Isoliquiritigenin (Figure 1C), a chalcone originated from dried roots of several Glycyrrhiza species (licorice plants), exhibits antioxidant, estrogenic, and anti-tumor activities (Guo et al., 2008). It has shown synergistic effect in combination with cisplatin in a xenograft mice model for CRC using CT-26 mouse CRC cells. In this mice model, an oral dose of 1 mg/kg of this chalcone, plus an intraperitoneal cisplatin injection of 5 mg/kg were able to reduce 79% the tumor growth. Also, addition of isoliquiritigenin to this cisplatin treatment was able to reduce liver and kidney damage, as transaminases (AST, ALT), creatinine and blood urea nitrogen levels were kept at normal concentrations, in contrast with control cisplatin treatment. With respect to oxidative damage, this combination therapy with isoliquiritigenin reduced nitric oxide serum levels, lipid peroxidation and GSH levels, in contrast to cisplatin treatment alone (Table 2; Lee et al., 2008). This point is very interesting, as major hepatic damages caused by cisplatin are bound to increased oxidative damage due to depletion in GSH levels and an increase in malonaldehyde and membrane peroxidation. Cisplatin treatment is also associated to increased serum levels of transaminases and bilirubin, two important markers for hepatic damage, following histopathological changes as necrosis and hepatocytes degeneration with infiltration of inflammatory cells around portal vein (Caro and Cederbaum, 2004; Dasari and Tchounwou, 2014). Increased liver damage due to cisplatin has been observed in patients with higher expression levels of cytochrome P450-2E1 (Caro and Cederbaum, 2004). The reduction achieved in cisplatin doses, if transferred to in vivo experiments, would contribute to a potential reduction in side effects caused by this drug, which usually are associated to ototoxicity, gastrotoxicity, myelosuppression, hepatotoxicity (due to ROS causing a reduction in GSH levels and an increase in malonaldehyde), cardiotoxicity (due to depletion in cardiac myocytes of lactate dehydrogenase and creatine kinase, following membrane peroxidation in these cells), and nephrotoxicity (due to inhibition of carnitine synthesis and its reabsorption by the proximal tube; Dasari and Tchounwou, 2014) (Figure 3).

Apigenin (Figure 1D) is one of the most widely distributed flavones in fruits and vegetables, such as parsley, Chinese cabbage, bell pepper, garlic, celery, and guava (Manach et al., 2004). It is a chemopreventive agent that has been shown to present strong cytostatic and anti-angiogenic effects in vitro (Hirano et al., 1989; Engelmann et al., 2002). In vitro, apigenin induces growth inhibition, cell cycle arrest, and apoptosis in CRC cells (Zhong et al., 2010; Lee Y. et al., 2014; Yang L. et al., 2015). Moreover, apigenin is a strong inhibitor of ABC transporters (ATP-Binding Cassette), which are responsible for the increase in the efflux of chemotherapeutic drugs in the lumen (apical) face of colonocytes, thereby significantly reducing its bioavailability and leading to its active detoxification (Katayama et al., 2007). Finally, apigenin is also responsible for NAG-1 [Nonsteroidal Anti-inflammatory Drug (NSAID) Activated Gene-1] overexpression in CRC cells, a member of the TGF-B (Transforming Growth Factor-B) superfamily which shows pro-apoptotic and antitumor activities (Yang et al., 2014). In fact, apigenin increases in a dose-dependent way in CRC cells, both in vivo and in vitro, NAG-1 and p53 expression, reducing intestinal tumor load and number (Zhong et al., 2010). Therefore, apigenin has a promising application as a safe antitumor agent. However, it has a modest antitumor activity against cancer cells when used alone, so new strategies are needed in order to enhance its effectiveness, as those based on combination therapy of this flavonoid with CRC drugs.

CD26 is a multifunctional cell-surface protein that is involved in the suppression of pathways responsible for tumor growth and metastasis. In fact, CD26 is down-regulated in several types of tumors including colon cancer and this protein is normally expressed in the epithelial cells of the human colon. Therefore, compounds which enhance CD26 levels are expected to have antitumor potential.

It has been shown that apigenin alone is able to cause an increase of 56.3% in the cell surface abundance and activity of CD26 in different CRC cell lines (HT-29 and HRT-18), so some authors have studied whether this flavone is able to enhance the up-regulation in CD26 cell surface expression of irinotecan, 5-FU and oxaliplatin, that are three chemotherapeutic agents used for the treatment of colorectal cancer. In the case of 5-FU and oxaliplatin, no specific interaction was reported with the action of apigenin; however, the ability of apigenin to potentiate CD26 was much more robust when was combined with increasing concentrations of irinotecan, generating a 4.2-fold increase in the potency of this drug (with a reduction of EC₅₀ for irinotecan from 4.68 to 1.26 μg/mL). An interaction was also observed when the experimental design was reversed, adding a fixed dose of irinotecan to a series of apigenin concentrations, increasing by 30 times the capacity of apigenin to enhance CD26 expression, lowering its EC₅₀ from 32.8 to 1.10 μM. Therefore, it was observed that in the case of irinotecan (a topoisomerase I inhibitor), but not of 5-FU or oxaliplatin, there is a specific interaction with the action of apigenin due to a cross-talk in the mechanism of action of apigenin with irinotecan, as apigenin is able to inhibit toposimorease I-DNA complex which overlaps with the primary mechanism of action of irinotecan (Lefort and Blay, 2011). This presupposes that part of the mechanism of action of apigenin is intimately related to topoisomerases.

TRAIL (tumor necrosis factor-related apoptosis-inducing protein), a member of the TNF superfamily, is able to induce apoptosis through interaction with the death receptor 5 (DR5), whose expression is regulated by the tumor suppressor p53. TRAIL is not toxic in normal cells because non-neoplastic cells express high levels of decoy receptors (DcR) for TRAIL, which could interfere with TRAIL signaling; it shows acquired resistance in cancer cells (Almasan and Ashkenazi, 2003; Du et al., 2016).

Apigenin induces the expression of DR5 in a dose-dependent manner preventing the degradation of this protein by acting as a proteasome inhibitor and increasing its expression in the membrane, so this up-regulation of DR5 acts in a synergic form sensitizing to the treatment with exogenous soluble recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in CRC DLD-1 cells, showing a greater apoptotic effect than the treatment with TRAIL alone. As said before, TRAIL is an attractive candidate for cancer therapy because it selectively induces apoptosis in cancer cells, and it has been shown that combination of TRAIL with apigenin did not induce expression of DR5 protein and enhanced TRAIL-induced apoptosis in normal human PBMCs cells (Horinaka et al., 2006). Therefore, the combined treatment of apigenin and TRAIL is a promising anticancer therapy.

Diverse anti-apoptotic proteins as Bcl-XL, Bcl-2, Bcl-w, and Mcl-1 can prevent cell death in tumor cells. ABT-263 (Navitoclax) is a novel oral inhibitor for Bcl-2 family proteins, acting as a Bcl-2 homology 3 (BH3) mimetic, and leading to apoptosis, except in tumor cells with Mcl-1 overexpression (Tolcher et al., 2015). As apigenin induces apoptosis in tumor cells by modulating different kinds of signaling pathways, including downregulation of Mcl-1 mRNA (Shi et al., 2015), this apigenin Mcl-1 downregulation may enhance the ABT-263 antitumor activity (Shao et al., 2013; Erdogan et al., 2016). Furthermore, the inhibitory effect of apigenin on ERK phosphorylation levels is significant when CRC HCT116 cells are cotreated with ABT-263 (Shao et al., 2013). All these findings were verified in vivo in a SCID mice model bearing HCT116 xenografts, in which treatment with ABT-263 or apigenin alone resulted in a 30% inhibition of tumor growth compared with untreated control, but this percentage was increased to 70% by combination therapy, with decreased Mcl-1 levels as well as phosphorylated prosurvival mediators ERK or AKT (Table 2; Shao et al., 2013).

Summing up, both apigenin and ABT-263 alone induced low apoptosis rates in HCT116, DLS1, SW48, HT29, and HCT-8 tumor cells, but in combination therapy, an increase of 80% in apoptosis was recorded via a caspase dependent mechanism. The combination index of these combinations was below 1.0, indicating a synergistic effect (Shao et al., 2013).

Chrysin (Figure 1E) is another flavone found in honey, propolis, and various plant extracts such as chamomile and blue passion flower (Passiflora caerulea; Renuka et al., 2016). Chrysin has multiple biological activities, including antitumor effects in diverse cancer cell lines and tumor animal models (Kasala et al., 2015).

Several studies in SW480 CRC cells have shown that this flavone is able to induce cell cycle arrest at G2/M transition in a dose-dependent manner. Combination of chrysin plus apigenin doubled the proportion of SW480 cells in G2/M arrest; indicating that both flavones cooperate in slowing down tumor progression (Wang et al., 2004). In vitro studies on DLD1 CRC cells have demonstrated a chemoprotective effect of chrysin due to induction of AhR activity (Aryl Hydrocarbon Receptor) accompanied by p21 overexpression, a cell cycle important inhibitor (Ronnekleiv-Kelly et al., 2012). At early tumorigenesis stages, chrysin shows a chemopreventive activity by modulating normal cryptal cells proliferation and by activating apoptosis in aberrant cryptal cells (as those generated in an azoxymethane animal model for CRC). These activities are carried out by downregulating PCNA (Proliferating Cell Nuclear Antigen) and growth factors such as IGF-1 (Miyamoto et al., 2006, 2010; Kasala et al., 2015).

Tumor necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine with a wide range of biological activities also including both cell progression and death. These TNFα conflicting activities rely on TNF receptor 1 (TNFR1) activation of two different pathways: a caspase cascade for induction of apoptotic events, and nuclear transcription factor kappa-β (NF-kβ), which is a cell survival mechanism. Generally, most tumor cells are refractory to TNFα-induced apoptosis if they keep a working NF-kβ pathway (Chen and Goeddel, 2002; Karin et al., 2004). But chrysin sensitizes HCT116 CRC cells (highly resistant to TNFα apoptosis induction) toward TNFα-induced apoptosis due to its blocking of NF-kβ/caspase 8 pathway, by downregulating its trigger, c-FLIP-I (Li et al., 2010). Combination therapy with chrysin and TNFα together showed a 40% increase in cell death compared to monotherapy, due to caspase 8 activation (Table 2; Chen et al., 2004; Romier et al., 2008; Li et al., 2010).

TRAIL binding to death receptors as DR5 results in adaptor protein FADD (Fas-associated protein with death domain) and procaspase 8 or 10 recruitment, which then activate this death pathway. The main negative regulator of this pathway is the cellular caspase-8 (FLICE) inhibitory protein (c-FLIP). Its overexpression causes resistance to this apoptotic process, thereby limiting the therapeutic use of TRAIL. In order to overcome these resistances, a combination with TRAIL pathway sensitizers targeting c-FLIP expression may be a promising approach. In this sense, chrysin is able to suppress c-FLIP expression and to enhance DR5 expression in HT-29 cells, enhancing TRAIL-induced cell death in CRC cells (Ding et al., 2012).

Flavonoids antioxidant activity can also reduce chemotherapy side effects. Cisplatin generates a wide variety of ROS that interact with DNA, lipids, and proteins in CRC cells, including Pt-DNA adducts that hinder cell division and DNA synthesis/repair, leading to apoptotic events (Dasari and Tchounwou, 2014). In this sense, the prophylactic effect of chrysin against colon toxicity due to cisplatin was tested in Wistar rats, confirming a protective effect by reducing oxidative stress (Khan et al., 2012). Although, cisplatin is not used in CRC patients' treatment, these experiments in rats show interesting effects of chrysin with respect to ROS inducing agents.

With a similar action to cisplatin, vanadium compounds are considered a new class of non-platinum metal compounds with eventual low toxicity, although they are not used in clinical praxis. In vitro, these compounds inhibit cell cycle even at low doses, by generating ROS, which leads to DNA cleavage and apoptosis. Chrysin complexation with vanadyl cation increased antitumor activity in HT-29 cells (cell cycle arrest in G2/M transition) compared to the monotherapy treatment. Chrysin vanadate complex reduced to 56% the HT-29 cell survival vs. 88% in the case of cisplatin. This chrysin potentiating effect may be due to a reduction in GSH levels, one of the most important antioxidant defenses in mammals (León et al., 2015). In order to avoid gastrointestinal damage in preclinical trials, different vanadium complexes have been generated with flavonoids (Evangelou, 2002).

Scutellarin is a glycoside of the flavone scutellarein (Figure 1F), isolated from the traditional Chinese medicine plant Scutellaria barbata (Xing et al., 2011). It has been used in HCT116 cells as chemosensitizing agent (at 100 μM) combined with resveratrol (at 200 μM) and 5-FU (at 500 μM). These experiments showed an increase in apoptosis, due co caspase 6 activation, which was absent in p53 (−/−) versions of this cell line (Chan et al., 2009).

Oroxylin A is a O-methylated flavone (Figure 1G) extracted from the herb Scutellariae radix. Oroxylin A inhibits iNOS and COX-2 gene expression by blocking NF-κB. Also, this flavone inhibits LPS-induced NF-kB activation by blocking IκB degradation, the protein which usually binds NF-kB in the cytosol, keeping it in its inactive form (Chen et al., 2000).

Combination of oroxylin A with 5-FU (1:5) both in vivo and ex vivo in a CRC model using HT-29 cells showed a synergistic action, with COX-2 inhibition and increased ROS generation, which led to HT-20 sensitization to 5-FU. 5-FU IC₅₀ in HT-29 is 4.63 mmol/L, but when combined with oroxylin A, this value diminishes to 764 μmol/L. To corroborate this synergistic effect, in a nude mice xenograft model for HT-29, 100 mg/kg oroxylin A plus 20 mg/kg 5-FU showed a 66% tumor size decrease, in comparison with 36 and 42% reduction in the monotherapies, respectively (Table 2; Ha et al., 2012). Therefore, oroxylin combination therapy could be a valuable tool in order to reduce 5-FU doses and subsequent in vivo side effects.

Kaempferol (Figure 1H) is a flavonol present in black tea, broccoli, propolis, grapefruit, and other plant sources. This compound has a marked antitumor potential on different types of cancer cells (Gutiérrez-del-Río et al., 2016). In CRC cells, it induces p53-dependent growth inhibition and, at the same time, apoptosis by inducing cytochrome c mitochondrial release and caspase-3 cleavage activation (Li W. et al., 2009; Lee H. S. et al., 2014b). In HT-29 cells, this flavonol induces apoptosis and inhibits IGF-IR and ErbB3 signaling (Lee H. S. et al., 2014a). Kaempferol is also able to induce G1 and G2/M cell cycle arrest by inhibiting the activity of CDK2, CDK4, and Cdc2 (Cho et al., 2013).

Monotherapy with kaempferol or TRAIL alone showed a slight effect on apoptosis induction in SW480 and DLD-1 CRC cells, while the combination therapy induced a dramatic apoptosis increase in a kaempferol dose-dependent manner. This means that kaempferol is able to sensitize these CRC cells to TRAIL-induced apoptosis. Interestingly, this combination of drugs showed very low cytotoxicity in PBMC normal cells (Yoshida et al., 2008).

Quercetin (Figure 1I) is an ubiquitous flavonol in nature, where it is found in onion, apples, and many other vegetables and fruits. Quercetin inhibits RASA1 expression in CRC cell lines, avoiding RAS activation and therefore its proliferative effects (Ranelletti et al., 2000). Quercetin was combined with TRAIL for treatment of three CRC cell lines, Caco-2 (adenoma), SW-620, and HT-29 (adenocarcinomas); demonstrating that this flavonol is a potent sensitizer to TRAIL-induced apoptosis in a synergistic manner (SW-620 and HT-29), whereas this combination resulted in an additive effect in the case of adenoma cells (Caco-2). These pro-apoptotic effects seem to be associated with a membrane distribution of quercetin in lipid rafts domains, regions which are rich in cholesterol, sphingolipids, and TRAIL death receptors. This membrane distribution could be the initiator for signal cascades causing TRAIL-mediated apoptosis (Psahoulia et al., 2007).

Quercetin has been also used in combination with 5-FU in vitro, treating CO115 (p53 positive) and HCT15 (p53 negative) CRC cell lines. This combination of drugs showed higher apoptosis levels in CO115 cell line, in a synergistic manner, but an additive effect in HCT15 cells. This enhanced apoptosis was even higher than with 100 times higher 5-FU concentration in monotherapy. p53 may elicit this synergistic pro-apoptotic effects by enhancing caspase 3 activation and diminishing Bcl-2 (anti-apoptotic) levels. This involvement of p53 is reinforced when a siRNA is used to silence p53 expression in CO115 cells, losing the synergistic effect of this combination of drugs (Table 2; Xavier et al., 2011).

(-)-Epigallocatechin-3-gallate (EGCG, Figure 1J) is the major polyphenolic constituent of green tea, representing 200–300 mg/brewed cup (Singh et al., 2011). The antitumor effects of this and other flavanols are widely supported by epidemiological, in vitro, animal and clinical studies (Singh et al., 2011). For example, different concentrations of grape seed extracts [rich in (-)-epicatechin] were tested in combination with 5-FU 100 μM in Caco-2 cells, showing a slightly synergistic effect on cell apoptosis (Cheah et al., 2014).

EGCG and related compounds are able to inhibit several critical signal transduction pathways in cancer cells. For example, EGCG inhibits multiple RTKs (Receptor Tyrosine Kinase) as the IGF/IGF1R system, EGFR, and HER2 receptors, which play key roles in CRC cell proliferation (Shimizu et al., 2005; Adachi et al., 2008, 2009). EGCG also blocks cell proliferation and cell migration in CRC cells, by inhibiting the signaling pathway TF (Tissue Factor)/VIIa/PAR2 (Protease-Activated Receptor 2) that usually mediates ERK1/2 phosphorylation and final activation of the pro-inflammatory NF-kβ. This lower activity of the transcription factor NF-kB induces an up-regulation of caspase-7 and a down-regulation of MMP-9 matrix metalloprotease expression, affecting proliferation and migration of tumor cells (Table 2; Zhou et al., 2012).

Furthermore, EGCG is an epigenetic regulator, which contributes to degradation of DNMT3A (DNA Methyltransferase 3A) and HDACs (Histone Deacetylases) through a process of ubiquitination in CRC cells sensitive to methylation. These effects, together with histones deacetylation, are very important epigenetic mechanisms in tumorigenesis, as they are responsible for silencing various tumor suppressor genes and other ones involved in cell cycle regulation and apoptosis (Moseley et al., 2013). Therefore, EGCG is able to restore the expression of genes involved in tumor suppression such as RXRalpha (Retinoid X Receptor alpha) that are silenced by epigenetic processes in tumor cells (Morris et al., 2016).

Also, it is remarkable the effect exerted by EGCG at the level of CSCs, downregulating Notch signaling, a membrane receptor, which is directly involved in the differentiation and proliferation of CRC stem cells (Jin et al., 2013).

Based on these anti-proliferative, anti-metastatic and epigenetic activities for EGCG, its effectiveness in combination with anticancer drugs has been tested. The combination of 5-FU with EGCG resulted in a synergistic growth inhibition in several human CRC cell lines (DLD-1, SW480, and COLO201) (Kumazaki et al., 2013). The same combination on HT-29 and HTC-116 CRC cells reduced cell viability significantly compared with the monotherapies. The EGCG potentiating effect on the 5-FU is supposed to be due to a downregulation in the expression of ABC transporters, which causes higher intracellular 5-FU concentrations (Hwang et al., 2007b; Wubetu et al., 2015).

Sodium butyrate is a non-toxic compound naturally produced in the colon after microbial fermentation of dietary fiber, which shows strong antitumor effects only on transformed colonocytes. Combination of EGCG and sodium butyrate on HT-29 tumor cells caused a synergistic reduction in survivin protein and mRNA levels, an anti-apoptotic protein highly expressed in CRC (Saldanha et al., 2014).

Another compound inducing apoptosis in tumor colonocytes, sulindac, is a NSAID inhibiting COX-1 (expressed constitutively in all tissues) and COX-2 (highly expressed in CRC and induced by cytokines). Although, sulindac has side effects due to this broad cyclooxygenases inhibition, its combination with EGCG reduces them due to an enhanced inhibition of COX-2 (Suganuma et al., 1999). This synergistic effect has been also observed in rats developing CRC by induction with the chemical inducer azoxymethane (Ohishi et al., 2002).

Genistein (Figure 1K) is an isoflavone which can be found in high concentrations in soybeans, lentils, beans, and chickpeas. Numerous epidemiological studies have reported a negative correlation between the incidence of CRC and diets rich in soybean (Spector et al., 2003; Rossi et al., 2006). This isoflavone has a growing interest as a pro-apoptotic agent because of its specific and almost exclusively activity against tumor cells (as CRC) rather than normal ones (Marín et al., 2015). Genistein acts by increasing the expression of pro-apoptotic proteins as Bax or p21 (Yu et al., 2004), by inhibiting NF-kβ (Luo et al., 2014) and topoisomerase II (Mizushina et al., 2013), by regulating ERB expression (Pampaloni et al., 2014), by suppressing the carcinogen induction of WNT/β-catenin signaling pathway (Zhang et al., 2013), by increasing the expression of antioxidant enzymes such as glutathione peroxidase (Ganai and Farooqi, 2015), and by preventing human CRC metastases due to MMP2 metalloproteases inhibition (Xiao et al., 2015). All these activities can be exploited by combinatory approaches in order to prevent or to treat CRC.

As it has been mentioned above, 5-FU is widely used in the treatment of solid tumors such as CRC, but its main clinical limitation is the development of resistant phenotypes by the over-expression of anti-apoptotic proteins or cell proliferation factors. In order to overcome this resistance problems, a combinated treatment with genistein on HT-29 cells resistant to 5-FU showed a significant reduction in cell viability compared to the monotherapy. These experiments demonstrated a synergistic effect on cell growth inhibition by over-expression of pro-apoptotic p53 and p21 genes and downregulation of survival genes such as Glut-1. However, the main mechanism involved in this combination was due to COX-2 expression inhibition (Hwang et al., 2005). In a similar way, combination of genistein with cisplatin also inhibited cell growth and induced apoptosis in a synergistic manner in HT-29 CRC cells, by inhibiting tyrosine kinases (Hu et al., 2014). Finally, combination with dexamethasone also shows synergistic effects by increasing p21 levels in Colo320 HSR cells, inhibiting their growth (Park et al., 2001).

Together with chemotherapy, radiotherapy also plays a crucial role in the treatment of rectal cancer, however, in more than 70% of patients, it causes side effects on the gastrointestinal system, as mucositis due to the generation of free radicals by ionizing radiation, which causes oxidative damage to normal colonocytes. A solution to this problem would be to combine radiotherapy with natural radioprotective agents (Jagetia, 2007). In this sense, the remarkable antioxidant activity of genistein, combined with its ability to activate antioxidant pathways, makes it a perfect candidate to protect against radiation cellular damage. Following this hypothesis, CT26 CRC cells were injected into BALB/c mice, and animals were treated with radiotherapy in the abdominal area. After a combination with genistein, this isoflavone reduced the apoptosis in normal cells and improved morphological changes in healthy intestinal mucosa. Also, tumors size was lower in mice subjected to combination therapy (Table 2; Son et al., 2013).

The epidermal growth factor receptor (EGFR) plays a very important role in tumor progression because binding of its ligands initiates a cascade of intracellular phosphorylations that ultimately triggers genes associated with cell proliferation, survival, or invasion. This receptor is over-expressed in tumor cells and diverse drugs inhibit this tyrosine kinase, although resistance phenotypes usually appear after irradiation (Singh et al., 2016). Interestingly, pretreatment with genistein during 24 h before irradiation was able to perform a synergistic effect on irradiated HTC116 cells survival (CI < 0.7), due to an enhanced EGFR inhibition (Gruca et al., 2014).

Silymarin is a flavolignan extract from milk thistle (Sylibum marianum). This flavolignans mixture contains silibinin (silybin A and B, the most active compounds, Figure 1L), isosilybin (A, B), silydianin, and silychrystin (Lee et al., 2006). Silybins induce cell cycle arrest and apoptosis by acting on cyclin dependent kinases (CDKs).

Silymarin has been tested in combination with doxorubicin and paclitaxel against CRC cells, in a cell line sensitive to doxorubicin (LoVo) and its multidrug resistant isogenic version (LoVo/DX). Twenty-four hour prior to treatment with both drugs, silymarin was used in these cells, showing a synergistic effect in LoVo cell line, but not in LoVo/DX cells, where an additive effect was observed. This additive effect may be valuable when dealing with in vivo experiments, as any contribution to reduce doxorubicin doses would also reduce its side effects, mainly associated to cardiotoxicity (Figure 3). This cardiotoxicity is due to formation of iron-related free radicals, as well as damages to mitochondrial NAD(P)H oxidase complex (Thorn et al., 2011). Silymarin causes higher intracellular drug concentrations in LoVo cells due to a repression of P-gp pump (P-glycoprotein, MDR1). However, in LoVo/DX cells, the strong P-gp overexpression prevents this sensitizing effect (Colombo et al., 2011).

Silibinin also enhances metformin antiproliferative effects. Combination of this antidiabetic agent at 10 mmol/L plus 100 μmol/L of silibinin in COLO205 CRC cell line showed a synergistic inhibition of 60% in cell survival, which did not affect normal HCoEpiC cells. Monotherapy at these concentrations had no effects. This combination of drugs increased caspase 3 activation and AIF expression, resulting in apoptosis activation by extrinsic and mitochondrial ways. A role for the PTEN/Akt pathway in this apoptosis induction in cancer cells was also observed, with increased PTEN levels and decreased phosphorylated protein kinase B (p-Akt) (Table 2; Tsai et al., 2015).

Vanadium (IV) complexes have been tested in combination with silibinin and chrysin against HT-29 cell line, showing increased cytotoxic effects at 100 μM vanadyl ion in comparison with monotherapies (280 μM vanadyl). In this combination, chrysin induces cell cycle arrest in G₂/M transition, while silibinin induces apoptosis due to caspases activation and NF-κB inhibition (León et al., 2015).

Flavopiridol (Alvocidib, Figure 1M) is a semi-synthetic flavonoid-like derivative, generated from rohitukine, an alkaloid from the bark of Dysoxylum binectariferum, a tree from India (Kelland, 2000). Flavopiridol inhibits cyclin-dependent kinases (CDKs), targeting the ATP-binding pocket of their catalytic subunit, as Cdk1, Cdk2, Cdk4, Cdk6, Cdk7, and Cdk9. Also, it inhibits other kinases like PKA, PKC, Erk-1, EGFR, and other receptor associated protein kinases. Flavopiridol blocks cell cycle in G₁/S and G₂/M transitions by lowering expression levels of cyclin D1, p27^Kip1, and p21^Waf1/Cip1. Low cyclin D1 levels cause a reduction in Cdk4 concentration, leading to an accumulation of hypophosphorylated retinoblastoma protein (Rb), which causes cell cycle arrest. Low p27^Kip1 or p21^Waf1/Cip1 levels also cause a reduction in Cdk2 concentration, inducing cell cycle arrest in G₁phase and inhibiting EEF1B2 elongation factor, which blocks RNApol II transcription of Newcomb (2004).

Flavopiridol is a potent apoptosis inducer in tumor cells, via the mitochondrial pathway (release of cytochrome c or caspases activation), but also via AIF (apoptosis-inducing factor) pathway (Achenbach et al., 2000). This pro-apoptotic effect is enhanced as flavopiridol also inhibits Akt activation, leading to NF-κB inactivation and therefore to an inhibition of proliferation processes (Takada and Aggarwal, 2004). Therefore, this flavonoid derivative triggers apoptosis and at the same time inhibits proliferation.

Irinotecan, a semisynthetic analog of the natural alkaloid camptothecin, and SN-38, the irinotecan bioactive metabolite, prevents DNA from unwinding by inhibiting topoisomerase 1. Combination of SN-38 treatment followed by flavopiridol in HCT116 cell line and its null isogenic p53 (−/−) equivalent showed apoptosis induction only in the p53 wild type cell line. Here, p53 produced Rad51 mRNA downregulation, a gene coding for a DNA-repair protein (Ambrosini et al., 2008).

In a mouse xenograft model with HCT116 cell line, irinotecan treatment followed by flavopiridol showed a significant decrease in tumor growth compared to monotherapies. This study suggests changes in the choline kinase activity and decreased phosphocholine (Motwani et al., 2001; Darpolor et al., 2011). Since diarrhea is one of the most common side effect associated to irinotecan treatment, this synergistic effect among flavopiridol and irinotecan may be a valuable combination for preventing or reducing this gastrointestinal toxicity associated to this camptothecin derivative (Fuchs et al., 2003) (Figure 3).

Sequential treatment with docetaxel, flavopiridol, and 5-FU in HCT116 cell line showed an 8-fold increase in caspase activity, with much lower increase if the three compounds were added simultaneously, in pairs or separately. Using this triple combination in mice xenografts with HCT116 caused a decrease in tumor volume by 95% (50% reduction for single drug treatment, 70% reduction for two drugs combination; Table 2; Guo et al., 2006).

A phase I trial of weekly, sequential docetaxel followed by flavopiridol (after 4 h first treatment) in patients with advanced solid tumors showed that this combination of drugs was well tolerated, with one dose-limiting toxicity occurring at 70 mg/m² flavopiridol. Docetaxel common toxicity effects are mostly associated to neutropenia (Ho and Mackey, 2014). Also, one complete response was observed in a patient with pancreatic carcinoma, as well as four partial responses in pancreatic (1), breast (2), and ovarian (1) tumors. Stable disease was observed in ten patients (27 patients in total; Fornier et al., 2007).

Artesunate (Figure 2C) is the hemisuccinate ester of artemisin, a sesquiterpene found in Artemisa annua (a traditional Chinese herb), widely used for malaria treatment as ROS inducer in the Plasmodium parasite (Meshnick, 2002). Artesunate is cytotoxic in HCT116 cells, inducing a cell cycle arrest at G₁, due to cyclin D1 downregulation and p21 overexpression. The treatment of these CRC cells with artesunate (1.9 μM) or oxaliplatin (another agent also causing ROS stress in cells, together with other alkylating activities on DNA; 4 μM) causes 50% cell killing. However, the same effect can be obtained with a combination of just 0.65 μM artesunate plus 1.6 μM oxaliplatin, which reinforces the used of artesunate as a possible adjuvant chemotherapy molecule (Liu et al., 2011).

The monoterpene geraniol (Figure 2A) is a main component in commercially important essential oils (rose, lemon, etc.), with wide use in perfumes. In vitro combination of geraniol (150 μM, IC₃₀) plus 5-FU (0.25 μM, IC₃₀) on Caco-2 cells increased the cell death in comparison with monotherapy, causing over 20% reductions cell survival. In a tumor xenograft model for TC118 CRC cell line, combination of geraniol (150 mg/kg) plus 5-FU (40 mg/kg) showed a clear 83% reduction in tumors size, whereas monotherapies with these concentrations caused only 26 and 30% tumor reductions, respectively (Carnesecchi et al., 2004).

In a CRC animal model using the mutagen dimethylhydrazine, oral geraniol (25 mg/100 g) was administered in order to test its effects in CRC prevention at early stages (aberrant crypt foci reduction in colon mucosa, ACF). After 9 weeks, a significant 37% reduction in colon ACF was observed with respect to control animals, together with enhanced apoptosis parameters. This protection was also accompanied by a 30% reduction in the cellular levels of Bcl-2 in geraniol treated animals, an anti-apoptotic protein, whose low levels may explain the geraniol antitumor effect in colon mucosa (Table 3). These results may open the path to the study of other non-cyclic monoterpenes as antitumor agents (Vieira et al., 2011).

Irofulven (Figure 2B) is a semi-synthetic derivative of illudin S, a sesquiterpene isolated from the mushroom Omphalotus illudens. It shows potent growth inhibition on a wide variety of human solid tumor cell lines and primary tumor cell types. In vivo testing has demonstrated excellent dose-related antitumor activity in several human tumor mice xenograft models.

Combination of irofulven with radiation or chemotherapeutic agents such as paclitaxel, irinotecan, 5-FU, mitomycin C, thiotepa, topotecan, and cisplatin have produced additive and/or synergistic inhibition of cellular proliferation in a variety of tumor types. With respect to CRC, simultaneous exposure to irofulven and cisplatin is at least additive for HCT116 cells, whereas simultaneous exposure to irofulven and 5-FU is additive for HT-29 cells and synergistic for the irofulven-resistant HCT116 cell line (Poindessous et al., 2003). Combination of irofulven with oxaliplatin also led to synergistic activity in HT-29 cell line (Serova et al., 2006). In a mice xenograft model for HT-29 cells, combination of irofulven and irinotecan, significant reduction in tumor weights occurred with partial responses in nearly all of the animals and some animals achieving complete responses (Table 3; Britten et al., 1999).

Triptolide (Figure 2D) is a diterpene from Tripterygium wilfordii tree, used as anti-inflammatory and antitumor in traditional Chinese medicine. Individual treatments with triptolide or oxaliplatin during 48 h in SW480 cell line showed IC₅₀ values of 16.7 ng/mL and 20.8 μg/mL, respectively. However, in combination with 10 μg/mL oxaliplatin, 8 ng/mL of this diterpene was able to induce 62% apoptosis. This synergistic effect was due to an inhibition of nuclear translocation of the transcription factor β-catenin under combinatory conditions, causing that this cell progression factor remains accumulated in the cytoplasm. Also, in a mice xenograft model for this cell line, combination of triptolide (0.1 mg/kg) with oxaliplatin (5 mg/kg) also showed this synergistic effect, reducing tumors growth by 60% (Table 3). This positive effect was not accompanied by a significant increase in ALT and AST transaminases (biomarkers for hepatic damage) nor in blood urea nitrogen (biomarker for renal damage; Liu et al., 2014). In a similar way, combination of triptolide (0.15 mg/kg) plus 5-FU (12 mg/kg) in another xenograft model with HT-29 cell line caused a reduction of 96% tumor growth for 3 weeks treatment, with no side effects observed (Tang et al., 2007). These results open the way for the use of triptolide in the treatment of solid tumors in preclinical trials (Jiang et al., 2001; Fidler et al., 2003).

The triterpene ursolic acid (Figure 2E) is found in diverse herb species as basil and rosemary. This antioxidant compound is able to modulate cellular redox status in normal cells, but in tumor cells it exerts pro-oxidative action. This is an important fact when dealing with tumor cells and radiotherapy, as ionizing radiation works by increasing cell oxidative damage in transformed cells, leading to apoptosis. In this sense, radio-resistance status can be avoided or reverted by using drugs able to increase ROS and its mitochondrial, DNA and membranes damages.

Using in vitro experiments with CT26 mouse CRC cells, a synergistic effect has been described for a combination of ursolic acid plus radiotherapy, where apoptosis was enhanced 55%, via caspase 3 activation. In these co-treated tumor cells, it was observed higher peroxides formation, lower GSH levels and extended mitochondrial damage (Koh et al., 2012). These pro-apoptotic effects of ursolic acid have been reproduced in other CRC cell lines as HCT116, where this compound was able to reduce the pro-inflammatory NF-kB cytokine, the pro-metastatic MMP-9 matrix metalloprotease, and the survival effectors Bcl-2 and survivin. All these changes in expression of key cancer modulators were reinforced when conducting these in vitro experiments with ursolic acid and capecitabine together (Table 3; Prasad et al., 2012).

In a mouse xenograft model with HCT116 cells, combination of ursolic acid with capecitabine caused a 68% reduction in tumor volume, also diminishing distant metastasis to lung around 60% (Prasad et al., 2012).

Panaxadiol is a ginsenosides (Figure 2F) triterpene found both in ginseng (Panax ginseng) and in notoginseng (Panax pseudoginseng). Previous studies have shown the antitumor activities of these compound on several cell lines and their targeting on multiple cancer signaling pathways (Park et al., 1999; Jin et al., 2003; Gao et al., 2013). Notoginseng extract, which contains high amounts of ginsenosides, enhances 5-FU induced apoptosis in human CRC cells (Wang et al., 2007a,b). Looking for specific bioactive components in these extracts, panaxadiol was found to be the component which caused enhanced apoptosis in HCT116 cell line (Li X. L. et al., 2009). Similar results were obtained with protopanaxadiol, another ginseng metabolite that significantly enhanced 5-FU effects on HCT116 cells by inducing arrest in G₁ phase and apoptosis. These in vitro data were confirmed by using an in vivo mice xenograft model, showing that protopanaxadiol and 5-FU co-administration very significantly reduced the tumor size in a dose-related manner (Table 3; Wang et al., 2015).

Another member of this family, ginsenoside Rg3, is able to repress NF-kB expression in HCT116 cell line, leading to apoptosis, with a IC₅₀ value of 100 μM. NF-kB is usually activated in these and other tumor cells. Twenty-four hour combination of 50 μM ginsenoside R3 plus 5 μM docetaxel in this cell line resulted in a synergistic NF-kB inhibition, absent in the monotherapy experiments at these concentrations. This pro-apoptosis effect was due to Bcl-2 repression and expression of the pro-apoptotic proteins caspase 3 and Bax, which demonstrated the chemosensitization of these tumor cells to docetaxel in the presence of ginsenosides R3 (Kim et al., 2009).

Celastrol (Figure 2G) is a triterpene from the bark of the T. wilfordii tree. This compound inhibits the heat shock protein HSP90, blocking its interaction with Cdc37. TRAIL addition to cultures of SW620 CRC cells shows an IC₅₀ value of 423.5 ng/mL, whereas this parameter is reduced to only 121.1 ng/mL in the presence of 2 μM celastrol during 72 h, due to apoptosis induction via caspase 3; therefore, celastrol shows a synergistic effect in combination with TRAIL (Zhu et al., 2010).

Betulinic acid (Figure 2H) is an anti-inflammatory and antimalarial triterpene isolated from diverse plants, as Betula pubescens tree (birch) and many others. This compound exerts apoptosis through caspase 3 induction in SNU-C5 CRC cell line with IC₅₀ value of 1 μg/mL. Three resistance variants were originated from this cell line, showing increased resistance to 5-FU (total resistance, instead IC₅₀ of 6 μg/mL in parental cell line), irinotecan (5.55-fold IC₅₀ instead of IC₅₀ of 18 μg/mL in parental cell line), and oxaliplatin (total resistance, instead IC₅₀ of 100 μg/mL in parental cell line). These three resistant variants were more sensitive to betulinic acid alone than the parental cell line, but more interestingly, combination of betulinic acid plus 5-FU reverted apoptosis induction in the 5-FU resistant cells. A similar reversion effect was observed with a combination of betulinic acid plus oxaliplatin in oxaliplatin-resistant cells. These results clearly demonstrate that in some cases it is possible to circumvent acquired chemoresistance by combination therapy of anticancer drugs with chemosensitizers as betulinic acid (Jung et al., 2007).

Fucoxanthin (Figure 2I) is a tetraterpenoid carotenoid found in the edible macroalga Undaria pinnatifida, which has been associated to prevention of CRC (Kim et al., 1998). In vitro studies with Caco-2 cell line have shown that this carotenoid is able to induce apoptosis after 72 h exposure at 22.6 μM. This apoptosis induction was due to an 80% reduction in Bcl-2 protein levels, a survival factor (Hosokawa et al., 2004).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.