Cancer has been one of the daunting causes of high morbidity in the human population for the past two decades. Systematic cancer research has revealed that the growth of malignant cancers with the ability to metastasize is caused by a complex collection of events, including the rapid spread and unregulated proliferation of aberrant cells [1]. Radiations, chemical substances, viruses, bacteria, genetic abnormalities, mutations, defective genes, lack of tumor-suppressive genes, defective cell cycle, apoptotic machinery, etc., have been found to majorly trigger carcinogenesis [2]. Although there are many different cancer therapy options available, some of them may be unsuccessful due to negative side effects or increased resistance to traditional anticancer medicines [3].

Moreover, conventional cancer therapies are found to affect healthy cells as well which leads to several unnecessary risks. Hence, natural compounds, that are having potential anticancer activity in vitro, could serve as a better alternative to the researchers as it regulates a wide range of cellular activities, namely cellular growth and differentiation, metastasis, apoptosis, DNA damage, and repair [4]. Natural compounds are a valuable asset in the research and development of new medications, primarily for cancer therapy as they have the benefits of having lesser toxicity and are easily tolerated by the human body at a higher dose compared to conventional chemotherapeutic drugs [5]. More than 5000 phytochemicals are reported in literature having antineoplastic properties which might offer vital resources for developing novel anti-cancer agents, and a safer alternative to a variety of synthetic medicines presently utilized in clinical treatments [6].

Amongst the natural compounds, essential oils from dietary plants and aromatic herbs play an important role in cancer therapy [7]. Eugenol, chemically allyl side chain guaiacol, is a natural monoterpene that belongs to the class of phenylpropanoids. It is a principal component of essential oils that are derived from basil, cinnamon, bay leaf, nutmeg, and clove, among which clove flower buds are the principal source. It acts as an anti-inflammatory [8], analgesic [9], antioxidant [10], antifungal [11], antimicrobial [12], antiviral [13], and antileishmanial [14] among other pharmacological properties. It has profound applications in the food, beverage, cosmetic, and pharma industry for its aroma ^[15][16][15,16]. Of note, eugenol has been evaluated as a versatile therapeutic molecule underscoring its anticancer activity [17]. Eugenol and its synthesized derivatives were reported to have an anti-proliferative effect against cancers of various anatomical origins. In vivo and in vitro investigations envisage specific molecular mechanistic pathways of antiproliferative activity of eugenol, like induction of apoptosis and cell cycle arrest ^[18][19][18,19].

2. Eugenol Characteristics

Eugenol (4-allyl-2-methoxy-phenol; C₁₀H₁₂O₂), often known as clove oil, is an aromatic oil derived from cloves (Syzygium aromaticum). The eugenol content in clove oil ranges from 70% to 96%. Eugenol is a pale yellowish liquid with an aromatic fragrance that dissolves well in organic solvents and mildly in water. Eugenol has limited chemical stability. Eugenol is prone to oxidation and a variety of metabolic reactions. When taken orally, it is promptly absorbed through several organs and metabolized in the liver. So, eugenol encapsulation seems to be the ideal method for minimizing early absorption, increasing water solubility, and thus boosting activity. Eugenol possesses antibacterial, antifungal, antioxidant, and anticancer properties. Low-dose eugenol appears to have little adverse effects other than local irritation, uncommon allergic responses, and contact dermatitis; however, high-dose eugenol can cause tissue injury and a syndrome of sudden onset seizures, coma, and liver and kidney damage ^[20][21].

3. Anticancer Potential of Eugenol

An assessment of existing literature clarified that eugenol exerts anticancer activity via different yet integrated pathways thus ameliorating the hallmarks of unprecedented cellular proliferation. The suggested techniques can be enlisted as induction of apoptosis, cell cycle arrest, reducing angiogenesis, interplaying dual roles as an oxidant and pro-oxidant, inhibiting inflammation, and stopping cellular invasion and metastasis. Autophagy and necroptosis are also reported in a few studies. The mechanisms involved vary according to the types of cancer dealt with, doses, and time dependence. Studies have also shown the chemopreventive role of eugenol when co-administered with other cytotoxic drugs ^{[21][22][23][24]}[22,24,25,26]. Figure 1 illustrates the basic molecular mechanism of eugenol in implementing anticancer effects. The application of nanotechnology has provided a great platform for improving the therapeutic vigor of many phytoconstituents and eugenol is no exception. Many nanocarriers for eugenol were constructed to increase the therapeutic efficiency such as liposomes, microemulsions, micelles, nanoparticles, magnetosomes, ethosomes, etc. ^[25][27]. The improved delivery of nanoengineered phytoconstituents to targeted cancer cells rather than healthy cells has been instrumental in reducing undesirable side effects and resistance to chemotherapeutic agents ^[26][27][28,29]. A detailed discussion of various nanoparticles containing eugenol and their applications in different cancers has been provided in the following sections.

3.1. Effect of Eugenol on Breast Cancer

The kind of breast cancer termed triple-negative breast cancer (TNBC) is notoriously deadly. The treatment with cisplatin alone resulted in higher toxicity to normal cells and drug resistance in malignant cells. Combining cisplatin (30 µM) with eugenol (1 µM) potentiated its chemotherapeutic activity by inhibiting aldehyde dehydrogenases (ALDH) enzyme activity, impeding the nuclear factor kappa B (NF-κB) and signaling cascade by reducing binding affinity of the nuclear factor to receptors interleukin 6 (IL-6) and interleukin-8 (IL-8), thus downregulating IL-6 and IL-8 mRNA (messenger ribonucleic acid). In vitro assays on MDA-MB-231, MDA-MB-468, and BT-20 cells and in vivo clarified the apoptotic activity of the eugenol and cisplatin combination via the mitochondrial pathway. Increased Bcl-2/Bax ratio, elevated levels of proapoptotic protein Bax, increased expression of cleaved caspases-3 and -9, cleaved poly (ADP-ribose) polymerase (PARP) on the higher side, and repression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) accounted for the apoptotic potential for the combination of eugenol and cisplatin. The repression of the expression level of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) explained the inhibition of the invasive tendency of the TNBCs by combination therapy ^[28][30]. In vivo studies revealed that the combination therapy of eugenol (50.0 mg/kg) and cisplatin (2.0 mg/kg body weight) exhibited potent anticancer activity, utilizing humanized tumor xenograft cells modeled in MDA-MB-231 (M.D. Anderson-Metastatic Breast 231) injected into nude mice after the treatment period of 4 weeks. Results revealed the diminution in the development of the tumor is associated with a decrease in the expression of Ki-67 by 95%. Increased apoptosis and angiogenesis of the combination therapy were revealed by the decreased levels of the blood vessel marker cluster of differentiation 31 (CD31). Reduced epithelial-to-mesenchymal transition (EMT) was evident from reduced expressions of N-cadherin and Snail1 and higher E-cadherin expression. Inhibition of pluripotency was evident by reduced expression of biomarker Sox-2 [(sex determining region Y)-box 2] ^[28][30]. Eugenol demonstrated anticancer activity by triggering apoptosis in Michigan cancer foundation 7 (MCF-7) (IC₅₀: 22.75 𝜇M) and MDA-MB-231 (IC₅₀: 15.09 𝜇M) breast cancer cells with increasing ROS levels which inhibited cell cycle at G₂/M phase, that leads to clastogenesis in vitro. Moreover, it downregulated the proliferation of the cell nuclear antigen (PCNA) associated with deceased mitochondria membrane potential (ΔΨm) and upregulation of Bcl-2 associated X protein (Bax) ^[29][31]. In a separate study, eugenol induced apoptosis in MCF-7 (EC₅₀ value 0.9 mM) adenocarcinoma breast cancerous cells by dose-dependently decreasing the proliferation and cellular viability. It is further associated with a higher level of reactive oxygen species (ROS) and a lower level of ATP and mitochondrial membrane potential (ΔΨm). There was a downregulation in Bcl-2 and Bax expression, but the relative ratio remained unchanged. The release of cytochrome-c and lactate dehydrogenase was also observed at a concentration of eugenol of more than 0.9 mM. Thus, as per observation, eugenol shows non-apoptotic Bcl-2 independent toxicity ^[30][32]. Some benzoxazine and aminoethyl derivatives of eugenol were synthesized and their cytotoxicity and cell viability against the MCF-7 cell line were evaluated and reported. Among the various analogs, 6-allyl-3-(furan-2-yl-methyl)-8-methoxy-3,4-dihydro-2H-benzo[e][1,3] oxazine (2) (IC₅₀: 21.7 ± 2.90 µg/mL), 6-allyl-3-benzyl-8-methoxy-3,4-dihydro-2H-benzo[e][1,3] oxazine (3) (IC₅₀: 26.4 ± 2.68 µg/mL), and 4-allyl-2-(benzylaminomethyl)-6-methoxy phenol (4) (IC₅₀: 29.2 ± 2.39 µg/mL) were found to be more potent cytotoxic candidates in comparison to eugenol ^[31][33]. Eugenol at an IC₅₀ value of 1.5 µg/mL is a potent cytotoxic agent which suppressed metastasis and cancer cell migration by downregulating 34.3% matrix metalloproteinase (MMP-9) and 13.7% paxillin mRNA expression levels, respectively, in MCF-7 breast cancer cells ^[32][34]. Eugenol-imposed, dose-specific (100–200 µM) preferential cytotoxicity for MCF10A-ras cells excluding ordinary MCF10A cells. Eugenol effectively regulates the mitochondrial pathway of apoptosis as evidenced by the dysregulation of oxidative phosphorylation machinery and reducing the expressions of the transcriptional factors that regulate beta-oxidation of fatty acids. These transcription factors include medium-chain acyl-coenzyme A dehydrogenase, peroxisome proliferator-activated receptor, and carnitine palmitoyl transferase 1 by downregulating c-Myc/peroxisome proliferator-activated receptor-γ-coactivator-1β (PGC-1β)/estrogen-related receptor-α (ERR-α) pathway ^[33][35]. Both eugenol and astaxanthin upgraded doxorubicin’s cytotoxic activity with a substantial lowering of its IC₅₀ (0.5 μM–0.088 μM) values. Doxorubicin, eugenol (1 mM), and astaxanthin (40 µM) combination synergistically improved the H3 and H4 histone acetylation by increasing histone acetyltransferase (HAT) protein expression, with decreased expressions of forkhead box P3 (FOXP3) protein, and tumor necrosis factor α (TNF-α) was associated with the elevated mRNA expression level of interferon-γ (IFN-γ). The combination of doxorubicin and eugenol further decreased the aromatase and epidermal growth factor receptor (EGFR) expression level in contrast to a single treatment of doxorubicin alone. The apoptosis induction by combination treatment of doxorubicin and eugenol was confirmed by increased expressions of caspase 3 and 8 which triggered cell cycle arrest of estrogen receptor-positive MCF-7 cell lines at the S and G₀/G₁ phase. These further downregulated the expression level of cytokeratin 7 (CK7). Eugenol induced intrinsic apoptosis through a higher BCl-2/Bax ratio, whereas astaxanthin resulted in the induction of extrinsic apoptosis via both caspase-8 and -3 but downregulated LC3B expression ^[34][36]. Upon treatment of eugenol in human triple-negative MDA-MB 231 breast cancer cells (4 µM and 8 µM) and HER2 positive SK-BR3 (5 µM and 10 µM) breast cancer cells, cell proliferating and migrating ability was inhibited. MDA-MB 231 cells were used to show the anti-metastatic activity of eugenol, whereas SK-BR3 cells had been utilized to evaluate the antiproliferative and apoptotic effect besides the anti-metastatic activity of eugenol. The biochemical interventions proved increased expressions of cleaved caspases-3, -7, and -9. Inhibition of angiogenesis and metastasis progression was proven by decreased expression of MMP2 and MMP9, respectively. This was collated with unusual increased levels of tissue inhibitor of metalloproteinases-1 (TIMP1) ^[35][37]. Eugenol induces apoptosis in Ehrlich ascites carcinoma and MCF-7 breast cancer cell lines momentously under the regulation of the Wnt signaling pathway. Both in vivo and in vitro studies dictated the cytoplasmic degradation of β-catenin due to impeded nuclear translocation by phosphorylation of N-terminal residue at ser37. Downregulation of cancer stem cell markers octamer-binding transcription factor 4 (oct4), Notch1 (Neurogenic locus notch homolog protein 1), epithelial cellular adhesion molecule (EpCAM), and CD44 was observed in the stem cells embedded in mammosphere culture. Docking studies in the in-silico binding analysis of eugenol with murine β-catenin showed good binding parameters ^[36][38]. A therapeutic dose (80 μM) of eugenol was shown to cease the proliferating of human epidermal growth factor of receptor 2 (HER-2) positive MCF-10AT cell lines by 32.8%. Insensitivity was observed in the MCF-10A and MCF-7 cell lines which had low levels of HER2 expressions. The major population of MCF-10AT cells showed signs of programmed cell death and remained clustered in the S phase of the cell cycle. Increased dose (180 μM) of eugenol treatment in MCF-10AT cells reduced the protein expressions of Ak strain transforming (AKT), HER2, 3-phosphoinositide-dependent kinase 1 (PDK1), BCL-2, p85, NF-κB, Cyclin D1, and BCL2 associated agonist of cell death (BAD), whereas the Bax, p21, and p27 expressions were augmented. In vivo animal models with breast precancerous lesions treated with 1 mg eugenol inhibited cellular invasion by 30.5% by obstructing HER2/phosphoinositide-3-kinase–protein kinase B (PI3K)-AKT signaling pathways ^[37][39]. Molecular hybrids of new sulfonamides were synthesized from eugenol and dihydroeugenol as precursors and cytotoxicity were measured in different cancer cell lines (HT-144, A549, HepG2 cells) including MCF-7 cell. The phenylpropanoid-based sulfonamide (4b) showed antitumor activity against MCF-7 breast cancer cell lines by inducing apoptosis and cell cycle arrest in G₁/S transition by downregulating cyclins D1 and E ^[38][40]. Eugenol (2 µM) induces apoptosis in breast adenocarcinoma cell lines by overexpression of antiapoptotic proteins independent of p53 modulation by downregulating the nuclear kappa factor and E2F family of transcription factors. The anticancer effect was mediated by targeting the E2F transcription factor 1/surviving oncogenic pathway. Upregulation of p21^wafi was also evident from the inhibition of cyclin D ^[39][41]. In MDA-MB-231 cell lines, eugenol exhibited cytotoxic activity that was dose-dependent, with an IC₅₀ value of 2.89 mM during a 48-h incubation period. Studies using reverse transcription–polymerase chain reaction (RT–PCR) showed that MMP-1, -3, -7, -9, and -11 had lower levels in cells that had been treated with eugenol as compared to cells that had not been treated. In a scratch wound healing experiment, a concentration of 25 µM scratch wound healing agent significantly inhibited cell migration and suppressed the metastasis of MDA MB-231 cells. According to the findings of this research, anti-breast cancer, anti-proliferative, and anti-metastatic effects of eugenol are mediated by targeting matrix metalloproteinase ^[40][42]. Eugenol indorses a spurt in reactive oxygen species levels triggering cell-cycle dysregulation, mitochondrial noxiousness, and clastogenesis eliciting apoptosis in breast cancer MCF-7 cell (IC₅₀: 22.75 µM) and MDA-MB 231 cells (IC₅₀:15.09 µM) in vitro. Oxidative stress in cells abrogates the cell cycle with proliferation cell nuclear antigen (PCNA) down-regulation and decreased mitochondrial transmembrane potential. Cancer cells when exposed to eugenol also showed an increased Bax expression level ^[29][31]. Eugenol displayed anticancer action in breast cancer MCF-7 cells at doses ranging from 1 to 4 mM. This activity was shown by a decrease in the intracellular glutathione level, as well as an increase in DNA fragmentation, intracellular H₂O_2, lipid peroxidation, and apoptosis ^[41][43]. Separate research found that at concentrations of 5 and 10 𝜇M, eugenol inhibited the growth of HER2-positive (SK-BR-3) and triple-negative (MDA-MB-231) breast cancer cells by lowering Nucleoporin 62, and elevating AKT serine/threonine kinase 1 (AKT), forkhead box O3 (FOXO3a), cyclin-dependent kinase inhibitor (p27), Caspase-3 and -9, cyclin-dependent kinase inhibitor 1A (p21), and apoptosis, and inhibiting PI3K/AKT/FOXO3a pathway ^[42][44]. Nanoformulations of eugenol have been reported to possess enhanced chemotherapeutic potential. Chitosan nanoparticles containing eugenol showed a good chemopreventive role against human breast cancer cell lines, MDA-MB 468 (IC₅₀: 51 μg/mL) and melanoma cell lines, A-375 (IC₅₀: 79 μg/mL) than their non-formulated states ^[43][45]. Magnetic field and pH-sensitive targeted drug delivery systems were developed by a group of scientists. They encapsulate eugenol and hesperidin in folic acid-conjugated bovine serum albumin and superparamagnetic calcium ferrite nanohybrids to enhance selective targeting of the hybrid nanoparticles to folate receptor overexpressed breast cancer cells with an encapsulation efficiency of 85.58% and 62.94%, respectively. The in vitro studies on MCF-7 breast cancer cell lines have indicated the reduction in IC₅₀ values by 20–30-fold for both of the herbal constituents (eugenol, IC₅₀ from 36.27 to 8.75 μg/mL and hesperidin, IC₅₀ from 39.72 to 9.08 μg/mL) ^[27][29].

3.2. Effect of Eugenol on Cervical Cancer

Eugenol is explored for its activities on cervical cancer and is reported to have a promising effect. The combination of methyl eugenol (60 µM) with myricetin (60 µM) synergistically enhanced the cancer cell growth inhibition of cisplatin (1 µM) by inducing strong apoptosis, arresting cells in the G₀/G₁ phase of the cell cycle, enhancing ΔΨm, and upregulating caspase-3 activity in the HeLa immortal cervical cell lines ^[44][46]. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay, flow cytometry analysis, and dual staining with acridine orange indicated cytotoxicity and apoptosis leading to inhibition of cell proliferation in Henrietta Lacks (HeLa) cervical cell lines with no apoptosis in the controls and normal cells treated with dichloromethane extract of eugenol isolated from Syzygium aromaticum ^[45][47]. A study showed that eugenol (150 µM) increased the antiproliferative and apoptotic capability of gemcitabine (15–25 mM) in a dose-dependent manner in tested HeLa cervical cell lines in comparison to normal cells. The analysis of genes expressed in the inflammatory process and apoptosis signaling showed downregulation of IL-1β, Bcl-2, and cyclooxygenase-2 (COX-2); indicating mediation of an anticancer effect by eugenol via apoptosis induction and inhibiting inflammation ^[22][24]. Another study showed that a synergistic increase in cancer inhibition was observed for the combination treatment of eugenol (350 µM), cisplatin (0.5–2.5 µM), and X-rays (4–6 Gy) in HeLa cervical cancer cell lines in a time- and concentration-dependent way. Increased expressions of Bax, caspase-3 and -9, cytochrome c, and reduced expressions of COX2 and IL-1β unleashed the fact that eugenol causes death via apoptosis and mediating anti-inflammatory action ^[46][48]. A comparative study of eight phenylpropanoids in combination with 5-fluorouracil (10.5 µM) illustrated eugenol (153 µM) as the most effective entity in minimizing drug-induced toxicity and resistance in normal and cancer cells, respectively. S phase and G₂-M phase arrest and induction of apoptosis were proposed mechanisms for eugenol’s antiproliferative activity. The combination therapy increased the cell numbers in the G₀/G₁ and G₂/M phase significantly, and the sub-G₁ phase as well. Involvement of p53, upregulation of caspase-3, dissipation of MMP9, PARP cleavage, etc. are reported in the apoptotic process. In vitro hemolytic activity studies confirm eugenol as less toxic to normal cells ^[47][49]. A combination of sulforaphane (6.5–8 µM) and eugenol (200–350 µM) produced an antagonistic and synergistic effect in low and higher doses, respectively. The combination produced synergistic cytotoxicity with gemcitabine (25 mM) at higher sublethal doses as well as in vitro. LD₅₀ of EUG and sulforaphane were found to be at 500 μM and 12 μM, respectively. Lowering the expressions of IL-β, Bcl-2, and COX-2 were major findings of western blot and RT–PCR studies ^[48][50]. Eugenol (50–200 µM) exhibited inhibition of HeLa cell migration. At a maximum concentration of eugenol (200 μM), the migratory rate was reduced by 3.38 ± 1.2 times compared to the control group. Furthermore, it was also discovered that the protein expression of vimentin and Snail-1 was downregulated, whereas that of E-cadherin was upregulated ^[49][51].

3.3. Effect of Eugenol on Colorectal Cancers

Eugenol induced apoptosis, necrosis, and slowing cell cycle in SW-620 and CACO-2 colon cancer cells after 72 h of treatment, but not in NCM-460 normal cell lines ^[50][52]. Eugenol inhibited the mRNA expression of the COX-2 enzyme, a major catalyst of prostaglandin synthesis, specifically PGE2, that plays a key function in producing inflammation and inducing colon carcinogenesis. The LPS-stimulated mouse macrophage HT-29 and RAW264.7 colon cancer cells showed decreased expressions of COX-2 ^[51][53]. The human colorectal carcinoma cell lines (HCT-116) showed signs of autophagy and apoptosis after treatment with an active fraction of clove which predominantly contains eugenol. The increased expressions of proteins LC3-II (microtubule-associated protein 1A/1B-light chain 3) and beclin-1 confirmed that eugenol induces autophagy in the colon cancer cells inhibiting their proliferation. Further, the induction of autophagy was synergistically increased following the combination treatment of 3-methyladenine and bafilomycin A1 as autophagy inhibitors with eugenol ^[52][54]. Eugenol induced apoptosis in HCT-15 and HT29 human colon cancer cell lines with IC₅₀ values of 300 and 500 µM, respectively, via MMP dissipation, activation of caspase-3 and PARP, and upregulation of the p53 tumor suppressor gene. The formation of ROS potentiated apoptotic action by augmenting higher deoxyribonucleic acid (DNA) fragmentation ^[53][55]. Eugenol-canola oil or Eugenol-medium chain triglyceride nanoemulsions were taken at a concentration of 750 µM (eugenol) to evaluate the anticancer effect against HTB37 cells; where apoptotic cell death has been observed via ROS generation, cell cycle was arrested at sub G₁/S phase ^[54][56]. Eugenol along with 4-trifluoromethyl benzoic acid (TFBA) was shown to increase cytotoxicity at concentrations of 20.7 and 20.1 µM, respectively, in HCT116 and WiDr cell lines, demonstrating anticancer action ^[55][57].

3.4. Effect of Eugenol on Gastric Cancers

In vivo studies in N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) that induced the rat gastric carcinogenesis model depicted the anti-proliferative and apoptotic activity of eugenol ^[56][57][58,59]. The suggested molecular mechanisms are involved in apoptosis induction, cell cycle arrest, inhibiting angiogenesis, and preventing metastasis progression. The increased expressions of p53, pro-apoptotic proteins, and cell cycle regulatory (inhibitory genes) advocated for the strong apoptotic potential of eugenol. The levels of anti-apoptotic proteins were found to be decreased. The downregulation of the NF-κB family of transcription factors was also profound. The decreased expressions of the matrix metalloproteinase (MMP-2 and MMP-9) with increased levels of tissue inhibitor of metalloproteinases 2 (TIMP-2) gene expression were correlated with the anti-angiogenic potential of eugenol. The anti-apoptotic Bcl-2 protein is upregulated while the pro-apoptotic Bax and caspase-3 proteins have been downregulated after eugenol therapy. The expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), and MMPs are all reduced by eugenol, while that of reversion-inducing-cysteine-rich protein with kazal motifs (RECK) and TIMP-2 is increased, resulting in apoptosis and a decrease in invasion and angiogenesis ^[57][59]. EUG inhibits cell proliferation, suppresses NF-κB, promotes cyclin B, cyclin D1, and PCNA expression, and also inhibits expression of the growth arrest and DNA damage-inducible 45 (Gadd45), p53, and p21 proteins in the MNNG-induced male Wistar rat gastric carcinogenesis model ^[56][58]. A comparative anticancer study involving capsaicin and eugenol indicated that eugenol can induce apoptosis and inhibit proliferation in human gastric carcinoma cell lines (AGS cells) and was independent of p53 which enhances caspase-8 and caspase-3 expression. In contrast, the apoptotic activity of capsaicin was p53-dependent, and capsaicin-induced the expression of proapoptotic proteins (Bax, caspase-3, and caspase-8) ^[58][60].

3.5. Effect of Eugenol on Lung Cancer

A study showed that aqueous infusion of clove effectively reduced lung cancer in strain A mice induced by benzo[a]pyrene. The clove infusion administered orally at a dose of 100 mL/mouse/day from the fifth week of benzo[a]pyrene administration and continued up to the 26th week was found to play a potential chemopreventive role due to its apoptogenic and anti-proliferative activities. Further studies showed the upregulation of the expression of pro-apoptotic p53 and Bax proteins and the downregulation of the expression of antiapoptotic Bcl-2 protein in the precancerous stages. Moreover, activation of caspase-3 by clove infusion was manifested from a very early stage of cancer. Clove infusion downregulated the expression of some growth-promoting proteins such as COX-2, Hras, and cMyc ^[59][61]. Studies on MRC-5 human embryonic lung fibroblast cells and A549 lung adenocarcinoma cells treated with eugenol (800 µM and 400 µM, respectively) in vitro demonstrated inhibition in cell viability, migration, and invasion. Biochemical findings demonstrated the antiproliferative and antimetastatic effect of eugenol by reducing the PI3/AKT pathway and reduction of MMP-2 ^[60][62]. The therapy with eugenol considerably reduced the growth of the xenograft tumor and significantly increased the overall survival rate of tumor-bearing mice. On a mechanistic level, eugenol was able to inhibit the expression of p65, which in turn led to a reduction in the expression of tripartite motif-containing protein 59 (TRIM59) protein. The antitumor phenotype induced by eugenol was completely reproduced by the absence of TRIM59 in the cells. It was shown that TRIM59 plays a predominant role in modulating the signaling that occurs downstream of eugenol therapy. Ectopic expression of TRIM59 was eliminated as a result of the tumor suppressive impact of eugenol. Through the suppression of the NF-κB-TRIM59 pathway, eugenol was able to inhibit non-small cell lung cancer ^[61][63]. The chemopreventive and antiproliferative role of eugenol utilizing an in vivo mice model has been studied. Downregulation of the WNT/beta-catenin signaling pathway was attributed to the anticancer role of eugenol. The decreased expressions of β-catenin-dependent cancer stem cell markers (CD44, EpCAM, Notch 1, and Oct4) advocated for the inhibition of metastatic progression ^[62][64]. The 1, 2, 3-triazole-isoxazoline derivatives of eugenol, when tested against A549 cells, showed anti-proliferative activity at 17.32–25.4 µM concentration ^[63][65].