Curcumin enhances ATG3-dependent autophagy and inhibits metastasis in cervical carcinoma

Abstract

Cervical carcinoma poses a significant health threat, with traditional treatments proving inadequate in advanced stages. Curcumin, a bioactive compound derived from turmeric, exhibits notable anti-inflammatory, antioxidant, and antineoplastic properties, potentially modulating autophagy, and metastasis in cancer cells. This study examines curcumin’s impact on autophagy and metastasis in cervical carcinoma, focusing on its interaction with autophagy-related gene 3 (ATG3). SiHa and HeLa cervical carcinoma cell lines were treated with curcumin, ATG3 knockdown (shATG3), and their combination. Cell migration was evaluated via wound healing assays, while cell proliferation was evaluated with CCK-8 assays. LC3 expression was assessed using immunofluorescence and western blotting. Molecular docking simulations identified curcumin’s binding interactions with key proteins. Curcumin and shATG3 significantly inhibited both cell migration and proliferation, with a synergistic effect observed when combined. LC3 expression was enhanced, indicating increased autophagy. Docking studies revealed curcumin’s potential binding to MMP2, MMP9, TGF-β, ATG3, LC3, and p62, suggesting modulation of these pathways. The combination of curcumin and ATG3 knockdown significantly inhibited cervical carcinoma cell migration and proliferation, while also enhancing autophagy, supporting the potential of curcumin as a therapeutic agent for cervical carcinoma. Further clinical research is needed to validate these findings.

Graphical abstract

Introduction

Cervical carcinoma is the fourth leading cause of mortality in women, with approximately 310,000 deaths globally each year, posing a severe threat to women’s health [1, 2]. While the promotion of the HPV vaccine has reduced the incidence in developed countries, it remains prevalent in low- and middle-income regions due to low vaccination coverage. Conventional treatments (surgery, chemotherapy, radiotherapy) are effective in early-stage cervical carcinoma but less so in advanced stages, where the risk of recurrence and metastasis is high [3] (Fig. 1B). Consequently, exploring new therapeutic mechanisms, targets, and methods for cervical carcinoma treatment is crucial.

Curcumin, a polyphenolic compound from the rhizomes of plants like Curcuma longa (Fig. 1A), exhibits a broad spectrum of pharmacological activities, including anti-inflammatory inflammatory [4], antioxidant [5], hypolipidemic [6], antihypertensive [7], antibacterial [8], hepatoprotective [6], and antitumor effects [9, 10]. Its therapeutic potential has been extensively studied in various cancers, including liver [11], gastric [12], lung [13], breast [14], prostate [15], colorectal [16], ovarian [17], and cervical cancers [18]. The updated hallmarks of cancer include 14 distinct characteristics, providing new insights for therapeutic interventions [19](Fig. 1C).

Autophagy, a cellular degradation process, intersects with many cancer hallmarks, influencing cellular metabolism, resistance to cell death, and phenotypic plasticity [20]. Understanding these pathways offers potential for targeted therapies. Curcumin’s role in cancer treatment involves evaluating clinical evidence and exploring its potential as a complementary agent alongside modern therapy. Listed as a third-generation cancer prevention drug in the United States [21], curcumin’s effects on cervical carcinoma include promoting apoptosis, inhibiting proliferation, metastasis, and invasion, and inducing autophagy [18]. This study aims to elucidate the mechanisms by which curcumin influences autophagy and metastasis in cervical carcinoma, promoting its potential as a therapeutic agent.

Fig. 1

Curcumin and Cervical Cancer: Mechanisms and Metastasis. (A) The source of curcumin, Curcuma longa, is shown with its flower and dried rhizome. The chemical structure of curcumin is illustrated. Curcumin, a bioactive compound extracted from turmeric. (B) Schematic representation of common metastasis sites for cervical cancer, including the lungs, liver, and distant lymph nodes. (C) The central hallmarks of cancer with a focus on autophagy-related pathways [19]

Fig. 2

Curcumin and ATG3 Knockdown Inhibit Migration and Proliferation of Cervical Cancer Cells. (A) Wound healing assay images of SiHa cells treated with control, ShATG3, curcumin, and ShATG3 combined with curcumin at 0, 24, and 48 h. The yellow lines indicate the wound edges. (B) Quantification of wound closure in Siha cells at 24 and 48 h. Wound closure percentage was significantly reduced in the ShATG3, curcumin, and ShATG3 + curcumin groups compared to the control group. (C) Wound healing assay images of HeLa cells treated with control, ShATG3, curcumin, and ShATG3 combined with curcumin at 0, 24, and 48 h. The yellow lines indicate the wound edges. (D) Quantification of wound closure in HeLa cells at 24 and 48 h. Wound closure percentage was significantly reduced in the ShATG3, curcumin, and ShATG3 + curcumin groups compared to the control group. (E) CCK-8 assays demonstrating reduced cell proliferation in both SiHa (left) and HeLa (right) cells following the combined treatment. Statistical significance is indicated by **P < 0.01, ***P < 0.001, ****P < 0.0001

Results

Curcumin and ATG3 knockdown synergistically inhibit both SiHa and HeLa cervical cancer cell migration and proliferation

The results clearly demonstrate that curcumin treatment and ATG3 knockdown (shATG3) exert significant inhibitory effects on the migration of cervical cancer cells (SiHa and HeLa), as evidenced by wound healing assays. Figure 2A and C illustrate representative images of wound closure at 0, 24, and 48 h in SiHa and HeLa cells, respectively. Both curcumin and shATG3 treatments individually impaired cell migration, with the combined treatment showing the most substantial inhibitory effect. Quantitative analyses of wound closure, presented in Fig. 2B and D, reveal that at 24 h, the Control group exhibited the highest wound closure percentage (SiHa: 32.2%; HeLa: 24.7%), followed by the Curcumin group (SiHa: 20.8%; HeLa: 16.7%), the shATG3 group (SiHa: 18.6%; HeLa: 16.6%), and the shATG3 + Curcumin group (SiHa: 17.3%; HeLa: 11.7%). By 48 h, the Control group continued to exhibit the highest closure rates (SiHa: 46.7%; HeLa: 46.5%), while the shATG3, Curcumin, and shATG3 + Curcumin groups displayed progressively lower closure percentages (SiHa: 36.5%, 31.2%, 24.7%; HeLa: 34.0%, 28.4%, 20.0%, respectively). These findings indicate that both shATG3 and curcumin, individually and in combination, significantly reduce the wound closure rate, suggesting a potent inhibitory effect on cell migration in both SiHa and HeLa cells. This underscores the therapeutic potential of curcumin and ATG3 inhibition in reducing cell migration in cervical cancer. Moreover, CCK-8 assays depicted in Fig. 2E further confirm that the combination of curcumin and shATG3 markedly reduces cell proliferation in both SiHa and HeLa cells, highlighting ATG3 as a viable therapeutic target in curcumin-enhanced cancer treatment strategies. Together, these results suggest that the combination of curcumin treatment and ATG3 knockdown may represent a promising therapeutic strategy for inhibiting migration and proliferation in cervical carcinoma.

Fig. 3

Curcumin and ATG3 Knockdown Modulate Autophagy in Cervical Cancer Cells (A) Immunofluorescence staining of LC3 in SiHa cells under control, ShATG3, curcumin, and ShATG3 combined with curcumin treatments. DAPI stains nuclei (blue), and LC3 is shown in green. Merged images indicate the co-localization of LC3 with DAPI. (B) Quantification of LC3 fluorescence intensity and protein levels in SiHa cells. The relative fluorescence intensity of LC3 significantly increased in curcumin and ShATG3 + curcumin groups compared to control and ShATG3 groups. Western blot analysis showing LC3-I and LC3-II levels in SiHa cells under different treatments. (C) Immunofluorescence staining of LC3 in HeLa cells under control, ShATG3, curcumin, and ShATG3 combined with curcumin treatments. DAPI stains nuclei (blue), and LC3 is shown in green. Merged images indicate the co-localization of LC3 with DAPI. (D) Quantification of LC3 fluorescence intensity and protein levels in HeLa cells. The relative fluorescence intensity of LC3 significantly increased in curcumin and ShATG3 + curcumin groups compared to control and ShATG3 groups. Western blot analysis showing LC3-I and LC3-II levels in HeLa cells under different treatments. Statistical significance is indicated by **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 4

Curcumin and ATG3 Knockdown Impact Protein Expression in Cervical Cancer Cells (A) Western blot analysis and quantification of MMP2, P62, and ATG3 protein levels in SiHa cells under control, ShATG3, curcumin, and ShATG3 combined with curcumin treatments. (B) Western blot analysis and quantification of MMP2, P62, and ATG3 protein levels in HeLa cells under control, ShATG3, curcumin, and ShATG3 combined with curcumin treatments. Statistical significance is indicated by **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Curcumin treatment and ATG3 knockdown significantly increase LC3 expression in both SiHa and HeLa cervical cancer cell lines

Explore the effects of curcumin and ATG3 knockdown (ShATG3) on LC3 expression in cervical cancer cells (SiHa and HeLa) using immunofluorescence staining and western blot analysis. Figure 3A shows representative immunofluorescence images of LC3 in SiHa cells, with DAPI staining the nuclei (blue) and LC3 (green) marking the autophagosomes. The merged images indicate the localization of LC3 within the cells. Figure 3B presents the quantification of the relative fluorescence intensity of LC3 and the relative protein levels of LC3 in SiHa cells. The results show a significant increase in LC3 fluorescence intensity in the curcumin (****P < 0.0001) and ShATG3 + curcumin (***P < 0.001) groups compared to the control and ShATG3 (*P < 0.05) groups. Western blot analysis shows corresponding increases in LC3-II protein levels, confirming enhanced autophagy.

Fig. 5

Molecular Docking Analysis of Curcumin with Key Autophagy and Metastasis Proteins (A) 2D interaction diagrams showing the binding of curcumin to MMP2, MMP9, TGF-β, ATG3, LC3, and P62. Key interactions, including hydrogen bonds and hydrophobic contacts, are indicated. (B) 3D binding poses of curcumin with MMP2, MMP9, TGF-β, ATG3, LC3, and P62, showing the spatial arrangement of interactions

Fig. 6

Schematic representation of curcumin’s dual role in inhibiting metastasis and enhancing autophagy in cervical cancer. Curcumin reduces pericyte detachment and recruitment, thereby decreasing angiogenesis and metastatic potential through the downregulation of PDGFRβ signaling. Additionally, curcumin enhances autophagy by modulating key proteins ATG3 and LC3, impacting the PI3K/AKT and MAPK/ERK pathways. These mechanisms highlight curcumin’s potential as a therapeutic agent in targeting both tumor growth and metastasis in cervical cancer, emphasizing its multifaceted approach to cancer treatment. Created with BioRender.com

Figure 3C shows representative immunofluorescence images of LC3 in HeLa cells under similar treatment conditions. Figure 3D provides the quantification of the relative fluorescence intensity of LC3 and the relative protein levels of LC3 in HeLa cells. The results show a significant increase in LC3 fluorescence intensity in the curcumin (****P < 0.0001) and ShATG3 + curcumin (****P < 0.0001) groups compared to the control and ShATG3 (**P < 0.01) groups. Western blot analysis confirms these observations with increased LC3-II levels. These results demonstrate that curcumin enhances autophagy in cervical cancer cells, and this effect is potentiated by ATG3 knockdown. The significant increase in LC3 expression, both in fluorescence intensity and protein levels, suggests a potential therapeutic role for curcumin in modulating autophagy in cervical cancer.

Curcumin and ATG3 knockdown modulate key proteins involved in autophagy and metastasis in cervical cancer cells

Figure 4A shows representative western blot images and quantification of MMP2, P62, and ATG3 protein levels in SiHa cells. MMP2 protein levels are significantly reduced by ShATG3 (**P < 0.01) and curcumin (****P < 0.0001) treatments compared to control. The combination treatment of ShATG3 and curcumin shows an additive effect (****P < 0.0001). P62 protein levels are also significantly reduced by ShATG3 (**P < 0.01), curcumin (**P < 0.01), and the combination treatment (****P < 0.0001) compared to control. ATG3 protein levels are significantly reduced in ShATG3 (***P < 0.001) and ShATG3 + curcumin (****P < 0.0001) groups compared to control. While Fig. 4B shows MMP2, P62, and ATG3 protein levels in HeLa cells. MMP2 protein levels are significantly reduced by ShATG3 (*P < 0.05), curcumin (**P < 0.01), and the combination treatment (****P < 0.0001) compared to control. P62 protein levels show no significant changes across the groups. ATG3 protein levels are significantly reduced in ShATG3 (**P < 0.01) and ShATG3 + curcumin (*P < 0.05) groups compared to control. These results highlight the potential of curcumin and ATG3 inhibition as therapeutic strategies in cervical cancer by modulating key proteins involved in autophagy and metastasis.

Curcumin shows potential binding affinity with several key proteins involved in autophagy and metastasis

Figure 5A displays 2D interaction diagrams, highlighting the hydrogen bonds and hydrophobic interactions between curcumin and each target protein. In MMP2, curcumin forms hydrogen bonds with Ala84 and other residues. In MMP9, it interacts with Glu427 among other contacts. TGF-β binding involves Lys232 and several hydrophobic contacts. Curcumin binds to autophagy-related proteins including ATG3 at Lys151, LC3 at Asp106, and P62 at Tyr128. The 3D poses (Fig. 5B) illustrate how curcumin fits within the active sites and the nature of the molecular interactions. These molecular docking results suggest that curcumin can interact with multiple targets involved in autophagy and metastasis, potentially modulating their activities, and contributing to its anticancer properties.

Discussion

Despite screening and vaccination, cervical carcinoma remains a major cause of mortality worldwide, with a 5-year OS rate of only 17% in metastatic/recurrent disease [22]. Therefore, there is an urgent need to identify new therapeutic combinations and mechanisms to address metastasis, prolong patient survival, and improve quality of life. Our study demonstrates that curcumin, a polyphenolic compound derived from turmeric, has significant potential as a therapeutic agent in cervical carcinoma by modulating autophagy and metastasis.

The wound healing assays reveal that curcumin treatment and ATG3 knockdown independently inhibit the migratory capacity of both SiHa and HeLa cervical cancer cell lines. Notably, the combination treatment exhibits a synergistic effect, suggesting that curcumin and ATG3 knockdown target overlapping pathways involved in cell migration [23]. These findings align with previous research indicating that curcumin can suppress cancer cell migration and invasion, potentially through the inhibition of matrix metalloproteinases (MMPs) and other migration-related proteins [24] [25]. In addition to these effects on cell migration, our study also reveals that curcumin and ATG3 knockdown significantly reduce cell proliferation [26, 27], as evidenced by CCK-8 assays. The reduction in cell viability was particularly pronounced when the treatments were combined, suggesting a potential cooperative interaction between curcumin and ATG3 knockdown that extends beyond migration to also impact cellular proliferation. This dual effect on both migration and proliferation underscores the therapeutic potential of targeting autophagy pathways alongside traditional anticancer strategies. The findings suggest that curcumin, particularly when used in conjunction with ATG3 inhibition, could serve as a potent approach to curtail both the spread and growth of cervical carcinoma cells.

A significant increase in LC3 expression following curcumin treatment and ATG3 knockdown, with the most pronounced effect observed in the combination treatment. Enhanced autophagy is evidenced by increased levels of LC3-II, a marker for autophagosome formation [28]. This enhancement of autophagy might contribute to the anti-tumor effects of curcumin by promoting the degradation of damaged cellular components and modulating cancer cell metabolism. Previous studies have suggested that autophagy can act as a double-edged sword in cancer, either suppressing tumorigenesis or promoting cancer cell survival depending on the context [29]. In our study, the promotion of autophagy appears to inhibit cancer cell migration, highlighting its potential therapeutic benefit. Western blot analyses demonstrate that curcumin and ATG3 knockdown significantly reduce the levels of MMP2 and ATG3 proteins in cervical cancer cells, with additive effects observed in combination treatments. The reduction in P62 levels by curcumin in SiHa cells, but not in HeLa cells, suggests a cell-line-specific response that warrants further investigation. These proteins are crucial in regulating autophagy and metastasis, and their modulation by curcumin supports its multifaceted role in cancer therapy [30]. Molecular docking studies provide further evidence for curcumin’s potential to interact with key proteins involved in autophagy and metastasis, including MMP2, MMP9, TGF-β, ATG3, LC3, and P62. These interactions suggest that curcumin can directly modulate the activity of these proteins, thereby influencing cancer cell behavior [31]. The binding affinity of curcumin to these targets underscores its capability to modulate multiple signaling pathways concurrently, which is advantageous in the context of complex diseases like cancer.

The dual role of curcumin in inhibiting tumor vascularization and modulating autophagy presents a compelling case for its inclusion in cancer treatment regimens. By preventing pericyte detachment and recruitment, curcumin reduces angiogenesis and metastasis, likely through the downregulation of PDGFRβ signaling [32]. Additionally, curcumin’s inhibition of the PI3K and mTOR pathways further supports its role in modulating autophagy and impacting key proteins such as ATG3 and LC3 [21]. In future, in vivo studies and clinical trials are essential to confirm the therapeutic efficacy of curcumin in cervical carcinoma. Investigating the combination of curcumin with other therapeutic agents could also provide a synergistic approach to cancer treatment, potentially overcoming the limitations of current therapies (Fig. 6).

Conclusion

In conclusion, our study highlights the promising potential of curcumin as a therapeutic agent in cervical carcinoma by enhancing ATG3-dependent autophagy and inhibiting metastasis. These findings contribute to the growing body of evidence supporting the use of curcumin in cancer therapy and underscore the need for further clinical research to fully realize its potential in treating cervical carcinoma.

Materials and methods

Drugs

Curcumin (HY-N0005) was purchased from MedChemExpress (Shanghai, China). Its chemical structure is shown in Fig. 1A.

Cell culture, transfection, and drug treatment

The SiHa cell line and HeLa cell lines were obtained from the National Collection of Authenticated Cell Cultures (China), All cell lines were inoculated in DMEM (containing 10% fetal bovine serum, 1% penicillin and 1% streptomycin) and cultured in an incubator at 37 °C with a 5% CO2 volume fraction. For all experiments, cells were seeded at equal densities to ensure consistency across different treatment groups. The siRNA oligonucleotide of ATG3 was synthesized by GenePharma Company (Suzhou, China) and transfection was performed using Lentivirus provided by GenePharma at a concentration of 1 × 10^⁹ TU/mL, following the manufacturer’s protocol. Then cells were collected for subsequent experiments. Curcumin was dissolved in DMSO, and the cells were treated with 25 µmol/L of curcumin, a concentration chosen based on previous studies that identified it as effective for inhibiting cell migration without causing significant cytotoxicity [33].

Cell migration and CCK8 cell proliferation assay

Wound healing assay was performed to assess cell migration. Cells were seeded into 6-well plates and cultured until reaching 80% confluence. A 200 µL sterile pipette tip was used to create a scratch through the cell monolayer. Images were captured at 0-, 24-, and 48-hours post-scratch to monitor wound closure. The percentage of wound closure was calculated using ImageJ software by measuring the area of the wound at each time point and comparing it to the initial wound area at 0 h. The wound closure percentage at each time point was calculated as [(initial wound area - wound area at time point) / initial wound area] * 100. The control group was transfected with a non-targeting shRNA to serve as a mock control. Cell proliferation was tested by Cell Counting Kit-8 assay (Dojindo, Shanghai, China) according to the manufacturer’s instructions. Briefly, 4000 cells/well seeded into 96-well plates were transfected and cultured for 24 h. At indicated time 0-, 24-, and 48-hours, each well was added using 10 µl CCK8 solution. The absorbance at 450 nm was read on a microplate reader (Waltham, MA, USA).

Immunofluorescence analysis by confocal laser-scanning microscopy

Cells were cultured and treated with curcumin on glass cover slips, then fixed in 4% paraformaldehyde in PBS for 15 min. After fixation, cells were permeabilized with 0.2% Triton X-100 for 5 min and blocked with 1% BSA in PBS for 1 h at room temperature. Subsequently, cells were incubated with the primary antibody LC3 (AB63817, Abcam) overnight at 4 °C. Following three washes with PBS-Tween, cells were incubated with the FITC-conjugated secondary antibody (A0562, Beyotime). Chromatin was counterstained with DAPI. Imaging was performed using confocal laser scanning microscopy (CLSM) with the LSM700 microscope, equipped with a Plan Apochromat 20×/0.8 M27 objective and ZEN 2009 software (Zeiss).

Western blot assay and antibodies

Total protein was extracted using RIPA buffer supplemented with protease inhibitors. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skimmed milk powder, followed by an overnight incubation at 4 °C with primary antibodies P62 (#8025, Cell Signaling Technology), ATG3 (EPR4801, Abcam), LC3 (ab63817, Abcam), and MMP2 (AB97779, Abcam). β-actin (AF0003, Beyotime) was used as a loading control. The membranes were then incubated with appropriate secondary antibodies for 1 h. Protein bands were visualized using an ECL detection reagent (Santa Cruz), and band intensities were quantified using ImageJ software.

Molecular docking

Docking simulations were conducted with Autodock Vina (version 1.1.2). The 3D structure of curcumin was obtained from the PubChem Compound database (http://pubchem.ncbi.nlm.nih.gov). Ligands were prepared in pdbqt format, and MGLTools-1.4.6 was used to prepare the protein (protein.pdbqt), and generate the grid parameter file (protein.gpf) and docking parameter file (ligand.dpf). The docking process involved two steps: first, a large grid box (75 Å × 60 Å × 60 Å) covered the entire protein to identify potential binding pockets. Then, a smaller grid box (22.5 Å × 22.5 Å × 22.5 Å) focused on the identified binding site for localized docking.

Statistical analysis

Statistical analysis was conducted using SPSS 27.0 (SPSS, Inc, USA) and GraphPad Prism 10 (GraphPad Software Inc, San Diego, CA, USA). Data were analyzed using one-way or two-way analysis of variance (ANOVA) and presented as mean ± standard deviation. Statistical significance was denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, with ns indicating no significant difference.