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

Background Cancer chemotherapy is still hampered by clinical failures due to multi-drug resistance (MDR) of tumor cells. In the present study, we have investigated the cytotoxicity of 20 methanol extracts from 10 medicinal plants against the sensitive leukemia CCRF-CEM cells. The most cytotoxic extracts were then further tested on a panel of 8 human cancer cell lines, including various MDR phenotypes. Methods The cytotoxicity of the 20 methanol extracts from 10 Cameroonian medicinal plants was determined using a resazurin reduction assay. Meanwhile, flow cytometry was used to measure cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS). Results In the preliminary assay using CCRF-CEM cells, 12 extracts from five plants displayed IC50 values below 80 μg/mL, namely Albizia adianthifolia, Alchornea cordifolia, Alchornea laxiflora, Pennisetum purpureum, and Spathodea campanulata. the four best extracts were from two plants: Albizia adianthifolia roots (AAR) and bark (AAB) as well as Alchornea cordifolia leaves (ACL) and bark (ACB) had respective IC50 values of 0.98 μg/mL, 1.45 μg/mL, 8.02 μg/mL and 12.57 μg/mL in CCRF-CEM cells. They were further tested in 8 other cell lines as well as in normal AML12 hepatocytes. IC50 values ranging from 2.71 μg/mL (towards glioblastoma U87MG.ΔEGFR cells) to 10.30 μg/mL (towards breast adenocarcinoma MDA-MB-231-BCRP cells) for AAB, from 3.43 μg/mL (towards U87MG cells) to 10.77 μg/mL (towards colon carcinoma HCT116 (p53−/−) cells) for AAR and from 0.11 μg/mL (towards CCRF-CEM cells) to 108 μg/mL (towards leukemia CEM/ADR5000 cells) for doxorubicin (as control drug) were obtained. ACL and ACB extracts displayed selective activities. AAR and ACL extracts induced apoptosis in CCRF-CEM cells, through caspases activation and loss of MMP, while apoptotic cell death was mediated by MMP diruption and increase ROS production for ACL. Conclusion Some of the tested plants namely Albizia adianthifolia, Alchornea cordifolia, Alchornea laxiflora, Pennisetum purpureum, Spathodea campanulata represent a potential source of novel anticancer drugs. Especially, Albizia adianthifolia and Alchornea cordifolia revealed considerable cytotoxic activities that could be exploited to develop phytomedicines to fight cancers including MDR phenotypes.

In our ongoing search of anticancer drugs from African medicinal plants, we undertook the present work to assess the cytotoxicity of 10 Cameroonian medicinal plants traditionally used to manage cancer or disease states bearing relevance to cancer or cancer-like symptoms, such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases [15]. The study was extended to the evaluation of the ability of extracts from two most active plants, Albizia adianthifolia and Alchornea cordifolia to alter the cell cycle distribution, caspases activity, mitochondrial membrane potential (MMP) and to increase reactive oxygen species (ROS) in leukemia CCRF-CEM cells.

Plant material and extraction
All medicinal plants parts used in the present study were collected in different regions of Cameroon in January 2014. These included leaves, bark and roots of Alchornea cordifolia, Alchornea laxiflora, Albizia adianthifolia and Spathodea campanulata, leaves and roots of Combretum hispidum and Laportea ovalifolia and the whole plant of Boerhavia diffusa, Eremomastax speciosa, Laportea aestuans and Pennisetum purpureum. The plants were identified at the National Herbarium (Yaoundé, Cameroon), where voucher specimens were deposited under the reference numbers shown in Table 1. The air-dried and powdered plant material was soaked in methanol for 48 h, at room temperature. The methanol extract was concentrated in vacuum under reduced pressure to give the crude extract. This extract was then conserved at 4°C until further use.

Chemicals
Doxorubicin 98.0 % and vinblastine ≥ 96 % from Sigma-Aldrich (Munich, Germany) were provided by the University Pharmacy of the Johannes Gutenberg University (Mainz, Germany) and dissolved in phosphate buffer saline (PBS; Invitrogen, Eggenstein, Germany) at a concentration of 10 mM. Geneticin > 98 % (Sigma-Aldrich) was stored at a stock concentration of 72.18 mM.

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 < 0.05 denoted significance in all cases.

Results
In the present investigation, the cytotoxicity of 20 methanol extracts from 10 plants was first determined at different concentrations in drug-sensitive CCRF-CEM leukemia cells. The results are summarized in Table 2. Twelve out of 20 (60 %) extracts displayed IC 50 values below 80 μg/ mL. These extracts were from Pennisetum purpureum, Spathodea campanulata bark, Spathodea campanulata roots, Alchornea laxiflora bark, Alchornea laxiflora leaves, Albizia adianthifolia leaves (AAL), Combretum hispidum leaves, Alchornea cordifolia roots (ACR), Alchornea cordifolia bark (ACB), Alchornea cordifolia leaves (ACL), Albizia adianthifolia bark (AAB) and Albizia adianthifolia roots (AAR). Extracts from Alchornea laxiflora roots, Boerhavia diffusa (whole plant), Combretum hispidum bark, Eremomastax speciosa (whole plant), Laportea aestuans (whole plant), Laportea ovalifolia leaves, Laportea ovalifolia roots, Spathodea campanulata leaves resulted in more than 50 % proliferation of CCRF-CEM cells at 80 μg/mL ( 14 μg/mL). It is worth noting that collateral sensitivity (or hypersensitivity: higher toxicity to resistant than to sensitive cells with a degree of resistance below 1) was observed in drug-resistant epidermal growth factor receptor-transfected U87MG.ΔEGFR cells to AAB (degree of resistance of 0.43fold), to AAR (0.39-fold), to ACL (0.83-fold) and to ACB (<0.40-fold) compared to its sensitive counterpart U87MG cells. Importantly, if cross-resistance to the tested extracts were observed, the degrees of resistance were in all cases lower than that of the reference compound, doxorubicin (Table 3). AAR and ACL were the most active extracts from Albizia adiathifolia and Alchornea cordifolia respectively, and were subsequently used for mechanistic studies. IC 50 values of AAR and ACL extracts as well as doxorubicin were used to treat CCRF-CEM cells for 6 h, and the cycle distribution was analyzed. The results are depicted in Fig. 1. Dose-dependent and significant modifications of the cell cycle phases were observed. Both AAR and ACL induced cell cycle arrest in the G0/G1 phase. After treatment with these two extracts, CCRF-CEM cells underwent apoptosis with a dose-dependent increase in the sub-G0/G1 phase. The percentages of cells in the sub-G0/G1 phase varied from 32.14 % (in 24 h) to 57.99 % (72 h) and from 31.69 % (24 h) to 59.67 % (72 h), respectively, for AAR and ACL treatments, while doxorubicin increased apoptosis in a range of 6.02 % (24 h) to 51.87 % (72 h). The highest percentage of sub-G0/G1 phase in non-treated cells was only 6.42 % after 72 h. After treating CCRF-CEM cells for 6 h at 2-fold IC 50 , AAR induced 4.35-fold, 2.02-fold and 1.52-fold increase of caspase 3/7, caspase 9 and caspase 8 activities, respectively, whereas no changes were observed upon ACL treatment (Fig. 2). AAR also induced significant MMP loss in a range of 35.5 % (1/2-fold IC 50 treatment) to 87.6 % (2-fold IC 50 ) (Fig. 3). ACL caused up 41.7 % MMP loss at 1/2-fold IC 50 treatment and complete rupture of the membrane (99.7 %) at 2-fold IC 50 (Fig. 3). A 48.6 % loss of MMP at 2-fold IC 50 of vinblastine was previously reported under similar experimental conditions in CCRF-CEM cells [12]. AAR did not induce ROS generation in CCRF-CEM cells contrary to ACL (Fig. 4). Dose-dependant increase in ROS production was also observed upon treatment of cells with ACL in a range of 0.73 % (1/2-fold IC 50 treatment) to 33.6 % (2-fold IC 50 ).  The degree of resistance was determined as the ratio of IC 50 value in the resistant divided by the IC 50 in the sensitive cell line; CEM/ADR5000, MDA-MB-231-BCRP, HCT116 (p53 −/− ), U87MG.ΔEGFR and AML12 were used as the corresponding resistant counterpart for CCRF-CEM (Table 1), MDA-MB-231-pcDNA, HCT116 (p53 +/+ ), U87MG and HepG2 cells, respectively. The tested methanol extracts were from AAB Albizia adianthifolia bark, AAR Albizia adianthifolia roots, ACL Alchornea cordifolia leaves, ACB Alchornea cordifolia bark. In bold: significant cytotoxic effects. Leukemia CEM/ADR5000 cells were tested in RPMI 1640 medium while carcinoma cells tested using DMEM medium, both containing 10 % FBS and 1 % penicillin-streptomycin

Discussion
The development of resistance by malignant cells remains a serious issue in cancer chemotherapy. Cancer cells rapidly develop chemoresistance, mainly due to the presence of adenosine triphosphate-binding cassette (ABC) transporters [2][3][4], such as the breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-gp/MDR1/ABCB1) [2] as well as the oncogene epidermal growth factor receptor (EGFR) [3,4,27] and the deletion or inactivation of tumor suppressor gene p53 [5]. Hence, identifying the mechanisms of resistance to different drugs is necessary, in order to efficiently prevent and overcome drug resistance. In this study, multi-factorial drug-resistant cancer cell lines such as leukemia CEM/ADR5000 cells overexpressing P-glycoprotein, breast adenocarcinoma MDA-MB-231-BCRP clone 23 expressing BCRP, p53 knockout HCT116 (p53 −/− ) colon cancer cells and EGFR-transfected U87MG.ΔEGFR glioblastoma cells [4,7,12,[20][21][22]28] were used to determine to assess the cytotoxicity the selected plant extracts. According to the US NCI plant screening program, botanicals with IC 50 values below of 20 μg/mL following incubation between 48 and 72 h [29] have been recognized as potential cytotoxic substances. In preliminary assays using the sensitive leukemia CCRF-CEM cells, AAB, AAR, ACL and ACB (Table 2) displayed IC 50 values below 20 μg/mL and were therefore selected for further assays against MDR phenotypes of other cell lines. Interestingly, AAB and AAR also displayed IC 50 values below or around 10 μg/mL and could therefore be considered as potential source for novel anti-cancer drugs. Most importantly, the degree of resistance of cells lines to AAB and AAR were in all cases lower than that of doxorubicin, highlighting their potential to combat MDR phenotypes. Though the IC 50 values recorded with ACL and ACB were all above 20 μg/mL, the cytotoxicity of these two samples on malignant cells can still be considered interesting, as they were much less toxic on normal AML12 hepatocytes, highlighting their good selectivity. It is also worth to note that the two best extracts, AAB and AAR were slightly toxic to normal AML12 hepatocytes (IC 50 values of 29.18 μg/mL and 29.14 μg/mL respectively for AAB and AAR). However, their high cytotoxicity towards cancer cells also suggests that they might be safely used in cancer chemotherapy. However, further evidence of the clinical efficacy of these extracts will be needed, as many phytochemicals are poorly bioavailable and they may be metabolized to more or less potent compounds by gut bacterial metabolism. MMP loss and increased ROS have been reported as a mode of apoptosis induction of plant extracts [29]. Hence, the ability of AAR and ACL to cause MMP breakdown in CCRF-CEM cells fits to this theory. The mode of action of AAR also includes the activation of caspases. Initiator caspases 9 (2.02-fold) and effector caspases 3/7 (4.35-fold) (Fig. 2) were significantly activated [29]. In addition to MMP alterations, ACL-induced apoptosis also include ROS production (Fig. 4).
To the best of our knowledge, the cytotoxicty of Albizia adiathifolia and Alchornea cordifolia towards the cell line panel tested in this study is being reported for the first time. Triterpenoid saponins such as adianthifoliosides A, B, and D isolated from Albizia adianthifolia exhibited cytotoxic effects towards Jurkat leukemia cells [30]. The presence of these compounds as well as other cytotoxic constituents such as prosapogenins [31] and  caspases activation and MMP loss. The mode of apotosis induction by ACL extract included MMP disruption and increased ROS generation in CCRF-CEM cells. The cytotoxicty of the two best plants, Albizia adiathifolia and Alchornea cordifolia towards the cell line panel tested in this study is being reported for the first time. Their purification will further be performed to identify their active constituents.