Introduction

Soft tissue sarcoma (STS) is a rare, heterogeneous group of distinct mesenchymal malignancies, accounting for 1–2% of all cancers in adults [1, 2]. Surgical resection is the primary curative treatment for patients with localized disease. However, STS frequently recurs as locally advanced or metastatic disease, which is not amenable to surgical resection, necessitating systemic therapies [3,4,5]. The chemotherapy agents most widely used in sarcomas include anthracyclines (doxorubicin), ifosfamide, gemcitabine, and trabectedin [6,7,8].

Trabectedin was the first marine-derived antineoplastic drug approved in 2007 for the treatment of patients with advanced STS after the failure of doxorubicin [9,10,11]. Trabectedin is a DNA-binding agent with a complex pleiotropic mechanism of action. It selectively targets monocytes and tumor-associated macrophages, downregulating the production of inflammatory mediators in the tumor microenvironment, along with conventional direct cytotoxic effects [12,13,14,15,16]. Specifically, trabectedin interacts with the minor groove of the DNA, interferes with gene transcription and DNA repair machinery, leading to DNA damage accumulation and cell cycle perturbation [12,13,14]. Additionally, trabectedin has a specific mechanism against some translocation-related sarcomas, such as myxoid liposarcoma, where it displaces the oncogenic fusion protein FUS-CHOP from its target promoter [17, 18].

Numerous studies have consistently shown that trabectedin confers long-lasting objective remissions and tumor control in about 20% of various STS types [4, 9, 19,20,21,22]. However, limited information is currently available on the mechanisms leading to secondary resistance. Thus, identifying novel therapeutic strategies to increase trabectedin efficacy is an unmet clinical need.

In this study, starting from an in silico approach, we investigated novel potential therapeutic strategies to enhance the anti-tumor effect of trabectedin using in vitro and in vivo models of STS.

Results

In silico analysis identifies Glyoxalase 1 (Glo1) as “druggable” target

To identify genes modulated by trabectedin exposure that are associated with clinical outcomes in STS patients, we performed a bioinformatic analysis combining two public datasets: GSEA M5190 [23] and TCGA SARC [24]. The GSEA M5190 dataset included cDNA microarray data from nine STS cell lines with varying sensitivity to trabectedin, collected both at baseline and after drug treatment. The TCGA SARC dataset included RNA-Seq data along with survival data from 206 STS patients.

We considered two classes of potential therapeutic targets: genes downregulated after trabectedin exposure (GSEA M5190) and upregulated in patients with good prognosis (TCGA SARC), which could potentially be targeted by agonist compounds (Fig. 1, Gene Set A); and genes overexpressed after trabectedin treatment (GSEA M5190) and upregulated in patients with poor prognosis (TCGA SARC), which could be targeted by specific inhibitors (Fig. 1, Gene Set B). We focused on glyoxalase-1 (GLO1) from Gene Set B, as it was the only gene encoding a “druggable” protein with a commercially available compound suitable for in vivo use.

Fig. 1
figure 1

Volcano Plot representing genes modulated by trabectedin exposure in STS cell lines and, simultaneously, associated with prognosis in STS patients.

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BBGC enhanced trabectedin anti-tumor activity in STS in vitro models

Firstly, we confirmed Glo1 expression in SK-LMS-1 leiomyosarcoma, SW872 liposarcoma, and HT1080 fibrosarcoma cell lines at both gene and protein levels (Supplementary Fig. 1). In contrast to trabectedin, that exhibited dose-dependent cytotoxicity, BBGC treatment at increasing doses (up to the limits of solubility) did not affect cell viability in any of the cell lines, as the half-maximal inhibitory concentration (IC50) was not reached (Fig. 2A–C, left-middle panel). Intriguingly, combination drug analysis revealed a strong synergistic activity between BBGC and trabectedin in all three cell lines (Synergy scores: SK-LMS-1: 38.058; SW872: 35.428; HT1080: 30.395) (Fig. 2A–C, right panel).

Fig. 2: Synergy analysis of BBGC and trabectedin.
figure 2

Cell viability percentage in SK-LMS-1 (A) SW872 (B), and HT1080 (C) cells treated with trabectedin or BBGC (left and middle panels). HSA synergy score of BBGC and trabectedin combined treatment in SK-LMS-1 (A), SW872 (B), and HT1080 (C) cells (right panel). The square in the plot indicates the highest synergy area.

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We then analyzed the potential synergistic effect of BBGC with two other chemotherapy drugs, doxorubicin, and gemcitabine, which are widely used in STS treatment. Similar to trabectedin, both doxorubicin and gemcitabine exerted dose-dependent cytotoxic activity in all three STS cell lines (Supplementary Fig. 3). However, no consistent synergy was observed with BBGC, except for a weak synergy with doxorubicin in HT1080 cells (Synergy score: 14.027) (Fig. 3A–C). These data suggest that BBGC specifically enhances the anti-tumor effect of trabectedin, while it does not enhance the cytotoxicity of doxorubicin and gemcitabine (Fig. 3D).

Fig. 3: Synergy analysis of BBGC and doxorubicin or gemcitabine.
figure 3

HSA synergy score of BBGC and doxorubicin or gemcitabine combined treatment in SK-LMS-1 (A) SW872 (B), and HT1080 (C) cells. The square in the plot indicates the highest synergy area. D Histogram summarizes the HSA synergy scores of SK-LMS-1, SW872, and HT1080 cells treated with BBGC in combination with trabectedin or doxorubicin or gemcitabine. The dashed line indicates the score above which the treatment is synergistic. *p < 0.05; **p < 0.01.

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BBGC restores trabectedin sensitivity in resistant myxoid liposarcoma cells

To investigate the effectiveness of BBGC in trabectedin-resistant cells, we used the myxoid liposarcoma cell lines 402-91 WT (sensitive to trabectedin) and 402-91 ET (resistant to trabectedin). We observed higher Glo1 gene and protein levels in 402-91 ET cells compared to 402-91 WT cells (Supplementary Fig. 3A). Data confirmed that 402-91 ET cells were less sensitive to trabectedin treatment compared to 402-91 WT cells (Fig. 4A). Similar to other STS cell lines, BBGC treatment did not affect the viability of either myxoid liposarcoma cell lines (Fig. 4B).

Fig. 4: Synergy analysis of BBGC and trabectedin in 402-91 ET and 402-91 WT cells.
figure 4

Percentage of cell viability of 402-91 ET and 402-91 WT cells treated with trabectedin (A) or BBGC (B). C HSA synergy score of BBGC and trabectedin combined treatment in 402-91 ET and 402-91 WT. The square in the plot indicates the highest synergy area (left panel). Histogram summarizes the HSA synergy scores of 402-91 ET and 402-91 WT and the dashed line indicates the score above which the treatment is synergistic (right panel). D The histogram shows IC50 value of trabectedin, alone or in combination with BBGC in 402-91 ET. The dashed line indicates IC50 trabectedin value in 402-91 WT. *p < 0.05; **p < 0.01.

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Drug combination analysis revealed a synergistic effect of BBGC and trabectedin in 402-91 ET cells compared to 402-91 WT cells (Synergy score: 402-91 ET: 37.218; 402-91 WT: 13.662) (Fig. 4C). Notably, BBGC treatment completely restored sensitivity to trabectedin in 402-91 ET cells, reducing the IC50 from 29.1 nM to 5.2 nM, similar to the trabectedin IC50 of 402-91 WT cells (IC50: 7.0 nM) (Fig. 4D).

Next, we investigated whether BBGC increased DNA damage of trabectedin. We identified the optimal trabectedin concentration (2 nM) that did not induce significant cytotoxicity, allowing us to better analyze DNA damage. Data showed that BBGC augmented DNA damage induced by trabectedin in both 402-91 WT and ET cells (Fig. 5A), confirming its ability to enhance trabectedin’s anti-tumor effect. Moreover, apoptosis analysis revealed that BBGC, in combination with trabectedin, significantly increased the percentage of annexin V-positive cells in the 402.91 ET line. In contrast, in the 402.91 WT cells, the combination treatment did not further increase the percentage of apoptotic cells, as trabectedin alone already induced apoptosis in nearly 90% of the cells (Fig. 5B).

Fig. 5: DNA-damage and apoptosis analysis in 402-91 ET and 402-91 WT cells.
figure 5

A Percentage of 402.91 WT (left) and 402.91 ET (right) H2A.X phospho positive cells after trabectedin treatment, alone or in combination with BBGC. B Percentage of 402.91 WT (left) and ET (right) annexin V-positive cells after drugs treatment. *p < 0.05; **p < 0.01; ***p < 0.001.

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Additionally, Glo1 silencing did not make 402-91 ET cells sensitive to trabectedin, as the effect is similar to 402-91 ET and 402-91 ET scramble cells (Fig. 6A), suggesting a compensation mechanism for MG detoxification.

Fig. 6: Anti-tumor effect of trabectedin and MG in 402.91 ET cells.
figure 6

A Cell viability percentage in 402.91 ET, ET lipofectamine and ET siGlo1 cells after trabectedin treatment. B Cell viability percentage in 402.91 ET cells treated with MG (left). HSA synergy score of MG and trabectedin combined treatment (right). The square in the plot indicates the highest synergy area.

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To confirm the pivotal role of MG in enhancing trabectedin activity, we treated 402-91 ET cells with MG alone or in combination with trabectedin. Data showed a strong synergistic effect of the two compounds (Synergy score: 25.328), similar to that observed with BBGC plus trabectedin (Fig. 6B).

BBGC enhanced trabectedin anti-tumor activity in mice

To test the potential synergy between BBGC and trabectedin, we used an orthotopic fibrosarcoma mouse model. One week after HT1080 cell implantation, tumor-bearing mice were randomized into four groups: control, BBGC (200 mg/kg), trabectedin (0.100 mg/kg), and BBGC (200 mg/kg) plus trabectedin (0.100 mg/kg). Mice treated with trabectedin showed a significant reduction in tumor volume, while the BBGC-treated group exhibited tumor growth similar to the control group (Fig. 7A). The combination of BBGC and trabectedin significantly enhanced the anti-tumor effect compared to trabectedin alone (p < 0.01, from day 2 to 7; p < 0.01, from day 11 to 16) (Fig. 7A). The combined treatment did not alter the body weight of the mice, suggesting that BBGC does not induce any additional toxic effects (Fig. 7B).

Fig. 7: Anti-tumor effect of BBGC and trabectedin alone or in combination in a mouse model.
figure 7

A Representative images of tumor growth in control and mice treated with BBGC plus Trabctedin. B Measurements of tumor volume (A) and the body weight (B) of mice after treatment with trabectedin or BBGC alone or in combination at different timepoints. **p < 0.01.

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Glo1 expression is associated with trabectedin response in patients affected by myxoid liposarcoma

We analyzed Glo1 expression in nine matched myxoid liposarcoma tumor tissue samples obtained from patients before and after trabectedin treatment. Then, patients were categorized into “high” and “low” Glo1 expression groups based on Glo1 staining in tissue samples collected before trabectedin treatment (score 3: “high”; score 1 or 2: “low”) (Fig. 8A). Immunohistochemical analysis showed greater tumor regression, in terms of radiological response and fibrosis percentage, in patients with low Glo1 expression (p < 0.01) (Fig. 8B), suggesting that Glo1 could be a predictive biomarker of trabectedin response.

Fig. 8: Glo1 expression analysis in human tissue sample.
figure 8

A Representative image of Glo1 staining in myxoid liposarcoma tissue pre trabectedin treatment (left panels) and hematoxylin/eosin (EE) staining in post-trabectedin treatment section (right panels). B Fibrosis/Hyalinosis percentage in tissue samples with low and high Glo1 expression. **p < 0.01.

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Discussion

While trabectedin is approved as a single agent for the treatment of all STS histotypes, it exhibits strong antitumor activity primarily in certain types, such as liposarcoma and leiomyosarcoma [25]. Preclinical and clinical evidence suggests a synergistic effect when trabectedin is combined with other agents, although this is associated with increased toxicity [25,26,27,28,29,30].

The aim of this study was to identify therapeutic strategies to enhance the antitumor effect of trabectedin using integrated in silico, in vitro, and in vivo approaches. Bioinformatics analysis identified Glo1 as a “druggable” target upregulated after trabectedin treatment and associated with poor prognosis in STS patients. Our retrospective analysis in a cohort of patients with myxoid liposarcoma showed that high Glo1 levels at baseline were associated with a lower response to trabectedin. Although based on a small number of cases, these results suggest a potential role of Glo1 in predicting trabectedin response in myxoid liposarcoma patients.

Glo1 is a key enzyme in the glyoxalase system involved in the detoxification of MG preventing its abnormal accumulation, which causes DNA strand breaks, mutagenesis, and cytotoxicity [31, 32]. Increased expression and activity of Glo1 have been found in various cancer types and are closely correlated with tumor aggressiveness and metastasis [33]. Cell-permeable Glo1 inhibitor prodrugs have been developed to induce severe dicarbonyl stress as a potential treatment for cancer, particularly for high Glo1-expressing, multidrug-resistant tumors. The prototype Glo1 inhibitor is BBGC, which has shown antitumor activity particularly in lung cancer and glioblastoma in vitro and in tumor-bearing mice in vivo models [32,33,34,35,36,37].

In our STS cell lines (leiomyosarcoma, fibrosarcoma, myxoid, and dedifferentiated liposarcoma cells), BBGC treatment did not affect cell proliferation when administered alone, but it was effective in increasing trabectedin activity. Conversely, BBGC did not exert any synergistic effect in combination with other chemotherapeutic agents, such as doxorubicin and gemcitabine, in the same cell lines. These data highlighted that the BBGC synergy was restricted to the combination with trabectedin, clearly suggesting a drug-specific enhancing effect in the four histotypes analyzed. Our in vitro results were confirmed in an orthotopic murine fibrosarcoma model, where the combination of BBGC and trabectedin significantly reduced tumor growth compared to trabectedin alone. Moreover, the combination of BBGC with trabectedin did not induce additional adverse effects, as assessed by monitoring body weight evolution, suggesting that the combined treatment could be effective without increasing toxicity.

This strong synergy could be related to the unique mechanism of action of trabectedin [38,39,40]. Specifically, trabectedin has higher cytotoxicity on cells in the G1 phase than other chemotherapy agents that act on proliferative cells [41,42,43,44,45,46,47]. Since MG accumulation causes cell cycle arrest in the G1 phase, we hypothesize that an increase in MG levels after BBGC treatment could induce a cellular quiescent state, making tumor cells more sensitive to trabectedin. In support of our hypothesis, we observed a significant arrest of the cell cycle at the G0-G1 phase following BBGC treatment (Supplementary Fig. 4).

Data available in the literature show that increased expression of Glo1 has a protective effect on tumor cells, contributing to their survival and multidrug resistance, and eventually leading to chemotherapy failure [31, 35]. Therefore, we investigated whether BBGC could enhance the antitumor effect of trabectedin in a well-characterized model of trabectedin resistance. Data showed that resistant cells expressed high levels of Glo1, and BBGC treatment was able to overcome the resistance in these cells. In contrast to BBGC treatment, Glo1 silencing in resistant cells did not resensitize them to trabectedin. The explanation for this differential effect may lie in the activation of compensatory detoxification mechanisms for MG in these cells. Indeed, differently from pharmacological inhibition by BBGC, Glo1 silenced models in several studies did not lead to elevated MG levels, indicating alternative detoxification routes during Glo1 loss [48]. Besides the glyoxalase system, other enzymes are also known to detoxify MG, such as aldo-keto reductases (AKRs), aldehyde dehydrogenases (ALDHs), and the Parkinson-associated protein DJ-1 [49, 50]. Moreover, it was recently discovered that the ketone body acetoacetate can scavenge endogenous MG in a nonenzymatic aldol reaction [51].

Since the intracellular measurement of free MG is known to be scarcely reliable with the currently available methods [52,53,54,55], we employed the strategy of administering MG at known concentrations (within a physiological range) in combination with trabectedin to verify their synergy. Data showed a synergy between MG and trabectedin in resistant cells similar to that observed with BBGC, suggesting that MG detoxification may play a key role in the establishment of resistance to trabectedin.

Some potential limitations should be considered. The study relies heavily on bioinformatic analysis using public datasets that may not fully capture the complexity and variability of real-world patient populations. The use of a single in vitro model of trabectedin resistance, albeit the most characterized, and the lack of in vivo validation represent a limitation of the study. Unfortunately, our trabectedin resistance model had several features that precluded its use in vivo evaluation: the engraftment rate was very low (about 10%), the tumor growth was very heterogeneous, and produced hind back paralysis in some animals. Despite HT1080 cells can not be considered a validated model for trabectedin resistance, they exhibit an IC50 of 30.2 nM, comparable to the 29.1 nM observed in 402.91 ET cells.

Finally, the mechanism by which BBGC potentiates the effect of trabectedin has not been fully elucidated, therefore, further analyses are necessary to deepen our understanding of this specific synergy.

Despite these limitations, the study identifies BBGC as a promising therapeutic approach for enhancing the antitumor effect of trabectedin in STS and overcoming trabectedin resistance.

Methods

Cell lines and drugs

SK-LMS-1 leiomyosarcoma, SW872 liposarcoma, and HT1080 fibrosarcoma cell lines were gifted by PharmaMar. The 402.91 WT and 402.91 ET cells were gifted by the D’Incalci research laboratory [56]. SK-LMS-1, SW872, and HT1080 cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning, New York, NY, USA), while the base medium for 402.91 WT and 402.91 ET was RPMI1640 (Corning, New York, NY, USA). All media were supplemented with 10% bovine serum (Sigma Aldrich, St. Louis, Missouri, USA), 2 mM L-glutamine (EuroClone, Milano, Italy), and 1% penicillin/streptomycin (EuroClone, Milano, Italy).

Drugs were used at the following concentrations: trabectedin (PharmaMar, Madrid, Spain) 6 nM, 12 nM, 25 nM, 50 nM; doxorubicin (Selleck Chemicals GmbH, Planegg, Germany) 0.5 μM, 1 μM, 2 μM, 4 μM; gemcitabine (Selleck Chemicals GmbH, Planegg, Germany) 0.1 μM, 0.2 μM, 0.5 μM, 1 μM; S-p-bromobenzyl-glutathione cyclopentyl diester (BBGC) (MedChemExpress, Monmouth Junction, NJ, USA) 2.5 μM, 5 μM, 10 μM, 20 μM; methylglyoxal (MG) (Sigma Aldrich, St. Louis, MO, USA) 0.25 μM, 0.5 μM, 1 μM, 2 μM.

Cell viability

STS cells were treated for 1 h with BBGC or chemotherapy agents (trabectedin, doxorubicin, gemcitabine), alone or in combination with BBGC, and then the drugs were washed out. Moreover, 402.91 ET cells were treated with MG plus trabectedin with the same treatment schedule. After 4 days, cell viability was analyzed using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. The synergistic effect of trabectedin/doxorubicin/gemcitabine and BBGC was calculated using SynergyFinder 2.0 software. The combination treatment was considered synergistic with a “synergy score” >10.

Flow cytometric γ-H2AX detection

STS cells were treated for 1 h with BBGC or chemotherapy agents (trabectedin, doxorubicin, gemcitabine), alone or in combination with BBGC, and then the drugs were washed out. After 24 h, DNA damage was analyzed by detecting phospho-histone H2AX (γ-H2AX) expression by flow cytometry. Briefly, cells were fixed and permeabilized with Foxp3/Transcription Factor Staining Buffer Set (eBioscience-Thermo Fisher Scientific, Waltham, MA, USA) for intracellular staining with anti-phospho-histone H2AX (γ-H2AX)-APC (clone 2F3) (Biolegend, San Diego, CA, USA). Moreover, for 402.91 ET cells, we evaluated apoptosis after 1 h of treatment with BBGC and trabectedin, alone or in combination, using the anti-human annexin V antibody (Becton Dickinson, New York, USA).

Samples were analyzed using a CytoFlex instrument (Beckman Coulter, Brea, CA, USA) and with FlowJo Software, v.10.8 (FlowJo, Ashland, OR, USA).

Gene silencing

Glo1 silencing was performed using the following 3 selected validated siRNA (Ambion, Life Technologies):

1) Sequence 5′- 3′: GAGUCAAAUUUGUGAAGAAtt

2) Sequence 5′- 3′: GGCUUAUGAGGAUAAAAAUtt

3) Sequence 5′- 3′: ACUGGAUUUUUAUACUAGAtt

Briefly, 402.91 ET cells were seeded to be 60–80% confluent at the time of transfection. The siRNA-lipidic complexes were prepared and added to the cells according to manufacturer’s instructions. 402.91 ET cells were incubated, for 48 h, with Glo1 siRNAs (siGlo1 402.91 ET cells) and then analyzed for Glo1 gene and protein expression by Real Time PCR and Western Blot, respectively (Supplementary Fig. 3B). siGlo1 ET cells were treated with trabectedin to analyze cell viability.

Immunohistochemical analysis

Representative tumor blocks (4 mm thickness) were obtained from nine patients with advanced localized myxoid liposarcoma enrolled in a phase II clinical trial of neoadjuvant trabectedin [57]. Immunohistochemistry (IHC) was performed by the streptavidin-biotin method using the recombinant rabbit monoclonal antibody against GLO1 protein (JU44-1, Invitrogen). The H-Score system was applied for GLO1 IHC evaluation [58], calculated by a semi-quantitative assessment of the intensity of staining (graded as: 0, non-staining; 1, weak; 2, medium; or 3, strong) and the percentage of positive cells. Immunostaining was assessed by two independent pathologists blinded to clinical characteristics and outcomes.

In vivo experiments

Female athymic nu/nu mice between 4 and 6 weeks of age were purchased from Envigo. Animals were housed in individually ventilated cages (Sealsafe® Plus, Techniplast) on a 12-h light-dark cycle at 21–23 °C and 40–60% humidity. Mice were allowed free access to irradiated standard rodent diet (Tecklad 2914C) and sterilized water. Animals were acclimated for 5 days prior to tumor implantation. To generate the fibrosarcoma model, animals were orthotopically implanted into the gastrocnemius muscle by intramuscular injection using a 26G needle and a 1 cc syringe with 1 × 107 HT1080 cells suspended in medium without serum or antibiotics. Tumor-bearing animals were randomly allocated into experimental groups, and treatment administration was initiated on Day 0. BBGC (200 mg/kg) treatment was intraperitoneally administered on days 0 and 8, and trabectedin (0.100 mg/kg) was intravenously injected on days 0 and 7. Tumor volume and body weight were measured twice a week from day 0 to day 18. Treatment tolerability was assessed by monitoring body weight evolution, clinical signs, and evidence of local damage at the injection site. Control and BBGC-treated mice were humanely sacrificed on day 14, while mice treated with trabectedin, alone or in combination with BBGC, were sacrificed between days 16 and 18. All experimental procedures were reviewed and approved by regional Institutional Animal Care and Use Committees (IACUC) and performed in accordance with national and international laws and policies (EU Directive 2010/63/EU).

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8.2.1 (San Diego, CA, USA). All in vitro experimental procedures consisted of at least three independent biological repeats. Comparisons were performed using a two-sided unpaired Student’s t-test or ANOVA test followed by Tukey’s test.