Abstract
Background
Leonurine (Leo), a bioactive alkaloid derived from Leonurus japonicus, has demonstrated anti-tumor potential in various malignancies; however, its specific role and molecular targets in renal cell carcinoma (RCC) remain to be elucidated. In this study, we aim to offer a novel perspective on Leo’s role in the treatment of RCC, and to provide guidance for the development of new anti-RCC drugs.
Methods
The anti-tumor efficacy of Leo was evaluated in RCC cell lines through Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, and Transwell assays. In silico prediction via the ChEMBL database was utilized to identify potential molecular targets, which were further validated using microscale thermophoresis (MST), molecular docking, and cell thermal shift assays (CETSA). Glycolytic flux was assessed by measuring the extracellular acidification rate (ECAR), glucose uptake, lactate secretion, and intracellular adenosine triphosphate (ATP) levels. Rescue experiments were employed to confirm the involvement of the pyruvate dehydrogenase kinase 1 (PDK1)-mediated metabolic axis. Subcutaneous xenograft mouse model was used to evaluate the role of Leo in RCC progression.
Results
Leonurine significantly inhibited the proliferation and migration of RCC cells while inducing apoptosis in a dose-dependent manner. Mechanistically, Leo was found to interact directly with PDK1 with high affinity, leading to the downregulation of PDK1 protein levels. Functional studies revealed that Leo effectively suppressed the Warburg effect in RCC cells. Notably, the ectopic overexpression of PDK1 significantly abrogated the inhibitory effects of Leo on cell growth, metastasis, and glycolytic metabolism. Consistently, in vivo experiments demonstrated that Leo administration markedly restricted tumor growth in xenograft mice.
Conclusions
Leo acts as a novel metabolic inhibitor in RCC by directly targeting the PDK1-mediated glycolytic pathway.
Keywords: Leonurine (Leo), renal cell carcinoma (RCC), pyruvate dehydrogenase kinase 1 (PDK1), glycolysis
Introduction
Renal cell carcinoma (RCC) remains one of the most lethal urological malignancies, characterized by a rising global incidence and a high propensity for systemic metastasis (1,2). Among various histological subtypes, clear cell RCC (ccRCC) accounts for the majority of cases and is frequently driven by profound genetic and metabolic alterations (3). Although the advent of targeted therapies, such as tyrosine kinase inhibitors (TKIs), and immune checkpoint blockades has significantly improved the clinical outlook for patients with advanced RCC, therapeutic resistance remains an almost inevitable challenge (4,5). The clinical management of RCC is further complicated by its inherent insensitivity to conventional radiotherapy and chemotherapy (6). Consequently, there is a pressing need to identify novel therapeutic agents that can effectively disrupt the oncogenic drivers of RCC progression.
Leonurine (Leo), a bioactive alkaloid primarily isolated from the traditional medicinal herb Leonurus japonicus Houtt., has been extensively recognized for its diverse pharmacological benefits, particularly in cardiovascular protection and anti-inflammatory responses (7,8). Recent pharmacological explorations have begun to unveil the potent anti-tumor properties of Leo across several cancer types (9,10). Studies have demonstrated that Leo can suppress proliferation and trigger apoptosis in prostate cancer and cervical cancer by modulating various signaling cascades (11,12). However, whether Leo exerts a tumor-suppressive effect on RCC, and the specific molecular mechanisms involved, remain to be fully elucidated.
A defining hallmark of RCC is its profound metabolic reprogramming, most notably the “Warburg effect” (13). Unlike many other solid tumors, this metabolic shift in RCC is predominantly driven by its unique genetic landscape, particularly the frequent inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene (14). VHL loss leads to the constitutive stabilization of hypoxia-inducible factors (HIFs), which in turn act as master transcription factors that hyperactivate the glycolytic cascade (15). Pyruvate dehydrogenase kinase 1 (PDK1) serves as a pivotal gatekeeper in this metabolic rewiring (16). By phosphorylating and inactivating the pyruvate dehydrogenase complex (PDC), PDK1 prevents pyruvate from entering the mitochondrial tricarboxylic acid (TCA) cycle, thereby facilitating lactate production and glycolytic flux (17,18). Given that PDK1 is a direct downstream target of the hyperactive HIF pathway and is critically upregulated in RCC, targeting this specific PDK1-mediated metabolic axis represents a highly innovative and context-dependent therapeutic strategy to exploit the intrinsic vulnerabilities of renal cancer.
In the present study, we provide the first evidence that Leo functions as a potent inhibitor of RCC progression both in vitro and in vivo. Through a combination of in silico prediction and biochemical validation, we identified PDK1 as a direct molecular target of Leo. Our findings demonstrate that Leo binds to PDK1, leading to its downregulation and the subsequent inhibition of the glycolytic program in RCC cells. This study not only uncovers a novel mechanism by which Leo dictates RCC cell fate but also highlights the Leo/PDK1 axis as a promising metabolic target for the treatment of renal cancer. We present this article in accordance with the MDAR and ARRIVE reporting checklists (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0088/rc).
Methods
Cells and culture conditions
Human RCC cell lines, 786-O and 769-P, were procured from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Cells were maintained in RPMI-1640 (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin. All cultures were incubated at 37 ℃ in a humidified atmosphere containing 5% CO2.
Cell transfection
PDK1 overexpression plasmids were constructed and produced by GeneChem (China). For Transfection, plasmids or empty vectors were introduced into cells using Lipofectamine 3000 (Invitrogen, CA, USA) according to the manufacturer’s instructions. Efficiency was verified by Western blot analysis 48 hours post-transfection.
Western blot
Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentrations were quantified using a bicinchoninic acid (BCA) assay kit (Beyotime, China). The same amount of protein was added and separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), following by transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After blocking with 5% non-fat milk, membranes were incubated overnight at 4 ℃ with primary antibodies against PDK1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Horseradish peroxidase (HRP)-conjugated secondary antibodies were applied, and protein bands were visualized using an enhanced chemiluminescence system. The detailed primary antibodies information was listed below: PDK1 (Abcam, Cambridge, UK, cat: #1901-1, 1:1,000, RRID: AB_765053); GAPDH (Cell Signaling Technology, MA, USA, cat: #2118, 1:5,000, RRID: AB_561053).
Cell Counting Kit-8 (CCK-8) assays and colony formation assays
For CCK-8 assay, cells were inoculated into 96-well plates (2,000 cells/well) and treated with indicated concentrations of Leo. Absorbance at 450 nm was measured at specific time points.
For colony formation, cells (1,000 cells/well) were seeded in 6-well plates and cultured for 10–14 days. Colonies were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and manually counted.
Apoptosis analysis
Cell apoptosis was evaluated using an Annexin V-FITC/PI Apoptosis Detection Kit (Multi Sciences, Hangzhou, China). Following Leo treatment for 48 hours, cells were harvested, washed with cold phosphate-buffered saline (PBS), and resuspended in binding buffer. Samples were incubated with Annexin V-FITC and PI for 15 minutes in the dark. Data were acquired using a flow cytometer and analyzed with FlowJo software.
Transwell assay
For Transwell migration assays, 2×104 RCC cells in serum-free medium were placed in the upper chamber of a Transwell insert (Corning, NY, USA). The lower chamber was filled with medium containing 10% FBS. After 24 hours, migrated cells were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet, and imaged.
Wound healing assay
For the wound healing assay, a sterile 200 µL pipette tip was used to create a linear scratch on a confluent cell monolayer. The width of the wound was recorded at 0 and 24 hours using an inverted microscope to calculate the migration rate.
Molecular docking and in silico prediction
The 3D chemical structure of Leo was retrieved from the PubChem database and subjected to geometry optimization. The high-resolution X-ray crystal structure of human PDK1 (PDB ID: 2Q8F) was downloaded from the RCSB Protein Data Bank. Protein preparation, addition of polar hydrogens, and assignment of Kollman charges, was executed using AutoDock Tools. Docking calculations were performed using AutoDock Vina to explore the optimal binding poses. A grid box was established to encompass the active site of the PDK1 kinase domain. The resulting lowest-energy protein-ligand conformations were visualized and analyzed using PyMOL to accurately map the hydrogen bond networks and hydrophobic interactions.
Microscale thermophoresis (MST) assay
The direct interaction between Leo and the PDK1 protein was quantified via MST. A constant concentration of the GFP-labelled PDK1 (20 nM) was incubated with 16 two-fold serial dilutions of Leo in PBS buffer for 30 minutes at room temperature in the dark. The equilibrated samples were loaded into Monolith NT.115 Premium Capillaries. The dissociation constant (Kd) was extrapolated by fitting the normalized fluorescence alterations to a mass action equation utilizing MO.Affinity Analysis software.
Cell thermal shift assay (CETSA)
RCC cells were treated with Leo (50 µM) or an equivalent volume of dimethyl sulfoxide (DMSO) vehicle for 4 hours. Following treatment, cells were harvested, washed, and resuspended in lysis buffer supplemented with a protease inhibitor cocktail. The cell suspensions were divided into equal aliquots and subjected to a rigorous temperature gradient (40, 44, 48, and 52 ℃) for exactly 3 minutes using a thermal cycler, followed by a 3-minute cooling period at room temperature. The soluble protein fractions were isolated from the precipitated, heat-denatured proteins at 20,000 g for 20 minutes at 4 ℃. The remaining soluble PDK1 protein in the supernatant was subsequently quantified using Western blotting.
Extracellular acidification rate (ECAR) assay
The ECAR was measured using a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies, CA, USA). Cells were seeded in XFe96 cell culture plates and equilibrated in base medium. Following sequential injections of glucose, oligomycin, and 2-deoxyglucose, ECAR values were recorded. Results were normalized to the total protein content of each well.
Oxygen consumption rate (OCR) assay
The OCR was assessed utilizing a Seahorse XFe96 Extracellular Flux Analyzer (Agilent Technologies, USA). Briefly, RCC cells were seeded into XFe96 microplates and equilibrated in base medium. Following sequential injections of oligomycin, FCCP, and antimycin A, ECAR values were recorded. Results were normalized to the total protein content of each well.
Measurement of glucose, lactate, and adenosine triphosphate (ATP)
Intracellular ATP levels and the concentrations of glucose and lactate in the culture supernatant were determined using specific assay kits (Beyotime, China) following the manufacturers’ protocols. All metabolic parameters were normalized to the cell count or total protein concentration to ensure inter-group comparability.
Xenograft animal model
Experiments were performed under a project license (No. 2023-Y-0739) granted by ethics committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, in compliance with institutional guidelines for the care and use of animals. Four-week-old nude mice (sex: female; species: BALB/C) were purchased from Sir Run Run Shaw Hospital Laboratory Animal Center and kept at 20–25 ℃ and 50–70% relative humidity. The mice were subcutaneously injected with 5×106 786-O cells into right flank. After 7 days, totally fourteen mice were randomly assigned to two groups: control group and Leo (100 mg/kg via oral gavage, twice weekly) group (each 7 mice). Total tumor volume was monitored every 7 days and calculated using the formula: V = 0.5 × length × width2. All methods were performed in accordance with relevant guidelines and regulations. A protocol was prepared before the study without registration.
Statistical analysis
Data are presented as the mean ± standard deviation (SD) from at least three independent experiments. Statistical differences between groups were evaluated using Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test using GraphPad Prism 9.0 software. A value of P<0.05 was considered statistically significant.
Results
Leo inhibits the proliferation of RCC cells
Given that Leo has been reported to suppress the progression of various malignancies, including prostate cancer and liver cancer (12,19), we sought to investigate its potential role in RCC (Figure 1A). RCC cells were treated with varying concentrations of Leo. The CCK-8 assay revealed that Leo significantly impaired cell viability in a dose-dependent manner (Figure 1B). Consistently, the colony formation assay demonstrated that Leo treatment markedly reduced the proliferative capacity of RCC cells (Figure 1C). Furthermore, flow cytometric analysis indicated a significant increase in the apoptotic rate of RCC cells following Leo treatment (Figure 1D). Collectively, these findings suggest that Leo exerts a potent anti-proliferative effect on RCC cells.
Figure 1.
Leonurine inhibits the proliferation of renal cell carcinoma cells. (A) The molecular structure of leonurine. (B) The cell viability of RCC cells was detected by CCK-8 assay at indicated condition. (C) The cell viability of RCC cells was detected by colony formation assay at indicated condition. (D) The apoptotic ratio of RCC cells was detected by apoptosis assays at indicated condition. The value was presented as mean ± SD. ***, P<0.001. CCK-8, Cell Counting Kit-8; NC, negative control; RCC, renal cell carcinoma; SD, standard deviation.
Leo suppresses the metastatic potential of RCC cells
We further explored the impact of Leo on the metastatic behavior of RCC cells. Transwell migration assays showed that Leo treatment significantly inhibited the invasive capacity of RCC cells in a dose-dependent fashion (Figure 2A). This observation was corroborated by wound healing assays, which indicated that Leo markedly delayed the rate of wound closure, reflecting an inhibition of cell motility (Figure 2B,2C). Taken together, these results demonstrate that Leo significantly attenuates the metastatic potential of RCC cells.
Figure 2.
Leonurine suppresses the metastatic potential of RCC cells. (A) The metastatic ability of RCC cells was detected by Transwell assays at indicated condition. Cells were stained with crystal violet and imaged under a light microscope at ×100 magnification. (B) The metastatic ability of 786-O cells was detected by wound healing assays at indicated condition. Cells were stained with crystal violet and imaged under a light microscope at ×100 magnification. (C) The metastatic ability of 769-P cells was detected by wound healing assays at indicated condition. Cells were stained with crystal violet and imaged under a light microscope at ×100 magnification. The value was presented as mean ± SD. ***, P<0.001. NC, negative control; RCC, renal cell carcinoma; SD, standard deviation.
Leo directly binds to and inhibits PDK1 expression
Recent studies have highlighted that small-molecule natural products can directly interact with specific proteins to modulate their biological functions. To identify the potential molecular targets of Leo, we utilized the ChEMBL database (https://www.ebi.ac.uk/chembl/) for target prediction (Figure 3A). Among the candidate proteins, PDK1 was prioritized for further investigation due to its high prediction score and relevance in cancer metabolism. MST analysis confirmed a direct interaction between Leo and PDK1, with a dissociation constant (Kd) of 25.4 µM, indicating a robust binding affinity (Figure 3B). This interaction was further supported by molecular docking data (Figure 3C). Subsequently, we assessed the biochemical consequences of this binding. Western blot analysis revealed that Leo treatment significantly downregulated PDK1 protein levels (Figure 3D). Additionally, the CETSA demonstrated that Leo treatment significantly altered the thermal stability of the PDK1 protein, providing further evidence of their direct physical interaction (Figure 3E). These data reveal that Leo directly binds to and inhibits PDK1 expression.
Figure 3.
Leonurine directly binds to and inhibits PDK1 expression. (A) The potential leonurine-interacted proteins predicted by ChEMBL database. (B) The binding affinity between PDK1 and leonurine was detected by MST assays. (C) The interaction between PDK1 and leonurine was analyzed by molecular docking. (D) The PDK1 expression in RCC cells was detected by western blotting assays after leonurine treatment. (E) Cell thermal shift assay showing the PDK1 expression after leonurine treatment. The value was presented as mean ± SD. DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Leo, leonurine; MST, microscale thermophoresis; NC, negative control; RCC, renal cell carcinoma; SD, standard deviation.
Leo inhibits RCC progression via the downregulation of PDK1
To establish the clinical significance of PDK1 in RCC, we first evaluated its expression profiles utilizing large-scale public cohorts. Analysis of The Cancer Genome Atlas (TCGA)-KIRC dataset revealed a significant upregulation of PDK1 mRNA levels in RCC tissues compared to adjacent normal tissues (Figure 4A). Consistently, interrogation of the CPTAC database confirmed that this upregulation translates to the macroscopic protein level, demonstrating an aberrant accumulation of PDK1 protein in primary RCC specimens (Figure 4B). Crucially, Kaplan-Meier survival analysis based on CPTAC database data demonstrated that RCC patients harboring high PDK1 protein levels experienced significantly poorer overall survival (OS), clearly defining PDK1 as an adverse prognostic indicator (Figure 4C). Prompted by these compelling clinical findings, we proceeded to elucidate whether Leo exerts its anti-tumor effects specifically by downregulating PDK1. First, CCK-8 assays showed that PDK1 overexpression significantly reversed the growth inhibition induced by Leo (Figure 4D). Similarly, colony formation assays revealed that restoring PDK1 expression rescued the long-term proliferative capacity of Leo-treated cells (Figure 4E). Furthermore, Transwell assays confirmed that PDK1 overexpression effectively neutralized the inhibitory effect of Leo on cell migration (Figure 4F). These data suggest that Leo inhibits RCC progression by targeting PDK1.
Figure 4.
Leonurine inhibits RCC progression via the downregulation of PDK1. (A) PDK1 mRNA expression was analyzed using TCGA-KIRC database. (B) PDK1 protein expression was analyzed using CPTAC database. (C) Kaplan-Meier survival analysis based on CPTAC database demonstrated the overall survival of RCC patients based on PDK1 expression. (D) The cell viability of RCC cells was detected by CCK-8 assays at indicated condition. (E) The cell viability of RCC cells was detected by colony formation assays at indicated condition. (F) The metastatic ability of RCC cells was detected by Transwell assays at indicated condition. Cells were stained with crystal violet and imaged under a light microscope at ×100 magnification. The value was presented as mean ± SD. *, P<0.05; ***, P<0.001; ****, P<0.0001. CCK-8, Cell Counting Kit-8; Leo, leonurine; NC, negative control; RCC, renal cell carcinoma; SD, standard deviation; TCGA, The Cancer Genome Atlas; TPM, transcripts per million.
Leo inhibits glycolysis in RCC through PDK1 modulation
In RCC, the constitutive activation of the HIF pathway establishes a rigid dependency on aerobic glycolysis while actively suppressing mitochondrial oxidative phosphorylation (OXPHOS) (20). PDK1 is the critical metabolic switch enforcing this specific Warburg phenotype by inhibiting the PDC (17,21). Consequently, overcoming this OXPHOS suppression by targeting PDK1 represents a highly specific vulnerability in RCC metabolism. We therefore investigated whether Leo disrupts this essential metabolic dependency. ECAR analysis showed that Leo significantly reduced the glycolytic rate, an effect that was largely reversed by PDK1 overexpression (Figure 5A). Consistently, the Leo-induced reduction in glucose uptake and lactate secretion was significantly restored upon PDK1 overexpression (Figure 5B,5C). Furthermore, Leo decreased intracellular ATP levels, which was also rescued by restoring PDK1 expression (Figure 5D). Because the complete Warburg phenotype is defined by the coexistence of enhanced glycolysis and impaired OXPHOS, we further performed mitochondrial stress testing to comprehensively evaluate the OCR. As hypothesized, Leo treatment triggered a marked increase in maximal respiratory capacity, while PDK1 overexpression reversed this phenomenon (Figure 5E,5F). Totally, these findings indicate that Leo inhibits glycolytic flux in RCC cells by targeting PDK1.
Figure 5.
Leonurine inhibits glycolysis in RCC through PDK1 modulation. (A) The glycolytic rate of RCC cells was detected by ECAR assays at indicated condition. (B) Glucose absorption was analyzed in RCC cells at indicated condition. (C) Lactate production was analyzed in RCC cells at indicated condition. (D) The cellular ATP level was calculated in RCC cells at indicated condition. (E,F) The mitochondrial respiratory capacity of RCC cells was detected by OCR assays at indicated condition. The value was presented as mean ± SD. ***, P<0.001. ECAR, extracellular acidification rate; Leo, leonurine; NC, negative control; OCR, oxygen consumption rate; RCC, renal cell carcinoma; SD, standard deviation.
Leo suppresses RCC growth in vivo
Finally, we evaluated the anti-tumor efficacy of Leo in vivo using a subcutaneous xenograft mouse model. Seven days post-implantation, mice were randomly assigned to either the control group or the Leo-treated group (100 mg/kg via oral gavage, twice weekly) until sacrifice at day 35 (Figure 6A). Our results demonstrated that Leo administration significantly restricted tumor growth, as evidenced by reduced tumor volume and weight compared to the control group (Figure 6B-6D). In conclusion, these in vivo data confirm that Leo significantly inhibits the progression of RCC.
Figure 6.
Leonurine suppresses RCC growth in vivo. (A) Schematic diagram showing the procedures of animal assays. (B-D) Xenograft animal assays showing the tumor volume and tumor weight of each group. The value was presented as mean ± SD. ***, P<0.001. Leo, leonurine; NC, negative control; RCC, renal cell carcinoma; SD, standard deviation.
Discussion
The pharmacological potential of natural alkaloids has gained significant attention in oncology due to their multi-targeted nature and favorable safety profiles. Leo, a prominent bioactive compound from Leonurus japonicus, has been traditionally recognized for its cardiovascular benefits (10,22). However, its role in RCC remained largely unexplored. In this study, we demonstrated for the first time that Leo serves as a potent tumor suppressor in RCC. Our in vitro data revealed that Leo induces apoptosis and impairs both the proliferative and migratory capacities of RCC cells in a dose-dependent manner. More importantly, these findings were corroborated by our in vivo xenograft models, where Leo administration significantly restricted tumor burden without apparent systemic toxicity. These results establish Leo as a promising therapeutic candidate for the management of RCC.
A critical challenge in natural product research is the precise identification of direct molecular targets. Through in silico screening and biochemical validation, we identified PDK1 as a primary target of Leo. Our MST analysis and molecular docking simulations provided robust evidence of a high-affinity interaction between Leo and the PDK1 protein. Notably, the CETSA confirmed that Leo treatment altered the thermal stability of PDK1, a hallmark of direct ligand-protein binding. These results indicate that Leo may induce conformational changes in PDK1 that render it more susceptible to degradation, a mechanism that warrants further proteomic investigation.
The metabolic hallmark of RCC is the “Warburg effect”, characterized by an over-reliance on aerobic glycolysis to sustain rapid proliferation (13). PDK1 sits at the nexus of this metabolic shift by phosphorylating the PDC, thereby diverting pyruvate away from mitochondrial oxidation and toward lactate production (23). Our study highlights the Leo/PDK1 axis as a critical regulator of this process. By targeting PDK1, Leo effectively restrains the metabolic program of RCC cells. This was evidenced by the significant reduction in ECAR, glucose uptake, and lactate secretion following Leo treatment. Furthermore, the decrease in intracellular ATP levels suggests that Leo disrupts the energy homeostasis essential for RCC cell survival. The fact that PDK1 overexpression effectively rescued these metabolic and functional phenotypes confirms that Leo’s anti-tumor efficacy is predominantly mediated through the suppression of PDK1-driven glycolysis.
From a translational perspective, evaluating Leo in comparison to existing metabolic interventions highlights its significant potential as a candidate drug. Currently, dichloroacetate (DCA) is the most extensively studied PDK inhibitor and has entered clinical trials for various solid tumors to target the Warburg effect (24). However, the clinical utility of DCA is severely restricted by its unfavorable pharmacokinetic profile; it requires administration at millimolar concentrations to achieve therapeutic efficacy, which inevitably leads to dose-limiting toxicities, including severe peripheral neuropathy and hepatotoxicity (25). In contrast, Leo, a well-characterized natural alkaloid, exhibits highly favorable pharmacological properties. Decades of pharmacological research have established its excellent biosafety profile and high tolerance in vivo, avoiding the neurotoxic pitfalls associated with synthetic PDK inhibitors like DCA.
Despite these promising findings, several limitations of this study should be acknowledged. First, while we demonstrated that Leo binds to PDK1 and reduces its protein levels, the exact pathway of degradation remains to be characterized. Second, although Leo showed high affinity for PDK1, its potential cross-reactivity with other PDK isoforms (PDK2-4) or related kinases was not extensively screened, which is essential for assessing off-target effects. Third, our in vivo studies utilized a subcutaneous xenograft model; however, orthotopic models or patient-derived xenografts (PDX) would provide a more clinically relevant microenvironment to evaluate Leo’s therapeutic index and its ability to penetrate renal tissues.
Conclusions
In summary, our study identifies Leo as a novel metabolic inhibitor that effectively suppresses RCC progression both in vitro and in vivo. We demonstrate that Leo directly binds to PDK1, leading to its downregulation and the subsequent inhibition of the Warburg effect in RCC cells. By disrupting the PDK1-mediated glycolytic program, Leo impairs the proliferative and metastatic potential of RCC. These findings provide a strong mechanistic rationale for the development of Leo-based therapies as a targeted metabolic intervention for patients with RCC.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Experiments were performed under a project license (No. 2023-Y-0739) granted by ethics committee of Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, in compliance with institutional guidelines for the care and use of animals.
Footnotes
Reporting Checklist: The authors have completed the MDAR and ARRIVE reporting checklists. Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0088/rc
Funding: The study was supported by Scientific Research Fund of Zhejiang Provincial Education Department (No. Y202044531); and Medical and Health Science and Technology Program Projects of Hangzhou (No. A20251464).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0088/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tau.amegroups.com/article/view/10.21037/tau-2026-1-0088/dss
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