African Flora Has the Potential to Fight Multidrug Resistance of Cancer

Background. Continuous efforts from scientists of diverse fields are necessary not only to better understand the mechanism by which multidrug-resistant (MDR) cancer cells occur, but also to boost the discovery of new cytotoxic compounds to fight MDR phenotypes. Objectives. The present review reports on the contribution of African flora in the discovery of potential cytotoxic phytochemicals against MDR cancer cells. Methodology. Scientific databases such as PubMed, ScienceDirect, Scopus, Google Scholar, and Web of Knowledge were used to retrieve publications related to African plants, isolated compounds, and drug resistant cancer cells. The data were analyzed to highlight cytotoxicity and the modes of actions of extracts and compounds of the most prominent African plants. Also, thresholds and cutoff points for the cytotoxicity and modes of action of phytochemicals have been provided. Results. Most published data related to the antiproliferative potential of African medicinal plants were from Cameroon, Egypt, Nigeria, or Madagascar. The cytotoxicity of phenolic compounds isolated in African plants was generally much better documented than that of terpenoids and alkaloids. Conclusion. African flora represents an enormous resource for novel cytotoxic compounds. To unravel the full potential, efforts should be strengthened throughout the continent, to meet the challenge of a successful fight against MDR cancers.


Introduction
Cancer is increasingly recognized as a critical public health problem in Africa [1,2]. The number of new cancer cases will reach 15 million every year by 2020 worldwide, 70% of which will be in developing countries, where governments are less prepared to address the growing cancer burden and where survival rates are often less than half of those in more developed countries [3]. Though communicable infections continue to burden African population, noncommunicable diseases also require the attention of health professionals in Africa [1]. Currently, limited funding is available to tackle cancer in African countries. Awareness of this impeding epidemic in Africa should be a priority today, and all possible resources should be mobilized to both prevent and efficiently treat cancers.
Cancer cells rapidly acquire multidrug resistance (MDR), mainly due to the presence of two adenosine triphosphatebinding cassette (ABC) transporters [4][5][6]. Continuous contributions of scientists from diverse fields are necessary not only to better understand the mechanisms of MDR, but also to boost the discovery of new cytotoxic drugs fighting drug resistance. The overexpression of ABC transporters contributes to MDR and participates in the failure of cancer chemotherapy [7]. MDR cancer cells reveal cross-resistance to a variety of chemically and functionally unrelated drugs [8][9][10]. The structural diversity of plant's secondary metabolites makes them an indispensable source for the discovery of new cytotoxic agents. Their use to combat drug resistance remains a challenging issue [11]. In the present review, we discuss the up-to-date prominent findings on anticancer plants and derived products from Africa.

Cancer Concern in Africa
Statistics of the International Agency for Research on Cancer (IARC) revealed that about 715,000 new cancer cases and 542,000 cancer deaths occurred in 2008 in Africa [12]. There will be about 1.28 million new cancer cases and 970,000 cancer deaths by 2030 solely in Africa, mainly due to aging and growth of the population [12]. The development might become even worse because of the adoption lifestyles associated with economic development, such as smoking, unhealthy diet, and physical inactivity [13]. The most occurring cancer types in Africa are those related to infectious agents (carcinoma of cervix, liver, and urinary bladder as well as Kaposi sarcoma) [1]. In 2008, cervical cancer accounted for 21% of the total newly diagnosed cancers in females and liver cancer for 11% of the total cancer cases in males [1]. The survival rates are considerably lower in Africa than in the developed world for most cancer types [1]. For example, the five-year survival rate for breast cancer is less than 50% in Gambia, Uganda, and Algeria, compared to nearly 90% in the United States [1]. According to the World Health Organization (WHO) government survey of national capacity for cancer control programs in 2001, anticancer drugs were only available in 22% and affordable in 11% of the 39 African countries that participated in the survey [2]. In parallel, efforts are being made by African scientists to search for new drugs from their most affordable resources, which are medicinal plants. Several plants from the flora of Africa were found to be active against various types of cancer cells. Even if not reported in the scientific literature for their antiproliferative potential, several medicinal plants of the African continent contain known antineoplastic compounds. Some of them include Plumeria rubra L. (Apocynaceae) with the well-reported cytotoxic compound plumericin or Diospyros crassiflora L. and Diospyros canaliculata L. (Ebenaceae) containing plumbagin [14]. Using a pharmacogenomics approach with Cameroonian flora as an example [14], it was demonstrated that African plants have an enormous and unstudied anticancer potential, as they contain an impressive arsenal of bioactive agents.

Cancer Cells and Drug Resistance
Cancer is caused by the accumulation of multiple genetic and epigenetic alterations, leading to abnormal expression of genes involved in initiation, progression, and promotion of carcinogenesis [15]. Cancer cells may rapidly acquire MDR, mainly due to the presence of adenosine triphosphatebinding cassette (ABC) transporters, such as the breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (Pgp/MDR1/ABCB1) [4] as well as the oncogene epidermal growth factor receptor (EGFR) [5,6,16] and the deletions or inactivation of tumor suppressor gene p53 [17]. P-Glycoprotein 1 (permeability glycoprotein, P-gp or Pgp), encoded by the multidrug resistance gene 1 (MDR1), also known as ATP-binding cassette subfamily B member 1 (ABCB1) or cluster of differentiation 243 (CD243), is an important protein of the cell membrane that pumps many foreign substances out of cells [4]. It is an ATP-dependent efflux pump with broad substrate specificity found in animals, fungi, and bacteria and likely evolved as a defense mechanism against harmful substances during evolution of life. Some cancer cells overexpress P-gp rendering these cancers multidrug resistant [5,6,16,[18][19][20].
The human epidermal growth factor receptor (EGFR/ ErbB-1/HER1) represents a cell-surface transmembrane glycoprotein that constitutes one of four members of the ErbB family of tyrosine kinase receptors [21]. EGFR is activated by binding of specific ligands, including epidermal growth factor and transforming growth factor (TGF ). Upon activation, EGFR undergoes a transition from an inactive monomeric form to an active homodimer that stimulates its intrinsic intracellular protein-tyrosine kinase activity [22]. This leads to autophosphorylation of several tyrosine residues in the C-terminal domain of EGFR [23]. This autophosphorylation elicits downstream activation and signaling by several other proteins that associate with phosphorylated tyrosines through their own phosphotyrosine-binding SH 2 domains, several signal transduction cascades (principally the MAPK, Akt, and JNK pathways) leading to DNA synthesis, and cell proliferation [24]. It was found that mutations leading to EGFR overexpression have been associated with many cancers, including lung cancer, anal cancers, and glioblastoma multiforme [25,26].
Human tumor suppressor protein p53 (a protein of 53 kDa), also known as p53, cellular tumor antigen p53, and phosphoprotein p53, is encoded by the TP53 gene [27]. The p53 protein is crucial in multicellular organisms, where it regulates the cell cycle and, therefore, functions as a tumor suppressor, preventing cancer [27]. Consequently p53 has been described as "the guardian of the genome" because of its role in conserving stability by preventing genome mutation, while TP53 is classified as a tumor suppressor gene [27][28][29].
Topoisomerase (Topo) inhibitors are compounds interfering with the action of topoisomerase enzymes (Topo I and II), which are enzymes that control the changes in DNA structure by catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle [30]. Topoisomerases have also become popular targets for cancer chemotherapy treatments, as their inhibitors block the ligation step of the cell cycle, generating single and double stranded breaks that harm the integrity of the genome leading to apoptosis and cell death [30].

Anticancer Activity of Plants and Derived Products
Screenings of medicinal plants used as anticancer drugs have provided modern medicine with effective cytotoxic pharmaceuticals. More than 60% of the approved anticancer drugs in United States of America (from 1983 to 1994) were in one or another way from natural origin [33,34]. The diversity of the biosynthetic pathways in plants has provided a variety of lead structures that have been used in drug development.
In the past decade, investigations on natural compounds have been particularly successful in the field of anticancer drug research. Early examples of anticancer agents developed from higher plants are the antileukemic alkaloids (vinblastine and vincristine), which were both obtained from the Madagascar periwinkle (Catharanthus roseus L.; Apocynaceae) [35]. A large number of plant extracts have shown in vitro and in vivo antitumor activities [36]. For in vitro anticancer screenings of plant extracts, IC 50 value of 30 g/mL represents a cutoff point to consider cytotoxic plant extracts for purification [36]. The IC 50 value of 20 g/mL is also considered for good cytotoxic extract [36]. However, there is still a lack of scientific references to define other bioactivities, for example, unspecific toxicity towards normal cells as well as toxicity for edible or culinary plant's part. Herein, we will set values tenfold higher than 30 g/mL as the point of tolerably low cytotoxicity to normal cells and 10-fold higher than 20 g/mL as corresponding values in cancer cells. In this report, we propose the following cutoff points.
Similarly for plant metabolites, the following cutoff points are proposed.
Nonetheless, the inhibitory potential of few terpenoids towards drug-resistant cells was also reported (Figure 1).
The bicyclic sesquiterpene esters jaeschkeanadiol p-hydroxybenzoate (4) isolated from the active fraction of the Egyptian medicinal plant Ferula hermonis exerted a strong cytotoxic effect towards breast cancer cell line MCF7 and moderate activities towards other cell lines (Table 1) [43]. However, this compound was as active on resistant leukemia CEM/ ADR5000 cell line as towards its sensitive parental CCRF-CEM cell line, showing a degree of resistance of 1.06 [43]. The labdane diterpenoids galanals A (2) and B (3) (Figure 1) isolated from the Cameroonian spice Aframomum arundinaceum demonstrated moderate, but selective, cytotoxicity towards cancer cell lines [37] (Table 1). However, compounds 2 and 3 were generally less active towards resistant cancer cells, with 3 showing collateral sensitivity towards resistant breast adenocarcinoma MDA-MB-231/BCRP cells (D.R.: <0.70 compared to its sensitive subline MDA-MB-231) [37].

Alkaloids.
Alkaloids are one of the most diverse groups of secondary metabolites found in plants, marine organisms, and microorganisms [2]. A well accepted definition is that alkaloids are naturally occurring, nitrogen-containing organic compounds with the exception of amino acids, peptides, purines and derivatives, amino sugars, and antibiotics. The nitrogen atom remains as a heterocyclic ring with some exceptions. Based upon biogenesis, the alkaloids are broadly classified as true alkaloids with heterocyclic nitrogen atom and pseudo alkaloids. They have an array of structural type, biosynthetic pathways, and pharmacological activities [2]. It is also worth noting that some alkaloids including anabasine, aristolochic acid I, nicotine, sanguinarine, and solanine are involved in plant side effects to humans and animals [59,60].
Compared to terpenoids and phenolics, a limited number of alkaloids isolated from African medicinal plants were reported for their cytotoxic effects on cancer cells. However, data available from the screening of some compounds isolated from African plants are rather moderate even when sensitive cell lines are involved. This is the case with the acridone alkaloids isolated from the fruits of Zanthoxylum leprieurii Guill. & Perr. (Rutaceae) collected in Cameroon, namely, helebelicine A, 3-hydroxy-1-methoxy-10-methyl-9acridone, 1-hydroxy-3-methoxy-10-methyl-9-acridone, and 1-hydroxy-2,3-dimethoxy-10-methyl-9-acridone that showed moderate activity against human lung carcinoma cells A549 (IC 50 values of 31 to 52 M) and colorectal adenocarcinoma cells DLD-1 (IC 50 of 27 to 74 M) [61].

Mode of Action of African Plant Extracts and Derived Products with Cytotoxic Effect on Drug-Resistant Cancer Cell Lines
The mode of action of many African plant extracts and isolated compounds having good antiproliferative activities on drug-resistant cells has been demonstrated. The documented modes of induction of apoptosis include activation of caspases, alteration of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS), and inhibition of angiogenesis. In this section, the synopsis of these mechanistic data will be provided.

Induction of Apoptosis and Cell Cycle Arrest. Several
African plant extracts and isolated compounds acting on MDR cancer phenotypes were found to induce apoptosis and cell cycle arrest in cancer cells. In this review, we proposed to classify the induction of apoptosis by plant extracts or derived molecules at not more than their twofold IC 50 values as follows: (i) very strong: if the percentage of induction is above 50%; (ii) strong: if the percentage of induction is between 20 and 50%; (iii) moderate: if the percentage of induction is between 10 and 20%; (iv) low: if the percentage of induction is between 4 and 10%; and (v) no induction: if the percentage of induction is below 4%.
The reported African medicinal plants with significant cytotoxic effects on MDR cancer cells and showing very strong induction of apoptosis include Echinops giganteus, Imperata cylindrica, Piper capense [40], Gladiolus quartinianus, Vepris soyauxii, and Anonidium mannii [38]. A moderate to strong induction of apoptosis was also recorded with the spice of Xylopia aethiopica [40]. It was also shown that most of the crude extracts from African medicinal plants induced cell cycle arrest mostly in G0/G1 and between G0/G1 and S phases. In fact, the cell cycle arrest in G0/G1 in leukemia CCRF-CEM cells was reported with the extracts from Vepris soyauxii, Anonidium mannii [38], Echinops giganteus, and Piper capense [40]. Arrest between G0/G1 and S phases in CCRF-CEM cells was reported with the extracts from Gladiolus quartinianus [38], Imperata cylindrica, Xylopia aethiopica [40], Polyscias fulva, and Beilschmiedia acuta [20].

Effects of African Plant Extract and Derived Molecules on
Caspase Activation. Caspases, a family of cysteine proteases, are central regulators of apoptosis [63]. Initiator caspases (caspases 2, 8, 9, 10, 11, and 12) are closely coupled to proapoptotic signals [63]. Upon activation, initiator caspases cleave and activate downstream effector caspases (caspases 3, 6, and 7), which in turn execute apoptosis by cleaving cellular proteins at specific aspartate residues [63]. In this report, we propose to classify the activation of caspases by plant extracts or derived molecules at not more than their twofold IC 50 values which will be as follows: (i) very high: if the increase is more than 64-fold; (ii) high: if the increase is between 8-and 64-fold; (iii) moderate: if the increase is between 4and 8-fold; (iv) low: if the induction is 1-4-fold; and (v) no induction: if the induction is less than 1-fold.
In general, several crude extracts having inhibitory effect on MDR cancer cells were reported not to induce the activation of caspase enzymes [20,40]. Benzophenones 19-21 were able to activate caspases in CCRF-CEM cells treated with concentrations equivalent to their IC 50 values [19]. A high activation was observed for caspases 3/7, whereas the effects on caspases 8 and 9 were moderate [19]. A high activation of caspases 3/7 activity and moderate activation of caspases 8 and 9 were also reported with the pterocarpan 39 as well as the xanthones 16 and 42 [18,54,55]. Low activation of caspases 3/7, 8, and 9 was reported with the pterocarpan 28, whilst no effect was obtained in similar experimental condition with 22, 23, and 25 [54].

Effects of African Plant Extract and Derived
Molecules on the Mitochondrial Membrane Potential. Apoptotic proteins target mitochondria and affect them in different ways. If cytochrome c is released from mitochondria due to formation of a channel in the outer mitochondrial membrane during the apoptosis process, it binds to apoptotic protease activating factor-1 (Apaf-1) and ATP, which then bind to procaspase-9 creating a protein complex known as apoptosome [64]. Herein, we propose to classify the extent of MMP alteration by plant extracts or derived molecules at not more than their twofold IC 50 values as follows: (i) very strong: if the percentage of MMP disruption is more than 50%; (ii) strong: if the percentage of MMP disruption is between 20 and 50%; (iii) moderate: if the percentage of MMP disruption is between 10 and 20%; (iv) low: if the percentage of MMP disruption is between 5 and 10%; and (v) no induction: if the percentage of induction is below 5%.
MMP disruption in cancer cells was reported as one of the likely mechanisms of induction of apoptosis by several African plant extracts and derived compounds. A strong depletion of MMP in CCRF-CEM cells was reported with crude extracts from Echinops giganteus, Xylopia aethiopica [40], and Anonidium mannii [38]. Moderate alterations of the MMP in CCRF-CEM cells were measured with extracts from Imperata cylindrica and Piper capense [40], Gladiolus quartinianus, Vepris soyauxii [38], and Polyscias fulva [20].

Effects of African Plant Extract and Derived
Molecules on Generation of Reactive Oxygen Species. The appearance of malignancies resulting in gain-of-function mutations in oncogenes and loss-of-function mutations in tumour suppressor genes leads to cell deregulation that is frequently associated with enhanced cellular stress [65,66]. In the present paper, we recommend to classify the extent of ROS production by plant extracts or derived molecules at not more than their twofold IC 50 values as follows: (i) very high: if the percentage of ROS production is more than 50%; (ii) high: if the percentage of ROS production is between 20 and 50%; (iii) moderate: if the percentage of ROS production is between 10 and 20%; (iv) low: if the percentage of ROS production is between 3 and 10%; and (v) no induction: if the percentage of ROS production is below 3%.

Antiangiogenic Effects of African Plant Extract and Derived
Molecules. Excessive angiogenesis represents an important pathogenic factor in many industrialized western countries [67]. Therefore, compounds with antiangiogenic properties are of importance in the treatment and prevention of malignancies as well as other chronic diseases [68,69]. Herein, we recommend to classify the extend of inhibition of angiogenesis by plant extracts or derived molecules at not more than their twofold IC 50 values as follows: (i) very strong: if the percentage of inhibition is more than 50%; (ii) strong: if the percentage of inhibition is between 20 and 50%; (iii) moderate: if the percentage of inhibition is between 10 and 20%; (iv) low: if the percentage of inhibition is between 5 and 10%; and (v) no induction: if the percentage of inhibition is below 5%.
The extracts from Xylopia aethiopica, Dorstenia psilirus, Echinops giganteus, and Zingiber officinale strongly inhibited angiogenesis in quail embryo [39]. A strong antiangiogenic activity on blood capillaries of the chorioallantoic membrane of quail eggs was also reported with compounds such as 16, 36, and 17 [18,57].  [57]. The two most upregulated genes by 36 were HSPA6 (heat shock 70 kDa protein 6) and HIST1H2BD (histone cluster 1, H2bd). In cooperation with other chaperones, HSPA6 stabilizes preexistent proteins against aggregation and mediates the folding of newly translated polypeptides in the cytosol as well as within organelles. They bind extended peptide segments with a net hydrophobic character exposed by polypeptides during translation and membrane translocation or following stress-induced damage [57]. Interestingly, 70 kDa heat shock protein protects cells from ischemia and its expression is increased in consequence to hypoglycemia [57], suggesting that 36 might cause hypoxic stress. Histone H2B type 1D is a protein that is in humans encoded by the HIST1H2BD gene. Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Levels of histone mRNA usually increase during S phase but decrease back to baseline level between the S phase and mitosis [57]. Certain histone mRNAs were upregulated after treatment with 36 confirming the S phases cell cycle arrest by this compound [57]. Other upregulated genes were FOSB and JUN, HIST2H2AC, HIST2H2AA4, and CD52.
Significantly downregulated genes were ACTB and ACTBL3, PGAM1, LOC728188, DHRS2, KPNA2, THOC4, RAB37 and TRAPPC6A, HNRNPK, LYAR and YBX1, LYAR, YBX1, MYCN, and RUVBL1 [57]. ACTB and ACTBL3 belonged to the most downregulated genes. Beta-actin mRNA levels are known to be disturbed after ischemia [57], which is in line with the assumption that 36 may mimic hypoxia. Another gene fitting to this hypothesis is PGAM1, which codes for phosphoglycerate mutase in glycolysis. Another gene coding for a protein similar to phosphoglycerate mutase processed protein was also downregulated by 36, LOC728188 [57]. Downregulation of glycolysis key molecules accompanied by hypoxic stress may destroy the entire energy production aperture ultimately leading to cell death. The misregulation in glyco-related mechanisms by 36 was also indicated by downregulation of DHRS2, whose encoded protein preferentially binds to glucose and related sugars [57].
KPNA2 codes for importin alpha. This protein is a key player in the nuclear transport of macromolecules [57]. THOC4 encoding a more investigated mRNA transporter molecule was also significantly downregulated by 36. The THOC4 protein is part of the TREX complex, which specifically associates with spliced mRNA [57]. THOC4 is especially involved in nuclear export of Hsp70 transcripts [57]. Interestingly, RAB37 and TRAPPC6A encode also two proteins which are also involved to transport mechanisms [57]. They were also misregulated in their transcriptional activity after 36 treatment. In summary, transport mechanisms were deregulated as consequence of treatment of cancer cells with 36 [57].

Structure-Activity Relationship of the Best Cytotoxic Compounds Identified in African Medicinal Plants
The general observation is that most of the terpenoids isolated from African medicinal plants such as compounds 1-6 were more toxic on leukemia than on carcinoma cells [20,37,41,50]. This observation is in accordance with the clinical situation, as it is well known that hematological tumors cells are frequently more sensitive than solid cancers [14]. Within the group of phenolics, this allegation varied, depending on the chemical structure. In fact, benzophenone moiety in compound 18 and the polyisoprenylated compounds 19, 20, and 21 were more active towards cell lines of different tumor types [19]. Furthermore, 18 had the lowest activity, indicating that the polyprenylation and other substitutions in the cycle B influenced the antiproliferative capacity of benzophenones [19]. Amongst the three isomers (19, 20, and 21), 19 and 20 were more active than 21 [19], suggesting that opening of the pyrone cycle in position C-31 may decrease the cytotoxic activity. The spatial configuration also influenced the cytotoxicity of benzophenones, as compounds 19 and 20 (two stereoisomers) revealed different degrees of activity on the majority of the studied cancer cell lines [19]. An analysis of the structure-activity relationship of flavonoids from Polygonum limbatum showed that the chalcones 30-32 revealed considerable cytotoxicity in contrast to the flavanones 33-35 [56]. The number of the hydroxyl (-OH) and methoxy (-OCH 3 ) substituents influences the activity of chalcones towards leukemia as well as carcinoma cell lines. In fact, chalcone 30 with two -OCH 3 substituents (in positions C-2 and C-6 ) together with -OH group (in C-4 ) demonstrated better activity than chalcones 31 and 32 with two -OH substituents and only one -OCH 3 substituent [56]. The position of the -OH and -OCH 3 did not significantly influence the activities of chalcones 31 and 32 [56].