Skip to main content
NCBI home page
Cytotechnology logoLink to Cytotechnology
. 2025 Sep 24;77(5):179. doi: 10.1007/s10616-025-00845-0

Resveratrol-rich extract from Vitis vinifera canes exhibits enhanced anticancer activity in A549 cells and 3D lung cancer co-culture model

Kenza Reguouat 1,✉, Saliha Boumaza Bourioune 1, Khadidja Aouragh 2, Sonya Allali 2, Asma Amalou 1, Souhila Aouichat Bouguerra 1
PMCID: PMC12460211  PMID: 41018138

Abstract

Resveratrol-rich extracts from Vitis vinifera cane have gained recognition as natural compounds with significant anticancer potential. This study examined the impact of this extract on A549 lung cancer cells using both 2D monolayer culture and a more physiologically relevant 3D co-culture model incorporating peripheral blood mononuclear cells (PBMCs). In monolayer culture, at 40 µg/mL we evaluated the extract’s impact on cell viability, colony formation ability, morphological changes, and oxidative stress markers. A 3D co-culture model was employed to examine the effects of the extract on matrix metalloproteinase ( MMP-2 and MMP-9) activity, c-Jun N-terminal kinase (JNK) signaling pathway, and nitric oxide (NO) production. Our results demonstrated that the extract significantly reduced A549 cell viability (IC50 39.11 ± 0.32 µg/mL) and colony formation while inducing morphological changes and cellular redox imbalance markers malondialdehyde (MDA) (p ≤ 0.001), protein carbonyl (p ≤ 0.0001), Advanced Oxidation Protein Products (AOPP) (p ≤ 0.01), nitric oxide (NO) (p ≤ 0.0001), Superoxide Dismutase (SOD) activity (p ≤ 0.01), and glutathione (GSH) level (p ≤ 0.01). and in monolayer cultures. In the 3D co-culture system, treatment with the extract significantly decreased the growth rate of the 3D structures (P ≤ 0.1), reduced MMP-2 and MMP-9 activities (P ≤ 0.1), and JNK phosphorylation, with an overproduction of nitric oxide (P ≤ 0.001). These findings reveal novel mechanisms by which resveratrol-rich extracts from Vitis vinifera cane may exert anticancer effects by modulating the JNK/MMPs/NO axis in lung cancer cells A549. This extract could represent a promising therapeutic alternative for lung cancer treatment.

Keywords: Resveratrol, Vitis vinifera, Cytotoxicity, Redox status, 3D co-culture, Matrix metalloproteinases

Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide, with 2.21 million new cases and 1.80 million deaths reported in 2022 (WHO, 2022). Non-small cell lung cancer (NSCLC) accounts for nearly 85% of all lung cancer cases (Leonetti et al. 2018)). Despite advances in chemotherapy, significant challenges such as drug resistance and metastasis remain (Sequist et al. 2011). These issues have spurred research into alternative therapeutic strategies.

Natural products have emerged as promising options for cancer treatment owing to their diverse bioactive properties. Extracts from Vitis vinifera have garnered particular interest, as viticulture generates substantial waste, including Vitis vinifera cane (VVC), which is a natural source of polyphenolic compounds (Goufo et al. 2020; Baroi et al. 2022; Filimon et al. 2023).

Recent studies investigating the anticancer properties of VVC extracts (Medrano-Padial et al. 2019; Aja et al. 2020; Pop et al. 2022; Tsantila et al. 2024). have predominantly focused on their antiproliferative and cytotoxic effects in two-dimensional (2D) cell culture models, using various cancer cell lines. However, to date, no study has specifically investigated the effects of VVC extracts on the oxidative balance in A549 lung cancer cells.

Excessive ROS production may damage important macromolecules, such as nucleic acids (DNA fragmentation), proteins (carbonylation), and lipids (lipid peroxidation), leading to cell death and tissue injury. To avoid such damage, cancer cells produce an antioxidant system, including superoxide dismutase (SOD), glutathione (GSH), and catalase (Pizzino et al. 2017; Zhao et al. 2023). Nitric oxide (NO), a small molecule synthesized from L-arginine, plays a dual role in inflammation, exhibiting both anti-inflammatory and proinflammatory properties. At high concentrations, NO induces apoptosis via oxidative stress. Conversely, physiological levels of NO have been shown to inhibit apoptosis in various cell types, including lung cancer cells. Additionally, NO is implicated in tumor immunity and tumor cell death, influencing lung cancer development and progression (Luanpitpong and Chanvorachote 2015; Mintz et al. 2021).

Trans-resveratrol (RSV) is a major component of VVC. It has demonstrated efficacy against various cancer types (Pozo-Guisado et al. 2005; Tyagi et al. 2005; Benitez et al. 2006). However, its therapeutic potential is limited by its poor bioavailability, rapid metabolism, and low solubility. Resveratrol-rich extracts (RRE) offer a promising alternative exhibiting enhanced anticancer activity, while potentially improving bioavailability and reducing toxicity (Jovanović Galović et al. 2022).

However, conventional 2D cell cultures inadequately mimic the complex tumor microenvironment, which significantly influences cancer progression and drug responses (Kapałczyńska et al. 2016). Three-dimensional (3D) culture systems better replicate the structural complexity and cell-extracellular matrix (ECM) interactions characteristic of in vivo tumors. Incorporating peripheral blood mononuclear cells (PBMCs) into 3D spheroid models further enhances their physiological relevance by including immune cell interactions that impact ECM remodeling, therapeutic resistance, and immune responses (Fontoura et al. 2020; Saraiva et al. 2020).

Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, play a critical role in tumor invasion and metastasis due to their role in degrading the extracellular matrix (ECM) (Wei 2023). Their high activity and overexpression are frequently observed in NSCLC (Han et al. 2020). Additionally, high MMP expression is associated with lung cancer progression and metastasis (Merchant et al. 2017).

c-Jun N-terminal Kinase (JNK) is a stress-related enzyme involved in cell functions such as growth and death. Studies have shown that it is often activated in non-small-cell lung cancer (NSCLC), playing a role in tumor development. While increased JNK activity is linked to early lung tumors, it can also suppress cancer by triggering protective processes, such as autophagy. Given its dual role, JNK is a key target for developing targeted cancer therapies (Stulpinas et al. 2024).

To address these knowledge gaps, our study is the first to evaluate the effects of RRE from Algerian VVC, obtained using a rapid extraction method, on A549 cell lines. First, we assessed cell viability using MTT and AO/PI assays and evaluated morphological changes and colony formation in A549 cells in two-dimensional (2D) monolayers. In addition, we measured the redox balance markers, including MDA, APOP, protein carbonyls, NO, GSH, and SOD. In the 3D co-culture model of A549 cells and PBMCs, we investigated the growth rate, MMPs activity, phosphorylated JNK, and NO production, providing a comprehensive understanding of the anticancer potential of the extract in an environment that closely mimics in vivo tumor conditions.

Material and methods

Reagents and equipment

HPLC–DAD system was from Shimadzu Prominence-I 2030C Plus equipped with a C18 column 5 μm, 4.6 mm internal diameter, 250 mm length, Shim-pack GIST (Japan),Cell culture media and reagents (RPMI-1640, FBS, penicillin–streptomycin, l-glutamine) were from Gibco (Thermo Fisher Scientific, USA); resveratrol standard (≥ 99% purity, R5010) and MTT reagent from Sigma-Aldrich (USA); A549 cells were kindly provided by the Pasteur Institute, Algiers; BRESSER inverted microscope (Germany); lysis buffer (Cat. no. FNN0011) and Instant One™ ELISA kit for phospho-JNK1/JNK2 [pT183/pY185] from Invitrogen (Thermo Fisher Scientific, USA); chemicals for redox assays (TCA, TBA, KI, DNPH, guanidine hydrochloride, DTNB) and histopaque 1077 (Cat. no. 10771) from Sigma-Aldrich (USA); PHA from Gibco (USA); electrophoresis system for gelatin zymography Owl P81 from Thermo Fisher Scientific (USA), microplaque reader ( BioTech, USA).

Resveratrol extraction and yield

Vitis vinifera cane (VVC) was collected from the BOUMERDES region of Algeria (36.8706°N, 3.7219°E) in November and air-dried at room temperature. Dry powder (3 g) was ground in 120 ml methanol for 15 min, centrifuged at 6000 × g for 15 min at 4 °C, and the supernatant was filtered through Whatman filter paper. The filtrate was concentrated using a rotary evaporator at 40 °C, and the dry extract was collected and stored at 4 °C in HPLC-grade methanol for HPLC analysis. The extraction yield was calculated using the following equation:

Yield%=weightofdryextract/weightofdryplantX100

Total phenolic content

The total phenolic content was quantified using the Folin-Ciocalteu colorimetric method, as described by Singleton and Rossi (1965). The extract was mixed with 0.25 ml of Folin’s reagent, and sodium carbonate was added after 3 min. The mixture was vortexed and incubated at 40 °C for 40 min, and the resulting blue color was measured at 760 nm. The total phenolic content was expressed as milligrams of gallic acid equivalent per gram of extract (mg GA/g extract).

Antioxidant activity DPPH

The DPPH assay was performed according to the procedure described by Tepe et al. (2007). Briefly, 1.5 ml of each extract dilution was combined with an equal volume of DPPH solution prepared in methanol (0.004%). The mixture was incubated in the dark for 30 min, and the absorbance was measured at 517 nm using a spectrophotometer (each test was repeated three times). A control consisting of DPPH without extract was used, and the absorbance value at the beginning of the reaction was taken as a blank. The percentage inhibition was calculated using the formula I % = ((A control—A sample) / A control) × 100. The concentration that resulted in 50% inhibition (IC50) was determined by analyzing the change in anti-radical activity at different extract concentrations.

Quantification of resveratrol (HPLC–DAD)

Resveratrol extract (1 mg/mL) was prepared in methanol and filtered for HPLC–DAD analysis using a mobile phase consisting of acetonitrile (A) and 0.2% acetic acid (B). The gradient elution was as follows: 0 min: 95% A + 5% B; 30 min: 5% A + 95% B. Detection was at 306 nm (Nour et al. 2012).

Cell culture and treatment

A549 cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin–streptomycin, and 1.2% L-glutamine at. Cells were plated at 1 × 106 cells/well in 6-well plates and treated with 40 µg/mL RRE or RSV at 0.2 µg/mL dissolved in DMSO (< 0.05%) for 24 h.

MTT assay

The MTT assay was performed to assess cell viability and determine the IC50 value. Cells were seeded at 10,000 cells/well in 96-well plates and treated with varying concentrations of RRE dissolved in DMSO (< 0.05%) for 24 h. After treatment, 20 µL of MTT solution (5 mg/mL) was added to each well and incubated for 3.5 h at 37 °C. The supernatant was removed, 100 µL of DMSO was added to dissolve the formazan crystals, and the absorbance was measured at 590 nm. Cell viability was calculated as follows:

%Viability=ODTreated-ODBlank/ODControl-ODBlank×100

The IC50 values were determined by plotting viability percentages against treatment concentrations using non-linear regression analysis in GraphPad prism 8 software.

Acridine orange/propidium iodide (AO/PI) assay

A549 cells were seeded in 96-well plates at 104 cells/well and treated with 40 µg/mL RRE or 0.2 µg/mL RSV. After 24 h, 1 µL of AO/PI reagent (0.5 mg/mL) was added and incubated for 10 min. Dual fluorescence was measured using a multi-detection microplate reader with excitation/emission wavelengths of 460/650 nm for AO and 525/595 nm for PI (Hussain et al. 2019). Cell viability and non-viability were calculated as follows:

%cellviability=AOemoftreated-AoofblankAOemofcontrol-AOemofblank×100
%cellnon-viability=100-PIemofsample-PIofblankPIemofcontrol-PIemofblank×100

Colony formation assay and morphological changes

A549 cells were seeded at a density of 1000 cells/well in 6-well plates. After overnight attachment, RRE (40 µg/mL) was added for 24 h. The cells were then incubated for 10 days, with medium refreshment every 2 days. Colonies were fixed with 95% ethanol, stained with Giemsa, and counted manually (colonies ≥ 50 cells).

For morphological changes, cells were treated with resveratrol extract for 24 h, fixed with 70% alcohol, and stained with Giemsa. Images were captured using a BRESSER inverted microscope at 400 × magnification.

Redox status parameter

Malondialdehyde (MDA)

MDA levels were quantified in A549 cells to evaluate lipid peroxidation. After cell lysis using Invitrogen lysis buffer (FNN0011), the lysates were mixed with Na2HPO4/NaH2PO4 buffer (0.2 M, pH 6.5), centrifuged at 1000 × g for 20 min at 4 °C, and the supernatant was combined with trichloroacetic acid (TCA) and thiobarbituric acid (TBA). The mixture was incubated at 100 °C, and the absorbance of the complex was measured at 532 nm (Heath and Packer 1968).

Advanced oxidation protein products (AOPP)

Quantification of Advanced Oxidation Protein Products (AOPP). Cell lysates were mixed with 1.16 M potassium iodide (KI) and acetic acid for 2 min. Absorbance was then measured at 340 nm, and the results were expressed in nanomoles of AOPP/mL of chloramines-T equivalents, normalized to total protein/106 cells (Witko-Sarsat 1999).

Protein carbonyl

Cell lysates were incubated with 10 mM 2,4-dinitrophenylhydrazine in 2.5 M HCl at room temperature in the dark for 1 h, with swirling every 15 min. Proteins were precipitated using 20% trichloroacetic acid (TCA), and the pellets were washed once with 10% TCA and three times with ethanol/ethyl acetate (v/v), mechanically disrupted during each wash, and centrifuged at 6000 rpm for 5 min. The pellets were dissolved in 6 M guanidine hydrochloride at 37 °C for 15 min. Absorbance was measured at 370 nm, and the results were expressed as nanomoles of carbonyl groups per µg of protein using a molar extinction coefficient of 22,000 M⁻1 cm⁻1 (Levine et al. 1994).

Nitric oxide (NO)

Nitric oxide (NO) levels were estimated by measuring nitrite concentrations using the Griess method Griess (1879). An equal volume (50 μL) of the sample and an equal mixture of Griess reagents A + B (A: 0.1% N-1-naphthylethylenediamine dihydrochloride in 20% phosphoric acid. B: 1% sulfanilamide in 20% phosphoric acid) were incubated for 15 min. Absorbance was measured at 540 nm using a microplate reader (BioTek, USA), NaNO2 was used as standard and results were expressed as µM/mg protein.

Superoxide dismutase activity (SOD)

SOD activity was measured in A549 cell lysates using the nitro blue tetrazolium (NBT) photoreduction method. Reagent A (50 mM K-phosphate buffer, pH 7.8, 13 mM methionine, 75 mM NBT, 0.1 mM EDTA, and 4 mM riboflavin) was added to the samples. The reaction mixture was exposed to a 15 W light bulb for 10 min, and NBT reduction to formazan was measured at 560 nm wavelength. SOD activity was expressed as IU/106 cells, with one unit defined as the enzyme amount causing 50% inhibition of NBT reduction (Beauchamp and Fridovich 1971).

Glutathione (GSH)

Reduced GSH levels were quantified using the reaction with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB). A549 cell lysates (50 µL) were mixed with 100 µL phosphate shielded from light, and the absorbance was measured at 405 nm using a microplate reader. The results are expressed as µM GSH per 106 cells (Riener et al. 2002).

3D Co-Culture of A549 Cells and PBMCs

PBMCs were isolated from 4 mL of diluted peripheral blood using Histopaque 1077 density gradient centrifugation at 1000 × g for 20 min. The interface layer containing PBMCs was collected, washed thrice with RPMI 1640, and stimulated overnight with 5 µg/mL phytohemagglutinin (PHA) in complete RPMI 1640 (10% FBS, 1% penicillin–streptomycin, 1.2% glutamine) at 37 °C, 5% CO₂. Cells were counted using trypan blue, adjusted to 103 cells/100 µL in supplemented RPMI 1640, and used for coculture. A549 cells (103 per well) were seeded in an agarose-coated 96-well plate and incubated for 6 days at 37 °C and 5% CO₂, with medium changes every 2 days. On day 6, 103 PBMCs were added per well, and after 48 h (Saraiva et al. 2020), 40 µg/mL of RRE in FBS-free RPMI 1640 was added. After 48 h of incubation, the spheroids were harvested for ELISA, and the supernatants were stored at − 40 °C for gelatin zymography.

Growth rate parameter

Spheroid imaging was performed using a BRESSER inverted microscope at 100 × magnification. Growth assessment was conducted using ImageJ software by quantifying the surface area of individual spheroids to determine the proliferation rates in both the treated and control groups. The growth parameter for treated spheroids (kc) was established by calculating area variations over the treatment period, while the baseline growth parameter for control spheroids (k0) was obtained by measuring area changes in the untreated cultures during equivalent time intervals. The relative growth index was computed as the ratio of treated to control growth rates (kc/k0), providing a standardized measure of treatment efficacy on spheroid development. This analysis was applied to each experimental condition (Shahi Thakuri et al. 2019).

Gelatin zymography

The supernatant obtained from a 3D co-culture of A549 and PBMCs was diluted in a non-reducing buffer to obtain the same amount of protein in each sample and subjected to migration on a 10% SDS-PAGE gel containing 0.1% gelatin-10% acrylamide for 1h30,150 V. The gel was washed twice with 2.5% Triton X-100 to remove SDS, followed by incubation in Tris buffer (pH 7.8) at 37 °C for 18 h. The gel was then stained with 0.5% Coomassie R250 and destained in acetic acid–methanol solution (10%). MMP-2 and MMP-9 activity was visualized as clear bands against a black background, and ImageJ was used for band intensity quantification (Tyagi et al. 1993).

JNK1/JNK2 (phospho) [pT183/pY185] measurement

We used an Invitrogen ELISA kit Instant One™ according to the manufacturer's protocol. The absorbance was measured at 450 nm.

Nitric oxide (NO)

The nitrite measurement protocol used for the 3D co-culture lysates was identical to that previously described for evaluating the redox status in 2D A549 cultures.

Statistical analysis

Data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, USA). Data normality was assessed using the Shapiro–Wilk test. Comparisons between two groups were performed using an unpaired Student’s t-test, and multiple group comparisons were analyzed by one-way ANOVA followed by appropriate post hoc tests. A value of p < 0.05 was considered statistically significant.

Results

Extraction yield and chromatographic analysis

The extraction yield was 9.8% w/w plant powder. the retention time was 28.079 min and 28.223 min for the standard (Fig. 1). The concentration of resveratrol was 4.8 ug per 1 mg of extract. The polyphenol content of the extract was 14.2 ± 0.23 mg/g gallic acid equivalent (GAE). the IC50 value of DPPH was 38.95 ± 1,743 µg/mL.

Fig.1.

Fig.1

Open in a new tab

a chromatogram of resveratrol standard. b chromatogram of resveratrol rich extract (RRE). a trans-resveratrol peak was detected with a retention time of 28.079 min

Cytotoxicity and viability

The IC50 value of RRE determined by the MTT assay was 39.11 ± 0.32 µg/mL RRE (Fig. 2a). Cell viability and mortality in A549 cells were assessed using dual spectrofluorometry with AO/PI. After 24 h of treatment with 40 µg/mL of RRE (approximately its IC50 value), the percentage of viable cells decreased to 58.48% ± 8.66, while treatment with 0.2 µg/mL of pure RSV (equivalent to the RSV content in 40 µg/mL of RRE) resulted in a viability of 59.30% ± 4.36. The proportion of dead cells increased to 26.55% ± 1.27 with RRE treatment and to 25.91% ± 5.52 with pure RSV (Fig. 2).

Fig. 2.

Fig. 2

Open in a new tab

a Viability of A549 cells treated with RRE for 24 h, assessed using the MTT assay. Data are presented as mean ± SD, n = 6. b, c Percentage of viable and non-viable A549 cells after 24 h of treatment with 40 µg/mL of RRE or 0.2 µg/mL of RSV. Asterisks indicate statistically significant differences compared to untreated cells (ANOVA test) n = 6, (***p ≤ 0.001), (****p ≤ 0.0001)

Colony formation and morphological changes

A549 lung cancer cell growth was significantly reduced by RRE treatment, as demonstrated by a colony formation assay. The number of colonies in the untreated control cells was 272.66 ± 24.83, which decreased markedly to 134 ± 16.09 following treatment with the extract (Fig. 3a). In addition to reduced colony formation, we observed distinct morphological alterations in A549 cells treated with RRE compared to the control group. Untreated control cells maintained their typical spindle-shaped (fusiform) morphology, with homogeneous cytoplasm and intact plasma and nuclear membranes, reflecting healthy epithelial cell characteristics (Fig. 3c and e). In contrast, RRE-treated cells displayed significant morphological changes and chromatin hypercondensation, which are hallmark features of apoptosis. Furthermore, treated cells exhibited disruptions in both the plasma and nuclear membranes, accompanied by nuclear fragmentation and the formation of apoptotic bodies (Fig. 3d and f). At 100 × magnification (Fig. 3c, d), RRE-treated cells showed significant cell loss compared to controls, with disrupted cellular monolayers and empty spaces indicating cell death and detachment.

Fig.3.

Fig.3

Open in a new tab

a, b Effect of 40 µg/mL of RRE on A549 cells in a clonogenic assay. The graph shows the number of colonies formed in control and treated cells with 40 µg/mL RRE for 24 h. (**P ≤ 0.01), n = 3. c, e Untreated A549 cells stained with Giemsa. d, f A549 cells were treated with 40 µg/mL of RRE for 24 h. and stained with Giemsa. Arrows indicate: red – apoptotic bodies; yellow– chromatin condensation; blue– plasma and nuclear membrane fragmentation. (c, d: X100; e, f: X400 magnification)

Redox status parameters

Exposure of A549 cancer cells to RRE (40 µg/mL) resulted in marked changes in redox homeostasis. Antioxidant markers, including SOD and GSH, showed a significant increase. Conversely, oxidative damage indicators were also elevated, as evidenced by the significant rise in lipid peroxidation (MDA levels), protein carbonyl content, and POAPP. Moreover, NO production was markedly increased. (Fig. 4).

Fig.4.

Fig.4

Open in a new tab

a MDA. b Protein carbonyl. c AOPP. d NO. (e) GSH. f SOD Activity; evaluation in control and treated cells with 40 μg/mL of RRE during 24 h. Data are expressed mean ± SD. n = 6, (***p ≤ 0.001), (**p ≤ 0.01). (****p ≤ 0.0001)

3D co-culture study

Growth rate parameter

3D A549 spheroids were cultured with PBMCs for 48 h before adding the treatment, and 3D coculture spheroids were cultured for 48 h with 40 µg/ml of RRE. (Fig. 5). the growth rates of treated 3D co-culture were calculated and compared to the control. The control group exhibited a mean growth rate of 0.41 ± 0.5, while RRE treated group showed a mean growth rate of − 0.11 ± 0.24 and RSV was about − 0.02 ± 0.08.The ratio of growth rates (treated/control) was calculated to be − 0.27, indicating a 27% reduction in the growth rate of the treated co-culture compared to the control (Fig. 5) and for RSV it was − 0.05, indicating that antiproliferative effects of RRE in 3D coculture model of A59 cells and PBMCs is more effective than RSV alone.

Fig.5.

Fig.5

Open in a new tab

a 3D A549 spheroid before adding PBMCs, b 3D A549 spheroid after being cultured with PBMCs for 48 h. c 3D co-culture before RRE treatment. d 3D co-culture after RRE treatment with. e the growth rate parameter of 3D co-culture of A549 and PBMCs before and after treatment with 40 µg/ml of resveratrol-rich extract (RRE) and resveratrol (RSV) 0.2 µg/mL during 48 h. Data are expressed mean ± SD, n = 6. The Asterix indicate significant difference (*P ≤ 0.1)

Gelatin zymography to study MMP-2 and MMP-9 activity, JNK1/JNK2 (Phospho) [pT183/pY185] and nitric oxide

We revealed the effect of RRE from VVC on the activity of MMP-2 and MMP-9 in 3D co-culture supernatants using gelatin zymography. The zymogram gel showed a clear band, indicating MMPs activity. The activity of both MMP-2 and MMP-9 decreased significatively in treated 3D co-culture compared to that in control (Fig. 6a, b). RRE significantly decreased JNK1/JNK2 (Phospho) [pT183/pY185] in treated 3D co-culture lysates (Fig. 6c). We evaluated NO level by estimating nitrate in 3D co-culture, RRE significantly increases NO level (Fig. 6d).

Fig.6.

Fig.6

Open in a new tab

a, b Gelatinolytic activity of MMP–9 and MMP-2 in the supernatant of a 3D co-culture of A549 cells and PBMCs, untreated (control) and treated with 40 µg/mL of RRE (*P ≤ 0.1), n = 4. c Optical density of phospho-JNK 1/2 (Thr183/Tyr185) normalized to total protein in a 3D co-culture of A549 cells and PBMCs, untreated (control) and treated with 40 µg/mL of RRE for 48 h (***P ≤ 0.001), n = 6. d Nitric oxide (NO) levels (µM/mg of protein) in the 3D co-culture of A549 cells and PBMCs, untreated (control) and treated with 40 µg/mL of RRE for 48 h, n = 6 (***P ≤ 0.001)

Discussion

In this study, we investigated the anticancer activity of a resveratrol-rich extract (RRE) from Vitis vinifera canes (VVC) against A549 lung cancer cells using monolayer and 3D co-culture models with PBMCs. VVC are a recognized source of stilbenes, particularly resveratrol, whose levels vary according to grape genotype, environmental conditions, and extraction procedures (Aaviksaar et al. 2003; Gorena et al. 2014; Goufo et al. 2020; Kodeš et al. 2021; Noviello et al. 2022).

In 2D culture, treatment with RRE at 40 µg/mL (close to its IC50 determined by MTT assay) reduced A549 cell viability to 58%, as confirmed by AO/PI staining and colony formation assays, and induced characteristic apoptotic features, including chromatin condensation, apoptotic bodies, and plasma/nuclear membrane disruption. Pure resveratrol at a concentration equivalent to that present in RRE was used as a reference standard in these assays. Comparable cytotoxicity between RSV and RRE in this simplified model indicates that RSV contributes to the observed effects, while additional bioactive compounds in the extract may also play a role.

These findings align with previous reports demonstrating that VVC extracts rich in stilbenes trigger apoptosis in various cancer cell lines, including HepG2 and Caco-2 (Medrano-Padial et al. 2019), HeLa, and MCF-7 (Jovanović Galović et al. 2022). The comparable efficacy between resveratrol-rich extracts and pure resveratrol observed in conventional 2D cultures is consistent with the findings of Jovanović Galović et al. (2022), who reported similar cytotoxicity levels for both treatments in malignant cells, while the extracts showed lower toxicity toward non-malignant fibroblasts (MRC-5). Resveratrol has been shown to induce apoptosis and inhibit the proliferation of A549 cells through multiple mechanisms, including the regulation of p53, Bax, Bcl-2, caspase-3 cleavage, and NF-κB inhibition, accompanied by important morphological changes (Kim et al. 2003; Wang et al. 2015). Additionally, resveratrol can induce cell cycle arrest in A549 cells (Yuan et al. 2015).

Although the pro-oxidant and antioxidant effects of pure resveratrol have been extensively studied in cancer cells, the redox-modulating potential of complex resveratrol-rich extracts (RRE) remains less explored. To better understand the mechanisms underlying the apoptosis observed in our 2D assays, we investigated the oxidative and nitrosative stress responses induced by RRE in A549 cells. RRE treatment induced significant oxidative stress markers in A549 cells, as evidenced by elevated MDA levels and increased protein carbonylation levels. Elevated MDA levels indicate lipid peroxidation, which compromises membrane integrity and promotes cell death (Kannan and Jain 2000; Jia et al. 2021; Arfin et al. 2021) while increased protein carbonylation reflects oxidative damage that impairs vital cellular functions (Rodríguez-García et al. 2020). The simultaneous increase in nitric oxide (NO) levels suggests enhanced nitrosative stress, which can promote antitumorigenic effects, including apoptosis induction (Mintz et al. 2021).

Interestingly, SOD activity and GSH levels also increased, likely representing an adaptive cellular response to counteract elevated ROS levels. These findings are consistent with those of Martins et al. (2014), who demonstrated that resveratrol increased SOD activity and MDA levels and reduced thiols in GRX cells.

Our results support the well-established dual role of polyphenols such as resveratrol, which act as pro-oxidants in cancer cells to disrupt redox homeostasis (Zheng et al. 2006; de la Lastra and Villegas 2007; León-González et al. 2015). This pro-oxidant mechanism is further supported by Vassi et al. (2022), who showed that Greek vine variety extracts modulated oxidative stress in HeLa and HepG2 cancer cell lines by altering intracellular GSH and ROS levels. Notably, the different extracts exhibited varying pro-oxidant activities: the Rhoditis extract had significant effects in both cell lines, the Muscat extract was active exclusively in HeLa cells, and the Mavrodaphne extract showed no measurable impact. Together, these findings demonstrate that RRE exerts its anticancer effects by inducing oxidative stress, overwhelming cellular antioxidant defense systems, and ultimately promoting cancer cell death.

To address the limitations of 2D monolayers, RRE was evaluated in a physiologically relevant 3D co-culture model incorporating PBMCs. This model better mimics the tumor microenvironment and allows assessment of tumor-immune interactions, which are critical for evaluating multi-target effects of natural extracts (Langhans 2018; Jensen and Teng 2020). In our study, RRE significantly reduced spheroid growth, showing a more pronounced effect than pure resveratrol and suggesting that the extract itself holds a higher therapeutic potential. The enhanced effect of RRE in this 3D system is likely related to the complex cellular interactions, which allow multi-target mechanisms to be expressed more effectively. As highlighted by Caesar and Cech (2019), simple 2D monolayer assays often fail to capture the synergistic effects of multi-component natural extracts, whereas more physiologically relevant models including co-cultures with immune cells enable the detection of combination effects and interactions between cancer and immune cells. The presence of PBMCs in the spheroids provides additional paracrine and cell–cell signaling cues, creating a tumor microenvironment that more closely resembles in vivo conditions (Hickman et al. 2014).

Recent studies have highlighted the effectiveness of resveratrol in 3D A549 spheroid models of lung cancer. Livraghi et al. (2024) demonstrated its inhibitory effect on A549 lung cancer spheroids at 30 µM, whereas Wang et al. (2020) found that resveratrol-loaded nanoparticles exhibited stronger anticancer activity in a 3D model. Barros et al. (2018) showed that 3D pancreatic cancer spheroids (PANC-1) are more resistant to and resveratrol than 2D cultures While resveratrol alone was less potent, combining it with doxorubicin where RSV was present at 4–5 times higher concentrations, significantly improved efficacy in the resistant 3D model. These findings highlight that 3D models better reflect tumor resistance mechanisms, and resveratrol’s activity is enhanced within complex mixtures. Our results, showing greater RRE efficacy than pure RSV in 3D co-cultures (27% vs 5% growth reduction), support this principle, suggesting that multiple bioactive compounds in RRE contribute to overcoming tumor resistance.

Gelatin zymography analysis revealed that RRE significantly reduced MMP-2 and MMP-9 activity in the 3D co-culture model, suggesting potential anti-metastatic effects. This finding is particularly significant because elevated MMPs activity is a hallmark of aggressive cancers, including NSCLC, and is associated with enhanced metastatic capacity and poor clinical outcomes (Sternlicht and Werb 2001; Merchant et al. 2017; Han et al. 2020; Wei 2023). The presence of immune cells in our model is crucial, as PBMCs can both stimulate and regulate MMPs (Parks et al. 2004). Previous studies have shown that resveratrol inhibits MMP-2 and MMP-9 activity in various cancer cell lines in 2D culture, including glioblastoma (Jiao et al. 2015), fibrosarcoma (Lee and Kim 2011), and in lung adenocarcinoma (Liu et al. 2010; Kim et al. 2012; Wang et al. 2023) suppressing metastasis and invasion. However, our study provides the first evidence of the MMP-inhibitory effects of resveratrol extract in a 3D co-culture system that incorporates immune-tumor interactions, which better represents the physiological tumor microenvironment where MMPs are produced by both cancer cells and infiltrating immune cells (Kessenbrock et al. 2010; He et al. 2023).

Additionally, RRE significantly reduced JNK phosphorylation, a key signaling pathway involved in tumor-immune interactions, cancer progression, and resistance to therapy (Insua‐Rodríguez et al. 2018; Tam and Law 2021). The JNK pathway regulates MMPs activity (Westermarck and Kähäri 1999), establishing a mechanistic link between JNK inhibition and the observed reduction in MMPs activity.

Our findings are supported by previous studies demonstrating the inhibitory effects of resveratrol on JNK signaling. Gweon and Kim (2014) showed that resveratrol inhibits both JNK phosphorylation and MMPs activity (MMP–2 and MMP–9) in chondrosarcoma cells. Additionally, resveratrol has been shown to reduce JNK1/2 phosphorylation in hepatocellular carcinoma (Huh7) cells (Yeh et al. 2013). Similarly, Yang et al. (2009) demonstrated that a resveratrol analog inhibited the invasion of A549 cells by reducing MMP–2 activity and JNK phosphorylation.

However, the effects of resveratrol on JNK signaling reported in the literature are inconsistent. While our study and others demonstrate JNK inhibition, some studies have shown that resveratrol can activate JNK (She et al. 2002; Puissant et al. 2010; Zhang et al. 2022). To the best of our knowledge, no study has specifically investigated the direct effect of resveratrol on JNK activity in A549 lung cancer cells using 3D co-culture models. These varying results underscore the importance of investigating the specific conditions and mechanisms that determine the effects of resveratrol on JNK signaling, particularly within physiologically relevant models that better recapitulate the tumor microenvironment (TME).

Elevated nitric oxide (NO) levels play a dual role in cancer biology. High NO concentrations inhibit cell proliferation and promote apoptosis in various cancer cell lines, including MCF–7 and HepG2 (Pozo-Guisado et al. 2005; Notas et al. 2006). Furthermore, NO modulates MMPs activity in a concentration-dependent manner; high NO concentrations inhibit MMPs activity, whereas low concentrations stimulate it (Utispan and Koontongkaew 2020).

Importantly, NO negatively regulates JNK signaling through multiple mechanisms. NO inhibits JNK1 activity via S-nitrosylation and thiol redox mechanisms, disrupting the physical interaction between JNK and its downstream substrates, such as c-Jun (So et al. 1998; Parks et al. 2004; Park et al. 2006). This leads to the suppression of JNK phosphorylation and downstream signaling pathways involved in tumor cell survival and apoptosis.

In our study, the simultaneous increase in NO levels and decrease in JNK phosphorylation following RRE treatment suggests a potential mechanism. Elevated NO levels may contribute to JNK inhibition through direct post-translational modifications, while both factors independently contribute to MMPs inhibition and the overall anti-metastatic effects observed. This interconnected network of NO-JNK-MMPs signaling supports the multifaceted anticancer potential of RRE. Although the 3D co-culture model with PBMCs is more realistic than standard 2D cultures, it has its limitations. The model lacks other important cells found in tumors, such as fibroblasts and endothelial cells, which prevents the full reproduction of the tumor environment. Additionally, RRE was not tested on normal lung cells, limiting our understanding of its selectivity and potential side effects on healthy tissue. Furthermore, while RRE may offer improved bioavailability compared to pure resveratrol, we did not assess the actual bioavailability or pharmacokinetic properties of the extract in our study.

Conclusions

This study demonstrates the potent anticancer activity of resveratrol-rich extracts from V. vinifera cane on A549 lung cancer cells in both 2D monolayer cultures and 3D co-cultures with PBMCs. By inducing cell death, modulating redox balance in 2D cultures, and inhibiting MMPs activity while decreasing phosphorylated JNK and increasing nitric oxide in 3D models, suggesting potential roles in limiting metastasis and tumor growth.

Our use of a simple, accessible, and cost-effective lung cancer 3D co-culture model represents a methodological advance for preclinical pharmacological and toxicological research, bridging the gap between basic 2D assays and in vivo studies.

Furthermore, leveraging vine canes a low-value agricultural byproduct enhances resource efficiency and economic sustainability. Resveratrol-rich extracts from Algerian vine canes may address bioavailability challenges while reducing toxicity, providing new avenues for cancer treatment. Future studies are needed to investigate additional bioactive compounds in vine canes, optimize extract formulations, and evaluate efficacy in vivo, leading to the development of novel solutions in health and agriculture.

Acknowledgements

Our thanks go to Djefal Assia and Gais Soumeya of Algiers nuclear research center, 02, Boulevard Frantz Fanon Algiers, Algeria for their valuable help. This work was funded by the General Directorate of Scientific Research of Development and Technology in a socio-economic project accepted in 2019 until 2023.

Author contribution

R.K.: Conceived and conceptualized the research, developed the study, organized and executed the experimental procedures, interpreted the resulting data, Writing article B.S.: Contributed to the experimental design and provided expertise in redox status assessment. A.K., Allali S and A.A.: participated in experimental, data collection. Aouichat S.: Supervision of the overall project, secured funding, and provided critical intellectual input throughout the study. Participated in data interpretation, manuscript revision, and final approval for publication.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

10/17/2025

The original online version of this article was revised due to tagging in the author affiliation.

References

  1. Aaviksaar A, Haga M, Püssa T et al (2003) Purification of resveratrol from vine stems. Proc Est Acad Sci Chem 52:155. 10.3176/chem.2003.4.02 [Google Scholar]
  2. Aja I, Ruiz-Larrea MB, Courtois A et al (2020) Screening of natural stilbene oligomers from Vitis vinifera for anticancer activity on human hepatocellular carcinoma cells. Antioxidants 9:469. 10.3390/antiox9060469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arfin S, Jha NK, Jha SK et al (2021) Oxidative stress in cancer cell metabolism. Antioxidants 10:642. 10.3390/antiox10050642 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baroi AM, Popitiu M, Fierascu I et al (2022) Grapevine wastes: a rich source of antioxidants and other biologically active compounds. Antioxidants 11:393. 10.3390/antiox11020393 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Barros AS, Costa EC, Nunes AS et al (2018) Comparative study of the therapeutic effect of doxorubicin and resveratrol combination on 2D and 3D (spheroids) cell culture models. Int J Pharm 551:76–83. 10.1016/j.ijpharm.2018.09.016 [DOI] [PubMed] [Google Scholar]
  6. Beauchamp C, Fridovich I (1971) Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal Biochem 44(1):276-287. 10.1016/0003-2697(71)90370-8 [DOI] [PubMed]
  7. Benitez DA, Pozo-Guisado E, Alvarez-Barrientos A et al (2006) Mechanisms Involved in Resveratrol-Induced Apoptosis and Cell Cycle Arrest in Prostate Cancer-Derived Cell Lines. J Androl 28:282–293. 10.2164/jandrol.106.000968 [DOI] [PubMed] [Google Scholar]
  8. Caesar LK, Cech NB (2019) Synergy and antagonism in natural product extracts: when 1 + 1 does not equal 2. Nat Prod Rep 36:869–888. 10.1039/C9NP00011A [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Christian K, Riener Gerald, Kada Hermann J, Gruber (2002) Quick measurement of protein sulfhydryls with Ellman's reagent and with 44′-dithiodipyridine. Anal Bioanal Chemis 373(4-5):266-276. 10.1007/s00216-002-1347-2 [DOI] [PubMed]
  10. de la Lastra CA, Villegas I (2007) Resveratrol as an antioxidant and pro-oxidant agent: mechanisms and clinical implications. Biochem Soc Trans 35:1156–1160. 10.1042/BST0351156 [DOI] [PubMed] [Google Scholar]
  11. Filimon RV, Bunea CI, Bora FD et al (2023) Physico-chemical characterization, phenolic compound extraction and biological activity of grapevine (Vitis vinifera L.) canes. Horticulturae 9:1164. 10.3390/horticulturae9111164 [Google Scholar]
  12. Fontoura JC, Viezzer C, Dos Santos FG et al (2020) Comparison of 2D and 3D cell culture models for cell growth, gene expression and drug resistance. Mater Sci Eng, C 107:110264. 10.1016/j.msec.2019.110264 [DOI] [PubMed] [Google Scholar]
  13. Gorena T, Saez V, Mardones C et al (2014) Influence of post-pruning storage on stilbenoid levels in Vitis vinifera L. canes. Food Chem 155:256–263. 10.1016/j.foodchem.2014.01.073 [DOI] [PubMed] [Google Scholar]
  14. Goufo P, Singh RK, Cortez I (2020) A reference list of phenolic compounds (including stilbenes) in grapevine (Vitis vinifera L.) roots, woods, canes, stems, and leaves. Antioxidants 9:398. 10.3390/antiox9050398 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gweon EJ, Kim S-J (2014) Resveratrol attenuates matrix metalloproteinase-9 and -2- regulated differentiation of HTB94 chondrosarcoma cells through the p38 kinase and JNK pathways. Oncol Rep 32:71–78. 10.3892/or.2014.3192 [DOI] [PubMed] [Google Scholar]
  16. Han L, Sheng B, Zeng Q et al (2020) Correlation between MMP2 expression in lung cancer tissues and clinical parameters: a retrospective clinical analysis. BMC Pulm Med 20:283. 10.1186/s12890-020-01317-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. He L, Kang Q, Chan KI et al (2023) The immunomodulatory role of matrix metalloproteinases in colitis-associated cancer. Front Immunol 13:1093990. 10.3389/fimmu.2022.1093990 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heath RL, Packer K (1968) Photoperoxidation in isolated chloroplasts. Archiv Biochem Biophy 125(1):189-198. 10.1016/0003-9861(68)90654-1 [DOI] [PubMed]
  19. Hickman JA, Graeser R, De Hoogt R et al (2014) Three-dimensional models of cancer for pharmacology and cancer cell biology: capturing tumor complexity in vitro/ex vivo. Biotechnol J 9:1115–1128. 10.1002/biot.201300492 [DOI] [PubMed] [Google Scholar]
  20. Hussain HLSR, Ahmad S et al (2019) Determination of cell viability using acridine orange/propidium iodide dual spectrofluorometry assay. Cogent Food Agric 5:1582398. 10.1080/23311932.2019.1582398
  21. Insua-Rodríguez J, Pein M, Hongu T et al (2018) Stress signaling in breast cancer cells induces matrix components that promote chemoresistant metastasis. EMBO Mol Med 10:e9003. 10.15252/emmm.201809003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jensen C, Teng Y (2020) Is it time to start transitioning from 2D to 3D cell culture? Front Mol Biosci 7:33. 10.3389/fmolb.2020.00033 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jia B, Zheng X, Wu M-L et al (2021) Increased reactive oxygen species and distinct oxidative damage in Resveratrol-suppressed glioblastoma cells. J Cancer 12:141–149. 10.7150/jca.45489 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jiao Y, Li H, Liu Y et al (2015) Resveratrol inhibits the invasion of glioblastoma-initiating cells via down-regulation of the PI3K/Akt/NF-κB signaling pathway. Nutrients 7:4383–4402. 10.3390/nu7064383 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Jovanović Galović A, Jovanović Lješković N, Vidović S et al (2022) The effects of resveratrol-rich extracts of Vitis vinifera pruning waste on HeLa, MCF-7 and MRC-5 cells: apoptosis, autophagia and necrosis interplay. Pharmaceutics 14:2017. 10.3390/pharmaceutics14102017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kannan K, Jain SK (2000) Oxidative stress and apoptosis. Pathophysiology 7:153–163. 10.1016/S0928-4680(00)00053-5 [DOI] [PubMed] [Google Scholar]
  27. Kapałczyńska M, Kolenda T, Przybyła W et al (2016) 2D and 3D cell cultures – a comparison of different types of cancer cell cultures. Arch Med Sci. 10.5114/aoms.2016.63743 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kessenbrock K, Plaks V, Werb Z (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141:52–67. 10.1016/j.cell.2010.03.015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kim Y-A, Lee W, Choi T et al (2003) Involvement of p21WAF1/CIP1, pRB, Bax and NF-κB in induction of growth arrest and apoptosis by resveratrol in human lung carcinoma A549 cells. Int J Oncol. 10.3892/ijo.23.4.1143 [PubMed] [Google Scholar]
  30. Kim YS, Sull JW, Sung HJ (2012) Suppressing effect of resveratrol on the migration and invasion of human metastatic lung and cervical cancer cells. Mol Biol Rep 39:8709–8716. 10.1007/s11033-012-1728-3 [DOI] [PubMed] [Google Scholar]
  31. Kodeš Z, Vrublevskaya M, Kulišová M et al (2021) Composition and biological activity of Vitis vinifera winter cane extract on Candida biofilm. Microorganisms 9:2391. 10.3390/microorganisms9112391 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Langhans SA (2018) Three-dimensional in vitro cell culture models in drug discovery and drug repositioning. Front Pharmacol 9:6. 10.3389/fphar.2018.00006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lee S-J, Kim M-M (2011) Resveratrol with antioxidant activity inhibits matrix metalloproteinase via modulation of SIRT1 in human fibrosarcoma cells. Life Sci 88:465–472. 10.1016/j.lfs.2011.01.005 [DOI] [PubMed] [Google Scholar]
  34. Leonetti A, Facchinetti F, Rossi G et al (2018) BRAF in non-small cell lung cancer (NSCLC): pickaxing another brick in the wall. Cancer Treat Rev 66:82–94. 10.1016/j.ctrv.2018.04.006 [DOI] [PubMed] [Google Scholar]
  35. León-González AJ, Auger C, Schini-Kerth VB (2015) Pro-oxidant activity of polyphenols and its implication on cancer chemoprevention and chemotherapy. Biochem Pharmacol 98:371–380. 10.1016/j.bcp.2015.07.017 [DOI] [PubMed] [Google Scholar]
  36. Levine RL, Williams JA, Stadtman EP, Shacter E (1994) Oxygen Radicals in Biological Systems Part C [37] Carbonyl assays for determination of oxidatively modified proteins Elsevier 346–357 [DOI] [PubMed]
  37. Liu P, Tsai J, Charles AL et al (2010) Resveratrol inhibits human lung adenocarcinoma cell metastasis by suppressing heme oxygenase 1-mediated nuclear factor-κB pathway and subsequently downregulating expression of matrix metalloproteinases. Mol Nutr Food Res. 10.1002/mnfr.200900550 [DOI] [PubMed] [Google Scholar]
  38. Livraghi V, Grossi A, Scopelliti A et al (2024) Stilbene treatment reduces stemness features in human lung adenocarcinoma model. Int J Mol Sci 25:10390. 10.3390/ijms251910390 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Luanpitpong S, Chanvorachote P (2015) Nitric oxide and aggressive behavior of lung cancer cells. Anticancer Res 35:4585–4592 [PubMed] [Google Scholar]
  40. Martins LAM, Coelho BP, Behr G et al (2014) Resveratrol induces pro-oxidant effects and time-dependent resistance to cytotoxicity in activated hepatic stellate cells. Cell Biochem Biophys 68:247–257. 10.1007/s12013-013-9703-8 [DOI] [PubMed] [Google Scholar]
  41. Medrano-Padial C, Puerto M, Moreno FJ et al (2019) In vitro toxicity assessment of stilbene extract for its potential use as antioxidant in the wine industry. Antioxidants 8:467. 10.3390/antiox8100467 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Merchant N, Nagaraju GP, Rajitha B et al (2017) Matrix metalloproteinases: their functional role in lung cancer. Carcinogenesis 38:766–780. 10.1093/carcin/bgx063 [DOI] [PubMed] [Google Scholar]
  43. Mintz J, Vedenko A, Rosete O et al (2021) Current advances of nitric oxide in cancer and anticancer therapeutics. Vaccines 9:94. 10.3390/vaccines9020094 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Notas G, Nifli A-P, Kampa M et al (2006) Resveratrol exerts its antiproliferative effect on HepG2 hepatocellular carcinoma cells, by inducing cell cycle arrest, and NOS activation. Biochimica Et Biophysica Acta (BBA) 1760:1657–1666. 10.1016/j.bbagen.2006.09.010 [DOI] [PubMed] [Google Scholar]
  45. Noviello M, Caputi AF, Squeo G et al (2022) Vine shoots as a source of trans-resveratrol and ε-viniferin: a study of 23 Italian varieties. Foods 11:553. 10.3390/foods11040553 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Park H-S, Mo J-S, Choi E-J (2006) Nitric oxide inhibits an interaction between JNK1 and c-Jun through nitrosylation. Biochem Biophys Res Commun 351:281–286. 10.1016/j.bbrc.2006.10.034 [DOI] [PubMed] [Google Scholar]
  47. Parks WC, Wilson CL, López-Boado YS (2004) Matrix metalloproteinases as modulators of inflammation and innate immunity. Nat Rev Immunol 4:617–629. 10.1038/nri1418 [DOI] [PubMed] [Google Scholar]
  48. Pizzino G, Irrera N, Cucinotta M et al (2017) Oxidative stress: harms and benefits for human health. Oxid Med Cell Longev 2017:8416763. 10.1155/2017/8416763 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pop A, Bogdan C, Fizesan I et al (2022) In vitro evaluation of biological activities of canes and pomace extracts from several varieties of Vitis vinifera L. for inclusion in freeze-drying mouthwashes. Antioxidants 11:218. 10.3390/antiox11020218 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Pozo-Guisado E, Merino JM, Mulero-Navarro S et al (2005) Resveratrol-induced apoptosis in MCF-7 human breast cancer cells involves a caspase-independent mechanism with downregulation of Bcl-2 and NF-?B. Int J Cancer 115:74–84. 10.1002/ijc.20856 [DOI] [PubMed] [Google Scholar]
  51. Shahi Thakuri P, Gary D Luker, Hossein TavanaV(2019) Cyclical Treatment of Colorectal Tumor Spheroids Induces Resistance to MEK Inhibitors. Trans Oncol 12(3) 404-416 10.1016/j.tranon.2018.11.009 [DOI] [PMC free article] [PubMed]
  52. Puissant A, Robert G, Fenouille N et al (2010) Resveratrol promotes autophagic cell death in chronic myelogenous leukemia cells via JNK-mediated p62/SQSTM1 expression and AMPK activation. Cancer Res 70:1042–1052. 10.1158/0008-5472.CAN-09-3537 [DOI] [PubMed] [Google Scholar]
  53. Riener CK, Kada G, Gruber HJ (2002) Quick measurement of protein sulfhydryls with Ellman's reagent and with 44′-dithiodipyridine. Anal Bioanal Chemis 373(4-5):266-276. 10.1007/s00216-002-1347-2 [DOI] [PubMed]
  54. Rodríguez-García A, García-Vicente R, Morales ML et al (2020) Protein carbonylation and lipid peroxidation in hematological malignancies. Antioxidants 9:1212. 10.3390/antiox9121212 [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Saraiva DP, Matias AT, Braga S et al (2020) Establishment of a 3D co-culture with MDA-MB-231 breast cancer cell line and patient-derived immune cells for application in the development of immunotherapies. Front Oncol 10:1543. 10.3389/fonc.2020.01543 [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Sequist LV, Waltman BA, Dias-Santagata D et al (2011) Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors. Sci Transl Med. 10.1126/scitranslmed.3002003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. She Q, Huang C, Zhang Y, Dong Z (2002) Involvement of c-jun NH2 -terminal kinases in resveratrol-induced activation of p53 and apoptosis. Mol Carcinog 33:244–250. 10.1002/mc.10041 [DOI] [PubMed] [Google Scholar]
  58. So HS, Park RK, Kim MS et al (1998) Nitric oxide inhibits c-Jun N-terminal kinase 2 (JNK2) via S-nitrosylation. Biochem Biophys Res Commun 247:809–813. 10.1006/bbrc.1998.8788 [DOI] [PubMed] [Google Scholar]
  59. Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 17:463–516. 10.1146/annurev.cellbio.17.1.463 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Stulpinas A, Tenkutytė M, Imbrasaitė A, Kalvelytė AV (2024) The role and efficacy of JNK inhibition in inducing lung cancer cell death depend on the concentration of Cisplatin. ACS Omega 9:28311–28322. 10.1021/acsomega.4c01950 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Tam SY, Law HK-W (2021) JNK in tumor microenvironment: present findings and challenges in clinical translation. Cancers 13:2196. 10.3390/cancers13092196 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tsantila EM, Esslinger N, Christou M et al (2024) Antioxidant and anticancer activity of Vitis vinifera extracts in breast cell lines. Life 14:228. 10.3390/life14020228 [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tyagi SC, Matsubara L, Weber KT (1993) Direct extraction and estimation of collagenase(s) activity by zymography in microquantities of rat myocardium and uterus. Clinical Biochem 26(3):191-198. 10.1016/0009-9120(93)90025-2 [DOI] [PubMed]
  64. Tyagi A, Singh RP, Agarwal C et al (2005) Resveratrol causes Cdc2-tyr15 phosphorylation via ATM/ATR–Chk1/2–Cdc25C pathway as a central mechanism for S phase arrest in human ovarian carcinoma Ovcar-3 cells. Carcinogenesis 26:1978–1987. 10.1093/carcin/bgi165 [DOI] [PubMed] [Google Scholar]
  65. Utispan K, Koontongkaew S (2020) High nitric oxide adaptation in isogenic primary and metastatic head and neck cancer cells. Anticancer Res 40:2657–2665. 10.21873/anticanres.14236 [DOI] [PubMed] [Google Scholar]
  66. Vassi E, Veskoukis A, Tekos F et al (2022) Biological effects of grape stem extracts on human cancer cell lines. Int J Funct Nutr 3:4. 10.3892/ijfn.2022.27 [Google Scholar]
  67. Wang X, Wang D, Zhao Y (2015) Effect and mechanism of Resveratrol on the apoptosis of lung adenocarcinoma cell line A549. Cell Biochem Biophys 73:527–531. 10.1007/s12013-015-0696-3 [DOI] [PubMed] [Google Scholar]
  68. Wang X, Parvathaneni V, Shukla SK et al (2020) Inhalable resveratrol-cyclodextrin complex loaded biodegradable nanoparticles for enhanced efficacy against non-small cell lung cancer. Int J Biol Macromol 164:638–650. 10.1016/j.ijbiomac.2020.07.124 [DOI] [PubMed] [Google Scholar]
  69. Wang W, Zeng Z, Zhang J, Sun Z (2023) A systematic study of traditional Chinese medicine for the treatment of lung adenocarcinoma using a reverse network of key targets based on bioinformatics and molecular docking: curcumin and trans-resveratrol as potential drug candidates for lung adenocarcinoma. Nat Prod Commun 18:1934578X231169370. 10.1177/1934578X231169370 [Google Scholar]
  70. Wei C (2023) The multifaceted roles of matrix metalloproteinases in lung cancer. Front Oncol 13:1195426. 10.3389/fonc.2023.1195426 [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Westermarck J, Kähäri VM (1999) Regulation of matrix metalloproteinase expression in tumor invasion. FASEB J off Publ Fed Am Soc Exp Biol 13:781–792 [PubMed] [Google Scholar]
  72. Witko-Sarsat V (1999) Advanced oxidation protein products as a novel molecular basis of oxidative stress in uraemia. Nephrol Dialysis Transplan 14(90001):76-78. 10.1093/ndt/14.suppl_1.76 [DOI] [PubMed]
  73. World Health Organization (2022) Global cancer observatory: Cancer today. International Agency for Research on Cancer. https://gco.iarc.who.int/today
  74. Yang Y, Weng C, Ho C, Yen G (2009) Resveratrol analog-3,5,4′-trimethoxy- trans -stilbene inhibits invasion of human lung adenocarcinoma cells by suppressing the MAPK pathway and decreasing matrix metalloproteinase-2 expression. Mol Nutr Food Res 53:407–416. 10.1002/mnfr.200800123 [DOI] [PubMed] [Google Scholar]
  75. Yeh C-B, Hsieh M-J, Lin C-W et al (2013) The antimetastatic effects of resveratrol on hepatocellular carcinoma through the downregulation of a metastasis-associated protease by SP-1 modulation. PLoS ONE 8:e56661. 10.1371/journal.pone.0056661 [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Yuan L, Zhang Y, Xia J et al (2015) Resveratrol induces cell cycle arrest via a p53-independent pathway in A549 cells. Mol Med Rep 11:2459–2464. 10.3892/mmr.2014.3100 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Zhang J, Ping J, Jiang N, Xu L (2022) Resveratrol inhibits hepatic stellate cell activation by regulating autophagy and apoptosis through the SIRT1 and JNK signaling pathways. J Food Biochem. 10.1111/jfbc.14463 [DOI] [PubMed] [Google Scholar]
  78. Zhao Y, Ye X, Xiong Z et al (2023) Cancer metabolism: the role of ROS in DNA damage and induction of apoptosis in cancer cells. Metabolites 13:796. 10.3390/metabo13070796 [DOI] [PMC free article] [PubMed] [Google Scholar]
  79. Zheng L, Wei Q, Cai Y et al (2006) DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: mechanism and structure-activity relationship. Free Radic Biol Med 41:1807–1816. 10.1016/j.freeradbiomed.2006.09.007 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No datasets were generated or analysed during the current study.

Articles from Cytotechnology are provided here courtesy of Springer Science+Business Media B.V.

RESOURCES