Triclabendazole inhibits PKM2 nuclear localization and glycolysis by enhancing HDAC6-mediated deacetylation in lung cancer

Abstract

Background

Metabolic reprogramming is a hallmark of cancer cells, enabling them to meet the heightened energetic and biosynthetic demands required for rapid growth and proliferation. Recently, non-canonical functions of metabolic enzymes have garnered significant attention in cancer research. Pyruvate kinase 2 (PKM2) has been identified as a key player in transcriptional regulation within the nucleus, presenting new opportunities for therapeutic interventions in cancer.

Methods

In this study, the cells (A549 and H1299) were treated with indicator concentration of triclabendazole. The effects of triclabendazole on proliferation was detected by CCK8 assay, colony formation assay, EdU staining, and cell count assay. A tumorigenesis study in nude mice was performed to demonstrate the inhibitory effect of triclabendazole on tumor growth. PKM2 nuclear translocation, HDAC6-mediated deacetylation, glycolytic flux downregulation, and activation of AMPK/mTOR signaling pathway were used to elucidate the mechanistic role of triclabendazole in lung cancer progression.

Results

This study discovered that triclabendazole, a novel benzimidazole derivative, commonly used against Fasciola hepatolithiasis, effectively inhibited the nuclear translocation of PKM2. This inhibition resulted in the downregulation of glycolytic flux, ultimately suppressing lung cancer cell proliferation. Notably, triclabendazole reduced PKM2 acetylation by promoting the interaction between PKM2 and histone deacetylase 6 (HDAC6), thus blocking PKM2 nuclear localization. Moreover, we also demonstrated that triclabendazole-mediated inhibition of cell proliferation is driven by the downregulation of glycolysis, which enhanced AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling. Consistently, triclabendazole administration significantly inhibited tumor growth in vivo, correlating with the blockade of PKM2 nuclear translocation and lactate production decreased.

Conclusion

Our findings revealed that triclabendazole inhibits PKM2 nuclear localization and glycolysis through an HDAC6-dependent mechanism, leading to the activation of AMPK/mTOR signaling and suppression of lung cancer cell proliferation. These results suggested that triclabendazole holds promise as a potential therapeutic agent, with the HDAC6-PKM2 axis representing a novel target for lung cancer treatment.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-06905-5.

Introduction

Benzimidazoles, a class of nitrogen-containing heterocyclic compounds, have demonstrated significant inhibitory effects on tumor cell development [1–3]. Triclabendazole, a novel benzimidazole derivative traditionally used for the treatment of Fasciola hepatolithiasis, has been shown to impair the intracellular transport mechanism by binding to tubulin, subsequently interfering with protein synthesis—similar to many antitumor drugs [4–7]. Our previous research has indicated that triclabendazole induces apoptosis-mediated pyroptosis via ROS/JNK/Bax-mitochondrial pathway [8]. However, its broader anti-tumor potential, particularly in lung cancer, and its underlying mechanisms, remain largely unknown.

Metabolic dysregulation is a critical feature of tumor development and progression [9]. Lung cancer, a heterogeneous disease, exhibits distinct metabolic characteristics across its subtypes, including alterations in glucose metabolism [10, 11]. For example, some metabolism-related proteins, such as glucose transporter-1 (GLUT1), lactate dehydrogenase (LDHA), glutamine transporter 1 (SLC1A5) and monocarboxylate transporter 1 (MCT1), are often overexpressed in lung cancer. Inhibition of these proteins, either by suppressing their expression or promoting their degradation, can effectively hinder lung cancer development and progression [12–14].

Pyruvate kinase 2 (PKM2) is a key player in cancer metabolic reprogramming, functioning as a rate-limiting glycolytic enzyme in cytoplasm of highly proliferative cancer cells. In recent years, PKM2 has also been found in the nucleus, mitochondria and extracellular space, where it performs non-canonical roles that promote cancer cell proliferation and other tumor-promoting processes [15, 16]. Nuclear translocated PKM2 enables its interaction with transcription factors, such as hypoxia-inducible factor-1 (HIF-1α) and β-catenin, regulating genes involving in glycolysis, such as GLUT1, LDHA, pyruvate dehydrogenase kinase isoenzyme 1 (PDK1), hexokinase 1 (HK1) [16–19]. These discoveries have spurred new avenues for cancer research and drug development. PKM2 translocation from the cytoplasm to the nucleus is regulated by post-translational modifications, including acetylation. Previous studies have shown that PKM2 acetylation is mediated by various proteins, including Testes-specific protease 50 (TSP50), SIRT1, SIRT2, SIRT3 and SIRT6 [20–24]. Our study aimed to identify additional proteins that interact with PKM2 and elucidate their role in lung cancer progression.

Histone deacetylase 6 (HDAC6), a member of the class IIb HDAC family, plays an important role in cancer development and progression [25]. HDAC6 deacetylates with a variety of proteins, including α-tubulin, HSP90, and TSC2 [26–29]. Inhibition of HDAC6 can promote α-tubulin acetylation, enhancing the resistance to paclitaxel-induced apoptosis in lung cancer [30]. In addition, the migration and invasion inhibitory protein (MIIP) has been shown to block HDAC6-mediated deacetylation of RelA (p65), thereby enhancing the transcriptional activity of RelA and promoting tumor metastasis [31]. However, the potential interaction between HDAC6 and PKM2, as well as its effect on PKM2 acetylation, has not been previously reported.

In this study, we investigated the effect of triclabendazole on lung cancer cells A549 and H1299. Our findings provided evidence that triclabendazole inhibited glycolysis and cancer cell proliferation by blocking PKM2 nuclear translocation, which suppressed the expression of key metabolic enzymes such as hexokinase 2 (HK2) and LDHA. Notably, the inhibitory effects of triclabendazole were mediated through an enhanced interaction between PKM2 and HDAC6, leading to a reduction in PKM2 acetylation. We also observed that triclabendazole-mediated glycolysis inhibition activated AMPK/mTOR signaling, potentially contributing to its anti-proliferative effects. These findings were further validated in a mouse xenograft tumor model, demonstrating triclabendazole’s potential as a therapeutic agent in lung cancer. Our results also suggested that the HDAC6-PKM2 axis represents a novel target for future cancer therapies.

Materials and methods

Reagents

Triclabendazole was purchased from Sigma-Aldrich (St. Louis, MO, USA), and dissolved in dimethyl sulfoxide (DMSO) at 100 mM and stored at − 20 ℃. Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute (RPMI) medium 1640, fetal bovine serum (FBS), streptomycin, penicillin and PVDF membranes were obtained from Thermo/Fisher/Invitrogen (Carlsbad, CA, USA). MG132 (S2619) and Trichostatin A (TSA) (S1045) was purchased from Selleck chemicals (Houston, TX, USA). The antibody to HDAC6 (PTM-6669) was purchased from PTM Bio (Hangzhou, China). The antibody against HDAC8 (ab187139) and PGD (ab129199) was the product of Abcam (Cambridge, UK). The antibodies against PKM2 (#4053), LDHA (#3582), HK2 (#2867), PDHA (#3205), PFKP (#8164), α-Tubulin (#3873), AMPK (#2532), Phospho-AMPK (#2535), mTOR (#2972), Phospho-mTOR (#2971), Acetylated-Lysine (#9441) and horse-radish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#7074) were obtained from Cell Signaling Technology (Danvers, MA, USA). The anti-β-actin (66009-1-Ig), anti-CyclinA2 (18202-1-AP), anti-Flag-tag (20543-1-AP) and anti-HA-tag antibody (51064-2-AP) were purchased from Proteintech (Wuhan, China). Antibodies to HDAC7 and HDAC9 were obtained from Santa Cruz (Dallas, TX, USA). The Lactate assay kit was the product of Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The EdU assay kit was purchased from Ribobio (Guangzhou, China). The annexin V-FITC/PI apoptosis assay kit were from Keygen Biotech Co., Ltd. (Jiangsu, China).

Cell culture

The human lung cancer cell lines (A549 and H1299) were obtained from American Type Culture Collection (ATCC). The A549 cells were cultured in complete DMEM medium and H1299 cells were cultured in RPMI medium 1640 (containing 10% FBS) at 37 °C in a humidified incubator with 5% CO₂, and sub-cultured every 2–3 days.

Cell viability and proliferation

Cell viability was detected by CCK-8 assay. Cells were seeded in 96-well plates and culture for overnight. Next day, the cells treated with indicated concentrations of triclabendazole for 24 h, and then co-incubated with CCK-8 solution for 1 h at 37 °C. The optical density (OD) values were measured at 450 nm using a Spectra Max iD5 microplate reader (Molecular Devices, San Jose, USA).

Cell proliferation was detected by cell count, colony formation assay or EdU assay kit. For cell count, the experiments were performed by culturing cells in 6-well plates overnight. The cells were treatment with triclabendazole or triclabendazole plus other inhibitors for 24 h, and then cell numbers were recorded. For colony formation assay, cells (1,000 per well) were seeded in 6-well plates, and they were treated with indicated concentrations of triclabendazole for 7–10 days, subsequently these cells were stained with Crystal violet. For EdU assay kit, cells were cultured in 24-well plates and treated with triclabendazole for 24 h, and then the cells were fixed by 4% paraformaldehyde in PBS, subsequently other steps according to the manufactures’ instructions (RiboBio, Guangzhou, China).

Apoptosis assay by flow cytometry

The cells were treated by triclabendazole for 24 h. Then, they were harvested and washed twice with PBS. Subsequently, the cells were stained with annexin V in binding buffer for 15 min at room temperature followed by staining with PI for 5 min without annexin V, which were then analyzed by flow cytometry. Data were acquired and analyzed by using the FlowJo software.

Lactate assay

The cells were seeded in 12-well plate. After triclabendazole or others treatment, the culture medium was replaced by Minimum Essential Medium (MEM) with phenol red free, and then cell culture supernatant was collected to measure lactate concentration. For lactate in tumor tissues, the tumor tissues were lysis in RIPA buffer followed by measuring lactate concentration. All the experiments were conducted according to the manufactures’ instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Real time PCR analysis

Cells were treated with triclabendazole for the indicated times, and then they were extracted using Trizol (Invitrogen), and reverse transcribed into cDNA using a cDNA synthesis kit (Takara). The cDNA levels were measured by SYBR green real-time in the Light-cycler. The house keeping gene β-actin was used as normalized in each individual sample and 2^−ΔΔCt method was used to quantify relative expression changes. The sequences of specific primers used for qRT-PCR assay were as follow: HK2 (Forward: 5′-GAGCCACCACTCACCCTACT-3′, Reverse: 5′-CCAGGCATTCGGCAATGTG-3′); LDHA (Forward: 5′-ATGGCAACTCTAAAGGATCAGC-3′, Reverse: 5′-CCAACCCCAACAACTGTAATCT-3′).

shRNA-mediated stable cell line generation

The HDAC6 knockdown cells were generated with shRNA which were cloned into the pLKO.1 vector. The shRNA sequences targeting human HDAC were listed in Supplementary Table. Lentivirus was produced by co-transfection of the lentiviral vector with psPAX and pMD2.G into 293 T cells using PEI transfection reagent. Lung cancer cells were seed in 6-well plates following by infecting with Lentivirus supernatant containing 5 μg/mL of polybrene. Cells were selected with 5 μg/mL of puromycin 48 h after infection. Western blot was used to detect the expression of the target protein and the infection efficiency.

Immunoprecipitation (IP)

The cells were rinsed once with PBS and lyzed with 0.5 ml ice-cold IP lysis buffer (containing 1 mM PMSF) for 15 min. Cell lysates were centrifuged at 15,000×g for 10 min at 4 °C and collecting supernatant. Subsequently, the supernatants were incubated with the 20 μl of anti-Flag beads (Sigma-Aldrich, USA) at 4 °C overnight. Next day, the precipitates were washed 3 times with 0.5 ml IP lysis buffer, and then boiled for 5 min in 2 × SDS-PAGE sample loading buffer.

Western blot analysis

Western blot analysis was performed as previously described [8]. The proteins samples were separated by SDS-PAGE, followed by electro-transferring to PVDF membranes (Millipore, Merck, MA, USA). The membranes were blocked in 5% skimmed milk in PBST (phosphate buffer solution contain tween 20) and then incubated with the primary antibody overnight at 4 °C. Subsequently, they were incubated with the secondary antibody for 1 h at room temperature. The blot images were captured by the Tanon 5200 Automatic Chemiluminescence Imaging System (Tanon, China).

Nude mouse xenograft assay

Female nude mice (BALB/c, 4 week of age) were bought from GemPharmatech Co., Ltd (Guangdong, China) and used for all studies. All animals were acclimatized for one week before experiments under 12 h dark/12 h light cycle condition. Animal experiments were performed according to the guidelines for the care and use of animals approved by the Committee on the Ethics of Animal Experiments of Jinan University.

Briefly, lung cancer H1299 cells (2 × 10⁶ cells/mouse) were injected subcutaneously into the right flanks of mice. When the tumor volumes reached approximately 100 mm³, nude mice were randomly divided into two groups (n = 5 per group), and intraperitoneally injected every two days with triclabendazole solution (20 mg/ kg body weight) or vehicle (5% ethanol in PBS). The tumor volume (V) was calculated as follows: V = [length × (width)²]/2. After 15 days, the mice were sacrificed, and the xenograft tumors were collected for assay.

Histological analysis

The liver and kidney tissues were fixed in a 4% neutral formaldehyde solution, embedded in paraffin, and subjected to conventional sectioning for hematoxylin and eosin (H&E) staining.

The immunohistochemistry was used to analyzed tumor tissues with Ki-67 levels and PKM2. VECTASTAIN®Universal Quick Kit, Avidin/Biotin Blocking Kit and DAB peroxidase substrate assay kit was used for immunohistochemical analysis according to the manufacturer’ s instructions. The images were captured by the Leica microscope.

Statistical analysis

Each experiment was performed at least three times independently. Data were presented as mean ± SD. Statistical analysis was performed using GraphPad Prism. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test and unpaired Student’s t-test were used to analyze the statistical significance among multiple groups or between two groups. P < 0.05 was considered statistically significant (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).

Results

Triclabendazole inhibits proliferation of lung cancer cells

To investigate the effect of triclabendazole in lung cancer, we treated two human cell lines A549 and H1299 with various concentrations of triclabendazole for 24 h, 48 h, and 72 h. The Cell Counting Kit-8 (CCK-8) assay showed that triclabendazole significantly reduce cell viability in a dose-dependent manner (Fig. 1A and Fig. S1A-C). However, triclabendazole was demonstrated low toxicity in normal lung epithelial cells BEAS-2B (Fig. S1A-C). We further confirmed these findings by performing cell counting, EdU incorporation, and colony formation assays, which revealed that triclabendazole inhibited the proliferation of A549 and H1299 cells in a dose-dependent manner. (Fig. 1B–E). Next, we used annexin V-FITC/PI staining to determine whether triclabendazole induced apoptosis in these cells. We found no significant differences in apoptosis between the control group and triclabendazole treatment group (Fig. S1D-E). In addition, triclabendazole reduced the expression of Cyclin A2, a protein required for the completion of mitotic prophase, further supporting its inhibitory effect on cell proliferation (Fig. 1F). Consistent with this, triclabendazole treatment disturbed cell cycle distribution, induced cell cycle arrest at the S phase (Fig. S1F-G). Together, these results demonstrate that triclabendazole effectively inhibits lung cancer cell proliferation without inducing apoptosis.

Fig. 1.

Triclabendazole inhibits proliferation of A549 and H1299 cells. The cells (A549 or H1299) were treated with triclabendazole (TRI) for 24 h, and then the cell viability was detected by CCK-8 assay A, cell proliferation was measured by cell counting B or EdU assay C. D Quantitative analysis of EdU positive cells (Red) to all cells (Blue). E Representative results of the colony formation. F Western blotting was used to detect the expression levels of CyclinA2. Data are shown as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, One way ANOVA

Triclabendazole blocks glycolysis by inhibiting PKM2 nuclear localization

We next aimed to investigate the mechanism by which triclabendazole inhibits the proliferation of lung cancer cells. Previous studies have shown that PKM2 plays a key role in tumor development and progression through both metabolic and non-metabolic functions [32]. Based on this, we explored the effect of triclabendazole on PKM2 expression and localization. Our results indicated that triclabendazole inhibited the nuclear translocation of PKM2 in both A549 and H1299 cells (Fig. 2A) without affecting its overall expression (Fig. 2B). This suggested that triclabendazole regulated the non-metabolic functions of PKM2 in lung cancer cells. Since nuclear-localized PKM2 interacts with hypoxia-inducible factor-1 (HIF-1α) transcription factor or β-catenin to activate the transcription of metabolic enzymes [16], we further investigated whether triclabendazole modulates metabolic processes by blocking PKM2 nuclear translocation. Western blot analysis showed that triclabendazole decreased the expression of key glycolytic enzymes, including lactate dehydrogenase A (LDHA) and hexokinase 2 (HK2), but had no effect on phosphofructokinase platelets (PFKP), phosphogluconate dehydrogenase (PGD), or pyruvate dehydrogenase alpha 1 (PDHA1) (Fig. 2B, C). RT-PCR analysis confirmed that triclabendazole significantly reduced the mRNA levels of LDHA and HK2 (Fig. 2D). We further explored whether the downregulation of LDHA and HK2 was mediated through protein degradation by co-incubating cells with MG132, a proteasome inhibitor. The results showed that MG132 didn’t reverse the inhibitory effect of triclabendazole on LDHA and HK2 expression (Fig. 2E), indicating that triclabendazole suppresses their transactivation rather than inducing their degradation. Support these results, shPKM2 could inhibit protein expression of LDHA and HK2 (Fig. S2). Moreover, we assessed glucose metabolic flux by measuring lactate production and found that triclabendazole significantly inhibited lactate levels in lung cancer cells (Fig. 2F). In addition, we further analyzed whether triclabendazole directly binds to PKM2 using a cellular thermal shift assay. The results showed that triclabendazole did not affect the thermal stability of PKM2 (Fig. S1).

Fig. 2.

Triclabendazole reduces levels of HK2 and LDHA to block glycolysis by inhibiting PKM2 nuclear localization. A PKM2 localization was detected by separating of cytosolic and nuclear fractions for western blotting in triclabendazole (TRI) treated cells. B Western blotting was used to detected the expression of metabolic enzymes. C Relative gray values of HK2 and LDHA blots were quantified in A. D The mRNA level of HK2 and LDHA were analyzed by RT-PCR. E The cells were co-incubated with triclabendazole and MG132 for 18 h, and then the proteins levels were detected by western blotting. F Lactate was measured in culture supernatant. Data are shown as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, One way ANOVA

In summary, these findings suggested that triclabendazole inhibited glycolysis by reducing the transactivation of LDHA and HK2 through the blockade of PKM2 nuclear translocation, but it did not directly target PKM2.

Triclabendazole inhibits deacetylation of PKM2 via enhancing the interaction between HDAC6 and PKM2

Since PKM2 translocation from the cytoplasm to the nucleus can be regulated by acetylation, we further investigate whether triclabendazole inhibition of PKM2 nuclear translocation involved this mechanism. Previous studies have shown that deacetylase function in the HDAC family is associated with cancer progression. We analyzed gene amplification frequencies of known HDACs in lung cancer uisng the cBioportal database, finding that HDAC9 showed the highest amplification frequency, followed by HDAC6 (Fig. 3A).

Fig. 3.

Triclabendazole promotes interaction of PKM2 and HDAC6 to inhibit PKM2 acetylation and nuclear localization. A HDACs gene amplification frequency analysis in Lung cancers using cBioportal database. B Co-immunoprecipitation (Co-IP) was used to assess the interaction between PKM2 and HDACs in H1299 and A549 cells with or without TRI treatment. C Western blotting was used to detected the expression of HDAC6 in cell lysis. D PKM2 and HDAC6 interaction was determined by Co-IP in 293 T cells transfected with indicated plasmids. E PKM2 acetylation levels was measured by immunoprecipitation, followed by western blotting. F PKM2 dimerization upon TRI treatment was determined in H1299 and A549 cells transfected with two distinct tagged PKM2 using Co-immunoprecipitation, followed by western blotting

To explore potential relationship between triclabendazole-mediated inhibition of PKM2 nuclear translocation and HDACs. We first performed co-immunoprecipitation to detect the interaction between PKM2 and HDACs upon triclabendazole treatment. The results revealed no interaction between HDAC9 and PKM2, as shown in Fig. 3B. Although HDAC7 and HDAC8 bound to PKM2 in lung cancer cells, triclabendazole did not influence their binding (Fig. 3B). Obviously, HDAC6 also bound to PKM2 (Fig. 3B, D), and triclabendazole significantly promoted this interaction (Fig. 3B). Notably, triclabendazole didn’t affect HDAC6 and HDAC9 expression levels in the cells (Fig. 3C and Fig. S4). These findings suggest that triclabendazole might regulate PKM2 nuclear translocation by enhancing HDAC6 binding to PKM2.

Next, we analyzed PKM2 acetylation levels. Immunoprecipitation and western blot analysis showed that triclabendazole reduced PKM2 acetylation in H1299 and A549 cells (Fig. 3E). It has been established that dimeric PKM2 exists in the nucleus of cancer cells, where it acts as a co-activator for transcription factors, regulating gene expression [15]. Furthermore, previous studies demonstrated that dimeric PKM2 formation is modulated by acetylation [33]. Consistent with this, triclabendazole inhibited the formation of dimeric PKM2 in lung cancer cells (Fig. 3F).

Altogether, these results indicated that triclabendazole blocks PKM2 nuclear translocation by HDAC6-mediated deacetylation of PKM2.

Triclabendazole-mediated deacetylation and nuclear localization of PKM2 reversed by inhibiting HDAC6

To further confirm the HDAC6-mediated deacetylation regulates the nuclear localization of PKM2, we generated HDAC6 stable knockdown cells using shRNA. Immunoprecipitation and western blot analysis revealed that absence of HDAC6 significantly increased PKM2 acetylation in H1299 cells (Fig. 4A). As expected, nuclear translocation of PKM2 was enhanced in shHDAC6 cells compared to control group (Fig. 4B). Additionally, in cells transfected with PKM2-Flag, treatment with the HDAC inhibitor trichostatin A (TSA) also increased the levels of PKM2-Flag in the nucleus (Fig. 4C). Furthermore, when the cells were pretreated with TSA and then co-incubated with triclabendazole, western blotting analysis showed that TSA reversed the inhibitory effect of triclabendazole on PKM2 nuclear translocation (Fig. 4D), indicating that HDAC6-mediated deacetylation plays a crucial role in triclabendazole’s suppression of nuclear PKM2 levels.

Fig. 4.

Triclabendazole inhibits PKM2 nuclear localization dependent on HDAC6-mediated deacetylation. A PKM2 acetylation levels were measured in HDAC6 knockdownH1299 cells expressing PKM2-Flagusing immunoprecipitation, following by western blotting. B Analysis of PKM2 nuclear localization by subcellular cell fraction in HDAC6 knockdown cells. C Analysis of PKM2 nuclear localization by subcellular cell fraction in cells treated with or without TSA (H1299: 2 μM; A549: 0.5 μM) for 24 h, and pan-acetylation of PKM2 was analyzed by immunoprecipitation for western blotting. D Analysis of PKM2 nuclear localization in cells treated with or without triclabendazole in the presence or absence of TSA. E–H Analysis of metabolic enzymes expression level (E, F) and cell proliferation (G, H) in cells treated with TSA or shHDAC6 in the presence of triclabendazole. I, J Analysis of expression levels of CyclinA2 by western blotting. Data are shown as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ns, not significant, One way ANOVA

In addition, shHDAC6 or TSA pretreatment reversed the of triclabendazole’s inhibitory effect on the expression of glycolytic metabolic enzymes, including HK2 and LDHA (Fig. 4E, F). Consistently, the inhibitory effects of triclabendazole on proliferation and reduction of CyclinA2 levels were also reversed by shHDAC6 or TSA treatment (Fig. 4G–I). These findings further demonstrate that triclabendazole promotes deacetylation and blocks PKM2 nuclear localization by enhancing HDAC6 binding to PKM2.

Triclabendazole enhances AMPK/mTOR signaling downstream of HDAC6/PKM2-mediated glycolysis inhibition

Our previous results demonstrated that triclabendazole inhibits glycolysis, and AMPK is a key regulator of energy metabolism that plays a critical role in tumor development and progression. Glucose starvation is known to activate AMPK signaling, and activated AMPK can reduce mTOR activity through phosphorylation, thereby inhibiting cell proliferation. Our data revealed that triclabendazole dose-dependently increased the phosphorylation of AMPK (p-AMPK) without affecting AMPK expression (Fig. 5A, B). Concurrently, triclabendazole treatment reduced the phosphorylation of mTOR (p-mTOR) (Fig. 5A, C).

Fig. 5.

Triclabendazole enhances AMPK/mTOR signaling pathway. A Representative western blots for proteins and phosphorylation levels including AMPK and mTOR. B, C Relative gray values of AMPK and mTOR phosphorylation levels in A were quantified. D The cells pre-treated with AMPK inhibitor compound C (20 μM) for 1 h, and then co-incubated with triclabendazole for 24 h. Western blotting was used to detected phosphorylation levels of AMPK and mTOR. E The cells were treated as D, and the cell proliferation was measured by EdU assay. F Quantitative analysis of EdU positive cells (Red) to all cells (Blue) in E. G Cell count was used to measure proliferation in A549 and H1299 cells. H The cells of shHDAC6 were treated by triclabendazole for 24 h compared to wild type, and analysis of AMPK and mTOR phosphorylation by western blotting. I The cells pre-treated with TSA for 1 h followed by co-incubating with triclabendazole for 24 h, the phosphorylation levels of AMPK and mTOR was measured by western blotting. Data are shown as mean ± SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, One way ANOVA

Furthermore, pretreatment with the AMPK inhibitor compound C, followed by co-incubation with triclabendazole, reversed triclabendazole-induced upregulation of p-AMPK (Fig. 5D). Under these conditions, p-mTOR levels were also restored (Fig. 5D). Additionally, compound C pretreatment counteracted triclabendazole’s inhibitory effect on cell proliferation (Fig. 5E–G). These findings indicate that triclabendazole enhances AMPK/mTOR signaling.

To confirm that the AMPK/mTOR pathway acts downstream of HDAC6/PKM2-mediated glycolysis inhibition, we examined AMPK and mTOR phosphorylation in shHDAC6 cells treated with or without triclabendazole. Western blot analysis showed that triclabendazole-induced upregulation of p-AMPK and downregulation of p-mTOR were reversed by shHDAC6 in both H1299 and A549 cells (Fig. 5H). Similarly, TSA treatment also reversed triclabendazole’s effects on p-AMPK and p-mTOR (Fig. 5I).

In summary, these results suggest that triclabendazole’s enhancement of AMPK/mTOR signaling may occur downstream of HDAC6/PKM2-mediated glycolysis inhibition.

Triclabendazole inhibits glycolysis and tumor growth in vivo by blocking PKM2 nuclear localization

To assess the in vivo effects of triclabendazole on tumor growth, we utilized a xenograft nude mice model. We subcutaneously injected H1299 lung cancer cells into the right flanks of BALB/C nude mice. Once the xenograft tumors reached approximately 100 mm³, we administered triclabendazole solution (20 mg/kg body weight) or solvent (control group) via intraperitoneal injection for two continuous weeks. The results showed a significant reduction in tumor volume in the triclabendazole-treated group compared to the control (Fig. 6A–C). Importantly, triclabendazole treatment did not affect the kidney or liver of the mice, indicating minimal toxicity (Fig. 6D).

Fig. 6.

Anti-tumor effect of triclabendazole on H1299 cells in vivo. A Tumor in BALB/C nude mice. B H1299-derived xenograft tumors were carefully dissected and photographed (n = 5), and showed in image. C Curves of tumor size. D Representative H&E staining images for kidney and liver (× 200). E Representative IHC staining images for Ki-67 expression and PKM2 nuclear localization in resected xenograft tumors (× 200). F Analysis of lactate levels in tumor tissue. G Western blotting was used to detected the expression of metabolic enzymes HK2 and LDHA in tumor tissue. H Analysis of PKM2 nuclear localization by subcellular cell fraction in tumor tissue. Data are shown as mean ± SD, n = 5, **p < 0.01, ***p < 0.001, Two way ANOVA in C and t test in F

We further analyzed tumor tissues through immunohistochemistry and found that Ki-67 expression was lower in the triclabendazole-treated group (Fig. 6E), consistent with reduced proliferation. Moreover, we observed that triclabendazole inhibited PKM2 nuclear translocation in tumor tissues, but didn’t affect the expression of PKM2 (Fig. 6E–H), aligning with in vitro findings. In addition, triclabendazole also inhibited the expression of HK2 and LDHA to reduce lactate levels in the tumors (Fig. 6F, G).

Overall, these results demonstrate that triclabendazole inhibits tumor growth by blocking PKM2 nuclear translocation and suppressing glycolysis in vivo.

Discussion

Chemotherapy remains one of the primary strategies for lung cancer treatment, but traditional chemotherapy drugs often exhibit high cytotoxicity and significant side effects. which limits their clinical application of chemotherapy drugs [34, 35].

Benzimidazoles a class of anti-parasitic drugs, are generally safe for humans, and their anti-cancer potential has been demonstrated [36, 37]. Triclabendazole, a benzimidazole derivative used for treating fluke infections, has been approved by the FDA [38]. However, its mechanism of action in cancer remains largely unknown. In this study, we found that triclabendazole could inhibit nuclear translocation of PKM2, reducing the expression of HK2 and LDHA. This leads to decreased glycolytic flux and lactate levels, ultimately suppressing the proliferation of A549 and H1299 lung cancer cells. Interestingly, triclabendazole reduced PKM2 acetylation by promoting its interaction with HDAC6, thereby blocking PKM2’s nuclear localization. Consistently, shHDAC6 or TSA treatment reversed triclabendazole’s effects on lung cancer cells. Triclabendazole-mediated inhibition of proliferation was also attributed to the activation of AMPK/mTOR signaling. In vivo, triclabendazole treatment reduced PKM2 nuclear translocation, lowered lactate production, and inhibited tumor growth. Collectively, this study suggests that HDAC6-dependent deacetylation of PKM2, leading to the blockade of PKM2 nuclear translocation, contributes to triclabendazole’s ability to suppress glycolysis and proliferation in lung cancer cells.

Lung cancer remains the leading cause of cancer-related deaths worldwide [39], underscoring the urgent need for new therapeutic strategies. Tumor cells often reprogram their metabolism to support their growth and progression, and this metabolic reprogramming has become a hallmark of cancer [40, 41]. Targeting cancer metabolism offers a promising therapeutic approach. Various signaling pathways and transcriptional networks regulate cancer metabolism [42, 43], including fascin, which promotes glycolytic flux by increasing the activity of phosphofructokinases 1 and 2. Pharmacological inhibition of fascin suppresses YAP1-PFKFB3 signaling and glycolysis, inhibiting lung cancer growth and metastasis [44]. ALDH3A1 enhances glycolysis and reduces oxidative phosphorylation, promoting cell proliferation by increasing LDHA expression [45]. Other studies have demonstrated that dihydroartemisinin and artesunate inhibit glycolysis by downregulating GLUT1, LDHA, and HK2 through suppression of c-Myc signaling in non-small cell lung cancer [46]. These findings support the notion that metabolic reprogramming can be targeted as a cancer treatment strategy. Consistent with these studies, we demonstrated that triclabendazole downregulated lactate levels by decreasing HK2 and LDHA expression, leading to suppressed proliferation in A549 and H1299 cells. Triclabendazole also reduced lactate production in vivo, suggesting that glycolytic flux was diminished. Supporting our findings, albendazole has been shown to inhibit glycolysis and lactate production [47], further suggesting that benzimidazole derivatives may modulate glycolysis.

Metabolic enzymes can acquire noncanonical functions based on their subcellular localization, such as in the nucleus, mitochondria, or endoplasmic reticulum. For example, HK2 localizes to mitochondria and binds to voltage-dependent anion channels (VDACs), inhibiting VDAC function and preventing apoptosis in tumor cells [48]. PKM2’s noncanonical localization to the nucleus, mitochondria, and extracellular environment enables it to perform novel biological functions in tumor progression [15, 16]. Nuclear PKM2 acts as a transcription factor, regulating the expression of metabolic enzymes and influencing glycolytic flux [49]. Long non-coding RNA (lncRNA)-AC020978 promotes PKM2 nuclear translocation, increasing the expression of metabolic enzymes such as HK2, GLUT1, and LDHA [49]. Similarly, lncRNA-MNX1-AS1 enhances glycolytic pathway components, including LDHA, GLUT1, and PDK1, by facilitating PKM2’s nuclear translocation [50]. Our findings show that triclabendazole blocks PKM2’s nuclear translocation, reducing mRNA levels of LDHA and HK2, which is consistent with our western blot results. In a xenograft tumor mouse model, triclabendazole also decreased nuclear PKM2 levels, suggesting that it may suppress glycolysis and lactate production by blocking PKM2’s nuclear translocation.

Recent evidence indicates that acetylation regulates PKM2 nuclear translocation. PKM2 acetylation by p300 promotes its nuclear localization [51], while deubiquitinating enzyme JOSD2 blocks PKM2’s nuclear translocation by deacetylating it [52]. Classical HDAC family members play a critical role in regulating the acetylation status of histones and numerous non-histone proteins, including transcription factors [53]. As a key regulator of histone and non-histone acetylation, HDAC6 has been implicated in various diseases, including cancer. For example, HDAC6 binds to and deacetylates cortactin, modulating its function [54]. HDAC6 deficiency leads to increased α-tubulin acetylation, dysregulating microtubule-dependent trafficking of endocytic vesicles and accelerating EGFR degradation [55]. In this study, we found that HDAC6 binds to PKM2, and triclabendazole treatment enhances their interaction. Triclabendazole reduced PKM2 acetylation, blocking its nuclear translocation. Furthermore, HDAC6 knockdown or TSA treatment confirmed that HDAC6 regulates PKM2 acetylation and mediates triclabendazole’s inhibitory effects on PKM2 acetylation. While previous studies have shown that PKM2 interacts with HDAC8 [56], we found that triclabendazole did not affect the PKM2-HDAC8 interaction. Interestingly, we observed that PKM2 might interact with HDAC7, although this interaction was not influenced by triclabendazole. This finding warrants further investigation into PKM2’s role in tumor progression.

AMP-activated protein kinase (AMPK) is a heterotrimeric protein that serves as a key sensor of energy homeostasis [57, 58]. Under conditions of nutrient or energy stress, such as glucose deprivation or glycolysis inhibition, the cellular AMP/ADP ratio increases, activating AMPK through phosphorylation at Thr172. In endothelial cells, GLUT1 inhibition reduces glucose uptake and glycolysis, leading to energy depletion and AMPK activation [59]. β-Boswellic acid (β-BA) activates AMPK by inhibiting glycolysis and reducing ATP production, thereby limiting MCF-10AT cell proliferation [60]. SLC45A4 knockdown also decreases glucose uptake and ATP production, activating the AMPK/ULK1 pathway to inhibit pancreatic cancer cell proliferation [61]. AMPK typically suppresses cell growth pathways and promotes cell cycle arrest by inhibiting mTOR signaling, which is crucial for maintaining metabolic homeostasis and cell proliferation[58, 62–64]. Recent studies have shown that STING promotes cell proliferation and drug resistance by downregulating AMPK-mTOR signaling in colorectal cancer [65]. Fisetin enhances AMPK/mTOR signaling to inhibit PANC-1 cell proliferation [66], while RBM4 depletion causes proliferation arrest by activating AMPK/mTOR signaling in glutamine-dependent esophageal cancer cells [67]. Isorhamnetin induces cell cycle arrest by enhancing AMPK/mTOR/p70S6K signaling in doxorubicin-resistant breast cancer cells [68]. Consistent with these findings, we found that triclabendazole activates AMPK while suppressing mTOR phosphorylation. Triclabendazole also reduces cyclin A2 expression, indicating cell cycle arrest. The inhibitory effects of triclabendazole on AMPK/mTOR signaling were partially reversed by AMPK inhibitor compound C, shHDAC6, and TSA, suggesting that AMPK/mTOR signaling mediates triclabendazole's anti-proliferative effects in lung cancer cells.

Benzimidazole derivatives, including triclabendazole, are considered relatively safe compounds [2, 69]. The doses of triclabendazole used in this study were based on previous research showing that 20 mg/kg or 100 mg/kg administered for two weeks did not cause significant toxicity in mice [8]. Human studies have demonstrated that doses between 5–20 mg/kg are safe. In agreement with these findings, our current study showed that triclabendazole (20 mg/kg) administered for two weeks did not cause significant changes in liver or kidney function, confirming its low toxicity.

In conclusion, our study demonstrates that triclabendazole inhibits glycolysis and cell proliferation by blocking PKM2’s nuclear translocation in lung cancer cells (Fig. 7). Triclabendazole’s inhibition of PKM2 nuclear translocation is mediated by HDAC6, which promotes PKM2 deacetylation. Additionally, triclabendazole suppressed lactate production and tumor growth in a xenograft mouse model, highlighting its potential as a therapeutic agent. These findings also suggest that the HDAC6-PKM2 axis may serve as a promising therapeutic target in lung cancer, warranting further investigation.

Fig. 7.

Schematic the effectiveness of triclabendazole on HDAC6-dependent deacetylation of PKM2 followed by blocking PKM2 nuclear translocation in lung cancer cells. Triclabendazole promoted the bind between PKM2 and HDAC6 contributing to deacetylation of PKM2, leading to block of PKM2 nuclear translocation, and then down-regulating the expression of LDHA and HK2, thereby inhibiting glycolysis and enhancing AMPK/mTOR signaling to suppress proliferation of lung cancer cells

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

LY, YS, SSS and YL performed in vitro experiments; LY, SY and YFZ performed animal experiments; LZQ and JL helped with reagents/materials/analysis tools. LY and JF analyzed the data and visualization; JF, QBZ and LY wrote the paper; JF, QBZ and YD conceived and supervised the research. All authors read and approved the final manuscript.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.