Glioblastoma (GBM) is the most frequent and aggressive primary malignant brain tumor and PI3K/Akt signaling pathway is activated in almost 90% of cases. The aim of this study was to evaluate the capacity of natural compounds to potentiate the activity of temozolomide (TMZ) - the standard pharmacological drug for GBM. U-87 MG (ATCC HTB-14) cells were treated with quercetin, resveratrol, curcumin and TMZ, alone or in combination of TMZ with either of them, for 30 minutes (for signaling protein phosphorylation) and 48 hours (for total signaling protein expression). LDH-based cytotoxicity and MTS assays were performed for dose selection. Signaling molecular patterns were studies by xMAP array technology. The effect of treatments was evaluated by assessing a 9-plex panel of total and phosphorylated signaling proteins. Based on the cytotoxicity testing, selected working concentrations were 30 μM for quercetin, 30 μM for resveratrol, 15 μM for curcumin and 50 μM temozolomide. We found strong increase in JNK Thr183/Tyr185, p38 Thr180/Tyr182 and STAT3 Ser727 phosphorylation and a moderate increase for ERK1/2 Thr185/Tyr187 phosphorylation after 30 minutes of treatment. Resveratrol treatment reduced Akt Ser473 phosphorylation, whereas the other treatments had no effect. After 48 hours treatment of U87 cells, the expression of most signaling proteins analyzed (CREB, JNK, ERK1/2, Akt and STAT3) was decreased upon treatment with natural products alone. When the natural compounds were combined with TMZ, an even stronger decrease in protein expression was observed: CREB fold-regulation 0.63-0.78), NFkB (fold-regulation 0.81-0.86) and p38 (fold-regulation 0.68-0.85). Our results revealed that quercetin and resveratrol performed best in potentiating TMZ effects on signaling protein expression. Natural compounds could be used as therapeutic adjuvants in GBM therapy. Given the complexity of signaling pathways in GBM treatment approaches, further studies are required to unravel the mechanisms for precision therapy.

3D tumor cell cultures are currently emerging as the novel standard for cytotoxicity testing as well as in vitro molecular studies, but various solutions are available to generate them. The aim of this study was to test whether there is one-fits-all solution to generate tumor spheroids for further studies. We used three breast tumor cell lines (MCF-7, MDA-MB-231, MDA-MB-361) and one glioblastoma cell line (U87), as comparison for MDA-MB-361 which is a secondary (metastatic) brain tumor. Different hydrogels (Matrigel® Corning, TrueGel3D -1, -6,-7 with or w/o RGD adhesion peptide® Merck, and TrueGel3D® HTS Hydrogel Plates®Merck) were tested for spheroid formation, as well as ultra-low attachment surface 24 well-plates ®Corning (no gels). Cells were seeded in two concentrations (300 and 3000/cm2) and documented daily for the first 5 days, than weekly for spheroid formation. Viability was tested using dual fluorescein diacetate/propidium iodide stain. For higher cell concentrations, presence of spheroids can be documented as early as 5 days, but at least 7 days are recommended. Once formed, spheroids can be maintained more than 28 days, with twice a week cell medium change. Presence of organic molecules (basement membrane matrix or RGD peptide) in the gel impaired formation of tumor spheroids for aggressive cell lines (MDA-MB-261 and U87) and yielded mixed cultures (2D and spheroids) for the rest. Also, higher stiffness and the presence of a non-degradable cell linker prevented proliferation of cells and formation of spheroids, regardless of cell type. A crosslinker gradient, although favored formation of all tested breast cancer spheroids, interfered with formation of spheroids for U87 cells. Finally, ultra-low adherence plates allowed consistent formation of spheroids only for MCF-7 cells, whereas for the rest, irregular cell aggregates were observed at higher cell concentrations. Dextran-based polymers can be used for tumor spheroid formation regardless of cell type, provided that no adhesion peptides are added.