4.1.1. Anti-Proliferative Activity

PAs derived from different plants exhibit potent cytotoxicity against a variety of cancer cells. These biologically active molecules engage directly or indirectly in biological processes, including cellular cycle arrest, HIF-1 (hypoxia-inducible factor-1 alpha) activation and signal transducer and transcriptional factor 3 (*STAT3*) suppression.

The anti-proliferative effects of FaOH isolated from carrots was initially shown in 2003. In addition, FaOH-type PAs show toxicity against human pancreatic carcinoma cells, but not against normal pancreatic cells, in vitro by modulating the expression of the genes involved in apoptosis, cell cycle, stress response and death receptors [42,98].

Treatment of leukaemia cell lines with carrot extract or isolated FaOH or FaDOH inhibited cell cycle progression, suggesting that carrots cause cell cycle arrest (G0/G1) in leukaemia cell lines [99]. Moreover, the cytotoxicity of FaOH, FaDOH and panaxydiol isolated from the dichloromethane extract of root celery was tested for its potential impact in a number of human cancer and leukaemia cell lines. All PAs examined exhibited moderate cytotoxicity against leukaemia, lymphoma and myeloma cell lines, although FaOH had significantly more activity against the lymphocytic leukaemia cells than FaDOH and panaxydiol [100]. In other studies, FaDOH also had less cytotoxic activity than FaOH and FaDOH3Ac [99,101].

#### 4.1.2. Pro-Apoptosis Activity

Cancer prevention and treatment depend on the use of a variety of bioactive compounds that inhibit the early stages of cellular transformation required for the development of the neoplastic phenotype, such as initiating autophagy, apoptosis or other forms of cell death such as oncosis or necrosis [102]. Apoptosis dysfunction is a major contributor to cancer development and progression. Tumour cells' ability to avoid apoptosis can play a significant role in their resistance to traditional therapies.

One study investigated the effects of FaOH on human pancreatic ductal adenocarcinoma cell lines compared with normal pancreatic cells. FaOH regulated the genes related to pro-apoptosis, anti-apoptosis, apoptosis, cell cycle, stress and death receptors in adenocarcinoma cells more preferentially than in normal pancreatic cells [98]. FaOH suppressed pro-apoptosis genes (*BAD* and *HTRA-2*), anti-apoptosis genes (*Livin* and *XIAP*), a cell cycle controller (Phospho-p53 at amino acids serine 15, 46 and 392 (S15, S46 and S392), stress-related genes (*Clusterin* and *Hsp60*) and death receptor genes (*TNFR1* and *TNFSF1A*). In addition, FaOH increased cell cycle checkpoint phosphorylation (Phospho-Rad17 (S635)) and induced stress-related genes (*HO-1, HMXO1, HP32 and Hsp27*). Furthermore, FaOHtype and other PAs are potent inhibitors of pancreatic cancer cell proliferation [98].

Tumour recurrence and drug resistance are both facilitated by cancer stem-like cells (CSCs) [103]. *Hsp90* is known to enhance cancer cell survival and their ability to acquire anti-cancer drug resistance; its overexpression has been linked to a poor prognosis in human malignancies [104,105]. An in vivo study showed that orally administered FaOH significantly suppressed the proliferation of lung cancer in a mouse model without overt symptoms of toxicity at concentrations of 50 mg/kg body weight [103], which would correspond to a human dose of 4 mg/kg [106]. FaOH selectively inhibited carcinogenesis cells but not normal cells both in vitro and in vivo by inhibiting the function and viability of cancer stem-like cells of non-small-cell lung cancer by triggering apoptosis without enhancing *Hsp70* expression. Moreover, FaOH, at a low dose of 1 μM, induced apoptosis in cancer stem-like cells [103]. The pro-apoptotic function of panaxydol from *P. ginseng* was also tested on different cell lines to check whether the induction of apoptosis occurred preferentially in cancer cells. Indeed, panaxydol selectively induced apoptosis in malignant cancer cells [85].

#### 4.1.3. Gut Microbiota Composition

A study aimed to investigate whether the antibacterial effects of FaOH and FaDOH may be a mechanism of action in the antineoplastic properties of FaOH and FaDOH. They tested the effect of FaOH and FaDOH on gut microbiota composition in an AOMinduced rat colorectal cancer model. Rats treated with AOM were fed either a normal rat diet or a diet enriched with FaOH and FaDOH. Analysis of cecum faecal samples revealed a significant change in the gut microbiota among the groups. FaOH and FaDOH, which suppressed the growth of neoplastic tumours in the colon in a rat colon cancer model, modified the composition of low-abundance gut microbiota GM members, such as *Lactobacillus reuteri,* and high-prevalence *Turicibacter*, which was also correlated with a reduction in the number of macroscopic sites of neoplasms. Thus, this study demonstrated that modifications in the gut microbiota may play a significant role in the colon-protective action of FaOH and FaDOH against neoplastic transformation [107].

#### 4.1.4. Other Effects

FaOH stimulated the differentiation of primary mammalian cells at concentrations as low as 0.004 to 0.4 μM, whereas cytotoxic effects were observed at concentrations of >4 μM [108]. Moreover, one study evaluated PAs (FaOH and FaDOH) isolated from carrots in non-cancerous human intestinal epithelial cells (FHs 74 Int. cells) and intestinal cancer cells (CaCo-2). The growth–inhibition response was seen in concentrations above 1 μg/mL (~4 μM), with maximum inhibition at 20 μg/mL (~80 μM). The FaOH showed a higher inhibitory potency compared with FaDOH. In addition, cancer cells treated with combinations of FaOH and FaDOH showed a synergistic response for the inhibition of cell growth [109]. FaOH purified from carrots inhibited caspase-3 expression to prevent cell death and reduced basal DNA strand breakage in CaCo-2 cells. Thus, FaOH is either prosurvival or pro-death in a concentration-dependent manner in CaCo-2 cells. The effects of FaOH on CaCo-2 cells appear to be biphasic, with low and high concentrations of falcarinol inducing pro-proliferative and apoptotic characteristics, respectively [110].

PAs have other effects relevant for cancer. PAs can be used to heal or relieve symptoms by interacting with other foods or drugs. Cisplatin, which has nephrotoxicity as a side effect, is a therapeutic drug for various solid tumours. FaDOH attenuates cisplatin-induced injury and down-regulates mRNA levels of *TNF-α, IL-1β* and the protein expression of p-NF-κB p65 in mice [111].

Another study demonstrated the effects of FaOH, FaDOH, FaDOH3Ac and falcarindiol 3,8-diacetate on breast cancer multidrug resistance protein (BCRP/ABCG2), a xenobiotic efflux transporter that causes chemotherapy resistance in cancer. PAs inhibited mitoxantrone efflux (an ABCG2 substrate) in HEK293 cells overexpressing ABCG2. In a vesicular transport assay, a concentration-dependent inhibition of methotrexate (another ABCG2 substrate) uptake into ABCG2-overexpressing Sf9 insect cell membrane vesicles was observed. PAs also inhibited baseline and sulfasalazine-stimulated vanadate-sensitive ATPase activities in ABCG2-overexpressing Sf9 insect cell membrane vesicles. This study suggested that PAs might mitigate multidrug resistance in chemotherapy. As ABCG2 may play a role in the absorption and disposition of PAs, there may be food–drug interactions between PA-rich foods and ABCG2 substrate drugs [112].
