*2.1. Chronic Inflammation Disease and Cancer*

Chronic inflammation is recognized as a leading promoting factor of diseases including carcinogenesis [24], which continues to be the leading cause of mortality and disability around the world [25–28]. Tumour-promoting inflammation is recognised as an enabling hallmark of cancer [29]. Cancer and inflammation are linked by intrinsic and extrinsic pathways. Intrinsically, oncogenes regulate the inflammatory microenvironment, whereas extrinsically, the inflammatory microenvironment promotes the growth and spread of cancer [30]. Various cell types involved in chronic inflammation can be found in tumours, both in the surrounding stroma and within the tumour itself. Neoplasms, including some of epithelial origin, contain a significant inflammatory cell component [31]. Multiple studies on human clinical samples have revealed that inflammation influences epithelial cell turnover [32,33]. Significantly, human susceptibility to breast, liver, large bowel, bladder, prostate, gastric mucosa, ovary and skin carcinoma is increased when proliferation occurs in the context of chronic inflammation [32–37].

Chronic inflammation is linked to approximately 25% of all human cancers and increases cancer risk [38] by stimulating angiogenesis and cell proliferation, inducing gene mutations and/or inhibiting apoptosis [38]. Chronic inflammation can develop from acute inflammation if the irritant persists, although in most cases the response is chronic from the start. Chronic inflammation is characterized by the infiltration of injured tissue by mononuclear cells such as macrophages, lymphocytes and plasma cells, as well as tissue destruction and attempts at repair [31]. *Helicobacter pylori* infections in gastric cancer, human papillomavirus infections in cervical cancer, hepatitis B or C infections in hepatocellular carcinoma and inflammatory bowel disease in colorectal cancer (CRC) are common causes of chronic inflammation associated with cancer development [39,40]. Inflammation also causes epigenetic changes that are linked to cancer development.

Natural PAs from diverse food and medicinal plants and their derivatives exert multiple bioactivities, including anti-inflammatory properties [41]. PAs can impact inflammation through known and unknown pathways. Evidence supports that PA compounds improve human health by stimulating anti-cancer and anti-inflammatory mechanisms [3]. These PAs contain triple bonds that functionality convert them into highly alkylating compounds that are reactive to proteins and other biomolecules. This unique molecular structure might be the key to understanding the beneficial effects of PAs such as their anti-inflammatory and cytoprotective function [41]. Recent research has suggested that the anti-cancer role of certain foods might be attributed to their anti-inflammatory function. Root vegetables, and particularly carrots, are promising natural sources in this respect thanks to their rich content of PAs [3,41–43]. The anti-inflammatory properties of purple carrots have been suggested to be due to the high levels of anthocyanin pigments [44]; however, another study showed that PAs, not anthocyanins, are responsible for the anti-inflammatory bioactivity of purple carrots [45]. In vitro and in vivo studies have demonstrated that the health-benefitting effects of carrots and other root vegetables might be attributed to PAs, such as FaOH and FaDOH [46]. Other dietary compounds, including several different phytochemicals, have been examined in the context of cancer chemoprevention; however, until now the measured effects [47–50] have been quite small and inconsistent compared with those found for PAs.

#### *2.2. Inhibition of Nuclear Factor Kappa B (NF-κB) Pathways*

NF-κB is a transcription factor that regulates the expression of many genes involved in the regulation of inflammation and autoimmune diseases [51,52]. Moreover, NF-κB plays a significant role in inflammation-induced cancers, as NF-κB is one of the major inflammatory pathways that are triggered by, for example, infections causing chronic inflammation [39,40,53]. Cellular immunity, inflammation and stress are all regulated by NF-κB signalling, as are cell differentiation, proliferation and apoptosis (Figure 2) [54,55]. Both solid and hematologic malignancies frequently modify the NF-κB pathway in ways that promote tumour cell proliferation and survival [56–58].

NF-κB, a key factor in the inflammatory process, provides a mechanistic link between inflammation and cancer, and the components of this pathway are targets for chemoprevention, particularly in CRC [59]. There are two major signalling pathways for NF-κB activation, namely the canonical and the non-canonical NF-κB signalling pathways. The canonical pathway activates NF-κB1 p50, RELA and c-REL, which are also called canonical NF-κB family members. The non-canonical NF-κB pathway, on the other hand, selectively activates p100-sequestered NF-κB members, mostly NF-κB2 p52 and RELB, which are also called non-canonical NF-κB family members [60]. LPS and proinflammatory cytokines, among other pathogenic substances, activate NF-κB through degrading inhibitors of κB (IκBs) [61] to release the common subunit P65 (RELA). In order to trigger the transcription of these genes, activated NF-κB travels into the nucleus and attaches to its associated DNA motifs. When activated, the NF-κB p65 subunit binds to the promoter regions of genes involved in inflammation, leading to the production of *IL-6*, *IL-1β* and *TNF-α* [62].

**Figure 2.** NF-κB target genes implicated in the onset and progression of inflammation. NF-κB is a transcription factor that is inducible. After activation, it can regulate inflammation by activating the transcription of several genes. NF-κB regulates cell proliferation, apoptosis and differentiation in addition to promoting the production of pro-inflammatory cytokines and chemokines. Endoplasmic reticulum (ER) stress results in an inflammatory unfolded protein response (UPR). Stress on the ER induces apoptosis by activating inflammation. This can be accomplished by stimulating IKK complex (element of the NF-κB) or (X-box binding protein 1) XBP1 and (protein kinase R-like ER kinase) PERK through a mediator. These trigger the release of pro-inflammatory molecules, hence accelerating cell death.

Carrot PAs, particularly FaOH and FaDOH, were studied for their anti-inflammatory properties [3,63], in part by inhibiting the transcription factor NF-κB [64]; however, their exact mechanism of action is still unknown. Mice fed a diet containing FaOH were less likely to develop severe inflammation after being exposed to LPS [5]. FaOH from *Saposhnikovia divaricata (S. divaricata)* significantly reduced the levels of LPS-induced *TNF-α* and *IL-6* in cultured BV-2 microglia cells and murine serum [61]. FaOH and FaDOH purified from carrots were demonstrated for their preventative effects on colorectal precancerous lesions in azoxymethane (AOM)-induced rats. Biopsies of neoplastic tissue were analysed for gene expression, and the results showed that FaOH and FaDOH inhibited NF-κB and the downstream inflammatory markers *TNF-α, IL-6 and COX-2* [46]. FaOH from Radix Saposhnikoviae (dried roots of *S. divaricata*, Apiaceae) inhibited LPS-induced NF-κB p65 activation and IκB-α phosphorylation in BV-2 microglia cells [61]. Treatment using FaOH from the roots of *Heracleum moellendorffii* (*H. moellendorffii*) inhibited LPS-induced NF-κB signalling activation by inhibiting IκB-α degradation and nuclear accumulation of p65 [65] in RAW264.7 cells. In addition, FaDOH reduced the level of LPS/IFNγ-induced NF-κB, IKK-α and IKK-β activation in rat primary astrocytes [64].

Prostaglandin (PG) synthesis is a hallmark of inflammation. Two enzymes, cyclooxygenase (COX) 1 and 2, catalyse the first step of PG synthesis, but *COX-2* is the major one that responds to inflammatory signals to produce PG at inflammatory sites [66]. However, *COX-2* can be suppressed by inhibiting the NF-κB translocation pathway (Figure 2) [67]. *COX-2* expressions in healthy tissues are low, but they can quickly increase in response to growth factors, cytokines and signals promoting tumour invasion, metastasis, aberrant proliferation and angiogenesis [68]. Malignancies, including colorectal [69], bladder [70], breast [71], lung [72], pancreatic [73], prostate [73] and head and neck cancer [74], tend to be associated with elevated levels of *COX-2*. Mechanistically, *COX-2* promotes carcinogenesis through the creation of prostaglandins (PGs), which suppress apoptosis and stimulate the development of blood vessels in tumour tissue, which helps in sustaining tumour cell

viability and growth [39,75], suggesting that anti-inflammatory drugs targeting *COX-2* might be beneficial in the treatment of different types of cancer.

PAs modulate inflammation via suppressing *COX-2* expression, which depends on NF-κB activation by inflammation [76]. FaOH inhibited LPS-induced *COX-2* expression in RAW264.7 cells, thus blocking PGE2 overproduction [65]. FaOH isolated from American ginseng (*P. quinquefolius*) effectively reduced the severity of colitis in mice treated by dextran sulphate sodium (DSS) induced for a week before FaOH treatment. FaOH reduced the number of CD11b+ macrophages in the lamina propria and the inflammation hallmark protein *COX-2*. These data suggest that macrophages expressing *COX-2* might be an essential factor for colitis development. Interestingly, FaOH treatment prior to DSS did not prevent colitis or reduce colitis severity in mice [8]. Quiescent macrophages in the lamina propria in a healthy mouse might offer protection against colitis induction, as depleting macrophages prior to induction of colitis may exacerbate DSS-induced colitis [77]. However, when colitis develops, there is an increase in the number of activated macrophages that secrete pro-inflammatory cytokines to boost the inflammatory response, thus exacerbating colitis. At this stage, an overactive macrophage response to enteric microbiota greatly contributes to the pathogenesis of colitis [78]. Treatment with FaOH to target macrophages was shown to be highly effective in suppressing colitis at this stage, highlighting the utility of FaOH in the treatment of a hyper-inflammatory disease (Figure 3) [79]. In an azoxymethane (AOM)-induced rat colorectal cancer model, FaOH and FaDOH downregulated *COX-2* in precancerous lesions of CRC [46] and also reduced the number of malignant tumour foci.

**Figure 3.** Schematic representation of the possible mechanism of immunoregulation activity of poly-acetylenes (PAs) in macrophages. (Interferon-γ) IFN-γ activates macrophage cells to M1, and PAs downregulate NF-κB activities in M1 macrophages by inhibiting *iNOS, COX-1* and *COX-2*. PAs suppress the inflammatory response by inhibiting cytokines (*IL-16, IL-1β* and *TNF-α*) and upregulating Nrf2 pathway (*HO-1* and *NQO1*) in macrophages. PAs structure modified from Mplanine (2022) https://www.wikiwand.com/en/Falcarinol#Media/File:Falcarinol\_3D\_BS.png (accessed on 1 January 2023).

#### *2.3. Oxidative Stress*

2.3.1. Inhibition of Nitric Oxide Synthase (NOS) and Pro-Inflammatory Cytokine Pathways

Nitric oxide (NO) is essential in a number of physiological functions, such as host defence, where it prevents the spread of disease-causing microbes within cells by stifling their reproduction [80]. The upregulation of NO expression in response to cytokines or pathogen-derived chemicals is a crucial part of the host's defence against different types of intracellular pathogens. Different cell types produce the enzyme NOS, which catalyses the synthesis of NO, at high levels in a number of different tumour types [81]. Inflammation induces a specific form of NOS, i.e., the inducible isoform of nitric oxide

synthase (iNOS), via activating *iNOS* gene transcription (Figure 2) [82]. iNOS is involved in complex immunomodulatory and antitumor mechanisms, which have a role in eliminating bacteria, viruses and parasites [83].

A considerable number of studies have been published on the role of PAs in *iNOS* expression in inflammation. Studies have demonstrated that FaOH extracted from *P. quinquefolius* inhibited *iNOS* expression in ANA-1 mΦ macrophage cells that were polarized to M1 [8] and LPS-induced iNOS expression in macrophages [84,85], leading to colitis suppression [8]. Moreover, FaDOH was tested on rat primary astrocytes for its impact on LPS/IFN-γ-induced *iNOS* expression. FaDOH blocked 80% of LPS/IFN-γ-induced *iNOS* by reducing *iNOS* protein and mRNA in a dose-dependent manner. FaDOH was shown to suppress *iNOS* expression, and it inhibited *IKK, JAK, NF-κB* and *Stat1* (Figures 2 and 3) [64].

Another study showed a dose-dependent reduction in nitric oxide production in macrophage cells, where treatment with an extract of purple carrots containing PAs significantly reduced iNOS activity and *iNOS* expression in macrophage cells [45]. PAs reduced nitric oxide production in macrophage cells without cytotoxicity [45]. In vivo, purple carrots also inhibited inflammation in colitic mice and reduced colonic mRNA expression of *iNOS* [44]. FaOH from *H. moellendorffii* roots inhibits the LPS-induced overexpression of *iNOS* in RAW264.7 cells [65]. FaOH and other PAs from *P. quinquefolius* such as panaxydiol have a suppressive effect on *iNOS* expression in macrophages treated with LPS [85].
