The activity of pyrazolo[4,3-e][1,2,4]triazine and pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulphonamide derivatives in monolayer and spheroid breast cancer cell cultures

Figures & data

Scheme 1. Reagents and conditions: (a) appropriate phenylalaninol derivative, acetonitrile, rt; (b) NaN₃, EtOH, reflux.

Figure 1. Viability of MCF-7 (A), MDA-MB-231 (B) breast cancer cells, and MCF-10A (C) normal breast cells incubated for 24 h with different concentrations of 2a, 2b and cisplatin. Mean values ± SD from three independent experiment (n = 3) done in duplicate are presented.

Figure 2. Viability of MCF-7 (A), MDA-MB-231 (B) breast cancer cells, and MCF-10A (C) normal breast cells incubated for 24 h with different concentrations of 3a, 3b and cisplatin. Mean values ± SD from three independent experiment (n = 3) done in duplicate are presented.

Table 1. The cytotoxic activity of new derivatives of pyrazolo[4,3-e][1,2,4]triazine.

Compound	Cytotoxicity IC50
	MCF-7	MDA-MB-231	MCF-10A
2a	6.0 ± 0.38 µM	9.2 ± 0.78 µM	>10.0 µM
2b	5.5 ± 0.39 µM	7.8 ± 0.53 µM	>10.0 µM
3a	0.35 ± 0.08 µM	0.71 ± 0.14 µM	3.5 ± 0.18 µM
3b	0.25 ± 0.07 µM	0.31 ± 0.14 µM	2.3 ± 0.04 µM
Cisplatin	>10.0 µM	>10.0 µM	>10.0 µM

Figure 3. Flow cytometric analysis of induction of apoptosis in MCF-7 (A) and MDA-MB-231 (B) breast cancer cells incubated for 24 h with 2b, 3b and cisplatin (0.25 μM and 0.5 μM) stained with annexin V-FITC and propidium iodide. Mean percentage values from three independent experiments done in duplicate are presented. *p < 0.05 vs. control group and ***p < 0.001 vs. control group.

Figure 4. Flow cytometric analysis of ROS induction in MCF-7 (A) and MDA-MB-231 (B) breast cancer cells incubated for 24 h with 3b and cisplatin (0.25 μM and 0.5 μM). Mean percentage values from three independent experiments done in duplicate are presented. *p < 0.05 vs. control group, **p < 0.01 vs. control group, ***p < 0.001 vs. control group.

Figure 5. Anti-NF-κB antibody flow cytometric analysis of MCF-7 (A) and MDA-MB-231 (B) breast cancer cells compared to a negative control cell after 24 h of incubation with 3b and cisplatin (0.25 μM and 0.5 μM). Mean percentage values from three independent experiments done in duplicate are presented. ***p < 0.001 vs. control group.

Figure 6. Anti-Bax antibody flow cytometric analysis of MCF-7 (A) and MDA-MB-231 (B) breast cancer cells compared to a negative control cell after 24 h of incubation with 3b and cisplatin (0.25 μM and 0.5 μM). Mean percentage values from three independent experiments done in duplicate are presented. ***p < 0.001 vs. control group.

Figure 7. Anti-p53 antibody flow cytometric analysis of MCF-7 (A) and MDA-MB-231 (B) breast cancer cells compared to a negative control cell after 24 h of incubation with 3b and cisplatin (0.25 μM and 0.5 μM). Mean percentage values from three independent experiments done in duplicate are presented. ***p < 0.001 vs. control group.

Figure 8. The activity of caspase 10 in MCF-7 (A) and MDA-MB-231 (B) breast cancer cells incubated with 3b (0.25 μM and 0.5 μM) in the absence and presence of Z-VAD-FMK (100 μM) for 24 h. Mean percentage values from three independent experiments done in duplicate are presented. ***p < 0.001 vs. control group.

Figure 9. The activity of caspase 9 in MCF-7 (A) and MDA-MB-231 (B) breast cancer cells incubated with 3b (0.25 μM and 0.5 μM) in the absence and presence of Z-VAD-FMK (100 μM) for 24 h. Mean percentage values from three independent experiments done in duplicate are presented. ***p < 0.001 vs. control group.

Figure 10. The activity of caspase 3/7 in MCF-7 (A) and MDA-MB-231 (B) breast cancer cells incubated with 3b (0.25 μM and 0.5 μM) in the absence and presence of Z-VAD-FMK (100 μM) for 24 h. Mean percentage values from three independent experiments done in duplicate are presented. ***p < 0.001 vs. control group.

Figure 11. Flow cytometric analysis of autophagy induction in MCF-7 (A) and MDA-MB-231 (B) breast cancer cells incubated with 3b (0.25 μM and 0.5 μM in the absence or presence of 3-MA (5 mM)). Mean percentage values from three independent experiments done in duplicate are presented. *p < 0.05 vs. control group, ***p < 0.001 vs. control group.

Figure 12. Anti-mTOR antibody flow cytometric analysis of MCF-7 (A) and MDA-MB-231 (B) breast cancer cells compared to a negative control cell after 24 h of incubation with 3b and cisplatin (0.25 μM and 0.5 μM). Mean percentage values from three independent experiments done in duplicate are presented. *p < 0.05 vs. control group, ***p < 0.001 vs. control group.

Figure 13. Anti-beclin-1 antibody flow cytometric analysis of MCF-7 (A) and MDA-MB-231 (B) breast cancer cells compared to a negative control cell after 24 h of incubation with 3b and cisplatin (0.25 μM and 0.5 μM). Mean percentage values from three independent experiments done in duplicate are presented. *p < 0.05 vs. control group, ***p < 0.001 vs. control group.

Figure 14. Microscopy images of cell invasion of spheroids incubated for 24 h with 2b, 3b and cisplatin (0.25 μM and 0.5 μM). Representative photographs are shown; scale bar: 100 μm. Microscopy images of cell invasion of spheroids incubated for 24 h with 2b, 3b and cisplatin (0.25 μM and 0.5 μM). Representative photographs are shown; scale bar: 100 μm. **p < 0.01 vs. control group, ***p < 0.001 vs. control group, ****p < 0.0001 vs. control group.

Data availability statement

Department of Synthesis and Technology of Drugs, Medical University of Bialystok, Kilinskiego 1, 15-089 Bialystok, Poland.