Profiling essential oils from leaf extract of Azadirachta indica inducing apoptosis in colorectal cancer cells and evaluating systems biology for drug discovery

This study aimed to assess cellular toxicity and to determine apoptosis-associated expression changes in adjacent colorectal cancer (HT-29) cell cultures treated with Azadirachta indica essential oil (AIEO). It also aimed to elucidate molecular targets and mechanisms of action in colorectal cancer (CRC) using network pharmacology and in silico approaches. Essential oil was extracted from AIEO leaves, and GC–MS was used to identify its components. Cell viability of treated HT-29 cancer cells was assessed using the MTT assay. Based on the essential oil’s toxicity, 5 and 10 μg/mL were used to treat HT-29 cells, and apoptosis was monitored by flow cytometry. The effect of the AIEO on HT-29 cell proliferation was evaluated using the reactive oxygen species (ROS) and BrdU assays. Expression of apoptosis-related mRNA and proteins was analyzed by qPCR and Western blotting. From GC–MS analysis, the seven most abundant phytochemicals were evaluated for drug-likeness and ADMET properties. Their anticancer targets and mechanisms were then investigated using network pharmacology and molecular docking. Primary apoptosis was 24.3 and 41.8% when the HT-29 cells were incubated for 24 h with 5 and 10 μg/mL of the essential oil, respectively. BrdU and ROS assays showed a significant inhibitory effect of AIEO on HT-29 cell proliferation. The expression of Caspase 3 and 9, as well as Bax, increased in the treatment group compared with the control group, whereas BCL-2 expression decreased significantly. Network pharmacology identified 135 overlapping gene targets between AIEO and CRC. Cathepsin D, STAT3, HSP90AA1, and NFKB1 were consistently among the top 10 hub proteins interacting with AIEO in CRC networks and were therefore selected for molecular docking with the seven AIEO compounds. Docking results showed that, among the seven compounds and four reference inhibitors, phytol produced the highest docking score for binding to the four oncoproteins.