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

Abstract

In the last decade, an increasing interest in compounds containing pyrazolo[4,3-e][1,2,4]triazine moiety is observed. Therefore, the aim of the research was to synthesise a novel sulphonyl pyrazolo[4,3-e][1,2,4]triazines (2a, 2b) and pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulphonamide derivatives (3a, 3b) to assess their anticancer activity. The MTT assay showed that 2a, 2b, 3a, 3b have stronger cytotoxic activity than cisplatin in both breast cancer cells (MCF-7 and MDA-MB-231) and exhibited weaker effect on normal breast cells (MCF-10A). The obtained results showed that the most active compound 3b increased apoptosis via caspase 9, caspase 8, and caspase 3/7. It is worth to note that compound 3b suppressed NF-κB expression and promoted p53, Bax, and ROS which play important role in activation of apoptosis. Moreover, our results confirmed that compound 3b triggers autophagy through increased formation of autophagosomes, expression of beclin-1 and mTOR inhibition. Thus, our study defines a possible mechanism underlying 3b-induced anti-cancer activity against breast cancer cell lines.

Keywords: Pyrazolo[43-e]tetrazolo[15-b][1,2,4]triazine; anticancer; pyrazolo[43-e][1,2,4]triazine; spheroid; sulphonamide.

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.

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.