Journal of Ethnopharmacology 153 (2014) 207–219

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Cytotoxicity and modes of action of five Cameroonian medicinal plants against multi-factorial drug resistance of tumor cells Victor Kuete a,b, Simplice B. Tankeo b, Mohamed E.M. Saeed a, Benjamin Wiench a, Pierre Tane c, Thomas Efferth a,n a

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon c Department of Organic Chemistry, Faculty of Science, University of Dschang, Dschang, Cameroon b

art ic l e i nf o

a b s t r a c t

Article history: Received 15 October 2013 Received in revised form 24 January 2014 Accepted 12 February 2014 Available online 28 February 2014

Ethnopharmacological relevance: Beilschmiedia acuta Kosterm, Clausena anisata (Willd) Hook, Fagara tessmannii Engl., Newbouldia laevis Seem., and Polyscias fulva (Hiern) Harms. are medicinal plants used in Cameroonian traditional medicine in the treatment of various types of cancers. The present study aims at investigating 11 methanolic extracts from the above Cameroonian medicinal plants on a panel of human cancer cell lines, including various drug-resistant phenotypes. Possible modes of action were analyzed for two extracts from Beilschmiedia acuta and Polyscia fulva and alpha-hederin, the representative constituent of Polyscia fulva. Materials and methods: Cytotoxicity was determined using a resazurin assay. Cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were measured by flow cytometry. Cellular response to alpha-hederin was investigated by a mRNA microarray approach. Results: Prescreening of extracts (40 mg/mL) showed that three of eleven plant extracts inhibited proliferation of CCRF-CEM cells by more than 50%, i.e. BAL (73.65%), the bark extract of Beilschmiedia acuta (78.67%) and PFR (68.72%). Subsequent investigations revealed IC50 values below or around 30 mg/mL of BAL and PFR in 10 cell lines, including drug-resistant models, i.e. P-glycoproteinoverexpressing CEM/ADR5000, breast cancer resistance protein-transfected MDA-MB-231-BCRP, TP53 knockout cells (HCT116 p53
/
), and mutation-activated epidermal growth factor receptor-transfected U87MG.ΔEGFR cells. IC50 values below 5 mg/mL of BAL were obtained for HCT116 (p53
/
) cells. IC50 values below 10 mM of alpha-hederin were found for sensitive CCRF-CEM and multidrug-resistant CEM/ ADR5000 cells. The BAL and PFR extracts induced cell cycle arrest between G0/G1 and S phases. PFR-induced apoptosis was associated with increased ROS generation and MMP breakdown. Microarray-based cluster analysis revealed a gene expression profile that predicted cellular response to alpha-hederin. Conclusion: BAL, PFL and alpha-hederin, an exemplarily taken constituent of Beilschmiedia acuta and Polyscia fulva extracts revealed cytotoxicity towards cancer cell lines. Hence, Beilschmiedia acuta and Polyscia fulva may be valuable to develop drugs against otherwise drug-resistant cancer cells. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Apoptosis Beilschmiedia acuta Cytotoxicity Polyscias fulva Mode of action Microarrays

1. Introduction Multidrug resistance is a major factor for the failure of cancer chemotherapy and affects patients with a variety of hematopoietic and solid tumors, including breast, ovarian, lung, and lower gastrointestinal tract cancers (Gottesman, 2002). Chemotherapy kills drug-sensitive cells, but drug-resistant subpopulations ultimately leading to treatment failure. Multidrug resistance is

n

Corresponding author. Tel.: þ 49 6131 3925751; fax: þ 49 49 6131 3923752. E-mail address: [email protected] (T. Efferth).

http://dx.doi.org/10.1016/j.jep.2014.02.025 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.

characterized by cross-resistance between chemically and functionally diverse drugs that are expelled out of tumor cells by drug efflux pumps of the ATP-binding cassette (ABC) transporter type. The best-known ABC transporter is P-glycoprotein (ABCB1/MDR1), a glycoprotein of 170 kDa which confers tumor cell resistance to a large array of anticancer drugs, including anthracyclines, Vinca alkaloids, taxanes and epipodophyllotoxins (Gottesman and Ling, 2006). Another ABC transporter that mediates multidrug resistance is breast cancer resistance protein (BCRP, MXR2, ABCG2) (Natarajan et al., 2012). Drug resistance is, however, even more complex, since not only one or few mechanisms, but many factors determine the failure of chemotherapy (Efferth et al., 1992; Efferth

208

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

and Volm, 1993; Volm et al., 1993). Drug resistance may be mediated by mechanisms upstream or downstream of the actual drug target sites (Efferth and Grassmann, 2000). Upstream mechanisms are not only ABC transporters, but also drugmetabolizing and -detoxifying enzymes (e.g. cytochrome P450 monooxigenases, enzymes of the glutathione redox cycle, antioxidant enzymes) (Azzariti et al., 2011; Sau et al., 2010; Singh et al., 2012). Proliferation-associated factors such as growth factors and their receptors (e.g. the epidermal growth factor receptor, EGFR) represent important determinants of response to chemotherapy (Abukhdeir and Park, 2008; Efferth and Volm, 1992; Lai et al., 2012; Lambert and Keyomarsi, 2007). Downstream mechanisms prevent cell death even after binding of drugs to their targets, e.g. by deregulated signaling cascades of apoptosis, autophagy, or necroptosis (Long and Ryan, 2012). The tumor suppressor gene TP53 is mutated in about half of all human tumors (
) and TP53 mutations confer resistance to apoptosis and, hence, to chemoand radiotherapy (Bush and Li, 2002; Lowe, 1995; Martinez-Rivera and Siddik, 2012). The multifactorial nature of drug resistance urgently necessitates the development of new treatment options to improve treatment outcomes and survival rates of cancer patients. There have been huge efforts to develop inhibitors of resistance proteins, e.g. inhibitors of ABC transporters, of drug detoxifying enzymes, or of apoptosis regulators (Binkhathlan and Lavasanifar, 2013; Sau et al., 2010; Sayers, 2011). While impressive results have been described in preclinical settings, clinical trials mainly failed because of non-tolerably high toxicities of these inhibitors in normal tissue and organs (Binkhathlan and Lavasanifar, 2013). Furthermore, the multifactorial nature of drug resistance would require the application of an entire cocktail of inhibitors leading to an even higher increase of toxic reactions in normal tissues. Natural products are frequently regarded as being less toxic than synthetic compounds. Therefore, we focused in recent years on phytochemicals as inhibitors of resistance proteins (Efferth and Greten, 2012; Eichhorn and Efferth, 2012). Another strategy is not to inhibit specific resistance mechanisms, but to bypass resistance by non-cross-resistant drugs. For example, drugs such as 5-fluorouracil that are not recognized by P-glycoprotein are not extruded leading to cell killing even in the presence of high amounts of P-glycoprotein in multidrug-resistant tumors. However, 5-fluorouracil is less active in tumors with mutated TP53 (Bunz et al., 1999). Therefore, an ideal compound would bypass several drug resistance mechanisms at the same time. Natural products are frequently multi-target compounds acting not only on one cellular target but on several (Efferth and Koch, 2011). Sometimes there have been attempts from scholars of conventional academic medicine to bring natural products into discredit because of this feature. The argument was that natural products would be “dirty drugs”, because they were too non-specific to inhibit one single target of interest. Fortunately, a change of mind can recently be observed concerning multi-targeted compounds (Schrattenholz et al., 2010). During evolution of life, the activity of natural products towards many different targets was a particular survival advantage of plants in the competition with predators (Wöll et al., 2013). Therefore, multitargeted natural compounds might be even better suited for cancer therapy than mono-specific drugs. For this reason, we hypothesized that it should be possible to identify medicinal plants that are capable to kill drug-resistant cells not only to one, but with different mechanisms of resistance. During the past few years, we focused on medicinal plants used in traditional Cameroonian medicine and found profound cytotoxic activities towards cancer cells (Kuete and Efferth, 2010, 2011; Kuete et al., 2011, 2012, 2013). In the present investigation, we analyzed the anti-proliferative activities of various parts of five Cameroonian medicinal plants

traditionally used to treat cancers in a panel of 10 cell lines, including different drug resistance phenotypes. We used (1) cells specifically over-expressing the ABC transporter P-glycoprotein (ABCB1/MDR1) or BCRP (ABCG2) exhibiting multidrug resistance phenotypes, (2) a p53 knockout cell line (HCT116 p53
/
) as a model for mutational loss-offunction of this tumor suppressor gene and (3) U87MG cells stably transfected with a deletion-mutation-activated EGFR gene as model for drug resistance by oncogenes. These cell lines were compared to their corresponding wild-type counterparts. All these cell models are known to exert resistance to anti-cancer drugs (Bragado et al., 2007; Doyle et al., 1998; Efferth et al., 2008; Lee et al., 2010; Nagane et al., 1998). The studied plants included Beilschmiedia acuta Kosterm (Lauraceae) locally known as Ndareh, Clausena anisata (Willd) Hook (Rutaceae) or Horsewood or Maggot killer (English) and locally known as Jumba, Fagara tessmannii Engl. (Rutaceae) or African pepper (English) and locally known as nashou, Newbouldia laevis Seem. (Bignoniaceae) or Boundary tree or Tree of life (English) and locally known as Faangum, and Polyscias fulva (Hiern) Harms. (Araliaceae) or Parasol tree (En), locally known as Ake-kwe. The use of Fagara tessmannii (Mbaze et al., 2007) and Newbouldia laevis (Bowen et al., 1985; Kupchan et al., 1971) is well-recorded whilst no information concerning that of the three other plants in the management of malignancies has been previously reported, despite the popular use for this purpose in Cameroon. However, it has been recommended that ethnopharmacological usages such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases should be taken into account when selecting plants used to treat cancer, since these reflect disease states bearing relevance to cancer or cancer-liker symptoms (Cordell et al., 1991; Popoca et al., 1998). The mode of action of the two most active plants against these drug-resistant tumor models, i.e. Beilschmiedia acuta Kosterm (Lauraceae) and Polyscias fulva (Hiern) Harms (Arabiaceae) were analyzed in more detail for their mode of action in terms of cell cycle distribution, apoptosis induction, generation of reactive oxygen species (ROS) and change of mitochondrial membrane potential (MMP). Bioinformatical COMPARE analysis and hierarchical cluster analyses of microarray-based transcriptomic mRNA expression data in a panel of 60 cell lines from the Developmental Therapeutics Program of the National Cancer Institute, USA (http://dtp.nci.nih.gov) were performed for alpha-hederin as an example for a cytotoxic compound of Polyscias fulva.

2. Materials and methods 2.1. Plant material All medicinal plants used in the present work were collected in different areas of Cameroon between January and April 2012 (Table 1). The plants were identified at the Cameroonian National Herbarium, Yaoundé where the voucher specimens were deposited under the accession numbers 37335/HNC [Beilschmiedia acuta Kosterm, roots, leaves, bark], 44242/HNC [Clausena anisata (Willd) Hook, roots, leaves, bark], 25124/SRF.Cam [Fagara tessmannii Engl., roots, leaves, bark], 29469/HNC [Newbouldia laevis Seem., roots, leaves, bark], and 60407/HNC [Polyscias fulva (Hiern) Harms., roots, leaves, bark]. The air-dried and powdered plant material was soaked in methanol at room temperature for 48 h. The methanol extract was concentrated under reduced pressure to give the crude extract. This extract was then conserved at 4 1C until further use. 2.2. Chemicals Doxorubicin and vinblastine were provided by the University Medical Center of the Johannes Gutenberg University (Mainz, Germany) and dissolved in PBS (Invitrogen, Eggenstein, Germany) at a concentration of 10 mM. Geneticin was purchased from

Table 1 Information on five Cameroonian medicinal plants. Plants (family) Traditional use and national herbarium codea

Polyscias fulva (Hiern) Harms. (Araliaceae) 60407/HNC

Cancer and gastrointestinal infections (Personal information).

Diabetes, anti-hypertensive, anti-nociceptic, malaria, fungal, bacterial and viral infections, inflammation, heart and mental disorders, constipation, convulsions, impotence and sterility (Adesinan and EI, 1982; Hutchings et al., 1996; Ito et al., 2009; Ojewole, 2002) Gonorrhea, cancers, swellings and inflammation (Mbaze et al., 2007)

Cancers, spasms, infectious diseases, male infertility and diabetes (Bowen et al., 1985; Kupchan et al., 1971), coagulant or antihemorrhagic properties; digestive threats, urogenital and pulmonary infections(Dandjesso et al., 2012; Gafner et al., 1996); Dysentery, worms, malaria, sexually transmitted diseases, dental caries and diarrhea (Eyong et al., 2005). Malaria, fever, mental illness (Tshibangu et al., 2002); venereal infections and obesity (Focho and Fonge, 2009; Jeruto et al., 2007) and cancer (Personal information).

Leaves (18.40%), fruits (20.22%) and barks (36.46%) Leaves (16.31%)

Bioactive (or potentially active) compounds isolated from plantsb,c

Biological activities of crude extractc,d

Lebialem, South-West – Region of Cameroon; (4110'N 9114'E/ 4.1671N 9.2331E)

–

Lebialem, South-West Essential oils (sabinene, β-pinene, pulegone, 1,8 -cineole, region of Cameroon estragole, (Senthilkumar and Venkatesalu, 2009); carbazole alkaloids, coumarins, limonoids (Ito et al., 2009; Ngadjui et al., 1991).

Antimicrobial: Essential oil active against Sa, Sp, Esp, St, Pa (Nelson, 1997; Senthilkumar and Venkatesalu, 2009)

Leaves (9.10%), Lebialem, South-West region of Cameroon roots (8.70%) and barks (10.65%)

Alkaloids (chelerythrine, dictamine), coumarins (scoparone, Antimicrobial: active against Bs, xanthotoxin) (Adesina, 2005); 2,6-dimethoxy-1,4-benzoquinone; Ec, Sa, Sv, Mm, Ca, Cv, Cs and Ss 3β-acetoxy-16β-hydroxybetulinic acid, 3β, 16β-hydroxybetulinic (Mbaze et al., 2007) acid (Mbaze et al., 2007), coumarins (Adesina, 2005; Mabry and Ulubelen, 1980).

Melon, Littoral region Tannins, triterpenoids, mucilages and reducing compounds, Leaves flavonoids, steroids, alkaloids, cardiac glycosides (Dandjesso (18.75%), and of Cameroon et al., 2012; Usman and Osuji, 2007). barks (19.35%) (04133'53“N 09138'04“E)

Antimicrobial: active against Ca, Ck, Sa, Sf, Ec, Pa, Sp, Pv, Kp, St, Sd, Ng Mtb, Ms (Dandjesso et al., 2012; Kuete et al., 2008).

Dschang, West region Polysciasoside A, kalopanax-saponin B, alpha-hederin (Bedir of Cameroon (6130'N et al., 2001; Kuete and Efferth, 2011) 10130'E/6.5001N 10.5001E)

Inhibition of microsomal lipid peroxidation (Njayou et al., 2008)

Leaves (15.62%), roots (17.56%) and barks (19.01%)

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

Beilschmiedia acuta Kosterm (Lauraceae) 37335/HNC Clausena anisata (Willd) Hook (Rutaceae) 44242/ HNC Fagara tessmannii Engl. (Rutaceae) 25124/ SRF. Cam Newbouldia laevis Seem. (Bignoniaceae) 29469/ HNC

Part used and Area of plant extraction collection yield (%)b (Geographic Coordinates)

a

Plants were identified at the Cameroon National Herbarium (HNC); ICNA: Voucher with no identification code at the HNC. The percentage of the methanol extract. (–): Data not available. d Microorganisms[Bs: Bacillus subtilis; Ca: Candida albicans; Ck: Candida krusei; Mm: Mucor miehei; Cv: Chlorella vulgaris; Cs: Chlorella sorokiniana; Ec: Escherichia coli; Esp: Enterococcus species; Mtb: Mycobacterium tuberculosis; Ms; Mycobacterium smegmatis; Ng: Neisseria gonorrhoeae; Pa: Pseudomonas aeruginosa; Sf: Streptococcus faecalis; Pv: Proteus vulgaris; Sa: Staphylococcus aureus; Sp: Streptococcus pneumoniae; St: Salmonella typhimurium; Kp: Klepsiella pneumoniae; Sd: Shigelle dysenteriae; Ss: Scenedesmus subspicatus; Sv: Streptomyces viridochromogene]. b c

209

210

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

Sigma-Aldrich (Munich, Germany) at a concentration of 50 mg/mL in sterile-filtered H2O and alpha-hederin was purchased from ChromaDex (Suite G Irvine, CA, USA). 2.3. Preliminary phytochemical investigations The major secondary metabolites classes such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins and triterpenes were screened according to the common phytochemical methods previously described by (Harbone, 1973). 2.4. Cell cultures Leukemia CCRF-CEM cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum in a humidified 5% CO2 atmosphere at 37 1C. Sensitive and drugresistant cells were kindly provided by Dr. J. Beck (Department of Pediatrics, University of Greifswald, Greifswald, Germany). Breast cancer cells transduced with control vector (MDA-MB231-pcDNA3) or with cDNA for the breast cancer resistance protein BCRP (MDA-MB-231-BCRP clone 23) were obtained from Dr. Douglas D. Ross (University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD) (Doyle et al., 1998) and maintained under standard conditions as described above for CCRF-CEM and HL-60 cells. Human wild-type HCT116 (p53 þ / þ ) colon cancer cells as well as knockout clones HCT116 (p53
/
) derived by homologous recombination were a generous gift from Dr. B. Vogelstein and H. Hermeking (Howard Hughes Medical Institute, Baltimore, MD). The generation of this knockout cell line has been previously described (Bunz et al., 1999). Human glioblastoma multiforme U87MG cells (non-transduced) and U87MG cell line transduced with an expression vector harboring an epidermal growth factor receptor (EGFR) gene with a genomic deletion of exons 2 through 7 (U87MG.ΔEGFR) were kindly provided by Dr. W. K. Cavenee (Ludwig Institute for Cancer Research, San Diego, CA) (Nagane et al., 1998). MDA-MB-231-BCRP, U87MG.ΔEGFR and HCT116 (p53
/
) were maintained in DMEM medium containing 10% FBS (Invitrogen) and 1% penicillin (100 U/ mL)-streptomycin (100 μg/mL) (Invitrogen) and were continuously treated with 800 ng/mL and 400 mg/mL geneticin, respectively. The multidrug resistance profile of these cell lines has been reported (Bragado et al., 2007; Doyle et al., 1998; Efferth et al., 2003b, 2008; Lee et al., 2010; Nagane et al., 1998). Human liver hepatocellular carcinoma HepG2 and the AML12 normal heptocytes were obtained from ATCC (USA). The above medium without geneticin was used to maintained MDA-MB-231, U87MG, HCT116 (p53 þ / þ ), HepG2 and AML 12 cell lines. The cells were passaged twice weekly. All experiments were performed with cells in the logarithmic growth phase. 2.5. Cell lines of the developmental therapeutics program of the NCI The human tumor cell lines of the Developmental Therapeutics Program of the National Cancer Institute (NCI, USA) consisted of leukemia, melanoma, as well as cancers of the lung, colon, kidney, ovary, breast, prostate, or brain. Their origin and processing have been described (Alley et al., 1988). Cells were examined by the sulforhodamine B assay (Rubinstein et al., 1990). 2.6. Resazurin reduction assay Resazurin reduction assay (O'Brien et al., 2000) was performed to assess the cytotoxicity of the test samples toward various sensitive and drug-resistant cancer cell lines. The assay is based on reduction of the indicator dye, resazurin, to the highly fluorescent resorufin by viable cells. Non-viable cells rapidly lose the

metabolic capacity to reduce resazurin and thus produce no fluorescent signal. Briefly, adherent cells were detached by treatment with 0.25% trypsin/EDTA (Invitrogen, Darmstadt, Germany). An aliquot of 1  104 cells was placed in each well of a 96-well cell culture plate (Thermo Scientific, Langenselbold, Germany) in a total volume of 200 mL. Cells were allowed to attach overnight and then were treated with different concentrations of the studied sample. For suspension cells, aliquots of 2  104 cells per well were seeded in 96-well-plates in a total volume of 100 mL. The studied sample was immediately added in varying concentrations in an additional 100 mL of culture medium to obtain a total volume of 200 mL/well. After 24 h or 48 h, 20 mL resazurin (Sigma-Aldrich, Schnelldorf, Germany) 0.01% w/v in double-distilled water (ddH2O) was added to each well and the plates were incubated at 37 1C for 4 h. Fluorescence was measured on an Infinite M2000 Pro™ plate reader (Tecan, Crailsheim, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Each assay was done at least two times, with six replicate each. The viability was evaluated based on a comparison with untreated cells. IC50 values represent the sample's concentrations required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel.

2.7. Flow cytometry for cell cycle analysis and detection of apoptotic cells The cell-cycle analysis was performed by flow cytometry using The Vybrants DyeCycle™ (Invitrogen). The Vybrants DyeCycle™ Violet stain is a DNA-selective, cell membrane-permeant, and nonfluorescent stain that uses the violet laser for DNA content analysis in living cells. The Vybrants DyeCycle™ Violet stain is fluorescent upon binding to double-stranded DNA. Leukemia CCRF-CEM cells (1  106) were treated with the concentrations equivalent to the IC50 values of the crude extract for 24 h, 48 and 72 h. Following incubation, 1 mL of Vybrants DyeCycle™ Violet stain was added to 1 mL of cell suspension and incubated for 30 min at 37 1C. Cells were measured on a LSR-Fortessa FACS analyzer (Becton-Dickinson, Germany). Ten thousand cells were counted for each sample. Vybrants DyeCycle™ Violet stain was measured with 440 nm excitation. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed at least in triplicate.

2.8. Analysis of mitochondrial membrane potential (MMP) The effects of extract on the MMP were analyzed by 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide) (JC-1; Biomol, Germany) staining. JC-1 is a dye that can selectively enter into mitochondria and exhibits an intense red fluorescence in healthy mitochondria with normal membrane potentials. In cells with reduced MMP, the red fluorescence disappears. Briefly, 1  106 CCRF-CEM cells treated with different concentrations compounds or DMSO (solvent control) for 24 h were incubated with JC-1 staining solution according to the manufacturer's protocol for 30 min. Subsequently, cells were measured in a LSR-Fortessa FACS analyzer (Becton-Dickinson). For each sample, 1  104 cells were counted. The JC-1 signal was measured with 561 nm excitation (150 mW) and detected using a 586/15 nm bandpass filter. The compounds signal was analyzed with 640 nm excitation (40 mW) and detected using a 730/745 nm bandpass filter. All parameters were plotted on a logarithmic scale. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed at least in triplicate.

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

2.9. Measurement of reactive oxygen (ROS) species by flow cytometry 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFH-DA) (SigmaAldrich, Germany) is a probe used for the highly sensitive and quantifiable detection of ROS. The non-fluorescent H2DCFH-DA diffuses into the cells and is cleaved by cytoplasmic esterases into 2',7'-dichlorodihydrofluorescein (H2DCF) which is unable to diffuse back out of the cells. In the presence of hydrogen peroxide, H2DCF is oxidized to the fluorescent molecule dichlorofluorescein (DCF) by peroxidases. The fluorescent signal emanating from DCF can be measured and quantified by flow cytometry, thus providing an indication of intracellular ROS concentration (Bass et al., 1983; Cossarizza et al., 2009). Briefly, 2  106 CCRF-CEM cells were resuspended in PBS and incubated with 2 mM H2DCFH-DA for 20 min in the dark. Subsequently, cells were washed with PBS and resuspended in RPMI 1640 culture medium containing different concentrations of extract or DMSO (solvent control). After 1 h of incubation, cells were washed and suspended in PBS. Subsequently cells were measured in a FACSCalibur flow cytometer (Becton-Dickinson, Germany). For each sample 1  104 cells were counted. DCF was measured at 488 nm excitation (25 mW) and detected using a 530/630 nm bandpass filter. All parameters were plotted on a logarithmic scale. Cytographs were analyzed using FlowJo software (Celeza, Switzerland). All experiments were performed at least in triplicate. 2.10. Statistical analysis Statistical analysis of all data was performed using a Student's t-test or Kruskal–Wallis test followed by Dunn's post-hoc multiple comparison test (Graph-Pad Prism 5.01; GraphPad Software, Inc., CA, USA). P o0.05 denoted significance in all cases. The NCI tumor cell line panel has been assayed for their transcriptome-wide mRNA expression by microarray analyses (Scherf et al., 2000; Staunton et al., 2001). The data are available through the NCI website (http://dtp.nci.nih.gov). We applied hierarchical cluster analyses (WARD method) using these microarray data and the WinSTAT program (Kalmia, Cambridge, MA, USA) as previously described (Efferth et al., 1997). Cluster analyses have been frequently used for gene expression profiling and molecular pharmacology of cancer (Scherf et al., 2000). COMPARE analyses were performed to produce rank-ordered lists of genes expressed in the NCI cell lines. The methodology has been described (Paull et al., 1989). Gene expression values of cells lines were ranked for similarity to the log10IC50 values for α-hederin. COMPAREbased correlation coefficients (R-values) were calculated. Pearson's correlation test was used to calculate significance values and rank correlation coefficients as a relative measure of the linear dependency of two variables (WINSTAT program, Kalmia). The Pearson correlation test was used as a measure for interval-scaled linear

211

correlations. We used Pearson's instead of Spearman's rank correlation test, because the values used for our analyses were not equidistant and equidistance of values is a precondition for Spearman's test. The chi-squared test was applied to bivariate frequency distributions of pairs of nominal scaled variables (WinSTAT program, Kalmia). It was used to calculate significance values (P-values) and rank correlation coefficients (R-values) as a relative measure of the linear dependency of two variables. To define cell lines as being sensitive or resistant to the test compounds, we took the median IC50 value as a cut-off threshold.

3. Results 3.1. Chemical composition of the studied extracts The results of the qualitative analysis showed that each of the studied plant extract contains at least one classes of secondary metabolites such as alkaloids, anthocyanins, anthraquinones, flavonoids, phenols, saponins and triterpenes (Table 2). Only the extracts from the roots of Fagara tessmannii and the bark of Newbouldia laevis contain anthraquinones, while anthocyanins were found in those from the leaves of Fagara tessmannii and the roots of Polyscia fulva. The extracts from the leaves and roots of Polyscia fulva, the leaves and bark of Beilschmiedia acuta, and the bark of Newbouldia laevis contained the most classes of the studied secondary metabolites, and phenols were presented in the extracts from the leaves of all the samples. 3.2. Cytotoxicity of the studied samples Cytotoxicity assays were performed using 11 extracts belonging to five medicinal plants and CCRF-CEM leukemia cells. The results in Fig. 1 show that the extracts from Clausena anisata, Fagara tessmannii, Newlbouldia laevis inhibited proliferation less than 50% leukemia CCRCEM cells at a concentration of 40 mg/mL. Only extracts from leaves (BAL; 73.65%) and bark of Beilschmiedia acuta (BAR; 78.67%) and the roots of Polyscias fulva (PFR; 68.72%) were able to display more than 50% inhibition of CCRF-CEM cell growth. The IC50 values of these three samples were further determined on a panel of cancer cell lines with drug-sensitive and -resistant phenotypes and compared with doxorubicin as control drug (Table 3). In addition to sensitive CCRF-CEM and multidrug-resistant P-glycoprotein (ABCB1/MDR1)-overexpressing CEM/ADR5000 cells, we tested MDA-MB-231 cells over-expressing breast cancer resistance protein (ABCG2/BCRP), HCT116 p53 knockout and wildtype cells, and transfectant U87MG cells harboring a mutation-activated EGFR gene (ΔEGFR) as examples for resistanceinducing ABC-transporters, tumor suppressors and oncogenes. Finally, we investigated HepG2 liver cancer cells and AML12 normal hepatocytes to compare carcinoma cells with normal cells. The BAL and PFR extracts displayed IC50 values below 30 mg/mL against all 10 studied

Table 2 Chemical constituents and extraction yield of five medicinal plant extracts from Cameroon. Chemical constituents

Plants, parts and constituents Polyscias fulva

Anthocyanins Flavonoids Triterpenes Anthraquinones Phenols Saponins Alkaloids

Beilschmiedia acuta

Fagara tessmannii

Clausena anisata

Newbouldia laevis

L

B

R

L

F

B

L

B

R

L

L

B





þ þ þ


þ þ
þ þ þ

þ þ þ
þ þ þ


þ þ
þ þ þ





þ þ



þ

þ þ þ

þ þ

þ
þ



þ


þ


þ þ þ

þ





þ
þ


þ þ
þ þ



þ þ þ þ þ þ

L: leaves; B: bark; R: roots; F: fruits, ( þ ): present; (
): absent

212

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

Growth proliferation (%control)

80 70

65.17±3.49g 64.25±2.96fg

62.68±0.71f 57.86±2.3e

60

55.28±2.01e 54.93±1.81e 51.15±1.04d

50

50.49±1.21d 31.28±4.78c

40

31.22±0.17c 26.35±3.70bc 21.33±3.49b

30

14.96±3.6

20 10 0

Fig. 1. Inhibition of CCRF-CEM leukemia cell growth (% of untreated control) by plant extracts at each 40 mg/mL, alpha-hederin and doxorubicin (each 10 mg/mL) as measured by the resazurin reduction assay. Data with different alphabetic letters are significantly different (Po 0.05). Table 3 Cytotoxicity of two medicinal plant extracts, alpha-hederin and doxorubicin towards sensitive and drug-resistant cancer cell lines and normal cells as determined by the resazurin reduction assay. Cell lines

CCRF-CEM CEM/ADR5000 MDA-MB-231 MDA-MB-231/BCRP HCT116 p53 þ / þ HCT116 p53
/
U87MG U87MG.ΔEGFR HepG2 AML12

Tested samples, IC50 valuesa and degrees of resistance (in brackets) PFRb

BALb

BARb

Alpha-hederinc

Doxorubucinc

7.79 70.73 22.63 7 2.44 (2.91) 3.277 0.41 16.677 2.04 (5.09) 14.667 1.38 5.98 7 0.47 (0.41) 4.157 0.51 16.357 1.26 (3.94) 12.99 7 0.94 (0.32) 4 40

8.22 7 0.71 19.767 2.04 (2.40) 6.45 7 0.58 21.09 72.34 (3.27) 21.127 1.76 4.79 7 0.61 (0.23) 7.46 7 0.64 17.85 7 1.18 (2.39) 23.09 7 2.34 (0.58) 4 40

14.727 2.06 26.74 72.42 (1.82) 6.667 0.74 22.75 7 1.97 (3.42) 11.62 7 1.17 21.177 2.37 (1.82) 7.277 0.65 32.53 7 2.43 (4.47) 4 40 4 40

7.43 70.99 (1.18) 21.3570.21 19.80 7 1.92 (0.93) 14.98 7 2.51 18.92 7 0.48 (1.26) 21.45 7 1.65 43.98 7 0.31 (2.05) 23.63 7 1.36 (o 0.59) 452.29

195.127 14.30 (975.60) 1.107 0.01 7.83 7 0.01 (7.11) 1.43 7 0.02 4.067 0.04 (2.84) 1.067 0.03 6.117 0.04 (5.76) 1.417 0.12 ( o 0.04) 473.59

a The degree of resistance was determined as the ratio of IC50 value of the resistant/IC50 sensitive cell line; the test extracts were PFR (from the roots of Polyscias fulva); BAL and BAR (respectively from the leaves and roots of Beilschmiedia acuta). b mg/mL. c mM.

Fig. 2. Cytotoxicity of alpha-hederin (A) and doxorubicin (B) toward sensitive and drug-resistant cancer cells and normal AML12 hepacytes. The corresponding drug resistance mechanisms are indicated.

cell lines. The BAR extract was active towards 9 of 10 cancer cell lines. The obtained IC50 values were in a range between 3.27 mg/mL (towards MDA-MB-231 cells) and 22.63 mg/mL (towards CEM/ ADR5000 cells) for PFR, 4.79 mg/mL (against HCT116 (p53
/
) cells) and 23.09 mg/mL (against HepG2 cells) for BAL, and 0.11 mg/mL (against CCRF-CEM cells) and 195.12 mg/mL (against CEM/ADR5000 cells) for doxorubicin. IC50 values below 30 mg/mL were obtained for BAR towards 8 of 10 tested cell lines, the lowest value being

6.66 mg/mL against MDA-MB-231. The constituent of Polyscia fulva, alpha-hederin also displayed doses-dependent growth proliferation of the cancer cell lines (Fig. 2) with IC50 values below 50 mM (Table 3). None of the studied extract inhibited growth more than 50% in normal hepatocytes AML12 cells at a concentration of 40 mg/mL. Transfection of cells with the genes for BCRP or mutation-activated EGFR as well as knock-out of p53 conferred resistance to doxorubicin (Table 3), indicating that these genetically modified cell lines are suitable models

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

Polyscia fulva (roots extract) 4.28 32.6 50%

0%

4.43 36.4 29.2

25.4

26.9

24 h

48 h

100%

23.9 31.6

34.4

Beilschmiedia acuta (leaves extract)

1.12

G2/M S

41.8

Go/G1 Sub-G1

72 h

Experimentation time

Cells percentage

Cells percentage

100%

213

3.21 37.2

1.89

1.45

23.8

25.5

45.1

50%

28.4

0%

24 h

S Go/G1

39.3 17.8

G2/M

28.8 48 h

43.3

Sub-G1

72 h

Experimentation time

Fig. 3. Cell cycle distribution of leukemia CCRF-CEM cells treated with extracts from Polyscias fulva roots, Beilschmiedia acuta leaves at their IC50 values for 72 h as determined by flow cytometry. Data of control and doxorubicin in similar experimental conditions were previously reported (Kuete et al., 2013) and are available as supportive information Figure S1).

Fig. 4. Effect of the extracts from Polyscias fulva roots and Beilschmiedia acuta leaves on MMP. Data of control and vinblastine in similar experimental conditions were previously reported (Kuete et al., 2013). Samples were tested at their 1/4  IC50(1), 1/2  IC50(2), IC50(3), and 2  IC50 (4) values. The IC50 values used for this experiment were 7.79 mg/mL (Polyscias fulva roots extract, PFR) and 8.22 mg/mL (Beilschmiedia acuta leaves extract, BAL). Loss of MMP (Q1), intact cells (Q2), ruptured cell membrane (Q3 and Q4).

to study drug resistance. The degrees of resistance were calculated by dividing the IC50 value of the resistant cell line by the corresponding parental sensitive wild-type cell line. Furthermore, normal AML12 hepatocytes were more resistant towards doxorubicin than HepG2 cancer cells. Collateral sensitivity (hypersensitivity) of otherwise drug-resistant cells was observed for PFR and BAL towards HCT116 (p53
/
) and HepG2 cells (Table 3). Furthermore, all drug-resistant cell lines exhibited much lower degrees of resistance to PFR, BAL and BAR compared to doxorubicin. All plant extracts showed higher IC50 values in normal AML12 hepatocytes compared to HepG2 liver cancer cells, indicating at least some tumor-specific action of these extracts. The two most active extracts, BAR and PFR, were subsequently studied for their effects on cell cycle distribution, induction of apoptosis, generation of ROS and breakdown of MMP. 3.3. Cell cycle distribution and apoptosis The effects of PFR, BAL and doxorubicin at their IC50 values after 72 h incubation on cell cycle distribution of CCRF-CEM cells are summarized in Fig. 3. Both extracts as well as doxorubicin considerably altered the distribution of the different cell cycle phases, with a time-dependent increase of sub-G0/G1 phase cells.

PFR and BAL extracts induced cell cycle arrest between G0/G1 and S-phases. Upon treatment of CCRF-CEM cells with concentrations equivalent to the IC50 value of each of the extracts, cells progressively underwent apoptosis, with percentages in sub-G0/G1 phase ranging from 25.4% (24 h) to 41.8% (72 h) for the PFR extract and from 17.8% (24 h) to 43.3% (72 h) for BAL. These values were higher than those obtained with non-treated cells [range from 3.82% (24 h) to 9.37% (72 h)] and lower than those obtained for doxorubicin [range from 42.9% (24 h) to 91.6% (72 h)].

3.4. Effect on the mitochondrial membrane potential (MMP) We assessed the effects of PFR, BAL and vinblastine on the MMP of CCRF-CEM cells treated with concentration equivalent to their 1/4  IC50, 1/2  IC50, IC50 and 2  IC50 values as obtain in resazurin assays. BAL induced a weak MMP reduction (5.17% at two-fold IC50) (Fig. 4), PFR induced up to 10.8% disruption of MMP after treatment with the highest tested concentration. These values were lower than that of the reference compound, vinblastine that yielded up to 48.6% alteration upon two-fold IC50 treatment.

214

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

Fig. 5. Effect of the extracts from Polyscias fulva roots, Beilschmiedia acuta leaves and H2O2 (at 50 mM) on the ROS production in CCRF-CEM cells after 24 h treatment. Samples were tested at their 1/4  IC50(1), 1/2  IC50(2), IC50(3), and 2  IC50 (4) values; The IC50 values used for this experiment were 7.79 mg/mL (Polyscias fulva roots ectract, PFR) and 8.22 mg/mL (Beilschmiedia acuta leaves extract, BAL).

3.5. Effects on reactive oxygens species (ROS) The effects of the crude extracts on ROS levels were investigated in CCRF-CEM cells after 24 h treatment as shown in Fig. 5. H2O2 as control compound increased the ROS level to 10.4%, while ROS production in non-treated cells was 0.94%. Among the two studied plant extracts, PFR considerably increased ROS production (up to 35.4% after treatment with two-fold IC50). 3.6. Role of ABC-transporters in resistance to alpha-hederin The IC50 values for alpha-hederin were 7.43 mM in CEM/ ADR5000 cells and 6.30 mM in CCRF-CEM cells in the resazurin assay (Table 3).The degree of resistance calculated from the IC50 values was 1.18, indicating no cross-resistance of CEM/ADR5000 cells to alpha-hederin (Fig. 2). For comparison, CEM/ADR5000 cells are highly resistant to standard drugs such as doxorubicin (975fold, Table 3). To further analyze the role of P-glycoprotein for resistance to alpha-hederin, we investigated a panel of 60 tumor cell lines. DNA gain/amplification at the chromosomal locus of the MDR1 gene (7q21) and mRNA expression of MDR1 (as assayed by Northern blot, RT-PCR or microarray hybridization) were correlated with the IC50 values for alpha-hederin. As a functional test for

P-glycoprotein function, the accumulation of the fluorescent dye rhodamine 123 (R123) was associated with the IC50 values for alpha-hederin. R123 is a specific substrate of P-glycoprotein and can be used to monitor P-glycoprotein function (Efferth et al., 1989; Sonneveld and Wiemer, 1997). The IC50 values for alphahederin did not correlate with any of these parameters with the Pearson's rank correlation test (Table 2), indicating that alphahederin is not involved in P-glycoprotein/MDR1-mediated multidrug resistance. By contrast, the control drug doxorubicin, which is a well-known substrate of P-glycoprotein, significantly correlated with all of these parameters (P o0.05; Table 2). 3.7. Role of TP53 in resistance to alpha-hederin Since the tumor suppressor TP53 is another important factor in drug resistance, we analyzed whether or not TP53 affects cellular response to alpha-hederin in HCT116 knockout (p53
/
) and wildtype (p53 þ / þ ) cells. The IC50 values were 18.92 mM for HCT116 (p53
/
) and 14.98 mM for HCT116 (p53 þ / þ ) cells, resulting in a degree of resistance of 1.26 (Table 1). Hence, HCT116 (p53
/
) were similar responsive to alpha-hederin than HCT116 (p53 þ / þ ) cells. To corroborate this result in a larger panel of cell lines, the mutational status of 60 tumor cell lines was correlated with their IC50 values for alpha-hederin. Again, no significant correlation was

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

215

Table 4 Genes identified by COMPARE analysis, whose mRNA expression correlated with log10IC50 values for alpha-hederin in a panel of 60 cell lines. COMPARE ExperiCoefficent mental ID

GenBank Accession

Gene Symbol

Name

Function

Immune system Suppresses apoptosis Receptor for neuropeptide Y and peptide YY Mediator of ubiquitination

0.703 0.676 0.667 0.665

GC89815 GC89506 GC48971 GC175100

M30894 M14745 AA725794 BF195460

TARP BCL2 NPY1R MARCH8

0.664 0.663

GC55070 GC51431

AF000981 AA872560

CDY1 REL

0.663 0.662 0.662

GC182353 NM003225 TFF1 GC27507 U78305 PPM1D GC29796 AB014530 HIPK1

TCRγ alternate reading frame protein B-cell CLL/lymphoma 2 Neuropeptide Y receptor Y1 Membrane-associated ring finger (C3HC4) 8, E3 ubiquitin-protein ligase Chromodomain protein, Y-linked, 1 V-rel reticuloendotheliosis viral oncogene homologue (avian) Trefoil factor 1 Protein phosphatase, Mg2 þ /Mn2 þ dependent, 1D Homeodomain interacting protein kinase 1

0.66

GC182304 NM003161 RPS6KB1

Ribosomal protein S6 kinase, 70 kDa, polypeptide 1

0.66 0.658

GC33855 GC37685

0.658 0.656

GC148258 AA213814 GC97572 U76248

XKRX SIAH2

Solute carrier family 39 (zinc transporter), member 6 Proteasome (prosome, macropain) 26S subunit, nonATPase, 6 XK, Kell blood group complex subunit-related, X-linked Seven in absentia homolog 2 (Drosophila)

0.656 0.656

GC38197 GC37808

AL080216 AB014610

TRIM33 PAN2

0.655 0.653 0.65 0.647

GC152443 GC27096 GC152624 GC173553

AF068220 AF023450 AF086735 BE644818

ATP2A3 DSCAM RDH16 SPPL2B

Tripartite motif-containing 33 PAN2 poly(A) specific ribonuclease subunit homologue (Saccharomyces cerevisiae) ATPase, Ca2 þ transporting, ubiquitous Down syndrome cell adhesion molecule Retinol dehydrogenase 16 (all-trans) Signal peptide peptidase-like 2B


0.7

GC81734

AJ243542

CCL27

Chemokine (C-C motif) ligand 27


0.598
0.598
0.587

GC97923 U91935 GC41382 AA150110 GC102364 X05615

POU6F2 CAV2 TG

POU class 6 homeobox 2 Caveolin 2 Thyroglobulin


0.584
0.575
0.571
0.551

GC85213 GC73393 GC80490 GC54594

AW043554 AI762843 AI971626 AB020653

ATP11A CTSZ HS3ST1 RASGRP3


0.551
0.55
0.549

GC90447 GC96628 GC54259

M89914 U31176 AB012043

NF1 GFER CACNA1G

ATPase, class VI, type 11A Cathepsin Z Heparan sulfate (glucosamine) 3-O-sulfotransferase 1 RAS guanyl releasing protein 3 (calcium and DAGregulated) Neurofibromin 1 Growth factor, augmenter of liver regeneration Calcium channel, voltage-dependent, T type, α 1G subunit


0.54
0.54
0.53
0.528
0.526

GC100588 GC89811 GC55402 GC62829 GC96578

X68879 M30607 AF022795 AI310198 U28043


0.524
0.522
0.519
0.518

GC55733 GC90573 GC97159 GC100109

AF040630 M97496 U55853 X14968

U41060 D14663

SLC39A6 PSMD6

Histone acetyltransferase Transcription factor, proto-oncogene Stabilizer of gastrointestinal mucosa. Relief of p53-dependent cell cycle arrest Serine/threonine-protein kinase involved in transcription and apoptosis Serine/threonine-protein kinase downstream of mTOR signaling Zinc-influx transporter Regulatory subunit of the 26S proteasome Membrane transporter Ubiquitination and proteasomal degradation of target proteins E3 ubiquitin-protein ligase Cytoplasmic mRNA decay ATP hydrolysis coupled with calcium transport Neuronal self-avoidance Oxidoreductase with preference for NAD Cleaves type II membrane signal peptides in the membrane Chemotactic factor attracting skin-associated memory T-lymphocytes Differentiation of amacrine and ganglion cells. Targeting lipid rafts and caveolae formation Precursor of the iodinated thyroid hormones thyroxine (T4) and triiodothyronine (T3) ATPase coupled to transmembrane ion movement Carboxy-monopeptidase and carboxy-dipeptidase activity Biosynthesis of heparan sulfate (HSact). Guanine nucleotide exchange factor (GEF) for Ras and Rap1 Stimulates GTPase activity of Ras

Liver regeneration Guanine nucleotide exchange factor (GEF) for Ras and Rap1 EMX1 Empty spiracles homeobox 1 transcription factor, brain development ZFY Zinc finger protein, Y-linked Transcriptional activator TGFBRAP1 Transforming growth factor, β receptor associated protein 1 Involved in TGF-β/activin signaling pathway CFD Complement factor D (adipsin) Complement activation SLC9A3 Solute carrier family 9 (sodium/hydrogen exchanger), Signal transduction member 3 GALR2 Galanin receptor 2 Receptor for the hormone galanin and for GALP GUCA2A Guanylate cyclase activator 2A (guanylin) Activator of intestinal guanylate cyclase GOLIM4 Golgi integral membrane protein 4 Endosome to Golgi protein trafficking PRKAR2A Protein kinase, cAMP-dependent, regulatory, type II, α Regulatory subunit of cAMP-dependent protein kinase

observed (Fig. 2), indicating that p53 does not affect resistance or sensitivity of tumor cells to alpha-hederin.

expression of EGFR mRNA or protein and the IC50 values for alpha-hederin in the panel of 60 tumor cell lines (Table 3). 3.9. Microarray, COMPARE, and cluster analyses

3.8. Role of EGFR in resistance to alpha-hederin EGFR represents another factor of drug resistance (Efferth, 2011; Konkimalla et al., 2009; Volm et al., 1992). We investigated alpha-hederin's activity in U87MG cells transfected with a mutation-activated EGFR cDNA (U87MG.ΔEGFR) in comparison to its activity in non-transfected U87MG control cells. The IC50 values were 43.98 mM and 21.45 mM, respectively, indicating that EGFR is not a resistance factor for alpha-hederin (Fig. 2, Table 1). Similarly, no significant correlation was found between the

To investigate further determinants of sensitivity and resistance to alpha-hederin, we mined the transcriptome-wide mRNA expression database of the NCI and correlated the data with the IC50 values for alpha-hederin. For this reason, COMPARE analyses were performed to identify genes whose expression was correlated with response of 60 tumor cell lines to alpha-hederin. Only variables with a correlation coefficient of R40.5 (standard COMPARE) or R o
0.5 (reverse COMPARE) were considered (Table 4). Among the candidates were genes involved in apoptosis (BCL2,

216

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

Fig. 6. Dendrogram of hierarchical cluster analysis (WARD method) obtained from microarray-based mRNA expression profiles of genes correlating with alpha-hederin. The dendrogram shows the clustering of 60 NCI cell lines.

Table 5 Separation of clusters of 60 cancer cell lines obtained by hierarchical cluster analysis for alpha hederin. The log10 IC50 median values (M) of alpha hederin was used as cut-off values to define cell lines as being sensitive or resistant. P40.05 was considered as not significant (χ2 test). Partition (log10IC50, M)

Cluster 1 Cluster 2 Cluster 3

Sensitive

Resistant

o
4.76

4
4.76

9 11 7

17 11 0

P¼ 0.00888 (χ2-test); Clusters 1, 2 and 3 are cell lines shown in Fig. 6.

HIPK1), growth and cell cycle regulation (PPM1D, GFER), signal transduction (HIPK1, RPS6KB1, SPPL2B, RASGRP3, NF1, CACNA1G, TGFBRAP1, SLCPA3, GUGA2A, PRKAR2A), transcription (REL, EMX1, ZFY), ubiquitination (MARCH8, PSMD6, SIAH2, TRIM33), transport processes (SCL39A6, XKRX, ATP2A3, ATP11A, CACNA1G, SLC9A3, GOLIM4), nerve cell functions (NPY1R, DSCAM, POU6F2), immunological functions (TARP, CCL27), or other functions (CDY1, TFF1, PAN2, RDH16, CAV2, TG, CTSZ, HS3ST1, CFD, GALR3). This gene expression profile was further investigated by hierarchical cluster analysis. The dendrogram of the cluster analysis could be divided into three major branches (Fig. 6). The distribution of sensitive and resistant cell lines differed significantly between the branches of the dendrogram (P ¼0.00888; χ2 test) (Table 5), indicating that this gene expression profile indeed determined the response of this cell line panel to alpha-hederin.

4. Discussion In plants, secondary metabolites attract beneficial and repel harmful organisms, serve as phytoprotectants and respond to environmental changes. Many of them are known for their anti-proliferative properties (Kuete and Efferth, 2011). The classes of secondary

metabolites detected in the tested plant extracts provide a preliminary explanation on their activities. In general, the phytochemical contents (Table 2) were in accordance with some previous reports for some of the studied extracts where data were available (Adesina, 2005; Ito et al., 2009; Kuete and Efferth, 2011; Mbaze et al., 2007; Ngadjui et al., 1991). It should, however, be mentioned that the detection of a certain bioactive phytochemical classes in a plant is not a guarantee for any biological property, as this will depend on the types of compounds, as well as their concentrations and possible interaction with other constituents. A saponin, alpha-hederin (Fig. 2) was reported in Polyscias fulva (Kuete and Efferth, 2011) and the presence of saponin in this plant is, therefore, consistent to previously reported studies (Bedir et al., 2001). The development of multidrug resistance remains a major challenge in the treatment of cancer (Efferth, 2001). In the present study, we evaluated the activities of different parts of five Cameroonian medicinal plants on a panel of cell lines, including both sensitive and MDR phenotypes. According to the criteria of the US-American National Cancer Institute, 30 mg/mL is the upper IC50 limit considered promising for purification of a crude extract (Suffness and Pezzuto, 1990). Consequently, the highest concentration tested (40 μg/mL) in our screening was slightly above this limit. Among 11 extracts from the five studied plants, the IC50 values were below or around 30 mg/mL for the PFR and BAL extracts on the 10 studied cell lines (Table 2). Interestingly, these extracts showed higher cytotoxicity to HepG2 liver carcinoma cells than to normal AML12 hepatocytes, indicating some degree of tumor specific action of PFR and BAL extracts. This might suggest a possible utility of the two plant for the treatment of malignant diseases with less harmful effects on normal cells. Besides, the degrees of resistance of the cells to the extracts PFR and BAL were in all cases lower than that of doxorubicin, speaking for the utility of these extracts for the management of drug-resistant cancer cells. Having in mind that drug resistance frequently occurs in the clinic leading to treatment failure and fatal outcome for patients, the search for novel non-cross-resistant cytotoxic sample from natural source is urgently warranted. Therefore, we have chosen

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

various models of drug-resistant cell lines. The collateral sensitivity seen with PFR and BAL extracts on colon HCT116 p53
/
and hepatocarcinoma HepG2, gives reason to hope for a future role as anticancer agents against phenotypes resistant to established anticancer drugs. Drug resistance due to overexpression of P-glycoprotein, BCRP, or EGFR or mutation of TP53 has been frequently reported (Choi and Yu, 2014; Knappskog and Lonning, 2012; Marchetti et al., 2013; Nakanishi and Ross, 2012; Santos and Paulo, 2013; Shien et al., 2013). Therefore, novel treatment options have to be deviced to improve the outcome of cancer chemotherapy. In the present investigation, we observed that, firstly, P-glycoprotein-expressing CEM/ADR5000 cells with very high degrees of doxorubicin resistance were only weakly resistant to the two plant extracts and, secondly, HCT116 TP53-knockout cells were collateral sensitive to extracts of Beilschmiedia acuta and Polyscias fulva. This result demonstrates that it may be indeed possible to seek for strategies treating otherwise drug-resistant cancer cells by medicinal plants. Since the breakdown of the MMP is amongst the sequences of events occurring during the apoptotic pathway, it can be deduced from the results of the present work that this can be one of the mechanism of PFR-induced apoptosis in CCRF-CEM cells. In addition, ROS generation was also associated with PFR-induced apoptosis. Other mechanistic studies are still to be explored for the BAL extract, as this plant rather show a lower MMP disruption potency and low level of induction of ROS. Furthermore, we analyzed molecular determinants of tumor cell sensitivity and resistance towards alpha-hederin. By microarraybased gene expression and COMPARE analyses, we correlated the IC50 values for alpha-hederin with transcriptomic mRNA expression levels in a panel of 60 tumor cell lines (Scherf et al., 2000). This approach has been successfully used to elucidate the mode of action of novel compounds in the past (Leteurtre et al., 1994). Cluster and COMPARE analyses are also useful for comparing gene expression profiles with IC50 values for investigational drugs to identify candidate genes involved in drug resistance (Efferth et al., 2003a) and for patient prognoses in clinical oncology (Volm et al., 2002). We identified genes closely associated with the response to alphahederin that belonged to diverse functional groups such as apoptosis, growth and cell cycle regulation, signal transduction, transcription, transport processes, nerve cell functions, nerve cell functions, or others. Although these genes have not, in the past, been associated with drug resistance, this approach identified several novel candidate genes that were significantly associated with sensitivity or resistance to alpha-hederin. These results merit further investigation to prove the contribution of these genes to alpha-hederin resistance. The fact that the genes associated with sensitivity and resistance to alpha-hederin may not associated with resistance to classical anticancer drugs indicates that alpha-hederin could bypass resistance to clinically established anti-cancer drugs. The genes identified by microarray and cluster analyses were from diverse functional groups, indicating that alpha-hederin may exert its cytotoxic effects by a multiplicity of mechanisms. It can be speculated that that alpha-hederin either has multiple cellular targets leading to multiple effects, or has one target that leads to activation and/or inactivation of multiple molecules downstream of this target. Multi-specificity is a frequent feature of natural products. Rather than acting on one single target, multiple targets and pathways are affected. Multi-specificity prevents the development of resistance towards single bioactive compounds, which likely was an important selection advantage during evolution (Efferth and Koch, 2011). In conclusion, the results of the present study provide evidence of the cytotoxic potential of two Cameroonian medicinal plants, Polyscias fulva and Beilschmeidia acuta. To the best of our

217

knowledge, the cytotoxic potential of Polyscia fulva and Beilschmiedia acuta is being reported here for the first time. The extract from the two plants induced apoptosis in CCRF-CEM cells. The mechanism of apoptosis induced by Polyscia fulva include the alteration of MMP and enhanced ROS production. The cytotoxicity of the plant extracts described in the present investigation deserves more detailed exploration in the future to develop novel anticancer drugs against drug-resistant tumors. Also, future experiments will be carry out on normal human cell lines to confirm the safety and selectivity of Polyscias fulva and Beilschmeidia acuta.

Acknowledgments VK is very grateful to the Alexander von Humboldt Foundation for an 18 months “Georg Foster Research Fellowship for Experienced Researcher” program in Germany.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.jep.2014.02.025. References Abukhdeir, A.M., Park, B.H., 2008. P21 and p27: roles in carcinogenesis and drug resistance. Expert Rev. Mol. Med. 10, e19. Adesina, S., 2005. The Nigerian zanthoxylum; chemical and biological values. Afr. J. Tradit. Comp. Altern. Med. 2, 282–301. Adesinan, S.K., EI, E., 1982. The isolation and identification of anticonvulsant agents from Clausena anisata and Afraegle paniculata. Fitoterapia 53, 63–66. Alley, M.C., Scudiero, D.A., Monks, A., Hursey, M.L., Czerwinski, M.J., Fine, D.L., Abbott, B.J., Mayo, J.G., Shoemaker, R.H., Boyd, M.R., 1988. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48, 589–601. Azzariti, A., Porcelli, L., Quatrale, A.E., Silvestris, N., Paradiso, A., 2011. The coordinated role of CYP450 enzymes and P-gp in determining cancer resistance to chemotherapy. Curr. Drug Metab. 12, 713–721. Bass, D.A., Parce, J.W., Dechatelet, L.R., Szejda, P., Seeds, M.C., Thomas, M., 1983. Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J. Immunol. 130, 1910–1917. Bedir, E., Toyang, N.J., Khan, I.A., Walker, L.A., Clark, A.M., 2001. A new dammaranetype triterpene glycoside from Polyscias fulva. J. Nat. Prod. 64, 95–97. Binkhathlan, Z., Lavasanifar, A., 2013. P-glycoprotein inhibition as a therapeutic approach for overcoming multidrug resistance in cancer: current status and future perspectives. Curr. Cancer Drug Targets 13, 326–346. Bowen, I.H., Jackson, B.P., Motawe, H.M., 1985. An investigation of the stem bark of Bersama abyssinica. Planta Med. 51, 483–487. Bragado, P., Armesilla, A., Silva, A., Porras, A., 2007. Apoptosis by cisplatin requires p53 mediated p38alpha MAPK activation through ROS generation. Apoptosis 12, 1733–1742. Bunz, F., Hwang, P.M., Torrance, C., Waldman, T., Zhang, Y., Dillehay, L., Williams, J., Lengauer, C., Kinzler, K.W., Vogelstein, B., 1999. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J. Clin. Invest. 104, 263–269. Bush, J.A., Li, G., 2002. Cancer chemoresistance: the relationship between p53 and multidrug transporters. Int. J. Cancer 98, 323–330. Choi, Y.H., Yu, A.M., 2014. ABC transporters in multidrug resistance and pharmacokinetics, and strategies for drug development. Curr. Pharm. Des. 20, 793–807. Cordell, G.A., Beecher, C.W., Pezzut, J.M., 1991. Can ethnopharmacology contribute to development of new anti-cancer? J. Ethnopharmacol. 32, 117–133. Cossarizza, A., Ferraresi, R., Troiano, L., Roat, E., Gibellini, L., Bertoncelli, L., Nasi, M., Pinti, M., 2009. Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry. Nat. Protoc. 4, 1790–1797. Dandjesso, C., Klotoé, J., Dougnon, T., Sègbo, J., Atègbo, J., Gbaguidi, F., Fah, L., Fanou, B., Loko, F., Dramane, K., 2012. Phytochemistry and hemostatic properties of some medicinal plants sold as anti-hemorrhagic in Cotonou markets (Benin). Indian J. Sci. Technol. 5, 3105–3109. Doyle, L.A., Yang, W., Abruzzo, L.V., Krogmann, T., Gao, Y., Rishi, A.K., Ross, D.D., 1998. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Nat. Acad. Sci. U.S.A. 95, 15665–15670. Efferth, T., 2001. The human ATP-binding cassette transporter genes: from the bench to the bedside. Curr. Mol. Med. 1, 45–65. Efferth, T., 2011. Natural products as inhibitors of epidermal growth factor receptor. Forum Immunopathol. Dis. Ther. 2, 281–301.

218

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

Efferth, T., Fabry, U., Osieka, R., 1997. Apoptosis and resistance to daunorubicin in human leukemic cells. Leukemia 11, 1180–1186. Efferth, T., Gebhart, E., Ross, D.D., Sauerbrey, A., 2003a. Identification of gene expression profiles predicting tumor cell response to L-alanosine. Biochem. Pharmacol. 66, 613–621. Efferth, T., Grassmann, R., 2000. Impact of viral oncogenesis on responses to anticancer drugs and irradiation. Crit. Rev. Oncogenesis 11, 165–187. Efferth, T., Greten, H., 2012. Autophagy by natural products in cancer cells. Biochem. Anal. Biochem. 1, 8. Efferth, T., Koch, E., 2011. Complex interactions between phytochemicals. The multitarget therapeutic concept of phytotherapy. Curr. Drug Targets 12, 122–132. Efferth, T., Konkimalla, V.B., Wang, Y.F., Sauerbrey, A., Meinhardt, S., Zintl, F., Mattern, J., Volm, M., 2008. Prediction of broad spectrum resistance of tumors towards anticancer drugs. Clin. Cancer Res. 14, 2405–2412. Efferth, T., Lohrke, H., Volm, M., 1989. Reciprocal correlation between expression of P-glycoprotein and accumulation of rhodamine 123 in human tumors. Anticancer Res. 9, 1633–1637. Efferth, T., Mattern, J., Volm, M., 1992. Immunohistochemical detection of P glycoprotein, glutathione S transferase and DNA topoisomerase II in human tumors. Oncology 49, 368–375. Efferth, T., Sauerbrey, A., Olbrich, A., Gebhart, E., Rauch, P., Weber, H.O., Hengstler, J.G., Halatsch, M.E., Volm, M., Tew, K.D., Ross, D.D., Funk, J.O., 2003b. Molecular modes of action of artesunate in tumor cell lines. Mol. Pharmacol. 64, 382–394. Efferth, T., Volm, M., 1992. Antibody-directed therapy of multidrug-resistant tumor cells. Med. Oncol. Tumor Pharmacother. 9, 11–19. Efferth, T., Volm, M., 1993. Reversal of doxorubicin-resistance in sarcoma 180 tumor cells by inhibition of different resistance mechanisms. Cancer Lett. 70, 197–202. Eichhorn, T., Efferth, T., 2012. P-glycoprotein and its inhibition in tumors by phytochemicals derived from Chinese herbs. J. Ethnopharmacol. 141, 557–570. Eyong, K.O., Krohn, K., Hussain, H., Folefoc, G.N., Nkengfack, A.E., Schulz, B., Hu, Q., 2005. Newbouldiaquinone and newbouldiamide: a new naphthoquinone
anthraquinone coupled pigment and a new ceramide from Newbouldia laevis. Chem. Pharm. Bull. (Tokyo) 53, 616–619. Focho, D., WT, N., Fonge, B., 2009. Medicinal plants of Aguambu—Bamumbu in the Lebialem highlands, southwest province of Cameroon. Afr. J. Pharm. Pharmacol. 3, 1–13. Gafner, S., Wolfender, J., Nianga, M., Stoeckli-Evans, H., Hostettmann, K., 1996. Antifungal and antibacterial naphtoquinones from Newbouldia laevis roots. Phytochemistry 42, 1315–1320. Gottesman, M.M., Ling, V., 2006. The molecular basis of multidrug resistance in cancer: the early years of P-glycoprotein research. FEBS Lett. 580, 998–1009. Harbone, J. (Ed.), 1973. Phytochemical Methods: A Guide to Modern Techniques of Plant Analysis. Chapman & Hall, London Hutchings, A., Scott, A., Lewis, G., Cunningham, A., 1996. Zulu Medicinal Plants. Natal University Press, Pietermaritzburg Gottesman, M.M., 2002. Mechanisms of cancer drug resistance. Ann. Rev. Med. 53, 615–627. Ito, C., Itoigawa, M., Aizawa, K., Yoshida, K., Ruangrungsi, N., Furukawa, H., 2009. Gamma-lactone carbazoles from Clausena anisata. J. Nat. Prod. 72, 1202–1204. Jeruto, P., Lukhoba, C., Ouma, G., Otieno, D., Mutai, C., 2007. Herbal treatments in Aldai and Kaptumo divisions in Nandi district, Rift valley province, Kenya. Afr. J. Tradit. Comp. Altern. Med. 5, 103–105. Knappskog, S., Lonning, P.E., 2012. P53 and its molecular basis to chemoresistance in breast cancer. Expert Opin. Ther. Targets 16 (Suppl 1), S23–30. Konkimalla, V.B., McCubrey, J.A., Efferth, T., 2009. The role of downstream signaling pathways of the epidermal growth factor receptor for Artesunate's activity in cancer cells. Curr. Cancer Drug Targets 9, 72–80. Kuete, V., Efferth, T., 2010. Cameroonian medicinal plants: pharmacology and derived natural products. Front. Pharmacol. 1, 123. Kuete, V., Efferth, T., 2011. Pharmacogenomics of Cameroonian traditional herbal medicine for cancer therapy. J. Ethnopharmacol. 137, 752–766. Kuete, V., Eichhorn, T., Wiench, B., Krusche, B., Efferth, T., 2012. Cytotoxicity, antiangiogenic, apoptotic effects and transcript profiling of a naturally occurring naphthyl butenone, guieranone A. Cell Div. 7, 16. Kuete, V., Mbaveng, A.T., Tsaffack, M., Beng, V.P., Etoa, F.X., Nkengfack, A.E., Meyer, J. J., Lall, N., 2008. Antitumor, antioxidant and antimicrobial activities of Bersama engleriana (Melianthaceae). J. Ethnopharmacol. 115, 494–501. Kuete, V., Tchakam, P.D., Wiench, B., Ngameni, B., Wabo, H.K., Tala, M.F., Moungang, M.L., Ngadjui, B.T., Murayama, T., Efferth, T., 2013. Cytotoxicity and modes of action of four naturally occuring benzophenones: 2,2',5,6'-tetrahydroxybenzophenone, guttiferone E, isogarcinol and isoxanthochymol. Phytomedicine 20, 528–536. Kuete, V., Wabo, H.K., Eyong, K.O., Feussi, M.T., Wiench, B., Krusche, B., Tane, P., Folefoc, G.N., Efferth, T., 2011. Anticancer activities of six selected natural compounds of some Cameroonian medicinal plants. PLoS One 6, e21762. Kupchan, S.M., Moniot, J.L., Sigel, C.W., Hemingway, R.J., 1971. Tumor inhibitors. LXV. Bersenogenin, berscillogenin, and 3-epiberscillogenin, three new cytotoxic bufadienolides from Bersama abyssinica. J. Org. Chem. 36, 2611–2616. Lai, D., Visser-Grieve, S., Yang, X., 2012. Tumour suppressor genes in chemotherapeutic drug response. Biosci. Rep. 32, 361–374. Lambert, L., Keyomarsi, K., 2007. Cell cycle deregulation in breast cancer: insurmountable chemoresistance or Achilles' heel? Adv. Exp. Med. Biol. 608, 52–69. Lee, Y.S., Yoon, S., Park, M.S., Kim, J.H., Lee, J.H., Song, C.W., 2010. Influence of p53 expression on sensitivity of cancer cells to bleomycin. J. Biochem. Mol. Toxicol. 24, 260–269.

Leteurtre, F., Kohlhagen, G., Paull, K.D., Pommier, Y., 1994. Topoisomerase II inhibition and cytotoxicity of the anthrapyrazoles DuP 937 and DuP 941 (Losoxantrone) in the National Cancer Institute preclinical antitumor drug discovery screen. J. Nat. Cancer Inst. 86, 1239–1244. Long, J.S., Ryan, K.M., 2012. New frontiers in promoting tumour cell death: targeting apoptosis, necroptosis and autophagy. Oncogene 31, 5045–5060. Lowe, S.W., 1995. Cancer therapy and p53. Curr. Opin. Oncol. 7, 547–553. Mabry, T.J., Ulubelen, A., 1980. Chemistry and utilization of phenylpropanoids including flavonoids, coumarins, and lignans. J. Agric. Food Chem. 28, 188–195. Marchetti, S., Pluim, D., van Eijndhoven, M., van Tellingen, O., Mazzanti, R., Beijnen, J.H., Schellens, J.H., 2013. Effect of the drug transporters ABCG2, Abcg2, ABCB1 and ABCC2 on the disposition, brain accumulation and myelotoxicity of the aurora kinase B inhibitor barasertib and its more active form barasertibhydroxy-QPA. Invest. New Drugs 31, 1125–1135. Martinez-Rivera, M., Siddik, Z.H., 2012. Resistance and gain-of-resistance phenotypes in cancers harboring wild-type p53. Biochem. Pharmacol. 83, 1049–1062. Mbaze, L.M., Poumale, H.M., Wansi, J.D., Lado, J.A., Khan, S.N., Iqbal, M.C., Ngadjui, B. T., Laatsch, H., 2007. alpha-Glucosidase inhibitory pentacyclic triterpenes from the stem bark of Fagara tessmannii (Rutaceae). Phytochemistry 68, 591–595. Nagane, M., Levitzki, A., Gazit, A., Cavenee, W.K., Huang, H.J., 1998. Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3-like proteases. Proc. Nat. Acad. Sci. 95, 5724–5729. Nakanishi, T., Ross, D.D., 2012. Breast cancer resistance protein (BCRP/ABCG2): its role in multidrug resistance and regulation of its gene expression. Chin. J. Cancer 31, 73–99. Natarajan, K., Xie, Y., Baer, M.R., Ross, D.D., 2012. Role of breast cancer resistance protein (BCRP/ABCG2) in cancer drug resistance. Biochem. Pharmacol. 83, 1084–1103. Nelson, R.R., 1997. In-vitro activities of five plant essential oils against methicillinresistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. J. Antimicrob. Chemother. 40, 305–306. Ngadjui, B., Mouncherou, S., Ayafor, J., Sondengam, B., Tillequin, F., 1991. Geranyl coumarins from Clausena anisata. Phytochemistry 30, 2809–2811. Njayou, F.N., Moundipa, P.F., Tchana, A.N., Ngadjui, B.T., Tchouanguep, F.M., 2008. Inhibition of microsomal lipid peroxidation and protein oxidation by extracts from plants used in Bamun folk medicine (Cameroon) against hepatitis. Afr. J. Tradit. Comp. Altern. Med. 5, 278–289. O'Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. Ojewole, J.A., 2002. Hypoglycaemic effect of Clausena anisata (Willd) Hook methanolic root extract in rats. J. Ethnopharmacol. 81, 231–237. Paull, K.D., Shoemaker, R.H., Hodes, L., Monks, A., Scudiero, D.A., Rubinstein, L., Plowman, J., Boyd, M.R., 1989. Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. J. Nat. Cancer Inst. 81, 1088–1092. Popoca, J., Aguilar, A., Alonso, D., Villarreal, M.L., 1998. Cytotoxic activity of selected plants used as antitumorals in Mexican traditional medicine. J. Ethnopharmacol. 59, 173–177. Rubinstein, L.V., Shoemaker, R.H., Paull, K.D., Simon, R.M., Tosini, S., Skehan, P., Scudiero, D.A., Monks, A., Boyd, M.R., 1990. Comparison of in vitro anticancerdrug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines. J. Nat. Cancer Inst. 82, 1113–1118. Santos, S.A., Paulo, A., 2013. Small molecule inhibitors of multidrug resistance gene (MDR1) expression: preclinical evaluation and mechanisms of action. Curr. Cancer Drug Targets 13, 814–828. Sau, A., Pellizzari Tregno, F., Valentino, F., Federici, G., Caccuri, A.M., 2010. Glutathione transferases and development of new principles to overcome drug resistance. Arch. Biochem. Biophys. 500, 116–122. Sayers, T.J., 2011. Targeting the extrinsic apoptosis signaling pathway for cancer therapy. Cancer Immunol. Immunother. 60, 1173–1180. Scherf, U., Ross, D.T., Waltham, M., Smith, L.H., Lee, J.K., Tanabe, L., Kohn, K.W., Reinhold, W.C., Myers, T.G., Andrews, D.T., Scudiero, D.A., Eisen, M.B., Sausville, E.A., Pommier, Y., Botstein, D., Brown, P.O., Weinstein, J.N., 2000. A gene expression database for the molecular pharmacology of cancer. Nat. Genet. 24, 236–244. Schrattenholz, A., Groebe, K., Soskic, V., 2010. Systems biology approaches and tools for analysis of interactomes and multi-target drugs. Methods Mol. Biol. 662, 29–58. Senthilkumar, A., Venkatesalu, V., 2009. Phytochemical analysis and antibacterial activity of the essential oil of Clausena anisata (Willd.) Hook. F. ex Benth. Int. J. Integr. Biol. 5, 116–120. Shien, K., Toyooka, S., Yamamoto, H., Soh, J., Jida, M., Thu, K.L., Hashida, S., Maki, Y., Ichihara, E., Asano, H., Tsukuda, K., Takigawa, N., Kiura, K., Gazdar, A.F., Lam, W. L., Miyoshi, S., 2013. Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells. Cancer Res. 73, 3051–3061. Singh, S., Khan, A.R., Gupta, A.K., 2012. Role of glutathione in cancer pathophysiology and therapeutic interventions. J. Exp. Ther. Oncol. 9, 303–316. Sonneveld, P., Wiemer, E., 1997. Assays for the analysis of P-glycoprotein in acute myeloid leukemia and CD34 subsets of AML blasts. Leukemia 11, 1160–1165. Staunton, J.E., Slonim, D.K., Coller, H.A., Tamayo, P., Angelo, M.J., Park, J., Scherf, U., Lee, J.K., Reinhold, W.O., Weinstein, J.N., Mesirov, J.P., Lander, E.S., Golub, T.R.,

V. Kuete et al. / Journal of Ethnopharmacology 153 (2014) 207–219

2001. Chemosensitivity prediction by transcriptional profiling. Proc. Nat. Acad. Sci. U.S.A. 98, 10787–10792. Suffness, M., Pezzuto, J., 1990. Assays Related to Cancer Drug Discovery. Academic Press, London Tshibangu, J.N., Chifundera, K., Kaminsky, R., Wright, A.D., Konig, G.M., 2002. Screening of African medicinal plants for antimicrobial and enzyme inhibitory activity. J. Ethnopharmacol. 80, 25–35. Usman, H., Osuji, J.C., 2007. Phytochemical and in vitro antimicrobial assay of the leaf extract of Newbouldia laevis. Afr. J. Tradit. Comp. Altern. Med. 4, 476–480. Volm, M., Efferth, T., Mattern, J., 1992. Oncoprotein (c-myc, c-erbB1, c-erbB2, c-fos) and suppressor gene product (p53) expression in squamous cell carcinomas of the lung. Clinical and biological correlations. Anticancer Res. 12, 11–20.

219

Volm, M., Kastel, M., Mattern, J., Efferth, T., 1993. Expression of resistance factors (Pglycoprotein, glutathione S-transferase-pi, and topoisomerase II) and their interrelationship to proto-oncogene products in renal cell carcinomas. Cancer 71, 3981–3987. Volm, M., Koomagi, R., Mattern, J., Efferth, T., 2002. Expression profile of genes in non-small cell lung carcinomas from long-term surviving patients. Clin. Cancer Res. 8, 1843–1848. Wöll, S., Kim, S., Efferth, T., 2013. Animal plant warfare and secondary metabolite evolution. Nat. Prod. Bioprospect. 3, 1–7.