage repair, as revealed by gene ontology (GO) analysis (Figure E,F). Consistently, the Gene Set Enrichment Analysis (GSEA) confirmed the findings as well, as characterized by the enrichment of the p53 signaling pathway and DNA repair processes when treated with Ten/VM-26 (Figure G,H). Furthermore, the PCA and heatmap cluster analyses indicated that the results of RNA sequencing were replicable (Figure S4). Other associated findings (e.g., BP and MF enrichment) can be found in Figure S5.

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Network pharmacology and RNA-sequencing analysis of the actions of Ten/VM-26 on lung cancer. (A) The Venn Diagram showed the intersection of Ten/VM-26 and nonsmall cell lung cancer (NSCLC). (B) The intersection of Ten/VM-26 and NSCLC is presented as the protein–protein interaction network (PPI). (C, D) Ten/VM-26-treated lung cancer cells exhibited DEGs, as displayed by volcano maps (C) and heat maps (D). (E–H) The DEGs were analyzed and enriched through GO (CC), KEGG (E, F) and GSEA (G, H) functional enrichment (sample replicates for each group, n = 3).

The significant DNA damage genes and DNA repair genes were presented. So, which pivotal molecule associated with DNA damage repair in lung cancer cells does Ten/VM-26 actually restrain? To answer this question, a screening was first carried out using bioinformatics databases. The results uncovered that APEX1, as previously described, was highly expressed in a variety of malignant tumors, including lung cancer (Figure A,B). In addition, the high APEX1 group of lung cancer patients had an obviously poor prognosis compared with the low APEX1 group (Figure C). We proceeded with further analysis using the single-cell database, which showed that APEX1 was highly expressed in the malignant cells and dendritic cells (Figure D). What’s more, APEX1 expression showed a negative correlation with the infiltration of immunocytes, including B cells, T cells, macrophages, neutrophils, and dendritic cells, as presented in Figure E. Based on the above, to screen the cell line utilized in the following work, the APEX1 expression in three kinds of lung cancer cell lines was detected. As exhibited in Figure F,G, all of the LLC, H1299, and A549 cell lines had high APEX1 basal expression, which demonstrated the appropriateness of the model selection.

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Ten/VM-26 suppressed APEX1 in lung cancer cells by ubiquitination. (A, B) The expression of APEX1 in lung cancer tissues was identified using the TIMER (A) and GEPIA (B) databases. (C) Highly expressed APEX1 showed a significantly poor prognosis. (D) The cell types in which APEX1 is predominantly highly expressed were analyzed using the single-cell database TISCH. APEX1 was highly expressed in malignant cells and dendritic cells (DC). (E) The correlation between APEX1 expression and the infiltration of immunocytes was rendered using the TIMER database. The APEX1 led to the decreased infiltration of B cells, T cells, macrophages, neutrophils, and dendritic cells, which was characterized. (F, G) The expression of APEX1 was detected in the LLC, H1299, and A549 lung cancer cell lines, wherein A549 cells had the highest APEX1 expression. (H–J) WB analysis ofexpression of APEX1 in Lewis and A549 cells treated with Ten/VM-26. (K) Ubiquitination of APEX1 in Ten/VM-26-treated Lewis cells was measured with CO-IP and WB. (L) Lewis cells were cotreated with proteasome inhibitor (MG-132) and Ten/VM-26. The APEX1 expression was detected by WB.

Notably and interestingly, the APEX1 expression in A549 and LLC was inhibited in response to Ten/VM-26 (Figure H–J), which corroborated the RNA-sequencing results, confirming that Ten/VM-26 impacted the DNA repair system of lung cancer cells. Consequently, the in vivo results of IHC also demonstrated that Ten/VM-26 treatment inhibited APEX1 in cancer tissues (Figure S6). We therefore proceeded to explore the mechanism of the inactivation of APEX1 induced by Ten/VM-26. The results indicated that Ten/VM-26 treatment promoted the degradation of APEX1, as evidenced by the increased ubiquitination of APEX1, as shown in Figure K. In addition, Ten/VM-26-mediated APEX1 inhibition could be rescued by proteasome inhibitor MG-132 (Figure L), further demonstrating that Ten/VM-26 may degrade APEX1 through the induction of ubiquitination. Thus, evidence has emerged suggesting a critical role of Ten/VM-26 in inhibiting APEX1 in lung cancer cells. Taken together, the above results strongly suggest that Ten/VM-26 could abolish APEX1 and thereby facilitate pronounced DNA damage in lung cancer cells.

Teniposide Regulate APEX1 Through Drug–Protein Interaction

Interaction of phytomedicine with protein is an essential approach for modulation. , Meanwhile, the data in Figures H and S7 suggested that Ten/VM-26 may be distributed in the nucleus. Based on the above, the regulation of Ten/VM-26 on APEX1 was first investigated thereupon. We therefore conducted molecular docking analysis. The results showed that Ten/VM-26 had favorable affinity with APEX1, wherein the binding energy was −7.73 kcal/mol by hydrogen bonding (Figure A–C). Further CETSA experiments evidenced that Ten/VM-26 treatment enhanced thermal stability, as confirmed by the decreased APEX1 degradation following treatment with Ten/VM-26 in the LLC (Figure D,E). Moreover, the pronaseE stability of APEX1 was increased when the extracted proteins were incubated with Ten/VM-26, as validated by DARTS (Figure F). More importantly, MST analysis calculated that increasing amounts of Ten/VM-26 impacted the thermophoretic motion of APEX1. The S/N was 8.0, and the K d value was 17 μM (Figure G). Finally, confocal laser scanning microscopy observed that 5-FMA-labeled Ten/VM-26 had strong colocalization with APEX1 in the nucleus of A549 and LLC (Figures H–J and S7). These findings indicated that Ten/VM-26 regulated APEX1 through interaction. However, instead of modulating the target molecule by binding, phytomedicine can also achieve regulation by acting on intracellular signaling or transcription factors. , Ten/VM-26 was fluorescently labeled and found to be distributed not only in the nucleus but also in the cytoplasm with a small part (Figures H–J and S7). Based on these findings, we hypothesized that it may also affect APEX1 by regulating transcriptional activity. Hence, there may be other indirect targets for Ten/VM-26 worth exploring in the future.

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Ten/VM-26 distributed in the cytoplasm and nucleus worked with APEX1 directly. (A–C) The binding sites (GLY-176, LEU-179, ALA-230, GLY-178, ASP-308, TYR-269) of Ten/VM-26 with APEX1 were analyzed by molecular docking and presented. (D, E) The thermal stability of APEX1 in Ten/VM-26-treated Lewis cells was detected by CETSA-WB. The thermal degradation curve was mapped. (F) The optimal concentration of Pronase was selected. The Pronase stability of APEX1 was measured by using DARTS. (G) The fluorescence-labeled APEX1 was incubated with different concentrations of Ten/VM-26, whose affinity was analyzed by MST. (H–J) The 5-FMA-leabeled Ten/VM-26 was utilized to treat Lewis cells. APEX1 was stained by immunofluorescence. The colocation of Ten/VM-26 and APEX1 was observed by confocal laser scanning microscopy. Fluorescence relevance in cells treated with Ten/VM-26 was analyzed.

APEX1 Supplementation Dampened Teniposide-Driven DNA Damage and Efficacy

As mentioned above, Ten/VM-26-mediated APEX1 inhibition led to robust oxidative DNA damage and anticancer efficacy. To uncover in-depth the critical role of APEX1 in Ten/VM-26-driven DNA damage, the APEX1 rescue experiments were carried out, wherein the successful overexpression of APEX1 was validated and utilized to perform the following explorations (Figure A–C). As shown in Figure D–H, the DNA damage in Lewis cells was weakened in the presence of Ten/VM-26 and the APEX1 plasmid, as evidenced by the attenuated expression of biomarkers of DNA damage (Figure D–F), the reduced accumulation of 8-oxo-dG (Figure G), and the loss of the comet tail (Figure H,I). In agreement with the results of DNA damage, the Lewis cells exhibited a decreased apoptosis rate when cotreated with the APEX1 plasmid and Ten/VM-26 (Figure J,K). Briefly speaking, APEX1 recovery dampened Ten/VM-26-mediated DNA damage and antilung cancer efficacy. APEX1 was the crucial target for Ten/VM-26-driven DNA damage and anticancer efficacy.

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APEX1 supplementation abolished Ten/VM-26-elicited DNA damage and cell viability inhibition. (A–C) The successful overexpression of APEX1 mediated by APEX1 protein was validated by qRT-PCR (A) and WB (B, C). (D–F) The expression of p53 and γ-H2A.X in Lewis cells coadded with APEX1 plasmid and Ten/VM-26 was detected. The mean gray value of the bands was calculated. (G) The 8-xox-dG accumulation in Lewis cells was observed by laser scanning confocal microscopy. (H, I) The DNA double-strand breakage was measured using a comet assay and the length of the comet tail was calculated. (J, K) The apoptosis rate of Lewis cells labeled by Annexin-V/7-ADD was assayed by flow cytometry. The Annexin-V positive cells were counted. Statistic values presented were means ± SD (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Discussion

Herein, our innovative results provided strong evidence for the idea that Ten/VM-26 could suppress APEX1 by directive combination, thereby enhancing DNA damage and stimulating anticancer efficacy (Figure ).

9.

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Presentation of the schematic diagram. Ten/VM-26-mediated inhibition of APEX1 contributes to DNA damage and thereby achieves favorable antilung cancer effects, wherein the Ten/VM-26 could down-regulate APEX1 by binding and ubiquitination.

The greatest innovation of the current study was the identification of the suppressive effect of Ten/VM-26 on APEX1. APEX1 plays a predominant role in DNA repair in cancer cells and is one of the most responsible antidamage molecules. Resistance to many chemotherapeutic medications after long-term administration occurs due to the activation of APEX1 while damaging DNA. For example, cisplatin, when used in the treatment of CNS tumors, can activate APEX1, thus leading to DNA repair. The damage to neurotumors may be increased if OGG1, which is responsible for excising 8-oxoG to leave a substrate for APEX1 and trigger the repair program, is repressed by APEX1 inhibitors. Moreover, APEX1 has also been identified for its ability to repair damaged mitochondrial DNA (mtDNA). Therefore, in-depth exploration and development of clinically ready APEX1 inhibitors are highly desirable.

In addition, unexpected findings of both direct and indirect regulatory effects of Ten/VM-26 on APEX1 were very interesting. Precisely because Ten/VM-26, which was attached to fluorescein, was distributed both in the nucleus and the cytoplasm (Figures H and S7), we carried out a deeper exploration of the targets. Although APEX1 is a type of cytosolic protein, there are also signaling pathways or transcription factors in the cytoplasm that indirectly regulate it. , Thus, we conducted a profound investigation of the intracellular target of Ten/VM-26 (Figure ). As a result of the complexity of the intracellular microenvironment in malignant cells, coregulation of intracellular molecules or processes by pharmaceutical agents through both direct targeting and indirect action is extremely common. Of necessity, there may even be many unknown targets for a single drug, which cannot all be explored based on current technological approaches. Hence, there may be other indirect targets for Ten/VM-26 worth exploring in the future.

Furthermore, APEX1 expression has been reported to be present in immunocytes, whose functions have been poorly understood. Few studies showed that APEX1 in monocytes increased their viability, achieved primarily through the maintenance of genetic stability and increased antioxidant capacity. In contrast, APEX1 expression in lymphocytes assists the host in increasing immunity against infections and affects the infiltration of CD4-positive cells. , APEX1 in endothelial cells, on the other hand, prevented the development of atherosclerosis by reducing inflammation. After Ten/VM-26 enters the tumor tissue from the circulation, it is likely to be distributed to the immunocytes as well. Whether Ten/VM-26 can suppress the function of APEX1 in immunocytes and whether this effect impacts the phenotype and function of immunocytes remains unknown. Our preliminary bioinformatic results so far have revealed that Ten/VM-26 can influence the infiltration of immunocytes (Figure E), which is very interesting. Additionally, the distribution of Ten/VM-26 in tumor-bearing mice was investigated in the present work (Figure S8), wherein the agent exhibited favorable accumulation in tumor grafts, which may be related to the high fat solubility of Ten (chemical structure incorporates thiophene). In the future, we will systematically investigate the effect of Ten/VM-26 on the immune microenvironment.

Of note, Ten/VM-26 has been reported to damage DNA by inhibiting DNA topoisomerases and thereby preventing the dehelicalization process during DNA replication, which in turn interferes with its replication. Although the above mechanism is classical, we hypothesized in the present workbased on the attenuation of DNA damage caused by Ten/VM-26 to lung cancer cells after inhibition of oxidative stress using NAC (Figures and )that Ten/VM-26 may generate oxidatively damaged DNA by mediating ROS as well. Overall, however, the major innovation of this paper was the screening and demonstration of Ten/VM-26 binding to the target APEX1, a previously discovered molecule that can repair DNA damage, which in turn inhibited its repair action to reinforce DNA damage. Most importantly, we also argued this logical relationship in depth by overexpressing APEX1 in Figure . To date, Ten has been widely clinically used in hematologic malignant cancers (e.g., acute lymphoblastic leukemia), but its use in solid cancers has been less prevalent. Elucidating the intracellular target and molecular mechanism of Ten is important for expanding its clinical indications.

Conclusion

In a nutshell, these data make a strong argument for the notion that Ten/VM-26-mediated inhibition of APEX1 contributes to DNA damage and thereby achieves favorable antilung cancer effects, wherein the Ten/VM-26 could down-regulate APEX1 by binding and ubiquitination. The current study presents a critical target and mechanism for Ten/VM-26-mediated antitumor therapy (Figure ).

Supplementary Material

pt5c00308_si_001.pdf (1.8MB, pdf)

Acknowledgments

The authors appreciated the instrument testing platform at the Institute of Biomedical Research, Hubei University of Medicine. This work was supported by the National Natural Science Foundation of China (82304552, 82303783), Hubei Provincial Natural Science Foundation (2024AFD100, 2025AFB636, 2025AFD198, 2025AFD210, and 2025AFD181), Natural Science Foundation of Hubei Provincial Department of Education (Q20232103, Q20242108), Foundation of Health Commission of Hubei Province (WJ2025Q088, WJ2025Q089), Cultivating Project for Young Scholars at Hubei University of Medicine (2020QDJZR002, 2021QDJZR015, 2023QDJZR29, and 2022QDJZR006), Advantages Discipline Group (Medicine) Project in Higher Education of Hubei Province (2024XKQY23), Project in Administration Bureau of Traditional Chinese Medicine of Hubei Province (ZY2023M073), Excellent Young and Middle-aged Scientific and Technological Innovation Team of Universities of Hubei Province (T2022022), Shandong Provincial Natural Science Foundation (ZR2023QH145), National Training Program of Innovation and Entrepreneurship for Undergraduates (S202410929013, S202410929021, S202410929029, X202410929044, X202410929072, and X202413249021).

Glossary

Abbrevations

LLC

Lewis lung cancer cells

ROS

reactive oxygen species

WB

Western blot

Ten/VM-26

Teniposide

APEX1

apurinic/apyrimidinic endonuclease 1

DEGs

differentially expressed genes

CC

cellular component

BP

biological process

CETSA

cellular thermal shift assay

DARTS

drug affinity responsive target stability

MST

microscale thermophoresis

GO

gene ontology

KEGG

Kyoto Encyclopedia of Genes and Genomes

TIMER

tumor immune estimation resource

GEPIA2

Gene Expression Profiling Interactive Analysis

GSEA

Gene Set Enrichment Analysis

IHC

immunohistochemistry

DDSB

DNA double-strand breakage

Data will be made available on reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.5c00308.

  • This part of the data consists mainly of the biodistribution and safety evaluation of Teniposide, basic information on RNA-sequencing, protein expression in tumor grafts, and the original bands of Western blots (PDF)

#.

N.H., J.H. and Z.X.X. contributed equally to this article. N.H., J.H., Z.-X.X., and L.Z. conducted the major data curation and collection in vitro and in vivo in the present study. X.-R.Y., L.-G.L., and C.H. performed the data analysis. C.K. and F.L. conducted part of the experiments. T.-F.L. wrote and conceptualized the research. T.-F.L. and M.-F.W. supervised this work. Q.Z. and T.-F.L. reviewed the manuscript.

The authors declare no competing financial interest.

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Associated Data

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Supplementary Materials

pt5c00308_si_001.pdf (1.8MB, pdf)

Data Availability Statement

Data will be made available on reasonable request.

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