Molecular mechanisms underlying the potential anticancer activity of Pulicaria crispa hexane fraction in HCT116 cancer cells

Abstract

Given the high mortality rate associated with tumors and the severe side effects of current treatments, scientists are exploring alternative therapies with fewer adverse effects. They are increasingly turning to natural remedies, much like our ancestors who used plant extracts to treat various ailments long before understanding the underlying mechanisms. Even though they did not know exactly why these plants treated those diseases then, we have the privilege of testing these plants and discovering the active ingredients responsible for these effects. This study aims to investigate the anticancer mechanisms of Pulicaria crispa hexane fraction (Hex F) against human colorectal cancer cells and elucidate its molecular pathways of action. The methanol extract of P. crispa and its fractions were evaluated for cytotoxic activity using MTT assay against HepG2, HCT116, and Hep-2 cancer cell lines, with oral epithelial normal cells (OEC) as controls. The most potent fraction (Hex F) was further analyzed using flow cytometry for cell cycle and apoptosis analysis, qRT-PCR for gene expression profiling, ELISA for protein quantification, and biochemical assays for oxidative stress and glycolytic enzyme activities. Hex F demonstrated significant cytotoxicity against HCT116 cells with an IC₅₀ of 39.4 μg/mL and a selectivity index of 1.76 indicating preferential toxicity toward cancer cells. Flow cytometry analysis revealed G₂/M phase cell cycle arrest and significant induction of apoptosis. Gene expression analysis showed significant upregulation of pro-apoptotic genes p53, caspase-8, and caspase-9, while anti-apoptotic Bcl2 was downregulated). Protein analysis confirmed increased caspase-3 and caspase-7 activities, accompanied by enhanced anti-inflammatory response with increased IL-10 and decreased IL-4 levels. Oxidative stress markers indicated cellular damage with decreased GSH and SOD levels, while MDA increased significantly. Glycolytic enzyme activities were substantially reduced, with PK, Aldolase, and LDH activities decreased, suggesting metabolic disruption. GC-MS analysis identified β-sitosterol (17.89%), phytol (15.65%), stigmasterol (13.13%), and lupeol (12.89%) as major bioactive compounds. These findings demonstrate that P. crispa Hex F exerts anticancer effects through multiple mechanisms including cell cycle arrest, apoptosis induction, oxidative stress generation, and metabolic disruption, supporting its potential as a natural anticancer therapeutic agent.

Keywords: Apoptosis; Cancer; Cytotoxicity; HCT116; Pulicaria crispa.

Fig. 1

Flow cytometric analysis of cell cycle distribution and apoptosis in HCT116 cells treated with P. crispa Hex F. (A-C) HCT116 cells were treated with 39.4 µg/mL Hex F. A Percentage of cell populations in each cell cycle phase, and viable, early apoptotic, late apoptotic cells, and necrotic cells. Data are presented as the means ± SD of triplicate experiments. *p < 0.05, **p < 0.005, and ***p < 0.001 vs. control. (B, C). Left panel: Typical cell cycle profiles. Right panel: representative scatter plots of PI (y-axis) vs. annexin V (x-axis). Quadrants represent: Q1 (necrotic cells, PI + /Annexin V-), Q2 (late apoptotic cells, PI + /Annexin V +), Q3 (viable cells, PI-/Annexin V-), Q4 (early apoptotic cells, PI-/Annexin V +). B Control untreated cells and C Cells treated with 39.4 μg/mL Hex F for 48 h

Fig. 2

Gene expression analysis of apoptosis-related and cell cycle regulatory genes in HCT116 cells treated with P. crispa Hex F. Relative mRNA expression levels were determined by qRT-PCR using the 2^(-ΔΔCt) method with GAPDH as the housekeeping gene. Cells were treated with 39.4 μg/mL Hex F for 48 h. Pro-apoptotic genes showing significant upregulation: p53 (tumor suppressor gene), caspase-8 (initiator caspase for extrinsic apoptosis pathway), and caspase-9 (initiator caspase for intrinsic apoptosis pathway). Anti-apoptotic gene Bcl2 shows significant downregulation, cell cycle regulatory genes showing downregulation: CDK2 (cyclin-dependent kinase 2, essential for S-phase progression) and TopBP1 (DNA topoisomerase II-binding protein 1, involved in DNA replication and repair). Data represent mean ± SD of three independent experiments performed in triplicate. Statistical significance was determined using an independent t test: **p < 0.005, and ***p < 0.001 compared to untreated control. Error bars represent standard deviation

Fig. 3

Protein expression analysis of caspases and inflammatory markers in HCT116 cells treated with P. crispa Hex F. Protein levels were quantified using specific ELISA kits according to manufacturer's protocols. Cells were treated with 39.4 μg/mL Hex F for 48 h. After treatment with P. crispa Hex F, cell lysates were prepared, and enzymatic activities were measured by a colorimetric assay for A Caspase activity analysis showing significant activation of executioner caspases: caspase-3 (Casp3) (primary executioner caspase responsible for DNA fragmentation and apoptotic body formation) and caspase-7 (Casp7) (secondary executioner caspase with similar function to caspase-3). B Inflammatory marker analysis demonstrating anti-inflammatory response: IL-10 (an anti-inflammatory cytokine that suppresses inflammatory responses) and IL-4 (pro-inflammatory cytokine involved in Th2 immune responses). Data represent mean ± SD of three independent experiments performed in triplicate. Statistical significance: **p < 0.005, and ***p < 0.001 compared to untreated control using independent t-test. Error bars represent standard deviation

Fig. 4

Oxidative stress marker analysis in HCT116 cells treated with P. crispa Hex F. Oxidative stress parameters were measured using specific colorimetric assay kits. Cells were treated with 39.4 μg/mL Hex F for 48 h. The antioxidant activities of A Reduced Glutathione (GSH) levels, a major intracellular antioxidant that protects cells from oxidative damage, B Superoxide Dismutase (SOD) activity, an enzyme that catalyzes the dismutation of superoxide radicals, C Malondialdehyde (MDA) levels, a biomarker of lipid peroxidation and oxidative damage, and D Catalase (CAT) activity, an enzyme that decomposes hydrogen peroxide. Data represent mean ± SD of three independent experiments performed in triplicate. Statistical significance: ***p < 0.001 compared to untreated control using independent t-test. Error bars represent standard deviation

Fig. 5

Glycolytic enzyme activity analysis in HCT116 cells treated with P. crispa Hex F. Key glycolytic enzymes were measured using specific activity assay kits to assess metabolic changes. Cells were treated with 39.4 μg/mL Hex F for 48 h. A Pyruvate Kinase (PK) activity, the rate-limiting enzyme that catalyzes the final step of glycolysis converting phosphoenolpyruvate to pyruvate, and Aldolase activity, an enzyme that cleaves fructose-1,6-bisphosphate into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, and B Lactate Dehydrogenase (LDH) activity, an enzyme that catalyzes the conversion of pyruvate to lactate in anaerobic conditions. The significant reduction in all three enzymes indicates substantial disruption of glycolytic metabolism and cellular energy production. Data represent mean ± SD of three independent experiments performed in triplicate. Statistical significance: *p < 0.05, and ***p < 0.001 compared to untreated control using independent t-test. Error bars represent standard deviation