Ethanol Extract of Ocimum Sanctum Leaves Exerts Apoptotic Activity on A549 Non-Small Lung Adenocarcinoma Cell Line through Down-Regulated of Nf-kb and Activated of Apaf-1

Abstract

Lung cancer was among the primary contributors to mortality within the realm of cancer-related ailments. Multiple research projects had demonstrated Ocimum sanctum’s diverse biological and pharmacological characteristics, including its antioxidant, neuroprotective, and anti-cancer capabilities. However, limited evidence supported the assertion that O. sanctum on human lung adenocarcinoma cancer cells. Here, we inspected ethanol extract O. sanctum’s (EEOS) impact on cellular apoptosis and the suppression of Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) in A549 human lung adenocarcinoma cancer cells. The chemical composition of EEOS was analyzed using gas chromatography-mass spectrometry (GC-MS), while antioxidant potential was assessed via the DPPH radical scavenging assay. Apoptotic activity was evaluated through Hoechst 33342 nuclear staining, mitochondrial membrane potential (JC-1) assay, and Reactive Oxygen Species (ROS) detection using DCFH-DA. Protein expression of NF-κB, Apoptotic Protease Activating Factor 1 (Apaf-1), caspase-9, and caspase-3 was quantified by ELISA. Molecular docking was performed to explore the interactions between linoleic acid - the predominant compound identified in EEOS - and key apoptotic proteins. Additionally, the in vivo chemoprotective effect of EEOS was evaluated in benzo(a)pyrene (B(a)P)-induced lung toxicity in C3H mice. GC-MS profiling identified 33 compounds in EEOS, with linoleic acid, phytol, and β-sitosterol as the major constituents. EEOS exhibited moderate antioxidant activity (IC₅₀ = 46.42 µg/mL). Treatment with EEOS significantly induced apoptosis in A549 cells, disrupted mitochondrial membrane potential, and elevated intracellular ROS levels. ELISA analysis showed downregulation of NF-κB and upregulation of Apaf-1, caspase-9, and caspase-3 in a dose-dependent manner. Molecular docking revealed strong binding affinity of linoleic acid to NF-κB and caspase proteins, comparable to cisplatin. In vivo, EEOS mitigated B[a]P-induced lung tissue damage. In conclusion, EEOS exerts potent pro-apoptotic effects through NF-κB inhibition and activation of the intrinsic Apaf-1/caspase-dependent pathway, supporting its potential as a promising adjunct therapy for lung cancer treatment.

HIGHLIGHTS

• This study investigates the potential of sanctum ethanol extract (EEOS) as a new therapy for lung cancer, given that lung cancer is the leading cause of cancer-related deaths.
• Gas Chromatography-Mass Spectrometry (GC-MS) analysis showed that linoleic acid is the most abundant component in EEOS, and this extract also has strong antioxidant activity.
• EEOS was found to inhibit Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and simultaneously increase the expression of (Apoptotic Protease Activating Factor 1) Apaf-1, Caspase 9, and Caspase 3. This process leads to the activation of apoptosis (programmed cell death) in A549 lung adenocarcinoma cancer cells.
• This study observed that EEOS reduced nuclear morphology and mitochondrial function in A549 cells, which are indicators of cancer cell damage.
• EEOS shows promise as an innovative therapy for the future treatment of lung cancer, paving the way for further medical interventions.

GRAPHICAL ABSTRACT