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Bi-targeting of thioredoxin 1 and telomerase by thiotert promotes cell death of myelodysplastic syndromes and lymphoma

Biology Direct volume 20, Article number: 7 (2025) Cite this article

Abstract

Thioredoxin1 (TRX1) and telomerase are both attractive oncology targets that are tightly implicated in tumor initiation and development. Here, we reported that the 6-dithio-2-deoxyguanosine analog thiotert exhibits an effective cytotoxic effect on myelodysplastic syndromes (MDS) cell SKM-1 and lymphoma cell U-937. Further studies confirmed that thiotert effectively disrupts cellular redox homeostasis, as evidenced by elevated intracellular reactive oxygen species (ROS) levels, increased MnSOD, accelerated DNA impairment, and activated apoptosis signal. Mechanistically, our present study revealed that thiotert treatment effectively inhibited the function of the TRX1/TRXR1 system and telomerase reverse transcriptase (TERT), rendering oxidative damage and impairment of telomeres. Meanwhile, pharmacological administration of glutathione (GSH), N-acetylcysteine (NAC), and mitoquinone (MitoQ), or genetic overexpression of TRX1 or TERT in MDS and cells could dampen the toxicity caused by thiotert. Remarkably, the in vivo mouse model of MDS demonstrated that thiotert administration exhibited greater efficacy in tumor reduction compared to the conventional chemotherapy drug cytarabine. Collectively, these results provide experimental insights into the mechanism of thiotert-induced MDS and lymphoma cell death and unveil that thiotert may be an effective and promising new drug for future MDS and lymphoma treatment.

Graphical Abstract

Introduction

Myelodysplastic syndromes (MDS) is a group of myeloid neoplasms characterized by clonal proliferation of hematopoietic stem cells, myeloid cell dysplasia, ineffective hematopoiesis, cytopenia, and high risk of progression to acute myeloid leukemia (AML) [1]. Patients with MDS have been stratified into 5 risk groups, which have different clinical outcomes in terms of survival and AML evolution, based on the revised International Prognostic Scoring System [2]. Despite immense progress in comprehending the molecular mechanisms of MDS in the last decade, the available therapeutic options for MDS are limited to transfusions and approved drugs that can only halt or defer the progression of the disease [3]. Meanwhile, most MDS patients exhibit a low response rate to a single drug and are prone to develop resistance to conventional chemotherapy [4,5,6,7]. Allogeneic hematopoietic stem cell transplantation is currently the only potentially curative therapy for MDS [8], but it is not suitable for all patients due to age and comorbidities [9]. Lymphoma represents an array of tumors originating from lymphocytes, which can be broadly classified into Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL) [10, 11]. Although the survival rate of HL patients has incrementally risen as the ameliorated chemotherapy regimen (e.g., doxorubicin, bleomycin, vinblastine, and others), not all lymphoma patients can benefit from the treatment modalities [12]. Unfortunately, NHL constitutes approximately 90% of all lymphoma cases, with a 5-year survival rate of roughly 57%; they often face dismal survival outcomes compared to HL [13]. In parallel, approximately 40% of NHL patients are incurable upon the combination of chemotherapies, like cyclophosphamide, doxorubicin, vincristine, and prednisone [14,15,16]. Therefore, it is imperative to seek novel therapeutic strategies to improve the dismal prognosis of MDS and NHL patients.

Thioredoxin 1 (TRX1), a cytosolic small molecular redox protein (12 kDa) and one of the components of the TRX system, is accepted as an essential regulator in redox signaling, which mediates the regeneration of active sites, ROS scavenging, reductive biosynthesis, and other biological processes [17]. Numerous scientific studies have reported the upregulation of the TRX in various malignancies such as MDS, AML, lymphomas, breast, liver, lung, stomach, colon, and rectal cancer, as well as skin cutaneous melanoma. TRX1 overexpression is associated with tumor growth, aggressiveness, metastasis, antiapoptosis, chemoresistance, and poor prognosis in patients [18,19,20,21,22,23]. For instance, TRX is proven to be upregulated in AML and can attenuate high ROS burden and oxidative stress (OS), which in turn is linked to faster relapse and adverse survival [24]. The above data mightily suggest that TRX1 represents a promising target for developing antineoplastic agents. 1-methylpropyl 2-imidazolyl disulfide (PX-12) is the TRX1-specific inhibitor, which could irreversibly suppress the redox function of TRX1 protein by combining the active site [25]. Studies have demonstrated that PX-12, particularly in several advanced solid tumors, displayed excellent safety and tolerance in phase I clinical trials [26]. Although PX-12 lacks significant therapeutic efficacy in phase II clinical trials, further investigation into its antitumor efficacy is warranted [27]. Considering its potential as a combinatorial reagent or adjuvant drug, exploring PX-12 in greater depth may help improve the prognosis of patients.

Telomeres are genomic portions localized at the cap chromosome ends and are completely crucial for the maintenance of chromosome stability. Human telomeres are comprised of telomeric DNA (tandemly repeated TTAGGG sequences) and a cohort of telomere-specific proteins (shelterin complex and telomerase complex) that defend the ends of chromosomes from being recognized as DNA damage sites [28, 29]. Telomerase composed of the telomerase reverse transcriptase (TERT), telomerase RNA template component (TERC), and several accessory proteins, is a ribonucleoprotein enzyme complex that prompts the TTAGGG sequences synthesis correctly [30]. Studies have consistently demonstrated that telomerase is a common characteristic of advanced cancers, exhibiting widespread expression in the majority of primary human cancers [31]. Importantly, elevated telomerase activity or increased expression of Tert is associated with an unfavorable prognosis in low-risk MDS patients [32]. Previous studies found that Tert gene mutations, which can prompt the progression of lymphoma are common in mantle cell lymphoma and that mutations are correlated with higher Tert mRNA expression [33]. Arakawa F et al. have observed high TERT expression in angioimmunoblastic T-cell lymphoma [34]. These findings suggest that telomeres and telomerase could serve as promising targets for therapeutic interventions in MDS and lymphoma [9]. Several telomerase inhibitors or telomerase substrates, including 6-Thio-2’-deoxyguanosine (6-thio-dG), imetelstat sodium (GRN163L), GV1001, and GRNVAC1 have been explored in previous studies. GRN163L is capable of targeting the RNA template of telomerase specifically, thereby modifying telomerase activity. Its remarkable efficacy has been demonstrated in enhancing transfusion independence and improving hematopoietic function in low-risk MDS patients [32]. However, the major limitation of GRN163L lies in its long treatment duration and associated adverse reactions, which significantly restrict its application as an antineoplastic agent [35]. In contrast to GRN163L, 6-thio-dG is a small-molecule telomerase substrate that selectively incorporates into newly synthesized telomeres. This leads to telomere uncapping, and destabilization of the genome, ultimately resulting in telomere dysfunction and cell death specifically in telomerase-positive cancer cells [36, 37]. 6-thio-dG may be a broad-spectrum drug as its potent effects and low-toxic.

The crosstalk between TRX and telomerase has been reported in previous studies. Mitsui et al. found that transgenic mice with human TRX overexpression were capable of counteracting OS and possessed longer lifespans than wild-type mice [38]. In addition, ascended telomerase activity in the spleen was also noted in Trx transgenic mice relative to wild-type mice. Dixit and colleagues found manumycin treatment renders the TRX1 and telomerase activity dampened in a ROS-dependent manner and results in apoptosis of glioma cells [39]. Given the pivotal roles of TRX1 and telomerase in the pathophysiology and progression of myelodysplastic syndromes (MDS) and lymphoma, it is indeed a promising therapeutic approach to develop novel inhibitors capable of simultaneously targeting both TRX1 and telomerase. Such inhibitors may potentially have a synergistic effect and improve patient outcomes. In the current study, we designed a dual-target antitumor agent thiotert that targets TRX1 and telomerase. Through a series of molecular, cellular, and pharmacological analyses, we demonstrate that thiotert effectively suppresses TRX1 and TERT, leading to the accumulation of reactive oxygen species (ROS), DNA damage, and ultimately eliciting cell apoptosis in SKM-1 and U-937 cells. Antioxidants or overexpression of TRX1 and TERT can mitigate the cytotoxic effects of thiotert. Overall, our study suggests that thiotert holds promise as a potential therapeutic agent for MDS and lymphoma treatment.

Method details

Cell lines and reagents

The MDS cell lines (SKM-1, and U-937) and 293T cells were passaged and cryopreserved in our laboratory. MDS cell lines were routinely grown in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (Gibco), and 293T cells were cultured in DMEM medium supplemented with 10% (v/v) fetal bovine serum (Gibco). All the mediums were supplemented with extra penicillin (100 U/mL) and streptomycins (100 U/mL) and incubated in 5% CO2 at 37 ℃.

Cell viability assay

CCK-8 (Boxbio Science & Technology, Beijing) was utilized to evaluate cell viability, which consists of tetrazolium salts that react with dehydrogenase in cells to produce yellow formazan dye [40]. Briefly, 3 × 104 cells (SKM-1 and U-937) or peripheral blood mononuclear cells (PBMC) were cultured in 96 well plates with a serum-free RPMI-1640 medium and treated with various concentrations of thiotert (0–40 µM) for 12 h. Subsequently, the microplate reader was used for detecting the absorbance at 450 nm wavelength upon 15 µL CCK8 reagent was added and incubated for 2 h.

EdU assay

The 5-ethynyl-2-deoxyuridine (EdU) is a pyrimidine analog that can be integrated into DNA double-strand during DNA synthesis, which was performed according to our previous publication [41]. Briefly, cells (2 × 106 cells/well) were cultivated in 6 well plates with a serum-free RPMI-1640 medium and treated with various concentrations of thiotert (0–40 µM) for 12 h. Subsequently, treated cells were first incubated with EdU (10 µM) for 2 h, and then paraformaldehyde with 0.3% triton was used for fixation and perforation. Then cells were stained with a prepared click reaction solution for 20 min after 5% BSA blockage for 2 h. Eventually, the laser confocal microscope (Leica TCS SP8 MP, Germany) was used for capturing images of nuclei co-stained with Hoechst 33,342.

Cell apoptosis assay

Cells (2 × 106 cells/well) were incubated in 6 well plates with a serum-free RPMI-1640 medium upon the indicated concentration of thiotert (0–40 µM) for 12 h. Then, cells were collected and washed with PBS. Each sample was stained with 500 µL 1 × binding buffer containing 10 µL Annexin V-FITC and 5 µL PI followed by the manufacturer’s instructions (Multi-Science, China). Ultimately, quantification analysis of apoptosis was carried out by flow cytometry (Beckman Coulter, Gallios, USA) after incubating for 5 min in the dark at room temperature.

Cytoplasm ROS and mitochondrial ROS analysis

Cells (2 × 106 cells/well) were plated in 6 well plates with a serum-free RPMI-1640 medium and stimulated with thiotert, and then cells were collected and washed with PBS. DCF-DA probe (5 µM) and MitoSOX probe (3 µM) were used to measure cytoplasm ROS and mitochondrial ROS. Flow cytometry (Beckman Coulter, Gallios, USA) or laser confocal microscopy (Leica TCS SP8 MP, Germany) was used to detect the fluorescence.

Total mRNA isolation, cDNA synthesis, and qPCR

Cells were treated with various concentrations of thiotert for 6 h, and total mRNA was extracted by RNA-Quick Purification Kit (Accurate Biology, China) according to the manufacturer’s instructions. NanoDrop One (Thermo Scientific, USA) was used for the quantification of mRNA. Subsequently, Evo M-MLV RT Mix Kit (Accurate Biology, China) was performed to reverse transcription followed by the manufacturer’s protocols. Quantitative PCR was then carried out using the SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biology, China), and the housekeeping gene GAPDH was used as endogenous control. The intron-spanning primers were designed from the National Center for Biotechnology Information (NCBI) database and synthesized from Genewiz (Suzhou, China). The 2−ΔΔCt method was used to analyze the relative mRNA expression levels of target genes. Detailed information on primers is shown in Table S1.

Western blot and quantification analysis

For western blot analysis, treated cells were lysed on ice for 10 min by RIPA buffer supplemented with protease and phosphatase inhibitors (Beyotime, China), and then centrifugation (14,000×g) for 10 min at 4 ℃. Quantification of protein concentration was measured by Pierce ™ BCA Protein Assay Kit (Thermo Scientific, USA). Lysed proteins (20 µg/lane) were run on 12% SDS-PAGE gels, and then electrotransferred to PVDF membranes (Bio-Rad, cat# 1620177). Then PVDF membranes were blocked with 5% skim milk for 1 h at room temperature and incubated with target antibodies overnight at 4 ℃, the dilution rate of primary antibodies used in this study was shown in Table S2. Subsequently, after being washed with TBST 3 times, the membranes were incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies at 1:2,000 for 1 h at room temperature. Finally, bands were detected by the chemiluminescence detection kit (FDbio Science, China) using ChemiDocn™ MP Imaging System (Bio-Rad). Image J (https://imagej.en.softonic.com/) was used for the quantification of western blot bands.

γH2AX and TRF2 immunofluorescence staining

Cells (1.5 × 106 cells/well) were seeded into 6 well plates with a serum-free RPMI-1640 medium and added various concentrations of thiotert for 12 h and then transferred on glass bottom culture plates (NEST Biotechnology, China) which were pretreatment with polylysine. Cells were washed with PBS 3 times for 15 min and fixed with 4% formaldehyde for 15 min. Next, cells were washed with PBS 3 times, followed by 1% Triton X-100 treatment for 5 min. Cells were rewashed with PBS and blocked by 5% BSA for 60 min at room temperature. Subsequently, cells were incubated with anti-gamma H2AX (Abcam, cat# ab81299) or anti-TRF2 (cat# ab108997) at 1:400 dilution rate at 4 ℃ overnight. After that, cells were washed with PBS 3 times and incubated with FITC-labeled Goat Anti-Rabbit IgG (H + L) (Beyotime, China) at a 1:200 dilution rate for 1 h at room temperature. Eventually, the laser confocal microscope was used to capture images upon PBS washing 3 times.

TRXR1/TRX1 activity analysis

For the analysis of TRXR/TRX1 activity, cells (1.5 × 106 cells/well) were plated into 6 well plates with a serum-free RPMI-1640 medium and treatment of thiotert for 12 h. Quantification of protein concentration was measured by Pierce ™ BCA Protein Assay Kit (Thermo Scientific, USA). TRXR1/TRX1 activity was determined using the Micro Oxidized Thioredoxin Reductase (TrxR) Assay Kit (Boxbio Science … Technology, Beijing) followed by the manufacturer’s protocols.

Lentiviral packaging and transduction

The cDNA of TRX1 and TERT was obtained from Sino Biological (Beijing, China) and subcloned into a pLVX-IRES-Neo lentivirus vector (Takara, Japan). The Lentiviral packaging was performed according to our previous publication [42, 43]. SKM-1 cells were seeded in 24-well plates and transfected with Trx1 and Tert overexpression lentivirus for 24 h. Polybrene was added to improve transfection efficiency. When the cell density reached 70%, 1 mg/mL G418 was added for screening tests. After 4 weeks of G418 selection, the stably transfected cell lines were determined using a western blot analysis.

Mice models

NSG mice (6 weeks) were obtained from Zhejiang Experimental Animal Center (Hangzhou, China), and kept in specific pathogen-free conditions. All mice were randomly divided into the vehicle (n = 6), thiotert (n = 6), and cytarabine (n = 6) treatment groups. SKM-1_GFP_ Luciferase cells (1.0 × 107) were injected into mice by tail veins, and treatment was started two weeks after injection. The vehicle group mice were treated with vehicle, thiotert (20 mg/kg), and cytarabine (50 mg/kg). Mice were injected i.p. every day for 1 week. The whole blood was derived from the orbital venous plexus. Mice tissues were resected and fixed with 4% paraformaldehyde (Leagene Biotechnology, China), and immunohistochemistry staining was conducted according to our previous publication [44]. Bioluminescence images were captured by a PerkinElmer In Vivo Imaging System (IVIS) Spectrum and total bioluminescence was analyzed using Living Image software.

Statistical analysis

Statistical analyses were calculated using Prism software (version 9). Data for in vitro experiments are expressed as mean ± SD. The t-test and one-way ANOVA were performed to determine the statistical significance between two or multiple groups, and P < 0.05 was considered significant.

Results

Thiotert treatment causes TRX1 and TERT inactivation

TRX1 and telomerase are pivotal factors in the pathogenesis and progression of most tumors, rendering them promising targets for drug therapy. We intend to investigate the potential synergy between thioredoxin 1 and telomerase in the treatment of MDS and lymphoma. To achieve this, we have devised a series of 6-dithio-2’-deoxyguanosine analogs, denoted as YLS00X, through the modification of the core structure of 2’‑deoxyguanosine. The chemical structure formulas of these analogs are depicted in Figure S1. We conducted an investigation into the antitumor effect of YLS analogs in vitro and in vivo, as well as their pharmacodynamics, pharmacokinetics, and toxicity in xenograft nude mice. Our findings confirmed that YLS004 effectively inhibits both TRX1 and telomerase simultaneously [45]. As a result, YLS004, also known as thiotert, was chosen for further studies. Mechanistically, thiotert interacts with the thiol groups of TRX1 protein, leading to its deactivation. Additionally, thiotert transforms into 6-thio-dG, which is recognized by telomerase and induces telomere dysfunction by incorporating it into newly synthesized telomeres. Finally, 6-thio-dG is metabolized to 6-thioguanine (6-TG), which is capable of being incorporated into DNA strands and contributes to DNA damage and cell cycle arrest (Fig. 1A).

Fig. 1
figure 1

Thiotert inhibited the expression of TERT and TRX1 in MDS and lymphoma cells. (A) Theoretical mechanism diagram of thiotert. (B, C) Relative mRNA expression levels of Tert or Trx1 upon different concentrations of thiotert induction in SKM-1 (B) and U-937 (C) cells, and the GAPDH gene as the reference for quantitative analysis. (D) Western blotting analysis of TERT, TRX1, and TXNIP expression treated with different periods and increased concentrations of thiotert. The quantification data are shown below, the protein levels were normalized to the β-actin. (E) Enzyme activity analysis of TRXR upon thiotert treatment in SKM-1 and U-937 cells. (F) Western blotting analysis showed the effect of GSH and MitoQ on the expression of TERT and TRX1 induced by thiotert for 12 h, and the quantification data were shown on the right. *P < 0.05, **P < 0.01, ***P < 0.001

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To elucidate whether thiotert exerts antitumor effects by targeting TRX1 and TERT, we first investigated their expression level by qPCR and western blot upon thiotert treatment. Results showed that the expressions of TRX1 and TERT were significantly decreased both in mRNA transcription and protein levels after thiotert treatment in SKM-1 and U-937 cells (Fig. 1B-D). It has been reported that thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of TRX, can bind and oxidize TRX1 or TRX2 to respond to the OS [46]. The results from the western blot showed that the expression of TXNIP was elevated, indicating a negative regulatory role of TXNIP in redox balance (Fig. 1D). TRX1 is an essential modulator in coordinating redox signals and defending against OS which is regulated by TRXR [47]. Further analysis suggested that thiotert treatment markedly inhibited the activity of TRXR in MDS cells (Fig. 1E). Furthermore, antioxidants (GSH and MitoQ) are capable of alleviating the inhibitory of thiotert on TRX1 and TERT expression (Fig. 1F). These results suggested that thiotert can suppress the expression of TRX1 and TERT.

The effects of thiotert on cell viability in SKM-1 and U-937 cells

To test the cytotoxicity effect of thiotert, PBMC, SKM-1, and U-937 cells were treated with various concentrations of thiotert for 12 h, and the cell viability was measured by CCK8 assay. We found that thiotert strikingly inhibited cell viability of SKM-1 and U-937 cells in a dose-dependent manner, and slight cytotoxicity on PBMC (Fig. 2A-C), and the IC50 values of thiotert for PBMC, SKM-1 and U-937 cells were 175.73 µM, 18.27 µM and 17.75 µM, respectively. In parallel, the Calcein-AM/PI staining results indicated that thiotert exhibits excellent cytotoxic effects on SKM-1 and U-937 cells as well. The number of green fluorescents labeled live cells was decreased which was accompanied by the increase of PI-positive death cells with the increase of thiotert concentration (Fig. 2D-G). Collectively, these results indicated the thiotert effectively restrained the viability of SKM-1 and U-937 cells and induced cell death in a dose-dependent manner.

Fig. 2
figure 2

The effects of thiotert on cell viability in MDS and lymphoma cells. (A-C) Cell viability and IC50 values of thiotert in PBMC (A), SKM-1 (B), and U-937 (C) cells. (D-E) SKM-1 (D) and U-937 (E) were treated with thiotert (0–40 µM) for 12 h and representative images of Calcein-AM/PI staining were captured. Hoechst was used for nuclear staining. (F-G) Quantification data of Calcein-AM/PI staining in SKM-1 and U-937 cells. **P < 0.01, ***P < 0.001

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Thiotert treatment increases intracellular ROS accumulation

ROS plays a crucial role in tumorigenesis, and it is worth noting that high levels of ROS can actually hinder tumor development [48]. Considering thiotert’s significant reduction in TRX1 activity, we aim to investigate the impact of thiotert on ROS homeostasis in the treated SKM-1 and U-937 cells. As shown in Fig. 3A, B, and Figure S2A, B, the ROS levels in the cytoplasm and mitochondria were dramatically elevated. As excessive ROS can cause oxidative damage, tumor cells have developed antioxidative defense mechanisms to counteract the increased ROS and maintain their survival and proliferation [47]. MnSOD is an endogenous antioxidant that subdues intracellular unrestrained ROS to homeostatic levels. We found that thiotert treatment significantly increased the expression of MnSOD (Fig. 3C). In addition, GSH and MitoQ could counteract the elevated levels of ROS and decrease OS caused by thiotert (Fig. 3D and Figure S2C). Furthermore, cell viability analysis showed that GSH, MitoQ, and NAC could salvage thiotert-triggered cell death (Fig. 3E-G). Overall, these results suggested that thiotert induces SKM-1 and U-937 cell death by elevating intracellular ROS levels.

Fig. 3
figure 3

Thiotert treatment induced oxidative stress in MDS and lymphoma cells. (A, B) Flow cytometric analysis showing the cytoplasm ROS or mitochondrial ROS levels induced by thiotert measured by DCF-DA and MitoSOX staining. The histograms of quantification data are shown on the right. (C) Western blot of MnSOD expression treated with different concentrations or temporal gradient of thiotert, and quantification data are shown on the right. (D) Representations of cytoplasm ROS levels in SKM-1 and U-937 cells subjected to the thiotert treatment which was pretreated with GSH and MitoQ. Hoechst was used for nuclear staining, and quantification data were shown on the right. (E-G) Cell viability of SKM-1 cells treated with thiotert in the presence or absence of GSH (1mM), MitoQ (0.5µM), and NAC (2µM). *P < 0.05, **P < 0.01, ***P < 0.001

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Thiotert treatment results in DNA damage

The inhibition of telomerase can cause the loss of telomeric DNA, which ultimately results in insufficient protection of the linear chromosome ends. This, in turn, activates the DNA damage response (DDR). Additional experiments conducted on various mammalian cells have indicated that treatment with the purine antimetabolite 6-TG can cause DNA single- and double-strand breaks, as well as chromosomal damage [49, 50]. Immunostaining results revealed a notable increase in the expression of the histone variant γH2AX, which is a known marker for DNA double-strand breaks (DSBs). This suggests that DNA in SKM-1 cells is indeed damaged following treatment with thiotert (Fig. 4A) [51]. In addition, we examined the levels of telomere-protective proteins and found a moderate increase in TRF2 (Fig. 4B). Through western blot analysis, we conducted further confirmation that thiotert treatment leads to the increased expression of γH2AX (Fig. 4C-E). Additionally, DNA agarose electrophoresis revealed that thiotert promoted nuclear DNA fragmentation(Fig. 4F). The above results suggest that thiotert treatment is capable of leading to severe DNA damage. Interestingly, our findings revealed that the thiotert-induced DNA damage signals could be alleviated by GSH, rather than the mitochondria-targeted antioxidant MitoQ (Fig. 4G).

Fig. 4
figure 4

Thiotert treatment induces DNA damage in MDS and lymphoma cells. (A) Representative images of γH2AX immunostaining upon various concentrations of thiotert treatment for 12 h in SKM-1 cells, and the histogram shows quantification data of DNA damage (γH2AX) signals. (B) Representative images of TRF2 immunostaining after thiotert (40 µM) administration for 12 h, quantification data were shown on the right. (C-E) Western blotting analysis of DNA damage-related protein in MDS and lymphoma cells induced by thiotert in different concentrations or temporal gradients, and the quantification data of γH2AX expression, all the protein signals were normalized to β-actin. (F) The result of DNA electrophoresis. 5-Fu was used as a positive control. (G) Western blotting analysis of γH2AX expression when exposed to a thiotert treatment (40 µM) with the presence of GSH (1 mM) and MitoQ (0.5 µM) in SKM-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001

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Thiotert treatment induces SKM-1 and U-937 cell apoptosis

Apoptosis, as the major pattern of programmed cell death, plays a central role in the growth and proliferation of tumor cells [52]. As aforementioned, DDR signaling can trigger the occurrence of senescence and apoptosis. To interrogate whether thiotert treatment activates apoptosis in the MDS and lymphoma cells, thiotert-treated SKM-1 and U-937 cells were subjected to flow cytometry for the detection of apoptosis. The results showed that apoptotic cells increased dramatically after thiotert treatment (Fig. 5A). Studies have demonstrated that pro-apoptotic members such as Bax and BID stimulate the release of cytochrome C, while anti-apoptotic members like Bcl-2 and Bcl-XL inhibit this release. The release of cytochrome C or the cleaved form of PARP acts as an indicator for the activation of caspase-3, which is capable of initiating apoptosis [53, 54]. We then measured the expression levels of apoptosis-related proteins and found that the expression of cleaved-Caspase-3, cleaved-PARP, Bax, and Cytochrome C were upregulated, and Bcl-2 was downregulated upon thiotert treatment (Fig. 5B-E). Likewise, the special inhibitor of apoptosis (Z-VAD), and antioxidants (GSH and MitoQ) could rescue the phenotypes of apoptosis induced by thiotert (Fig. 5F-H). Taken together, these data indicated that thiotert promotes cell apoptosis via upregulating pro-apoptotic proteins and inhibiting anti-apoptotic proteins.

Fig. 5
figure 5

Thiotert treatment induces MDS and lymphoma cell apoptosis. (A) Flow cytometric analysis showing apoptosis levels of SKM-1 and U-937 cells exposed to various concentrations of thiotert for 12 h. The quantification of apoptosis was displayed on the right. (B) Western blot analysis of apoptosis-related protein expression in SKM-1 and U-937 cells treated with different concentrations of thiotert for 12 h. (C-E) Histograms show densitometric analysis of apoptosis-related protein expression which were normalized to the β-actin. (F) Cell viability was analyzed under the treatment of thiotert with or without Z-VAD in SKM-1 cells. (G, H) Western blot analysis of apoptosis-related proteins under the treatment of thiotert with or without GSH (1 mM) and MitoQ (0.5 µM) in SKM-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001

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Overexpression of TERT or TRX1 alleviates the cytotoxicity of thiotert

To examine whether the cytotoxicity effects of thiotert were specific for TERT and TRX1, TERT and TRX1 were overexpressed in SKM-1 cells (Figure S3A, B). Cell viability analysis revealed that cells overexpressing TERT or TRX1 exhibited enhanced proliferation and increased resistance to thiotert-induced cytotoxicity compared to the control groups (Fig. 6A, B). Previous research demonstrated that TERT could protect mitochondrial DNA from oxidative damage and maintain antioxidant enzyme levels [55]. To verify whether TERT or TRX1 overexpression was sufficient to ameliorate the accumulated ROS levels induced by thiotert. Flow cytometry and laser confocal microscopy analysis were conducted, and marked reductions of cytoplasm and mitochondrial ROS were observed in TERT and TRX1 overexpressed SKM-1 cells when exposed to the treatment of thiotert (Fig. 6C-E, S3C-D). Western blot analysis demonstrated that TERT and TRX1 overexpression significantly alleviated apoptotic cell death with the mitigation of Cleaved-Caspase-3 and Cleaved-PARP (Fig. 6F, G). These findings suggest that increased expression of TERT or TRX1 may enhance the cells’ ability to combat elevated levels of ROS, and apoptosis induced by thiotert.

Fig. 6
figure 6

TERT or TRX1 overexpression enhances resistance to thiotert in SKM-1 cells. (A) Proliferation assay in SKM-1 cells with the overexpression of TERT or TRX1. (B) Cell viability upon thiotert treatment for 12 h in control, TERT, and TRX1 overexpressed SKM-1 cells. (C) Representative images of cytoplasm ROS or mitochondrial ROS levels induced by thiotert in control and TERT, TRX1 overexpressed SKM-1 cells. (D, E) Quantification data of cytoplasm ROS and mitochondrial ROS were shown. (F, G) Western blotting analysis of TERT, TRX1, and apoptosis-related protein expression under the treatment of thiotert in control, TERT, and TRX1 overexpressed SKM-1 cells. *P < 0.05, **P < 0.01, ***P < 0.001

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Thiotert treatment suppresses the development of SKM-1 in vivo

To investigate the antitumor effect of thiotert in vivo, we injected NSG mice with SKM-1_GFP_LUC cells by tail vein to establish the tumor model and evaluate the therapeutic effect of thiotert. Cytarabine, the first-line drug for MDS patients, was used for treatment in parallel to compare the antitumor effect of thiotert (Fig. 7A). Results showed that the mice in both treated groups had no obvious changes in body weight compared with the mice in the vehicle group (Fig. 7B). Interestingly, we observed a significant decrease in fluorescence intensity, an indicator of tumor proliferation in vivo, in the treatment groups, particularly in the thiotert treatment group (Fig. 7C). This suggests that thiotert treatment effectively suppressed tumor cell proliferation in vivo, which is more efficient than the treatment of cytarabine. Flow cytometry analysis indicated a significant reduction in the number of CD45+ GFP+ MDS cells in the whole blood upon thiotert or cytarabine administration (Fig. 7D). H&E staining analysis revealed that cytarabine treatment resulted in edema and damage to hepatocytes in liver tissues (Fig. 7E). However, treatment with thiotert significantly reduced the infiltration of SKM-1_GFP_LUC cells in liver tissues without causing pathological injury to the lung, kidney, heart, brain, or liver tissues (Fig. 7E, S4). This suggests that thiotert treatment specifically targets and inhibits the infiltration of SKM-1_GFP_LUC cells in the liver without causing adverse effects on other organs. Additionally, our findings demonstrated that thiotert treatment effectively suppressed the expressions of Ki67, TERT, and TRX1 in vivo (Fig. 7E). Taken together, these results showed that thiotert might be a promising drug for MDS treatment.

Fig. 7
figure 7

Thiotert treatment suppresses the growth of SKM-1 cells in vivo. (A) The treatment schema for the tumor xenografts model in NSG mice. (B) The body weight after 20 mg/kg thiotert or 50 mg/kg cytarabine injection, i.p. every day for 7 days. (C) Representative images of fluorescence intensity upon 20 mg/kg thiotert or 50 mg/kg cytarabine treatment. (D) Flow cytometry analysis showing the number of GFP+/CD45+ cells in whole blood when treated with thiotert or cytarabine, quantification data were shown on the right. (E) H&E staining of liver and lung; IHC analysis for TRX1, TERT, and Ki67 in SKM-1 cell upon thiotert and cytarabine treatment. *P < 0.05, **P < 0.01

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Discussion

ROS are indeed metabolic byproducts that are generated during aerobic life. They play a crucial role in regulating cellular redox homeostasis and function as important signaling molecules, which are implicated in various cellular processes, including gene and protein expressions, cell proliferation and differentiation, and epigenetic modifications [56,57,58,59]. Pathogenesis of various diseases, including MDS, lymphoma, and AML is associated with ROS [60]. Iron overload (IO) is the typical phenomenon in low-risk MDS patients and is closely associated with inferior clinical outcomes in MDS due to iron toxicity and its negative effect on organ function [61]. Free labile ferrous iron ions are potent generators of ROS, which are capable of impairing hematopoietic stem cells and their capability of hematopoietic reconstruction in the bone marrow microenvironment [61]. It is not surprising that ROS accumulation and hematopoiesis failure were observed in MDS patients with excess iron [62]. A growing slew of literature supports that excessive ROS augments genomic instability of the pre-leukemic clone and expedites the transformation of MDS to AML [63, 64]. Abnormal iron homeostasis was also reported in lymphoma, which renders the augmented peroxidation and ultimately ferroptosis of lymphoma cells [65]. Meanwhile, there is plenty of evidence proving that the survival and proliferation signal axis of lymphoma cells are controlled by intracellular ROS level [10, 66, 67]. Therefore, targeting ROS or ROS-mediated signaling pathways may present a viable strategy for MDS and lymphoma therapy [68]. Here, we reported that the 6-dithio-2-deoxyguanosine analog thiotert could induce ROS accumulation, and oxidative stress, and ultimately promote MDS and lymphoma cell apoptosis by inhibiting TRX1 and TERT.

In our previous study, a series of 6-dithio-2’-deoxyguanosine analogs, including 6-sec-butyl dithio-2’-deoxyguanosine (YLS001), 6-p-fluorobenzyl dithio-2’-deoxyguanosine (YLS002), 6-tert-butyl dithio-2’-deoxyguanosine (YLS003), and 6-isopropyl dithio-2’-deoxyguanosine (YLS004, also named thiotert), have been designed and synthesized, and in colon cancer and melanoma cells, we found thiotert significantly dampens TRXR1/TRX1 system activity and telomerase expression more than the other 3 analogs [45]. A recent study confirmed that thiotert can induce glioblastoma cell death by wrecking cellular redox homeostasis [69]. To assess whether thiotert has antitumor effects in MDS and lymphoma cells, we conducted a series of experiments and observed that thiotert could suppress SKM-1 and U-937 cell viability, and proliferation. Because ROS is tightly associated with numerous cellular processes, additional experiments have suggested that thiotert is capable of disrupting cellular redox homeostasis, resulting in massive ROS accumulation.

We observed that the expression of MnSOD increased with the elevation of intracellular ROS, which is required to deduce oxidative injury and maintain cellular ROS balance. Studies have shown that excessive ROS can lead to DNA DSBs and activate the DDR pathway to repair damaged DNA in the form of cell cycle blockage [46, 70]. Our results showed that the expression of the DNA damage marker γH2AX was significantly heightened, suggesting that administration of thiotert induced DNA damage. Moreover, antioxidants such as GSH, NAC, and MitoQ can effectively mitigate ROS levels and reduce oxidative damage caused by thiotert. Intriguingly, the mitochondria-targeted antioxidant MitoQ does not impede the occurrence of DNA impairment that may be on account of the active form of MitoQ being oxidized exclusively by the respiratory chain. Significantly, the apoptosis inhibitor Z-VAD was employed to determine if apoptosis is the mechanism of cell death induced by thiotert. It was observed that Z-VAD could effectively counteract the cytotoxicity and restore the viability of SKM-1 cells. Collectively, our present work emphasizes the potential of thiotert as a therapeutic agent for MDS/lymphoma treatment by targeting TRX1 to regulate ROS accumulation and promote apoptosis of MDS and lymphoma cells.

Telomeres shortened lead to replicative senescence throughout all somatic normal human cells, in contrast, tumor cells are able to escape senescence and become immortalized by reactivating the telomerase to maintain the length of telomeres [71]. 6-thio-dG could be recognized by telomerase and induced telomere dysfunction by incorporating into de novo synthesized telomeres [36]. Here we present evidence that 6-dithio-2-deoxyguanosine analog thiotert not only inhibits TRX1 but also induces TERT inactivation in SKM-1 and U-937 cell lines. In addition, overexpressed TERT caused resistance to oxidative damage and apoptosis. Furthermore, we observed S-phase arrest and DNA impairment as a result of the incorporation of 6-thio-2’-deoxy-guanosine-5’-triphosphate (6-TGTP) into DNA synthesis. This incorporation, which occurs after a series of transformations from 6-TG, may lead to DNA damage, cell cycle arrest, and apoptosis [72]. Mechanically, we found that thiotert dramatically inactivates the TRX1 and TERT, which facilitates redox system disequilibrate and telomeres dysfunction.

In conclusion, our present study demonstrates that thiotert can lead to telomerase inactivation and inhibition of TRX1 expression, resulting in the accumulation of ROS. This, in turn, induces DNA damage, cell cycle arrest, apoptosis signaling, and exhibits a synergistic antitumor effect in MDS and lymphoma cells. We further confirmed that pharmacological administration of GSH, NAC, and MitoQ, or genetic overexpression of TRX1 or TERT in SKM-1 and U-937 cells, could dampen the toxicity caused by thiotert. Our results show that thiotert may act as a promising therapeutic agent for the treatment of MDS and lymphoma.

Data availability

No datasets were generated or analysed during the current study.

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Funding

This research was supported by the National Natural Science Foundation of China (No. 82202429); Zhejiang Public Welfare Technology Application Research Project (Grant Nos. LGF22H160027); Medical and Health Science and Technology Project of Zhejiang Province (Grant Nos. 2024XY184, 2023KY1275); Outstanding Youth Foundation of Zhejiang Provincial People’s Hospital (No. ZRY2020B001, ZRY2023J008).

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  1. Qiangan Jing, Yunyi Wu and Yanchun Li contributed equally to this work.

Authors and Affiliations

  1. Laboratory Medicine Center, Department of Clinical Laboratory, Zhejiang Provincial People’s Hospital(Affiliated People’s Hospital), Hangzhou Medical College, Hangzhou, Zhejiang, China

    Qiangan Jing, Yunyi Wu, Chaoting Zhou, Jun Xia, Keyi Li, Yuhuan Shen & Jing Du

  2. Department of Clinical Laboratory, Affiliated Hangzhou First People’s Hospital, School of Medicine, Westlake University, Hangzhou, Zhejiang, China

    Yanchun Li, Xiangmin Tong & Ying Wang

  3. Department of Hematology, Lishui Central Hospital, Lishui, Zhejiang, 323000, China

    Junyu Zhang

  4. Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China

    Lushan Yu

  5. Westlake Laboratory of Life Sciences and Biomedicine of Zhejiang Province, Hangzhou, 310024, China

    Lushan Yu

  6. Department of Clinical Laboratory, Zhuji People’s Hospital of Zhejiang Province, Zhuji, Zhejiang, 311800, China

    Hongfeng Yao

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Contributions

Conception and design of the study: Xiangmin Tong, Jing Du, and Ying Wang. Provision of study material: Lushan Yu. Collection and assembly of data: Qiangan Jing, Yanchun Li and Yunyi Wu. Analysis and interpretation of data: Chaoting Zhou, Junyu Zhang, Jun Xia, Keyi Li, Yuhuan Shen, Hongfeng Yao. Manuscript writing: Qiangan Jing, and Jing Du. Final check and approval of manuscript: All authors.

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Correspondence to Xiangmin Tong, Jing Du, Lushan Yu or Ying Wang.

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Jing, Q., Wu, Y., Li, Y. et al. Bi-targeting of thioredoxin 1 and telomerase by thiotert promotes cell death of myelodysplastic syndromes and lymphoma. Biol Direct 20, 7 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13062-025-00594-2

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