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.
The problem of creating antitumor drugs with new mechanisms of action that predominantly induce apoptosis is still topical. The extract of Gratiola officinalis is a potential antitumor agent containing mainly flavonoids. The aim of this research is to study the effects of Gratiola officinalis extract on activation of apoptosis and autophagy in breast adenocarcinoma SK-BR-3 and kidney carcinoma A-498 lines. Apoptotic activity of the extract was studied by flow cytofluorometry using Hoechst stain and double staining with annexin V plus propidium iodide. There was 96.3% of cells in SK-BR-3 culture in late apoptosis phase detected by flow cytofluorometry method at the extract concentration of 0.88 mg/ml, 86.3% of cells were in apoptosis by Hoechst stain. The concentration of 0.82 mg/ml caused apoptosis in half of the cells. The extract has cytoprotective activity at low concentration (0.0352 mg/ml). The cytoprotection mechanism is realized through the activation of autophagy. The maximum number of autophagosomes in kidney carcinoma cells is observed at the extract concentration of 0.056 mg/ml. Thus, Gratiola officinalis extract is able to block cytoprotective autophagy with increasing the extract concentration and to activate apoptosis in 85% of tumor cells. Detailed research should be continued to understand the mechanisms of antitumor activity of Gratiola officinalis extract.
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