Antimetastatic Effects of a Griffonia simplicifolia Seed Extract in Osteosarcoma Cell Lines

TL;DR

A natural extract from Griffonia simplicifolia seeds may help reduce osteosarcoma metastasis when used with chemotherapy.

Contribution

The study identifies a natural extract that blocks metastatic signals and enhances chemotherapy effects in osteosarcoma cells.

Findings

The extract blocks proliferation and migration signals induced by interleukin-1β in osteosarcoma cells.

The extract epigenetically inhibits interleukin-6 gene expression by blocking promoter demethylation.

The extract synergizes with doxorubicin to enhance antimetastatic effects without reducing chemotherapy efficacy.

Abstract

Osteosarcoma is one of the most common malignant tumors that develop in the bone. Currently, surgery is often the best and most used approach, often preceded and followed by chemotherapy, which, however, carries serious short- and long-term side effects. Recently, much attention has been paid to natural compounds capable of inducing tumor cell death, reducing tumor and metastatic activity, and interacting with selective chemotherapy targeting tumor cells. Griffonia simplicifolia, a tropical African plant, has attracted attention because its extracts with bioactive chemicals have demonstrated multiple therapeutic uses. We show the antitumor properties of a Griffonia seed extract, obtained by maceration in a hydroalcoholic mixture (ethanol/water, 70/30, v/v, Gri70), on osteosarcoma cell lines, evaluating cytotoxicity, interaction with a pro-inflammatory signal (interleukin-1β), epigenetic activity of this signal on interleukin-6 gene expression, and interactions with an elective chemotherapeutic agent, doxorubicin. Although the extract did not have strong antiproliferative activity in the cell lines analyzed, we nevertheless observed that it was able to block proliferative and migration signals induced by interleukin-1β, as well as acting epigenetically by blocking the de-methylation of the interleukin-6 promoter and its expression. Furthermore, the extract did not appear to interfere with the antitumor activity of doxorubicin, and the interaction potentiated antimetastatic effects. These results indicate that Gri-70 extract may be useful as adjuvants to enhance the effect of doxorubicin, reducing the adverse effects associated with the increased EMT process of osteosarcoma cells that manage to overcome cell death induction. Indeed, metastasis represents the main cause of poor prognosis.

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1. Introduction

Osteosarcoma (OS) is one of the most common bone tumors, characterized by cells with mesenchymal characteristics involved in the formation of a non- or poorly mineralized bone matrix [1,2]. Although osteosarcoma is predominantly a pediatric disease, with a peak in adolescence, with a significant age of onset between 10 and 14 years, it also has a second peak in adults over 65 years of age, resulting in a bimodal distribution across age groups [3,4]. Worldwide, the incidence of osteosarcoma is approximately two to four cases per million people per year, although the International Incidence of Childhood Cancer has indicated that the total number of cases per year in Europe is 1135, with an incidence of five cases per million in adolescents (between 15 and 24 years) [5,6,7].

OS tumor tissue is composed of several cellular subpopulations, including bone cells (osteoblasts, osteocytes, and osteoclasts), immune cells (macrophages and lymphocytes), and stromal cells (mesenchymal stem cells and fibroblasts), surrounded by an extracellular matrix (ECM), which together with tumor cells constitutes a complex and dynamic environment. Within this complex, often pro-inflammatory tumor microenvironment, tumor cells, in response to various stimuli, develop drug resistance and lead to tumor recurrence and metastasis, with a high capacity for self-renewal and adaptability. Furthermore, genomic instability, as well as epigenetic alterations and metabolic plasticity, plays a fundamental role in tumor progression, which can manifest even after an apparently very effective initial treatment [8,9,10,11].

The most commonly used chemotherapeutic drugs for the treatment of OS are doxorubicin (Adriamycin, DOX), cisplatin (cis-diaminodichloroplatinum II, CDDP), ifosfamide (IFO), and high-dose methotrexate (HDMTX), often in combination with each other or sequentially in a very complex chemotherapy regimen, due precisely to the development of resistance, linked to the high adaptability of these tumor cells. Furthermore, the complexity of these chemotherapy regimens is compounded by the many serious short- and long-term side effects, such as cardiac toxicity [12,13,14,15], nephrotoxicity [16,17,18,19,20], neurotoxicity [21,22,23,24,25], infertility [26,27,28], secondary osteoporosis [29,30,31] and even secondary malignancies and metastasis [32,33,34,35], which reduce both the life quality and life expectancy of patients.

In recent years, several studies have shown that natural compounds, individually or in complex mixtures (extracts, herbal teas, etc.), have demonstrated various protective effects in several orthopedic diseases, including in the fight against tumor progression. The observed effects involved antioxidant, anti-inflammatory, and proapoptotic activities and entailed various signaling pathways, such as NF-κB and MAPKs (ERK, JNK, and p38-MAPK). In addition, they have displayed epigenetic effects, including expression of miRNAs, DNA methylation changes, and post-translational modifications of histones (methylation and acetylation), which may contribute to explaining their potential therapeutic benefits [36,37,38]. In fact, some natural compounds enhance the antitumor effects when used in combination with traditional chemotherapy, and this reveals their role as promising therapeutic adjuvant agents for the treatment of OS cells [39,40,41,42,43,44]. Most of them enhance therapeutic effects by increasing drug bioavailability and drug accumulation in cancer cells, modulating apoptosis pathways, inhibiting cell proliferation, and activating the immune system. Therefore, natural compounds act as chemotherapeutic adjuvants by sensitizing tumor cells to be more sensitive to chemotherapy drugs and thus enhancing the tumoricidal effect. In addition to sensitizing tumor cells to chemotherapy, phytochemicals also protect normal cells that are inevitably exposed to chemotherapeutic agents, promoting DNA repair mechanisms. In this way, combination therapy could reduce the toxicities associated with traditionally used chemotherapy drugs [45,46,47]. The use of natural compounds in combination with cancer therapies plays a significant role due to their synergistic effects, but a promising strategy would be to aim at using them as chemotherapeutics for cancer treatment [47,48].

Griffonia simplicifolia is a tropical plant endemic to West Africa, widely used in traditional medicine and nutraceuticals due to its seeds’ high content of 5-hydroxytryptophan (5-HTP), which can reach up to 20% of the dry weight. Seeds can be processed to obtain pastes or powders to be incorporated into various foods and beverages [49]. On the other hand, seed extracts have been employed for decades in the preparation of dietary supplements aimed at treating conditions associated with altered serotonin levels, such as mood modulation and the management of gastrointestinal disorders. Despite its extensive use, studies investigating biological activities of Griffonia seeds beyond serotonin modulation remain extremely limited. Nevertheless, it has been demonstrated that the seeds are not only a source of 5-HTP but also contain numerous other bioactive molecules, which may contribute to known biological effects as well as to potential new ones yet to be demonstrated [50]. Moreover, no standardized extraction protocol currently exists, although hydroalcoholic mixtures are the most commonly adopted for commercial supplements.

In this context, in a previous study, Mannino et al. evaluated the impact of different extraction methods on the biological activities of Griffonia seeds, with a particular focus on antioxidant and antiproliferative effects, which are still poorly characterized. Among the studied extracts, a hydroalcoholic seed extract obtained by maceration in 70% ethanol (Gri70) emerged as particularly promising [50]. Gri70 consistently showed the highest content of antioxidant polyphenols, as well as significant antioxidant activity and exhibited antiproliferative effects on three human epithelial cancer cell lines (HeLa, HepG2, and MCF-7). Its phytochemical profile, obtained by high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS/MS) in Mannino et al., is highly enriched in flavonoids, including glycosylated forms of kaempferol, myricetin, quercetin, and naringenin, supporting a multifaceted bioactivity beyond that of 5-HTP, suggesting potential other therapeutic applications [50].

Given the initial premises, this study evaluated the nutrigenomic and antitumor activity, and the potential adjuvant role of the extract of Griffonia simplicifolia seeds, obtained by 70% ethanol maceration (Gri70), which has already been previously fully characterized in Mannino et al. [50], on two OS cell lines, MG63 and SAOS2, demonstrating the potential contribution of the aforementioned extract as “epi-drug” in adjuvating doxorubicin treatment against OS.

2. Materials and Methods

2.1. Reagents

Cell culture reagents, including Dulbecco’s modified Eagle’s medium with high glucose (DMEM), fetal bovine serum (FBS), L-glutamine, and the penicillin/streptomycin antibiotic mixture, were obtained from Lonza (Verviers, Belgium).

2.2. Griffonia simplicifolia Seed Extract Preparation

The Griffonia simplicifolia seed extract, referred to as Gri70, was obtained using a hydroalcoholic extraction procedure based on ethanol and water (70:30, v/v), following an established protocol [50]. Briefly, seeds supplied by BioResources International Inc. (Lafayette, LA, USA) were kept at room temperature and protected from light prior to processing. For extraction, seeds were finely milled and incubated with the ethanol/water solvent at a ratio of 1:10 (w/v). The maceration was carried out for 24 h at 4 °C under continuous agitation. After incubation, samples were centrifuged at 10,000× g for 10 min at 4 °C to separate the soluble fraction. The remaining solid residue was subjected to two additional extraction cycles under identical conditions. All recovered supernatants were pooled, passed through a 0.45 µm Millex HV membrane filter (Millipore, Billerica, MA, USA), and stored at −80 °C until further use.

2.3. Total Polyphenolic Content and Cellular Antioxidant Activity (CAA) Assay of Griffonia simplicifolia Seed Extract

For consistency with previously reported results, the Griffonia simplicifolia seed extract used in this study (Gri70) was characterized in terms of total polyphenol content (TPC), determined by the Folin–Ciocalteu assay, and radical scavenging capacity, measured using the DPPH method, according to the procedures described by Mannino et al. [50]. In parallel, the intracellular antioxidant potential of Gri70 was further investigated by means of the Cellular Antioxidant Activity (CAA_50_) assay, as described below. The antioxidant properties of Gri70 at the cellular level were assessed using a well-established CAA assay [51]. HepG2 cells (ATCC, Rockville, MD, USA) were maintained in RPMI culture medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (50 IU/mL), and streptomycin (50 µg/mL), and incubated at 37 °C in a humidified atmosphere containing 5% CO_2_. Cells were plated in 96-well microplates and allowed to attach for 24 h prior to treatment.

Subsequently, cells were exposed to increasing concentrations of Gri70 (50–2500 µg/mL) in the presence of 2′,7′-dichlorofluorescin diacetate (DCFH-DA; Sigma-Aldrich, St. Louis, MO, USA). Oxidative stress was initiated by the addition of 2,2′-azobis(2-amidinopropane) dihydrochloride (ABAP; Sigma-Aldrich, St. Louis, MO, USA). Fluorescence signals were monitored over time, and the area under the fluorescence–time curve was calculated to determine CAA values at each concentration. The extract concentration producing a 50% reduction in DCF formation (CAA_50_) was derived from the corresponding concentration–response curve. Data are expressed as the mean of three independent experiments, each performed in quadruplicate.

2.4. OS Cell Cultures

Two human osteosarcoma (OS) cell lines, MG63 (ATCC^®^ CRL-1427™) and SAOS2 (ATCC^®^ HTB-85™), were purchased from ATCC^®^ (Manassas, VA, USA). Cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL) (Gibco, Invitrogen Corp., Carlsbad, CA, USA). Cultures were incubated at 37 °C in a humidified atmosphere containing 5% CO_2_, according to previously established conditions [52].

2.5. Viability Assay (MTT Assay)

Cell viability of osteosarcoma (OS) cell lines was assessed using the MTT assay, as previously described [53]. Briefly, OS cells were seeded at a density of 1 × 10^5^ cells per well in 96-well plates and treated with varying concentrations of Gri70 (5–100 ng dry weight [DW]/mL of culture medium). Cell viability was also evaluated under exposure to 25 ng/mL of Interleukin-1β (IL-1β), both alone, in co-treatment with the highest concentration of Gri70, or in cells pre-treated with Gri70 for the same duration. After the treatment period, absorbance at 540 nm was measured using a Bio-Rad microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Results are expressed as a percentage relative to untreated control cells.

2.6. Wound-Healing Assay

OS cells were seeded in 6-well plates and allowed to grow until reaching confluence. A scratch was then made across the cell monolayer using a 10 μL pipette tip, followed by gentle rinsing with phosphate-buffered saline (PBS) to remove detached cells. Images of the scratch were acquired at 0 and 24 h using a Nikon Eclipse Ti microscope (Nikon Europe B.V., Amsterdam, The Netherlands). The wound closure, reflecting cell migration and proliferation under different treatments, was quantified by measuring the distance between the edges of the wound using ImageJ software (1.41o version), as previously described [54].

2.7. Nucleic Acid Extractions

Total RNA was extracted from OS cells subjected to the different treatments using Trizol reagent (Invitrogen™, Waltham, MA, USA) following the manufacturer’s instructions. The resulting RNA pellets were resuspended in diethyl pyrocarbonate (DEPC)-treated water (0.01% DEPC). Genomic DNA (gDNA) was isolated from the same cell samples using the PureLink Genomic DNA Mini Kit (Invitrogen™, Waltham, MA, USA) according to the provided protocol. The quantity and purity of both RNA and gDNA were assessed using a Nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). Integrity was further evaluated by electrophoresis on 0.8% agarose gels stained with GelRed (Biotium, Hayward, CA, USA), as previously described [55].

2.8. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

First-strand cDNA was synthesized from 250 ng of total RNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Fisher Scientific Italia), following the manufacturer’s instructions. Each cDNA sample was run in duplicate. Quantitative real-time PCR (qRT-PCR) was performed on a StepOne Real-Time PCR System (Applied Biosystems™) using the SYBR^®^ Green Real-Time PCR Master Mix (Applied Biosystems™). Primer sequences are listed in Table 1. Relative gene expression was calculated using the 2^–ΔΔCt^ method, normalizing to the housekeeping gene β-actin and to untreated OS cell line controls [56].

2.9. Methylation Analysis of IL-6 Promoter by MSRE-PCR

The methylation status of six CpG-rich sites within the Interleukin-6 (IL-6) promoter was assessed using Methylation-Sensitive Restriction Endonuclease–PCR (MSRE–PCR). Experimental procedures were conducted following the methods we have previously described [39,54,55,57]. PCR amplicons were separated on 2% agarose gels stained with GelRed (Biotium, Hayward, CA, USA) and visualized using a ChemiDoc imaging system (Bio-Rad Laboratories, Hercules, CA, USA). Densitometric analysis of the bands was performed with the Bio-Rad “Image Lab” software, version 5.2.1.

2.10. ELISA Assay

For the ELISA assay, 2 × 10^4^ cells were seeded per well in 96-well plates and allowed to adhere for 24 h. Cells were then treated under the following conditions: IL-1β (25 ng/mL) alone, Gri70 (100 ng/mL) alone, co-treatment with IL-1β (25 ng/mL) and Gri70 (25 ng/mL), or pre-treatment with Gri70 for 24 h followed by IL-1β (25 ng/mL). Levels of IL-6 secreted into the conditioned medium were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Europe, Ltd., Abingdon Science Park, Abingdon, UK) according to the manufacturer’s instructions.

2.11. Statistical Analysis

The statistical analysis was performed using SPSS v.19.0 (IBM Corp., Armonk, NY, USA). All the data are shown as expected mean and 95% confidence intervals. The generalized linear models with a post hoc Sidak test for pairwise comparisons, based on Gamma distribution and loglink function with treatment and follow-up as fixed effects, were used to test the difference among treatments along the follow-up for MTT analyses. The ANOVA test with Thamane post hoc pairwise comparisons evaluated using the bootstrap method for small samples was used to assess the differences among treatments in wound-healing assays, transcription levels of genes, and percentage of methylation in MSRE PCR analyses. The non-parametric Kruskal–Wallis test with Mann–Whitney post hoc pairwise comparisons with Bonferroni correction for multiple comparisons, evaluated with the Montecarlo method for small samples, was used to assess the differences among treatments in relative IL-6 mRNA expression and ELISA of secreted IL-6 after 48 h. For all tests, significant differences among tested treatments versus untreated cell lines, versus IL-1β-treated cell lines, or versus doxorubicin-treated cell lines are reported by asterisk in the graphs (1 symbol, p < 0.05; 2 symbols, p < 0.005; 3 symbols, p < 0.0005).

3. Results

3.1. Polyphenol Content and Antioxidant Properties of Gri70

Gri70 was first evaluated for total polyphenolic content (TPC) and radical scavenging activity (DPPH assay) as indicators of its functional potential. The extract showed a TPC value of 103 ± 9.7 mg GAE/100 g DW and a DPPH value of 3.581 ± 0.106 mmol Trolox equivalents/100 g DW. These values closely match those reported previously [50], indicating that the extract used in the present study can be considered equivalent to that previously characterized, with the same phytochemical contents, enriched in kaempferol, myricetin, quercetin and naringenin and their glycosylated forms.

To assess the capacity of Gri70 antioxidant components to function within cells, their intracellular antioxidant activity was evaluated using a whole-cell lipid peroxidation model. The CAA assay, unlike other lipid peroxidation models, not only evaluates the ability of redox-active compounds to interact with biological membranes but also measures their stability under cellular metabolic conditions [58]. Cellular antioxidant protection involves not only direct scavenging of reactive species but also modulation of antioxidant enzyme activity and expression. For this reason, HepG2 cells, which possess a full complement of cellular antioxidant defenses, were chosen for the CAA assay. Gri70 exhibited a CAA_50_ of 722.49 ± 34.31 µg DW/mL cell medium, indicating relatively high intracellular antioxidant activity (Figure 1), particularly considering its moderate polyphenol content compared with other plant matrices with much higher TPC values [59].

These results indicate that Gri70 contains bioactive compounds capable of exerting antioxidant effects within cells, supporting its potential to modulate redox-sensitive pathways involved in cell proliferation, migration, and differentiation.

3.2. Gri70 Treatments Interfere with IL-1β-Induced Proliferation and Migration Signals in OS Cell Lines

To investigate the effects of Gri70 treatment on OS cell lines, we have previously carried out MTT analyses in the presence of various concentrations of Gri70 extract ranging from 5 to 100 ng/mL. In Figure 2, both in MG63 (A) and SAOS2 (B), the proliferation was not influenced by Gri70 treatments, indicating that no cytotoxicity is induced in this type of tumor, different from other cancer cells previously tested [50], although a reduction in proliferation rates is visible in higher concentrations (Gri70—100 ng/mL).

Successively, we have tested the influence of Gri70 extract on a pro-inflammatory signal that induces both proliferation and migration in OS cell lines, such as IL-1β. We have then treated these cell lines with IL-1β (25 ng/mL), the Gri70 pre-treated cells (24 h, with higher concentration of Gri70 tested, 100 ng/mL), or co-treated OS cells with Gri70 (100 ng/mL) and tested viability with MTT assay. The results shown in Figure 3 indicate that the Gri70 extract, both in pre-treatment and in co-treatment, is able to block IL-1β-induced proliferation.

To test migration, wound-healing assays under the same treatments were carried out. After 24 h, analysis of the wound-healing assay showed that IL-1β treatment was able to accelerate, with a statistically significant difference, the process of wound edge closure, reaching approximately 85% for both MG63 (Figure 4) and SAOS2 (Figure 5) cells, compared to the control, which was approximately 50% for both cell lines. Gri70 treatment, however, blocked the healing process, remaining below 7%. This indicates a significant reduction in the migration process of the Gri70-treated OS cell lines.

To confirm the inhibition of IL-1β-induced motility and migration of OS cells pre-treated or in co-treatment with Gri70, we have carried out an RT-PCR of TGF-β, implicated in the induction of the epithelial–mesenchymal transition (EMT) process, Snail, a transcription factor, highly expressed during EMT, and Mmp9, a matrix metallo-proteinase implicated in matrix degradation and cell invasion during tumor progression. Figure 6 showed a transcriptional induction of these factors under IL-1β treatment. This induction is blocked by Gri70 pre- or co-treatments that determine the return to basal transcription levels of TGF-β, Snail and MMP9, indicating that Gri70 administration interferes with IL-1β-dependent tumor progression, slowing motility of OS cells.

3.3. Gri70 Acts as an “Epidrug” Inhibiting Demethylation of the IL6 Gene Promoter

A previous study of ours highlighted how IL-1β acts epigenetically by demethylating the IL-6 promoter, resulting in increased IL-6 expression and the release of both MG63 and SAOS2 cell lines [55]. IL-6 is known to be a key factor in inducing proliferation and the EMT process in OS. Analysis of methylation status of the IL-6 promoter gene, shown in Figure 7A and B, indicates that treatment with IL-1β induces strong demethylation of the proximal promoter both in MG63 (Figure 7A) and SAOS2 (Figure 7B) cell lines and that this demethylation corresponds to increased IL-6 transcriptions (Figure 7C,D) and protein releases (Figure 7E,F). However, treatment with Gri70 interferes with this demethylation (Figure 7A,B) and consequently with IL-6 transcriptions (Figure 7C,D) and protein releases (Figure 7E,F) with results comparable to those of untreated cells.

3.4. Gri70 Extract Interacts with Doxorubicin Treatment

Because chemotherapy drugs often act by inducing strong oxidative stress, we investigated whether the antioxidant power of Gri70 extract could interfere or increase the chemotherapeutic effects of doxorubicin. We initially performed an MTT assay using different concentrations of doxorubicin to identify the IC50 of this chemotherapeutic agent. Figure 8 shows graphs identifying the concentration of 0.5 μM doxorubicin as the IC50 at 48 h in the MG63 and SAOS2 cell lines.

This concentration is used in co-treatment or after 24 h of Gri70 (100 ng/mL) pre-treatment to evaluate cell viability. The analyses are shown in Figure 9, where the MTT assays indicate that no significant differences, neither positive nor negative, were identified with either the Gri70 pre-treatments followed by doxorubicin or the Gri70/doxorubicin co-treatments compared to treatment with doxorubicin alone.

Furthermore, analyses through RT-PCR of genes implicated in migration and invasions indicate the interference of Gri70, both in pre-treatments and co-treatment with doxorubicin, in these doxorubicin-induced processes [41]. Figure 10 showed how TGFβ, Snail, IL-6 and MMP9 transcription are increased in doxorubicin treatment, while the presence of Gri70 is able to reduce these expressions, indicating then a reduction in doxorubicin-induced cell mobility and then metastasis formation. Furthermore, statistically significant reductions between Gri70-pre-treated cells with doxorubicin or co-treated Gri70/doxorubicin cells with respect to untreated cells were identifiable in the expression of the genes TGFβ (Figure 10A,B), Snail (Figure 10C,D) and MMP9 (Figure 10G,H), suggesting an enhancement of the antimetastatic effects of Gri70, at least for these genes in all two OS cell lines tested. While, for IL-6 (Figure 10E,F), doxorubicin treatment in Gri70-pre-treated cells or in co-treatment with Gri70 resulted exclusively in the maintenance of the basal transcription levels of the untreated cells.

4. Discussion

This in vitro study evaluated the effects of treating OS cell lines with a Griffonia simplicifolia seed extract obtained by maceration in 70% ethanol, investigated the underlying mechanisms, and assessed whether these effects could be mediated through epigenetic mechanisms. Gri70 inhibited tumor-associated phenotypes in OS cell lines, substantially reducing IL-1β-induced proliferation and migratory activity, as assessed by MTT and wound-healing assays and by transcriptional analyses of genes implicated in the epithelial–mesenchymal transition (EMT). Specifically, an in vitro “inflamed tumor” model was set up by stimulating OS cells with exogenous IL-1β to mimic the in vivo tumor microenvironment and promote migration and proliferation. Under these conditions, Gri70 effectively counteracted tumor-promoting inflammatory signals.

Consistent with our previous study, which showed that IL-1β-induced proliferation and motility in OS cell lines are driven by epigenetic activation of the IL-6 gene via promoter demethylation and that inhibition of this pathway blocks these processes [55,60], we now observe for the first time that Gri70 acts epigenetically by preventing IL-6 promoter demethylation. RT-PCR analysis of IL-6 mRNA and ELISA measurements of IL-6 in conditioned medium revealed a marked and consistent reduction, corroborating the strong inhibitory effect of Gri70 on IL-6 expression. These findings support the hypothesis that Gri70 directly interferes with the epigenetic regulation of pro-inflammatory cytokines in tumor cells. Wound-healing assays further highlighted a substantial decrease in cell motility in Gri70-treated cells, to levels lower than those of untreated controls, suggesting that Gri70 may exert additional, as yet unidentified, antimetastatic activities. This observation is particularly relevant, as limiting cell migration is critical for preventing metastasis.

OS cells experience pronounced oxidative stress, rendering them susceptible to the pro-oxidant mechanisms of chemotherapeutics, which induce oxidative modification and denaturation of key proteins and extensive nucleic acid damage, thereby triggering apoptosis [34,61,62,63,64]. Accordingly, the potent antioxidant action of Gri70 could, in principle, antagonize chemotherapeutic agents such as doxorubicin [33,34,35]. To test this possibility, we conducted pre-treatment and co-treatment experiments combining Gri70 with doxorubicin. Gri70 did not attenuate doxorubicin cytotoxicity under either regimen. In addition, increasing evidence indicates that doxorubicin resistance in OS is driven primarily by enhanced migratory and invasive behavior and cellular dedifferentiation, which markedly undermines drug efficacy. In this context, similar to other natural compounds [40,41,42,43,44], Gri70 significantly reduces the doxorubicin-induced expression of EMT genes, as shown by RT-PCR analyses of Snail, TGF-β, IL-6, and MMP9 mRNAs. Notably, for all targets except IL-6, EMT gene expression in both Gri70 pre-treated and Gri70–doxorubicin co-treated cells significantly differed from that in untreated controls, often falling below basal levels, indicating a robust antimetastatic effect arising from the Gri70–doxorubicin combination.

These observed effects may be consistent with the well-documented anti-inflammatory actions of antioxidant phytochemicals, given the central role of oxidative stress in promoting inflammatory signaling and the sensitivity of pathways such as NF-κB to redox imbalance [65,66]. Despite its moderate polyphenolic content, Gri70 exhibited relatively high intracellular antioxidant activity in the CAA assay, suggesting that the extract contains compounds capable of sustaining cellular redox homeostasis not only by directly scavenging reactive species but also by upregulating the expression of enzymatic antioxidant defenses. In this context, the transcription factor Nrf2 serves as the master regulator of the cellular antioxidant response, and its activation depends on redox-sensitive targets [67,68]. Many phytochemicals are known to activate Nrf2 either by direct binding or by modulating intracellular redox status [66]. Importantly, extensive experimental evidence indicates that Nrf2 and NF-κB are reciprocally regulated: inflammatory conditions activate NF-κB while repressing Nrf2 signaling, and this cross-talk includes the Nrf2-dependent induction of antioxidant enzymes such as HO-1 and SOD, which suppress NF-κB activity [68,69]. Several components of Gri70, including kaempferol and naringenin, have been shown to inhibit NF-κB activation and stimulate Nrf2 signaling, thereby exerting anti-inflammatory effects in multiple experimental models [70,71,72]. Therefore, it is plausible that the effects of Gri70 within an inflammatory tumor microenvironment are, at least in part, mediated by the modulation of redox-sensitive transcriptional pathways, which may underlie its observed anti-inflammatory and antimetastatic properties.

These preliminary findings indicate that Gri70 can be safely co-administered with doxorubicin, enhancing its therapeutic profile by inhibiting metastatic activity while preserving the cytotoxic action of chemotherapy. Importantly, Gri70’s antioxidant capacity may also help mitigate doxorubicin-induced side effects in patients, a consideration particularly relevant for inoperable OS cases, where chemotherapic treatment remains the only therapeutic option [73,74,75,76].

5. Conclusions

In conclusion, Gri70 effectively counteracts inflammation-driven tumor-associated phenotypes in osteosarcoma cell lines, significantly reducing IL-1β-induced proliferation and migratory capacity within an in vitro model that recapitulates key features of the inflamed tumor microenvironment. Beyond its anti-proliferative and anti-migratory effects, the observed modulation of EMT-related gene expression suggests that Gri70 may interfere with transcriptional programs sustaining tumor progression. Notably, the emerging evidence of epigenetic involvement points to a potential regulatory role of the extract at the chromatin or gene-expression level, providing a mechanistic framework that extends beyond simple cytotoxic activity. While further in vivo studies and deeper molecular investigations are warranted, these findings support the therapeutic potential of Griffonia simplicifolia seed extract as a biologically active compound capable of targeting inflammation-associated and possibly epigenetically regulated pathways in osteosarcoma progression.