The Potential Anti-Cancer Effects of Polish Ethanolic Extract of Propolis and Quercetin on Glioma Cells Under Hypoxic Conditions

Abstract

Tissue hypoxia is commonly observed in head cancers and contributes to both molecular and functional changes in tumour cells. It is known to stimulate erythropoiesis, angiogenesis, and metabolic alterations within tumour cells. Glioblastoma, a type of brain tumour, is characterized by rapid proliferation and aggressive growth. Recent studies have indicated that natural products may hold potential as components of cancer therapy. Among these, Polish propolis and its active compound, quercetin, have demonstrated promising anti-cancer properties. The aim of this study was to evaluate the concentrations of selected cytokines-specifically IL-6, IL-9, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB), interferon gamma-induced protein 10 (IP-10), and monocyte chemoattractant protein-1 (MCP-1)-produced by astrocytes of the CCF-STTG1 cell line. The cytotoxic effects of ethanolic extract of propolis (EEP) and quercetin were assessed using the MTT assay. Astrocytes were stimulated with lipopolysaccharide (LPS, 200 ng/mL) and/or IFN-α (100 U/mL), followed by treatment with EEP or quercetin (25-50 µg/mL) under hypoxic conditions for two hours. Cytokine concentrations were measured using the xMAP Luminex Multiplex Immunoassay and the Multiplex Bead-Based Cytokine Kit. Our study demonstrated that Polish propolis and its component quercetin modulate the tumour microenvironment in vitro, primarily by altering the levels of specific cytokines. The HCA analysis revealed that IL-6 and MCP-1 formed a distinct cluster at the highest linkage distance (approximately 100% of Dmax), suggesting that their expression patterns are significantly different from those of the other cytokines and that they are more similar to each other than to the rest. PCA analysis showed that EEP-PL (50 μg/mL) with IFN-α and EEP-PL (50 μg/mL) with LPS exert similar activities on cytokine secretion by astrocytes. Similar effects were demonstrated for EEP-PL 50 μg/mL + LPS + IFN-α, EEP-PL 25 μg/mL + IFN-α and EEP-PL 25 μg/mL + LPS + IFN-α. Our findings suggest that Polish propolis and quercetin may serve as promising natural agents to support the treatment of stage IV malignant astrocytoma. Nonetheless, further research is needed to confirm these results.

Keywords: astrocytes; cytokine; glioma cells; hypoxia; propolis; quercetin.

Figure 1

Viability [%] of astrocyte cell line CCF-STTG1 under hypoxic conditions after treatment with ethanolic extract of Polish propolis (EEP-PL) or quercetin (Q), alone or in combination with lipopolysaccharide (LPS, 200 ng/mL), interferon-alpha (IFN-α, 100 U/mL), or both (LPS + IFN-α). Cells were incubated with EEP-PL or quercetin at final concentrations of 25 and 50 µg/mL. Cell viability was assessed by MTT assay, and results are expressed as mean ± standard deviation (SD) from three independent experiments (n = 3). Statistical significance was evaluated using the two-tailed Student’s t-test, with comparisons made between treated groups and their respective stimulated controls (i.e., EEP + LPS vs. LPS alone, Q + IFN-α vs. IFN-α alone, etc.). Statistical analysis was performed using STATISTICA v13.1 software (StatSoft, Tulsa, OK, USA); * p < 0.05, ** p < 0.01, *** p < 0.001 vs. corresponding control group.

Figure 2

Concentrations of IL-6 (a,b), VEGF (c,d), PDGF-BB (e,f), IP-10 (g,h), MCP-1 (i,j), and IL-9 (k,l) [pg/mL] secreted by CCF-STTG1 astrocytes cultured under hypoxic conditions and treated with Polish ethanolic extract of propolis (EEP-PL) or quercetin (Q), at 25 and 50 µg/mL in combination with IFN-α, LPS, or both. Data are expressed as mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Fisher’s LSD post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001) in STATISTICA 13.1 software. Comparisons were made versus the respective stimulated control groups (LPS, IFN-α or LPS + IFN-α alone). Further details can be found in the Supplementary Materials.

Figure 2

Concentrations of IL-6 (a,b), VEGF (c,d), PDGF-BB (e,f), IP-10 (g,h), MCP-1 (i,j), and IL-9 (k,l) [pg/mL] secreted by CCF-STTG1 astrocytes cultured under hypoxic conditions and treated with Polish ethanolic extract of propolis (EEP-PL) or quercetin (Q), at 25 and 50 µg/mL in combination with IFN-α, LPS, or both. Data are expressed as mean ± SD of three independent experiments. Statistical significance was determined using one-way ANOVA followed by Fisher’s LSD post hoc test (* p < 0.05, ** p < 0.01, *** p < 0.001) in STATISTICA 13.1 software. Comparisons were made versus the respective stimulated control groups (LPS, IFN-α or LPS + IFN-α alone). Further details can be found in the Supplementary Materials.

Figure 3

Dendrogram obtained via HCA analysis of data regarding the behaviour of IL-6, VEGF, PDGF-BB, MCP-1, IP-10 and IL-9 secretion by CCF-STTG1 cells stimulated by LPS, IFN-α and LPS + IFN-α treatment with EEP-PL in hypoxia conditions. Dlink denotes the linkage distance, or the measured separation between two clusters. Dmax indicates the maximum possible distance that may occur between any clusters.

Figure 4

PCA score plot illustrating data regarding the impact of EEP-PL (25–50 μg/mL) on the secretion of IL-6, VEGF, PDGF-BB, MCP-1, IP-10 and IL-9 by astrocytes cell line CCF-STTG1 stimulated with LPS and/or IFN-α. Abbreviation: PC—principal component.

Figure 5

Dendrogram obtained via HCA analysis of data regarding the behaviour of IL-6, VEGF, PDGF-BB, MCP-1, IP-10 and IL-9 secretion by CCF-STTG1 cells stimulated by LPS, IFN-α and LPS + IFN-α treatment with quercetin in hypoxia conditions. Dlink denotes the linkage distance, or the measured separation between two clusters. Dmax indicates the maximum possible distance that may occur between any clusters.

Figure 6

PCA score plot illustrating data regarding the impact of quercetin (25–50 μg/mL) on the secretion of IL-6, VEGF, PDGF-BB, MCP-1, IP-10 and IL-9 by astrocyte cell line CCF-STTG1 stimulated with LPS and/or IFN-α treatment with quercetin in hypoxia conditions. Abbreviation: PC—principal component.