Comprehensive Study on Thiadiazole-Based Anticancer Agents Inducing Cell Cycle Arrest and Apoptosis/Necrosis Through Suppression of Akt Activity in Lung Adenocarcinoma and Glioma Cells

Abstract

Objectives: Akt is considered as an attractive target for anticancer drug discovery and development and therefore extensive efforts have been devoted to the discovery of new potent anticancer agents targeting Akt.

Materials and methods: Due to the importance of thiadiazoles for anticancer drug discovery, herein eight 1,3,4-thiadiazole derivatives were investigated for their cytotoxic effects on C6 rat glioma and A549 human lung adenocarcinoma cell lines using the MTT assay. The effects of the most promising anticancer agents on apoptosis, caspase-3 activation, mitochondrial membrane potential, and cell cycle arrest were determined on a BD FACSAria (I) flow cytometer. Akt activity was measured in the C6 and A549 cell lines using an ELISA colorimetric method. Schrödinger's Maestro molecular modeling package was used to explore the possible binding modes of compounds 3 and 8 in the active site of Akt enzyme (PDB code: 3OW4).

Results: N-(4-Chlorophenyl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (3) and N-(6-nitrobenzothiazol-2-yl)-2-[(5-((4- nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (8) induced apoptosis and cell cycle arrest in the C6 cell line through inhibition of Akt activity (92.36% and 86.52%, respectively). The docking results of compounds 3 and 8 indicated that π-π interactions, H bonds, and salt-bridge formation were responsible for the observed Akt inhibitory activity.

Conclusion: According to in vitro and docking studies, compounds 3 and 8 stand out as promising antiglioma agents.

Keywords: Akt activity; Apoptosis; Cancer; Molecular docking; Thiadiazole.

Figure 1

Typical quadrant analysis of Annexin V-FITC/PI flow cytometry of C6 cells treated with compounds 1, 2, 3, 4, 5, 8, and cisplatin. At least 10,000 cells were analyzed per sample and quadrant analysis was performed. The portion (%) of cell number is shown in each quadrant. Q1, necrotic cells; Q2, late apoptotic cells; Q3, viable cells; Q4, early apoptotic cells. C6 cells were cultured for 24 h in medium with IC₅₀ concentrations of the compounds, cisplatin, and untreated control cells

Figure 2

Typical quadrant analysis of Annexin V-FITC/PI flow cytometry of A549 cells treated with compounds 1, 3, 5, 8, and cisplatin. At least 10,000 cells were analyzed per sample and quadrant analysis was performed. The portion (%) of cell number is shown in each quadrant. Q1, necrotic cells; Q2, late apoptotic cells; Q3, viable cells; Q4, early apoptotic cells. A549 cells were cultured for 24 h in medium with IC₅₀ concentrations of the compounds, cisplatin, and untreated control cells

Figure 3

Caspase 3 activity of C6 cells treated with IC₅₀ concentrations of compounds 1, 3, 5, 8, and cisplatin

Figure 4

The reduction in the MMP in the C6 cell line by the compounds. The cells treated or untreated with IC₅₀ doses of the compounds for 24 h were stained with mitochondrial-selective JC-1 dye and analyzed by flow cytometry. P1: mitochondrial membrane polarized cells, P2: mitochondrial membrane depolarized cells

Figure 5

The reduction in the MMP in the A549 cell line by the compounds. The cells treated or untreated with IC₅₀ doses of the compounds for 24 h were stained with the mitochondrial-selective JC-1 dye and analyzed by flow cytometry. P1: mitochondrial membrane polarized cells, P2: mitochondrial membrane depolarized cells

Figure 6

Cell cycle distribution of C6 cells treated with IC₅₀ concentrations of the compounds for 24 h. At least 10,000 cells were analyzed per sample. Blue color shows debris, green color shows aggregates, and red color shows cells in G1 and G2 phases

Figure 7

Cell cycle distribution of A549 cells treated with IC₅₀ concentration of compound 8 and cisplatin for 24 h. At least 10,000 cells were analyzed per sample. Blue color shows debris, green color shows aggregates, and red color shows cells in G1 and G2 phases

Figure 8

Docking positions and interactions of compounds 3 and 8 in the active site of Akt enzyme, respectively (Ligand custom carbons are colored orange for compound 3 and turquoise for compound 8)