Hesperidin increases the in vitro anticancer activity of Pentagamavunon-1 in triple‐negative breast cancer with limited effects on normal cells

Abstract

Pentagamavunon-1 (PGV-1) is a potential anticancer candidate due to its specific targeting of cancer cells with minimal toxicity to normal cells. This study examined the potency of hesperidin (HSD) in improving the anticancer effects of PGV-1 for triple-negative breast cancer (TNBC). The 4T1 and NIH-3T3 cell lines were used as the model of TNBC and normal fibroblast, respectively. The cytotoxicity was quantified using MTT and clonogenic assays, whereas the cell cycle and apoptosis were measured by flow cytometry. Cell migration was assessed by scratch-wound healing assay. Senescence was measured using the SA-β-gal assay, and the secretion of matrix metalloproteinase (MMP)-2 and 9 was examined using gelatin zymography. Protein expression level was analyzed through capillary Western blot. The collected data were quantified using one-way ANOVA. PGV-1 significantly reduced 4T1 cell viability, with IC₅₀ values of 7 and 2 µM at 24 and 48 h, respectively. Its cytotoxic effect persisted up to 7 days after removal. HSD alone showed no cytotoxicity against 4T1 cells, but in combination with 1 µM of PGV-1, it synergistically enhanced cell growth inhibition and apoptosis induction. This synergism is likely due to ERK1/2 phosphorylation inhibition, leading to reduced levels of Bcl-2 and elevated levels of Bax. In NIH-3T3 cells, higher concentration PGV-1 (10 µM) induced early senescence-associated features, but 100 µM HSD prevented this and suppressed MMP-2 and 9 secretion. PGV-1 and HSD combination demonstrates potential as a co-treatment strategy for effective anticancer therapy while preserving normal cell integrity.

INTRODUCTION

The therapeutic landscape for triple-negative breast cancer (TNBC) continues to evolve due to its poor prognosis and high metastatic potential.[1] Its treatment has shifted from monotherapy to combination strategies, which are more effective yet remain challenging.[1] Combination therapy is favored for reducing side effects on normal cells while enhancing cytotoxicity in cancer cells.[2] However, many anticancer agents target shared mechanisms in both cancerous and normal cells,[3,4] often causing toxicity and immune-related adverse effects.

Synthesized compound, Pentagamavunon-1 (PGV-1), structurally related to curcumin, has revealed promising anticancer effects, including activity against TNBC,[5,6,7,8,9,10] by disrupting the cell cycle.[10] PGV-1 has relatively low toxicity in normal cells and animal models.[11,12,13,14] Despite these favorable features, optimization of its therapeutic potency and safety profile is needed for further application. Natural bioactive compounds − such as diosmin,[15,16] hesperidin (HSD),[17] hesperetin,[18] galangin,[19] and piperine[20] – have been reported to enhance anticancer responses when combined with PGV-1, suggesting the potential of adjunctive strategies.

HSD, abundant in citrus peels,[21] has antioxidant and anticancer properties without significant adverse effects.[22] Previous studies highlight the ability of HSD to enhance anticancer effects and protect normal cells from therapy-induced senescence when combined with another chemotherapeutics such as doxorubicin[23] or cisplatin.[24] In a recent study, HSD synergistically improves the effect of PGV-1 in suppressing the growth of luminal breast cancer cell.[17]

Despite the increasing focus on combination therapies, a key challenge in TNBC treatment is the rational integration of senescence-modulating agents with cytotoxic drugs to improve anticancer efficacy while minimizing therapy-induced damage, including senescence, in normal cells. Accordingly, this study aims to investigate the PGV-1 and HSD combination as a senescence-modulating adjunctive strategy for TNBC therapy, integrating anticancer efficacy with protection of normal cells. Specifically, this study aims to (i) evaluate the cytotoxic and growth-suppressive effects of PGV-1 and HSD, individually and in combination, in 4T1 TNBC cells; (ii) assess the selectivity of this combination toward cancer cells relative to NIH-3T3 fibroblasts; and (iii) determine whether HSD can modulate PGV-1–associated stress responses in normal cells, particularly therapy-induced senescence. Through this framework, the PGV-1–HSD combination is positioned not merely as a cytotoxic enhancer, but as an adjunctive approach designed to optimize the balance between tumor suppression and preservation of normal cell function to address a critical gap in TNBC treatment development.

MATERIALS AND METHODS

Compounds and cells

PGV-1 (PubChem CID: 10760152) with a purity of ≥95% was synthesized,[25,26] while HSD (PubChem CID: 0621) was obtained from Sigma (H5254, purity ≥80%). 4T1 (ATCC CRL-2539) and NIH-3T3 cells were cultured per standard protocol[27] in DMEM (Gibco) enriched with 10% fetal bovine serum and 1% antibiotics (10,000 U/mL penicillin-streptomycin, Gibco).

MTT assay

4T1 cells (5 × 10³/well) were plated in 96-well plates and subsequently treated with the compound at a serial concentration for 24 and 48 h. After removing media and rinsing with phosphate-buffered saline, MTT reagent was added for 4 h. The reaction was stopped with 0.01 M HCl in 10% SDS. Formazan absorbance was read at 595 nm (BioRad) to calculate cell viability and inhibitory concentration 50% (IC₅₀). For combination assays, PGV-1 and HSD at sub-IC₅₀ concentrations were tested. The combination index (CI) was calculated using Chou’s method.[28] All experiments were performed in triplicate.

Clonogenic assay

4T1 cells (1 × 10⁴/well) were plated in 6-well plates for treatment with sub-IC₅₀ compound concentrations for 48 h, then incubated in fresh media for 7 days, with media changes every 2 days. Due to the strong antiproliferative effects of the treatments, this density resulted in well-separated, countable colonies at the end of the assay. Colonies were fixed in 4% paraformaldehyde, stained with 0.1% crystal violet (Sigma), and observed with the aid of an inverted microscope (Olympus).

Flow cytometry

4T1 cells were treated with PGV-1 (1 μM), HSD (100 μM), or their combination for 24 h. For cell cycle analysis, cells (~1 × 10⁵) were stained with propidium iodide (PI, BD Pharmingen, 556547) and analyzed (BD FACS Diva 8.0.2). For apoptosis, Annexin-V and PI staining were used per the manufacturer’s protocol. Triplicates were used for all experiments.

Capillary Western blot

4T1 cells were subjected to PGV-1 (1 μM), HSD (50 μM), or combination for 24 h. Lysates were prepared with RIPA buffer and protein concentrations adjusted to 23 μg/mL. Capillary Western blotting[29] was performed using the ProteinSimple Abby System as described.[30] The details can be found in Supplementary File. Primary antibodies used included β-actin, Bax, Akt, p-Akt (Cell Signaling), Bcl-2, ERK1/2, and p-ERK1/2 (Santa Cruz), diluted 1:100. Data were interpreted using Compass software.

Senescence detection

NIH-3T3 cells were exposed to PGV-1 (10 μM), HSD (50/100 μM), or their combination (24 h), fixed, and subjected to senescence-associated β-galactosidase (SA-β-gal) assay (0.2% X-gal, Sigma, B4252). Senescent cells (blue) were documented under a microscope. Triplicate wells and five fields/well were analyzed.

Gelatin zymography

Conditioned media from senescence assays were collected. Protein was standardized to 20 µg/sample and run on 10% gelatin-containing PAGE. Gels were renatured, stained with Coomassie blue, and de-stained. Gelatinase bands were quantified with ImageJ in triplicate.

Data analysis

Data were analyzed using GraphPad Prism v7.0 (GraphPad Software, Inc., San Diego, CA, USA). Tukey’s post hoc test following one-way ANOVA was used for statistical analysis with 95% confidence level. Results are expressed in average value with standard deviation (SD).

RESULTS

Cytotoxicity of the single exposure of pentagamavunon-1 or hesperidin in 4T1 cells

PGV-1 has shown strong cytotoxicity against various cancer cell lines, including 4T1 TNBC cells.[5,6,7,8,10] However, its safety and sustained effects need further study. We combined PGV-1 with HSD and evaluated their effects on cancer and normal cells. PGV-1 showed IC₅₀ values of 7 μM (24 h) and 2 μM (48 h) [Figure 1a and c]. HSD alone was noncytotoxic at 500 μM (24 h) and weakly cytotoxic at 48 h (IC₅₀ = 270 μM) [Figure 1b and c]. Clonogenic assays revealed that 1 μM PGV-1 completely suppressed colony formation, whereas 100 μM HSD had no effect [Figure 1d]. These results confirm PGV-1’s potent, irreversible cytotoxicity, whereas HSD has minimal impact on 4T1 cell viability.

Figure 1.

Pentagamavunon-1 (PGV-1) and hesperidin (HSD) cytotoxicity in 4T1 cells. MTT assays evaluated cytotoxicity of PGV-1 (a) and HSD (b), and IC₅₀ values after 24 and 48 h (c). Clonogenic assay (d) assessed long-term effects after 48 h treatment, followed by 7 days recovery. Data are shown as mean ± standard deviation (n = 3)

Synergistic cytotoxicity of pentagamavunon-1 and hesperidin in 4T1 cells

This combination strategy aims to maximize PGV-1’s anticancer effect at minimal concentrations by pairing it with the less cytotoxic HSD to reduce adverse effects on normal cells. Cytotoxicity tests using sub-IC₅₀ doses (PGV-1: 0.5–2 μM; HSD: 50–200 μM) showed increased effects, especially with 100 μM HSD [Figure 2a]. CI values were all <1, indicating synergism, particularly at 50 and 100 μM HSD [Figure 2b], supporting further development of this combination for TNBC treatment. Based on cytotoxicity results, PGV-1 (1 μM) combined with HSD (50 or 100 μM) was used in further tests. The combination with 100 μM HSD fully suppressed colony formation, supporting HSD as a potential co-treatment agent [Figure 1d].

Figure 2.

Synergistic cytotoxicity of Pentagamavunon-1 and hesperidin in 4T1 cells. MTT assay measured cell viability under combination treatments for 24 h (a), and Combination Index (CI) values were calculated (b). Each bar graph indicates mean ± standard deviation (n = 3)

Pentagamavunon-1 and hesperidin combination induces 4T1 cell apoptosis

To explore the synergistic effect of PGV-1 and HSD, we examined cell cycle and apoptosis using flow cytometry with PGV-1 (1 μM) and HSD (100 μM). While individual treatments showed no cell cycle changes [Figure 3a], their combination increased the sub-G1 population [Figure 3b] and significantly induced apoptosis [Figure 3c and d]. These results suggest their synergistic cytotoxicity is linked to enhanced apoptosis.

Figure 3.

Apoptotic induction of Pentagamavunon-1 (PGV-1) and hesperidin (HSD) combination in 4T1 cells. Cells received PGV-1, HSD, or combined treatment for 24 h. Cell cycle (a) and apoptosis (c) were analysed by flow cytometry. Quantification (b and d) shown as mean ± standard deviation (n = 3)

The effect of pentagamavunon-1 and hesperidin combination on signaling and apoptotic protein expressions in 4T1 cells

HSD enhances the cytotoxic, apoptotic, and antimigration effects of PGV-1 on 4T1 cells, even at low PGV-1 concentrations. Their impact on MAPK and PI3K/Akt/mTOR pathways, which regulate proliferation,[31] was examined. While neither treatment affected total Akt or its phosphorylation (p‐Akt), both PGV‐1 and HSD suppressed ERK1/2 phosphorylation, with the combination showing the strongest inhibition [Figure 4a and Supplementary 1 ^{(4.4MB, tif)} ]. This downregulation indicates disrupted proliferative signaling. In addition, the combination resulted in increased Bax expression and reduced Bcl‐2 levels, promoting apoptosis [Figure 4b and Supplementary 2 ^{(5MB, tif)} -4 ^{(3.1MB, tif)} ].

Figure 4.

Regulation of the ERK/Akt pathway (a) and apoptotic proteins (b) in 4T1 cell by Pentagamavunon-1 and hesperidin, measured via capillary Western blot. The band density was quantified automatically by Abby system, normalized with β-actin, and the relative expression level of each protein was presented below the blot

The cytoprotective property of hesperidin in combination with pentagamavunon-1 on NIH-3T3 cells

PGV-1 induces senescence in 4T1 cancer cells, contributing to its cytotoxic effects, while exhibiting low toxicity to normal cells like NIH-3T3 fibroblasts (IC₅₀ >100 μM).[14] The senescence-inducing effects of PGV-1 on NIH-3T3 cells and whether HSD could diminish these effects were assessed. PGV-1 at 10 μM significantly triggered senescence-associated features, as indicated by increased SA-β-gal activity (formation of blue-colored substrate), but HSD (100 μM) reduced it to baseline levels [Figure 5a and b]. In addition, PGV‐1 and HSD combination suppressed matrix metalloproteinase (MMP)‐9 and MMP‐2 secretion [Figure 5c and d], both linked to senescent cell activity and ECM degradation,[32] suggesting a potential correlation in reducing senescence in NIH‐3T3 cells.

Figure 5.

Cellular safety of pentagamavunon-1 and hesperidin combination in NIH-3T3 cells. Cells were treated for 24 h; senescence was detected by SA-β-gal staining (a) and quantified (b). Gelatin zymography showed matrix metalloproteinase activity (c), analyzed via ImageJ (d). Bar graphs represent mean ± standard deviation (n = 3)

DISCUSSION

The safety and selectivity of targeted cancer therapies remain key concerns in cancer treatment. This study investigates the efficacy and safety of PGV-1 combined with HSD, emphasizing their synergistic effects. PGV-1 showed robust cytotoxicity against 4T1 cells, which was enhanced by HSD. Even at a low concentration (1 μM), PGV-1 exhibited persistent cytotoxic effects, confirmed by the clonogenic assay. While HSD alone does not inhibit cancer cell growth, it significantly enhances PGV-1’s effects, as previously observed with doxorubicin on 4T1[23] and T47D cells.[33]

PGV-1 did not alter the cell cycle in this study, though previous studies showed G2/M arrest.[9,34] The PGV-1 and HSD combination, even when PGV-1 was used at low concentrations, significantly induced apoptosis, supporting the mechanism of persistent cell growth inhibition. This apoptosis was likely due to the combination’s impact on MAPK signaling and its ability to regulate the expression of pro- and anti-apoptotic proteins, including Bax and Bcl-2.[35]

In terms of safety, PGV-1 induced early senescence-associated features in NIH-3T3 fibroblast cells at concentrations of 10 μM, but HSD prevented this effect, maintaining fibroblast health. The combination also suppressed MMP-9 and MMP-2 secretion, indicating that HSD helps protect normal tissue integrity while enhancing the anticancer effects of PGV-1. Senescence triggers MMPs, part of the senescence-associated secretory phenotype, that degrade ECM components and disrupt tissue architecture.[36] Senescence is a physiological response that permanently halts cell division in response to stress or damage, and although beneficial for tissue repair, it can impair normal tissue function if uncontrolled.[37] These results underline the anticancer potential of PGV-1 and HSD as a safe and effective combinational strategy, particularly for TNBC, which remains a challenge for therapeutic development. Further studies, including in vivo experiments, would be valuable to confirm these findings.

CONCLUSION

PGV-1 exhibits a sustained cytotoxic effect and demonstrates a synergistic interaction with HSD, effectively inhibiting the growth of TNBC cells. The combined exposure of PGV-1 and HSD induces apoptosis by reducing p-ERK1/2, downregulating Bcl-2, and upregulating Bax expression levels. While PGV-1 induces cellular senescence in fibroblast normal cells, HSD mitigates this effect. Therefore, the combination of PGV-1 and HSD holds promise as a combination strategy for TNBC, enhancing cytotoxicity against cancer cells while minimizing harm to normal cells.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Universitas Gadjah Mada (grant ID: 5634/UN1/DITLIT/PT.01.03/2024).

SUPPLEMENTARY FILES

Hesperidin increases the in vitro anticancer activity of Pentagamavunon-1 in triple-negative breast cancer with limited effects on normal cells

Journal of Advanced Pharmaceutical Technology & Research

Muthi’ Ikawati*, Mila Hanifa, Nadzifa Nugraheni, Novia Permata Hapsari, Anif Nur Artanti, Dhania Novitasari, Rohmad Yudi Utomo, Mukh Syaifudin, Muchtaridi Muchtaridi, Endah Puji Septisetyani, Okid Parama Astirin, Edy Meiyanto

*Corresponding author:

Affiliations:

Cancer Chemoprevention Research Center, Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta 55281, Indonesia

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Universitas Gadjah Mada, Sekip Utara,

Yogyakarta 55281, Indonesia

E-mail address:

muthi_ikawati@ugm.ac.id

Supplement

Capillary Western blot

4T1 cells were treated for 24 h with PGV-1 (1 μM), HSD (50 μM), or their combination for 24 h. The pellets of treated cells were mixed with RIPA buffer (Sigma) with the addition of 1 mM PMSF, 2 mM NaF, and protease inhibitor cocktail (Sigma), followed by incubation on ice for approximately 15 min and centrifugation at 13,500 rpm (10 min, 4 °C). Cell lysates were mixed with a sample preparation solution containing 5× fluorescent master mix in 400 mM DTT 20 μL and 10× sample buffer 20 μL to obtain a sample loading concentration of 23 μg total protein/μL. The samples were then denatured by heating it to 95 °C for 5 min.

Capillary Western blotting (Lu et al. 2017), instead of conventional Western blot, was performed on the Protein Simple Abby System as previously described (Nugraheni et al. 2024) utilizing a 12-230 kDa separation module with a voltage of 375 V, followed by a 10 min blocking step, a 30 min incubation with primary antibodies, and a 30 min incubation with secondary antibodies (Abby protocol, USA). Primary antibodies obtained from Cell Signaling, i.e. β-actin (cat. no. #12620), Bax (#2772S), Akt (#9272), phospho-Akt (#4056), caspase-3 (#9662) PARP (#9532), and cleaved-PARP (#5625) were conjugated to rabbit secondary antibodies. Primary antibodies Bcl-2 (Cell Signaling, cat no. #15071), ERK1/2 (Santa Cruz, #SC514302), and p-ERK1/2 (Santa Cruz, #SC81492) were conjugated to mouse secondary antibodies. All these antibodies were diluted 100 times. The running analysis was started by loading the plate for electrophoresis and immunodetection, controlled by the biotinylated ladder's molecular weight standard. Virtual blot-like images obtained according to the molecular weight of each protein were interpreted using Compass software.

Supplementary 1

Supplementary 2

Supplementary 3

Supplementary 4