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

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(cyclo-phosphamide).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 demon-strates 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 fiavonoid-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.

Optical in vivo and ex vivo imaging of glioma cells migration via the cerebral vessels:Prospective clinical application of the beta2-adrenoreceptors blockade for glioma treatment

Malignant gliomas are highly invasive tumors that 1use the cerebral vessels for invasion due to high vascular fragility of the blood--brain barrier(BBB).On one hand,glioma is characterized by the BBB disruption,on the other hand,drug brain delivery via the BBB is a big challenge in glioma therapy.The limited information about vascular changes associated with glioma growth is a reason of slow progress in prevention of glioma development.Here,we present in vrivo and er vrivo study of the BBB disruption and glioma cells(GCs)migration in rats using fuorescence and confocal microscopy.We uncovered a local breach in the BBB in the main tumor mass but not within the border of normal and malignant cells,where the BBB was impermeable for high weight molecules.The migr ation of GCs were observed via the cerebral vessels with the intact BBB that was associated with macrophages infiltration.The mechanisms underlying glioma progression remain umknown but there is an evidence that the sympathetic nervous system(SNS)via activation of vascular beta2-adrenoreceptors(B2-ADRs)can play an important role in tumor metastasis.Our results clearly show an increase in the expression of vascular B2-ADRs and production of the beta arrestin-1-co-factor of B2-ADRs signaling pathway in rats with glioma.Pharmacological blockade of B2-ADRs reduces the BBB disruption,macrophages infiltration,GCs migration and increases survival rate.These data suggest that the blockade of B2-ADRs may be a novel adjuvant therapeutic strategy to reduce glioma progression and prevent metastasis。