Cytotoxicity of medicinal plants of the West-Canadian Gwich׳in Native Americans towards sensitive and multidrug-resistant cancer cells.

Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Cytotoxicity of medicinal plants of the West-Canadian Gwich'in Native Americans towards sensitive and multidrug-resistant cancer cells Asuman Karadeniz a,b, Gladys Alexie c, Henry Johannes Greten d,e, Kai Andersch f,g, Thomas Efferth a,n a

Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Mainz, Germany Mehmet Akif Ersoy University, Biology Department, Burdur, Turkey c Fort McPerson, Northwest Territories, Canada d Abel Salazar Biomedical Sciences Institute, University of Porto, Portugal e Heidelberg School of Chinese Medicine, Heidelberg, Germany f Wilderness International, Dresden, Germany g Wilderness International, Stony Plain, Alberta, Canada b

art ic l e i nf o

a b s t r a c t

Article history: Received 5 January 2015 Received in revised form 11 March 2015 Accepted 23 March 2015

Ethnopharmacological relevance: Traditional medicine of the Native Americans has a long tradition of medicinal plants, which also influenced modern oncology. For instance, podophyllotoxin the active ingredient of Podophyllum peltatum L. (Berberidaceae) used by Native Americans to treat warts led to the development of etoposide and teniposide. In the present investigation, we studied 10 medicinal plants used by the Gwich'in First Nation of West-Canada, which have been used against diverse diseases including cancer. Material and methods: Sensitive and multidrug-resistant (MDR) tumor cell lines expressing various ATPbinding cassette (ABC) transporters (P-glycoprotein/ABCB1/MDR1, MRP1/ABCC1, or BCRP/ABCG2) have been used. Cytotoxicity was determined by the resazurin assay. Results: Arctium minus Bernh. (Asteraceae). Lysichiton americanus Hultén & St. John (Araceae), and Maianthemum dilatatum (Alph.Wood) A.Nelson & J.F.Macbr.(Asparagaceae) were cytotoxic with IC50 values ranging from 2.40 to 86.35 mg/mL. The MDR cell lines did not exert cross-resistance to these extracts. Conclusion: As these medicinal plants of the West-Canadian Gwich'in First Nation were not involved in classical drug resistance mechanisms and might therefore be valuable to bypass anticancer drug resistance in refractory tumors. & 2015 Published by Elsevier Ireland Ltd.

Keywords: ABC transporter Cancer First nations Multidrug resistance Pharmacognosy

1. Introduction Medicinal plants of indigenous tribes were frequently the only primary health care for thousands of years. It can thus be expected that the millennia-old experience-based knowledge led to the selection of pharmacologically active plants. By using modern pharmacological and biological methods, it should therefore, be possible to determine the bioactivity and to identify underlying cellular and molecular modes of action of medicinal plants. Indeed, ethnopharmacological investigations delivered a large body of experimental

Abbreviations: MDR, multidrug resistance; ABC, ATP-binding cassette; MRP1, multidrug resistance related protein 1; BCRP, breast cancer resistance protein; MDR1, multidrug resistance gene 1 (P-glycoprotein) n Corresponding author. Tel.: þ 49 6131 3925751; fax: þ 49 6131 3923752. E-mail address: [email protected] (T. Efferth).

evidence for the therapeutic activity of plants used in traditional medicine systems worldwide. While much emphasis has been put on traditional Chinese medicine, Ayurveda, Kampo medicine, Arabic medicine, European phytotherapy and traditional African medicine (Efferth et al., 2007a, 2007b, 2007c; Konkimalla and Efferth, 2010; Kuete and Efferth, 2010, 2011; Khalid et al., 2012), systematic analyses on traditional medicine of the First Nations of North America are rare, although traditional medicine of the Native Americans has a long tradition of medicinal plants and prominent examples illustrate their therapeutic potential. For instance, podophyllotoxin the active ingredient of Podophyllum peltatum L. (Berberidaceae) used by Native Americans to treat warts led to the development of etoposide and teniposide, two clinically established anticancer drugs. Few reports on the medicinal plants of Native Americans indicated that this represents an exciting field of research awaiting to be explored in more detail (Chandler et al., 1979; Kolb, 2002; Nauman, 2007).

http://dx.doi.org/10.1016/j.jep.2015.03.052 0378-8741/& 2015 Published by Elsevier Ireland Ltd.

Please cite this article as: Karadeniz, A., et al., Cytotoxicity of medicinal plants of the West-Canadian Gwich'in Native Americans towards sensitive and multidrug-resistant cancer cells. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.03.052i

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A. Karadeniz et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Table 1 Traditional use (in addition to cancer treatment) of selected medicinal plants by the first Nations in North America (according to Moerman, 1998). Scientifics name (Common name) (Family)

Traditional use by first nations in North America

Arcticum minus Bernh. (Lesser Burrdock) (Asteraceae)

Leaves: analgesic, anti-rheumatic, against ulcers, cough, boils, blue skin swellings. Roots: antirheumatic, febrifuge, tonic, stimulant, blood purifier, against cough, boils, abscesses, stomach pain, gynecological ailments No handed down records

Boykinia elata (syn. B. occidentalis) Torr & A.Gray (Coastal Brookfoam) (Saxifragaceae) Epilobium angustifolium L. (Fireweed) (Onagraceae)

Hypericum formosum Kunth (Western St. John’s Wort) (Hypericaceae) Hypopitys monotropa Crantz (Dutchman’s Pipe) (Ericaceae) Ledum groenlandicum Oeder (Bog Labrador Tea) (Ericaceae)

Lysichiton americanus Hultén & St. John (American Skunk-gabbage) (Araceae) Maianthemun dilatatum (Alph.Wood) A. Nels. & J.F. Macbr. (Twoleaf False Solomons’s Seal) (Asparagaceae) Plantago major L. (Common Plantain) (Plantaginaceae)

Polypodium glycyrrhiza D.C. Eat. (Licorice Fern) (Polypodiaceae)

Seeds: for wound healing after cutting open tumors Roots: laxative, against boils, carbuncles, abscesses, wounds, swellings, sore throats, tuberculosis, internal injuries from lifting, urination problems Leaves: analgesic, laxative, to remove sliver, against bruise, stomachache, intestinal discomfort Whole plant: bath for invalids, against gastritis, sores No handed down records No handed down records Flowers: anti-rheumatic, against insect sting pain, for tender feet Leaves: anti-rheumatic, blood purifier, narcotic, tonic for mothers after childbirth, disinfectant wash against itchy skin, hand sores and chapped skin, against umbilical scabs, rashes in skin folds, cracked nipples, burns, scalds, colds and respiratory ailments, sore throats, asthma, tuberculosis, kidney problems, scurvy, appetite stimulant, emetic Wood: against chafed skin Roots: against burns, ulcers Whole plant: analgesic, tonic, against colds, nasal inflammation, whooping cough, pneumonia, fever and chills, stomach pain, jaundice, eye sores, blindness, antidot for poison ivy Blossoms: anti-rheumatic Leaves: analgesic, stimulant, against stomach trouble, burns, cuts, swellings, cores with scrofula, carbuncle, chest pain Roots: anti-hemorrhagic, anti-rheumatic, blood purifier, abortive, for bladder cleansing, emetic, against sores, boils, burns, bloody urine, carbuncles, swellings, blood poisoning Fruits: against tuberculosis Leaves: against boils, cuts, burns, sores, wounds, eye sores Roots: against childlessness Seeds: facilitate digestion, reduce intestinal inflammation, regulate menses Leaves: analgesic, antirheumatic, disinfectant, diuretic, for wound healing without scars, orthopedic aid, against burns, bruises, cuts, sores, abscesses, blisters, swellings, contusions, boils, fever, carbuncles, hemorrhoids, ear aches, ulcer, stomach tonic Roots: antidiarrheal, antidote (against poisonous bites), febrifuge, laxative, blood purifier, against chest pain, pneumonia, colds, skin inflammations Whole plant: laxative, stomach and bowel complaints, female diseases, cough, colds, to draw out poisonous thorns and splinters Rhizomes: antiemetic, anti-hemorrhagic, antidiarrheal, analgesic, against colds, cough, sore throats, chest pain, blood vomiting, to sweeten mouth flavor Whole plant: alterative and veneral aid

In an effort to investigate the medicinal plants of the First Nations of North America, we started to analyze the cytotoxic activity of plants of the Gwich'in in West-Canada (Deeg et al., 2012). In the borderland of Alaska and Canada, three tribes belong to the Kutchin Athapaskans, i.e. Gwich'in, Loucheux and Takudh. The Athapaskans are a large language family with many tribes living in Alaska, Canada, West and Southwest USA. The name Athapaskaw can be translated as “everywhere is pasture and reed” and reminds to a region westward of the Athabasca Lake (Alberta, Canada). In the present study, we investigated medicinal plants collected in a collaboration project between the Gwich'in American Natives (Northwest Territories, Canada) and the non-governmental organization, Wilderness International (Stony Plain (Alberta, Canada). These plants were widely used by many First Nations of North America for a broad range of diseases and ailments (Table 1). It is important to point out here that some these plants have been traditionally also used for cancer treatment. The Thompson Native Americans used Twoleaf leave decoctions to treat cancer (Ward Cameron, 2005). For comparison, the tightly related M. racemosum (L.) Link (Asparagaceae) was also used by several North American tribes to treat cancer, indicating that this genus contains several species which may reveal anticancer activity (Moerman, 2009). Fireweed (Epilobium angustifolium (L.) Holub, Onagroideae) has

been used by the Kwakiutl tribe in West Canada to treat cancer (Turner et al., 1983). Labrador Tea (Ledum groenlandicum (Oeder) Kron & Judd, Ericaceae) has traditionally been used against tumors throughout North America (Duke et al., 2010). The traditional use of Common Plantain (Plantago major L., Plantaginaceae) in North and South America to treat cancer has been reported too (Lithander, 1992; Johnson, 1998; Duke, 2001). Western St. John's Wort (Hypericum formosum Kunth, Hypericaceae) and Licorice Fern (Polypodium glycyrrhiza, D.C.Eaton (Polypodiaceae) have been applied to treat sores by the Native Americans (Spjut, 1978; Vizgirdas and Rey-Vizgirdas, 2006). Since the diagnosis of cancer was not well developed in former times, cancer might be described in traditional medicine by terms such as sores or swellings, etc. The ethnobotanical use of these two drugs against sores might indicate its possible activity against cancer. In addition to locally restricted and endemic plants of Alaska and Canada, the Gwich'in also utilized plants for therapeutic purposes, which are commonly distributed in temperate climate zones not only in North America but also worldwide. This fact facilitates comparisons between traditional uses of these medicinal plants in different cultures across different continents. It is fascinating to observe that many of these plant species were used in different traditional medicines worldwide, although these folks did not know from each other. This indicates that the need to cure


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Table 2 Traditional use (in addition to cancer treatment) of selected medicinal plants in human and veterinary ethnomedicine. Scientific name

Traditional use outside North America

Arcticum minus

Leaves: against rheumatic pain, fever, sunstroke (Erdemoğlu et al. 2009), blood purification, rheumatism, kidney stones, skin disease, snake and scorpion bites (Mosaddegh et al. 2012), herpes (Cavero et al. 2011), skin inflammations, alopecia (Neves et al. 2009), wounds, whitlow, scabies, ringworms (Cavero et al. 2011) N/A Upper parts: against benign prostate hyperplasia (Hiermann et al. 1986; Vitalone et al., 2001; 2003; Kosalec et al., 2013); Tinea captitis (Kiliç et al., 2001) N/A

Boykinia elata Epilobium angustifolium Hypericum formosum Hypopitys monotropa Ledum groenlandicum Lysichiton americanus Maianthemun dilatatum Plantago major

Polypodium glycyrrhiza

N/A N/A N/A Leaves: against boils, burns, cuts, wounds, tuberculosis (Ziarovky, 2014) Roots: against sterility, sore eyes (Ziarovky, 2014) Leaves: against cancer (Gálvez et al., 2003; Chiang et al., 2003), wound healing, analgesic, antioxidant, and antibacterial, immune modulation, antiviral, antifungal, anti-inflammatory (Hetland et al., 2000; Samuelsen, 2000; Chiang et al., 2002; 2003; Beara et al., 2010; Gómez-Estrada et al., 2011; Reina et al., 2013) Whole plant: against of cutaneous leishmaniasis (França et al., 1996), snakebite antidote (Vásquez et al., 2013) Ethnoveterinary use: against diarrhea and scurs (Lans et al., 2007) N/A

diseases led to experience-based millennia-lasting selections of therapeutically active plants, which are ubiquitously distributed in temperate zones of this globe. This can be exemplarily illustrated by medicinal plants chosen in the present investigation, which were not only used as medicinal plants of the Gwich'in and other North-American First Nations, but also outside America. Tables 1 and 2 demonstrate that these plants were traditionally used for comparable diseases and ailments in America, Europe and Asia. This can be taken as a strong hint for the therapeutic activity of the plants, which has indeed been corroborated by modern pharmacological research (Table 3). We also have some experimental clues that these medicinal plants may also exert cytotoxic activity towards cancer cells. This does not come as a surprise, since (1) inflammation causes carcinogenesis (Chiba et al., 2012; Vendramini-Costa and Carvalho, 2012; Yehuda-Shnaidman and Schwartz, 2012; Nathan and Cunningham-Bussel, 2013) and since (2) the anti-proliferative effects of medicinal plants against bacteria, fungi, and infectious protozoans (e.g. Plasmodia, Leishmania) can be understood as part of a more general cytotoxicity, which also affects cancer cells. Rather than focusing solely on the cytotoxic activity of medicinal plants of the Gwich'in, we were interested, whether these plants are also active against tumor cell lines resistant to established anticancer drugs. Development of drug resistance prevents treatment of cancer patients with drug doses high enough to kill all cell subpopulations in a tumor, which ultimately leads to therapy failure and death of patients. Therefore, novel drugs capable to kill otherwise drug-resistant tumor cells are urgently required. Aggressive forms of drug resistance represent crossresistance patterns among functionally and chemically different anticancer drugs, termed multidrug resistance (MDR). Efflux pumps of the ATP-binding cassette (ABC) transporter family extrude drugs out of cancer cells and keep intracellular drug concentrations at sub-lethal concentrations. Among the 48 ABC transporter genes in the human genome, the P-glycoprotein (ABCB1/MDR1), MRP1(ABCC1), and BCRP (ABCG2) are the best characterized to confer MDR. Each of these efflux pumps confers distinct, but overlapping cross-resistance profiles to anticancer drugs (Efferth, 2001; Gillet et al., 2007).

The aims of the present investigation were, firstly, to identify medicinal herb extracts, which reveal cytotoxicity to cancer cells and, secondly, to compare the activity of wild-type cell lines to their corresponding MDR1-, MRP1- or BCRP-expressing multidrugresistant sublines.

2. Materials and methods 2.1. Plant material The plants were collected during a nature conservation expedition of Wilderness International Foundation in the Three Rivers Region (Peel River Watershed) in the border area between the Yukon Territory and the Northwest Territories in the northeast of Canada. The extraction was performed by ethanol and the botanical identification was done by two of the authors (K.A. and G.A.). Voucher specimens are deposited at the Department for Pharmaceutical Biology, Johannes Gutenberg University, Mainz, Germany (specimen numbers CAN1-17). Plants were preserved in absolute ethanol and stored until further processing. Between 3 and 39 g plant material was taken up in 35–80 mL alcohol. For preparation of extracts, alcohol volumes were rotated to dryness and the dry pellets were solved in DMSO in a final concentration of 1 mg/mL. 2.2. Cell lines Leukemic CCRF-CEM and HL60 cells were maintained as previously described (Efferth et al., 2003a). Drug resistance of P-glycoprotein/ABCB1/MDR1-overexpressing CEM/ADR5000 cells was maintained in 5000 ng/mL (¼ 8.62 nM) doxorubicin. The ABCC1/MRP1-expressing HL60/AR subline was continuously treated with 100 nM daunorubicin. The establishment of the resistant sublines has been previously described (Kimmig et al., 1990; Brügger et al., 1999). Breast cancer cells transduced with a control vector (MDA-MB-231-pcDNA3) or with cDNA for the breast cancer resistance protein, ABCG2/BCRP (MDA-MB-231-BCRP clone 23) were generated and maintained as reported (Doyle et al., 1998). The mRNA expression of MDR1, MRP1, and BCRP in the resistant cell lines has been reported (Efferth et al., 2003; Gillet et al., 2004).


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Table 3 Phytochemical composition and pharmacological activities of selected medicinal plants. Scientific name

Phytochemical composition

Pharmacological activities

Arcticum minus

Leaves: unsaturated lactones (Cavallito and Kirchner, 1947), flavonoids (Saleh and Bohm, 1971). Seed oil: trans-3-enoic acids (Morris et al., 1968)

Boykinia elata Epilobium angustifolium

N/A Upper parts: oenothein B, quercetin-3-Oglucuronide, myricetin-3-O-rhamnoside (Hiermann et al, 1991; Kiss et al. 2006a; Bazylko et al., 2007; Stolarczyk et al., 2013b)

Hypericum formosum Hypopitys monotropa

N/A Upper parts: sterols and fatty acids (Stanley and Patterson, 1977) Upper parts: ursolic acid (Dufour et al., 2007)

Leaves: antibacterial (Cavallito et al., 1945). Antinociceptive, antiinflammatory, antioxidant (Erdemoğlu et al., 2009), antifungi, vulnerary (Neves et al., 2009), antiasthmatic, anticholagogue, antiherpetic (Agelet and Vallès, 2003) N/A Upper parts: inhibition of prostate growth (Hiermann and Bucar, 1997), antimicrobial (Battinelli et al., 2001; Rauha et al., 2000; Bartfay et al., 2012; Huttunen et al., 2013; Kosalec et al., 2013), antiinflammatory (Juan et al., 1988; Kiss et al., 2011), antioxidant (Shikov et al., 2006); Kiss et al., 2011), antiproliferative against cancer cells (Vitalone et al., 2001; 2003; Kiss et al., 2006a; Stolarczyk et al., 2013a); antifungal (Webster et al., 2008); analgesic (Tita et al., 2001), modulation of metallopeptidases: neutral endopeptidase (NEP), angiotensin-converting enzyme (ACE), and aminopeptidase N (APN) (Kiss et al., 2004; 2006b), anti-ageing, photo-protecting (Ruszová et al., 2013), estrogen receptor modulation (Kujawski et al., 2010) N/A N/A

Ledum groenlandicum

Lysichiton americanus Maianthemun dilatatum Plantago major

Leaves: 2-(4-methoxyphenyl)-1-nitroethane, 2-(4hydroxyphenyl)-1-nitroethane (Hanawa et al., 2000) N/A N/A Leaves: phenylethanoid glycosides (acteoside ¼verbascoside, plantamajoside), iridoid glycosides (aucubin, catalpol) (Taskova et al., 2002; Gonda et al., 2013)

Whole plant: phenylpropanoid (Ravn and Brimer, 1988), Ursolic acid, oleanolic acid, 18betaglycyrrhetic acid (Bakker et al., 1998; Ringbom et al., 1998), pectin (Samuelsen et al., 1996), heteroxylan poyisaccharides (Samuelsen et al., 1999), iridoid glucosides (Taskova et al., 1999), polysaccharides, lipids, caffeic acid derivative, alkaloids (Michaelsen et al., 2000; Samuelsen, 2000), flavonoids (luteolin7-O-glucoside, apigenin-7-O-glucoside, luteolin, apigenin, rutin, and quercetin) and terpenes (Andrade-Cetto and Heinrich, 2005; Beara et al., 2009)

Polypodium glycyrrhiza

Root: steroidals (Kim and Kinghorn, 1989), flavonoid glycosides (Kim and Kinghorn,1987), calagualine (saponin) (Aggarwal and Shishodia, 2006), polypodoside A (Kim et al., 1988)

The expression of doxorubicin resistance in the three resistant cell lines has been verified by us (Efferth et al., 2008; Kuete and Efferth, 2013). Human CCRF-CEM, CEM/ADR5000, HL60 and HL60/ AR leukemia cell lines were maintained in RPMI medium (Gibco BRL, Eggenstein, Germany) supplemented with 10% fetal calf serum (Gibco BRL) in a 5% CO2 atmosphere at 37 1C. Cells were passaged twice weekly. Breast cancer cells transduced with a control vector(MDA-MB-231-pcDNA3) or withcDNAfor the breast cancer resistance protein BCRP (MDA-MB-231-BCRP clone 23) were maintained under standard conditions as described above

Upper parts: antioxidant, anti-inflammatory, anticancer (Dufour et al., 2007), antimutagenic (Idaomar et al., 2002), weak antimicrobial (Cybulska et al., 2011) Leaves: antifungal (Hanawa et al., 2000). Root: antiviral (McCutcheon et al.,1995) N/A Seeds: antinoceptive (Atta et al., 2004) Leaves: NO- and TNF-alpha production ( GomezFlores et al., (2000)), antihistaminic (Ikawati et al., 2001), antinoceptive (Atta and Abo EL-Sooud, 2004), inhibition of angiotensin I-converting enzyme (Nhiem et al., 2011), increased cellular proliferation and migration (Zubair et al., 2012) Whole plant: anti-atherosclerotic (Angarskaia and Sokolova, 1962), uterotonic (Shipochliev, 1981), against chronic bronchitis (Matev et al., 1982), cytotoxic against cancer cells (Lin et al., 2002; Ruffa et al., 2002; Gálvez et al., 2003, VelascoLezama et al., 2006), anticancer activity in vivo (Ozaslan et al., 2007), anticarcinogenic (Oto et al., 2012), immunomodulation (Chiang et al., 2003), antiviral (Chiang et al., 2002), antifungal (Ramos Ruiz et al., 1996; Holetz et al., 2002), antibacterial (Holetz et al., 2002; Velasco-Lezama et al., 2006), antioxidant (Ren et al., 1999; Beara et al., 2009; Oto et al., 2011), anti-inflammatory (Shipochliev et al., 1981; Türel et al., 2009), hepatoprotective (Türel et al., 2009), antimalarial (Sangian et al., 2013), COX-1/2 and 12-LOX inhibition (Beara et al., 2010; Stenholm et al., 2013) Root: antiviral (McCutcheon et al.,1995), not acutely toxic, non-mutagenic (Kim et al.,1988)

for CCRF-CEM and HL60 cells and additionally supplemented with in 800 ng/ml geneticin (Invitrogen, Karlsruhe, Germany). All experiments were performed with logaritmically growing cells. 2.3. Resazurin cell growth inhibition assay The resazurin (Promega, Mannheim, Germany) reduction assay (O’Brien et al., 2000) was used to assess the cytotoxicity as previously described (Kuete and Efferth, 2013). The assay tests cellular viability and mitochondrial function. Briefly, adherent cells


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Fig. 1. Cytotoxicity of plant extracts towards CCRF-CEM tumor cells and their P-glycoprotein-expressing, multidrug-resistant subline CEM/ADR5000 at a fixed concentration of 10 mg/mL as determined by the resazurin assay. The bars show mean values7 SD of two independent experiments with each four parallel measurements.

Fig. 2. Cytotoxicity of plant extracts towards HL-60 tumor cells and their MRP1-expressing, multidrug-resistant subline HL-60AR at a fixed concentration of 10 mg/mL as determined by the resazurin assay. The bars show mean values 7 SD of two independent experiments with each four parallel measurements.

were grown in tissue culture flasks, and then harvested by treating the flasks with 0.025% trypsin and 0.25 mM EDTA for 5 min. Once detached, cells were washed, counted and an aliquot (5 103 cells) was placed in each well of a 96-well cell culture plate in a total volume of 100 mL. Cells were allowed to attach overnight and were then treated with the samples. After 48 h, 20 mL of 0.01% w/v resazurin solution was added to each well and the plates were incubated at 37 1C for 1–2 h. Fluorescence was measured on an automated 96-well Infinite M2000 ProTM plate reader (Tecan, Crailsheim, Germany) using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. For leukemia cells, aliquots of 5 104 cells/mL (obtained from overnight suspension) were seeded in 96-well plates, and extracts were added immediately. After 72 h incubation, plates were treated with resazurin solution as described above. Doxorubicin was used as positive control. Each assay was conducted at least three times, with two replicates each. Cell viability was evaluated based on a comparison

with untreated cells. IC50 values were taken to be the concentration of sample required to inhibit 50% of cell proliferation, and were calculated from a calibration curve by linear regression using Microsoft Excel.

3. Results 3.1. Plant extract screening Sixteen extracts from 10 plants were tested for their cytotoxic activity at a fixed concentration of 10 mg/mL towards human CCRFCEM wild-type leukemia cells and their P-glycoprotein-(ABCB1/ MDR1)-expressing multidrug-resistant subline, CEM/ADR5000. As shown in Fig. 1, three extracts from two plants (Maianthemum dilatatum whole plant, Arctium minus Bernh. leaves (Asteraceae) of the 16 tested extracts revealed inhibition rates in the range of


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Fig. 3. Cytotoxicity of plant extracts towards MDA-MB-231 pcDNA tumor cells and their BRCP-expressing, multidrug-resistant subline MDA-MB-231 BCRP at a fixed concentration of 10 mg/mL as determined by the resazurin assay. The bars show mean values7 SD of two independent experiments with each four parallel measurements.

Table 4 Cytotoxicity of plant extracts toward sensitive and drug-resistant cancer cell lines as determined by the resazurin assay. Plant

Extract (IC50 in lg/mL): Lysichiton americanum (stem and leaves) Arctium minus (leaves) Maianthmum dilatatum (whole plant) Maianthmum dilatatum (whole plant) Control drug (IC50 in lM): Doxorubicinnn Doxorubicinnn n

Cell model

IC50 values sensitive

Resistant

Degree of resistancen

CCRF-CEM vs. CEM/ADR5000 CCRF-CEM vs. CEM/ADR5000 CCRF-CEM vs. CEM/ADR5000 MDA-MB-231 pcDNA vs. BCRP

4.64 (7 1.83) 2.93 ( 7 0.9) 2.40 ( 7 0.63) 86.35 ( 7 19.49)

3.19 ( 7 1.21) 4.53 ( 7 2.09) 3.14 ( 7 2.44) 64.77 ( 7 9.26)

0.69 1.55 1.31 0.75

CCRF-CEM vs. CEM/ADR5000 MDA-MB-231 pcDNA vs. BCRP

0.20 ( 7 0.06) 1.10 ( 70.09)

195.12 ( 7 14.30) 7.83 ( 7 0.01)

975.60 7.12

C50 of resistant divided by IC50 of sensitive cell line. taken from Kuete et al. (2013) for comparison.

nn

21.67 2.5% and 39.37 6.7% cell viability compared to untreated control cells. The stem and leaves extract of Lysichiton americanus Hultén & St. John (Araceae) reduced cell viability between 57.276.7% and 57.77 6.7% of control. Next, the extracts were tested in human HL-60 wild-type leukemia cells and their MRP1-expressing multidrug-resistant subline, HL-60 AR. Extracts of Maianthemum dilatatum (leaves or whole plant), Arctium minus (leaves), and Lysichiton americanus (root) strongly inhibited these two cell lines with cell viability rates between 21.870.7% and 33.0 70.7% compared to untreated control cells, while the Ledum groenlandicum (stem and leaves) extract did not show any inhibitory activity. The other extracts revealed cytotoxic activities in the range of 26.3 79.1% and 89.0 75.4% of controls (Fig. 2). Furthermore, the activity of the extracts was tested using human MDA-MB-231pcDNA and BCRP-transfected multidrugresistant MDA-MB-231 BCRP breast cancer cells. The most cytotoxic extracts were from Arctium minus (leaves) and Maianthemum dilatatum (leaves or whole plant) (range of 21.670.5% to 25.0 70.6% of control), while all others showed weaker inhibitory

activities compared to untreated control cells (between 37.272.4% and 77.37 7.7% of control) or were inactive (values above 100% of control) (Fig. 3). Interestingly, none of the extracts exerting activity with cell viability rates below 30% of control against the wild-type cells in Figs. 1–3, was resistant in the MDR1-, MRP1- or BCRP-expressing sublines,at a fixed concentration of 10 mg/mL, indicating that these multidrug-resistant cells did not exert cross-resistance to these cytotoxic plant extracts. 3.2. Dose response curves of plant extracts We performed dose-response curves using sensitive and multidrug-resistant cell lines and cytotoxic extracts identified in Figs. 1–3. The extracts of Arctium minus (leaves), Lysichiton americanum (stem and leaves), and Maianthemum dilatatum (whole plant) revealed IC50 values in a range between 2.93 (7 0.9) and 86.35 (7 19.49 mg/mL) in the sensitive and drug-resistant cell lines (Table 4). The multidrug-resistant cell lines were not crossresistant to these extracts (Fig. 4A–C, Table 4). For comparison,


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Fig. 4. Dose response curves of selected plant extracts in sensitive and multidrug-resistant tumor cell lines as determined by the resazurin assay. The dose response curves show mean values 7 SD of two independent experiments with each four parallel measurements.

P-glycoprotein-expressing CEM/ADR5000 cells were 975–fold, and BCRP-expressing MDA-MB-231 BCRP cells 7-fold resistant to the control drug doxorubicin as compared to their corresponding drug-sensitive counterparts (Kuete et al., 2013). Interestingly, the Maianthemum dilatatum extract was even more cytotoxic in BCRPexpressing cells than in the corresponding drug-sensitive counterpart (hypersensitivity, collateral sensitivity (Fig. 4D).

4. Discussion The medicinal plants of the First Nations of North America and especially of the Gwich'in have not been investigated in much detail yet. As a part of our ongoing studies in ethnopharmacology, we analyzed the cytotoxic activity of medicinal plants used since ages in this Athbascan tribe. From a total of 17 extracts derived from 11 plants, we found that exgtracts of Arctium minus (leaves) and Maianthemum dilatatum (leaves or whole plant) revealed considerable inhibitory activities towards cancer cells. Several secondary metabolites have been isolated in Arctium minus and Lysichiton americanus, but not in Maianthemum dilatatum as of yet (Table 3). While these phytochemicals have been shown to exert diverse bioactivities (e.g. antibacterial, antinoceptive, anti-inflammatory, antifungal, antiviral and others), their cytotoxic activity towards cancer cells is sparsely known. Rather than only looking for the general cytotoxicity towards tumor cells, we were interested to see whether multidrug-resistant tumor cells could be killed by medicinal plants of the Gwich'in. MDR is a pressing problem in clinical management of human cancers and many clinically established anticancer drugs lose their activity, if tumors are getting refractory – with fatal consequences for patients. It is extremely unfortunate that many tumors are not only resistant to

one anticancer drugs, but to many at the same time. This dramatically reduces the chances for successful treatment by changing drug regimens. The reason is that drug efflux pump of the ABC transporter type (e.g. P-glycoprotein/MDR1/ABCB1, MRP1/ ABCC1, and BCRP/ABCG2) recognize a large number of anticancer drugs and expel them out of cancer cells preventing their cytotoxic effects. Characteristic cross-resistance profiles to established anticancer drugs have been described for these ABC transporters: Pglycoprotein extrudes anthracyclines, Vinca alkaloids, taxanes, epipodophyllotoxins out of cancer cells, but not platinum compounds, alkylating agents or antimetabolites. MRP1 translocates anthracyclines, Vinca alkaloids, camptothecin derivatives, methotrexate and antifolates, epipodophyllotoxones, arsenite and others. BCRP expels antracyclines, mitoxantrone, Vinca alkaloids, taxanes, platinum compounds, and camptothecin derivatives out of cancer cells (Efferth, 2001; Gillet et al., 2007). Therefore, there is a desperate search for novel compounds to kill multidrug-resistant tumors and to improve treatment outcomes. On the other hand, it is well-known that the majority of anticancer drugs developed during the past half century are natural products, derivatives of them or drugs based on biological mechanisms taken from them (Newman and Cragg, 2012). For this reason, it might be promising to investigate medicinal plants with a millennia-old history of ethnomedical use to identify and develop new treatment principles for oncology. It is pleasing that our cell line panel expression the three major MDR-conferring ABC-transporters were not crossresistant to extracts of Arctium minus (leaves) and Maianthemum dilatatum (leaves or whole plant). Remarkably, Maianthemum dilatatum extract was even more cytotoxic in BCRP-expressing cells than in their drug-sensitive counterpart. The phenomenon of hypersensitivity or collateral sensitivity has been occasionally observed for few compounds in


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P-glycoprotein-expressing cells (Saeed et al., 2013). To the best of our knowledge, hypersensitivity to naturally occurring xanthones as recently described by us (Kuete et al., 2014) and to Maianthemum dilatatum whole plant extract in the present investigation are the only described examples of collateral sensitivity in BCRPexpressing cells. Future studies are warranted to isolate the active phytochemicals from these three plants and to investigate the mechanisms of cytotoxicity in more detail. Our results that some of the extracts were involved in classical resistance mechanisms such as ABC transporters, also might imply that they affect cancer cells by multiple pathways. The exact nature of this multi-specificity is, however, elusive as of yet. This should be investigated in the future, for instance by transcriptome- and proteome-wide analyses to unravel the pathways, which are specifically affected by these medicinal plants. Such pharmacogenomics studies are currently being performed in our laboratory. Multi-specificity is a frequent feature of many natural products and multi-specificity prevents the development of resistance towards single bioactive compounds, which likely was an important selection advantage during evolution of life on this globe (Efferth and Koch, 2011). This may be taken as another hint on the high potentials of medicinal plants for the development of novel treatment strategies to fight cancer.

Conflict of interest There is no conflict of interest.

Uncited references Erdemoglu et al., (2009); Khuroo et al., (1988); Lans et al., (2008); Turner and Bell (1973).

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Cytotoxicity of selected Cameroonian medicinal plants and Nauclea pobeguinii towards multi-factorial drug-resistant cancer cells.

Cytotoxicity of the methanol extracts of Elephantopus mollis, Kalanchoe crenata and 4 other Cameroonian medicinal plants towards human carcinoma cells.

Cytotoxicity potentials of eleven Bangladeshi medicinal plants.

Cytotoxicity of methanol extracts of 10 Cameroonian medicinal plants towards multi-factorial drug-resistant cancer cell lines.

Cytotoxicity and Modes of Action of the Methanol Extracts of Six Cameroonian Medicinal Plants against Multidrug-Resistant Tumor Cells.

Cytotoxicity of methanol extracts of Annona muricata, Passiflora edulis and nine other Cameroonian medicinal plants towards multi-factorial drug-resistant cancer cell lines.

Antibiotic screening of medicinal plants of the British Columbian native peoples.

Cytotoxicity and modes of action of five Cameroonian medicinal plants against multi-factorial drug resistance of tumor cells.

Cytotoxicity of three South African medicinal plants using the Chang liver cell line.

Apoptosis-inducing effect of three medicinal plants on oral cancer cells KB and ORL-48.

Stomach cancer incidence rates among Americans, Asian Americans and Native Asians from 1988 to 2011.

Cytotoxicity of selected medicinal plants used in Mt. Frere District, South Africa.

Use of medicinal plants by breast cancer patients in Algeria.

Neuroprotective effects of medicinal plants.

Antifertility activity of medicinal plants.

Cancer mortality in Native Americans in North Carolina.

Phytodentistry: use of medicinal plants.

Virtualizing the p-ANAPL library: a step towards drug discovery from African medicinal plants.

Indian Medicinal Plants.

Indian Medicinal Plants.

Poverty, suicide, and homicide among Native Americans.

Multidrug resistant bacteria are sensitive to Euphorbia prostrata and six others Cameroonian medicinal plants extracts.

An ethnobotanical study of medicinal plants used by tribal and native people of Madhupur forest area, Bangladesh.

Medicinal plants of the family Caryophyllaceae: a review of ethno-medicinal uses and pharmacological properties.