1. Introduction
Colorectal cancer constitutes a major global health burden and the second leading cause of cancer mortality worldwide (1). Despite advances in diagnosis and treatment, metastasis remains the leading cause of CRC-related mortality (2). Approximately 25% of patients present with metastatic disease at diagnosis, and nearly half develop metastases during their illness (3).
Tumor progression toward invasive and metastatic phenotypes is a multifaceted process involving several sequential events, with matrix metalloproteinases (MMPs) and the urokinase-type plasminogen activator (uPA) system serving as key mediators of extracellular matrix degradation and tissue invasion (4). Overexpression of MMP-2, MMP-9, uPA, and uPAR promotes extracellular matrix (ECM) degradation, facilitating tumor spread. Conversely, tissue inhibitors of metalloproteinases (TIMPs) particularly TIMP-1 and TIMP-2 suppress MMP activity and limit metastasis (5).
Natural products have been increasingly studied as potential sources of anticancer compounds due to their multitargeted mechanisms and lower toxicity (6). Ziziphora clinopodioides (family Lamiaceae) has been used widely for various therapeutic purposes in Iranian folk medicine (7). Its essential oil and extracts contain several biologically active components including thymol, carvacrol, 1,8-cineole, limonene, and rosmarinic acid known for antimicrobial, antioxidant, and anticancer effects (8).
Given these properties and the growing interest in plant-derived anticancer agents, this study aimed to evaluate the cytotoxic and antimetastatic potential of Z. clinopodioides extract on colorectal cancer cells.
2. Materials and Methods
2.1 Preparation of Z. clinopodioides extract
The aerial parts of Z. clinopodioides were purchased from a verified medicinal plant supplier (Giah Kala, Tehran, Iran). Following botanical identification and authentication, the collected plant material was air-dried at ambient temperature and subsequently ground into a fine powder. Forty grams of powder were macerated in 300 mL of 70% ethanol (selected for its efficiency in extracting both polar and non-polar phytochemicals) for 72 h on a shaker at room temperature in the dark. After filtration using Whatman No. 1 paper, the extract was subjected to solvent evaporation under controlled conditions at 45 °C for 48 h. The concentrated extract obtained was maintained at −20 °C until use in the experimental procedures. 
2.2 Cell culture and treatment
Human SW742 colorectal carcinoma cells were purchased from the Pasteur Institute (Tehran, Iran) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. Upon completion of four passages, the cells were subjected to treatment with graded concentrations of the extract (6.25, 12.5, 25, 50, 100, 200, 400, and 800 µg/ml) for viability and cytotoxicity assessments. The IC₅₀ value derived from these experiments was selected for use in subsequent analyses.
2.3 Viability assay
The cells were seeded in 96-well plates (15×103 cells/well) and placed in an incubator for 24 hr to allow the cells to adhere to the bottom of the plate. Then, the culture medium was replaced with the medium containing the extract. The control cells received culture medium. After 24, 48, 72, and 96 hr, 50 μL of MTT solution (5 mg/ml) was added. After 4 hr, the medium was removed and 200 μl of DMSO was added to each well. The plate was subsequently placed on an orbital shaker and mixed for 15 min. Absorbance values were measured at 570 nm, while readings at 650 nm were used for background correction. Cell viability was calculated as the percentage of absorbance in treated cells relative to control cells according to the formula below:
Cell viability (%) = (OD treated / OD control) × 100
IC₅₀ values were estimated using GraphPad Prism 5.0 (USA) based on the dose–response curves generated from the viability data.
2.4 Cytotoxicity assay
The extent of cell membrane damage was determined by analyzing LDH release at 24, 48, 72, and 96 h after treatment. Supernatants were collected and subjected to LDH quantification using a commercially available kit (Sigma, St. Louis, MO, USA) following the manufacturer's guidelines. Absorbance was measured at a wavelength of 490 nm using an ELISA plate reader.
2.5 Cell migration assay
The effect of the extract on cell migration was investigated using a wound-healing (scratch) assay. Cells were plated in 6-well culture plates at a density of 150 × 10³ cells/well and cultured until a uniform monolayer was established. A scratch was created in the cell layer with a sterile pipette tip, after which the wells were gently washed with PBS to eliminate detached cells. The cultures were then maintained in medium containing 2% FBS in the presence or absence of the extract and incubated for 24 h at 37 °C. The migratory capacity of the cells was quantified by monitoring wound closure under a microscope and analyzing the images with T-Scratch software.
2.6 Cell invasion assay
Cell invasion was determined using Transwell chambers (Cell Biolabs, Inc. San Diego, CA, USA) with 6.5 mm polycarbonate filters of 8 μm pore size. 50 μL of Matrigel was pipetted into the upper chamber and incubated at 37 ° C for 30 min to solidify the gel. Cells were seeded at 1×106 cells/mL in 100 μL of the serum-free medium in the upper chamber. Immediately, this chamber was transferred to the lower chamber consisting of 600 μl of medium (2% FBS with and without the extract). After a 24-h incubation period at 37 °C, the upper inserts were processed to remove cells that had not migrated through the membrane. The cells attached to the external surface of the chamber were fixed with 70% methanol for 10 min and stained with 0.2% crystal violet. Following rinsing with distilled water, the samples were dried at ambient temperature and examined under an inverted microscope. The number of migrated cells was quantified by extracting the bound dye with an acetic acid solution and measuring the absorbance at 570 nm.
2.7 Cell adhesion assay
The wells of 6-well culture plates were pre-coated with 0.1% gelatin and dried at 37 °C for 30 min. Subsequently, harvested cells obtained by trypsin/EDTA treatment were suspended in fresh medium in the presence or absence of the extract prior to the adhesion assay. Then, the cells were distributed in the respective wells and incubated at 37 ° C for 24 hr. Finally, the cells were fixed using 70% methanol for 10 min. Then, they were stained with 0.2% crystal violet and washed with distilled water. The cells were air-dried. Then, they were observed under an inverted microscope. The color of the cells was extracted with acetic acid solution and the intensity of the extracted color of each group was measured at a wavelength of 570 nm.
2.8 Gene expression analysis
After treatment, total RNA from cells was extracted using RNXplus solution. The absorbance of each sample was measured at wavelengths 260, 280, and 230, and the ratio of absorbance of 260 to 280 and 260 to 230, as well as RNA concentration, was calculated. The quality of extracted RNA was determined by agarose gel electrophoresis. Considering that RNA cannot be directly used as a template for a Real-time PCR assay, it must first be reverse transcribed into complementary DNA (cDNA). The Ekta Tehiz Teb kit was used to prepare cDNA according to the kit protocol. The resulting cDNA was kept in a freezer at -80 ° C. The specific primers for each gene were designed using Allel ID6 software. GAPDH gene was used as a housekeeping gene to determine the relative expression level of the studied genes. The primer efficiencies were tested and confirmed to be within acceptable limits (90–110%) prior to use (9). The SYBR kit produced by Ben Yakhte Company (Iran) was used for Real-time PCR.
2.9 Statistical analysis
Data are reported as mean values with corresponding standard deviations (mean ± SD). Group differences were analyzed by one-way ANOVA with Tukey’s multiple comparison test, and statistical significance was defined as p < 0.05.
3. Result
3.1 The effect of the extract on cell viability and calculation of IC50
The effects of different concentrations of the extract on cell viability after 24, 48, 72, and 96 h are presented in Figure 1A. After 24 hr, the decrease in cell survival was significant at concentrations of 50, 100, 200, 400, and 800 µg/ml (p<0.05). After 48 hr, it was significant in concentrations of 12.5, 25, 50, 100, 200, 400, and 800 µg/ml (p<0.05) and after 72 and 96 hr, at all concentrations used (p<0.05). The IC50 values of the extract were 760.59 ± 32.45, 395.16 ± 12.85, 241.87 ± 27.77, and 119.05 ± 11.05 for 24, 48, 72, and 96 hr.
3.2 The cytotoxic effect of the extract
After 24 hr, cytotoxicity was significant at concentrations of 100, 200, 400, and 800 µg/ml (p<0.05). After 48 and 72 hr, this effect was significant in concentrations of 25, 50, 100, 200, 400, and 800 μg/ml (p<0.05), and after 96 hr it was significant in concentrations of 12.5, 25, 50, 100, 200, 400 and 800 μg/ml (p<0.05) (Figure 1 B).
3.3 The effect of the extract on cell migration, invasion, and adhesion
Treatment with the extract caused a significant decrease in the ability of cells to migrate and invade (p<0.05) (Figures 2 and Figure 3). It also significantly reduced the adhesion potential of cells (p<0.05) (Figure 4).

A

B

Figure 1. Effect of Ziziphora clinopodioides extract on (A) cell viability and (B) plasma membrane integrity in SW742 colorectal cancer cells. Cells were treated with various concentrations of extract (6.25–800 µg/ml) for 24–96 hr. Cell viability was measured by MTT assay, and plasma membrane integrity was evaluated using the LDH release assay. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control. (Prepared by Authors, 2026).

Figure 2. Effect of Ziziphora clinopodioides extract on cell migration ability (scratch assay). (A) Control cells and (B) extract-treated cells after 24 hr. (C) Quantitative analysis of wound closure using T-Scratch software. *** p < 0.001 vs. control. (Prepared by Authors, 2026).

Figure 3. Inhibitory effect of Ziziphora clinopodioides extract on cell invasion. (A) Control cells and (B) extract-treated cells after 24 hr. (C) Quantitative absorbance of crystal-violet-stained invading cells measured at 570 nm. * p < 0.05 vs. control. (Prepared by Authors, 2026).

Figure 4. Effect of Ziziphora clinopodioides extract on cell adhesion. (A) Control cells and (B) extract-treated cells after 24 hr. (C) Mean absorbance of attached cells stained with crystal violet at 570 nm. *** p < 0.001 vs. control. (Prepared by Authors, 2026).