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Secondary Metabolites from Plants Inhibiting ABC Transporters and Reversing Resistance of Cancer Cells and Microbes to Cytotoxic and Antimicrobial Agents

Fungal, bacterial, and cancer cells can develop resistance against antifungal, antibacterial, or anticancer agents. Mechanisms of resistance are complex and often multifactorial. Mechanisms include: (1) Activation of ATP-binding cassette (ABC) transporters, such as P-gp, which pump out lipophilic compounds that have entered a cell, (2) Activation of cytochrome p450 oxidases which can oxidize lipophilic agents to make them more hydrophilic and accessible for conjugation reaction with glucuronic acid, sulfate, or amino acids, and (3) Activation of glutathione transferase, which can conjugate xenobiotics. This review summarizes the evidence that secondary metabolites (SM) of plants, such as alkaloids, phenolics, and terpenoids can interfere with ABC transporters in cancer cells, parasites, bacteria, and fungi. Among the active natural products several lipophilic terpenoids [monoterpenes, diterpenes, triterpenes (including saponins), steroids (including cardiac glycosides), and tetraterpenes] but also some alkaloids (isoquinoline, protoberberine, quinoline, indole, monoterpene indole, and steroidal alkaloids) function probably as competitive inhibitors of P-gp, multiple resistance-associated protein 1, and Breast cancer resistance protein in cancer cells, or efflux pumps in bacteria (NorA) and fungi. More polar phenolics (phenolic acids, flavonoids, catechins, chalcones, xanthones, stilbenes, anthocyanins, tannins, anthraquinones, and naphthoquinones) directly inhibit proteins forming several hydrogen and ionic bonds and thus disturbing the 3D structure of the transporters. The natural products may be interesting in medicine or agriculture as they can enhance the activity of active chemotherapeutics or pesticides or even reverse multidrug resistance, at least partially, of adapted and resistant cells. If these SM are applied in combination with a cytotoxic or antimicrobial agent, they may reverse resistance in a synergistic fashion.


INTRODUCTION EVOLUTIONARY AND ECOLOGICAL BACKGROUND
Plants are sessile organisms which cannot run away when attacked by an herbivore nor do they have an immune system to combat infesting parasites, bacteria, fungi, or viruses. From early days of the evolution of land plants they had to cope with these environmental challenges. Plants developed a number of mechanical traits, such as resistant epidermal and bark tissues but also spines and thorns as defense tools. In addition, plants evolved a high diversity of defense chemicals, the so-called secondary metabolites (SM ; Table 1). Besides defense, some SM function as signal compounds or protect against oxidative or UV stress (Wink, 1988(Wink, , 2003(Wink, , 2008b(Wink, , 2010a.
The structures of SM have been optimized during evolution in such a way that they can interfere with molecular targets in herbivores and microbes. The main group of targets include (1) proteins, (2) DNA, RNA, and (3) the biomembrane (Wink, 2008a,b;Wink and Schimmer, 2010). Neuronal signal transduction is a central and specific target in animals and many SM, especially alkaloids and amines are directed toward it (Wink, 1993(Wink, , 2000. SM which interfere with proteins, such as polyphenols, biomembranes (saponins and other lipophilic terpenoids), or DNA (alkylating or intercalating mutagens) affect a wider range of organisms, including animals and microbes. In general, membrane and DNA active SM have cytotoxic properties. Affected cells usually undergo apoptosis (Wink, 2007). Several SM interfere with the neuronal signal transduction in animals and are thus potent neurotoxins (Wink, 1993(Wink, , 2000. A large number of SM have lipophilic properties which enable them to readily pass biomembranes in target organisms by simple diffusion. These SM are also dangerous for the producing plants. Therefore, they are usually stored in dead tissue away from living cells, such as resin ducts, oil cells, trichomes, or cuticles (Wink, 2010b). The absorption of polar SM is usually slower or does not take place at all, with the exception of SM that can use transporters for sugars or amino acids or endocytosis as a kind of "stowaway." Furthermore, SM usually occur in complex mixtures which may contain SM (such as saponins) that can facilitate the uptake of polar SM (Hebestreit and Melzig, 2003).

THE RESPONSE OF HERBIVORES AND PATHOGENS AGAINST PLANT DEFENSE CHEMICALS
In the evolutionary arms race herbivores and microbes evolved mechanisms to avoid or inactivate the defense chemistry of plants. Mechanisms of resistance in animals and humans are complex and often multifactorial. Mechanisms include: (1) Activation of ATPbinding cassette (ABC) transporters, such as p-gp, which pump out lipophilic compounds that have entered a cell, (2) activation of cytochrome p450 oxidases (CYP) which can oxidize lipophilic agents to make them more hydrophilic and accessible for conjugation reaction with glucuronic acid, sulfate, or amino acids, and (3) activation of glutathione transferase (GST), which can conjugate xenobiotics with glutathione. The reactions of CYP, GST, and conjugation are well known in pharmacology and categorized as phase I and phase II reactions. These reactions are important in the metabolism of medicinal drugs and toxins. This evolutionary history also applies for humans which enables us to metabolize a large number of xenobiotics.
In phase I, a lipophilic SM is made more hydrophilic by introducing hydroxyl groups. This reaction is catalyzed by CYP and CYP1A1, CYP1A2, CYP3A4, and CYP2D6 are the most important enzymes. Furthermore, these CYP can cleave N -methyl, O-methyl, or methylene groups in order to obtain a more hydrophilic or better accessible substrate (Guengerich, 2001). In the human genome, about 57 active CYP genes are known (Ingelman-Sundberg and Gomez, 2010). A substantial polymorphism of CYPs exists which enables them to metabolize a wide range of xenobiotics. The regulation of the corresponding genes is only partly known. The genes encoding these enzymes, which occur in intestinal epithelia and in the liver, are inducible by SM that have entered the body. In phase II, the hydroxylated xenobiotics are conjugated with polar molecules, such as glutathione, sulfate, or glucuronic acid. These conjugates are eliminated via the kidneys and urine. That means, on exposure to lipophilic SM, genes which encode these enzymes are often induced and that activation can inactivate the toxins. Several SM carry methylenedioxy groups on their phenolic rings, such as in the isoquinoline alkaloids berberine and hydrastine, which are assumed to be inhibitors of CYP (Wink, 2007). Alkaloids which can inhibit CYP have been summarized by Wink (2007).
Resistance mechanisms in bacterial pathogens are even more evident because several pathogens already have evolved resistance against medicinally used antibiotics. The main mechanisms include: • Direct inactivation of the antibiotic, e.g., by cleavage of the beta-lactam ring of penicillin by beta-lactams or acetylation, methylation of other antibiotics • Target site modification: molecular change of the target molecule (proteins, rRNA) in such a way that the antibiotic cannot bind any longer • Bypass or alteration of metabolic pathways in cases where an antibiotic blocks a pathway (e.g., as for sulfonamides) • Prevention of drug uptake • Export out of the cell by ABC transporters so that the intracellular concentration of an antibiotic (e.g., tetracycline) are reduced. In Bacteria, this is one of several factors responsible for multidrug resistance (MDR).

ABC TRANSPORTER
Resistance against defense chemicals can be obtained through the expression of ABC transporters that are present in most cells and organisms. They are especially active in epithelia of intestinal, liver, kidney, and endothelia (Twentyman and Bleehen, 1991;Nielsen and Skovsgaard, 1992;Nooter and Stoter, 1996;Steinbach et al., 2002;You and Morris, 2007). Three types of ABC transporters have been studied in detail: 1. P-glycoprotein (P-gp; molecular weight 170 kD) or MDR1 protein (multiple drug resistance protein) was the first cloned ABC transporter. It is encoded by the ABCB1 gene. P-gp is composed of two similar moieties and each half contains one transmembrane and one ATP-binding domain. P-gp is an efflux pump directed to the gut lumen. The substrate molecules bind to transmembrane domains and then are exported to extracellular space, driven by the energy of ATP hydrolysis. A wide range of lipophilic chemotherapeutical agents, such as anthracenes, anthracyclines, epipodophyllotoxins, taxanes, and Vinca alkaloids, which can enter tumor cells by free diffusion, are substrates of P-gp and can be extruded by the transporter (Loo and Clarke, 2005).
Breast cancer resistance protein and P-gp are highly expressed at the apical membrane of blood-brain barrier (BBB), placenta, liver, intestine, and other organs (Schinkel and Jonker, 2003). These ATP-driven transporters can pump lipophilic compounds out of the cell, either back to the gut lumen or into the blood system, thus reducing the intracellular concentration of potentially toxic compounds. ATP-binding cassette transporters are also important at the BBB. The BBB only allows the entry of small lipophilic substances by passive diffusion. However, the uptake of lipophilic compounds in the brain is relatively low due to the high activity of P-gp, MRP, and organic anion transporting polypeptides (OATPs). These transporters catalyze a rapid efflux of lipophilic xenobiotics from the CNS (Elsinga et al., 2004;Mahringer and Fricker, 2010).
Multidrug resistance was discovered during chemotherapy of cancer patients who developed resistance against a cytotoxic drug. It transpired that the tumor cells were able to pump out the lipophilic alkaloids (such as Vinca alkaloids, taxanes, and anthracycline derivatives) at almost the same speed as they were entering the tumor cells. Activated cells became resistant to vincristine but also to several other lipophilic drugs. This means that a cross-resistance or MDR had occurred. As a consequence, a major obstacle to the successful chemotherapy of tumors is MDR. Upon exposure to xenobiotics MDR genes can become upregulated. Overexpressed ABC transporters (P-gp, MRP1, or BCRP) can mediate resistance of tumor cells against a variety of anticancer drugs (Schinkel and Jonker, 2003). This phenomenon is called MDR, which is one of the most important reasons of chemotherapy failure (Gottesman, 2002).
Several of human protozoal parasites (Plasmodium, Leishmania, Trypanosoma) can develop resistance against prophylactic and therapeutic agents, such as quinolines, naphthoquinones, sesquiterpene lactones, and others. The underlying mechanism includes membrane glycoproteins that are orthologous to human P-gp. These ABC transporters can also be induced and activated.
ATP-binding cassette transporters are also present in bacteria and fungi in which they confer resistance to antibiotics and fungicidal compounds (Steffens et al., 1996). A medicinally important issue is the increasing resistance of bacteria toward antibiotics, and ABC transporters can be involved in bacterial MDR (besides other mechanisms discussed above). Apparently, ABC transporters are an old invention of nature, which occur from E. coli to Homo sapiens.

OVERCOMING RESISTANCE CAUSED BY ABC TRANSPORTERS
Multidrug resistance reversal agents are also called chemosensitizers or modulators. They can inhibit the efflux activity of transporters and other relevant MDR targets (see above); in consequence they can potentiate cytotoxicity, and are therefore important alternatives to overcome MDR (Watanabe et al., 1995;Dantzig et al., 1996;Robert and Jarry, 2003).
Multi-resistant tumor cells frequently express different ABC transporters simultaneously, e.g., P-gp, MRP1, BCRP, and others (Annereau et al., 2004;Gillet et al., 2004). Because the substrate spectra of ABC transporters only partly overlap, co-expression of transporters might produce more diverse resistance profiles than those of any one member alone. Thus broad-spectrum reversal agents are needed and some compounds exhibit this property (Hyafil et al., 1993;Maliepaard et al., 2001;Brooks et al., 2003).
A number of natural or synthetic compounds have been discovered that can inhibit P-gp and re-sensitize resistant tumor cells in vitro (Chauffert et al., 1990;Genne et al., 1992;He and Liu, 2002;Wink, 2007). Although these agents work successfully in some patients, most results of clinical trials were disappointing (Solary et al., 2000;Dantzig et al., 2001). Some of these reversal agents did not work in vivo or some had too severe side effects. Therefore, new and better reversal agents are still needed.
Most modulators of ABC transporters act by binding to membrane transport proteins (especially P-gp, MRP1, and BCRP) as competitive inhibitors, or by indirect mechanisms related to phosphorylation of the transport proteins, or the expression of the mdr1 and mrp1 genes. Other inhibitors not only act at the level of the transporter gene but influence their expression; for example, the alkaloid piperine lowered the expression levels of ABCB1, ABCC1, and ABCG2 genes which encode P-gp, MRP1, and BCRP (Li et al., 2011b).

INHIBITORS OF ABC TRANSPORTERS FROM PLANTS
For this review we carried out a comprehensive literature research. Table 2 summarizes the search results for SM from plants, which can serve as ABC transporter substrates and might be useful in strategies to reverse drug resistance in cancer cells, fungi, and parasites. Compounds affecting other resistance mechanisms, which are important and which were discussed above, were not considered in this review.
Lipophilic SM, such as monoterpenes, diterpenes, triterpenes (including saponins), steroids (including cardiac glycosides), and tetraterpenes (carotenoids; Table 2) function as substrates for P-gp in cancer cells. The ABC transporter from fungi, AtrB (Andrade et al., 2000), or the NorA efflux pump in Staphylococcus aureus can also be affected (Smith et al., 2007). Because of their lipophilicity, these terpenoids most likely are substrates for P-gp and other ABC transporter. If administered as a chemosensitizer in combination with a cytotoxic agent they function as inhibitors competing for binding to the active side of the transporters. www.frontiersin.org      ) www.frontiersin.org   (1994,1996) Hesperidin, neohesperidin,

nobiletin, Tangeretin
Citrus jambhiri, Citrus pyriformis    ) www.frontiersin.org    ) www.frontiersin.org Among the structurally heterogenous group of alkaloids, a large number of the more lipophilic substances from the classes of isoquinoline, protoberberine, quinoline, indole, monoterpene indole, and steroidal alkaloids ( Table 2) can serve as substrates whereas the more polar alkaloids with a tropane, quinolizidine, piperidine, and pyrrolizidine skeleton do not bind to ABC transporters (Wink, 2007). Similar to the situation of terpenoids, the active alkaloids probably function as competitive inhibitors of Pgp and BCRP in cancer cells, and NorA in bacteria and fungi ( Table 2).
It is remarkable on the first sight that also quite a large number of more polar phenolic SM (phenolic acids, flavonoids, catechins, chalcones, xanthones, stilbenes, anthocyanins, tannins, anthraquinones, and naphthoquinones) inhibit P-gp, MRP1, BCRP, and OATP in cancer cells with MDR. Some of them can reverse MDR when given in combination with cytotoxic agents ( Table 2). Bacteria and fungi appear to be sensitive as well (Guz et al., 2001;Falcao-Silva et al., 2009). Some of these phenolics are lipophilic enough to be competitive inhibitors of ABC transporters.
Polyphenols are exciting tethering compounds of proteins. They can effectively interact directly with proteins by forming hydrogen and ionic bonds with amino acid side chains. They can thus interfere with the 3D structure of proteins (conformation) and inhibit their activities (details in Wink, 2008b;Wink and Schimmer, 2010). We speculate therefore, that the inhibition seen in polyphenols is caused by a direct binding and complex formation (not necessarily the active side) of ABC transporters. Since many polyphenols have no or very low toxicity (e.g., many of them are ingredients of our food, such as flavonoids or tannins), they might be excellent candidates as reversal agents, both in chemotherapy and in agriculture.
We have focused on ABC transporters in this review. But as mentioned above, resistance can be due to other mechanisms as well and is often multifactorial. Faria et al. (2011) and Kim et al. (2007Kim et al. ( , 2010 have successfully employed thymol (a phenolic monoterpene), salicyl aldehyde, and the alkaloid berberine to enhance the activity of fungicides in Candida, Aspergillus, Penicillium, and Cryptococcus. These experimental data can be regarded as a proof of concept that plant secondary products can be interesting candidates for chemosensitization (even if they not interfere with ABC transporters) of pathogenic fungi in agriculture and food technology to improve the fungicidal activity of certain fungicides.

CONCLUSION
This review summarizes the evidence that selected SM of plants can be interesting candidates to inhibit ABC transporters in MDR cancer cells or to chemosensitize pathogenic fungi and other microbes for treatment with antimicrobial agents. Whereas lipophilic terpenoids and alkaloids appear to be substrates of P-gp, MRP1, or BCRP and thus competitive inhibitors, the more polar phenolic compounds (flavonoids, tannins, quinones) can bind to the transporter proteins and inhibit their activity by disturbing protein conformation. A combination of a cytotoxic agent, antibiotic, or fungicide with a natural chemosensitizer (not necessarily an inhibitor of ABC transporters) might provide an interesting strategy to overcome MDR in cancer patients and to improve antibiotic or antifungal efficacy in medicine, agriculture, or food industry.