Integrative In Vitro and In Silico Evaluation of Flavonoid-enriched Carica papaya Linn. Extract Targeting Oncogenic Pathways in A549 Lung Cancer Cells

Abstract

Non-Small Cell Lung Carcinoma (NSCLC) specifically is still one of the top causes of cancer-related death globally. The multi-targeted mechanisms and lower toxicity of natural products make them intriguing pharmacological candidates. Using a combination of in vitro and in silico methods, the current study assessed the anticancer potential of the Ethanol Extract of Carica papaya Linn. roots (EECP). Phytochemical profiling and High-Performance Thin-Layer Chromatography (HPTLC) analysis confirmed the presence of flavonoids, including rutin. The 2,2-diphenyl-1- picrylhydrazyl (DPPH) assay was used to assess antioxidant activity. Cytotoxicity against Human lung adenocarcinoma cell line (A549) and normal human lung fibroblast cell line (WI-38) cells was evaluated, along with apoptosis via nuclear condensation, Deoxyribonucleic Acid (DNA) fragmentation, and Reactive Oxygen Species (ROS) accumulation. Molecular docking (SwissDock) and Absorption, Distribution, Metabolism, and Excretion (ADME) predictions were performed to assess protein interactions, drug-likeness, and oral bioavailability. EECP exhibited strong antioxidant activity (IC₅₀ = 4.07 μg/mL) and selective cytotoxicity toward A549 cells (IC₅₀ = 89.96 ± 0.24 μg/mL) with lower toxicity in WI-38 fibroblasts. Treatment induced significant ROS generation (213% at 300 μg/mL) and apoptotic changes. Docking studies revealed strong interactions of quercetin (–9.758 kcal/mol) and kaempferol (–9.353 kcal/mol) with oncogenic proteins, comparable to staurosporine (–10.439 kcal/mol). ADME predictions supported favorable bioavailability and drug-likeness. Ethanolic extract demonstrated potent antioxidant activity (IC50 = 4.07 μg/mL) and selective cytotoxicity against A549 cells (IC50 = 89.96±0.24 μg/mL), with comparatively lower toxicity in WI-38 cells (IC50 = 75.83±0.34 μg/mL). Apoptosis was associated with nuclear condensation, DNA fragmentation, and a 213% increase in ROS at 300 μg/mL. Docking studies indicated strong binding of quercetin (-9.758 kcal/mol) and kaempferol (-9.353 kcal/mol) to oncogenic proteins, comparable to that of staurosporine (-10.439 kcal/mol), mediated by hydrogen bonds and hydrophobic interactions. Phenolic acids showed moderate interactions, and carpaine/ ergosta derivatives showed weaker interactions. ADME analyses revealed favourable oral bioavailability and high gastrointestinal absorption for key flavonoids. Ethanolic extract induces oxidative stress-mediated apoptosis and regulates key signalling proteins, including Protein Kinase B (Akt) and tumor suppressor p53 (p53). Overall, the EECP exhibits strong anticancer potential by inducing ROS-mediated apoptosis and regulating Akt and p53, supporting further preclinical evaluation of C. papaya Linn. root flavonoids for lung cancer.