Introduction: Cancer is increasingly a global health issue. Each year, millions of people are diagnosed with cancer around the world and it is still a leading cause of death. Despite the significant improvement in treatment and prevention, there is a critical need for more effective strategies, like the use of synthetic or natural agents to inhibit, retard, or reverse the process of carcinogenesis. Biologically active compounds (BACs), such as polyphenols and flavonoids, are well known in nonconventional medicine due to their anti-proliferative, anti-inflammatory or anti-bacterial properties. The aim of our study was to evaluate the anti-proliferative properties of BACs, extracted from bio-waste products, on epidermoid carcinoma cells in vitro.
Methodology: Samples of the biomass were collected, dried and used for BACs extraction. After the extractions and filtration, the extracts were analysed to determine the total phenolic and flavonoids content and polyphenols composition. Anti-cancer investigations were carried out using PBS extracts containing BACs (BACs-PBS) with the use of epidermoid carcinoma cells (A-431) and mesenchymal stem cells immortalized with hTERT (ASC52telo). MTT assay was used to measure cell viability according to the ISO 10993-5:2009. Both cell lines were incubated with extracts for 24-72h. To determine cell viability, LIVE/DEAD™ Cell Imaging Kit was used. Additionaly, cells’ proliferation was measured with the BrdU assay.
Results: After conventional extraction, high equilibrium values of the extracted species was observed (i.e. 24 mg/(g solid) for total phenolics and 21mg/(g solid) for total flavonoids, respectively). The ESI-MS analysis showed that the obtained extracts are mixtures of different types of phenolic compounds. The predominant one is Luteolin. After 24 h incubation, only minor changes in mitochondrial dehydrogenase activity were found. 72 h incubation with 20-fold diluted BACs-PBS extract reduced ASCs viability by 17.7 % (p<0.01) compared to control. Interestingly, cancer cells viability was reduced by 40.7 % (p<0.0001). It was noticed that the number of actively proliferating cancer cells was reduced by 25 % during the first 24 h. As measured using the thymidine analog, the total cell number decreased to 53 %. No changes were observed for ASCs. It was observed that BACs-PBS extract reduced ASCs and A-431 cells viability after 72 h by respectively 38.2 % (p<0.01) and 53.4 % (p<0.001) in comparison to control. To detect newly synthesized DNA of actively proliferating cells, BrdU assay was used. Co-staining with DAPI allowed for quantification of total cell number. It was observed that 72 h incubation with BACs-PBS extract reduced proliferation of A-431 cells by 26.7 % in comparison to control. Furthermore, cell number was reduced by 46.8 %, which could be attributed to Luteolin effect from BACs-PBS. Interestingly, no effects were observed for ASCs.
Conclusion: Based on the conducted research, we proved that BACs-PBS extract, which contains a high amount of Luteolin, induces cytotoxicity toward cancer cells, which, together with its high selectivity, robustness and nontoxicity make them very promising materials for health applications.
Acknowledgments: This work was supported by the H2020_JTI: PHENOLEXA grant and the “Excellence Initiative Research University” (Emerging Field “Multifactorial Molecular-Behavioral Cancer Profiling Team”).