Sixteen guaianolides have been identified in C. intybus L. (Fig. 1A) (21–33) of which only two, 13-dihydro-8-deoxylactucin (jacquilenin) and 11,13-dihydrolactucopicrin, have chemotherapeutic properties. Both were isolated from a fractionated ethanol extract of leaves through a combination of column, thin layer chromatography, and HPLC (23). Leclercq (21) and Van Beek et al (27) also purified 11,13-dihydrolactucopicrin from C. intybus root methanolic extract. Jacquilenin showed inhibitory activity on the induction of ICAM-1 induced by IL-1α and TNF-α in alveolar basal epithelial adenocarcinoma cells (A549; IC₅₀ values of 16.1 and 20.1 µM for IL-1α and TNF-α, respectively) and cytotoxicity against human lung fibroblast cells (WI38 and VA13; IC₅₀ values of 2.7 and 8.5 µM, respectively) and hepatocellular carcinoma cells (HepG2; IC₅₀ of 25 µM) in vitro (34). Isolated 11,13-dihydrolactucopicrin from Mulgedium tatarica by Ren et al (35) demonstrated antitumor activity in human nasopharyngeal cancer cells (KB; IC₅₀ of 22 µM) and human liver cancer cells (Bel 7402; IC₅₀ of 30 µM).

Structural activity relationship (SAR) studies by Ren et al (35) revealed that the position 8 ester group (γ-butyrolactone) and the methylene group at exocyclic position 11 (α) (Fig. 1A) play a major role in antitumor activities of lactucin-like guaianolides (35). α-methylene-γ-lactone, the ‘-enone’ or unsaturated carbonyl (O=C-C=CH₂) system was found to increase the toxicity towards tumor cells (36). Both reported chemoprotective guaianolides in C. intybus (Jacquilenin and 11,13-dihydrolactucopicrin) share these features, confirming the structural basis of the activity. Further studies have shown that this structural motif works as a monofunctional alkylate and selectively deactivates the p65 dimer of NF-κB preventing NF-κB transcription in treated cells (37,38). This targeting of specific signaling pathways is also observed with other metabolites.

Polyacetylenes. C. intybus contains five polyacetylenes (Fig. 1B) (21,23,26,31,39,40). Putrescine and spermidine extracted from leaves by Krebsky et al (39) show potency as antitumor agents (41–45). Putrescine is the precursor of spermidine, and both are ubiquitous constituents of eukaryotic cells (41). Their cellular concentration is associated with the effectiveness of many anticancer therapeutics. The in vitro antitumor activity of putrescine has been demonstrated in breast cancer cell line MDA-MB-231 (IC₅₀ of 110 µg/ml) (43,45), and it showed antiproliferative effect on T-47D breast cancer cells (0.1 mM) (44). Inhibition of proliferation of human alveolar basal epithelial adenocarcinoma cells (A549; IC₅₀ of 4.54 µg/ml), prostate adenocarcinoma cells (LNCaP; IC₅₀ of 1.1 µg/ml), breast epithelial cancer cells (T47D; IC₅₀ of 0.97 µg/ml), bone osteosarcoma cells (U2-OS, IC₅₀ of 4.47 µg/ml), HeLa cells, skin fibroblast cells (Malme-3M), prostate cancer cells (PC-3) and embryonic kidney cells (HEK-293) was demonstrated by Cheng et al (42). Putrescine and spermidine were found to be able to cross the cell membrane by a single unique channel or by separate ones (41). The higher lipophilicity of polyacetylene compounds facilitates plasma membrane penetration but often lowers their bioavailability in vivo (36). A comparison between different plant-derived polyacetylenes by Kinjo et al (46) suggested an inverse relationship of the presence of bulky side chains (hydroxy, methoxy, amino or other groups) with activity and effectiveness. The authors also found that C. intybus polyacetylenes were more effective against MK-1 cells than HeLa and B16F10 cells, and their antiproliferative effects were more profound than other chemoprotective characteristics (46). In estrogen-responsive breast cancer cells (ZR-75-1), both putrescine and spermidine were internalized via the same or similar transporter and both V_max was rapidly upregulated by estrogens and insulin (47). The presence of positively charged primary and secondary amino groups (at physiologic pH) and hydrophobic methylene bridging groups indicate their capability of acting as ligands at multiple locations of DNA, RNA, proteins, phospholipids and nucleotide triphosphate. Few of these connections are electrostatic and replaced easily by inorganic cations. The rest are specific to the extent of the aliphatic carbon chain (Fig. 1B) (48,49). Although polyamines are critical for cell proliferation, their high concentration can lead to interruption in transcriptions and protein-protein interactions, and thus inhibition of tumor growth.

Phytometabolites containing a 3,4-dihydroxyphenethyl group (Fig. 1C) are the most frequent type of metabolites found in C. intybus. Furthermore, 19 of the 28 phytometabolites (5,24,50–52) were found to exhibit antitumor properties. Major members of this group include hydroxycinnamic acids, quercetins, kaempferols, and cyanidins, among others.

Among the hydroxycinnamic acids, chlorogenic acid and caffeic acid have both been extracted from C. intybus hairy root (Agrobacterium rhizogenes-induced) culture (24) and leaves [MeOH-HCOOH (99:1, v/v) extract] (5,24,51). Caffeic acid is highly effective against human promyelocytic leukemia cells (HL-60; 90% inhibition at 10 µM) (53,54), mammary duct carcinoma cells (T-47D; IC₅₀ of 2.17×10⁻⁹ M) (55) and liver carcinoma cells (Hep3B) (56). It also demonstrated high antioxidant as well as anti-inflammatory activity (57). Caffeic acid was found to be moderately effective against epidermal DNA synthesis, epidermal ornithine decarboxylase activity, and skin tumors induced by 12-O-tetradecanoylphorbol-13-acetate (TPA; IC₅₀ of 72.3 µM) (58). Chlorogenic acid showed in vivo inhibitory effects on 8-hydroxydeoxyguanosine (8-OH-dG) formation induced by lipid peroxides in 4-nitroquinoline-1-oxide (an oxygen radical-forming carcinogen)-treated animal tongue, but not the endogenous 8-OH-dG (59). Chlorogenic acid was found to protect against environmental carcinogen-induced carcinogenesis (60). An in vitro study also demonstrated the antiviral potency of chlorogenic acid in human hepatoblastoma cells (Hep-G2.2.15; IC₅₀ >1,000 µM) (61), antitumor potency against mouse preadipocyte cells (3T3-L1; IC₅₀ of 72.3 µM) (62,63), and increased insulin secretion in rat insulinoma cells (INS-1E) (64). Caffeic acid contains both phenolic (Fig. 1I) and acrylic functional groups. The amount of absorption in the small intestine (most of the caffeic acid and one-third of the chlorogenic acid) indicates that most chlorogenic acid reaches the colon and only a fraction enters the blood circulation (65).

5-Caffeoylquinic acid, 5-caffeoylshikimic acid, di-caffeoyl tartaric acid (also called chicoric acid), trans-caftaric acid and 4-O-feruloylquinic acid have been purified from C. intybus leave MeOH-HCOOH (99:1, v/v) extract (5). A previous study showed that 5-caffeoylquinic acid inhibited non-small cell lung cancer cell (H1299) invasion (66). Trans-caftaric acid has antioxidant properties and protects against DNA damage caused by ROS. A trans-caftaric acid rich extract was found to demonstrate cytotoxicity in HepG2 (liver cancer cells; IC₅₀ of 50±12 µg/ml) and HeLa cells (IC₅₀ of 32±16 µg/ml) (67). 5-Caffeoylshikimic acid displayed antioxidant and antitumor properties on rat skeletal myoblast (L6; IC₅₀ of 90 µg/ml) cells (68). A 4-O-feruloylquinic acid rich extract from Oplopanax horridus (Sm.) Miq. exhibited anti-proliferative effects against human colon adenocarcinoma cells (HT-29; 56.5% inhibition with a 0.2 mg/ml extract) (69). Chicoric acid, in synergy with luteolin was found to act as an anti-oxidant and anti-inflammatory agent in mouse macrophage cells (RAW 264.7; luteolin:chicoric acid =1:1, 1:2, 1:4 where IC₅₀ of luteolin was 11.6, 9.8 and 9.8 µM, respectively) (70). Chicoric acid caused the apoptosis of mouse 3T3-L1 preadipocytes (71) and displayed antiproliferative activity in MCF-7 cells but promoted the proliferation of the HeLa cell line (72).

Among the eight quercetins (a polyphenolic flavonoid compound) found in C. intybus, six were found to inhibit the growth of several malignant tumors (73). Both leaf and flower extracts of C. intybus contain quercetin 3-O-β-D-glucoside (52,74), which promoted the apoptosis of human gastric carcinoma cells (BGC-823; 12.1±0.03% inhibition at 100 µM) (75), HepG2 (IC₅₀ of 150 µg/ml), Caco-2 (IC₅₀ of 79 µg/ml), HEK-293 (IC₅₀ of 186 µg/ml) cells (76). Quercetin-7-O-galactoside, quercetin-3-O-(6″-O-malonyl)-glucoside, quercetin-7-O-glucoside, quercetin-7-O-glucuronide, quercetin-7-O-(6″-O-acetyl)-glucoside, quercetin-7-O-p-coumaroylglucoside, and quercetin-3-O-glucuronide-7-O-(6″-O-malonyl)-glucoside have been extracted from the leaf (5). Most of the compounds containing 3,4-dihydroxyphenethyl are potent antioxidant and specific inhibitors of NF-κB and Akt. The functionality of quercetin depends largely on the positioning of glycosylation and derivatization of a sugar molecule (77). With the different glycation sites, glucoside derivatives of quercetin show ex-vivo 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) free radical scavenging capacity at a varying degree (77). Quercetin isomers with a pentyl group in 7 positions were found to significantly inhibit CT26 cell proliferation (78). Quercetin hydrate demonstrated cytotoxicity against a human liver cancer cell line (HepG2) (73) and demonstrated antitumor activity against N2a, a mouse neuroblastoma cell line whereas C3 quercetin showed excellent antioxidant property in ex-vivo trials (77,79).

Leaf extract from C. intybus also yields phytochemicals such as 1,3-dicaffeoylquinic acid (5), 3,4-dicaffeoylquinic acid (5), cyanidin-3-O-galactoside (5) and cyanidin-3-O-glucoside (50). 1,3-Dicaffeoylquinic acid was found to exhibit antioxidant properties and inhibit oxidative damage created by FeSO₄ and AAPH [2,20-azobis(2-amidinopropane) dihydrochloride] and scavenge ROS. 1,3-Dicaffeoylquinic acid is required in low concentrations than other known antioxidants (80). 3,4-Dicaffeoylquinic acid inhibited the growth of Kato III (human stomach cancer; 20–40% inhibition at 100–500 µM), DLD-1 (20–40% inhibition at 1,000 µM) (53) and HL-60 (40–90% inhibition at 1,000 µM) cells in vitro (53). Cyanidin-3-O-glucoside inhibited tumor cell growth, induced apoptosis in vitro, and suppressed tumor growth in vivo (81). It protected MIN6 (pancreatic β-cells; cell viability 86.6% at 200 µg/ml) cells against apoptosis induced by oxidative stress (82), and showed dose-dependent growth inhibition on tumors derived from HS578T, LLC (Lewis lung carcinoma) (81) and MDA-MB-453 (human breast cancer) cells in a xenografted animal model (83). Cyanidin-3-O-galactoside inhibited the development of the A431 cell line (human vulva carcinoma; IC₅₀ >100 µM) (84).

The second largest group of phytochemicals is the 6-methoxyflavone (also known as 6-hydroxyflavone) group (Fig. 1D), with 18 members (5,52). Six have shown antitumor properties. Among them, apigenin-7-O-glucoside, kaempferol-7-O-glucoside, isorhamnetin-7-O-glucoside, and isorhamnetin-7-O-glucuronide have been isolated from leaf extract (5). Apigenin-7-O-glucoside was found to be cytotoxic against colon cancer cells (HCT116) (85), androgen-refractory PC-3 cells, and other cancer cell lines (86). Interestingly, there are 11 known isomers of kaempferols in C. intybus, but only kaempferol-7-O-glucoside was found to induce G2/M mitotic phase arrest and cell death in a p53-independent manner in HeLa cells (87). Among the three derivatives of isorhamnetin (a 6-O-methylated flavanol) (5), only C7 glucoside and glucuronide isomers were demonstrated to exert antitumor properties (88,89). In lipopolysaccharide-challenged mouse Abelson murine leukemia virus-transformed macrophage cells (RAW264.7), isorhamnetin-3-O-glucuronide increased heme-oxygenase-1 but suppressed p38 and c-Jun N-terminal kinase (JNK) activation (89). This indicates that derivatives of isorhamnetin from C. intybus can be modified to generate chemoprotective agents. Among four delphinidins isolated by Nørbaek et al (52) from C. intybus flower extract, only delphinidin 3,5-di-O-(6-O-malonyl-β-D-glucoside) exerted an antitumor effect, and the mono sugar substitute was effective against breast epithelial cells (MCF10A) (90).

Another study showed that 6-methoxyflavone (Fig. 1D) was associated with HeLa cell growth inhibition (46). However, the precise mechanism is not yet known.

Phytosterol. C. intybus contains four phytosterols (Fig. 1G), all with chemoprotective properties. Campesterol and stigmasterol are two of the major phytosterols found in the leaves (39). Campesterol shows antitumor and antiproliferative activity against HepG2 and MCF-7 cells (91). Stigmasterol was found to exhibit antitumor activity against EAC in swiss albino mice (92). The cytotoxicity of stigmasterol was demonstrated by Kangsamaksin et al (93) in KKU-M2123, RMCCA-1 and MMNK-1 cell lines; by Syed Abdul Rahman et al (94) in MCF-7, CaSki (cervical carcinoma), and HCT-116 cell lines; and by Dutra et al (95) in a chronic myelogenous leukemia cell line (K-562). In vivo, stigmasterol showed antitumor efficacy against a 1,3-dimethylbutylamine-induced skin carcinoma mouse model (96) and antioxidant activity against EAC in swiss albino mice (92). β-sitosterol is the most abundant phytosterol, present in leaves (39), roots (97), and total areal part extract (26). β-sitosterol increased the activity of antioxidant enzymes, glutathione peroxidase, and superoxide dismutase in cultured macrophage cells with phorbol 12-myristate 13-acetate-induced oxidative stress. This indicates that phytosterols can protect cells from ROS induced damage (98). In in vitro experiments, β-sitosterol was found to be cytotoxic to HeLa (99), intrahepatic cholangiocarcinoma (KKU-M213, RMCCA-1) (93), immortalized normal cholangiocyte (MMNK-1) (93), and breast cancer cells (MCF-7) (100,101). In in vivo experiments, β-sitosterol reduced tumor growth in 17 β-estradiol-treated mice (98). A nonmalignant enlargement of the prostate known as benign prostatic hyperplasia (BPH) was reduced by β-sitosterol treatment (102). β-sitosterol was also found to inhibit the proliferation and thus reduce the viability of mouse fibrosarcoma (98). β-sitosterol-3-O-glucoside from the areal part of C. intybus isolated by Satmbekova et al (26) exerted an anticancer effect against three cancer cell lines, MCF-7, HL-60 and HepG2 (91,103).

The phytosterols containing an unsaturated ring structure (Fig. 1G) are susceptible to oxidation under certain conditions. Comparison of the corresponding phytosterol and cholesterol oxidation-products (POP) in four cell lines demonstrated that phytosterol induces oxidation-independent apoptosis (104), which is in contrast to a previous report by O'Callaghan et al (105). These authors suggested that POP induced apoptosis by high oxidative stress, glutathione reduction, mitochondrial dysregulation and elevated caspase activity (105). From Table I, we can see that phytosterols show specificity towards fatty tissue-related cancer lines such as those derived from breast, cholangiocyte, and cervix cancer.

All four delphinidins (Fig. 1E) found in C. intybus are di-glucoside isomers, and none has antitumor properties. However, mono-glucoside substitutes of delphinidins are cytotoxic (88). Delphinidin-3-glucoside was found to be chemopreventive against breast epithelial cells (MCF10A) (90). Derivatives of C. intybus derived delphinidins can serve as potent chemoprotective agents.

The aglycons are the richest anthocyanins (Fig. 1F) found in food. Cyanidin and delphinidin were found to inhibit the growth of human tumor cells in vitro in small quantities (84). C. intybus fresh leaf extract [MeOH-HCOOH (99:1, v/v)] contains malvidin-3-O-glucoside and pelargonidin-3-O-monoglucuronide (5). Malvidin-3-O-glucoside was demonstrated to have antitumor activity against MCF-7 (106) and A431 (84) cell lines. Pelargonidin-3-O-monoglucuronide increased the concentration of IL-6 and monocytes in vitro, affecting tumor prognosis of THP-1 cells (human monocytic leukemia) (107). Its glucoside derivative, pelargonidin-3-O-glucoside, acts as an anti-inflammatory agent (108).

Seong et al (109) demonstrated that delphinidin treatment induced hypoacetylation of histone acetyltransferase (HAT) and inhibited p65 acetylation in a human rheumatoid fibroblast-like synoviocyte cell line (MH7A) (Fig. 4). TNF-α stimulation increases NF-κB expression, and thus promotes the functions of NF-κB target genes (109). Delphinidin also inhibited the release of pro-inflammatory cytokines IL-6 and TNF-α in lipopolysaccharide-treated Jurkat T lymphocytes (109). The chemotherapeutic property of delphinidin-3-glucoside against MCF10A cells is related to downregulation of non-coding RNA (lncRNA) and HOX transcript antisense RNA (HOTAIR) expression (90). Pelargonidin-3-O-monoglucuronide treatment was found to increase the IL-6 and monocytes concentration (107). Bioactivity of these compounds is related to the presence of hydroxyl group in position 3 of the C ring and 3, 4, 5 in the B ring (Fig. 1E and F) which corresponds to the report by Wang and Stoner (110), showing that methylation on these positions decreases the activity.

4-O-Feruloylquinic acid is one of the two 4-hydroxy-3-methoxyphenyl groups (Fig. 1J) containing phytochemicals found in C. intybus. It was demonstrated to scavenge oxygen radical absorbance capacity (ORAC) and DPPH radical in an in vitro experiment (69).

Researchers also identified several other types of phytochemicals in C. intybus. Artesin (also known as artemisinin, an antimalarial agent) was identified in the root extract fraction by Kisiel and Zielińska (23). Its antitumor property has been well studied (111–115). The derivatives of artesin have in vivo chemosensitizing effects in breast, lung, pancreas, and glioma cancer cells (112). Artesin and its derivatives activated by heme [Fe (II)] showed selective inhibition of colon (HCT116, SE480), leukemia (HL-60), breast (MCF-7), melanoma (KM, MJT3), lung (NSCLC), pancreas (PANC-1, MIAPaCa), and glioma (U87MG, A172) cancer cell lines (113–115). In primary cancer culture, cell lines and xenograft models, artesin inhibited tumor proliferation, metastasis, and angiogenesis (114). Several antitumor mechanisms of artesin have been proposed, including apoptosis, cell cycle arrest at G0/G1, and oxidative stress (111). Rapidly proliferating cancer cells express more transferrin receptors on their cell surface, leading to higher iron uptake. Artesin/artemisinin is the only phytochemical found in C. intybus with a peroxide bridge (Fig. 1H). When artesin binds to Fe (II), the endoperoxide bridge (Fig. 1H) is disrupted, resulting in the production of toxic C-4 and seco-C-4 free radicals that destroy tumor cells (Fig. 2) (36).

3-O-p-Coumaroyl quinic acid was isolated by Nørbaek et al (52) from an extract of C. intybus flower. 3-O-p-Coumaroyl quinic acid was demonstrated to show antiproliferative activity against PC-3 and undifferentiated non-cancerous 3T3L1 fibroblast cells (116). Usnic acid purified from the areal part extracts (26) was reported to exhibit chemoprotective effects against the wild-type p53 MCF-7 cell line along with a breast cancer cell line with non-functional p53 (MDA-MB-231), lung cancer cell line (H1299) (117) and prostate cancer cell line (LNCaP) (118). Mechanistically, (+)-usnic acid treatment dose-dependently decreases β-catenin-mediated transfection grade T-cell factor reporter plasmid activity and KAI1 COOH-terminal interacting tetraspanin-mediated AP-1 activity. In addition, (+)-usnic acid decreases the mRNA levels of CD44 (a cell-surface glycoprotein), cyclin D1 (a mitotic regulatory protein) and c-myc (a transcription factor). These are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Furthermore, (+)-usnic acid treatment was found to decrease the functionality of Rac1 and RhoA. Interestingly, cotreatment of (+)-usnic acid and cetuximab showed higher inhibition of cell proliferation then the single cetuximab treatment. These results indicate the potential antitumor activity and metastasis inhibitory quality of (+)-usnic acid and suggest (+)-usnic acid can be used for anticancer therapy with distinct mechanisms of action (119).

C. intybus root is a major source of inulin, a heterogeneous collection of fructose polymers primarily used as a prebiotic (120). The chemoprotective property of inulin has been confirmed in colon cancer colonic preneoplastic aberrant crypt foci inhibition (121). Its antitumor property was also demonstrated on transplantable liver tumor cells (TLT) and mouse mammary carcinoma cells (EMT6) (121–123). Low fermentation of inulin indicates its possibility to reach the distal part of the intestine (124). Inulin extracted from Cichorium endivia L. (a related species) was found to reduce the occurrence of intestinal tumors in an APC^MIN mouse model (121).

Most of the phytochemicals identified in C. intybus appear to show potent activity as inhibitors in specific tumor cell lines; only a few phytochemicals work on all cell lines. A screening study of anti-proliferative activity revealed the specificity of purified C. intybus phytochemicals against different cancerous cell lines. Kinjo et al (46) and other researchers showed that groups of phytochemicals had specificity towards specific cancer cell lines (listed in Table II). In general, polyacetylenes are potent antiproliferative agents against MK-1 cells, and compounds with 3,4-dihydroxyphenethyl against B16F10 cells, and some 6-methoxyflavone derivatives and 8-hydroxy furanocoumarins against HeLa cells are potent anti-proliferative agents (46). This indicates that the structural features of these phytochemicals directly interact with the cytochemistry of specific cancerous cells. The biological activities of the guaianolides, 6-hydroxyflavone, and anthocyanin depend broadly on the following: i) reactivity of alkylation center; ii) lipophilicity of the side chain; iii) electronic features and molecular genomics (36).

Similarly, every phytochemical has its own conserved structural features responsible for bioactivity. The antitumor property of flavonoids is partly due to their ability to counteract fatty acid synthase (FASN). Cancerous cells have an elevated metabolic rate compared with healthy cells, which enables cancer cells to grow and proliferate at a faster rate (18). Rapid cell division often leaves trails of both biochemically and structurally irregular cells. FASN is overexpressed in cancerous tissues of the breast, prostate, and colon (18), and is also related to angiogenesis and metastasis (125); therefore, FASN is recognized as an important antitumor target.

Previous studies have reported that phytochemicals of C. intybus exert antitumor effects by affecting many critical overactive signaling pathways in cancerous cells, such as NF-κB, p53-associated cell cycle, and CYP-mediated inactivation. The various effects of these phytochemicals confer the advantages to effectively target more than one cell type, which is often encountered during metastasis.

The nuclear factor-κB (NF-κB) transcription factor plays a critical role in cell development, growth, and survival as well as various biological processes, including immune response and inflammation. Numerous inflammatory stimuli such as growth factors and infectious microbes lead to NF-κB activation (126). Activated NF-κB in turn regulates the expression of genes governing cell growth, proliferation, survival, and apoptosis as well as immune responses, stress responses, embryogenesis, and development of a variety of stimuli (127). Abnormal NF-κB activation causes various autoimmune, inflammatory, and malignant disorders such as rheumatoid arthritis, atherosclerosis, inflammatory bowel diseases, multiple sclerosis, and malignant tumors. Thus, inhibition of NF-κB signaling is a key target in the treatment of tumors and inflammatory diseases (127). The mammalian NF-κB family is composed of five members that form various dimeric complexes. Among these complexes, the p50/65 heterodimer is most abundant. Overexpression of this complex in cancer cells leads to the aberrant levels of cell cycle control factors. Several phytochemicals of C. intybus disrupt different stages of NF-κB activation and NF-κB-DNA complex formation. Guaianolides, germacranolides, heliangolides, pseudo guaianolides, hypocretenolides, and eudesmanolides are collectively classified as sesquiterpene lactones. Sesquiterpene lactones bind to the p65 dimer in the NF-κB transcription factor to prevent NF-κB-DNA binding (Fig. 3). The three-dimensional structure created by Arg 33, Arg 35, Tyr 36, Cys 38, Glu 39 and Arg 187 is crucial for DNA binding of the p65 dimer. Rüngeler et al (37) proposed that lactucin creates cross linkage of Cys 38 to Cys 120 in the p65 molecule (Fig. 3) that changes the DNA binding motif structure and affects subsequent transcription, eventually leading to apoptosis. This mechanism was further demonstrated in a computer-generated model by García-Piñeres et al (38). The authors showed the change in native confirmation of p65 caused by a sesquiterpene lactone that led to its inability of NF-κB-DNA complex formation.

Búfalo et al (57) demonstrated that caffeic acid mediated cell viability independent anti-inflammatory activity and proposed that caffeic acid exhibited an inhibitory effect in lipopolysaccharide (LPS)-induced NF-κB activity. The chemoprotective effect of chlorogenic acid may be accomplished through its increase of cellular antioxidant enzymes and suppression of ROS-mediated NF-κB, activator protein 1 (AP-1), and mitogen-activated protein kinase (MAPK) activation (60). 5-Caffeoylquinic acid inactivates ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB/Akt) activity and thus affects multiple cellular processes and signal transduction pathways in cancerous cells. Another study showed that 1,3-dicaffeoylquinic acid scavenged hydroxyl radical and superoxide radicals as measured by electron spin resonance (ESR) (80). Chicoric acid was found to exert its anti-inflammatory function by halting the phosphorylation of NF-κB (70). Upon co-treatment with luteolin, chicoric acid simultaneously reduced the concentration of nitric oxide and prostaglandin E2 (PGE2) in cells and also inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression (70).

Genomic instability is one of the fundamental cause of tumor development. p53, commonly known as TP53 or tumor protein 53, is a cell cycle regulatory protein that functions as a tumor suppressor. p53 responds to DNA damage and other types of genotoxic stress and functions to maintain genomic stability. The close involvement of p53 in maintaining genomic stability is why nearly half of human cancers lack functional p53. In the other half of cancers, the p53-independent regulatory mechanism is absent or the p53-dependent pathway gets disabled at different key points. For instance, the p53 inhibitor MDM2 is overexpressed in tumors that lack p53 gene mutation. p53 is a crucial component of a complex network of signaling pathways. However, other components of this pathway can be alternately targeted for inactivation in cancer. C. intybus extract, in particular, usnic acid, was found to induce levels of factors such as ataxia telangiectasia mutase (ATM) or reductase (ATR) that phosphorylates p53 at the MDM2 binding site (Fig. 5) (117). However, kaempferol-7-O-glucoside arrests cell division in a p53-independent manner (87).

Cytochrome P450 enzymes (CYPs) are a superfamily of enzymes that are important for the metabolism of endobiotics and xenobiotics (128). Rodriguez-Antona and Ingelman-Sundberg (129) described the pharmacogenetics of CYPs in cancer formation and treatment. CYPs are linked to the metabolic activation of numerous pre-carcinogens and participate in the activation and inactivation of antitumor drugs (129). Hepatic CYP expression and activity can be upregulated or downregulated by bioactive phytochemicals (130). Direct foddering of dried C. intybus roots to pigs resulted in increased activities of CYP1A2 and CYP2A, which in turn reduced the skatole concentration in plasma and fat (128), reducing the chances for colon cancer occurrence (131). CYP1A2 is one of the class I CYPs, which are distinguished by a well-conserved sequence and lack of functional polymorph. CYP1A2 activity has interindividual (genetic) difference, and this polymorphism is triggered by external factors such as smoking (129). Although downregulated by total extract, artemisinin upregulates the mRNA expression of CYP1A2, CYP2C33, CYP2D25 and CYP3A29 in porcine hepatocyte culture (132), suggesting a shared regulatory mechanism of CYP transcription and an inverse agonist effect of C. intybus.