Ameliorative inhibition of sirtuin 6 by imidazole derivative triggers oxidative stress-mediated apoptosis associated with Nrf2/Keap1 signaling in non-small cell lung cancer cell lines

Abstract

Background: Redox homeostasis is the vital regulatory system with respect to antioxidative response and detoxification. The imbalance of redox homeostasis causes oxidative stress. Nuclear factor-erythroid 2 p45-related factor 2 (Nrf2, also called Nfe2l2)/Kelchlike ECH-associated protein 1 (Keap1) signaling is the major regulator of redox homeostasis. Nrf2/Keap1 signaling is reported to be involved in cancer cell growth and survival. A high level of Nrf2 in cancers is associated with poor prognosis, resistance to therapeutics, and rapid proliferation, framing Nrf2 as an interesting target in cancer biology. Sirtuins (SIRT1-7) are class III histone deacetylases with NAD + dependent deacetylase activity that have a remarkable impact on antioxidant and redox signaling (ARS) linked with Nrf2 deacetylation thereby increasing its transcription by epigenetic modifications which has been identified as a crucial event in cancer progression under the influence of oxidative stress in various transformed cells. SIRT6 plays an important role in the cytoprotective effect of multiple diseases, including cancer. This study aimed to inhibit SIRT6 using an imidazole derivative, Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate, to assess its impact on Nrf2/Keap1 signaling in A549 and NCI-H460 cell lines. Method: Half maximal inhibitory concentration (IC₅₀) of Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate was fixed by cell viability assay. The changes in the gene expression of important regulators involved in this study were examined using quantitative real-time PCR (qRT-PCR) and protein expression changes were confirmed by Western blotting. The changes in the antioxidant molecules are determined by biochemical assays. Further, morphological studies were performed to observe the generation of reactive oxygen species, mitochondrial damage, and apoptosis. Results: We inhibited SIRT6 using Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate and demonstrated that SIRT6 inhibition impacts the modulation of antioxidant and redox signaling. The level of antioxidant enzymes and percentage of reactive oxygen species scavenging activity were depleted. The morphological studies showed ROS generation, mitochondrial damage, nuclear damage, and apoptosis. The molecular examination of apoptotic factors confirmed apoptotic cell death. Further, molecular studies confirmed the changes in Nrf2 and Keap1 expression during SIRT6 inhibition. Conclusion: The overall study suggests that SIRT6 inhibition by imidazole derivative disrupts Nrf2/Keap1 signaling leading to oxidative stress and apoptosis induction.

Keywords: Epigenetics; HDAC; HDACi; KEAP1; Nrf2; SIRT6.

FIGURE 1

Illustration of the cytotoxicity activity of Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate in (A) A549 and (B) NCI-H460 cell lines.

FIGURE 2

SIRT6 expression in A549 and NCI-H460 NSCLC cell lines: (A) Represents the qRT-PCR analysis and (B) Represents the Western blot analysis. GAPDH for gene expression and β-actin for protein expression were used as an internal control for normalization (Data represent mean values ±SD. **p < 0.01, ***p < 0.001 and ****p < 0.0001).

FIGURE 3

The effect of imidazole derivative Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl] acetate on antioxidant regulators (A) GSH Activity, (B) GPx Activity, (C) Catalase Activity, and (D) % Radical Scavenging Activity in A549 and NCI-H460 cell lines. (Data represent mean values ±SD. *p < 0.05, **p < 0.01, and ***p < 0.001).

FIGURE 4

The protein expression of cytochrome-c (cyt-c) in NSCLC cell lines A549 and NCI-H460. β-actin is used as an internal control for normalization. (Data represent mean values ±SD. **p < 0.01, and ***p < 0.001).

FIGURE 5

Gene and protein expression of caspase 9: (A) Caspase 9 gene expression, (B) pro-caspase 9, and (C) active-caspase 9 protein expression in A549 and NCI-H460 cell lines. GAPDH for gene expression and β-actin for protein expression were used as an internal control for normalization. (Data represent mean values ±SD. *p < 0.05, **p < 0.01 and ***p < 0.001).

FIGURE 6

The expression of Caspase 3: (A) Caspase 3 gene expression, (B) protein expression of pro-caspase 3, and (C) active-caspase 3 in A549 and NCI-H460 cell lines. GAPDH for gene expression and β-actin for protein expression were used as an internal control for normalization (Data represent mean values ±SD.**p < 0.01, ***p < 0.001 and ****p < 0.0001).

FIGURE 7

Morphological assessment of NSCLC cell lines A549 and NCI-H460 cells stained by (A) AO/EtBr, (B) Propidium iodide, (C) DCFH-DA, (D) Rhodamine-123, and (E) Hoechst 33258 were captured using a fluorescent microscope with ×20 magnification (Scale Bar - 125 μm). (F) AO/EtBr, (G) Propidium iodide, (H) DCFH-DA, (I) Rhodamine-123, and (J) Hoechst 33258 represent the analysis of morphological staining by image (J) (Data represent mean values ±SD. *p < 0.05, **p < 0.01 and ***p < 0.001).

FIGURE 8

Gene and protein expression of Keap1 in A549 and NCI-H460 NSCLC cell lines: (A) Keap1 gene expression and (B) Western blot analysis of Keap1. GAPDH for gene expression and β-actin for protein expression were used as an internal control for normalization (Data represent mean values ±SD. **p < 0.01 and ***p < 0.001).

FIGURE 9

Gene and protein expression of Nrf2 in A549 and NCI-H460 NSCLC cell lines: (A) Nrf2 gene expression and (B) Western blot analysis of Nrf2. GAPDH for gene expression and β-actin for protein expression were used as an internal control for normalization (Data represent mean values ±SD. *p < 0.05, **p < 0.01 and ***p < 0.001).