Phytochemicals: Potential Lead Molecules for MDR Reversal

Multidrug resistance (MDR) is one of the main impediments in the treatment of cancers. MDR cancer cells are resistant to multiple anticancer drugs. One of the major mechanisms of MDR is the efflux of anticancer drugs by ABC transporters. Increased activity and overexpression of these transporters are important causes of drug efflux and, therefore, resistance to cancer chemotherapy. Overcoming MDR is a fundamental prerequisite for developing an efficient treatment of cancer. To date, various types of ABC transporter inhibitors have been employed but no effective anticancer drug is available at present, which can completely overcome MDR. Phytochemicals can reverse MDR in cancer cells via affecting the expression or activity of ABC transporters, and also through exerting synergistic interactions with anticancer drugs by addressing additional molecular targets. We have listed numerous phytochemicals which can affect the expression and activity of ABC transporters in MDR cancer cell lines. Phytochemicals in the groups of flavonoids, alkaloids, terpenes, carotenoids, stilbenoids, lignans, polyketides, and curcuminoids have been examined for MDR-reversing activity. The use of MDR-reversing phytochemicals with low toxicity to human in combination with effective anticancer agents may result in successful treatment of chemotherapy-resistant cancer. In this review, we summarize and discuss published evidence for natural products with MDR modulation abilities.


INTRODUCTION
Besides surgery and radiation, chemotherapy is one of the standard treatments of cancer. Drugs used in chemotherapy usually disturb cell division by inhibition of microtubule formation or disassembly (vinca alkaloids, paclitaxel), DNA topoisomerase (camptothecin and derivatives) or they intercalate or alkylate DNA (doxorubicin, cisplatin) (Wink, 2007;Wink et al., 2012). Furthermore, chemotherapy often causes extreme side effects as these drugs also affect the division of normal cells or they cause mutations, which can lead to secondary cancers. This additionally leads to restriction of the therapeutic applications; both dosage and application interval must be kept limited. When chemotherapeutic agents are used, it is often a matter of time before the cancer cells develop resistance against them. One of the major resistance mechanisms is the overexpression of ABC transporters, which can pump out the chemotherapeutic from cancer cells. Because these ABC transporters have a wide substrate spectrum, they not only confer resistance to a single drug but to several others, therefore, the term Multidrug Resistance (MDR). Since multiple drug resistance is a major issue in tumor therapy, new strategies are necessary to overcome this obstacle. One strategy involves the combination of anticancer drugs with modulators of ABC transporters. Our review presents a summary about various modulating effects of phytochemicals.

Multidrug Resistance in Cancer
One of the major difficulties in suppressing growth and survival of cancer cells is multidrug resistance. MDR is the resistance of cancer cells to various types of anticancer drugs which may have an intrinsic or acquired origin. Acquired resistance is induced after the administration of chemotherapy, whereas, intrinsic resistance already exists prior to drug application in cancer cells . Several mechanisms in cancer cells can lead to MDR (Coley, 2008). These include changes in target enzymes, such as DNA topoisomerases (Brown et al., 1995), alteration in microtubule-associated proteins (Zhang et al., 1998), mutations or changes in tubulin (Kamath et al., 2005;Wang and Cabral, 2005), alteration in microtubules (Kavallaris et al., 2001), mitotic arrest (Kamath et al., 2005), mutated protein p53 (O'Connor et al., 1997), disruption in DNA repair due to the damaging effect of an anticancer drug (Bernstein et al., 2002) and the impairment of apoptosis or genes involved in apoptosis and necrosis (Tanaka et al., 2000;Simstein et al., 2003).
A widespread mechanism of MDR is drug efflux via transmembrane transporters known as ATP-binding cassette transporters (ABC transporter). Overexpression of these transporters is the most important cause of drug resistance in many cancer cells. The family of ABC transporter proteins has 48 members in humans and far more in nature (Chen et al., 2016). The most well-known and widely-studied ABC transporters include P-Glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), and breast cancer resistance protein (BCRP). These transporters are expressed in healthy cells of various mammalian tissues having physiological tasks for translocating small molecules. They are found especially in intestinal epithelial cells, endothelial cells of blood capillaries and epithelia of renal proximal tubules being involved in the excretion and clearance of endogenous and exogenous cytotoxic substances (Borst et al., 2000;Durmus et al., 2015;Chen et al., 2016). ABC transporters evolved in nature millions of years ago to eliminate toxic phytochemicals that herbivores would obtain from their plant diet (Wink, 2007). Anticancer drugs also can be substrates of these transporters and if being considered as foreign they get exported to the extracellular space by cells expressing ABC transporters. Cancer cells which are not resistant to anticancer agents yet can develop the ability by overexpressing these transporters for saving themselves from the substances being toxic to them. This leads to an increased efflux, leading to low intracellular drug concentrations, insufficient to kill a cancer cell.
Once this overexpression has occurred, the efflux also affects other chemotherapeutics and thus, makes the cancer cell resistant to chemotherapy (Coley, 2008;Durmus et al., 2015).

P-gp (ABCB1)
P-gp is a 170 kDa protein which is encoded by the MDR1 gene. This transporter is found in normal cells of various tissues including the brain, liver, kidney, gastrointestinal tract and pancreas. P-gp transports anticancer drugs such as paclitaxel, doxorubicin, daunorubicin, epirubicin, mitoxantrone, vincristine, and vinblastine against the concentration gradient using energy derived from hydrolysis of ATP (Chen et al., 2016). Chemotherapeutic agents can stimulate P-gp expression in cancer cells and thereby cause resistance to chemotherapy. Chemotherapy has been reported to increase the proportion of P-gp-expressing tumors by approximately 1.8-fold in breast cancer. Moreover, in patients with activated P-gp transporter in their tumors, the risk of failure of chemotherapy is 3 times higher than in patients who do not express P-gp transporter (Trock et al., 1997).

Multidrug Resistance Proteins (MRPs)
Another class of membrane transporters which causes MDR is MRPs. Nine members of this class have been identified so far (König et al., 2005;Coley, 2008). MRPs are found in normal cells of some mammalian tissues and expel drugs as a complex with glutathione, glucuronate, or sulfate (Borst et al., 2000;Coley, 2008). Among the MRP transporters, MRP1 (ABCC1) is the most important and most studied one regarding MDR. The MRP1 protein has a molecular weight of 190 kDa. Similar to Pgp, MRP1 expression has been reported to be considerably higher expressed in cancer cells after chemotherapy than before chemotherapy (Trock et al., 1997). Therefore, MRP1 enhances resistance to chemotherapy and to anticancer drugs such as doxorubicin, daunorubicin, epirubicin, vincristine, and vinblastine (Coley, 2008).

BCRP (ABCG2)
Breast cancer resistance protein, also called mitoxantrone transporter (MXR1), has a molecular weight of 72 kDa. BCRP is extensively expressed in MCF-7 breast cancer cells (Doyle et al., 1998). This protein is also expressed in other tissues including the liver, kidney, and intestine (Chen et al., 2016). The anticancer drugs doxorubicin, daunorubicin, epirubicin and mitoxantrone have been described as substrates of BCRP transporter (Coley, 2008). Thus, cancer cells overexpressing BCRP transporter become resistant to these drugs.

MDR Modulators
One of the essential requirements for developing better anticancer therapies is overcoming multidrug resistance. Much research has been carried out on cancer treatment and development of anticancer drugs in recent years but MDR to cytostatics is still a great impediment. Although our knowledge about the mechanisms of multidrug resistance has increased, there is no effective drug which can completely overcome or reverse resistance at non-toxic concentrations. Since ABC transporters play a fundamental role in resistance to chemotherapy, the ability to inhibit them in a combination with conventional treatments will greatly help to treat cancer (Chen et al., 2016).
Until now, different types of ABC transporter inhibitors have been examined. The use of the first generation of these compounds, including verapamil and cyclosporine A, in combination with anticancer drugs had poor clinical success and toxic effects (Daenen et al., 2004). Second generation of MDR modulators included dexverapamil, valspodar, and dexniguldipine. Even though less toxic and with a higher therapeutic index than the first generation, this group of modulators is not well suited for a therapy either, both because of its interactions with other drugs and ABC transporters, as well as due to the inhibition of enzymes like CYP3A (Wandel et al., 1999;Syed and Coumar, 2016). The third-generation ABC transporter modulators do not have the disadvantages of the first and second generation. They are potent and noncompetitive inhibitors of P-gp, and also less toxic. Tariquidar (XR9576) and zosuquidar are members of the third generation of MDR modulators but unfortunately they were not efficative in clinical trials (Cripe et al., 2010;Kelly et al., 2011).

Phytochemicals
Alkaloids (Figure 1) are the most widely studied group of secondary metabolites in terms of MDR, not only because of their quantity but also because of their great diversity (Wink, 2007;Wink et al., 2012). As alkaloids have a wide distribution among angiosperms (Wink, 2020) and represent a diversity of structures, they differ in pharmacological and toxicological properties. Alkaloids contain heterocyclic nitrogen, which mostly has its origin in amino acids (Cseke et al., 2006). Alkaloids are subdivided into many subcategories of special functional groups, similarities of skeleton or biosynthetic pathways.
Quinoline and isoquinoline alkaloids for example have a benzopyridine ring differing in the position of their nitrogen. Quinazoline alkaloids have a similar aromatic structure but with two nitrogen atoms instead of one. Each of these structures has found several uses depending on molecular structure. There are many examples for quinoline alkaloids such as mefloquine as antimalarial agent, fluoroquinolone antibiotics and topotecan as anticancer drug, just to name a few (Collin and Höke, 2000;Tiwary et al., 2015). The latter two work as inhibitors of different DNA topoisomerases (Lemmer and Brune, 2004).
Quinolizidines also are cyclic nitrogen-containing compounds but unlike the previously mentioned subgroups they are not aromatic. A natural representative is sparteine, which is used as an antiarrhythmic agent blocking sodium channels (Ruenitz and Mokler, 1977;Körper et al., 1998;Gawali et al., 2017).
Among other groups of alkaloids, indole, monoterpene indole and b-carboline alkaloids show many pharmacological activities (Gilbert, 2001). A typical basic structure of b-carboline alkaloids consists of benzene fused with a five-membered pyrrole and is consequently similarly structured to some endogenous hormones and neurotransmitters such as serotonin and melatonin. In addition to benzene, the pyrrole ring of bcarboline is fused to a pyridine, another six-membered nitrogen-containing ring. This structure by itself is an inverse agonist of GABA-receptors, which involves psychological influence on humans (Aktories et al., 2017). Substrates among the indoles target many receptors, for example PDE-receptors e.g. by tadalafil, 5-HT receptors e.g. by naratriptane and HMG-CoA reductases e.g. by fluvastatin (Wink, 2000; de Sa et al.,   (Wink, 2000;Wink and van Wyk, 2008). As the nitrogen of steroidal alkaloids does not originate from amino acids they belong to pseudoalkaloids. A member of steroidal alkaloids is the teratogenic cyclopamine, which can cause cyclopean eyes in vertebrates (Roberts and Wink, 1998;Incardona et al., 1998).
Piperidine and diketopiperazine are six-membered nonaromatic moieties in alkaloids whereby diketopiperazine is a cyclic dipeptide having two oppositely located nitrogen. Pyrazine has the same position of nitrogen, though aromatic. Here we also can find medicinal use of active ingredients such as the oxytocin antagonist retosiban and plinabulin which is still in clinical trial against multiple drug resistant non-small cell lung cancer (Borthwick and Liddle, 2011;Mohanlal et al., 2019).
Tropane alkaloids are widespread and their plants one of the oldest medicines to use because of spasmolytic, mydriatic and hallucinogenic properties (Wink and van Wyk, 2008;van Wyk and Wink, 2017). They contain a special bicyclic moiety which is made of a seven-membered ring and a nitrogen atom which is linked to its C-1 and C-5 and forms the second ring (Osman et al., 2013). Due to their spasmolytic effect nowadays we use tropane alkaloids such as scopolamine or atropine for digestive tract spastic conditions and for ophthalmological purposes (Kukula-Koch and Widelski, 2017).
The most popular alkaloid caffeine belongs to purine alkaloids, which are consumed by many people on all continents. Theobromine, theophylline and caffeine are common members found as main ingredients in chocolate, mate, cola, green and black tea or coffee (Baumann and Frischknecht, 1988). By inhibiting adenosine receptors and cAMP phosphodiesterase they can mediate a stimulant effect (van Wyk and Wink, 2017).
Flavonoids ( Figure 2) are another complex but also often colored group of secondary metabolites. Unlike alkaloids, we can make a general statement about their common origin and basic skeleton. Flavonoids can be classified as polyphenols which share a common biosynthesis. They contain aromatic rings with phenolic hydroxyl groups. These phenolic hydroxyl groups can dissociate under physiological condition and form negatively charged phenolate ions. Because of these properties flavonoids and polyphenols can interact with proteins forming multiple  hydrogen and ionic bonds (Wink, 2015). Flavonoids are widely distributed in plants and are responsible, inter alia, for their pollinator attracting colors, ultraviolet light protection, antioxidant and antimicrobial functions, and mediating symbiosis with bacteria. Including the main subgroups flavones, isoflavones, flavonols, flavanones, anthocyanins, chalcones and catechins, they derive from flavan, a benzopyran structure with a phenyl ring in position 2. Flavonoids with a phenyl ring in position 3 and 4 are called iso-and neoflavonoids, respectively. The variety of flavonoids comes from many functional groups and different states of oxidation of the heterocycle (Koes et al., 1994;Wink and van Wyk, 2008;Hänsel and Sticher, 2009). Although there has been a lot of research on the antioxidant capacity of flavonoids, the mechanism is not fully understood yet. Many studies have reported anti-inflammatory, anti-carcinogenic, anti-mutagenic, antiviral, anti-allergic and osteogenetic potentials of flavonoids in vitro. There is evidence, that polyphenols are also important for the pharmacological activity of many medicinal plants (van Wyk and Wink, 2017). Still there is a lack of information about how the necessary bioavailability is achieved in the human body as polyphenols are polar compounds (Panche et al., 2016). Most common representatives in food are luteolin and apigenin.
Isoflavones are known for their estrogenic properties (Wink, 2015). In addition to flavonoids, there are smaller but also important groups of polyphenols, for example stilbenoid with resveratrol as its main member known for its potential as anti-cancer , antioxidant and anti-aging agent (Alamolhodaei et al., 2017). Curcuminoids (Figure 3) have been widely studied and have been found to have many functions such as antioxidative, anti-cancer, anti-microbial and antiinflammatory effects in humans. They can interact with many targets (Fantini et al., 2015).
Terpenes ( Figure 4) are widely distributed in plants, fungi and animals. They are composed of different numbers of isoprene units, forming mono-(C10), di-(C20), sesqui-(C15), tetra-(C40) and triterpenes (C30). Many mono-and sequiterpenes are volatile and aromatic and typical ingredient of essential oils. These compounds are often lipophilic and can thus modulate the fluidity and permeability of biomembranes in animals and microbes. Many plants with essential oil have been used in traditional medicine for treatment of microbial infections and inflammation (Wink, 2015;van Wyk and Wink, 2017).
Known for their skin permeation enhancing ability, terpenes have been used as moieties of synthetic structures for topical  use (Smith and Maibach, 1995). In vitro studies have shown anti-cancer, antimicrobial and antioxidant activities but for practice and use in humans this class of secondary metabolites must be further investigated (Lu et al., 2012;Cör et al., 2018). Squalene is a standard triterpene produced in plants and animals, and it is the precursor for steroid synthesis. Glycosides of steroids or triterpenes, so-called saponins have one or more polysaccharides attached saponins are amphiphilic and react as a detergent. They generally form stable foams and complex cholesterol in biomembranes. As a consequence, saponins can lyse biomembranes (Bloch, 1983). In traditional medicine they have been used for example as expectorants and anti-infectants (Wink, 2015). As effective components of vaccine adjuvants they enhance the cellular immune response (Sun et al., 2009). Carotenoids are tetraterpenes with many conjugated double bonds. They often exhibit yellow to purple colors and can function as anti-oxidants and precursors for vitamin A.
As the name implies polyketides contain carbonyl groups positioned between methylene groups. Still they vary in shape and volume. Although they appear with an impressive structural variety, they have their source from the same biosynthetic pathway. The most famous members among drugs may be the antibiotic erythromycin or the antifungal amphotericin B.

Phytochemicals Modulating MDR Targeting ABC-Transporters
A considerable number of secondary metabolites, which affect ABC transporters has already been discovered ( Table 1) and several of them will be discussed in the following; glaucine (an isoquinoline alkaloid) increased the efflux of substrates such as ADR and MTX in the P-gp over-expressing cell lines of MCF-7/ ADR and reduced DOX resistance in Caco-2 and CEM/ ADR5000 (Eid et al., 2013;Lei et al., 2013). Tetrandrine (a benzolisoquinoline alkaloid) also caused an inhibition of efflux in Caco-2 and CEM/ADR5000 cells (Sun and Wink, 2014). A 5substituted derivative of it named PY35 was tested for the MDR reversal activity, and showed more MDR reversal than the natural compound in resistant K562/ADM and MCF-7/ADM cells (Cao et al., 2014). Hernandezine, a bisbenzyl-isoquinoline, is a potent inhibitor of P-gp in MDR19-HEK293 cells and was able to resensitize MDR19-HEK293 and KB-V-1 cells to DOX after entering the cell membrane (Hsiao et al., 2016). High MDRreversing activities of the quinoline derivatives were linked to the                   presence of two aryl rings in the hydrophobic moiety, deviation of the aryl rings from a common plane, basicity of nitrogen atom in piperazine, as well as to the distance between the hydrophobic moiety and the basic nitrogen of piperazine which must be no less than 5 Å (Suzuki et al., 1997). The quinolyl group was also suggested to have a key role in the activity of quinolines, because substitution of a quinoline ring by a naphthyl ring or a phenyl ring resulted in the reduction of MDR-reversing activity of the compounds (Suzuki et al., 1997). Among indole alkaloids, antofine showed synergistic effects with PTX in A549-PA cells and overcame resistance to PTX (Kim et al., 2012). The bcarboline harmine reversed the resistance of MTX and CPT in MDA-MB-231 cells with BCRP as overexpressed transporter but it could not affect the P-gp over-expressing CEM/ADR5000 cells the same way in this study (Ma and Wink, 2010). Not only has harmine been tested in combination with DOX but also as threedrug-combination with DTN both of which showed here an increase of effect on cells and a reduction of DOX resistance in Caco-2 and CEM/ADR5000 cells (Eid et al., 2013). As an Nacylpiperidine, piperine inhibited the efflux of the tested substrate which led to an increase of its concentration in Caco-2 and CEM/ADR5000 cells (Li et al., 2018a). Tests of piperine on MCF-7/DOX and A-549/DDP cells resulted in an increase in cy totoxi cit y of MTX a nd DOX ( L i e t a l . , 2 0 1 1). Gravacridonetriol, gravacridonediol and its monomethyl ether, all had an inhibitive effect on P-gp L5178/MDR1 cells, which also led to a higher cytotoxicity of DOX (Rethy et al., 2008). Substitution of methyl groups at the positions C-2 and C-4 of acridone led to the increased lipophilicity, which can enhance the binding affinity of acridone derivatives to P-gp (Mayur, 2015). Murahari et al. (2017) studied on 2,4-dimethylacridone derivatives and showed that these compounds were potential modulators of P-gp-mediated MDR. 2,4-dimethylacridones are tricyclic and hydrophobic and have methyl groups at C-2 and C-4 positions with a propyl or butyl side chain containing terminally substituted tertiary amino groups. They found that an alkyl side chain and hydroxyl substituted secondary amine are necessary for the acridones to reverse the P-gp-mediated multidrug resistance. Moreover, an alkyl side chain of length four (butyl) was found to have higher biological activity (Murahari et al., 2017). Hegde et al. (2004) reported that replacing the hydrogen atom at position C-4 by a methoxy group slightly enhanced the lipophilicity of the acridone derivatives. Furthermore, they investigated the MDR-reversing activity of N-10-substituted acridones and N-10-substituted 4methoxyacridones relative to their corresponding unsubstituted counterparts where the C-4 positions of the acridone rings are occupied by a hydrogen atom and a methoxy group, respectively. The parent acridone and 4-methoxyacridone had the least effect in inhibiting drug efflux suggesting that N-10-substitution is necessary for an ideal activity. It was also found that 4methoxyacridone derivatives are more efficient than their acridone derivatives' counterparts in increasing drug accumulation. Several N-10-substituted acridones and N-10substituted-4-methoxyacridones showed MDR-reversing activity greater than the P-gp inhibitor verapamil (Hegde et al., 2004). In another study eighteen N-10-substituted-2bromoacridones were examined for the anti-MDR activity and compared to the parent compound 2-bromo-10H-acridin-9-one. N-10-substitution was suggested to be necessary for optimal activity of 2-bromoacridones because the parent compound had the least effect in efflux inhibiting activity (Mayur et al., 2006). Research on olomoucine, purvalanol and roscovitine revealed amongst other things the inhibitive potential of purine alkaloids on P-gp in the form of MDCKII-ABCB1 cells and human HCT-8 and HepG2 cells while olomoucine had the strongest effect of all (Cihalova et al., 2013). Several groups have run experiments with the benzazepine alkaloid capsaicin on different cell lines such as KB-C2, Caco-2 and CEM/ADR5000. The most relevant result was the inhibition of P-gp efflux in the presence of digoxin as substrate at non-toxic concentrations Han et al., 2006;Li et al., 2018a).
Isofl avonoids and some fl avonoids often act as phytoestrogens. Investigations revealed that hydroxyl groups at position C-5 and C-7 are an important property for the P-gp inhibitory effect of flavonoids, although hydrophobicity usually promotes the affinity (Sheu et al., 2010). Acacetin, genistein, kaempferol and naringenin, were examined by Imai et al. (2004) and it resulted in positive effects on K562 cells expressing BCRP. The cytotoxicity of MTX and 7-ethyl-10-hydroxycamptothecin was enhanced using phytoestrogens. In addition, genistein and naringenin enhanced the accumulation of topotecan in K562/ BCRP cells. Also apigenin had strong reversal effect on BCRPmediated MDR (Imai et al., 2004). In MCF-7 MX100 cells, which also overexpressed BCRP, apigenin had similar effect on the accumulation of the anticancer agent MTX . However, naringenin, which differs from apigenin only in the saturation of C-2 and C-3, had a significant loss of potency in comparison to the latter due to the lack of a double bond (Conseil et al., 1998). Several groups experimented with the isoflavone and phytoestrogen biochanin A and its outcome on BCRP and P-gp in cell lines such as Panc-1, MCF-7 MX100, MDA435/LCC6, MCF-7/ADR, MDA435/LCCMDR1 and Caco-2. In all studies, an inhibition of drug efflux, an increase of accumulation and a potentiation of cytotoxicity were observed. A combination of biochanin A with some other flavonoids yielded additive effects (Nguyen et al., 2003;Zhang and Morris, 2003;Zhang et al., 2004;Sergent et al., 2005). Nobiletin and tangeretin prevented the P-gp mediated efflux of [ 3 H]vincristine (Ikegawa et al., 2000). Among other flavones with methoxyls they were examined by Ohtani et al. (2007) for the uptake potential of [ 3 H]vincristine as a P-gp substrate. The MDR-reversing effect increased with the number of methoxyl moieties but with the exception of both at C-3′ and C-5′ position on B-ring. In this case there was even a decrease of MDR-reversing potency. Chrysin is a hydroxyflavone with simple structure which has also been examined by a number of groups. The competitive inhibition of the MRP-2 pumps in Caco-2 cells and an antiproliferative effect of chrysin are certain (Gyeḿańt et al., 2005;Sergent et al., 2005). Moreover, a prevention of BCRP and P-gp mediated efflux took place in MCF-7 MX100 cells and L5178 cells, respectively Zhang et al., 2005;Gyeḿańt et al., 2005). A prenyl or geranyl group at C-6 and C-8 of chrysin, and a prenyl in both positions ensured an improvement of the binding affinity to P-gp. In addition, there was more potency for inhibition after geranylation than prenylation which could be due to its level of lipophilicity . In contrast, glycosylation taking place at any position tested, dramatically decreased the binding affinity of flavonoids (Sheu et al., 2010). Another way to gain binding affinity toward the C-terminal nucleotide-binding domain of Pgp, were other kinds of alkylation of chrysin including methyl, benzyl, isopropyl and 3,3-dimethylallyl (Boumendjel et al., 2002).
Several experiments have been carried out for the flavan epigallocatechin gallate (EGCG), a derivative of epigallocatechin (EGC) abundant in green tea. EGCG has been tested on different cells such as liver cancer cells Bel-7404/DOX, the colon cancer cell line Caco-2, leukemia cells CEM/ADR5000, endocervical adenocarcinoma cells KB-C2 and others. An increase of drug accumulation and sensitization in these and several other cell lines was observed (Kitagawa et al., 2004;Liang et al., 2010;Eid et al., 2013;Reyes-Esparza et al., 2015). Particularly substitutions at Dring had positive effect on EGC and derivatives. A derivative of EGC with three methoxy groups in cis-configuration at D-ring and an oxycarbonylvinyl as its connection to C-3 led to a higher potency. This structure only regulated P-gp and could not affect BCRP or MRP1 (Wong et al., 2015).
While curcumin is a promising agent for liver protection and inhibition of cancerous cell growth, other curcuminoids like demethoxycurcumin and tetrahydrocurcumin have being studied more closely. They inhibited the efflux of chemptherapeutics and consequently increased cellular accumulation of drugs (Limtrakul et al., 2007;Ampasavate et al., 2010).
Ephedrine K562/A02 MDR1 gene expression using semiquantitative RT-PCR, P-gp expression using western blot Decrease in MDR1 mRNA and P-gp expression Gao et al. (2008) Indole-3-carbinol Hepatocytes of mouse treated with combination of indole-3-P-gp expression using western blot, quantitative stereology using immunohistochemical staining with anti-Pgp antibody Inhibition of VBL/VCR-induced P-gp expression Arora and Shukla (2003) (Continued)   Among phenylpropanoids, some lignans ( Figure 5) showed an inhibitory effect on P-gp. Schisandrin A was used on Caco-2 cells resulting in an increase in apical-to-basal transport and cytotoxicity Yoo et al. (2007b). A study which examined the structure activity of lignans showed the benefit of the absence of a hydroxyl group in position C-8 as in schisandrin A and gschizandrin for the function as p-gp inhibitor. In addition, a higher effect was seen in R-configurated biaryl than in the Sconfiguration (Slaninováet al., 2009). The reversal of cytotoxicity in CEM/ADR 5000 and Caco-2 cells was moreover successful by using menthol or thymol, monoterpenes obtained from volatile oils. They could reduce the IC 50 value of doxorubicin which enhances the effectiveness of the drug (Eid et al., 2013). Reis et al. (2016) reported that jatrophane diterpenes including esulatin M, epoxywelwitschene, welwitschene, and euphowelwitschine A were more efficative in MDR cells than the positive control verapamil. In comparison to the known MDR modifier verapamil, euphoboetirane C, D, E, F, and G extracted from Euphorbia boetica showed multifold P-gp modulatory activity in L5178Y-MDR cells (Neto et al., 2019). Three types of latilagascenes (G, H and I), macrocyclic diterpene esters, were effective in L5178 cells expressing P-gp as transport inhibitors (Duarte et al., 2008). All successfully tested diterpenes mentioned above had a cyclopentane moiety in their structure. An enormous increase of activity was observed due to their saturation. Moreover, there was lower MDR-reversing activity mostly when the number of three hydroxyls was exceeded, especially in position 5, 7, 9 and 12. Nevertheless hydropxyls at C-1, C-13, C-14 and C-15 had not any negative effect on their function . Vasas et al. (2011) observed that the lack of oxygenation at C-2 increased the MDR-reversing activity in diterpene jatrophanes such as 3b,5a,15b-triacetoxy-7bisobutanoyloxyjatropha-6(17),11E-diene-9,14-dione and 3b,5a,15btriacetoxy-7b-isobutanoyloxy-9a-nicotinoyloxyjatropha-6(17),11dien-14-one. Furthermore, they stated that the presence of benzoyl or nicotinoyl group at C-2 instead of an acetyl group increases the potency of jatrophane diterpenes. The reversal of MDR was noticed in some triterpenoids including ursolic acid tested in KB-C2 cells (Nabekura et al., 2010), and deacteylnomilin and limonin in CEM/ADR5000 cells (El-Readi et al., 2010). A study on triterpenoids such as tormentic acid and derivatives suggests that an acetylation of C-2 of a terpenoid can cause an increase of activity, while this is not the case with the acetylation of C-3 (da Graca Rocha et al., 2007). Carotenoids, a group derived from tetraterpenes, are substrates for ABC transporters and can effectively modulate MDR in cancer cells (Eid et al., 2012). A few types such as b-carotene (Eid et al., 2013) or the xanthophyll member capsorubin showed MDR reversal activity . To mention another important group with aromatic members, coumarins and coumaric acids are benzopyrones which we presented in Tables 1 and 2. Auraptene for example not only caused an enhancement of drug uptake (Nabekura et al., 2008b); its effect even was of a synergistic kind with drugs such as cisplatin and VCR (Moussavi et al., 2017). Kasaian et al. (2015) examined fifteen sesquiterpene coumarins. Considering the structure-activity relationship they described that ring-opened drimane-type sesquiterpene coumarins such as lehmferin, farnesiferol B and farnesiferol C showed the best P-gp inhibitory effects. Their study also revealed that farnesiferol C inhibited the Rh-123 efflux even more than the positive control verapamil.
The studies introduced phytochemicals with completely different basic structures, each showing both strong and ineffective members in targeting ABC-transporters. The molecules mentioned were a selection chosen due to their effectiveness, especially in relation to the substances to which they were compared.

Phytochemicals Modulating Transporter or Protein Expression
The following discussion focusses on the question how far phytochemicals affect the expression of ABC transporters or proteins in cancer cell lines. A list of relevant publications is documented in Table 2. Although many investigations addressed the inhibitory effect of secondary metabolites on transporter activity, many phytochemicals regulate the expression of corresponding genes, including alkaloids, polyphenols, lignans, terpenes and benzopyrones. Berbamine caused a downregulation of Mdr-1 expression in K562/ADR cells (Dong et al., 2004), and so did its derivative O-(4-ethoxylbutyl)-berbamine (Cheng et al., 2006). Glaucine not only functioned as inhibitor of transporters but also reduced the expression of MDR1 and MRP1 (Lei et al., 2013) while the isoquinoline alkaloids berberine and coptisine, mediated the expression of P-gp (Lin et al., 1999a). Tetramethylpyrazine downregulated the expression of MDR1, MRP2, MRP3 and MRP5 in BEL-7402/ADM cells . It has also reduced the expression of MDR1 and MRP1 in MCF-7/Dox and Pumc-91/ADM cells, respectively (Zhang et al., 2012;Wang et al., 2016). MDR1 mRNA and P-gp expression were reduced in response to clitocine and sulfinosine in several cell  Liu et al. (2017) lines, such as R-HepG2, MES-SA/Dx5, NCI-H460/R and U87-TxR (Sun et al., 2012;Dacěvićet al., 2013). Among polyphenols, EGCG mediated a reduction of P-gp and BCRP expression resulting in drug accumulation (Farabegoli et al., 2010). Curcumin enhanced the cytotoxicity of PTX and ADR, respectively against SKOV3(TR) and K562/A02 cells by reducing the expression of P-gp in both cell lines (Chang et al., 2006;Ganta and Amiji, 2009). While some diterpenes and sesquiterpenes induced the transporter expression (Li et al., 2009), others like triptolide suppressed the expression of MDR1 and MRP1 proteins in KB-7D and KB-tax cells .
Beside the positive effect of some coumarins on the activity of ABC transporters, pyranocoumarin was tested on KB-V1 cells and showed a reduction of P-gp protein and MDR1 expression (Wu et al., 2003). Athough no clear relationship could be established between the structure of a modulator with thre expression of a transporter protein, these findings can be relevant for clinical applications.

Synergistic Combinations of Chemotherapeutics With Phytochemicals
In addition to affecting the activity or expression of transporters, phytochemicals can also reverse MDR in cancer cells through synergism. Phytochemicals causing synergistic interactions with anticancer drugs are documented in Tables 1 and 2. The twodrug combination of harmine with DOX not only showed an enhancement of cytotoxicity but a synergistic effect. Glaucine potentiated DOX toxicity in Caco-2 and CEM/ADR5000 cells and reversed their resistance to anticancer drugs. Glaucine exerted synergistic interaction with DOX and even more in a three drug combination with DTN (Eid et al., 2013). Euphoboetirane C, D, F, G, H and I extracted from Euphorbia boetica showed strong synergistic interactions with DOX in L5178Y-MDR cells (Neto et al., 2019). Nobiletin and antofine showed synergistic interactions with PTX (Kim et al., 2012;Ma et al., 2015). Jatrophane diterpenes including euphowelwitschine A, euphowelwitschine B, epoxywelwitschene, esulatin M isolated from Euphorbia welwitschii demonstrated synergism with DOX in L5178Y-MDR cells (Reis et al., 2016). Fangchinoline, tetrandrine, and pyranocoumarins has been shown to synergistically increase the cytotoxicity of DOX (Wu et al., 2003;Sun and Wink, 2014). These synergistic effects are probably caused by interference of the phytochemicals with ABC transporters and additional molecular targets in cancer cells . These studies show that a combination of substances can have a great advantage over using single drugs against cancer cells by exploiting synergism.

In Silico Analysis of Phytochemicals as MDR Reversing Agents
In Silico experiments are promising methods for making predictions about interactions of molecules to the drugbinding site of a target such as P-gp, BCRP and MRP1. M o l e c u l a r d o c k i n g s t u d i e s c a n h e l p fi n d i n g t h e phytochemicals with best affinity for the drug-binding site which should be used as lead molecules for further studies. The nucleotide-binding domains (NBD) of these ABC-Transporters are hydrophilic protein parts (Jones and George, 2002). In addition to essential hydrogen bonds which play a crucial role in maintaining the stability and function of biomolecules, modulators with hydrophobic moieties often show affinties to NBDs.
Regarding in silico investigations, another subtype of alkaloids, piperine was examined by Syed et al. (2017) revealing that hydrophobic interactions with P-gp took place in following positions: Leu339, Met69, Met986, Phe72, Phe336, Phe728, Phe983, Tyr953 and Val982. Moreover, an H-bond with Tyr307 was established. Two piperine analogs were designed which showed better hydrophobic interaction with most of the amino acids mentioned (Syed et al., 2017).
Pharmacophore modeling which was carried out for acridones indicated that three aromatic rings and two Hacceptors which are given in position C-9 bearing a carbonyl group and N-10 were conducive for stable docking. Including designed analogs of acridone the oxygen of a morpholine moiety at a phenyl ring built a water bridge with Ser309 of P-gp and a carboxamide at C-4 helped docking with Phe343. This analog showed hydrophobic interactions with Ala229, Ala302, Ala342, Ile218, Ile221, Ile299, Ile306, Leu225 with even more than two interactions, Leu339, Phe343, and Val345 of P-gp (Gade et al., 2018). Badhan and Penny (2006) worked on the main characteristics of the pharmacophore of flavonoids by in silico modeling. Flavonoids tested get located within the C-terminal NBD of Pgp binding to amino acids of the ATP pocket such as Tyr1044. The hydroxyl moiety at C-5 of natural flavonoids can form a water bridge with Lys1076 while the hydroxyl group in position 3 and the carbonyl group get into hydrogen bonds within the NBD. Hydroxyl groups at position C-7 led to important hydrogen bonds with amino acids for several flavonoids such as EGC and chrysin (Wongrattanakamon et al., 2017). Ring B gets involved in p-p interaction with Tyr1044. If benzyl, geranyl or prenyl groups added at C-6 and several substitutes added at C-8 position, the docking capability was enhanced. Molecular docking analysis demonstrated that apigenin binds to the ATP-binding site of P-gp through a hydrogen bond with LYS408 and therefore, interferes with binding and cleavage of ATP which are vital for the function of ABC transporters (Saeed et al., 2015). Comparing flavonols to their corresponding flavones such as kaempferol to apigenin, better docking properties have been found in flavonols due to the number of hydrogen bonds (Badhan and Penny, 2006). A number of naringenin derivatives such as hydrazones and azines were produced by Ferreira et al. (2018) to achieve an improved MDR reversal in P-gp and BCRP. There was a high selectivity for most members of these two groups. It has been shown that the active compounds bind to hydrophobic residues such as transmembrane helix 4 and 5 in the center of an active pocket of BCRP similar to fumitremorgin C, a common inhibitor of BCRP. The hydrazone of these derivatives, a continuation of imine, is located in a pocket in p-p interaction with Phe515 and a dipoledipole interaction with Met541. The B-ring is in another lipophilic bag made of Phe545, Pro574 and Val516. An improvement in the interaction between the transporter MRP1 and the carbohydrazide derivatives of flavonones due to the resulting flexibility was also observed (Ferreira et al., 2018).

Conclusions and Future Perspectives
Due to the increase of cancer cases and restrictions in therapy owing to the degree of harmfulness, effectiveness and the associated high costs, the development of new substances with less side effects and higher efficacy is required. So far, many structures from the plant kingdom have been discovered and used, such as, diterpene derivatives as taxanes and vinca alkaloids are prime examples with regard to antitumor therapy. Therefore, numerous secondary metabolites which already had positive effect on cell lines should be taken into account for further investigations, which is why we have tried to list them extensively in our tables.
Many phytochemicals including alkaloids, flavonoids, curcuminoids, stilbenoids, terpenes, carotenoids, lignans, and polyketides were examined for their pharmacological activity against MDR. In most of the studies it was possible to differentiate between more effective and less effective molecules. As the next step these molecules must be further studied to determine molecular mechanisms and to identify the pharmacophores. Some valuable results have been provided by structure-activity relationship studies or in silico modeling as shown in this article. Subtances which were superior to others should be selected for further research, so possible lead molecules can be developed. A further step to increase efficacy could be the use of new drug delivery methods and controlled release as with nanotechnologies. As shown in this review, the combination of two and more modulators of differing structures and mode of actions together with chemotherapeutics is another interesting approach. The resulting additive, but above all synergistic effects can be of great importance. They would allow reducing the dose of chemotherapeutics resulting in less side effects and a higher compliance of patients.
Summin up, many phytochemicals in the group of alkaloids, flavonoids, curcuminoids, stilbenoids, terpenes, carotenoids, lignans, and polyketides have been investigated for MDRreversing activity. Phytochemicals can be a promising source of adjuvant chemicals against cancer, not at least because of their generally low cytotoxicity in the human body. The adjuvant uses of MDR reversing phytochemicals in combination with anticancer drugs may improve the treatment of multidrug resistant cancer types. The present review summarized reports of several secondary metabolites that are capable of synergistically reversing MDR and inhibiting chemotherapyresistant cancer cells by affecting transporter activity and the expression of ABC transporter genes. Synergism would allow reducing the dose of chemotherapeutics resulting in less side effects and a higher compliance of patients. The efficacy of phytochemicals needs to be confirmed clinically, but nevertheless they already can be considered as the fourth generation of ABC transporter modulators.

AUTHOR CONTRIBUTIONS
BT and IS performed the literature search and wrote the first draft of the manuscript. MW revised and edited the manuscript. All the authors approved the final version of the manuscript.

FUNDING
Ruprecht-Karls-Universität Heidelberg provided financial support within Open-Access Publishing Program.