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

Background Cancer still constitutes one of the major health concerns globally, causing serious threats on patients, their families, and the healthcare system. Methods In this study, the cytotoxicity of the methanol extract of Elephantopus mollis whole plant (EMW), Enantia chlorantha bark (ECB), Kalanchoe crenata leaves (KCL), Lophira alata bark (LAB), Millettia macrophylla leaves (MML) and Phragmanthera capitata leaves (PCL) towards five human solid cancer cell lines and normal CRL2120 fibroblasts, was evaluated. Extracts were subjected to qualitative chemical screening of their secondary metabolite contents using standard methods. The cytotoxicity of samples was evaluated using neutral red uptake (NR) assay meanwhile caspase activation was detected by caspase-Glo assay. Flow cytometry was used to analyze the cell cycle distribution and the mitochondrial membrane potential (MMP) whilst spectrophotometry was used to measure the levels of reactive oxygen species (ROS). Results Phytochemical analysis revealed the presence of polyphenols, triterpenes and sterols in all extracts. The IC50 values of the best samples ranged from 3.29 μg/mL (towards DLD-1 colorectal adenocarcinoma cells) to 24.38 μg/mL (against small lung cancer A549 cells) for EMW, from 2.33 μg/mL (mesothelioma SPC212 cells) to 28.96 μg/mL (HepG2 hepatocarcinoma) for KCL, and from 0.04 μg/mL (towards SPC212 cells) to 0.55 μg/mL (towards A549 cells) for doxorubicin. EMW induced apoptosis in MCF-7 cells mediated by MMP loss and increased ROS production whilst KCL induced apoptosis via ROS production. Conclusion This study provides evidences of the cytotoxicity of the tested plant extract and highlights the good activity of Elephantopus mollis and Kalanchoe crenata. They deserve more exploration to develop novel cytotoxic drugs.


Background
Cancer still constitutes a major health concern globally, causing serious threats on patients, their families, and the healthcare system. The related economic impact is significant and is increasing, with annual cost in 2010 being estimated at about 1.16 trillion US dollars [1].
About 70% of deaths caused by cancer occur in low-and middle-income countries. Chemotherapy is recognized as the major mode of treatment of malignant diseases, and the plant kingdom has been the origin of many cytotoxic drugs such as paclitaxel (from Taxus brevifolia) and Vinca alkaloids (from Catharanthus roseus) [2][3][4][5]. The potential of African flora as a source of a variety of cytotoxic agents is intensively being demonstrated [6,7]. In fact, various cytotoxic plants of the continent were reported amongst which are Anthocleista schweinfurthii, Morus mesozygia, Nauclea latifolia, Erythrina sigmoidea [8], Erythrina sacleuxii, Albizia gummifera, Strychnos usambarensis, Zanthoxylum gilletii, Bridelia micrantha, Croton sylvaticus, Albizia schimperiana, Erythrina burttii, Erythrina sacleuxii, Bridelia micarantha, Zanthoxylum giletii and Solanum aculeastrum [9]. In our continuous search for cytotoxic agents from African flora, this study was undertaken to evaluate the antiproliferative activity of the methanol extracts of six Cameroonian plants used traditionally to treat cancers or disease states with symptoms related to cancer. These plants included Enantia chlorantha Oliv. (Annonaceae), Elephantopus mollis Kunth (Asteraceae), Kalanchoe crenata (Andrews) Haworth (Crassulaceae), Lophira alata Banks ex C.F.Gaertn.(Ochnaceae), Millettia macrophylla Benth. (Fabaceae) and Phragmanthera capitata (Spreng.) Balle (Loranthaceae). The study was extended to the assessment of the mode of action of the best extracts, namely those from Elephantopus mollis whole plant (EMW) and Kalanchoe crenata leaves (KCL).

Plant material and extraction
Plants studied in this work are used in the traditional medicine to treat cancer or disease states with symptoms related to cancer (Table 1). They were collected in different parts of Cameroon in February 2015 and included barks of Lophira alata and Enantia chlorantha, leaves of Phragmanthera capitata, Kalanchoe crenata and Millettia macrophylla and the whole plant of Elephantopus mollis. The identification of palnts was done by the Cameroon National Herbarium (HNC; Yaounde) and voucher specimens are availaible under accession numbers (Table 1). The powder obtained from each air dried plant sample (300 g) was macerated in methanol (MeOH, 1 L) for 48 h at room temperature. The macerate was further concentrated under reduced pressure to obtain the crude extract. All extracts were then conserved at 4°C.

Chemicals
The reference drug used in this work was doxorubicin 98.0%, purchased from Sigma-Aldrich (Munich, Germany).

Cell lines and culture
Five carcinoma and one normal cell lines were tested in this work. They were SPC212 human mesothelioma cell line obtained from American Type Culture Collection (ATCC) and provided by Dr. Asuman Demiroğlu Zergeroğlu (Gebze Technical University, Kocaeli, Turkey), A549 human non-small cell lung cancer (NSCLC) cell line, obtained from the Institute for Fermentation, Osaka (IFO, Japan) and provided by Prof. Dr. Tansu Koparal (Anadolu University, Eskisehir, Turkey), HepG2 hepatocarcinoma cells obtained from ATCC and MCF-7 breast adenocarcinoma cells obtained from ATCC and provided by Prof. Dr. Tansu Koparal (Anadolu University, Eskisehir, Turkey), DLD-1 colorectal adenocarcinoma cell lines obtained from ATCC and the normal CRL2120 human skin fibroblasts obtained from ATCC. The cells were maintained as a monolayer in DMEM medium (Sigma-aldrich, Munich, Germany), supplemented with 10% fetal calf serum and 1% penicillin (100 U/mL)streptomycin (100 μg/mL) in a humidified 5% CO 2 atmosphere at 37°C.

Neutral red (NR) uptake assay
The cytotoxicity of samples was performed by the cheaper and sensitive NR uptake assay as previously described [14][15][16]. Samples were added in the culture medium so that dimethylsufoxide (DMSO) used prior for dilution, did not exceed 0.1% final concentration. Briefly, cells were detached by treatment with 0.25% trypsin/EDTA (Invitrogen, USA) and an aliquot of 1 × 10 4 cells was placed in each well of a 96-well cell culture plate (Thermo Scientific, Germany) in a total volume of 200 μL. The cells were allowed to attach overnight and subsequently treated with different concentrations of the extracts and doxorubicin. Each of the studied samples were immediately added in varying concentrations in additional 100 μL of culture medium to obtain a total volume of 200 μL/well. After 72 h incubation in humidified 5% CO 2 atmosphere at 37°C, the medium was removed and 200 μL fresh medium containing 50 μg/mL NR was added to each well and incubation continued for an additional 3 h at 37°C in 5% CO 2 atmosphere. The dye medium was then removed and each well was then washed rapidly with 200 μL phosphate buffer saline (PBS) followed by addition of 200 μL of acetic acid-water-ethanol in water (1:49:50). The plates were kept for 15 min at room temperature to extract the dye and then shaken for a few minutes on a GFL 3012 shaker (Gesellschaft für Labortechnik mbH, Burgwedel, Germany). Absorbance was measured on ELx 808 Ultra Microplate Reader (Biotek) equipped with a 540 nm filter. Each assay was done at least three times, with three replicates each. The viability was evaluated based on a comparison with untreated cells. The IC 50 Antimicrobial activity [36]; analgesic and anticonvulsant effects [38] and antihyperglycaemic [37] Lophira alata Banks ex C.F.Gaertn. ( values represented the sample's concentrations required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel [17].
Flow cytometry for cell cycle analysis and detection of apoptotic cells The cell-cycle analysis was performed by flow cytometry using BD cycletest™ Plus DNA Kit Assay (BD Biosciences, San Jose, USA). The BD Cycletest™ Plus DNA kit provides a set of reagents for isolating and staining cell nuclei. Flow cytometric analysis of differentially stained cells is used to estimate the DNA index (DI) and cellcycle phase distributions. Briefly, MCF-7 cells (3 mL, 1 × 10 5 cells/mL) were seeded into each well of 6-well plates and allowed to attach for 24 h. The cells which were treated with ¼ × IC 50 , ½ × IC 50 and IC 50 concentrations of Elephantopus mollis whole plant (EMW) and Kalanchoe crenata leaves (KCL) extracts and the standard drug, doxorubicin, and grown for 72 h. The untreated cells (control) were also included in the assay. They were further trypsinized and suspended in 1 mL PBS, then centrifuged at 400 g for 5 min at room temperature (RT). The cells were further processed according to the manufacturer's protocol [16]. The cells were further measured on a BD FACS Aria I Cell Sorter Flow Cytometer (Becton-Dickinson, Germany). For each sample 10 4 cells were counted. For PI excitation, an argon-ion laser emitting at 488 nm was used. Cytographs were analyzed using BD FACSDiva™ Flow Cytometry Software Version 6.1.2 (Becton-Dickinson).

Caspase-Glo 3/7 and caspase-Glo 9 assay
Caspase activity in MCF-7 cells was detected using Caspase-Glo 3/7 and Caspase-Glo 9 Assay kits (Promega, Mannheim, Germany) as previously reported [18][19][20]. Cells were treated with EMW and KCL at their ½ × IC 50 and IC 50 values with DMSO as solvent control for 6 h. Luminescence was measured using an BioTek Synergy™ HT multi-detection microplate reader. Caspase activity was expressed as percentage of the untreated control.

Cytotoxicity
The results of the antiproliferative activity of the tested extracts and doxorubicin as determined by the NR uptake assay are shown in Table 2. The selectivity index (   The selectivity index was determined as the ratio of IC 50 value in the CRL2120 normal fibroblasts divided by the IC 50 in the cancer cell lines. In bold: significant activity [7,23,24,45] distribution, caspases activity, MMP loss and ROS production in MCF-7 cells.

Mechanistic studies
Cell cycle distribution in MCF-7 cells treated with EMM, KCL and doxorubicin is depicted in Fig. 1. EMW and KCL induced dose-dependent cell cycle modifications with progressive increase of sub-G0/G1 phase cells. Both EMW and KCL induced cell cycle arrest in G0/G1. Upon treatment of MCF-7 cells with the selected samples, they progressively underwent apoptosis; the increase of sub-G0/G1 cells ranged from 11.8% (¼ IC 50 ) to 31% (IC 50 ) for KCL, from 28.8% (¼ IC 50 ) to 83.4% (IC 50 ) for EMW, from 27.6% (¼ IC 50 ) to 60% (IC 50 ) for doxorubicin and only 3.1% in non-treated cells. Upon treatment of MCF-7 cells with EMW, KCL and doxorubicin with equivalent (eq.) to the ½ × IC 50 and IC 50 for 6 h, no activation of caspase 3/7 and caspase 9 activities was observed. MCF-7 cells were also treated with EMM, KCL and doxorubicin, and the integrity of the MMP was analyzed. Data shown in Fig. 2 [7,23,24]. IC 50 values below 20 μg/mL were recorded with EMW, KCL, MML and ECB respectively in 4, 3, 2 and 1 of the 5 tested carcinoma cells. Importantly, IC 50 values below 5 μg/mL were obtained with EMW in 4/5 carcinoma cell lines as well as KCL towards SPC212 cells and MML against HepG2 cells. These data highlight the usefulness of these extracts in the fight against solid cancers. This hypothesis is strengthened by the good selectivity index (SI > 1; Table 2) of the tested extract, which is compatible with their possible use in cancer chemotherapy. Elephantopus mollis and Millettia macrophylla are traditionally used in the treatment of cancers [25,26]. The two plants, Especiallye. mollis, had cytotoxic effects on the tested carcinoma cells, validating their traditional use in the management of malignancies. In this study, plants used traditionally to treat disease states with symptoms related to cancer, were Lophira alata, Enantia chlorantha, Phragmanthera capitata and Kalanchoe crenata. Amongst them, only P. capitata was not active on the tested cancer cell lines. This also consolidates the recommandations that ethnopharmacological usages such as immune and skin disorders, inflammatory, infectious, parasitic and viral diseases should be taken into account when selecting plants that treat cancer [27].
To the best of our knowledge, the anticancer activity of Enantia chlorantha, Lophira alata and Kalanchoe crenata is being reported herein for the first time. The antiproliferative effect of ethyl acetate extract of Elephantopus mollis, collected from Penang Agriculture Department, Relau, Malaysia), on HepG2 cells, with the lowest IC 50 value of 9.38 μg/mL, NCI-H23 cells (13.17 μg/mL), T-47D cells (12.57 μg/mL) and Caov-3 cells (42.11 μg/mL) [25,28], was reported. A much more lower IC 50 value of 3.74 μg/mL was obtained with samples from Cameroon. This could be explained by possible geographic variations in the chemical constitution of the plant. However, both studies confirm the cytotoxic potential of this plant. The cytotoxicity of methanolic extract and chalcone dimers from L. alata on Ehrlich Ascites carcinoma cells [29] was also reported in the present work. This plant was moderately active against HepG2 cells, providing additional information on the anticancer activity of the plant. The poor cytotoxic effects of compounds and phenolic fractions of M. macrophylla towards breast cancer cells MCF-7 and MDA-MB-231, was reported [26]. Data obtained herein are in accordance with this previous study, as a moderate effect of MML was obtained in MCF-7 cells. However, MML had good effect against SPC212 lung adenocarcinoma and HepG2 adenocarcinoma cells, highlighting its possible use in the fight against cancers.
Finally, evidences of the antiproliferative effects of the tested plant extract, highlights the good activity of Elephantopus mollis, Kalanchoe crenata and in lesser extent Millettia macrophylla have been provided. Extract of E. mollis, induced apoptosis in MCF-7 cells, mediated by MMP loss and increased ROS production whilst Kalanchoe crenata leaves extract induced apoptosis via ROS production (Figs. 2 and 3). It should be noted that only ROS production is not enough to identify cell apoptosis. Therefore, additional studies including detection of other molecules related to apoptosis such as BCL2, BAX, PRPP, etc., will be performed. Purification of the most active plants (Elephantopus mollis, Kalanchoe crenata and Millettia macrophylla) will also be performed to identify their cytotoxic constituents.

Conclusions
In this work, the antiproliferative activity of extracts from six Cameroonian medicinal plants, Lophira alata, Enantia chlorantha, Phragmanthera capitata, Kalanchoe crenata, Elephantopus mollis and Millettia macrophylla was reported on five human solid cancer cell lines and normal CRL2120 fibroblasts. The three most active extracts were those from E. mollis whole plant, K. crenata leaves and M. macrophylla leaves. They can be used in the management of malignant diseases and deserve more exploration to isolate their active constituents in order to develop novel cytotoxic drugs.