Tumor cell death visualization of renal cell carcinoma under the combined effect of the Gratiola officinalis extract and cyclophosphamide using fluorescent staining methods

Abstract

Objective of the study: We used fluorescence imaging methods of apoptosis and necrosis in human renal carcinoma A498 tumor cells in vitro to reveal the indicated forms of cell death under the combined effect of flavonoid-containing extract of Gratiola officinalis and cytostatic (cyclophosphamide). Materials and methods: The dyes were propidium iodide and acridine orange, which were used in the “alive and dead” test. This test helped us to identify the total number of dead cells in the forms of necrosis and apoptosis and the number of cells in which apoptosis had started, it was characterized by the appearance of apoptotic bodies or nucleus pyknosis. Results: We found the most pronounced cytotoxic activity at the ratio of extract of Gratiola officinalis and cyclophosphamide concentrations of 1:1. The number of living cells decreased when exposed to the ratio of extract and cytostatic concentrations of 2:1. When the ratio of concentration of the extract relative to the cytostatic increased to 3:1, the cytostatic activity of the extract began to appear, the total number of tumor cells decreased. The number of cells with nucleus pyknosis and the number of cells with apoptosis signs significantly increased at a 3:1 ratio of extract and cytostatic concentrations, which confirms the presence of pro-apoptotic activity of the studied combination. This trend indicates the dependence of a certain form of cell death (apoptosis, necrosis) on the ratio of extract and cytostatic doses, and it also demonstrates the cytostatic and cytotoxic effects of this combination. Conclusion: Fluorescence methods of investigation in the “alive and dead” test allowed us to visualize the forms of cell death of human kidney carcinoma A498 by combined exposure to the flavonoid-containing extract of Gratiola officinalis and cytostatic (cyclophosphamide) 24 h after exposure. We found that the combination with a concentration ratio of the extract and cyclophosphamide of 3:1 has the greatest effectiveness due to stimulation of the cytostatic effect and cytotoxic effect.

Activation of Apoptosis and Autophagy by Gratiola Officinalis Extract in Human Tumor Cell Lines

“…We used the concentration of the extract at which its apoptotic effect and the ability to cause (or not to cause) autophagy were obvious, preliminary work on the analysis and selection of extract concentrations was carried out earlier [36].…”

“…The extract of G. officinalis obtained us by the author's method contains flavonoids and has no toxic effects [26]. Earlier, we revealed antioxidant, antitumor, antiexical and immunomodulatory properties of G. officinalis L. extract in laboratory animals with transplanted tumors [27][28][29][30][31][32][33][34][35][36]. This extract also has antituberculous, anti-inflammatory, antipyretic, and antimicrobial activity, which indicates its polyvalent action [29,31].…”