Anti-Cancer Potential of Cannabinoids, Terpenes, and Flavonoids Present in Cannabis

2.1. Delta9-tetrahydrocannabinol (THC)

Δ⁹-tetrahydrocannabinol (THC) is the major psychoactive component present in Cannabis sativa L. cultivars, mediating its effects in the central nervous system via CB1 receptors [126]. THC binds and activates CB1 receptors in the central nervous system (CNS), leading to the intoxicating feelings associated with cannabis use. THC can be administered via multiple routes, including orally, intravenously, intramuscularly and inhalation. The most common method of administration in humans is orally, and due to its high lipophilicity, it is highly bound by plasma proteins and is readily distributed to vascularized tissues such as the liver, heart and lungs. Fat tissues have also been shown to be reservoirs for THC accumulation. Due to the psychoactive effects of THC mediated in the CNS, there are concerns in terms of prescribing THC for medicinal use in cancer patients. There are also other undesirable side effects of THC use, such as dependence, tolerance and issues surrounding abuse [27]. Despite the limitations and concerns associated with THC treatment, there are many studies regarding THC’s potential as an anti-cancer therapy and we highlight these studies herein.

2.2. Cannabidiol (CBD)

Cannabidiol (CBD) is one of the major and most extensively researched phytocannabinoids present in cannabis species. Cannabidiol binds to a large array of physiological targets within the body’s endocannabinoid system. In medical settings, CBD is most commonly administered orally and is commonly prepared as an oil. Cannabidiol is non-intoxicating, which is why its potential as a therapeutic agent is more appealing than some other cannabinoids that do possess psychoactive effects, like THC. To date, there are many studies surrounding the anti-cancer potential of cannabidiol. Two recent reviews by Afrin et al. and Kis et al. [27,136] thoroughly highlighted research studies investigating the anti-cancer effects of cannabidiol. Treatment with CBD exhibited a multitude of beneficial anti-cancer effects in lung, breast, colon, prostate, melanoma, leukemia, cervical, brain, neuroblastoma and multiple myeloma cancer cells (Reviewed in [27,136]), and we highlight these studies here.

2.3. Cannabigerol (CBG)

Cannabigerol (CBG) is one of the main active phytocannabinoids produced by Cannabis Sativa L. plants; however, it is considered a minor phytocannabinoid due to its lower abundance relative to THC and CBD. Cannabis cultivars that tend to have higher cannabigerol content are referred to as Type IV cannabis [138]. Cannabigerol is derived from its acidic precursor cannabigerolic acid (CBGA), which also serves as the precursor molecule for the production of THC and CBD. Recently, cannabigerol has attracted more attention for its use in therapeutics due to its lack of intoxicating effects, and more commercial hemp varieties have been developed with CBG and CBGA as the main phytocannabinoids present [139]. As of yet, only a handful of studies have been done to investigate the anti-cancer potential of cannabigerol. Two early studies by Baek et al. [124,125] looked at the potential therapeutic benefits of cannabigerol in mouse skin melanoma cells and oral epithelioid carcinoma (KB) cells. In mouse skin melanoma cells, CBG was found to have significant inhibitory effects on proliferation, with an IC₅₀ of 3 µg/mL. In KB cell lines, CBG over a concentration range of 1–100 µM was the most effective of the cannabinoids tested at reducing cell viability. In MDA-MB-231 breast carcinoma cells, 25 µM CBG was shown to inhibit the uptake of [¹⁴C]anandamide and activate vanilloid receptor TRPV1 [30]. CBG also stimulated apoptosis, ROS production, up-regulated C/EBP homologous protein (CHOP) mRNA and inhibited cell proliferation in colorectal cancer (CRC) cells. In vivo, CBG was shown to decrease the growth of xenograft tumours in a murine model, and that this effect was largely due to its activity as an antagonist at TRPM8 receptors on CRC cells [123].

2.4. Cannabichromene (CBC)

Cannabichromene is considered another one of the minor phytocannabinoids produced by Cannabis sativa L. plants, due to its lower abundance than the major cannabinoids THC and CBD. In the United States, cannabichromene has been found to be the second most abundant type of cannabinoid present in some strains of cannabis, particularly abundant in dry-type cannabis material. Though CBC is commonly found in many cannabis strains, there is much to be discovered about its pharmacology. Like CBD, cannabichromene lacks intoxicating effects and is therefore appealing to researchers in terms of its potential therapeutic effects in human health and medicine [140]. The potential anti-cancer effects of cannabichromene have not been extensively studied. In prostate carcinoma (PCC) cells DU-145 and LNCaP, cannabichromene was found to be the second most potent inhibitor of cell viability behind CBD, and CBC at 10 µM had very little effect on caspase 3/7 activity. In serum deprived PCC 22RV1 and PC-3 cells, treatment with CBC at 20 µM resulted in significant activation of caspase 3/7, and CBC caused elevated intracellular Ca²⁺ in all four PCC cell lines mentioned [87]. In colorectal cancer Caco-2 cells, CBC was able to inhibit cell growth, but only at a concentration of 30 µM [123]. In MDA-MB-231 and MCF-7 breast cancer cell lines, CBC demonstrated high potency as an inhibitor of cell viability [30].

2.5. Cannabidivarin (CBDV)

Cannabidivarin’s structure is similar to cannabidiol except with a shortened side chain. Cannabis cultivars with relatively high levels of CBDV have been identified in India and Nepal. In one study, CBDV was assessed for potential cytotoxic effects on various human prostate carcinoma cell lines. Results showed an IC₅₀ of around 20 µM [87]. In colon cancer cells, CBDV inhibited cell viability in a concentration-dependent manner, with an IC₅₀ of 10 µM [123].

2.6. Cannabinol (CBN)

Cannabinol (CBN) is present in the cannabis plant and, particularly in aged cannabis, is the degraded product of tetrahydrocannabinolic acid. While it was the first of the phytocannabinoids to be isolated, it remains poorly studied. Cannabinol’s psychoactive effects are estimated to be 10 times lower than that of THC [141]. Some cytotoxic effects were observed for cannabinol in prostate cancer cell lines DU-145 and LNCaP. The observed IC₅₀ was reported to be superior to the highest dose tested (25 µM) in most experiments [87]. CBN has also been shown to have some antiproliferative effects in aggressive breast cancer cells [96]. Multi-drug transporters are an ongoing issue in the treatment of cancers due to their ability to confer resistance to multiple anti-cancer agents. In one study, CBN inhibited multidrug transporter ABCG2 and promoted the accumulation of mitoxantrone, a substrate for this transporter [92].

2.7. Cannabivarin (CBV)

Cannabivarin (CBV), also known as cannabivarol, is found in minor amounts in some cannabis cultivars, and it is an analog of cannabinol with a shortened side chain. It is considered an oxidization product of tetrahydrocannabivarin and is rarely found in fresh cannabis. There does not appear to be any published literature surrounding the biological effects of cannabivarin (or cannabivarol) in cancer.

2.8. Tetrahydrocannabivarin (THCV)

Tetrahydrocannabivarin (THCV) is a homologue of THC, where different side chains contribute to a variety of effects that are distinct from THC. Most cannabis cultivars only contain trace amounts of THCV, but some sativa cultivars from hybridized African genetics may have higher levels of THCV. As for most other minor cannabinoids, little has been demonstrated regarding the effects of THCV in cancer. Some cytotoxic effects were observed for tetrahydrocannabivarin in prostate cancer cell lines DU-145 and LNCaP, with IC₅₀ values above 17.5 µM [87].

4.1. Kaempferol

Kaempferol is a well-characterized natural flavonol that is commonly found in dietary items like tea, apples, strawberries, broccoli, and beans [327]. It is also produced by the Cannabis plant and has attracted much research surrounding its potential health benefits, including its potential as an anti-cancer agent. A plethora of research in recent years has demonstrated many of kaempferol’s anti-cancer effects in vitro and in vivo on a variety of cancer subtypes. Two comprehensive reviews by Irman et al. [328] and Kashyap et al. [327] discussed the anti-cancer effects of kaempferol in various cancers. Here we highlight the main review findings and discuss more recent studies that looked at kaempferol’s potential as an anti-cancer agent. Kaempferol treatment inhibited cell viability in a dose-dependent manner in a multitude of cancer subtypes. Most of the studies reviewed indicated that kaempferol’s inhibitory effects on cell viability in cancer cells were as a result of cell cycle arrest or apoptosis. Kaempferol was able to induce cell cycle arrest at the G2/M phase in a multiple cancers, including leukemia, breast, liver, stomach and ovarian cancers [329,336,337,338]. In oral cancers, kaempferol also induced cell cycle arrest, but alternatively at the G0/G1 phase [339]. In glioblastoma, hepatic, colorectal, pancreatic, lung, renal and breast cancer cell lines, kaempferol was able to significantly reduce migration and/or invasion in vitro [341,342,343,344,345]. In lung and breast cancer cell lines (A549 and MDA-MB-231/MCF-7), studies have found that treatment with kaempferol was able to inhibit the epithelial-mesenchymal-transition (EMT), resulting in decreased metastasis and resistance to chemotherapeutics in these cells [343,347]. The detailed mechanisms of the anti-cancer actions of kaempferol in breast cancer can be found thoroughly reviewed by Wang et al. [422]. Several studies have demonstrated the ability of kaempferol to reduce angiogenesis; this has been demonstrated in ovarian cancer cells as a result of altered expression of vascular endothelial growth factor [348]. Studies investigating the anti-cancer effects of kaempferol in vivo were also reviewed by Irman et al. [328,423]. In in vivo mouse models of various cancers, including bladder, oral, prostate, lung and bone cancers, treatment with kaempferol was able to increase survival and reduce the growth and metastasis of tumours [339,352,353,354,355]. In a rat model of leukemia, treatment with kaempferol resulted in degranulation in basophilic leukemia cells (RBL-2H3) and increased the accumulation of mediators in human leukemic mast cells (HMC-1) [357,358].

In addition to the studies highlighted in the reviews mentioned previously, several studies have since been published that investigated the anti-cancer potential of kaempferol. In gastric, colon, prostate, colorectal and neuroblastoma cancer cells, kaempferol was able to significantly decreased cell viability and proliferation [330,331,332,333,334]. Kaempferol treatment induced autophagy in gastric cancer cells [330]. In breast cancer cells, kaempferol was able to induce apoptosis, cell cycle arrest at the G2/M phase, and suppress cell proliferation [329]. In a mouse model of breast cancer, kaempferol treatment was able to suppress primary tumour growth and lung metastasis [356]. In ovarian cancer cell lines, treatment with kaempferol inhibited growth with IC₅₀ values between 25–50 µM [335]. Further investigation revealed that kaempferol caused cell cycle arrest at the G2/M phase and induced apoptosis in OVACAR-3 ovarian cancer cells due to upregulation of apoptotic proteins such as caspase 3 and Bax [335]. Kaempferol exhibited anti-proliferative effects in endometrial carcinoma cells through apoptosis and cell cycle arrest, and was able to decrease migration and invasion trends in these cells [340]. In immortalized human retinal pigment epithelial (ARPE-19) cells, treatment with kaempferol was able to decrease cell migration through ERK1/2 signaling [346]. Kaempferol-conjugated gold nanoclusters (K-AuNCs) were developed by Govindaraju et al. [424] as a potential anti-cancer drug delivery system, and they demonstrated that K-AuNCs targeted A549 lung cancer cells and exhibited toxicity via nucleus damage.

A few studies have investigated the potential of combination treatment with kaempferol and previously established anti-cancer agents or other compounds. A study by Seydi et al. [349] using cancerous hepatocytes from a rat model of hepatocellular carcinoma found that kaempferol combined to luteolin (another common flavonoid) was able to inhibit cell proliferation, induce cell death and inhibit migration and invasion. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) stimulates apoptosis through binding death receptors 4 and 5 in a variety of cancers, however resistance to TRAIL has been known to occur [350]. Hassanzadeh et al. [350] found that co-treatment of lymphoblastic leukemia (MOLT-4) cells with TRAIL and kaempferol was able to induce apoptosis by inhibiting the expression of anti-apoptotic proteins and up-regulation of death receptors 4 and 5, and they suggested that this co-treatment could be used as a potential solution to overcome resistance to TRAIL in cancers. Li et al. [351] looked at the potential of combining 5-fluorouracil (5-FU) with kaempferol in colorectal cancer. They found that the combination of 5-FU and kaempferol was superior at inhibiting cell viability than either agent alone, and the anti-cancer effects were mediated through reduction in cell proliferation and induction of apoptosis. Another study looked at the combination of 5-FU with kaempferol in 5-FU-resistant colon cancer cells [341]. They found that combination treatment of 5-FU with kaempferol had synergistic effects on cell viability and was able to chemo-sensitize the resistant cells.

4.2. Apigenin

Apigenin is a natural flavone found in many fruits and vegetables, and predominantly found in parsley, celery, and in the flower of chamomile plants, among others. Apigenin is a pigment, yellow in color. A comprehensive review by Imran et al. [425] provides details about the anticancer effects of apigenin in various types of cancer such as breast, lung, liver, brain, skin, blood, bone, colon, prostate, pancreatic, cervical, ovarian, oral, and stomach. Detailed mechanisms of action of apigenin in each of these cancer types can be found within the review however, the induction of apoptosis, upregulation of caspases-3, -8 and TNF-α, downregulation of MMP-2, -9, NFkB, PI3K, Akt and pAkt, and modulation of kinases are mechanisms frequently involved. Since the review’s publication, further studies have investigated apigenin’s anticancer effects. Apigenin reduced cell viability in MCF-7, A549, HepG2 and normal HEK 293 cell lines with the greatest activity against HepG2 liver cancer cells (EC₅₀ of 12 µg/mL) [359]. Li et al. [362] further noted that in three hepatocellular carcinoma cell lines, apigenin was cytotoxic and induced G1 phase cell cycle arrest in a dose dependent manner through the regulation of CyclinD1 and CDK4. Additionally, hypoxia-inducible factor 1α (HIF-1α) is associated with hypoxia-induced resistance in cancer cells but has been shown to be inhibited by apigenin. Two pathways involved in suppressing the HIF-1α expression in hypoxic tumors are through the inhibition of the AKT/p-AKT pathway and HSP90, which also enhance the activity of the chemotherapeutic paclitaxel. Apigenin and paclitaxel also acted synergistically in a liver cancer cell line and murine models [362]. When combined with the chemotherapeutic sorafenib, apigenin decreased cell viability of liver cancer cells to a greater extent than either drug alone. This combination caused an increase in apoptosis and decreased the migration and invasion capability of the cells [364].

In a colon cancer and lymph-endothelial cell model, treatment with apigenin reduced the formation of circular chemorepellent-induced defects in the endothelial barriers [426]. In a cervical cancer model, apigenin inhibited cell growth in vitro and tumour growth in vivo through the ER-mediated PI3K/Akt/mTOR pathway [365]. Similarly, in a model of diffuse large B-cell lymphoma, apigenin inhibited proliferation and colony formation by activating pro-apoptotic proteins, downregulating cell cycle proteins to increase G2/M phase arrest and inhibiting the PI3K/mTOR pathway. Apigenin also acted synergistically in vitro and in vivo with Abivertinib, a bruton tyrosine kinase inhibitor, which can provide new options for patients who have developed resistance to traditional therapies [360]. Apigenin altered the tumor necrosis factor and IL-10 release by microglia. When treated with conditioned medium of microglia treated with apigenin, C6 glioma cells exhibited reduced tumor migration and viability, due to the reduction in IL-6 levels. Apigenin also preferentially reduced viability of C6 glioma cells when co-cultured with microglia [363]. One study showed that apigenin inhibited cell proliferation, induced apoptosis, reduced vascular endothelial growth factor (VEGF) expression, and reduced tumor-induced angiogenesis in two human esophageal cancer models [361]. The inhibition of IL-6 transcription further potentiated these effects, suggesting that the inhibition of IL-6 transcription was how apigenin exhibited its anticancer effects in esophageal cancer cells. Similar effects were seen in a murine xenograft model [365].

4.3. Cannflavins

Cannflavins are a group of prenylflavonoids uniquely found in cannabis. Cannflavins A and B are formed by a derivative of luteolin, chrysoeriol [427]. Recently, a study examined the potential of cannflavin B derivatives for the treatment of pancreatic cancer. In vitro results showed an increase in apoptosis in two pancreatic cancer cell lines treated with concentrations of FBL-03G (or caflanone), the cannflavin B derivative. In vivo local and metastatic tumor progression were delayed in pancreatic cancer animal models as well as an increase in survival compared to control cohorts [366]. In 2019, caflanone was granted orphan drug status by the United States Food and Drug Administration and clinical trials with the drug were scheduled to begin as potential treatment for pancreatic cancer. Caflanone has been identified in a rare, flavonoid-rich cannabis cultivar native to Jamaica known as Black Swan. Little is known about the potential actions of Cannflavin A and Cannflavin C in cancer.

4.4. Silymarin

Silymarin is a flavonoid derived from milk thistle, but is also present in artichokes, cilantro, coriander, and cannabis. Silymarin consists of three phytochemicals, silybin, silidianin, and silicristin, and has a long medicinal tradition. Silybin is its most active phytochemical and is largely responsible for the effects of silymarin. A recent review by Delmas et al. [428] provided extensive details about mechanistic actions of silymarin in various models of cancer. The review highlighted silymarin’s ability to synergize with anticancer drugs, induce cell death through both the intrinsic and extrinsic pathways, cause cell cycle arrest in the G0/G1 and G2/M phase, modulate metabolizing enzymes and drug transporters which alter cellular sensitivity to chemotherapeutics, as well as several clinical trials currently in progress. More recently, in a Burkkett’s lymphoma model, silymarin induced apoptosis and caused a reduction in toll-like receptor 8 (TLR8) mRNA expression, implicating toll-like receptors in silymarin’s anticancer activity [367]. Silymarin further decreased cell viability, increased apoptosis, and changed the mitochondrial membrane potential in glioblastoma cells [368]. Silymarin reduced cell viability and diminished migration of stomach cancer cells through the induction of apoptosis, inhibition of p-ERK and activation of p-p38 and p-JNK. In vivo, silymarin at a concentration of 100 mg/kg reduced tumor volume and induced apoptosis [369]. Additionally, silymarin dose-dependently inhibited cell growth in prostate cancer cells by initiating apoptosis. After treatment, the expression of Slit Guidance Ligand 2 (SLIT2) and Roundabout Guidance Receptor 1 (ROBO1) were increased and the expression of CXCR4 was decreased [429]. Silymarin also had antiproliferative, antimetastatic and pro-apoptotic effects in a dose-dependent manner on liver cancer cells, and also acted through the Slit-2/Robo-1 pathway [370]. Co-treatment with cold atmospheric plasma (CAP) and a silymarin nanoemulsion (SN) decreased intracellular ATP levels and downregulated the PI3K/AKT/mTOR survival and RAS/MEK transcriptional pathways in melanoma cells [371]. Silibinin (30–90 μM), a main active component of silymarin, inhibited the epithelial-mesenchymal transition (EMT) in breast cancer cells. Silibinin also inhibited cell migration and increased mitochondrial fusion, which contributed to silibinin’s inhibitory effect on cell migration. Additionally, silibinin decreased ROS production, which decreased the NLRP3 inflammasome activation [372]. In silico, silymarin was shown to inhibit the proto-oncogene B-Raf (BRAF) and the smoothened gene (SMO), two targets in anticancer therapy [430]. A phase I clinical trial studied the effects of high dose silibinin (13 g daily) and found it to be well tolerated in patients with advanced prostate cancer. The most commonly seen adverse event was asymptomatic liver toxicity (hyperbilirubinemia and elevation of alanine aminotransferase) [373].

4.5. Luteolin

Luteolin is a flavone commonly found in several plants including broccoli, pepper, thyme, and celery. Luteolin has been used as a source of yellow dye since at least the first millennium B.C. and originally obtained from the plant Reseda luteola, a common weed. A recent review by Imran et al. [328] provides extensive information about the anticancer effects of luteolin in many cancers including breast, prostate, oral, lung, kidney, cervical, placental, ovarian, skin, liver, esophageal, bladder and glioblastoma. This review provides insight about mechanisms involved in luteolin’s anticancer effects ultimately leading to reductions in cell proliferation, cell survival signaling, angiogenesis, and metastasis and an increase in apoptosis in many of these types of cancers [431]. Since this review’s publication, more studies have further evaluated luteolin’s anticancer effects.

In vivo models of melanoma showed that luteolin inhibited cell growth through the extracellular matrix pathways, the oncogenic signaling pathway, and the immune response pathways, but not through ROS induction [378]. Furthermore, luteolin reduced proliferation, migration, invasion, adhesiveness, and tube forming potential in a metastatic melanoma model. HIF-1α/VEGF signaling-mediated epithelial to mesenchymal transition and angiogenesis was implicated in the anti-metastatic effects demonstrated by luteolin [379]. Luteolin caused double-strand DNA breaks and prevented nonhomologous end joining (NHEJ) and homologous recombination (HR) in a bursal lymphoma model, and additionally caused G2/M phase cell cycle arrest in BRCA-deficient cells and inhibited Poly [ADP-ribose] polymerase 1 (PARP1) [362,385].

Luteolin decreased cell viability in breast cancer cells as well as inhibited migration and invasion by decreasing the expression of matrix metalloproteinase-9 (MMP9) [378,379,432]. Apoptosis was induced through the extrinsic and intrinsic pathways and the epithelial-mesenchymal transition (EMT) was prevented. This was mediated by increased expression of miR-203, reduced Ras/Raf/MEK/ERK signaling, cell cycle arrest at the S phase, and by reducing telomerase levels through suppressing human telomerase reverse transcriptase (hTERT) expression [374]. S100 calcium-binding protein A7 (S100A7) has been implicated in the EMT, promoting metastasis, and was inhibited by luteolin through Src/Stat3 signaling in epidermoid carcinoma cells. This reduced migration and invasion of A431-III cells and decreased metastasis in a xenograft zebrafish model [384]. Similarly, miRNA-301-3p was downregulated in pancreatic cancer cells following treatment with luteolin, causing a decrease in cell growth [380]. In several ovarian cancer cell lines, luteolin induced apoptosis through the extrinsic and intrinsic pathways. The cell cycle was disrupted and cell invasion on the collagen was altered [375]. In non-small cell lung carcinoma, luteolin induced G2/M cell cycle arrest and reduced EMT by reducing the expression of absent in melanoma 2 (AIM2), leading to decreased AIM2 inflammasome activation which was also seen in lung cancer mouse xenograft models [376]. Masraksa et al. [389] reported that luteolin showed no cytotoxic activity on lung cancer cells up to 40 µM, however 20–40 µM was able to reduce migration, invasion and the formation of filopodia in a concentration dependent manner.

Luteolin has also had various anticancer effects in colon cancer cells. Luteolin reduced the viability and proliferation of colon cancer cells and increased the expression of pro-apoptotic and pro-autophagic proteins in a concentration-dependent manner. The EMT process was reversed after treatment, and the ERK/FOXO3a-dependant mechanism was implicated in these anticancer effects [381]. Luteolin reduced cell viability by inducing apoptosis and this effect was increased in p53-expressing cells. Treatment also reduced oxaliplatin-treated p53-null cell viability and colony counts further and may provide a new treatment for colon cancer cells resistant to oxaliplatin [386]. Luteolin increased the expression of pro-apoptotic proteins and antioxidant enzymes in colon cancer cells [433]. Treatment increased the number of sub-G1 phase cells and cells with fragmented nuclei and increased the expression of the Nrf2 promoter and altered its interaction with the tumor suppressor p53 [433]. Interestingly, one study found that luteolin did not impact cell proliferation of colorectal cancer in vitro or in vivo; however, it inhibited cell migration and invasion. The downregulation of pleiotrophin (PTN) via miR-384 was implicated in luteolin’s effects [390]. In liver cancer cells, luteolin reduced NF-κB transcription factor activation and decreased COX-2 gene expression. It also promoted cytotoxic effects including inhibition of proliferation, ER stress, and induction of apoptosis in a model lacking p53 [382].

The serine-threonine kinase CK2–overexpressed in all cancers where it promotes proliferation, spread, and survival–is inhibited by luteolin [383]. Luteolin induced apoptosis and inhibited the progression of rat prostate carcinogenesis in a transgenic rat for adenocarcinoma of prostate (TRAP) model in addition to a xenograft prostate cancer model where angiogenesis was also inhibited. In another human and rat cell model of prostate cancer, luteolin induced apoptosis by activation of caspases-3 and 7 [387]. Additionally, migration and tumorigenesis were inhibited, and apoptosis was induced in a glioblastoma model. Apoptosis was caused by depolarization of the mitochondrial membrane, ERK protein phosphorylation, cleavage of PARP and caspase-9, causing DNA damage by H2AX phosphorylation [388]. Bladder cancer cell survival was inhibited by luteolin by inducing G2/M cell cycle arrest, p21 upregulation and inhibition of mTOR signalling. In xenograft models, tumor volumes and dimensions were decreased after oral administration of luteolin [377]. When combined with the anticancer agent oxaliplatin, luteolin inhibited gastric cancer cell proliferation and induced apoptosis through the cytochrome-c and caspase pathways and by altering the cell cycle [391].

4.6. Orientin

Orientin is a flavone and glucoside derivative of luteolin. Orientin is found in various plants and flowers including the passion flower, bamboo leaves, açaí palm, buckwheat sprouts and in millets. Orientin had anti-migratory and anti-invasive effects in TPA-treated MCF-7 cells. Orientin downregulated TPA-induced membrane translocation of protein kinase C-α, phosphorylation of extracellular signal regulated kinase, and nuclear translocations of activator protein-1 and signal transducer and activator of transcription 3 and inhibited matrix metalloproteinase-9 and IL-8 expression. This resulted in reduced migration and invasion of the MCF-7 cells [392]. Similar inhibitory effects were shown in EC-109 esophageal squamous carcinoma cells in a time and concentration-dependent manner. Orientin-induced apoptosis occurred as a result of upregulated p53 and downregulated pro-apoptotic protein Bcl-2 [393]. Orientin also had cytotoxic and antiproliferative effects against human colorectal cancer HT29 cells. In these cells, orientin induced apoptosis and cell cycle arrest at the G0/G1 phase through the regulation of cyclin and cyclin-dependent protein kinases [394]. Additionally, orientin inhibited T24 human transitional cell bladder carcinoma cell proliferation, induced cell cycle arrest, and decreased the expression of inflammatory mediators such as NF-ĸB and components of the Hedgehog signaling pathway, at a concentration of 100 µM [395]. In vivo, orientin administration (10 mg/kg) resulted in antiproliferative effects on 1,2-dimethyl hydrazine (DMH)-induced colorectal cancer in rats, and improved tumor marker levels while decreasing proliferative marker levels, such as proliferating cell nuclear antigen (PCNA) and Ki67 [396]. Orientin also reduced the occurrence of colonic polyps and aberrant crypt foci and increased the antioxidant defense in rats with DMH-induced colorectal cancer, demonstrating its antiproliferative and antioxidant properties in vivo [397].

4.7. Vitexin and Isovitexin

Vitexin and Isovitexin are apigenin flavone glucoside, a chemical compound formed by the combination of apigenin with sugars found in the passion flower, chasteberry, and some bamboo leaves, among other plants. Isovitexin is also known as homovitexin or saponaretin. Anticancer effects have been observed for both these compounds. A review by Ganesan and Xu [434] provides insight into vitexin and isovitexin’s anticancer effects in various in vitro and in vivo models of cancer. The review highlights vitexin and isovitexin’s ability to inhibit cell growth, induce apoptosis, reduce autophagy, reduce cell migration, and provides further mechanistic details about the specific pathways involved. More recently, vitexin has been shown to reduce the viability of adenocarcinomic human alveolar basal epithelial cells dose-dependently, with virtually no toxicity to normal human bronchial epithelial cells. Vitexin induced apoptosis in these cells by increasing pro-apoptotic protein expression, reducing mitochondrial membrane potential and through Akt signalling [398]. In a melanoma model, vitexin inhibited growth in vitro and in vivo, arrested the cell cycle in G2/M phase, induced apoptosis, caused DNA damage, and increased ROS accumulation. Vitexin increased the ROS levels, causing DNA cytotoxicity leading to G2/M cell cycle arrest and apoptosis in BRAFi (BRAF inhibitor)-resistant melanoma cells [399]. Vitexin has shown anticancer effects in several colon cancer models. Chen et al. [402] showed that vitexin promoted apoptosis through p53, p53 upregulated modulator of apoptosis (PUMA) and Bax (Bcl-2-associated X protein) activation. When vitexin was combined with the anticancer agent 5-fluorouracil, a synergistic antitumor effect via PUMA induction was observed. In a multidrug resistant human, colon cancer cell line vitexin had cytotoxic effects by inhibiting autophagy and inducing apoptosis through decreasing autophagy related protein expression and increasing pro-apoptotic protein expression. Additionally, it induced apoptosis and suppressed tumor growth in a multidrug resistant human colon cancer xenograft model [403]. It was also noted that heat shock factor 1 (HSF-1) is a potential target of vitexin in this multidrug resistant model [435]. Both vitexin and isovitexin exhibited anticancer effects in a liver cancer model. These effects were achieved via blocking the STAT3 signaling cascade and reducing survival and invasion. When combined with doxorubicin and sorafenib, vitexin had apoptotic effects [405]. Isovitexin had antiproliferative effects in prostate cancer cells and osteosarcoma cells [400,401]. In the osteosarcoma model, isovitexin further induced apoptosis and caused epigenetic regulation through the DNA methyltransferase 1 (DNMT1)/miR-34a/Bcl-2 axis, causing the suppression of stemness and inducing apoptosis in the spheres derived from osteosarcoma cells. Isovitexin also decreased tumor growth and size in a murine xenograft model [401]. In liver cancer, isovitexin decreased sphere and colony formation rates and stemness-associated markers by downregulating FoxM1 via inhibition of MnSOD. Additionally, isovitexin inhibited liver tumor growth in a murine model [406]. In another hepatocarcinoma model, isovitexin caused miR-34a upregulation, which induced apoptosis and suppressed the stemness of SK-Hep-1 spheroids [404].

4.8. Quercetin

Quercetin, a flavonol, is commonly present in red onions, kale, grapes, berries, cherries, broccoli, citrus fruits, as well as in a variety of leaves, seeds, and grains. Quercetin is one of the most abundantly consumed dietary flavonoids, and has a bitter flavor [436]. A recent review by Tang et al. [437] has outlined in detail the anti-cancer actions of quercetin on many types of cancer including, breast, prostate, leukemia, ovarian, gastric, osteosarcoma, melanoma, glioma, lung, colon, and liver in vitro and in vivo. The review highlights the ability of quercetin to inhibit the cell cycle, induce apoptosis through the intrinsic and extrinsic pathways, and inhibit angiogenesis and metastasis in vitro. Quercetin was shown to have the ability to decrease tumor volume and increase survival rate of tumor bearing animals in vivo through apoptosis, and inhibit proliferation, angiogenesis and metastasis [437]. To complement this recent review, we will focus on the anticancer effects of quercetin demonstrated after its publication.

Quercetin treatment reduced cell viability and induced apoptosis in primary and metastatic colon adenocarcinoma cell lines [407]. Quercetin dose-dependently suppressed HGF- and TGFα-induced migration of hepatocellular carcinoma cells by inhibiting the signaling pathway of AKT, but not p38 MAPK [413]. In non-small cell lung carcinoma models, quercetin inhibited proliferation and anchorage-independent growth by inhibiting the Src-mediated Fn14/NF-ĸB pathway both in vitro and in vivo [408] and improved the radiosensitivity of these cells by altering the expression of miR-16-5p and Wee1 [414]. In vivo, lung adenocarcinoma appearance was delayed and increased non-neoplastic body weight gain in mice with tumor oxidative stress after daily treatment with quercetin was observed [420]. In a melanoma model, quercetin treatment decreased proliferation and promoted apoptosis in vivo and in vitro and was found to upregulate IFNα and IFNβ expression through activation of the retinoic acid-inducible gene I promoter [409]. In human prostate cancer cells, in vitro exposure to quercetin downregulated the expression of Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1), suppressed epithelial to mesenchymal transition, promoted apoptosis and inactivated the PI3K/Akt signaling pathway in both a time and dose dependent manner. Similarly, quercetin inhibited the proliferation of prostate cancer in a xenograft model [411,412].

Quercetin in combination with various compounds has been shown to produce enhanced cytotoxic effects in various cancer cells. When combined with the polyphenol resveratrol, quercetin inhibited cell growth and DNA damage, induced S phase cell cycle arrest and caused cell death in oral and pharyngeal cancer cells in a synergistic manner [410]. The combination of curcumin and quercetin lead to synergistic alterations in the expression of genes related to proliferation, apoptosis, cell cycle, inflammation, hypoxia and oxidative stress in myeloid leukemia cancer cells [438] and increased anticancer effects including apoptosis and reactive oxygen species production in breast cancer cells when loaded into apoferritin nanoparticles compared to either free compound alone [439]. Quercetin in combination with Lycopodium clavatum extract resulted in reduced cell growth in colon cancer cells. The combined treatment impacted mRNA expression of pro-apoptotic proteins [440].

Quercetin has been shown to have anticancer effects in cell lines resistant to traditional chemotherapeutic agents and in some cases improved the cytotoxicity of those agents. When combined with paclitaxel in a prostate cancer model, quercetin reduced cell proliferation and migration, while apoptosis, G2/M cell cycle arrest, ER stress and ROS were increased. Beneficial effects in a prostate cancer murine model were also observed, where co-treatment of quercetin with paclitaxel increased the effects of paclitaxel, while causing nearly no additional side effects [416]. The combination of doxorubicin and quercetin also produced favorable effects. In colon adenocarcinoma cells overexpressing the P-gp, quercetin improved the cytotoxicity of doxorubicin by inhibiting the ATP-driven transport activity of P-gp and increasing the intracellular accumulation of doxorubicin. It also downregulated the expression of the glutamine transporter solute carrier family 1, member 5 (SLC1A5) [417]. In multi-drug resistant breast cancer cells, the combination of doxorubicin with quercetin reduced cell viability and was mediated through doxorubicin-induced apoptosis. This was shown to decrease in vivo xenograft tumors without producing toxic effects [418]. Similarly, when combined with docetaxel, quercetin caused a reversal of docetaxel resistance in prostate cancer cells through decreased activation of the androgen receptor and PI3K/Akt pathway, fewer mesenchymal and stem-like cell phenotypes and lower P-gp expression. Features altered after treatment included decreased proliferation, colony formation, migration, invasion, and apoptosis. In vivo, xenograft tumors treated with the combination of quercetin and docetaxel had decreased growth [411]. Treatment with quercetin also decreased lymphoid enhancer-binding factor-1 (Lef1) in docetaxel-resistant breast cancer cells and re-sensitized these cells to docetaxel, acting synergistically to reduce the viability of the drug-resistant cells [415]. Quercetin acted in vitro in HeLa cells and in vivo as an inhibitor of the breast cancer resistance protein (BCRP) [421] while it induced cell death in HL60 cells and multidrug resistant HL60/VINC cells [441]. The cell death was mediated through cell cycle arrest, production of reactive oxygen species, caspase mediated apoptosis, and lysosome membrane permeabilization-dependent mechanisms. Finally, Quercetin exhibited cytotoxic and pro-apoptotic effects on gemcitabine-resistant hepatocellular carcinoma and pancreatic cancer cells. The combined treatment of quercetin and gemcitabine lead to an increased pro-apoptotic response, particularly through S phase cell cycle arrest, the upregulation of tumor protein p53 and downregulation of cyclin D1 [419].