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

Abstract

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.

Keywords: Alnus incana; Apoptosis; Cell cycle arrest; Cervical cancer; Cytotoxicity; HeLa cells.

Fig. 1

Cytotoxic effects and IC₅₀ values of the A. incana DCM fraction and doxorubicin on various cell lines over 48 h, as determined by the MTT assay. Panels (a–e) show the cytotoxic effects of the A. incana DCM fraction on (a) normal human skin fibroblasts (HSF), (b) cervical cancer cells (HeLa), (c) breast cancer cells (MCF-7), (d) liver cancer cells (HepG2), and (e) colon cancer cells (HCT-116). Panels (f) and (g) illustrate the cytotoxic effects and IC₅₀ values of doxorubicin on (f) HSF and (g) HeLa cells after 48 h

Fig. 2

Flow cytometric analysis of cell cycle distribution in HeLa cells treated with the A. incana DCM fraction. HeLa cells were treated with the IC₅₀ concentration (135.6 µg/mL) of the A. incana DCM fraction for 48 h, and cell cycle phase distribution was analyzed via flow cytometry. Panels: (a) Control (untreated) HeLa cells, (b) HeLa cells treated with the DCM fraction, and (c) A graphical comparison of cell percentages across each cell cycle phase between treated and untreated groups

Fig. 3

Flow cytometric analysis of apoptosis and necrosis in HeLa cells treated with the A. incana DCM fraction. Detection of apoptotic and necrotic cell populations was conducted using Annexin V-FITC and PI dual staining after treating HeLa cells with the IC₅₀ concentration (135.6 µg/mL) of the A. incana DCM fraction for 48 h. Quadrant distribution: upper left (necrotic cells), lower left (viable cells), lower right (early apoptotic cells), and upper right (late apoptotic cells). Panels: (a) Control (untreated) HeLa cells, (b) DCM-treated HeLa cells, and (c) Graphical representation comparing cell percentages in each quadrant between treated and untreated cells

Fig. 4

Gene expression of (a) Pro-apoptotic genes p53 and Bax, and (b) Anti-apoptotic gene Bcl-2 in HeLa cells treated and untreated with the A. incana DCM fraction

Fig. 5

Chromatographic analysis of Alnus incana leaves’ DCM fraction: (a) GC-MS chromatogram for unsaponifiable matter, and (b) GC-FID chromatogram for saponifiable matter (fatty acids)