Antitumor Phenylpropanoids Found in Essential Oils

The search for new bioactive substances with anticancer activity and the understanding of their mechanisms of action are high-priorities in the research effort toward more effective treatments for cancer. The phenylpropanoids are natural products found in many aromatic and medicinal plants, food, and essential oils. They exhibit various pharmacological activities and have applications in the pharmaceutical industry. In this review, the anticancer potential of 17 phenylpropanoids and derivatives from essential oils is discussed. Chemical structures, experimental report, and mechanisms of action of bioactive substances are presented.


Introduction
Cancer is a global health concern that causes mortality in both children and adults. More than 100 distinct types and subtypes of cancer can be found within specific organs [1]. Despite the success of several cancer therapies, an ideal anticancer drug has not been discovered, and numerous side effects limit treatment. However, research into new drugs has revealed a variety of new chemical structures and potent biological activities that are of interest in the context of cancer treatment.
Essential oils are natural products that are a mixture of volatile lipophilic substances. The chemical composition of essential oils includes monoterpenes, sesquiterpenes, and phenylpropanoids, which are usually oxidized in an aliphatic chain or aromatic ring. Several studies have shown that this chemical class has several biological activities, including analgesic, anticonvulsant, and anti-inflammatory effects [2][3][4]. Antitumor activity has been reported for essential oils against several tumor cell lines [5][6][7], and these oils contain a high percentage of phenylpropanoids, which are believed to contribute to their pharmacological activity [8,9]. This paper presents a literature review of phenylpropanoids from essential oils with respect to antitumor activity, with chemical structures and names of bioactive compounds provided. The phenylpropanoids presented in this review were selected on the basis of effects shown in specific experimental models for evaluation of antitumor activity and/or by complementary studies aimed at elucidating mechanisms of action ( Table 1). The selection of essential oil constituents in the database was related to various terms, including essential oils and phenylpropanoids, as well as names of representative compounds of chemical groups, and refined with respect to antitumor activity, cytotoxic activity, and cytotoxicity. The search was performed using scientific literature databases and Chemical Abstracts Service (CAS) in November 2013.
Synergistic effects have been demonstrated for eugenol with gemcitabine and fluorouracil, which potentiated its cytotoxic effect on HeLa cells (human cervical carcinoma) [19,20,38]. Eugenol also significantly decreased expression of Bcl-2, COX2, and IL-1b in the HeLa cell line [20]. Atsumi and collaborators [39] demonstrated that the effects of eugenol on ROS production were biphasic, with production enhanced at lower eugenol concentrations (5-10 M) and inhibited at higher concentrations (500 M). Suzuki and collaborators [40] demonstrated that eugenol stimulated production of superoxide (O 2 − ) free radicals in guinea pig neutrophils without lag time.
Eugenol halts cells in the replication phase, suggesting that cells stop to repair DNA damage and either reenter the cell cycle or, in cases of massive DNA damage, activate apoptosis. Melanoma cells treated with eugenol remain in the S phase and undergo apoptosis, and eugenol treatment upregulates numerous enzymes involved in the base excision repair pathway, including E2F family members [15].
In another study, eugenol at higher doses induced chromosomal aberrations, with significant increases (3.5%) in aberrant cells at a concentration of 2500 M in V79 cells (Chinese hamster lung fibroblast). Eugenol was also assayed for genotoxic activity via inhibition of topoisomerase II and showed dose-dependent inhibition [41].
The chemopreventive potential of eugenol was also studied [10]. Using in vivo methods, Pal and collaborators [10] showed that eugenol inhibits skin carcinogenesis induced by dimethylbenz[a]anthracene (DMBA) croton oil in mice, probably due to inhibition of proliferation-associated genes (c-Myc and H-ras) and antiapoptotic gene Bcl2, along with upregulation of proapoptotic genes Bax, p53, and active caspase-3 [10]. Kaur and collaborators [42] studied the chemopreventive effect of eugenol in DMBA/TPA-induced carcinogenesis in murine skin. They showed that topical application of eugenol resulted in a marked decline in hyperplasia, epidermal ODC activity, protein expression of iNOS and COX-2, and secretion of proinflammatory cytokines, all of which are classical markers of inflammation and tumor promotion [42]. In addition, eugenol has been shown to produce antioxidant effects via free radical scavenging activity and reduction of ROS [22,36,43]. Atsumi and collaborators [36] showed that visible-light irradiation and elevation of the pH of the eugenol-containing medium resulted in significantly lower cell survival in HSG cultures in comparison with eugenol alone.
In vivo murine assays have also demonstrated the antitumor potential of eugenol. Treatment of female B6D2F1 mice bearing B16 melanoma allografts with 125 mg/kg of eugenol resulted in a small, but highly significant ( = 0.0057), 2.4day tumor growth delay. Furthermore, the treated animals had no fatalities that were attributed to metastasis or tumor invasion, which is indicative of the ability of eugenol to suppress melanoma metastasis [15]. Jaganathan and collaborators [44] also demonstrated the antitumor potential of eugenol using an in vivo assay, in which a dose of 100 mg/kg caused 24.35% tumor growth inhibition and inhibited the growth of Ehrlich ascites by 28.88%. In contrast, Tangke Arung and collaborators [45] showed that 100 g/mL eugenol inhibited melanin formation by more than 42% in the B16 melanoma cell line in vitro, with cytotoxicity in 5% of cells. At a higher concentration of 200 g/mL 23% cytotoxicity was observed, which demonstrated that eugenol could be useful as a skinwhitening agent for the treatment of hyperpigmentation [45].
Furthermore, it has been demonstrated that eugenol, when mixed with zinc oxide, has a restorative effect on dental erosion and demineralization [46]. Using human dental pulp cells (D824) it was observed that eugenol had a cytotoxic effect, with reduction of cell growth and inhibition of colonyforming cell [35]. D824 cells have the potential for metabolic activation, because they are a mixed-cell population composed of many types of cells, and thus the cytotoxic activity of eugenol could be attributable to eugenol metabolites. However, Marya and collaborators [46] showed a hemolytic effect of eugenol, which could be a possible side effect of this drug. In addition, Anpo and collaborators [35] showed that eugenol reduced growth and survival of human dental pulp cells, as well as collagen synthesis and bone sialoprotein (BSP) expression, which play a critical role in physiological and reparative dentinogenesis. Eugenol is a phenylpropanoid with promising antitumor drug profile. Further studies to elucidate the mechanisms that mediate the adverse effects of eugenol are necessary.

Methyleugenol, Isoeugenol, Methylisoeugenol, and 1 -Hydroxymethyleugenol.
Methyleugenol is a substituted alkenylbenzene found in a variety of foods and essential oils. It is structurally similar to eugenol and found in many plant species [47]. Methyleugenol produced cytotoxic effects in rat and mouse hepatocytes [47,48] and leukemia [48]. Methyleugenol also produced genotoxicity in mice [47] and in cultured cells [49] and caused neoplastic lesions in the livers of Fischer 344 rats and B6C3F1 mice [47].
Isoeugenol is a phenylpropanoid produced by plants. As a flavoring agent, isoeugenol is added to nonalcoholic drinks, baked foods, and chewing gums. In male F344/N rats,  ESR analysis Activity of the production of phenoxyl radicals with most efficiently scavenged reactive oxygen HSG cells [29] Laser cytometry analysis Production of ROS induced by VL-irradiated is significantly affected by pH

Cormation of dimers
Dichlorofluorescein assay Reduction in the intracellular level of GSH [39] 6 BioMed Research International  Boyden-chamber assay  Induction of skin papillomas Carcinogenic in the induction of skin papillomas CD-1 mice

TNF--induced apoptosis
In vivo assay Anticancer effects with no toxic effects NOD/SCID mouse isoeugenol showed carcinogenic effects, causing increased incidence of rarely occurring thymoma and mammary gland carcinoma. There was no evidence of carcinogenic activity due to isoeugenol in female F344/N rats. However, there was clear evidence of carcinogenic activity due to isoeugenol in male B6C3F1 mice, including increased incidence of hepatocellular adenoma, hepatocellular carcinoma, and hepatocellular adenoma with carcinoma. Carcinogenic activity due to isoeugenol in female B6C3F1 mice was observed in the form of increased incidence of histiocytic sarcoma. Exposure to isoeugenol resulted in nonneoplastic lesions of the nose in male and female rats, of the kidney in female mice, and of the nose, forestomach, and glandular stomach in mice of both sexes [50]. However, methyleugenol is minimally cytotoxic for hepatocytes and leukemia cells compared to eugenol [48,49]. The structural similarity of these substances with eugenol stimulates advances in pharmacological studies to explore their therapeutic potential in cancer treatment.
Fan and collaborators [58] showed that safrole promoted the activities of macrophages and NK cells in BALB/c mice. While promoting macrophage phagocytosis, safrole increased abundance of cell markers such as CD11b and Mac-3. Additionally, NK cell cytotoxicity was remarkably suppressed in mice treated with safrole, as were levels of cell markers for T cells (CD3) and B cells (CD19). Safrole was also cytotoxic against primary human buccal mucosal fibroblasts (BMFs) [52]. Ni and collaborators [52] demonstrated that safrole increased NF-B expression, which may have been involved in the pathogenesis of oral submucous fibrosis. NF-B expression induced by safrole in fibroblasts may be mediated by ERK activation and the COX-2 signal transduction pathway.
A study by Chang and collaborators [53] investigated the effect of safrole on intracellular Ca 2+ mobilization and viability of human PC3 prostate cancer cells. Cytosolic free Ca 2+ levels ([Ca 2+ ] i ) were measured using fura-2 as a probe. Safrole increased [Ca 2+ ] i by causing Ca 2+ release from the endoplasmic reticulum in a phospholipase C-and protein kinase C-independent manner, which decreased cell viability in a concentration-dependent manner. In HL-60 leukemia cells, safrole promoted the expression of glucose-regulated protein 78 (GRP78), growth arrest-and DNA damageinducible gene 153 (GADD153), and activating transcription factor 6 (ATF-6 ) [51]. In the unscheduled DNA synthesis (UDS) assay described by Howes and collaborators [55], safrole exhibited genotoxic activity in freshly isolated rat hepatocyte primary cultures.
Safrole-2 ,3 -oxide (SAFO) is a reactive electrophilic metabolite of safrole. SAFO is the most mutagenic metabolite of safrole that has been tested in the Ames test, but data on the genotoxicity of SAFO in mammalian systems is scarce. SAFO induced cytotoxicity, DNA strand breakage, and micronuclei formation in human cells in vitro and in mice [56]. In addition, safrole produced mutagenicity in Salmonella TA 98 and TA 100 in the Ames test [59].
Myristicin (1-allyl-3,4-methylenedioxy-5-methoxybenzene) is an active constituent of nutmeg, parsley, and carrot. A study by Lee and collaborators [60] investigated the cytotoxic and apoptotic effects of myristicin on human neuroblastoma SK-N-SH cells. Apoptosis triggered by myristicin was caused by cleavage of PARP, which was accompanied by accumulation of cytochrome c and activation of caspase-3. These results suggested that myristicin induced cytotoxicity in human neuroblastoma SK-N-SH cells by an apoptotic mechanism [60].
Ahmad and collaborators [61] investigated the effect of myristicin on activity of glutathione S-transferase (GST) and NADPH:quinone oxidoreductase (QR) in four mouse strains. The authors showed that activity of GST and QR was significantly increased in the livers of all four mouse strains, GST activity was increased in the intestine of three out of four strains, and QR activity was significantly increased in the lungs and stomachs of three out of four stains. Thus myristicin, which is found in a wide variety of herbs and vegetables, shows strong potential as an effective chemoprotective agent against cancer.
Safrole, safrole-2 ,3 -oxide, and myristicin are bioactive substances in antitumor models that can be used as starting materials for the preparation of derivatives with improved pharmacological profile.

Estragole, Anethole, and trans-Anethole Oxide.
Estragole has been isolated from essential oils of Artemisia dracunculus and Leonotis ocymifolia. Howes and collaborators [55] demonstrated the genotoxic activity of estragole via UDS assay, in which estragole induced dose-dependent increases in UDS up to 2.7 times that of the control in rat hepatocytes in primary culture.
Chainy and collaborators [66] reported that anethole reduced apoptosis by inhibiting induction of NF-B, activator protein 1 (AP-1), c-jun N-terminal kinase (JNK), and mitogen-activated protein kinase kinase (MAPKK) by tumor necrosis factor (TNF). Choo and collaborators investigated the antimetastatic activity of anethole [63] and showed that anethole inhibited proliferation, adhesion, and invasion of highly metastatic human HT-1080 fibrosarcoma cells. Anethole also inhibited the activity of metalloproteinases (MMP-2 and MMP-9) and increased the activity of MMP inhibitor TIMP-1 [63]. Nakagawa and Suzuki [62] showed that anethole induced a concentration-and time-dependent loss of cell viability in isolated rat hepatocytes, which was followed by decreases in intracellular levels of ATP and total adenine nucleotide pools. Howes and collaborators [55] demonstrated that anethole did not induce unscheduled DNA synthesis (UDS) in rat hepatocytes in primary culture. In Ehrlich ascites tumor-bearing mice,anethole increased survival time and reduced tumor weight, tumor volume, and body weight [65].
Anethole is metabolized through 3 pathways: O-demethylation, -hydroxylation followed by side chain oxidation, and epoxidation of the 1,2-double bond. The cytotoxicity of trans-anethole oxide in rat hepatocytes has been shown to be due to its metabolism to epoxide [67]. In addition, transanethole oxide produced a positive result in the Salmonella mutation assay and induced tumors in mice. These results suggest that epoxidation of the side chain of anethole in vivo could be a carcinogenic metabolic mechanism. Kim and collaborators [67] found that trans-anethole oxide is more toxic to animals than trans-anethole and was mutagenic in point mutation and frameshift mutation Ames test models. trans-Anethole did not induce hepatomas in male B6C3F1 mice, but the highest dose of trans-anethole oxide tested (0.5 mol/g) significantly increased the incidence of hepatomas.
Ng and Wu [80] showed that cinnamaldehyde induced lipid peroxidation in hepatocytes isolated from male Sprague-Dawley rats with glutathione depletion. Adding NADH generators, for example, xylitol, prevented cytotoxicity induced by cinnamaldehyde, but decreasing mitochondrial NAD + with rotenone markedly increased cinnamaldehyde cytotoxicity. The authors showed that cinnamaldehyde-induced cytotoxicity and inhibition of mitochondrial respiration were markedly increased by ALDH inhibitors and in particular by cyanamide [80].
Chew and collaborators [78] used flow cytometric analysis to show that 80 M of cinnamaldehyde caused cell cycle arrest at the G 2 /M phase in HCT 116 cells and induced cleavage of caspase-3 and PARP. It has also been proposed that cinnamaldehyde induced apoptosis by ROS release with TrxRinhibitory and Nrf2-inducing properties [78]. Ka and collaborators [71] demonstrated that cinnamaldehyde induced ROS-mediated mitochondrial permeability and cytochrome c release in human leukemia cells (HL-60).
Using hepatoma cells, Wu and collaborators [81] demonstrated that cinnamaldehyde upregulated Bax protein, downregulated Bcl-2 and Mcl-1, and caused Bid to cleave upon the activation of caspase-8. These events consequently led to cell death. JNK, p38, and ERK were activated and phosphorylated after cinnamaldehyde treatment in a time-dependent manner, which suggested that apoptosis was mediated by activation of proapoptotic Bcl-2 family (Bax and Bid) proteins and MAPK pathways [81]. Cinnamaldehyde can also activate TRPA1 expression in melanoma cells [77].
Cinnamaldehyde caused a time-dependent increase in CD95 (APO-1/CD95) protein expression in HepG2 cells (human hepatoma), while also downregulating antiapoptotic proteins (Bcl-XL) and upregulating proapoptotic (Bax) proteins in a time-dependent manner [80]. Preincubation of HepG2 cells with cinnamaldehyde effectively inhibited the expression of Bax, p53, and CD95, as well as the cleavage of PARP. This pretreatment also prevented downregulation of Bcl-XL [80]. Using the HepG2 and Hep3B human hepatoma cancer cell lines, Chuang and colleagues [88] demonstrated that cinnamaldehyde had a potent inhibitory effect against human hepatoma cell growth. They observed that the JAK2-STAT3/STAT5 pathway might be an important target of cinnamaldehyde. Cinnamaldehyde also altered apoptotic signaling. Cinnamaldehyde significantly decreased protein levels of cyclin D1 and proliferative cell nuclear antigen (PCNA) but increased the protein levels of p27 Kip1 and p21 Waf1/Cip1 [86]. In an assay of thioredoxin reductase (TrxR) action, cinnamaldehyde showed a TrxR inactivation effect that could contribute to its cytotoxicity [89]. Furthermore, cinnamaldehyde had an antitumor effect in Sarcoma 180bearing BALB/c mice and a protective effect on immune function [89].
2 -Hydroxycinnamaldehyde, a cinnamaldehyde derivative, was studied for its immunomodulatory effects. The chemopreventive effects of cinnamaldehyde derivatives were demonstrated on hepatocellular carcinoma formation in H-ras12V transgenic mice, where they probably produced a long-term immunostimulating effect on T cells, because immune cell infiltration into hepatic tissues was increased [90].
2 -Hydroxycinnamaldehyde has immunomodulatory effects in vivo, but in vitro studies showed that secreted IgM level was depressed in the culture supernatants of splenocytes. Decreased IgM produced by cinnamaldehyde treatment in vitro appeared to be due to lower levels of B-cell proliferation, rather than direct inhibition of IgM production [74]. Koh and collaborators [74] also demonstrated that cinnamaldehyde induced T-cell differentiation from CD4CD8 double positive cells to CD4 or CD8 single positive cells.
Cinnamic acid had an inhibitory effect on uterocervical carcinoma (U14) cells in mice, causing tumor cell apoptosis [92]. In vitro assay of U14 cells demonstrated a shortened G 2 -M period, lengthened cell cycle, and inhibited cell proliferation, which supported the conclusion that cinnamic acid influenced tumor cell cycle [92].
Ekmekcioglu and collaborators [79] showed that cinnamic acid inhibited proliferation and DNA synthesis of Caco-2 (human colon) cells. Treatment with cinnamic acid modulated the Caco-2 cell phenotype by dose-dependently stimulating sucrase and aminopeptidase N activity, while inhibiting alkaline phosphatase activity. In melanoma cells cinnamic acid induced cell differentiation with morphological changes and increased melanin production. Cinnamic acid reduced the invasive capacity of melanoma cells and modulated expression of genes implicated in tumor metastasis (collagenase type IV and tissue inhibitor metalloproteinase 2) and immunogenicity (HLA-A3, class-I major histocompatibility antigen) [94].
Using in vivo and in vitro assays, Zhang and collaborators (2010) [92] showed that cinnamic acid influenced the cell cycle of uterocervical carcinoma cells (U14); the G 2 -M period was shortened, cell cycle was lengthened, and cell proliferation was inhibited. Cinnamic acid also induced differentiation of human osteogenic sarcoma cells and caused a higher percentage of cells in S phase [95].

2.7.
Hydroxychavicol and 1 -Acetoxychavicol Acetate. Hydroxychavicol (1-allyl-3,4-dihydroxybenzene) is a major component in Piper betle leaf, which is used for betel quid chewing in Asia, and is also a major metabolite of safrole, which is the main component of sassafras oil, in rats and humans. A study by Nakagawa and collaborators [54] demonstrated the biotransformation and cytotoxic effects of hydroxychavicol in freshly-isolated rat hepatocytes. In hepatocytes pretreated with diethyl maleate or salicylamide, hydroxychavicol-induced cytotoxicity was enhanced and was accompanied by a decrease in the formation of conjugates and inhibition of hydroxychavicol loss.
Other studies indicate that mitochondria are the target organelles for hydroxychavicol, which induces cytotoxicity through mitochondrial failure related to mitochondrial membrane potential at an early stage, and lipid peroxidation through oxidative stress at a later stage. Furthermore, the onset of cytotoxicity depends on the initial and residual concentrations of hydroxychavicol, rather than its metabolites.
1 -Acetoxychavicol acetate is obtained from the rhizomes of Languas galanga (Zingiberaceae), a traditional condiment in Thailand. Recent studies have revealed that 1acetoxychavicol acetate has potent chemopreventive effects against rat oral carcinomas and inhibits chemically induced tumor formation and cellular growth of cancer cells. 1 -Acetoxychavicol acetate inhibited NF-B and induced apoptosis of myeloma cells in vitro and in vivo. Therefore, 1acetoxychavicol acetate is a novel NF-B inhibitor and represents a new therapy for the treatment of multiple myeloma patients [99]. The isolation and identification of 1acetoxychavicol acetate, an inhibitor of xanthine oxidase, may induce antitumor activity by inhibiting generation of anions during tumor promotion [100] (Figure 1).

Conclusions
The studies presented in this review reveal the anticancer therapeutic potential of bioactive constituents found in essential oils and medicinal plants, the phenylpropanoids. The research on the clinical studies of these natural products is required to the development of new drug candidates with applications in the therapy of cancer.