Pistacia integerrima, known in traditional medicine systems for its distinctive leaf galls, is a rich source of flavonoids, steroids, terpenoids and phenolic compounds. Previous pharmacological work has hinted at anti-inflammatory and cytotoxic properties, but the molecular basis of any anticancer effect has remained poorly defined. In the new study, the researchers prepared an ethyl acetate fraction of the plant, a fraction enriched for moderately polar phytochemicals, and tested it against A549 cells, a widely used human lung adenocarcinoma cell line that models NSCLC. The choice of fraction was deliberate: ethyl acetate extracts typically concentrate flavonoids such as kaempferol, quercetin and luteolin, along with the phytosterol beta-sitosterol, all of which have been implicated in anticancer activity in earlier literature.

The cytotoxic results were striking. In the MTT assay, a colorimetric test that measures metabolic activity as a proxy for cell viability, the ethyl acetate fraction produced significant, dose-dependent killing of A549 cells, with statistically robust effects at a concentration of 100 micrograms per millilitre (p < 0.0001). In other words, as the dose increased, progressively fewer cancer cells survived, a dose-response relationship that is a hallmark of genuine cytotoxic activity rather than experimental noise. The team then examined whether the fraction could stop cancer cells from replicating over longer periods using a colony formation assay, which tests the ability of individual cells to divide repeatedly and establish new colonies, a key measure of proliferative capacity.

Colony formation collapsed after treatment. The ethyl acetate fraction reduced colony formation to just 18.41 percent of control levels, a statistically significant decrease (p < 0.002) indicating that surviving cells had lost much of their ability to seed new populations. This distinction matters clinically: a drug that merely slows growth may delay tumour expansion, but one that erodes clonogenic capacity strikes at the self-renewing behaviour that drives relapse. The researchers interpret the combined cytotoxicity and anti-clonogenic data as evidence that the plant fraction attacks fundamental proliferative machinery in NSCLC cells rather than exerting a transient, non-specific toxic effect.

Perhaps more important for metastasis, the fraction also crippled the migratory behaviour of the cancer cells. Using two complementary assays, the wound healing assay, in which a scratch is made across a confluent cell layer and the rate of closure is measured, and the Transwell migration assay, in which cells are challenged to move through a porous membrane, the team showed that treated A549 cells migrated markedly less than untreated controls (p < 0.01). Migration is the cellular behaviour that underpins invasion and metastatic spread, the processes responsible for most cancer deaths. Suppressing it suggests the phytochemicals may interfere with the epithelial-mesenchymal transition and the extracellular matrix remodelling programmes that lung tumours exploit to colonise distant tissues.

To understand what was happening at the molecular level, the researchers turned to proteomic profiling with the Human XL Oncology protein array, a platform that simultaneously quantifies dozens of cancer-relevant proteins. Treatment with the ethyl acetate fraction significantly downregulated three proteins: endoglin (CD105), kallikrein-related peptidase 5 (KLK5) and matrix metalloproteinase-2 (MMP-2). Each of these tells a coherent story. Endoglin is a co-receptor in the TGF-beta signalling pathway that promotes angiogenesis, the formation of new blood vessels that feed tumours. KLK5 is a protease associated with tumour progression, and MMP-2 degrades the extracellular matrix, clearing a physical path for invading cells. Their coordinated downregulation indicates that the plant fraction suppresses both the angiogenic and metastatic signalling networks that NSCLC depends on for spread.

The team then asked which individual phytochemicals might be responsible, and against which protein targets they act. Molecular docking, a computational technique that predicts how small molecules fit into the binding pockets of proteins, revealed favourable binding affinities for the major Pistacia integerrima compounds against several oncogenic targets central to NSCLC biology. Kaempferol bound AKT1 with a docking score of -7.6 kcal/mol, while beta-sitosterol showed strong affinity for PI3K at -9.4 kcal/mol, quercetin engaged KRAS at -8.5 kcal/mol, and luteolin docked to MMP9 at -8.1 kcal/mol. These are not arbitrary targets. The PI3K/AKT1 axis is a master regulator of cell survival and proliferation that is frequently hyperactivated in lung cancer, KRAS is one of the most notorious oncogenes in NSCLC and has historically been considered nearly undruggable, and MMP9 drives matrix degradation and invasion.

Docking scores alone can be misleading, because a molecule may fit well in a static protein structure yet fail to remain bound in the dynamic environment of the cell. To address this, the researchers ran molecular dynamics simulations, which track the physical motion of atoms over time using the laws of classical mechanics. The kaempferol-AKT1 complex remained structurally stable throughout a 100-nanosecond simulation, with root mean square deviation and fluctuation analyses indicating that the ligand stayed anchored in the binding pocket without destabilising the protein fold. This kind of sustained stability strengthens the argument that kaempferol is a plausible direct modulator of AKT1 rather than an artefact of the docking algorithm, and it provides a structural starting point for medicinal chemists interested in optimising flavonoid-based AKT inhibitors.

Taken together, the study weaves a consistent narrative from cell culture to proteomics to computational structural biology. A plant fraction rich in flavonoids and phytosterols kills NSCLC cells, blocks their ability to form new colonies, suppresses their migration, and pushes cancer-relevant proteins away from an angiogenic and metastatic state, all while its principal constituents show computationally predicted and dynamically stable interactions with the PI3K/AKT1/KRAS signalling core and matrix metalloproteinases. The authors conclude that Pistacia integerrima bioactives exhibit significant anti-proliferative, anti-migratory and anti-metastatic activities in vitro, providing a scientific rationale for identifying newer promising candidates for NSCLC.

Important caveats remain. All of the experimental evidence comes from a single cell line in vitro, and the concentrations used, particularly the 100 micrograms per millilitre dose in the cytotoxicity assay, are far removed from anything a patient could achieve through an extract or supplement. The docking and dynamics work, however rigorous, generates hypotheses about direct target engagement that still need confirmation with techniques such as surface plasmon resonance, cellular thermal shift assays or kinase activity measurements. No animal data or pharmacokinetic information exists yet, and the fraction itself is a complex mixture whose active constituents and their relative contributions have not been disentangled. Nevertheless, the convergence of phenotypic, proteomic and computational evidence makes Pistacia integerrima a credible candidate for further preclinical development, and it adds to a growing body of work suggesting that flavonoids such as kaempferol and quercetin, and phytosterols such as beta-sitosterol, deserve systematic evaluation as leads against some of the most stubborn signalling pathways in lung cancer. As the search for new weapons against NSCLC intensifies, an old medicinal tree may yet yield distinctly modern drug candidates.

Subject of Research: Anticancer activity of Pistacia integerrima phytochemicals against non-small cell lung cancer via PI3K, AKT1 and KRAS signalling

Article Title: Proteomic regulation of anti-proliferative and anti-migratory activity by potent phytochemicals from Pistacia integerrima J.L. Steward Ex Brandis via PI3K, AKT1, and KRAS for Lung Cancer

Article References: Jamwal, A., Paudel, K., Dua, K., Kulkarni, M. P., Mujwar, S., Dhiman, S., Dalwal, V., Negi, P., & Goyal, R. (2026). Proteomic regulation of anti-proliferative and anti-migratory activity by potent phytochemicals from Pistacia integerrima J.L. Steward Ex Brandis via PI3K, AKT1, and KRAS for Lung Cancer. Molecular Biology Reports, 53(1), Article 1571. https://doi.org/10.1007/s11033-026-12743-5

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12743-5

Keywords: non-small cell lung cancer, Pistacia integerrima, A549 cells, PI3K/AKT1 signalling, KRAS, molecular docking, molecular dynamics simulation, flavonoids, kaempferol, quercetin, MMP-2, anti-migratory activity