Chemical profiling and anticancer activity of Alnus incana dichloromethane fraction on HeLa cells via cell cycle arrest and apoptosis.

Background: Cervical cancer remains a global health challenge with persistently high incidence and mortality rates despite advancements in conventional treatments. The therapeutic potential of natural products has gained attention, particularly for their selective cytotoxicity and ability to modulate cancer pathways. Alnus incana (L.) Moench, a species-rich in bioactive compounds, shows potential as an anticancer agent; however, the cytotoxic effects of its leaves dichloromethane (DCM) extract remain underexplored. This study investigates the DCM fraction's cytotoxicity on various cancer cell lines, with a primary focus on HeLa cells.

Methods: The cytotoxic effects of the A. incana DCM fraction were evaluated in a dose-dependent manner using the MTT assay on several cancer cell lines, with particular emphasis on HeLa cells. Flow cytometry was used to assess cell cycle arrest and apoptosis, while RT-qPCR quantified changes in the expression of apoptotic markers (Bax, Bcl-2, and p53). Chemical composition analysis was conducted using gas chromatography-mass spectrometry/flame ionization detection (GC-MS/FID) to identify the major bioactive compounds within the fraction.

Results: The DCM fraction exhibited dose-dependent cytotoxicity in HeLa cells, with an IC₅₀ value of 135.6 µg/mL and a selectivity index (SI) of 2.72 relative to normal cells. Flow cytometry analysis revealed G0/G1 cell cycle arrest, significantly hindering progression through the S and G2/M phases. Moreover, there was a significant increase in both early and late apoptotic cell populations, correlating with the upregulation of pro-apoptotic genes (Bax and p53) and the downregulation of the anti-apoptotic gene Bcl-2. The chemical analysis identified 22 compounds in the unsaponifiable fraction, chiefly terpenoids such as phytol (65.74%). The saponifiable fraction presented a balanced composition of saturated (48.69%) and unsaturated (51.29%) fatty acids, with palmitic acid, linolenic acid, and linoleic acid as the predominant compounds.

Conclusion: While the DCM fraction's relatively high IC₅₀ value may limit its utility as a standalone treatment, its ability to induce cell cycle arrest and apoptosis demonstrates its promise as a co-therapeutic agent with conventional anticancer drugs. Further research is essential to elucidate its precise mechanisms of action and to evaluate its efficacy in combination therapies, potentially advancing its role in cervical cancer treatment.