Cis-3-hexen-1-ol suppresses bladder cancer progression by inducing mitophagy via the PCK2/AMPK/mTOR signaling pathway

Abstract

Background

Cis-3-hexen-1-ol is a naturally occurring green leaf volatile widely distributed in aromatic and medicinal plants. However, its antitumor activity and underlying mechanisms in bladder cancer (BLCA) remain largely unexplored.

Methods

The antitumor effects of cis-3-hexen-1-ol were evaluated using human BLCA cell lines and mouse xenograft and metastasis models. Cell proliferation, migration, invasion, mitochondrial function, and mitophagy were assessed using CCK-8, EdU incorporation, transwell assays, mitochondrial membrane potential and ROS assays, transmission electron microscopy, and immunofluorescence. Transcriptomic profiling, molecular docking, co-immunoprecipitation, immunoblotting, and in vitro kinase assays were performed to elucidate the underlying molecular mechanisms.

Results

Cis-3-hexen-1-ol significantly inhibited BLCA cell proliferation, migration, and invasion in vitro and suppressed tumor growth and lung metastasis in vivo without overt toxicity. Treatment induced mitochondrial dysfunction and robust mitophagy, as evidenced by mitochondrial fragmentation, loss of membrane potential, increased mitochondrial ROS, and enhanced recruitment of autophagy markers. Mechanistically, cis-3-hexen-1-ol activated mitophagy through the PCK2/AMPK/mTOR signaling axis. Genetic silencing of PCK2 or pharmacological inhibition of autophagic flux using 3-methyladenine markedly attenuated the antitumor and pro-mitophagic effects. Notably, biochemical analyses demonstrated that PCK2 undergoes autophosphorylation and directly phosphorylates AMPK in vitro, supporting a kinase-dependent regulatory mechanism.

Conclusions

These findings identify cis-3-hexen-1-ol as a novel mitophagy-inducing phytochemical that suppresses BLCA progression via activation of the PCK2/AMPK/mTOR pathway, highlighting its potential as a natural-product lead for targeted bladder cancer therapy.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-026-01233-2.

Introduction

Bladder cancer (BLCA) is a prevalent malignancy worldwide within the urinary system, posing a significant global health burden [1, 2]. According to GLOBOCAN 2020, BLCA accounted for approximately 573,000 new cases and 213,000 deaths worldwide [3, 4]. Clinically classified by invasion depth as non-muscle invasive (NMIBC) or muscle-invasive (MIBC) [5], current therapeutic strategies rely on surgical resection combined with pharmacotherapy [6, 7]. High-risk NMIBC is most often managed with intravesical BCG immunotherapy, while treatment of advanced stages has evolved to include immune checkpoint inhibitors, molecularly targeted agents, and antibody-drug conjugates [8, 9]. Despite these advancements, therapeutic efficacy is frequently compromised by treatment resistance, disease recurrence, and systemic toxicity, contributing to poor patient prognosis [1, 10]. These findings underscore the critical need for novel therapeutic agents with improved safety profiles and mechanistic innovations.

Mitophagy, a selective form of autophagy responsible for removing damaged mitochondria, is essential for maintaining mitochondrial homeostasis and cellular metabolic balance [11]. Increasing evidence suggests that dysregulated mitophagy contributes to cancer initiation, progression, and therapeutic resistance, although its biological role can vary depending on tumor context [12]. Given the reliance of many cancers on altered mitochondrial function, pharmacological modulation of mitophagy has emerged as a promising therapeutic strategy [13].

Natural products remain a rich source of bioactive molecules with anticancer potential due to their structural diversity and favorable safety profiles [14, 15]. Many natural compounds have been shown to influence mitochondrial homeostasis or modulate mitophagy pathways, thereby affecting tumor survival and metabolic adaptation. Cis-3-hexen-1-ol (leaf alcohol, CAS: 928–96-1), a volatile organic compound ubiquitously distributed in green plants [16, 17] is conventionally employed in fragrance formulations and ecological pest management because of its insect-repellent properties [18, 19]. Our preliminary screening of natural product libraries identified cis-3-hexen-1-ol as a potent inhibitor of BLCA progression in preclinical models. Notably, no prior studies have investigated its antitumor potential or elucidated its molecular targets, which represents a significant knowledge gap in onco-pharmacology.

This study pioneers the investigation of cis-3-hexen-1-ol as a mitophagy-inducing agent against BLCA. Through integrated transcriptomic profiling and functional validation, we identified phosphoenolpyruvate carboxykinase 2 (PCK2) as the critical mediator of the antitumor effects of cis-3-hexen-1-ol, functioning through the modulation of mitochondrial autophagy. These results not only reveal potential therapeutic targets for BLCA but also provide a framework for developing plant-derived mitophagy modulators for precision oncology.

Materials and methods

Pharmacological reagents

Cis-3-hexen-1-ol (S6059), and 3-methyladenine (S2767) were obtained from Selleck Chemicals. Subcellular fractionation was performed using a mitochondrial isolation kit (C3601) and a cytoplasmic/nuclear extraction kit (C0028) from Beyotime Biotechnology.

Cell proliferation assays

Cell viability was quantified using Cell Counting Kit-8 (CCK-8; C6005, NCM Biotech, China). DNA replication activity was assessed via BeyoClick™ EdU Cell Proliferation Kit (AlexaFluor 594; C0078S, Beyotime, China). For colony formation analysis, 1 × 10³ cells/well were seeded in 6-well plates (Corning, USA) and cultured in complete RPMI-1640 medium with cis-3-Hexen-1-ol (0–4 μM) for 14 days. Colonies (>50 cells) were fixed with 4% paraformaldehyde (30 min), stained with 0.01% crystal violet (10 min), and quantified using ImageJ (NIH, USA). All assays were performed in biological triplicate (n = 3). Cells were used at passages 3–12. Cis-3-hexen-1-ol (S6059, Selleck; purity ≥ 98%) was dissolved in DMSO; final DMSO concentration did not exceed 0.1% (v/v) in any assay.

Migration and invasion analysis

Scratch Wound Assay: Confluent monolayers were scratched using sterile pipette tips (200 μl). Migration in 0.1% FBS medium containing cis-3-Hexen-1-ol (0–4 μM) was monitored at 0/24 h using Leica DMi8 microscope (10×), at identical fields. Percent wound closure was quantified with ImageJ. Experiments were performed in triplicate (n = 3).

Transwell System: 5 × 10⁴ serum-starved cells in 200 µL serum-free medium were seeded into upper chambers (8 µm pore; Corning). For invasion assays, inserts were coated with 50 µL of Matrigel (BD Biosciences, USA). Migrated cells (24 h post-treatment) were fixed/stained as above and counted from 5 random fields (20×).

Mitochondrial function assessment

JC-1 Assay: T24/5637 cells treated with cis-3-Hexen-1-ol (2 μM) ± 3-MA (10 mM) were analyzed using Mitochondrial Membrane Potential Assay Kit (C2006, Beyotime). Red/green fluorescence ratios were quantified by flow cytometry (BD FACSCanto II).MitoSOX Red Detection: Mitochondrial superoxide levels were measured using MitoSOX™ Red reagent (S0061S, Beyotime) following manufacturer protocol.Confocal Imaging: Mitochondrial morphology was visualized using MitoTracker Red CMXRos (200 nM, C1035, Beyotime) on Talos L120C confocal microscope (Thermo Scientific).

Metabolic profiling

Cellular oxygen consumption rate (OCR) was measured using XF24 Extracellular Flux Analyzer (Seahorse Bioscience, USA). Seahorse XF24 mitochondrial stress tests were performed with 5 technical replicates per condition and at least 3 biological replicates. Cells were seeded at 1 × 10^4 cells/well and equilibrated 24 h before assay. Sequential injections: oligomycin (2.5 μM), FCCP (optimized; 1.0 μM), rotenone/antimycin A (1 μM). Basal respiration, ATP production, maximal respiration, and spare respiratory capacity were calculated per manufacturer instructions. OCR values were normalized to total protein measured from each well by BCA after XF assay; protein quantification was performed on the same wells immediately after assay using Pierce BCA reagent (Thermo Fisher). Where FCCP optimization was required, we performed separate titration experiments (data not shown).

Ultrastructural analysis

Transmission electron microscopy (TEM) samples were processed using standard protocols: 3% glutaraldehyde primary fixation → 1% OsO₄ post-fixation → graded acetone dehydration → Epox 812 embedding. Ultrathin sections (70 nm) stained with uranyl acetate/lead citrate were imaged on Talos L120C TEM (Thermo Scientific). At least 10 random fields per sample and 3 biological replicates were imaged. Images were analyzed blinded to treatment. The number of mitochondria engulfed by autophagic vesicles was quantified per field.

Immunoblotting

Protein extracts from tissues/cells were prepared using RIPA buffer (WB3100, NCM Biotech) with protease/phosphatase inhibitors (P002, NCM Biotech). Mitochondrial/cytoplasmic fractions were isolated using commercial kits (C3601/P0028, Beyotime). After BCA quantification (WB6501, NCM Biotech), 20 μg protein/lane was resolved on 4–12% gradient gels (ACE Biotechnology) and transferred to PVDF membranes (Merck Millipore). Membranes were probed with primary antibodies (4 °C, overnight) and HRP-conjugated secondaries (1 h, RT). Signals were developed using ECL substrate (Bio-Rad) and quantified via ImageLab. All immunoblotting experiments were independently repeated at least three times.

Antibodies

Primary antibodies were procured from various suppliers as follows: PCK2 antibody (#8565) and LC3A/B antibody (#4108) were acquired from Cell Signaling Technology, Massachusetts, USA. Antibodies including P62 (A7758), Beclin1 (A7353), PCK2 (A21529), AMPK (A1229), phosphorylated AMPK (p-AMPK, AP1441), mTOR (A2445), phosphorylated mTOR (p-mTOR, AP0115). Additionally, HSP60 antibody (#15282–1-AP), COXⅣ antibody (#66110–1-lg), VDAC1 antibody (#66345–1-lg), TOM20 antibody (#11802–1-AP), ACTB antibody (#166009-1-lg), and GAPDH antibody (#60004–1-lg) were obtained from Proteintech, Sanying, W.

Molecular modeling

Protein-Ligand Docking: The crystal structures of PCK2 (UniProt: Q16822) and AMPK isoforms (PDB: 4RED/8BIK) were prepared in AutoDockTools-1.5.7. Ligand cis-3-hexen-1-ol (PubChem CID: 13,587) was energy-minimized with MMFF94, protonated at pH 7.4. Docking grids (20 × 20 × 20 Å) were centered on the catalytic site; 100 Lamarckian GA runs were performed with default exhaustiveness. Top poses were subjected to MD simulations in GROMACS 2020.3 using the CHARMM36m force field and TIP3P water model. Systems were neutralized with Na+/Cl− to 0.15 M ionic strength. Energy minimization was followed by 1 ns NVT equilibration and 5 ns NPT equilibration at 310 K (V-rescale thermostat) and 1 bar (Parrinello-Rahman barostat). Production runs were 100 ns; trajectories were analyzed for RMSD, RMSF, hydrogen bonds, radius of gyration, and MM-PBSA binding free energy.

Cell lines and transfection

Human BLCA cell lines (T24, 5637, RT112, BIU87, SW780) and HEK293T cells (Procell Life Science, China) were maintained in RPMI-1640/DMEM (Gibco) supplemented with 10% FBS (Thermo Fisher) and 1% penicillin/streptomycin at 37 °C/5% CO₂. PCK2-targeting siRNAs (GeneChem, China) were transfected using siRNA-mate (GenePharma, China), and functional assays were conducted 48 h post-transfection. All transfection experiments were performed in at least three independent biological replicates.

Animal experiments

All animal experiments were approved by the Laboratory Animal Ethics Committee of Xiangya Hospital of Central South University (Approval No. 202,412,259; approved on 03 January 2025). This study was designed, conducted, and reported in accordance with the ARRIVE 2.0 guidelines for reporting animal research. During bioluminescence imaging and other procedures requiring restraint, mice were anesthetized using inhalational isoflurane (2–3% for induction and 1–2% for maintenance). At the experimental endpoint or when predefined humane endpoints were reached, animals were euthanized by carbon dioxide (CO₂) inhalation followed by cervical dislocation to ensure death. All anesthesia and euthanasia procedures were performed in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. Male BALB/c nude mice (4–6 weeks old; GemPharmatech) were randomized into groups (n = 6/group) using a random number generator. Investigators were blinded to treatment during data collection and analysis. For subcutaneous xenografts, 5 × 10^6 T24 cells in 100 μL PBS were injected subcutaneously. Treatments: vehicle (0.1% DMSO in PBS, i.p., q3d), cis-3-hexen-1-ol 5 mg/kg (i.p., q3d), cis-3-hexen-1-ol 10 mg/kg (i.p., q3d). Tumour volume was measured by caliper and calculated as (length × width^2)/2; body weight and clinical signs were monitored twice weekly. Humane endpoints: tumour volume > 2000 mm^3 or severe distress led to euthanasia. All tissues were collected after euthanasia for analysis. For experimental metastasis, mice (n = 6/group) were pretreated for 3 days, then injected with 1 × 10^6 T24-luc cells via tail vein. Bioluminescence imaging (IVIS Lumina II) was performed weekly; total flux (photons/sec) per mouse was quantified using Living Image software. Metastatic nodules in lungs were confirmed by histology and counted.

Transcriptome analysis

Cells were treated with cis-3-hexen-1-ol for 24 hours, and RNA-seq was performed on three biological replicates per group (n = 3). Total RNA was extracted using a TransZol Up Plus Kit (TransGen Biotech) and subjected to paired-end sequencing (2 × 150 bp) on an Illumina HiSeq 2000 (Personalbio). Quality control was performed with FastQC; adapters were trimmed with Trimmomatic; reads were aligned to GRCh38 with STAR v2.7.10a with >90% mapping rate. Differential expression was analyzed with DESeq2 v1.30 with Benjamini-Hochberg FDR < 0.05. GO enrichment used clusterProfiler.

In vitro phosphorylation assays

Recombinant GST-PCK2 (500 ng) and His-AMPK (500–1000 ng) were incubated in kinase buffer (25 mM Tris-HCl, 10 mM MgCl₂, 1 mM CaCl₂/DTT, 100 μM ATP, pH 7.5) at 30 °C for 30 min. Controls included GST alone, His-AMPK alone, and reactions without ATP. Phosphorylation was detected with pIMAGO kit (TMR-800–10, Tymora Analytical) and validated by autoradiography when applicable. The purity and integrity of recombinant GST-PCK2 and AMPK-HIS proteins were verified by Coomassie blue-stained SDS-PAGE following affinity purification. All experiments were repeated independently at least three times.

Statistical analysis

Data are presented as mean ± standard deviation (SD) from at least three biological replicates unless otherwise specified. Comparisons between two groups used two-tailed Student’s t-test; multiple group comparisons used one-way ANOVA with Tukey’s post-hoc test. Statistical analyses were performed in GraphPad Prism 8.0. A p-value < 0.05 was considered significant.

Results

In vitro, cis-3-hexen-1-ol reduces the proliferation, migration, and invasion of BLCA cells

Through a comprehensive screening of the natural compound library provided by SELLECK (Fig. 1A), we identified cis-3-hexen-1-ol as a compound exhibiting notable inhibitory activity against BLCA cell proliferation. The chemical structure of cis-3-hexen-1-ol is depicted in Fig. 1B. To assess its antiproliferative properties, we employed CCK8 assays across various human BLCA cell lines, including T24, 5637, RT112, BIU87, and SW780. The BLCA cells were exposed to various concentrations of cis-3-hexen-1-ol (0, 0.25, 0.50, 1.25, 2.50, 5, and 10 μM) for 24 and 48-hour periods. As shown in Fig. 1C, the results indicate that cis-3-hexen-1-ol suppresses BLCA cell proliferation in a dose- and time-dependent manner. Additionally, colony formation (Fig. 4B; Fig. S1. A) and EdU assays (Fig. 1D; S1. B) further confirmed that cis-3-hexen-1-ol) inhibited BLCA cell proliferation in a dose-dependent manner (Fig. 1D, Fig. S1A-B). To evaluate the impact of cis-3-hexen-1-ol on the migratory ability of BLCA cells, we conducted cell scratch tests (Fig. 1E, Fig. S1C) and transwell migration assays (Fig. 1F, Fig. S1D), both of which revealed a dose-dependent reduction in BLCA cell migration. Similarly, transwell invasion assays (Fig. 1G, Fig. S1E) demonstrated that cis-3-hexen-1-ol inhibited invasion capabilities in a concentration-dependent manner. Given that cis-3-hexen-1-ol had the most significant inhibitory effects on the T24 and 5637 cell lines, subsequent experiments were conducted in these two cell lines (Fig. 1J,K).

Fig. 1.

Cis-3-hexen-1-ol suppresses proliferation, migration, and invasion of BLCA cells in vitro. (A) Schematic illustration of the screening workflow used for identifying bioactive compounds from the natural product library. (B) Chemical structure of cis-3-hexen-1-ol (CAS: 928–96-1). (C) Cell viability of T24, 5637, RT112, BIU87, and SW780 cells treated with increasing concentrations of cis-3-hexen-1-ol for 24 h or 48 h, as measured by CCK-8 assay. IC₅₀ values are shown in the accompanying table. (n = 3; mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001). (D) EdU incorporation assay demonstrating inhibition of DNA synthesis in T24 and 5637 cells treated with cis-3-hexen-1-ol (0, 1, 2, 4 μM) for 24 h. Representative images and merged channels are shown. Scale bar = 100 μm. (E) Representative wound-healing images of T24 and 5637 cells treated with cis-3-hexen-1-ol (0–4 μM). Images were taken at 0 h and 24 h to assess migration capacity. Scale bar = 100 μm. (F) Transwell migration assay showing dose-dependent inhibition of cell motility after cis-3-hexen-1-ol treatment. Migrated cells were crystal-violet stained and quantified. Scale bar = 100 μm. (G) Transwell invasion assay using Matrigel-coated inserts demonstrating reduced invasive ability after cis-3-hexen-1-ol exposure. Scale bar = 100 μm. (H-K) Quantification of EdU-positive cells (H), wound-healing closure percentage (I), migrated cell counts (J), and invaded cell counts (K) across different cis-3-hexen-1-ol concentrations. (Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001); LA: Leaf Alcohol

Fig. 4.

Inhibition of mitophagy attenuates cis-3-hexen-1-ol-induced suppression of proliferation, migration, and invasion in BLCA cells (A) Cell viability was assessed by CCK-8 assay in T24 and 5637 cells treated with cis-3-hexen-1-ol (2 μM) with or without 3-methyladenine (3-MA). IC₅₀ values were calculated using nonlinear regression. (B) Colony formation assays showing clonogenic survival of T24 and 5637 cells under the indicated treatments (NC, 2 μM, 2 μM + 3-MA). Colonies were stained with crystal violet and quantified. Scale bar = 100 μm. (C) EdU incorporation assays evaluating DNA synthesis in T24 and 5637 cells following cis-3-hexen-1-ol ±3-MA treatment. Representative images and quantification of EdU-positive cells are shown. Scale bar = 100 μm. (D) Wound-healing assays assessing migratory capacity. Images were taken at 0 h and 24 h following linear scratch formation. Percent wound closure was quantified for each group. Scale bar = 100 μm. (E) Transwell migration assays evaluating cell motility after cis-3-hexen-1-ol ±3-MA exposure. Migrated cells were stained and counted in five random fields per well. Scale bar = 100 μm. (F) Transwell invasion assays performed using Matrigel-coated inserts to determine invasive potential under each treatment condition. Invaded cells were stained and quantified. Scale bar = 100 μm. (G) Mitochondrial mass was assessed using MitoTracker Red staining and quantified by flow cytometry in T24 and 5637 cells treated with cis-3-hexen-1-ol ±3-MA. (H) Mitochondrial reactive oxygen species (mtROS) were measured by MitoSOX Red staining in T24 and 5637 cells following the indicated treatments. Representative histograms and quantification are shown. (I) Mitochondrial membrane potential (MMP) was examined using JC-1 staining. The JC-1 red/green fluorescence ratio was quantified to determine mitochondrial depolarization. (**Data are presented as mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA with Tukey post-hoc test: *p < 0.05, **p < 0.01, *p < 0.001)

Cis-3-hexen-1-ol induces mitophagy in BLCA cells

To elucidate the mechanism of the inhibitory effects of cis-3-hexen-1-ol on BLCA, we performed RNA-seq analysis. GO analysis of DEPs revealed enhanced mitophagy-related pathways upon 2 μM treatment compared to DMSO (Fig. 2A). Thus, we speculated that the antitumor activity is predominantly due to mitophagy. As expected, we observed the mitochondrial rupture and engulfment, and loss of crista structure, which showed that the mitochondria were surrounded by autolysosomes (Fig. 2B, Fig S2A). Additionally, the colocalization of mitochondria with LC3B and lysozyme further demonstrated mitophagy induction by cis-3-hexen-1-ol in BLCA cells (Fig. 2C,D; Fig S2B,C). Western blot assays revealed that the cis-3-hexen-1-ol induced autophagy in the mitochondrial fractions of BLCA cells in the mitochondrial fractions by a time- and dose-dependent manner (Fig. 2E–H, Fig S2D,E). Volcano plot analysis revealed significant up- and down-regulation of multiple genes following cis-3-hexen-1-ol treatment (Fig. 2I). Taken together, these results indicate that cis-3-hexen-1-ol induces mitophagy-associated responses in BLCA cells.

Fig. 2.

Cis-3-hexen-1-ol induces mitophagy in BLCA cells. (A) Bubble plot of GO pathway enrichment of differentially expressed proteins (DEPs) in T24 cells treated with cis-3-hexen-1-ol (2 μM). Mitophagy-related pathways were significantly enriched. (B) Transmission electron microscopy (TEM) images showing mitochondrial ultrastructural changes after cis-3-hexen-1-ol treatment. Red arrows indicate swollen or damaged mitochondria and mitochondria enclosed within double-membrane autophagic vesicles. Scale bar = 2 μm. (C) Colocalization of mitochondria (TOM20) with lysosomes (Lysozyme) in T24 and 5637 cells after 24 h treatment with cis-3-hexen-1-ol (2 μM). Increased TOM20-lysosome colocalization indicates enhanced mitochondrial degradation. Scale bar = 10 μm. (D) Colocalization of mitochondria (TOM20) with autophagosomes (LC3B) following cis-3-hexen-1-ol treatment. The increased overlap of TOM20 and LC3B signals demonstrates mitochondrial sequestration during mitophagy. Scale bar = 10 μm. (E) Time-dependent immunoblot analysis of mitophagy-related proteins (P62, Beclin1, LC3-I/LC3-II) in mitochondrial fractions from T24 and 5637 cells treated with cis-3-hexen-1-ol (2 μM) for 0–72 h. VDAC1 served as the mitochondrial loading control. (F) Dose-dependent immunoblotting of mitochondrial P62, Beclin1, and LC3-I/LC3-II levels following exposure to cis-3-hexen-1-ol (0–4 μM) for 24 h. VDAC1 served as the mitochondrial loading control. (G-H) Densitometric quantification of mitochondrial P62, Beclin1, and LC3-II relative to VDAC1 for the time-course (G) and dose-response (H) experiments. (I) Volcano plot showing differentially expressed genes following cis-3-hexen-1-ol treatment. (Data are presented as mean ± SD (n = 3); *p < 0.05, **p < 0.01, ***p < 0.001.)

Fig. 3.

Cis-3-hexen-1-ol induces mitochondrial dysfunction in BLCA cells. (A) T24 and 5637 cells were treated with cis-3-hexen-1-ol (2 μM) for the indicated durations and stained with MitoTracker Red (200 nM) and Hoechst 33,342 (2 μg/mL). Mitochondrial fragmentation and punctate morphology were visualized by fluorescence microscopy. Scale bar = 10 μm. (B) Mitochondrial ROS levels were assessed using MitoSOX Red staining and quantified by flow cytometry after 24 h treatment with cis-3-hexen-1-ol (2 μM). (C) Mitochondrial membrane potential (MMP) was evaluated by JC-1 staining in T24 and 5637 cells following 24 h treatment with cis-3-hexen-1-ol (2 μM). Representative JC-1 red/green fluorescence scatter plots are shown. (D) Immunoblotting analyses of mitochondrial proteins (HSP60, VDAC1, COX IV, TOM20) in whole-cell lysates of T24 and 5637 cells treated with 0, 2, or 4 μM cis-3-hexen-1-ol for 24 h. VDAC1/COX IV/TOM20 were used as mitochondrial loading controls, and ACTB was used as the whole-cell loading control. (E) Oxygen consumption rate (OCR) measurements were performed using a Seahorse XF24 Analyzer. Basal respiration, ATP-linked respiration, maximal respiration, and spare respiratory capacity were evaluated following sequential injections of oligomycin, FCCP, and rotenone/antimycin A in T24 and 5637 cells treated with or without cis-3-hexen-1-ol (2 μM) for 24 h. (F-H) Quantification of mitochondrial fragmentation (F), MitoSOX fluorescence intensity (G), and JC-1 red/green ratio (H) in T24 and 5637 cells treated with cis-3-hexen-1-ol (2 μM). Data are presented as mean ± SD (n = 3). (I) Densitometric quantification of mitochondrial protein expression (HSP60, VDAC1, COX IV, TOM20) in T24 and 5637 cells after treatment. (Data normalized to mitochondrial loading controls. Mean ± SD; n = 3)

Cis-3-hexen-1-ol causes mitochondrial dysfunction in BLCA cells

Mitophagy has been widely recognized as a key process in mitochondrial quality control, particularly in response to mitochondrial damage [20–22]. Consequently, we hypothesized that cis-3-hexen-1-ol treatment would induce alterations in the level of mitochondrial damage in BLCA cells. Compared with those in control cells, Mitotracker red staining assays revealed a substantial increase in fragmented and punctuated mitochondria in cells treated with cis-3-hexen-1-ol (Fig. 3A, F). Furthermore, treatment with cis-3-hexen-1-ol in BLCA cells significantly elevated MitoSox production (Fig. 3B, G, Fig S3A) and markedly reduced the mitochondrial membrane potential (MMP) (Fig. 3C, H; Fig S3B). Western blot analysis demonstrated that cis-3-hexen-1-ol significantly compromised mitochondrial quality in BLCA cells in a time- and dose-dependent manner (Fig. 3D, J). Treatment with cis-3-hexen-1-ol also significantly suppressed basal OCR, ATP levels, peak respiration, and respiratory reserve (Fig. 3F,G; Fig S3C). Collectively, these findings indicate that cis-3-hexen-1-ol treatment induces mitochondrial damage.

Inhibition of mitophagy attenuates the antitumour effects of cis-3-hexen-1-ol

T24/5637 cells were pretreated with 3-methyladenine (3 MA), a classical inhibitor of mitophagy [23], for 24 h prior to exposure to cis-3-hexen-1-ol. We found that the proliferation (Fig. 4A–C), migration (Fig. 4D, E) and invasion (Fig. 4F) abilities of BLCA cells were alleviated in the 3 MA pretreated group. Moreover, mitochondrial mass (Fig. 4G) and mitochondrial function (Fig. 4H, I) were restored.

Cis-3-hexen-1-ol induces mitophagy via the PCK2/AMPK/mTOR axis in BLCA cells

To investigate the effect of cis-3-hexen-1-ol on mitophagy in BLCA cells, we analyzed the gene expression heatmap (Fig. 5A). Among all expressed genes, PCK2 showed the most significant change, with a 2.852-fold increase. A review of the literature revealed that PCK2 regulates mitophagy via the AMPK/mTOR pathway [24, 25], which is consistent with the RNA-seq results. Furthermore, molecular docking analysis demonstrated a strong fit between cis-3-hexen-1-ol and the binding pocket of the PCK2 receptor, with a binding energy of −4.4 kcal/mol, suggesting a potential binding interaction (Fig. 5B). Detailed results from the molecular dynamics simulations are provided in the Supplementary Results. Furthermore, the colocalization of mitochondria with PCK2 was significantly enhanced by cis-3-hexen-1-ol in BLCA cells (Fig. 5C; Fig. S6). To elucidate the underlying mechanisms by which cis-3-hexen-1-ol induces mitophagy, we initially assessed the expression levels of PCK2, phosphorylated AMPK (p-AMPK), AMPK, phosphorylated mTOR (p-mTOR), and mTOR in the mitochondrial fractions of BLCA cells following cis-3-hexen-1-ol treatment. The results demonstrated that PCK2 and p-AMPK were upregulated, whereas p-mTOR was downregulated, in a time- and dose-dependent manner (Fig. 5D,E). Conversely, the expression patterns of p-AMPK, p-mTOR, and mitophagy marker proteins were reversed upon PCK2 knockdown in BLCA cells (Fig. 5F,G). Basal LC3B expression was detectable in shNC cells, reflecting constitutive autophagic activity. Notably, cis-3-hexen-1-ol significantly enhanced LC3B-II accumulation, whereas this effect was markedly attenuated upon PCK2 silencing. Collectively, these findings indicate that cis-3-hexen-1-ol facilitates the activation of mitophagy through the modulation of the PCK2/AMPK/mTOR signaling pathway.

Fig. 5.

Cis-3-hexen-1-ol induces mitophagy through the PCK2-AMPK-mTOR signaling axis in BLCA cells. (A) Heatmap of differentially expressed mitophagy-related genes in cis-3-hexen-1-ol-treated T24 cells compared with controls. (B) Molecular docking analysis of cis-3-hexen-1-ol with PCK2 using AutoDock. The predicted binding pose of cis-3-hexen-1-ol within the catalytic pocket of PCK2 is shown. (C) Immunofluorescence staining of TOM20 (mitochondria) and PCK2 in T24 and 5637 cells treated with cis-3-hexen-1-ol (2 μM, 24 h). Increased colocalization of TOM20 and PCK2 indicates mitochondrial enrichment of PCK2. Scale bar = 10 μm. (D) Time-dependent changes in mitochondrial PCK2, p-AMPK, AMPK, p-mTOR, and mTOR protein levels in T24 and 5637 cells following cis-3-hexen-1-ol (2 μM) treatment for 0–72 h. VDAC1 served as the mitochondrial loading control. Quantitative densitometry is shown on the right. (E) Dose-dependent regulation of mitochondrial PCK2, p-AMPK, AMPK, p-mTOR, and mTOR in T24 and 5637 cells treated with cis-3-hexen-1-ol (0–4 μM, 24 h). VDAC1 served as the mitochondrial loading control. Densitometric analysis is provided on the right. (F) Western blot analysis of mitochondrial PCK2, p-AMPK, AMPK, p-mTOR, and mTOR following PCK2 knockdown (shPCK2) with or without cis-3-hexen-1-ol treatment (2 μM). VDAC1 served as the mitochondrial loading control. The functional loss of cis-3-hexen-1-ol-mediated AMPK activation and mTOR inhibition after PCK2 silencing is shown in the corresponding quantifications. (G) Immunoblot analysis of mitochondrial autophagy-related proteins (P62, Beclin1, LC3-I/LC3-II) under the same shPCK2 ± cis-3-hexen-1-ol treatment conditions. VDAC1 served as the mitochondrial loading control. Quantification is displayed on the right. (**Data are presented as mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. *p < 0.05; **p < 0.01; *p < 0.001)

PCK2 regulates AMPK through direct phosphorylation

PCK2 is a key enzyme in mitochondrial glucose metabolism; however, whether it regulates AMPK through metabolic intermediates or via a direct molecular mechanism remained unclear. To address this, we first examined AMPK activation following treatment with phosphoenolpyruvate (PEP), the immediate catalytic product of PCK2. PEP treatment induced a modest dose-dependent increase in AMPK Thr172 phosphorylation in both T24 and 5637 bladder cancer cells; however, the magnitude and consistency of this activation were markedly weaker than those observed with cis-3-hexen-1-ol treatment, suggesting that PCK2-mediated AMPK activation is not primarily dependent on downstream metabolic products (Fig. 6A). These findings support a predominantly direct regulatory mechanism between PCK2 and AMPK.We next explored the possibility of a direct spatial and physical interaction between PCK2 and AMPK. Immunofluorescence analysis revealed clear colocalization of PCK2 and AMPK in both T24 and 5637 cells, which was markedly enhanced following cis-3-hexen-1-ol treatment (Fig. 6B). This observation is consistent with previous reports describing mitochondrial localization of AMPK and supports the potential for direct interaction between these proteins [26, 27]. To validate this interaction biochemically, co-immunoprecipitation assays were performed. Endogenous PCK2 robustly pulled down AMPK in T24, 5637, and HEK-293T cells, whereas no interaction was detected with IgG controls, confirming the specificity of the PCK2-AMPK complex (Fig. 6C). These results demonstrate that PCK2 physically associates with AMPK in multiple cellular contexts. We then investigated whether PCK2 could directly phosphorylate AMPK. Prior to functional analysis, the successful expression and affinity purification of recombinant GST-PCK2 and AMPK-HIS proteins were confirmed by Coomassie blue-stained SDS-PAGE (Fig. S5). In-vitro kinase assays using purified recombinant proteins showed that GST-PCK2 induced phosphorylation of AMPK-HIS, as detected by phospho-serine/threonine antibodies, while GST alone had no effect (Fig. 6D; Fig. S5B). These findings provide direct biochemical evidence that PCK2 functions as an upstream kinase capable of phosphorylating AMPK independently of cellular metabolic processes. Finally, molecular docking analyses were performed to further characterize the interaction between PCK2 and AMPK catalytic subunits. Docking simulations revealed stable binding conformations between PCK2 and both AMPKα1 and AMPKα2, with predicted interaction interfaces involving conserved residues critical for kinase activity (Fig. 6E, F). Together, these data support a model in which PCK2 directly binds to and phosphorylates AMPK, thereby linking mitochondrial metabolic enzymes to AMPK signaling regulation in bladder cancer cells.

Fig. 6.

PCK2 interacts with and phosphorylates AMPK in BLCA cells. (A) Western blot analysis of phosphorylated AMPK (p-AMPK, Thr172) and total AMPK in T24 and 5637 bladder cancer cells treated with phosphoenolpyruvate (PEP; 0–50 μM) for 24 h. ACTB was used as a loading control. Quantification of p-AMPK/AMPK ratios is shown on the right. (B) Immunofluorescence staining showing the subcellular colocalization of PCK2 (green) and AMPK (magenta) in T24 and 5637 cells treated with cis-3-hexen-1-ol (2 μM, 24 h). Nuclei were counterstained with DAPI (blue). Merged images are shown. Scale bar = 10 μm. (C) Co-immunoprecipitation (co-IP) assays demonstrating endogenous interaction between PCK2 and AMPK in T24, 5637, and HEK-293T cells. Cell lysates were immunoprecipitated with anti-PCK2 antibody or IgG control and immunoblotted with anti-AMPK and anti-PCK2 antibodies. Input lysates are shown as controls. (D) In vitro kinase assay showing that recombinant GST-PCK2 phosphorylates AMPK-HIS. Phosphorylation was detected using anti-phospho-serine/threonine antibodies (α-Phos). α-GST and α-HIS immunoblots confirm protein inputs. GST alone served as a negative control. (E-F) Molecular docking simulations of PCK2 with AMPKα1 (E) and AMPKα2 (F), illustrating predicted binding modes, interaction interfaces, and key amino acid residues involved in the PCK2-AMPK interaction

Cis-3-hexen-1-ol prevents the progression and metastasis of bladder cancer in vivo

The anti-BLCA effect of cis-3-hexen-1-ol was tested in vivo using a T24 tumor-bearing nude mouse model. In brief, a total of 18 nude mice received intraperitoneal injections of low-dose cis-3-hexen-1-ol (5 mg/kg), high-dose cis-3-hexen-1-ol (10 mg/kg), or a vehicle control every two days. According to the results, cis-3-hexen-1-ol had a concentration-dependent effect on lowering the weight (Fig. 7A, B) and decreasing the volume of bladder tumors (Fig. 7C), but it did not significantly affect the body weight of the nude mice (Fig. 7D).

Fig. 7.

Cis-3-hexen-1-ol suppresses tumour growth and metastasis of bladder cancer in vivo. (A) Representative photographs of excised subcutaneous T24 xenograft tumours from mice treated with vehicle (Con), cis-3-hexen-1-ol (5 mg/kg), or cis-3-hexen-1-ol (10 mg/kg). (B) Tumour weights measured at the endpoint of the study. Data show dose-dependent suppression of tumour growth by cis-3-hexen-1-ol. (C) Body weight curves of mice during treatment. No significant body-weight loss was observed, indicating minimal systemic toxicity. (D) Tumour volume progression during treatment. Tumour volumes were measured every 3 days and calculated using the formula: (length × width²)/2. (E) Western blot analysis of PCK2, p-AMPK, AMPK, p-mTOR, and mTOR expression in tumour tissues from each treatment group. ACTB served as the loading control. Quantification of protein band intensities is shown on the right. (F) Western blot analysis of mitophagy-related proteins (P62, Beclin1, LC3-I/LC3-II) in tumour lysates. Densitometric quantification normalized to ACTB is presented on the right. (G) In vivo bioluminescence imaging of lung metastases following tail-vein injection of T24-luciferase cells. Representative images and gross lung tissues are shown. (H) Hematoxylin and eosin (H&E) staining of major organs (heart, liver, spleen, lung, kidney) from each group. No overt histopathological toxicity was observed. Scale bar = 100 μm. (**Data are presented as mean ± SD (n = 6 per group). Statistical significance was determined using one-way ANOVA with Tukey’s post-hoc test. *p < 0.05; **p < 0.01; *p < 0.001.)

Western blotting (Fig. 7E, F) of tumor tissue revealed that cis-3-hexen-1-ol treatment increased mitochondrial PCK2, p-AMPK, Beclin1 and LC3B levels and decreased p-mTOR. Total-cell P62 decreased (consistent with increased autophagic flux), whereas mitochondrial-enriched P62 levels increased, consistent with recruitment of P62 to damaged mitochondria during mitophagy. We quantified P62 in both whole-cell lysates and mitochondrial fractions to distinguish overall autophagic flux from mitochondrial recruitment (see Methods and Fig. S2B-C). Furthermore, in the lung metastasis model, cis-3-hexen-1-ol treatment significantly decreased the number of metastatic nodules in bladder cancer (Fig. 7G). In summary, cis-3-hexen-1-ol has the potential to suppress both the development and metastasis of bladder carcinoma in a living body. Moreover, the major organs of the cis-3-hexen-1-ol-treated nude mice did not exhibit significant toxicity (Fig. 7H).

Discussion

BLCA has become a critical global health concern because of its high incidence and mortality rates [28, 29]. Although there have been substantial advancements in systemic treatments for bladder cancer in recent years, the risk of tumor progression remains considerable among affected patients [29, 30]. Therefore, the development of novel therapeutic strategies for bladder cancer is imperative [31, 32].

Advancements in technologies for extracting and identifying natural products have underscored their promise in counteracting multidrug resistance in cancer, thereby garnering growing academic interest [33, 34]. In our previous research, we reported that the natural product cis-3-hexen-1-ol can significantly inhibit the progression of BLCA both in vitro and in vivo through a comprehensive screening of a natural product library. Cis-3-hexen-1-ol is an essential aromatic compound predominantly extracted from green plants [16, 17]. It is utilized in fruit and vegetable flavorings and perfumes and also functions as an attractant for predatory insects, playing various defense-related roles in response to insect stress [18, 19]. While previous studies on cis-3-hexen-1-ol have focused primarily on its role in plants, to the best of our knowledge, there are currently no research reports on its role in tumors. Accordingly, this study aimed to elucidate the underlying mechanisms through which cis-3-hexen-1-ol suppresses bladder cancer progression, with the goal of offering novel perspectives for therapeutic intervention.

Mitochondria are dynamic organelles with diverse functions, as evidenced by their involvement in metabolism, necrosis, and programmed cell death (apoptosis), thereby playing a pivotal-roles in cellular metabolism and survival [35–39]. Mitophagy, a specialized form of macro autophagy, facilitates the selective transport of mitochondria to lysosomes for degradation [40, 41]. This mechanism acts as a critical quality control system by removing excess or damaged mitochondria, thus maintaining mitochondrial homeostasis and overall cellular balance [42, 43]. Activation of mitophagy occurs when mitochondrial injury exceeds the capacity of other mitochondrial quality control pathways or when mitochondrial clearance is required to meet specific metabolic demands [44, 45]. Unlike bulk autophagy, mitophagy relies on the recruitment of distinct receptor proteins on the mitochondrial surface and engages specialized signaling cascades [35, 46].

The involvement of mitophagy in cancer progression is complex and not fully understood [47]. Similar to nonselective autophagy, its role appears context-dependent, varying with tumor type and stage [40, 47]. While mitophagy is implicated in tumor suppression during the initial stages of tumor development, it may facilitate tumor progression in later stages [48, 49]. Alterations in mitochondrial function are crucial drivers of tumor growth and malignancy [50]. Cancer cells often exhibit aerobic glycolysis, coupled with disrupted mitochondrial quality control, altered redox states, increased ROS generation, and modulated apoptotic signaling to adapt to their microenvironment [51, 52]. The literature indicates that mitophagy can either promote or inhibit tumorigenesis, with deficiencies in mitophagy contributing to oncogenic stress and tumorigenesis [53–55]. Despite its potential significance, targeting mitophagy for therapeutic purposes remains an underexplored area [53, 56].

Compounds obtained from various natural sources have garnered considerable interest in biomedical research due to their potential benefits for health [57]. Research into the potential cancer-preventive and therapeutic roles of various natural compounds has rapidly expanded. Numerous natural compounds have been shown to exert a substantial effect on cancer cell apoptosis and exhibit anticancer properties by modulating mitophagy. Recent studies have increasingly elucidated the critical role these naturally occurring substances play in cancer prevention and treatment [58, 59].

In this study, we determined that cis-3-hexen-1-ol suppressed tumor growth and cellular phenotypes both in vitro (Fig. 1) and in vivo (Fig. 7) by screening natural product libraries. The results of transcriptome sequencing (Fig. 2A,) suggest that mitophagy may be the mechanism by which cis-3-hexen-1-ol inhibits BLCA, which was supported by TEM (Fig. 2B, Fig. S2A), immunofluorescence (Fig. 2C, D; Fig S2B,C) and WB (Fig. 2E, F, Fig. S2B-C) assays. While these findings support activation of mitophagy-associated processes, we acknowledge that the current evidence is largely based on static observations and does not directly assess mitophagic flux. Therefore, we cannot fully exclude the possibility that these changes reflect mitochondrial damage accompanied by autophagosome recruitment rather than complete mitochondrial degradation. Future studies employing flux-based assays, such as lysosomal inhibition with bafilomycin A1 or chloroquine, or mitophagy-specific reporters (e.g., mt-Keima or mito-QC), as well as genetic perturbation of key mitophagy regulators, will be necessary to definitively establish functional mitophagy and its mechanistic contribution to the observed antitumor effects. The transcriptomic analysis highlighted enrichment of mitophagy-related pathways were not fully explored in this study. Moreover, we observed decreased mitochondrial mass (Fig. 3D, I), MMP (Fig. 3C, H, Fig. S3B), and baseline OCR (Fig. 3F, G) in the cells, along with increased MitoSOX (Fig. 3B, G, Fig. S3A) levels and mitochondrial fragmentation (Fig. 3A, F), after cis-3-hexen-1-ol treatment of BLCA cells. To verify the results of the rescue experiment, BLCA cells were pretreated with 3 MA, a typical mitophagy inhibitor [23] for 24 hours. These findings are consistent with mitochondrial dysfunction following cis-3-hexen-1-ol treatment. Notably, mitochondrial fragmentation, ROS accumulation, and membrane depolarization can also arise downstream of apoptotic signaling. Although the Seahorse assay indicates impaired oxidative phosphorylation, clearer normalization strategies and complementary assessment of mitochondrial content (e.g., mtDNA copy number or citrate synthase activity) would help distinguish between reduced mitochondrial function and decreased mitochondrial mass. Moreover, while TEM analysis revealed morphological alterations, quantitative ultrastructural evaluation was not performed and could further strengthen these observations. We observed that the proliferation (Fig. 4A–C), migration (Fig. 4D, E), and invasive capabilities (Fig. 4F) of BLCA cells were significantly reduced in the group pretreated with 3 MA. Additionally, the mitochondrial mass (Fig. 4G) and function (Fig. 4H, I) were restored to normal levels. 3-methyladenine (3-MA) is a broad inhibitor of class III PI3K and does not specifically target mitophagy. While it is widely used to inhibit autophagic flux, the observed rescue effects may reflect broader cytoprotective mechanisms rather than selective inhibition of mitophagy. More specific approaches, such as genetic perturbation of key mitophagy regulators (e.g., PINK1, Parkin, BNIP3) or alternative pharmacological modulators, would help further clarify the role of mitophagy in this context.

Our findings suggest that cis-3-hexen-1-ol induces a mitochondrial stress response characterized by impaired oxidative phosphorylation, increased ROS production, and activation of AMPK signaling, which is commonly recognized as a central sensor of cellular energy status [60]. In this context, AMPK activation may not be exclusively driven by PCK2 but could also arise as a consequence of metabolic stress induced by mitochondrial dysfunction. Furthermore, although our data support the involvement of mitophagy-associated processes, the role of mitophagy in bladder cancer is likely context-dependent, potentially contributing to either tumor suppression or adaptive survival depending on the cellular environment and stage of disease. These considerations highlight that the observed signaling changes may reflect an integrated stress response rather than a strictly linear pathway, and underscore the need for further studies to dissect the precise mechanistic relationships

To explore how cis-3-hexen-1-ol affects the mitophagy in BLCA cells, we reviewed the results of the gene heatmap (Fig. 5A) and found that the change in PCK2 was the most significant, with a 2.852-fold increase. The molecular docking results revealed that the binding energy between cis-3-hexen-1-ol and PCK2 was −4.4 kcal/mol (Fig. 5B), suggesting a potential binding interaction between the two. The docking binding energy is modest and that computational predictions alone are insufficient to establish a definitive functional interaction. In this study, molecular docking was used as a supportive approach and was complemented by biochemical assays, including co-immunoprecipitation and in vitro kinase assays. Nevertheless, further structural and biophysical studies will be required to fully validate this interaction. Furthermore, immunofluorescence suggested a significant increase in colocalization after cis-3-hexen-1-ol treatment (Fig. 5C; Fig S6). PCK2 is located primarily in mitochondria, and is a subtype of phosphoenolpyruvate carboxykinase (PEPCK), a crucial enzyme in gluconeogenesis [61]. Recently, studies have shown that PCK2 can regulate autophagy [62] and mitophagy [24, 25] through the AMPK signaling pathway. The WB results revealed a decreasing trend in the expression of PCK2 and p-AMPK, as well as a decline trend in the expression of p-mTOR, which was time- and dose-dependent (Fig. 5D, E). The expression of p-AMPK, p-mTOR and mitophagy marker proteins trended in the opposite direction when PCK2 was knocked down in BLCA cells (Fig. 5F,G). Given the mitochondrial localization of PCK2, we investigated whether AMPK activation occurs through metabolic intermediates or via a direct protein-protein interaction. Treatment of T24 and 5637 cells with phosphoenolpyruvate (PEP), the immediate catalytic product of PCK2, induced a modest dose-dependent increase in AMPK Thr172 phosphorylation; however, the magnitude of this activation was weaker than that observed with cis-3-hexen-1-ol, suggesting that PCK2-mediated AMPK activation is not primarily dependent on downstream metabolic products (Fig. 6A). We next examined whether PCK2 and AMPK interact spatially within BLCA cells. Immunofluorescence analysis revealed clear colocalization of PCK2 and AMPK, which was markedly enhanced upon cis-3-hexen-1-ol treatment, supporting a potential functional association between these proteins (Fig. 6B, Fig S5A). Consistently, co-immunoprecipitation assays demonstrated that endogenous PCK2 physically associates with AMPK in T24, 5637, and HEK-293T cells, whereas no interaction was detected in IgG controls, confirming the specificity of the PCK2-AMPK complex (Fig. 6C). Notably, the observed colocalization of PCK2 with mitochondrial marker TOM20 and with AMPK suggests a potential mitochondrial signaling context following cis-3-hexen-1-ol treatment. However, AMPK is known to translocate to mitochondria under conditions of metabolic stress, where it contributes to mitochondrial quality control and mitophagy [63, 64]. Therefore, the increased PCK2-AMPK colocalization observed in this study may also reflect stress-induced recruitment of AMPK to mitochondria, rather than exclusively indicating a direct upstream regulatory interaction. These findings support a functional association between PCK2 and AMPK within a mitochondrial context, while the precise mechanistic hierarchy remains to be further defined.

Importantly, the use of highly purified recombinant PCK2 and AMPK proteins (Fig. S5) enabled direct demonstration of PCK2-mediated AMPK phosphorylation, supporting the possibility that PCK2 may function as an upstream regulator independent of metabolic intermediates. To determine whether PCK2 directly phosphorylates AMPK, we performed in vitro kinase assays using purified recombinant proteins. GST-PCK2 robustly induced phosphorylation of AMPK-HIS, whereas GST alone failed to do so, providing biochemical evidence supporting PCK2-mediated phosphorylation of AMPK (Fig. 6D; Fig S5B). In line with previous reports, PCK2 also exhibited autophosphorylation activity, a characteristic property of this enzyme [65, 66]. Finally, molecular docking simulations revealed stable binding conformations between PCK2 and both AMPKα1 and AMPKα2, with predicted interaction interfaces involving conserved residues critical for kinase activity (Fig. 6E, F). Collectively, these findings support a functional association between PCK2 and AMPK activation through direct interaction and phosphorylation; however, the precise hierarchical relationship and the extent to which cis-3-hexen-1-ol directly targets PCK2 require further clarification. Furthermore, it remains possible that PCK2 upregulation reflects a secondary response to metabolic stress, and that AMPK activation may occur upstream of or in parallel with PCK2 regulation.

The relative molecular dynamics simulation results are detailed in the Supplementary Results, suggesting potential binding interactions between them. However, although our in vitro kinase assay indicates that PCK2 can phosphorylate AMPK. The in vitro kinase assays demonstrate that PCK2 can directly phosphorylate AMPK. Consequently, additional phospho-proteomic or site-directed mutagenesis studies will be required to conclusively establish PCK2-mediated AMPK phosphorylation in vivo. The use of established cell lines and immunodeficient mouse models does not fully recapitulate the complexity of the human tumor microenvironment, particularly immune interactions. Future studies incorporating patient-derived organoids, PDX models, and immunocompetent systems will be essential to validate the clinical relevance of these findings. Therefore, PCK2 may engage in additional interactions under certain conditions to form more interactions with AMPK. Subsequently, molecular dynamics simulations (Fig. S4A-R) demonstrated the possibility of interactions between the three entities. Finally, we used T24 tumor-bearing nude mouse models and lung metastasis models to further verify the results, which were consistent with the previous findings (Fig. 7). However, as these analyses were performed on bulk tumor lysates, they do not provide spatial or cell-type specific information regarding pathway activation. Therefore, it remains unclear whether the observed signaling changes occur predominantly within tumor cells or reflect contributions from the tumor microenvironment. Approaches such as immunohistochemistry or immunofluorescence-based localization would help further resolve the spatial and cellular context of these signaling events. Given its natural origin and ability to target mitochondrial metabolism and mitophagy, cis-3-hexen-1-ol may represent a promising phytotherapeutic lead or adjuvant candidate for bladder cancer treatment. However, the pharmacokinetic properties, metabolic stability, and long-term toxicity of cis-3-hexen-1-ol were not evaluated in this study. Future investigations focusing on pharmacological profiling and optimization will be essential for clinical translation. In addition, as a volatile plant-derived compound widely used in fragrance and ecological applications, its physicochemical properties may pose challenges for therapeutic development, including limited stability, bioavailability, and systemic delivery. Furthermore, although our study demonstrates inhibitory effects in short-term assays and colony formation experiments, additional evaluation using long-term growth models, such as three-dimensional spheroid cultures or extended clonogenic assays, will be necessary to determine whether these effects reflect durable tumor suppression. These aspects will be important considerations for future translational studies.

Conclusions

To our knowledge, this study provides a new insight into the therapeutic potential of cis-3-hexen-1-ol as a phytotherapeutic candidate for bladder cancer. We demonstrate that cis-3-hexen-1-ol inhibits the growth, proliferation, and migration of BLCA cells by disrupting mitochondrial function and activating mitophagy. Mechanistically, this mitophagic response is mediated through the PCK2/AMPK/mTOR signaling axis. Our findings further indicate that PCK2 regulates AMPK activation predominantly through a direct phosphorylation mechanism rather than through downstream metabolic intermediates. Genetic silencing of PCK2 or pharmacological inhibition of mitophagy markedly attenuated the cytotoxic and pro-mitophagic effects of cis-3-hexen-1-ol, highlighting mitophagy as a potential therapeutic target in BLCA.

Despite these advances, additional studies are warranted. Future work should aim to identify selective mitophagy modulators, further delineate the molecular events downstream of PCK2, and validate these mechanisms under physiological conditions. In particular, mapping the precise AMPK phosphorylation sites regulated by PCK2 in vivo will be essential to fully establish the endogenous signaling circuitry. Collectively, our findings position cis-3-hexen-1-ol as a promising natural-product candidate for the development of innovative therapeutic strategies against bladder cancer.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

J.P. and Z.T. conceived the study and designed the overall research strategy. Y.H. and S.L. conducted the experiments and curated the resulting data. J.P., Q.Z., and B.X. carried out the formal data analysis, interpreted the findings, and contributed to the methodological development. J.P., Q.Z., and B.X. prepared the original draft of the manuscript, while J.P. and Z.T. provided critical revisions and supervised all stages of the project. All authors contributed to reviewing and editing the manuscript, approved the final version, and agree to be accountable for the integrity and accuracy of the work.

Funding

This work was supported by the Natural Science Foundation of Hunan Province (S2024JJQYLH09) and Jishou University Research Project (JDZD25038).

Data availability

Further details and additional raw data are available from the corresponding author upon reasonable request. For any restrictions regarding access to specific data, please contact the corresponding author.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Laboratory Animal Ethics Committee of Xiangya Hospital, Central South University (Approval No. 202,412,259; approved on January 3, 2025). All procedures were conducted in accordance with the ARRIVE 2.0 guidelines and complied with institutional guidelines for the care and use of laboratory animals. Euthanasia procedures were performed following the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals. This study did not involve human participants; therefore, the Declaration of Helsinki is not applicable.

Consent for publication

All authors agree to publish

Competing interests

The authors declare no competing interests.