Anti-oral Squamous Cell Carcinoma Activity, DNA Damage, and Apoptosis Induced by Nectaroscordum tripedale Essential Oil

1. Introduction
Cancer, commonly referred to as a malignant neoplasm or tumor, encompasses a broad spectrum of diseases characterized by uncontrolled cell proliferation and the potential to invade or spread to distant organs [1]. According to the World Health Organization (WHO), cancer remains a leading global cause of mortality, with approximately 7.6 million deaths annually—a number projected to rise beyond 11 million by 2030 [2].
Among the various cancer types, oral cancer ranks as the eighth most prevalent in men and the fifteenth in women [3]. This category includes malignancies of the lips, tongue, oral mucosa, gingiva, floor of the mouth, hard and soft palates, tonsils, salivary glands, and regions such as the oropharynx, nasopharynx, and hypopharynx. Over 90% of these oral cancers are diagnosed as squamous cell carcinomas (SCC), while the remaining cases comprise salivary gland neoplasms, sarcomas, lymphomas, and metastases from other primary sites like the lungs, breast, prostate, and kidneys [4]. Histologically, SCC originates from dysplastic epithelium and is marked by the presence of infiltrative malignant epithelial clusters [5].
Conventional treatments for cancer include surgical intervention and chemoradiotherapy, both of which are associated with significant side effects [6]. Radiation therapy may lead to xerostomia, mucosal sensitivity, rampant dental decay, and dysphagia. On the other hand, chemotherapy can cause mucositis, gastrointestinal disturbances, immunosuppression, and general systemic toxicity. In advanced-stage cases requiring extensive surgery, patients often face functional impairments in speaking, mastication, and swallowing [7]. Despite notable progress in multimodal treatment strategies, the five-year survival rate for SCC remains suboptimal, ranging from 50% to 59% [8]. Given these limitations, alternative approaches, including traditional and herbal medicine, have gained increased attention worldwide for their role in disease prevention and as complementary therapy [9-11].
Among these, essential oils (EOs) derived from plants have been extensively explored for their anticancer properties. These oils are rich in bioactive constituents such as monoterpenes, sesquiterpenes, oxygenated derivatives, and phenolic compounds. Their anticancer potential is linked to mechanisms including anti-mutagenic and anti-proliferative activities, enhancement of immune surveillance, induction of detoxifying enzymes, and antioxidant effects [12].
Nectaroscordum tripedale, a perennial species in the Amaryllidaceae family native to Central Asia, has been recognized for its medicinal value. It is characterized by a tall, sturdy stem (50–90 cm), bearing an umbrella-like inflorescence composed of around 30 bell-shaped flowers. Its foliage, reminiscent of garlic, emits a strong, distinctive odor [13, 14]. Biochemically, the plant is notable for its cysteine-rich profile, containing compounds such as O-phthaldialdehyde (OPA), (+)-S-(1-butenyl)-L-cysteine sulfoxide, its γ-glutamyl derivatives, and other related sulfur-containing metabolites [15]. Prior studies have demonstrated a range of pharmacological activities for N. tripedale, including antioxidant, antimicrobial, antidiabetic, hepatoprotective, and nephroprotective effects [13, 14, 16]. Building on these properties, the current study was designed to evaluate the anticancer potential and molecular mechanisms of N. tripedale EO in human oral SCC models.

2. Materials and Methods

2.1 Ethical approval 
This experimental protocol was reviewed and approved by the Ethics Committee of Lorestan University of Medical Sciences, Khorramabad, Iran.

2.2. Plant collection and identification 
Aerial parts of N. tripedale were harvested in May 2022 from mountainous regions surrounding Khorramabad, located in western Iran. Following botanical authentication, a voucher specimen was deposited at the Herbarium of the Razi Herbal Medicines Research Center under accession number 1402244. The plant material was air-dried and stored in light-protected containers until further processing.

2.3. EO extraction
The EO was extracted from the dried aerial parts of N. tripedale using a Clevenger-type apparatus via hydrodistillation for 2 hours. The resulting oil was dried over anhydrous sodium sulfate to remove moisture and subsequently stored at 4 °C in sealed vials until analysis and bioassays were performed [17].

2.4. Gas chromatography-mass spectrometry (GC-MS) analysis
To identify the chemical composition of the EO, GC-MS analysis was conducted using a gas chromatograph (model 7890A) coupled with a mass spectrometer (model 5975A). Components were identified by comparing their retention indices and mass spectra with reference compounds and data from the NIST library. Quantification of individual constituents was achieved by integrating the peak areas in the chromatograms.

2.5. Cell culture conditions
Normal human gingival fibroblasts (HGF1) and oral squamous carcinoma cells (KB) were procured from the Pasteur Institute of Iran. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS; Merck, Germany), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained at 37 °C in a humidified incubator with 5% CO2.

2.6. MTT cytotoxicity assay 
The cytotoxic potential of N. tripedale EO was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay [18]. Cells were seeded into 96-well plates at a density of approximately 7,000 cells/well and allowed to adhere for 24 hours. Following incubation, the culture medium was replaced with serial dilutions of the EO (3.125–200 µg/mL), and cells were exposed for 48 hours. After treatment, 10 µL of MTT solution (Sigma-Aldrich, Germany) was added to each well, followed by 4 hours of incubation. Next, 150 µL of DMSO was added to solubilize formazan crystals, and absorbance was measured at 570 nm using a microplate reader. The 50% cytotoxic concentration (CC50) was determined via probit analysis using SPSS software, version 25.0. The selectivity index (SI) was calculated as the ratio of CC50 in normal cells to CC50 in cancer cells.

2.7. Gene expression analysis of apoptosis markers
To assess the impact of the EO on apoptotic pathways, the expression levels of caspase-3, Bcl-2, and Bax genes were quantified using real-time polymerase chain reaction (PCR). Total RNA was extracted from both untreated and treated HGF1 and KB cells using a commercial RNA isolation kit (Qiagen, USA), according to the manufacturer’s instructions. Cells were detached with trypsin, pelleted, and subjected to RNA extraction, followed by cDNA synthesis using a complementary kit (Qiagen, USA).
PCR amplification was conducted using synthesized cDNA, gene-specific primers (Table 1), and Maxima™ SYBR Green Master Mix (Fermentas, USA). The thermal cycling conditions were as follows: initial denaturation at 96 °C for 7 minutes, followed by 40 cycles of denaturation at 95 °C for 10 seconds, annealing at 56 °C for 30 seconds, and extension at 72 °C for 30 seconds. Gene expression changes were quantified using the 2-ΔΔCt method, with β-actin serving as the internal control. Analysis was performed using IQ™5 software (Bio-Rad, Hercules, CA) [19].

2.8. Assessment of DNA synthesis inhibition
To evaluate the effect of N. tripedale EO on DNA synthesis, cell treatment was carried out in 96-well plates following the protocol used in the MTT assay. The BrdU (5-bromo-2′-deoxyuridine) incorporation assay was performed using a commercial ELISA kit (Roche, Germany) as per the manufacturer’s instructions [20]. Briefly, after 24 hours of EO treatment, 5 μL of BrdU labeling solution was added to each well and incubated for 3 hours. The culture medium was then removed, and 100 μL of fixation/denaturation solution was added to each well, followed by incubation at room temperature (25 °C) for 30 minutes. Subsequently, 50 μL of anti-BrdU-POD conjugate was added and incubated for 90 minutes at 25 °C. After washing the wells thoroughly with phosphate-buffered saline (PBS), 50 μL of the substrate solution was added. Absorbance was measured at 405 nm and 490 nm using a microplate reader to determine DNA synthesis levels.

2.9. Statistical analysis
All experiments were performed in triplicate. Data were analyzed using SPSS software (version 25.0). A P<0.05 was considered statistically significant.

3. Results 

3.1. GC-MS analysis of N. tripedale EO
The chemical composition of the EO extracted from N. tripedale was determined using GC-MS. As summarized in Table 2, the analysis revealed that Germacrene-D was the most abundant constituent, comprising 32.3% of the total oil content. Other major components included hexadecanoic acid (13.2%) and diphenylamine (10.7%), along with several minor compounds contributing to the overall phytochemical profile.

3.2. Cytotoxic activity of N. tripedale EO 
As depicted in Figure 1, the MTT assay demonstrated that treatment with N. tripedale EO led to a concentration-dependent reduction in cell viability in both KB oral squamous carcinoma cells and normal human gingival fibroblasts (HGF1-RT1) (P<0.001). The half-maximal cytotoxic concentration (CC50) was calculated to be 58.6 μg/mL for KB cancer cells and 136.4 μg/mL for HGF1-RT1 cells. Based on these values, the SI—calculated as the ratio of CC50 in normal cells to CC50 in cancer cells—was greater than 2, suggesting that N. tripedale EO exhibited selective cytotoxicity against cancerous cells while exerting minimal toxicity on non-cancerous cells.

3.3. Effect of N. tripedale EO on apoptosis-related gene expression
Quantitative real-time PCR analysis revealed a significant upregulation of caspase-3 and Bax gene expression in both KB oral cancer cells and normal gingival fibroblast cells (HGF1-RT1) following treatment with N. tripedale EO at concentrations corresponding to ½ CC50 and CC50 (P<0.05). Conversely, the expression of the anti-apoptotic gene Bcl-2 was markedly downregulated in both cell types, with the most pronounced reduction observed at the higher concentration (CC50) of the EO (P<0.05), as shown in Figure 2. These findings suggest that N. tripedale EO may induce apoptosis through a caspase-dependent pathway and by modulating the Bax/Bcl-2 regulatory axis.

3.4. Inhibition of DNA synthesis by N. tripedale EO
The analysis of DNA synthesis using the BrdU incorporation assay revealed a concentration-dependent inhibition of DNA replication in both KB oral cancer cells and normal HGF1 fibroblasts following exposure to N. tripedale EO. As shown in Figure 3, treatment at the CC50 concentration led to a marked suppression of DNA synthesis in both cell types, with a more substantial effect observed in the cancerous cells. These results suggest that N. tripedale EO may interfere with cell proliferation by impairing DNA synthesis mechanisms.

4. Discussion
N. tripedale is a medicinal plant known for its diverse array of bioactive compounds, contributing to a range of biological effects such as antioxidant, antimicrobial, anti-inflammatory, and anticancer activities [14, 16]. In the present study, GC-MS analysis revealed that Germacrene-D, hexadecanoic acid, and diphenylamine were the major constituents of the EO derived from N. tripedale. Each of these compounds has been previously associated with pharmacological properties. For instance, Germacrene-D, a sesquiterpene, has demonstrated antimicrobial and anti-inflammatory effects [21]. Hexadecanoic acid, a saturated fatty acid also known as palmitic acid, is abundant in plant oils and has been reported to exhibit cytotoxic and anti-inflammatory actions [22]. Diphenylamine, a nitrogen-containing aromatic compound, is recognized for its antioxidant properties, which may contribute to its potential antitumor activity [23].
In line with these biochemical profiles, our study demonstrated that N. tripedale EO exerted cytotoxic effects on KB oral squamous carcinoma cells in a dose-dependent manner, while maintaining relative safety toward normal human gingival fibroblasts. The calculated SI>2 further supports the selective toxicity of the EO toward malignant cells. These results are consistent with previous findings by Ezatpour et al. (2016), who reported the cytotoxicity of N. tripedale extracts against leukemic cell lines, with limited toxicity to normal cells [15]. Moreover, the low systemic toxicity of N. tripedale in vivo has been previously confirmed in animal models, where no significant alterations in liver and kidney function biomarkers were observed [14]. Collectively, these findings suggest the potential of N. tripedale EO as a relatively safe and natural anticancer agent.
To explore the underlying mechanisms of its anticancer activity, we assessed the expression of key apoptosis-related genes following treatment with N. tripedale EO. Notably, exposure to the EO resulted in a significant upregulation of the pro-apoptotic genes caspase-3 and Bax, along with downregulation of the anti-apoptotic gene Bcl-2. These findings align with the established roles of these genes in the regulation of programmed cell death: caspase-3 functions as a central executioner of apoptosis [24], Bax promotes mitochondrial membrane permeabilization, and Bcl-2 acts as a suppressor of apoptosis by stabilizing mitochondrial integrity [25]. The observed gene expression pattern indicates activation of the intrinsic apoptotic pathway, suggesting that N. tripedale EO may trigger mitochondrial-mediated cell death in KB cells.

5. Conclusion 
Additionally, our data showed that DNA synthesis was markedly suppressed in both normal and cancer cells treated with the EO, with the greatest inhibition observed in KB cells. This inhibitory effect on DNA replication could contribute to reduced cell proliferation and tumor progression, further reinforcing the potential of N. tripedale EO as an antiproliferative agent.
Given the limitations of conventional therapies—such as chemotherapy and radiotherapy—which are often associated with adverse side effects and limited specificity, the development of plant-derived compounds offers an attractive alternative. In this context, our study adds to the growing body of evidence supporting the application of EOs in cancer treatment. The promising in vitro effects observed for N. tripedale EO warrant further investigation in animal models to validate its safety and efficacy under physiological conditions. Ultimately, such studies could pave the way for clinical trials aimed at developing novel, plant-based therapeutics for oral cancers.

Acknowledgements
The authors would like to thank the Vice Chancellor for Research of the Faculty of Dentistry, Lorestan University of Medical Sciences, Khorramabad, Iran.

Compliance with ethical guidelines
The present study was approved by the Research Ethics Committee of Lorestan University of Medical Sciences, Khorramabad, Iran (Code: IR.LUMS.REC.1402.244).

Data availability  
All data analyzed during this study are included in this article.

Funding
This research did not receive any grant from funding agencies in the public, commercial, or non-profit sectors. 

Authors' contributions
Conceptualization, study design, and writing the original draft: Pegah Shakib; Experiments: Mohammad Rezaei, Asma Sepahdar, and Aida Hemmati; Data acquisition and analysis: Zeinab Sharafi and Fatemeh Tavakol; Review and editing: Roshanak Abbasi.

Conflict of interest
The authors declared no conflict of interest.

References

1. di Camillo Orfali G, Duarte AC, Bonadio V, Martinez NP, De Araújo MEMB, Priviero FBM, et al. Review of anticancer mechanisms of isoquercitin. World J Clin Oncol. 2016; 7(2):189-199. [DOI:10.5306/wjco.v7.i2.189] [PMID]
2. Bhalla KN, Rao R, Sharma P, Das Gupta S, Chauhan L, Stecklein S, et al. Abstract S3-7: Treatment with histone deacetylase inhibitors creates ‘BRCAness’ and sensitizes human triple negative breast cancer cells to PARP inhibitors and cisplatin. Cancer Res. 2012; 72(24_Supplement):S3-7.[DOI:10.1158/0008-5472.SABCS12-S3-7]
3. Johnson NW, Jayasekara P, Amarasinghe AA. Squamous cell carcinoma and precursor lesions of the oral cavity: Epidemiology and aetiology. Periodontol 2000. 2011; 57(1):19-37. [DOI:10.1111/j.1600-0757.2011.00401.x] [PMID]
4. Rhodus NL, Kerr AR, Patel K. Oral cancer: Leukoplakia, premalignancy, and squamous cell carcinoma. Dent Clin North Am. 2014; 58(2):315-40. [DOI:10.1016/j.cden.2013.12.004] [PMID]
5. Shu Z, Guo J, Xue Q, Tang Q, Zhang B. Single-cell profiling reveals that SAA1+ epithelial cells promote distant metastasis of esophageal squamous cell carcinoma. Front Oncol. 2022; 12:1099271. [DOI:10.3389/fonc.2022.1099271][PMID]
6. Zugazagoitia J, Guedes C, Ponce S, Ferrer I, Molina-Pinelo S, Paz-Ares L. Current challenges in cancer treatment. Clin Ther. 2016; 38(7):1551-66. [DOI:10.1016/j.clinthera.2016.03.026] [PMID]
7. Amjad MT, Chidharla A, Kasi A. Cancer chemotherapy. 2023 Feb 27. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025. [PMID]
8. Kacher JE. Oral and maxillofacial pathology. Case of the month. Histoplasmosis. Tex Dent J. 2013; 130(3):198, 232. [PMID]
9. Kuruppu AI, Paranagama P, Goonasekara CL. Medicinal plants commonly used against cancer in traditional medicine formulae in Sri Lanka. Saudi Pharm J. 2019; 27(4):565-73. [DOI:10.1016/j.jsps.2019.02.004][PMID]
10. Kim S, Kim N, Jeong J, Lee S, Kim W, Ko SG, et al. Anti-cancer effect of Panax ginseng and its metabolites: From traditional medicine to modern drug discovery. Processes. 2021; 9(8):1344. [DOI:10.3390/pr9081344]
11. Assaf AM, Haddadin RN, Aldouri NA, Alabbassi R, Mashallah S, Mohammad M, et al. Anti-cancer, anti-inflammatory and anti-microbial activities of plant extracts used against hematological tumors in traditional medicine of Jordan. J Ethnopharmacol. 2013; 145(3):728-36. [DOI:10.1016/j.jep.2012.11.039] [PMID]
12. Bhalla Y, Gupta VK, Jaitak V. Anticancer activity of essential oils: A review. J Sci Food Agric. 2013; 93(15):3643-53. [DOI:10.1002/jsfa.6267] [PMID]
13. Mahmoudvand H, Ezatpour B, Rashidipour M, Mirbadie SR, Mahmoudvand H. Evaluation of the scolicidal effects of Nectaroscordum tripedale extract and its acute toxicity in mice model. Pak J Pharm Sci. 2016; 29(6):2125-8. [Link]
14. Kiani AA, Ezatpour B, Niazi M, Jahanbakhsh S. Toxicity effect of Nectaroscordum tripedale extract on hematological and biochemical parameters in mice. Entomol Appl Sci Lett. 2018; 5(2):22-5. [Link]
15. Ezatpour B, Azami M, Motamedi M, Rashidipour M, Mahmoudvand H, Alirezaei M, et al. Chemical composition, in vitro antibacterial and cytotoxicity effect of Nectaroscordum tripedale extract. Herbal Med J. 2016; 1(1):29-36. [Link]
16. Jahanbakhsh S, Ebrahimi K, Sepahvand M, Niazi M, Yari F, Shakarami A, et al. Chemical composition and antifungal effects of nectaroscordum tripedale extract against some of pathogenic yeast strains. Anti-Infective Agents. 2021; 19(1):36-40. [Link]
17. Ullah H, Wilfred CD, Shaharun MS. Comparative assessment of various extraction approaches for the isolation of essential oil from polygonum minus using ionic liquids. J King Saud Univ Sci. 2019; 31(2):230-9. [DOI:10.1016/j.jksus.2017.05.014]
18. Alizadegan F, Aghaei M, Kumar SJ, Saadatmand M, Kumar SA. In vitro and in vivo antileishmanial effects of Nectaroscordum koelzi extract against Leishmania major. J Parasit Dis. 2023; 47(3):683-8. [DOI:10.1007/s12639-023-01614-6][PMID]
19. Saad Al Shehri Z, Alanazi AD, Alnomasy SF. Anti-cancer effects of queen bee acid (10-hydroxy-2-decenoic acid) and its cellular mechanisms against human hepatoma cells. Molecules. 2023; 28(4):1972. [DOI:10.3390/molecules28041972][PMID]
20. Muir D, Varon S, Manthorpe M. An enzyme-linked immunosorbent assay for bromodeoxyuridine incorporation using fixed microcultures. Anal Biochem. 1990; 185(2):377-82. [DOI:10.1016/0003-2697(90)90310-6] [PMID]
21. Telascrea M, de Araújo CC, Marques MO, Facanali R, de Moraes PL, Cavalheiro A, et al. Essential oil from leaves of Cryptocarya mandioccana Meisner (Lauraceae): Composition and intraspecific chemical variability. Biochem Syst Ecol. 2007; 35(4):222-32. [Link]
22. Clarke SR, Mohamed R, Bian L, Routh AF, Kokai-Kun JF, Mond JJ, et al. The Staphylococcus aureus surface protein IsdA mediates resistance to innate defenses of human skin. Cell Host Microbe. 2007; 1(3):199-212. [DOI:10.1016/j.chom.2007.04.005] [PMID]
23. Sugihara T, Rao G, Hebbel RP. Diphenylamine: an unusual antioxidant. Free Radic Biol Med. 1993; 14(4):381-7. [DOI:10.1016/0891-5849(93)90087-B] [PMID]
24. Brentnall M, Rodriguez-Menocal L, De Guevara RL, Cepero E, Boise LH. Caspase-9, caspase-3 and caspase-7 have distinct roles during intrinsic apoptosis. BMC Cell Biol. 2013; 14:32. [DOI:10.1186/1471-2121-14-32][PMID]
25. Czabotar PE, Lessene G, Strasser A, Adams JM. Control of apoptosis by the BCL-2 protein family: Implications for physiology and therapy. Nat Rev Mol Cell Biol. 2014; 15(1):49-63. [DOI:10.1038/nrm3722.] [PMID]