γ-Elemene Impairs Mitochondrial Biogenesis in Breast Cancer Cells by Upregulating GCN5-Mediated PGC-1α Acetylation

Abstract

Mitochondrial biogenesis represents a promising therapeutic target in triple-negative breast cancer (TNBC) due to its essential role in cancer cell metabolism and survival. The natural compound γ-Elemene exhibits potent anti-tumor activity, but its effects on mitochondrial regulation in TNBC remain unclear. In this study, we demonstrate that γ-Elemene induces dose-dependent cytotoxicity in MDA-MB-468 and HCC1806 TNBC cells while significantly impairing mitochondrial function, as shown by reduced membrane potential, oxidative phosphorylation capacity, and ATP production. γ-Elemene treatment markedly suppressed mitochondrial biogenesis, decreasing mitochondrial DNA content and downregulating key mitochondrial genes and proteins. These effects were associated with reduced expression of the master regulators NRF1 and TFAM, but independent of PGC-1α expression levels. Mechanistically, γ-Elemene upregulated the acetyltransferase GCN5, leading to enhanced PGC-1α acetylation. This upregulation occurs primarily through increased GCN5 transcription. Genetic ablation of GCN5 completely reversed γ-Elemene-induced PGC-1α acetylation and restored mitochondrial biogenesis and cell viability, establishing a critical role for GCN5 in mediating these effects. Our findings reveal a novel mechanism whereby γ-Elemene disrupts mitochondrial function in TNBC through GCN5-mediated PGC-1α acetylation, providing new insights into its anti-cancer properties and potential therapeutic applications against TNBC.

INTRODUCTION

Triple-negative breast cancer (TNBC) is a highly aggressive and clinically challenging subtype of breast cancer, defined by the lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) (Zhu et al., 2025). Representing approximately 15-20% of all breast cancer cases, TNBC is associated with a poor prognosis, high rates of recurrence and metastasis, and limited treatment options due to the absence of well-defined molecular targets (Ensenyat-Mendez et al., 2021; Chen et al., 2025b). TNBC cells exhibit heightened reliance on mitochondrial metabolism for energy production and survival, making mitochondrial pathways a promising therapeutic target (Wang et al., 2020). In recent years, increasing attention has been paid to the metabolic reprogramming of TNBC cells, particularly in relation to mitochondrial function (Wang et al., 2020; Xu et al., 2021; Chen et al., 2025a). Mitochondria are not only the central organelles for ATP production through oxidative phosphorylation, but also play essential roles in regulating reactive oxygen species (ROS) levels, intrinsic apoptosis, and cellular biosynthesis—all of which are critical for cancer cell survival and proliferation (Spinelli and Haigis, 2018; Nolfi-Donegan et al., 2020; Suomalainen and Nunnari, 2024).

Mitochondrial biogenesis, the cellular process that increases mitochondrial mass and function, is governed by a coordinated transcriptional network. Among the key regulators of this process is peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), which orchestrates the expression of downstream targets such as nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) (Jornayvaz and Shulman, 2010; Popov, 2020; Liu et al., 2023). PGC-1α has emerged as a master regulator of cellular energy metabolism and mitochondrial homeostasis. Dysregulation of PGC-1α expression or activity has been implicated in the progression and therapy resistance of various cancers, including TNBC (Fernandez-Marcos and Auwerx, 2011; Wenz, 2013; Abu Shelbayeh et al., 2023). Moreover, post-translational modifications of PGC-1α, particularly acetylation, have been shown to significantly influence its transcriptional activity and, consequently, mitochondrial biogenesis (Housley et al., 2009; Luo et al., 2019).

γ-Elemene, a naturally occurring sesquiterpene derived from Curcuma wenyujin, has been widely studied for its antitumor effects in several malignancies, such as glioma, lung cancer, and breast cancer. It has been shown to inhibit tumor growth through multiple mechanisms, including the induction of apoptosis, inhibition of proliferation and metastasis, and suppression of angiogenesis (Tan et al., 2021; Song et al., 2023; Wu et al., 2025). Recent studies suggest γ-Elemene’s efficacy in vivo, supporting its therapeutic potential (Tan et al., 2021). Despite these promising findings, the effects of γ-elemene on mitochondrial function and metabolic regulation in TNBC cells remain largely unexplored. Given the central role of mitochondrial biogenesis in tumor cell adaptation and survival, targeting this pathway offers a promising therapeutic approach. We hypothesized that γ-elemene exerts its anticancer effects in TNBC by modulating mitochondrial biogenesis through the regulation of PGC-1α activity. Specifically, we investigated whether γ-elemene disrupts mitochondrial biogenesis by promoting the acetylation and inactivation of PGC-1α. This study aims to elucidate the underlying mechanisms by which γ-elemene impacts mitochondrial metabolism in TNBC, thereby providing new insights into the development of mitochondrial-targeted therapies for this challenging breast cancer subtype.

MATERIALS AND METHODS

Cell culture, treatment, and transduction

Human triple-negative breast cancer MDA-MB-468 and HCC1806 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco, Thermo Fisher Scientific, USA) and HCC1806 cells in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin, and maintained at 37°C in a humidified 5% CO₂ incubator. MCF-7 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in DMEM with 10% FBS for comparative studies. γ-Elemene (≥98% purity, Dalian Jingang Pharmaceutical Co., Ltd., China) was dissolved in DMSO as a stock solution and diluted with culture medium for treatment. For gene knockdown, cells were transduced with adenoviral vectors carrying shRNA targeting GCN5 (Ad-shGCN5) or a non-targeting control (Ad-shNC) (Hanbio Biotechnology, Shanghai, China) at a multiplicity of infection (MOI) of 50. For NRF1 overexpression, cells were transduced with adenoviral NRF1 (Ad-NRF1, Hanbio Biotechnology) at an MOI of 50. After 48 h of infection, cells were collected for subsequent experiments.

Cell viability, LDH release assay, and γ-glutamyl transpeptidase (GGT)

Cell viability was determined using the CCK-8 kit (Dojindo, Japan) according to the manufacturer’s instructions. MDA-MB-468 cells and HCC 1806 cells were seeded into 96-well plates (5×10³ cells/well) and treated for 48 h. Absorbance at 450 nm was measured using a microplate reader (BioTek Synergy H1, USA). LDH and GPT activities in culture supernatants were quantified using commercial kits (Nanjing Jiancheng Bioengineering Institute, China) according to the protocols provided. Absorbance was recorded at 450 nm and 510 nm, respectively.

RH123 staining

Mitochondrial membrane potential (ΔΨm) was assessed using Rhodamine 123 (RH123; Sigma-Aldrich, USA). Cells were incubated with 10 μM RH123 at 37°C for 30 min in the dark. After washing with PBS, fluorescence was visualized using a fluorescence microscope (excitation 488 nm, emission 525 nm).

Determination of complex IV activity

Complex IV activity was measured using the Complex IV Enzyme Activity Microplate Assay Kit (Abcam, ab109911, UK). Cell lysates were prepared according to the manufacturer’s instructions. Absorbance was read kinetically at 550 nm, and the enzymatic activity was normalized to total protein concentration measured by a BCA assay (Beyotime Biotechnology, China).

ATP production measurement

Cellular ATP content was quantified using the Enhanced ATP Assay Kit (Beyotime Biotechnology, China) based on a luciferase/luciferin bioluminescence method. Luminescence intensity was measured using a microplate luminometer (Promega GloMax, USA) and normalized to total protein levels.

Measurement of mitochondrial respiration

The oxygen consumption rate (OCR) was analyzed using the Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies, USA). Cells were seeded at 20,000 cells/well in XF96 microplates and treated with γ-Elemene. Mitochondrial respiration parameters, including basal respiration, ATP-linked respiration, and maximal respiration, were assessed using the Seahorse XF Cell Mito Stress Test Kit (Agilent).

Assessment of mtDNA/nDNA

Total genomic DNA was extracted using a genomic DNA purification kit (Qiagen, Germany). Quantitative real-time PCR (qPCR) was performed to assess mtDNA copy number using primers targeting mitochondrial ND1 and nuclear β-actin genes. The mtDNA/nDNA ratio was calculated using the 2^-ΔΔCt method with SYBR Green Master Mix (Takara, Japan) on a CFX96 Real-Time PCR System (Bio-Rad, USA).

MitoTracker red staining

Mitochondrial mass was evaluated using MitoTracker Red CMXRos (Invitrogen, Thermo Fisher Scientific). Cells were incubated with 100 nM MitoTracker Red for 30 min at 37°C in the dark, washed with PBS, and observed using a fluorescence microscope (Olympus IX73, Japan). Fluorescence intensity was also quantified using a microplate reader (excitation 579 nm, emission 599 nm).

Real-time PCR

Total RNA was extracted using Trizol reagent (Invitrogen, USA), and reverse transcription was performed with the PrimeScript RT Reagent Kit (Takara, Japan). qRT-PCR was conducted using SYBR Premix Ex Taq II (Takara) on a Bio-Rad CFX96 system. GAPDH was used as an internal control. Primers for GCN5 (Forward: 5’- GCAAGGCCAATGAAACCTGTA-3’, Reverse: 5’- TCCAAGTGGGATACGTGGTCA-3’), NRF1 (Forward: 5’- AGGAACACGGAGTGACCCAA-3’, Reverse: 5’- TATGCTCGGTGTAAGTAGCCA-3’), and TFAM (Forward: 5’- ATGGCGTTTCTCCGAAGCAT-3’, Reverse: 5’-TCCGCCCTATAAGCATCTTGA-3’) were sourced from PrimerBank (https://pga.mgh.harvard.edu/primerbank/).

Western blot

Cells were lysed in RIPA buffer (Beyotime, China) supplemented with protease and phosphatase inhibitors (Roche, Switzerland). Proteins (30-40 μg/lane) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). After blocking with 5% non-fat milk, membranes were incubated overnight at 4°C with specific primary antibodies (anti-COX1, Abcam, ab14705; anti-COX IV, Cell Signaling Technology, #4850; anti-GCN5, Cell Signaling Technology, #3305), followed by HRP-conjugated secondary antibodies (Cell Signaling Technology, USA). Signals were visualized using enhanced chemiluminescence (ECL) reagents (Thermo Fisher) and imaged with the ChemiDoc XRS+ System (Bio-Rad).

Co-immunoprecipitation (Co-IP)

Co-IP assays were conducted to evaluate PGC-1α acetylation. Cells were lysed with IP lysis buffer (Beyotime, China) and incubated overnight at 4°C with anti-PGC-1α antibody (Abcam, ab54481), followed by Protein A/G agarose bead incubation (Santa Cruz Biotechnology, USA) for 4 h. After extensive washing, the immune complexes were analyzed by Western blot using anti-acetyl-lysine antibody (Cell Signaling Technology, #9441).

Statistical analysis

All experiments were performed in triplicate unless otherwise stated. Data are presented as mean ± standard deviation (SD). Statistical comparisons were conducted using ANOVA followed by Tukey’s post hoc test or Student’s t-test using GraphPad Prism 9.0 (GraphPad Software, USA). A p-value <0.05 was considered statistically significant.

RESULTS

γ-elemene exhibits dose-dependent cytotoxicity in triple negative breast cancer cells

To investigate the cytotoxic effects of γ-Elemene (Fig. 1A) on TNBC cells, MDA-MB-468 and HCC1806 cells were treated with increasing concentrations (0-800 mg/L) for 48 h. Cell viability was significantly reduced in a dose-dependent manner, decreasing to 15% and 23% of control at the highest concentration (Fig. 1B, Supplementary Fig. 1A). Concurrently, lactate dehydrogenase (LDH) release, a marker of membrane integrity loss, was significantly elevated (Fig. 1C). γ-Glutamyl transpeptidase (GPT) levels also increased in response to γ-Elemene, further indicating cellular damage (Fig. 1D). Based on these results, 100 and 200 mg/L γ-Elemene were used for subsequent studies.

Fig. 1.

Cytotoxicity of γ-Elemene in MDA-MB-468 TNBC cells. MDA-MB-468 cells were stimulated with 0, 25, 50, 100, 200, 400, 800 mg/L γ-Elemene for 48 h. (A) Molecular structure of γ-Elemene. (B) Cell viability of MDA-MB-468 cells. (C) LDH release from MDA-MB-468 cells. (D) The level of γ-glutamyl transpeptidase (GPT) (*p<0.05, **p<0.01, ***p<0.005, ****p<0.001 vs. Control group).

γ-elemene induces mitochondrial dysfunction in triple negative breast cancer cells

Given the critical role of mitochondria in cell survival, we assessed mitochondrial function following γ-Elemene treatment (0, 100, 200 mg/L, 48 h). Mitochondrial membrane potential (MMP) measured by RH123 staining was significantly decreased in γ-Elemene-treated MDA-MB-468 cells, indicating mitochondrial depolarization (Fig. 2A). In HCC1806 cells, γ-Elemene similarly reduced MMP in a dose-dependent manner, with approximately 55% reduction at 200 mg/L (Supplementary Fig. 1B). Comparative analysis showed greater MMP reduction in MDA-MB-468 and HCC1806 than in MCF-7 cells at 200 mg/L (Supplementary Fig. 2). Enzymatic activity of mitochondrial respiratory chain Complex IV was also markedly inhibited (Fig. 2B). Furthermore, oxygen consumption rate (OCR) analyses revealed significant reductions in both basal and maximal respiration (Fig. 2C, 2D). These impairments culminated in a substantial decrease in cellular ATP production (Fig. 2E).

Fig. 2.

γ-Elemene induced the “Loss of mitochondrial function” in MDA-MB-468 TNBC cells. MDA-MB-468 cells were stimulated with 0, 100 and 200 mg/L γ-Elemene for 48 h. (A) The levels of mitochondrial membrane potential (MMP) were measured using RH123. (B) Complex IV activity. (C) Basal respiration: OCR. (D) Maximal respiration: OCR. (E) ATP production (**p<0.01, ***p<0.005 vs. Control group).

γ-elemene suppresses mitochondrial biogenesis

Next, we examined the effects of γ-Elemene on mitochondrial biogenesis. The mtDNA to nuclear DNA ratio (mtDNA/nDNA) in MDA-MB-468 cells was significantly reduced in a dose-dependent manner (Fig. 3A). In HCC1806 cells, γ-Elemene treatment similarly decreased the mtDNA/nDNA ratio by approximately 50% at 200 mg/L, confirming impaired mitochondrial biogenesis across TNBC cell lines (Supplementary Fig. 1C). Correspondingly, transcription of mitochondrial marker genes TOMM22, ATP5D, and NDUFS3 was downregulated (Fig. 3B), and protein expression of Complex IV subunit NDUFA4, COX1, and COX IV in MDA-MB-468 cells was decreased (Fig. 3C). Consistent with these findings, mitochondrial mass assessed by MitoTracker red staining was significantly diminished following γ-Elemene treatment (Fig. 3D).

Fig. 3.

γ-Elemene impaired mitochondrial biogenesis of MDA-MB-468 TNBC cells. MDA-MB-468 cells were stimulated with 0, 100, and 200 mg/L γ-Elemene for 48 h. (A) The ratio of mtDNA to nDNA (mtDNA/nDNA). (B) The mRNA expression of TOMM22, ATP5D, and NDUFS3. (C) Protein levels of the Complex IV subunit (NDUFA4, COX1, and COX IV). (D) Mitochondrial mass was measured with MitoTracker red staining (**p<0.01, ***p<0.005 vs. Control group). NDUFA4, 10 KDa; COX1, 68 KDa; COXIV, 17 KDa; β-actin, 43 KDa.

γ-elemene downregulates NRF1 and TFAM expression

Mitochondrial biogenesis is tightly regulated by nuclear transcription factors. We observed significant suppression of NRF1, TFAM, and TFB1M expression at both mRNA and protein levels after γ-Elemene exposure (Fig. 4A, 4B) in MDA-MB-468 cells. To assess the role of NRF1, we overexpressed it in MDA-MB-468 cells treated with 200 mg/L γ-Elemene. NRF1 overexpression partially restored the expression of TFAM and TFB1M (Fig. 4C) as well as the mtDNA/nDNA ratio (Fig. 4D), though not to the level of the control group. These results suggest that PGC-1α acetylation remains the primary driver of the observed effects. These results suggest that γ-Elemene inhibits the transcriptional machinery necessary for mitochondrial biogenesis.

Fig. 4.

γ-Elemene inhibited the expression of nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) in MDA-MB-468 TNBC cells. MDA-MB-468 cells were stimulated with 0, 100, and 200 mg/L γ-Elemene for 48 h. (A) The mRNA of NRF1, TFAM, and TFB1M. (B) Protein expression of NRF1, TFAM, and TFB1M. (C, D) NRF1 overexpression partially restores TFAM and TFB1M expression and the mtDNA/nDNA ratio in γ-Elemene-treated cells (**p<0.01, ***p<0.005 vs. Control group; ^##p<0.01 vs. 200 mg/L γ-Elemene group). Nrf1, 68 KDa; TFAM, 24 KDa; TFB1M, 40 KDa; β-actin, 43 KDa.

γ-elemene does not affect PGC-1α expression but enhances its acetylation via GCN5 upregulation

PGC-1α is a master regulator of mitochondrial biogenesis. Notably, γ-Elemene treatment did not alter PGC-1α mRNA or total protein expression in MDA-MB-468 cells (Fig. 5), indicating that its inhibitory effect is not due to decreased PGC-1α abundance. Instead, the increase in GCN5 expression induced by γ-Elemene was observed at both the transcriptional (mRNA, Fig. 6A) and translational (protein, Fig. 6B) levels. Interestingly, cycloheximide chase assays showed no change in GCN5 protein stability, indicating transcriptional upregulation (Data not shown). Co-immunoprecipitation assays demonstrated that γ-Elemene dose-dependently enhanced PGC-1α acetylation (Fig. 6C), a post-translational modification known to repress PGC-1α transcriptional activity. In HCC1806 cells, a similar dose-dependent increase in PGC-1α acetylation was observed, with an increase at 200 mg/L compared to control (Supplementary Fig. 1D).

Fig. 5.

γ-Elemene had no impact on the expression of PGC-1ɑ in MDA-MB-468 TNBC cells. MDA-MB-468 cells were stimulated with 0, 100, and 200 mg/L γ-Elemene for 48 h. (A) The mRNA levels of PGC-1ɑ. (B) Protein levels of PGC-1ɑ. PGC-1α, 100 KDa; β-actin, 43 KDa.

Fig. 6.

γ-Elemene induces GCN5 upregulation and stimulates PGC-1α acetylation in MDA-MB-468 TNBC cells. MDA-MB-468 cells were stimulated with 0, 100, and 200 mg/L γ-Elemene for 48 h. (A) The levels of GCN5 as measured by real time PCR. (B) The levels of GCN5 as measured by western blot analysis. (C) The levels of acetylated PGC-1α as measured by co-Immunoprecipitation (Co-IP) (**p<0.01, ***p<0.005 vs. Control group). GCN5, 94 KDa; β-actin, 43 KDa; Ace-lysine PGC-1α, 100 KDa; PGC-1α, 100 KDa.

Silencing of GCN5 reverses γ-elemene-induced PGC-1α acetylation and restores mitochondrial biogenesis

To confirm the role of GCN5 in mediating γ-Elemene’s effects, GCN5 was knocked down using adenoviral shRNA in MDA-MB-468 cells before γ-Elemene treatment. Western blot confirmed efficient GCN5 silencing (Fig. 7A). Importantly, GCN5 knockdown abolished γ-Elemene-induced PGC-1α acetylation (Fig. 7B). Furthermore, restoration of NRF1, TFAM, and TFB1M mRNA levels (Fig. 7C) and mtDNA/nDNA ratio (Fig. 7D) was observed, indicating rescue of mitochondrial biogenesis. To confirm the role of GCN5 in mediating γ-Elemene’s effects, we knocked down GCN5 using adenoviral shRNA in MDA-MB-468 cells prior to γ-Elemene treatment. Western blot analysis confirmed efficient GCN5 silencing (Fig. 7A). Importantly, GCN5 knockdown abolished γ-Elemene-induced PGC-1α acetylation (Fig. 7B). Furthermore, it restored the mRNA levels of NRF1, TFAM, and TFB1M (Fig. 7C) and the mtDNA/nDNA ratio (Fig. 7D), indicating the rescue of mitochondrial biogenesis. Correspondingly, GCN5 knockdown reversed the γ-Elemene-induced reduction in mitochondrial membrane potential (MMP), suggesting improved mitochondrial function (Fig. 7E). Notably, it also restored cell viability (Fig. 7F) and reduced LDH release (Fig. 7G), suggesting that GCN5 depletion and inhibition of PGC-1α acetylation attenuates γ-Elemene-induced cytotoxic effects. These data strongly suggest that γ-Elemene suppresses mitochondrial biogenesis via GCN5-mediated PGC-1α acetylation, leading to mitochondrial dysfunction and cytotoxicity in TNBC cells.

Fig. 7.

Silencing of GCN5 abolished the effects of γ-Elemene in PGC-1α acetylation and mitochondrial biogenesis in MDA-MB-468 TNBC cells. MDA-MB-468 cells were transduced with Ad-viral shGCN5, followed by stimulation with 200 mg/L γ-Elemene. (A) Western blot analysis revealed successful knockdown of GCN5. (B) The levels of acetylated PGC-1α as measured by co-Immunoprecipitation (Co-IP). (C) The mRNA of NRF1, TFAM, and TFB1M. (D) The ratio of mtDNA to nDNA (mtDNA/nDNA). (E) The levels of MMP. (F) The levels of cell viability. (G) The levels of LDH release (***p<0.005 vs. Control group; ^##p<0.01, ^###p<0.005 vs. γ-Elemene+sh-NC group). GCN5, 97 KDa; β-actin, 43 KDa; Ace-lysine PGC-1α, 100 KDa; PGC-1α, 100 KDa.

DISCUSSION

This study is the first to demonstrate that γ-elemene suppresses mitochondrial biogenesis in TNBC MDA-MB-468 and HCC1806 cells by promoting GCN5-mediated acetylation of PGC-1α, thereby disrupting mitochondrial function and inhibiting cell proliferation. Our findings reveal that γ-elemene significantly reduces mitochondrial membrane potential, complex IV activity, ATP production, and mitochondrial DNA copy number. These effects are more pronounced in TNBC cells compared to luminal MCF-7 cells, highlighting TNBC’s mitochondrial dependency (Wang et al., 2020). Moreover, it downregulates the expression of key mitochondrial proteins, indicating a comprehensive impairment of mitochondrial function. These results align with previous studies highlighting mitochondrial dysfunction as a hallmark of metabolic stress and a key contributor to tumor progression (Roca-Portoles and Tait, 2021; Prabhu et al., 2023; Wen et al., 2025).

γ-Elemene reduced MMP by approximately 55% in TNBC cell lines MDA-MB-468 and HCC1806 (approximately 45% of control, p<0.005) but only 20% in non-TNBC MCF-7 cells (approximately 80% of control, p<0.05), as shown in Supplementary Fig. 2. This heightened mitochondrial sensitivity in TNBC cells, driven by GCN5-mediated PGC-1α acetylation, supports γ-Elemene’s potential as a targeted TNBC therapy.

Mitochondrial biogenesis is tightly regulated by transcriptional coactivators and factors such as PGC-1α, NRF1, and TFAM, which coordinate the replication and transcription of mitochondrial DNA (Picca and Lezza, 2015; Ploumi et al., 2017; Halling and Pilegaard, 2020). In our study, γ-elemene treatment markedly suppressed both mRNA and protein levels of NRF1 and TFAM, leading to compromised mitochondrial biogenesis. However, NRF1 overexpression only partially restored biogenesis, confirming that PGC-1α acetylation is the primary mechanism. These findings are consistent with the work of Nanjaiah et al., which demonstrated that reduced NRF1/TFAM expression leads to impaired mitochondrial replication and function (Nanjaiah and Vallikannan, 2019).

PGC-1α, as the master regulator of mitochondrial biogenesis, exerts its function largely through post-translational modifications, especially acetylation. GCN5, a known histone acetyltransferase, negatively regulates PGC-1α activity by enhancing its acetylation, thereby reducing its transcriptional coactivation potential (Fernandez-Marcos and Auwerx, 2011; Yao et al., 2016; Mihaylov et al., 2023). Our study demonstrates that γ-elemene upregulates GCN5 expression at the transcriptional level and significantly increases PGC-1α acetylation, leading to downregulation of downstream genes critical for mitochondrial biogenesis. Inhibition of GCN5 with shRNA restored cell viability and mitochondrial function, confirming the GCN5/PGC-1α axis’s role. Importantly, the total protein level of PGC-1α remained unchanged, suggesting that γ-elemene modulates its activity rather than its expression. However, the mechanism by which γ-elemene upregulates the expression of GCN5 is complicated. It might act through potential mechanisms such as modulation of transcription factors (e.g., NF-κB or STAT3) or alteration of the epigenetic landscape at the GCN5 promoter. Further studies are warranted to elucidate the precise upstream signaling pathways involved in this regulatory process.

To our knowledge, this is the first report identifying the GCN5/PGC-1α axis as a key target of γ-elemene in TNBC cells. These findings provide a novel mechanistic insight into how γ-elemene exerts its anti-cancer effects through metabolic reprogramming and mitochondrial disruption. Validation in HCC1806 cells, including cell viability (Supplementary Fig. 1A), MMP (Supplementary Fig. 1B), mtDNA/nDNA ratio (Supplementary Fig. 1C), and PGC-1α acetylation (Supplementary Fig. 1D), strengthens the generalizability of this mechanism across TNBC models. They also offer a promising therapeutic avenue for targeting mitochondrial metabolism in aggressive breast cancers.

Nonetheless, this study has certain limitations. The data are derived from in vitro experiments, and in vivo validation is warranted to confirm the anti-tumor efficacy and metabolic impact of γ-elemene in TNBC models. Previous studies have shown γ-Elemene’s efficacy in vivo in other cancers, supporting its potential (Tan et al., 2021). Future in vivo studies will assess tumor growth inhibition and mitochondrial effects in TNBC xenografts. Additionally, the influence of γ-elemene on other metabolic regulators and its potential interactions with the tumor microenvironment remain to be investigated. Future studies should explore whether γ-elemene affects mitochondrial dynamics, ROS generation, or immune cell infiltration in the tumor context.

In conclusion, γ-elemene inhibits mitochondrial biogenesis and disrupts energy metabolism in TNBC cells via GCN5-mediated acetylation of PGC-1α. These results not only deepen our understanding of the anti-tumor mechanisms of γ-elemene but also underscore the therapeutic potential of targeting mitochondrial biogenesis in triple-negative breast cancer.

This study is the first to reveal that γ-elemene suppresses mitochondrial biogenesis in triple-negative breast cancer (TNBC) cells by promoting GCN5-mediated acetylation of PGC-1α. This mechanism leads to impaired mitochondrial function, including reduced oxidative phosphorylation, membrane potential, and ATP production. γ-Elemene also decreases mitochondrial DNA content and downregulates key regulators such as NRF1 and TFAM. Although total PGC-1α levels remain unchanged, its functional inactivation through acetylation is a critical outcome. Notably, GCN5 knockdown reverses these effects, underscoring the central role of GCN5 in mediating γ-elemene’s mitochondrial impact.

These findings unveil a novel mitochondrial regulatory mechanism targeted by γ-elemene and support its potential as a mitochondrial-targeted therapeutic agent for TNBC. Future studies should investigate its in vivo efficacy, potential synergy with existing chemotherapeutics, and applicability in other cancer types or drug-resistant TNBC models.