Dose-Dependent Cytotoxic Effects of Lavandula angustifolia L. Oil on A549 Human Lung Carcinoma Cells

Introduction

The prevalence of lung cancer is increasing, making it the second most common cancer worldwide after breast cancer. Lung cancer accounted for about 12.4% of all cancers in 2022, with an estimated 2.5 million new cases diagnosed globally. In 2022, nearly 1.8 million people died from lung cancer [1]. In most countries, lung cancer is the leading cause of cancer-related deaths. The rates are highest in industrialized regions such as Eastern Asia, North America, and Europe [2].

Many chemical compounds with diverse biological activities are found in aromatic medicinal plants [3]. One of the most well-known medicinal herbs in aromatherapy, L. angustifolia (Lamiaceae), has a long history of use as a sedative. In addition to reducing skeletal muscle tone in rats, the plant also possesses anti-inflammatory, anti-flatulent, antispasmodic, spasmolytic, and antimutagenic properties [4,5]. Lavender oil is used to treat several rheumatic, neurological, and gastrointestinal diseases [6]. Essential oil from lavender, or lavandin, exhibits antimicrobial, antifungal, and desensitizing properties [7]. A 0.25% (v/v) dosage of lavender oil was found to be cytotoxic to human skin cells in vitro [8]. Essential oils from L. angustifolia include compounds that help calm nerves and promote relaxation in both humans and animals [9–11].

The mint family, which includes L. angustifolia Mill. (Lamiaceae), originated in the western Mediterranean [12–14]. The L. angustifolia plant is used as a medicinal herb through oil extraction from its flowers and leaves [15]. Recent years have seen a growing body of scientific evidence suggesting that L. angustifolia extracts has antibacterial, antioxidant, and anticancer properties [15,16]. It has a history of use in relieving irritation, rheumatism, and gastrointestinal problems. Animal models of lung (A-549), breast (MCF-7), cervical (HeLa), and prostate (PC-3) cancers have shown cytostatic and apoptotic effects from distillation extracts of L. angustifolia [17]. Linalool has been shown to induce apoptosis and activate antitumor immunity in cell line studies, acting as an antiproliferative agent against SW 620, Hep G2, A-549, and T-47D cells [18].

This preliminary in vitro cytotoxicity screening study was conducted to investigate the dose-dependent cytotoxic effects of L. angustifolia L. oil on human lung cancer cells. We used A-549 cells for its prior use in essential oil screening [18].

Material and Methods

MATERIALS:

The A549 human lung carcinoma cell line was purchased frozen from ATCC, with the catalog number CCL-185 (ATCC CCL-185).

CELL CULTURE:

A549 human lung carcinoma cells (ATCC CCL-185) were obtained in cryopreserved form and cultured using F-12K medium (Kaighn’s modification of Ham’s F-12, ATCC 30-2004), supplemented with 10% fetal bovine serum (ATCC 30-2020) and 1% antibiotic-antimycotic solution (Gibco 15240062). Frozen vials were thawed in a 37°C water bath for about 1 min, decontaminated with 70% ethanol, and handled under sterile conditions. Cells were transferred into 9 mL of complete medium, centrifuged at 1500 rpm for 5 min, and the pellet was resuspended in 1 mL of fresh medium. This suspension was seeded into T75 flasks (Corning #430641) containing 11 mL of complete medium and incubated at 37°C in a humidified 5% CO₂ atmosphere. When cells reached approximately 80% confluency, they were detached using trypsin-EDTA (ATCC 30-2101) after being washed with PBS. The detached cells were resuspended with medium, centrifuged at 1500 rpm for 3 min, and resuspended in 5 mL of fresh medium. Cell counting was performed using a Scepter™ 2.0 Handheld Automated Cell Counter (Merck, C85360).

:

Lavender essential oil (L. angustifolia L.) was obtained from Sigma-Aldrich (Cat. No: 8000-28-0, Product No: 61718; appearance: colorless to yellow liquid; refractive index at 20°C: 1.450–1.470; ¹H-NMR: conforms to structure). The stock solution (0.879 g/mL) was first dissolved in dimethyl sulfoxide (DMSO) to facilitate homogeneous dispersion and subsequently diluted with complete culture medium to obtain 5 working concentrations (10, 50, 100, 150, and 200 μg/mL).

The final DMSO concentration in all treatment wells was 0.1% (v/v) and was kept constant across concentrations. A vehicle control group containing 0.1% DMSO without essential oil was included and incorporated into all statistical analyses. Cells incubated only with complete medium served as the negative control, while cells treated with Triton X-100, a membrane-lysing detergent, served as the positive control.

To exclude optical interference, oil-containing wells without cells were prepared for each concentration and measured at 570 nm. These blank values were subtracted from corresponding experimental readings before normalization.

MTT CELL VIABILITY ASSAY:

Cells were seeded in 96-well plates at a density of 1×10⁴ cells per well and incubated for 24 h to allow attachment. At approximately 70% confluence, medium was replaced with fresh medium containing the designated concentrations of L. angustifolia oil. Each concentration was tested in triplicate wells in 3 independent biological experiments.

After 24-h exposure, medium was removed and MTT solution (0.5 mg/mL) was added at a 1: 20 ratio relative to total volume and incubated for 3 h at 37°C in 5% CO₂. The solution was discarded and formazan crystals were solubilized using a 1: 1 mixture of DMSO and culture medium. Plates were shaken in the dark for 1 h and absorbance was measured at 570 nm using a microplate spectrophotometer.

Absorbance values were first corrected by subtracting oil-only blanks and then normalized to the negative control. The half-maximal inhibitory concentration (IC₅₀) was calculated from normalized viability values using nonlinear regression analysis in GraphPad Prism (version 9.1). Additionally, morphological evaluation of cells exposed to the IC₅₀ concentration for 24 h was performed using an inverted microscope (Carl Zeiss™ Axio Vert.A1), and representative images were recorded.

STATISTICAL EVALUATION:

Dose–response relationships were analyzed using nonlinear regression in GraphPad Prism (GraphPad Software, USA). Concentration–response curves were fitted using a 4-parameter logistic inhibitory model (4-parameter logistic, 4PL) with variable slope, defined as: Y = Bottom + Top − Bottom1 + 10 (LogIC50 − X) × HillSlope Y=Bottom+ 1+10 (LogIC 50 − X)×HillSlope Top–Bottom where: X represents log₁₀ (concentration), Y represents normalized cell viability (%), Top and Bottom correspond to the upper and lower asymptotes of the curve. HillSlope describes the curve steepness curve, and fitting was performed using least-squares nonlinear regression without weighting (each concentration equally weighted). Top and Bottom parameters were not constrained and were estimated freely by the model. The analysis was performed using normalized viability percentages calculated relative to untreated control, rather than raw absorbance values. The half-maximal inhibitory concentration (IC₅₀) was derived from the fitted curve. Estimated parameters: LogIC₅₀=2.247 IC₅₀=176.6 μM 95% CI for IC₅₀=146.7–221.0 μM, Hill slope=−0.6618 (95% CI: −0.8131 to −0.5439) R²=0.9783, Degrees of freedom=19, Number of concentrations analyzed=21, Goodness of fit was evaluated using coefficient of determination (R²) and residual distribution. All experiments were performed in at least 3 independent replicates and data are presented as mean±SEM. Statistical comparisons between groups were performed using one-way ANOVA followed by Tukey’s post hoc test. A P value <0.05 was considered statistically significant. The positive control group (Triton X-100) was included as an independent group within the same ANOVA model as all treatment and negative control groups and was therefore incorporated into post hoc multiple comparison testing.

Results

CELL MORPHOLOGY:

Representative phase-contrast images of A549 cells are shown in Figure 3 to illustrate gross morphological differences between untreated cells (Figure 3A) and cells exposed to L. angustifolia L. oil at the IC50 concentration for 24 h (Figure 3B). Control A549 cells exhibited typical adherent growth with an epithelioid morphology, forming a confluent monolayer with uniform cell shape and close cell–cell contacts (Figure 3A). After 24-h exposure to L. angustifolia L. oil, cells showed reduced adhesion, increased rounding, and decreased cell density, with many areas of cell detachment and widened intercellular spaces (Figure 3B).

Discussion

LIMITATIONS:

This study has several limitations that should be considered when interpreting the findings. First, the experiments were conducted using a single lung adenocarcinoma cell line (A549). Therefore, the observed cytotoxic response may not fully represent the heterogeneity of lung cancer subtypes or other tumor models. Second, cytotoxicity was primarily evaluated using the MTT assay and morphological observations. Although these methods reliably demonstrate loss of metabolic activity and cell viability, they do not distinguish among apoptosis, necrosis, and other forms of cell death. Confirmation of the underlying mechanism would require assessment of specific molecular markers such as caspase activation, annexin V binding, mitochondrial membrane potential, or DNA fragmentation. Third, no normal lung epithelial cell line was included; therefore, the selectivity of L. angustifolia oil toward malignant versus non-malignant cells could not be determined. Fourth, despite the use of vehicle controls and blank correction, essential oils are complex mixtures that can interact with tetrazolium-based assays. Thus, metabolic assays alone may not fully reflect clonogenic survival or long-term proliferative capacity. Finally, in vitro exposure conditions do not reproduce the pharmacokinetic and metabolic processes present in vivo; consequently, the reported IC₅₀ values should be interpreted as demonstrating biological activity rather than therapeutic efficacy.

Conclusions

Our results suggest that the oil of L. angustifolia L. can decrease the viability of human lung cancer cells (A549). The antiproliferative effect of L. angustifolia L. is quite strong. The concentration determines how much the oil inhibits A549 cell growth; higher doses lead to more significant cytotoxicity. It alters cell shape, resulting in a sharp decline in cell density and the formation of large gaps between cells, which are both signs of widespread cell death.