Screening for Selective Anticancer Activity of 65 Extracts of Plants Collected in Western Andalusia, Spain

Finding cytotoxic drugs with a high selectivity towards cancer cells is crucial to improve the low survival rates of patients diagnosed with metastatic cancers. Since plants are an important source of anticancer drugs, we have screened 65 extracts from 45 plants collected in several areas of Western Andalusia (Spain) for cytotoxic activity on lung cancer cells versus lung normal cells. An extract from the leaves of Tetraclinis articulata (Vahl) Mast. (Cupressaceae) showed a marked cytotoxicity (IC50 = 0.37 ± 0.03 μg/mL) and selectivity (selectivity index = 378.3) against the lung cancer cells; cisplatin, 5-fluorouracil, and an extract from the leaves of Taxus baccata L. (Taxaceae) were less cytotoxic and selective. Extracts from Cascabela thevetia (L.) Lippold (Apocynaceae), Frangula alnus Mill. (Rhamnaceae), Iberis ciliata subsp. contracta (Pers.) Moreno (Brassicaceae), Juniperus macrocarpa Sm (Cupressaceae), and Pancratium maritimum L. (Amaryllidaceae) also showed selective cytotoxicity (selectivity index > 10). Active extracts were also tested against a panel of cancer cell lines from a variety tissues. The plants identified in this work are potential sources of natural compounds with selective toxicity towards cancer cells.


Introduction
Despite the recent approval of new anticancer treatments, metastasis continues to be an incurable disease for most cancer patients. The limited efficacy of the existing therapies is reflected in the poor survival rates of patients diagnosed with the most common metastatic cancers [1]. For example, distant metastases occur in 57% of patients diagnosed with lung cancer, and only 6% of them survive 5 years after diagnosis [1]. The percentages are also low for other common metastatic cancers. The five-year relative survival rates for patients with distant metastases are 30% in prostate cancer, 28% in breast cancer, 14% in colorectal cancer, 27% in melanoma, 13% in renal cancer, 30% in ovarian cancer, 17% in cancers of the uterine corpus, 17% in cancers of the uterine cervix, 6% in bladder cancer, 5% in esophageal cancer, 3% in liver cancer, and 3% in pancreatic cancer [1]. Many patients with metastases do not overcome the disease despite surviving five years after diagnosis.
Understanding why the current treatments rarely cure patients with disseminated disease is important to discover better therapies. When one treats cancer cells with specific concentrations of the available anticancer drugs and examines the cells under the microscope, one generally observes a massacre. All cancer cells die in response to most treatments. However, these drugs also kill normal cells at similar concentrations. The consequence of this limited selectivity is that patients cannot receive the drug doses needed to kill all their cancer cells; such doses would also kill their normal body cells and would be lethal. Although oncology patients generally receive the maximum tolerated doses, these doses are usually insufficient to reach the drug concentrations required to eradicate their cancer cells. The surviving cancer cells continue to proliferate and eventually lead to a fatal outcome. Finding drugs with a high selectivity towards cancer cells is crucial to develop more effective treatments for patients with metastasis [2][3][4].
Several plants have provided useful drugs for the treatment of a variety of cancers, including lung cancer [5][6][7]. For example, the diterpene paclitaxel (isolated from the bark of Taxus brevifolia Nutt., Taxaceae) and its semisynthetic derivative docetaxel are FDA-approved drugs for the treatment of non-small-cell lung cancer [8,9]. Vinorelbine, a semisynthetic analog of the vinca alkaloids (isolated from Catharanthus roseus G. Don., Apocynaceae), is also approved for patients with this type of cancer [9]. Etoposide (a semi-synthetic analogue of the natural lignan podophyllotoxin, isolated from Podophyllum species, Podophyllaceae) and topotecan (an analogue of the quinoline alkaloid camptotethin, isolated from Camptotheca acuminata Decne, Nyssaceae) are approved for patients with small cell lung cancer. Due to the fact that several plants have provided useful anticancer agents, we recently used lung cancer cells and lung normal cells to evaluate the selective anticancer activity of 57 extracts from plants collected in Grazalema Natural Park (Andalusia, Spain) [10]. Using a similar experimental approach, we have evaluated the selective anticancer activity of 65 extracts from 45 new plants collected in several areas of Western Andalusia, and report the results in this article.

Results and Discussion
Due to the fact that patients with metastatic cancers need selective anticancer drugs, we have searched for potential sources of selective anticancer drugs in 45 plants growing in Western Andalusia, Spain. After collecting the plant material and preparing 65 extracts, we used lung cancer cells (A549) and lung normal cells (MRC-5) to evaluate their selective cytotoxicity with the MTT assay. A549 cells are epithelial cells and MRC-5 cells are fibroblastic cells. However, since normal epithelial cells are difficult to maintain and expand in culture, we selected this widely used normal cell line for our screening [11][12][13].
We exposed both cell lines for 72 h to at least five concentrations of the extracts and the anticancer drugs cisplatin and 5-fluorouracil (5-FU). Table 1 shows the botanical names in alphabetical order, the plant families and other pertinent information on the 45 plant species. It also shows an identification number for each extract, the IC50 value for each cell line, and the selectivity index. Dose-response curves for the 65 extracts and positive controls are provided in Figures 1-6; these curves allow for the visualization of their cytotoxic profile over a wide concentration range.        Table 1). The extract from the leaves of Tetraclinis articulata (Vahl) Mast. (58) (Figure 5) showed the most relevant activ-ity; it was more cytotoxic against the lung cancer cells (IC50 = 0.37 ± 0.03 µg/mL) than against the lung normal cells (IC50 = 129.5 ± 64.0 µg/mL), displaying a selectivity index of 378.3 ± 178.1. No significant differences were observed when the cytotoxicity of this extract was tested with the MTT assay and the resazurin assay ( Figure S1). Photographs of this plant and the plant material used to prepare the extract are shown in Figure S2. The extract from the leaves of Taxus baccata L. (Taxaceae) was also more cytotoxic against the lung cancer cells (IC50 = 0.86 ± 0.27 µg/mL) than against the lung normal cells (IC50 = 146.9 ± 87.8 µg/mL), displaying a selectivity index of 157.3 ± 110.6. Several extracts (e. g., 4, 6, 9, 11, 12, 17, 20, 23, 34, 35, 41, 42, 48, 49, 53) were cytotoxic but were not selective against the A549 lung cancer cells.
Extracts (10, 18, 37, 46, 57, and 58), and the anticancer drugs cisplatin and 5-fluorouracil, were tested with the resazurin assay against eleven additional cell lines: two leukemia cell lines, six cancer cell lines derived from solid tumors of different tissues (liver, colon, bone, cervix, prostate, and breast), and three genetically modified skin cell lines with increasing degrees of malignancy (Table 2 and Figure 7). Due to the fact that the leukemia cell lines grow in suspension, we determined cell viability with the resazurin assay. Unlike the MTT assay, this assay does not require removal of the culture medium. The extract from the leaves of Tetraclinis articulata (Vahl) Mast. (58) displayed the highest cytotoxic activity, with IC50 values between 0.2 and 2.1 µg/mL against seven of the eight cancer cell lines. No clear differences in cytotoxicity were found for any extract or compound in the three skin cell lines with increasing degrees of malignancy (BJ-hTERT, BJ-SV40T, and BJ-RASV12) [14] ( Table 2). Due to the fact that Tetraclinis articulata (Vahl) Mast. extract (58) showed a high cytotoxicity against several cancer cell lines, we acquired additional cancer cell lines from a variety of tissues to further evaluate the activity of the extract. Fourteen human cancer cell lines were treated with several concentrations of 58 for 96 h and cell viability was estimated with the resazurin assay. Results show that this extract was cytotoxic against all the cancer cell lines, with IC50 values in the range 4.3-4.9 µg/mL (Table 3).  (10, 18, 37, 46, 57, and 58), cisplatin, and 5-FU against six cancer cell lines derived from solid tumors and two acute promyelocytic leukemia cell lines. Cells were treated for 72 h, and cell viability was evaluated with the resazurin assay.
Several types of phytochemicals may be involved in the selective anticancer activity shown by the most active extracts. Cardiac glycosides may be responsible for the selective cytotoxicity shown by the extracts from the leaves of Cascabela thevetia (L.) Lippold (10) and the leaves of Digitalis purpurea L. (18). We, and others, have previously observed that plants with cardiac glycosides, and several cardiac glycosides (e.g., digitoxin), induce potent and selective cytotoxic effects against several types of cancer cells, including lung cancer cells [15][16][17][18][19][20][21][22][23]. Isoquinoline alkaloids may participate in the cytotoxicity and selectivity observed for the extract from the bulb of Pancratium maritimum L. (46) [24,25]. The cytotoxicity and selectivity of our extract from the leaves of Taxus baccata L. (57) were probably mediated by different taxane-type diterpenes, including paclitaxel [26,27]. Diterpenes (e.g., ferruginol and sandaracopimaric acid.), monoterpenes (e.g., limonene and carveol), and sesquiterpenes (e.g., β-caryophyllene and humulene) may participate in the cytotoxicity of the extract from the leaves of Tetraclinis articulata (Vahl) Mast. [28][29][30][31][32][33]. In summary, because patients with advanced cancers need selective anticancer drugs, we have searched for potential sources of selective anticancer drugs in a variety of plants.

Plant Material
All plants were collected by F. García between November 2012 and April 2013 in several areas of Sevilla, Cadiz, and Huelva (Andalusia, Spain). Collection was non-destructive and plant specimens (5-110 g) were carefully selected to avoid any damage that could affect the conservation of any species. A voucher specimen was deposited in the herbarium at the CITIUS II Celestino Mutis (Center for Research, Technology, and Innovation of the University of Seville). The botanical names, plant parts, and voucher specimen numbers are listed in Table 1. Collection coordinates are provided in Table 4.

Cell Viability Assays
Exponentially growing cells (3000-5000 cells per well) were seeded in 96-well plates and were allowed to grow for 24 h. The cells were then exposed to several concentrations of the extracts or the positive controls cisplatin and 5-fluorouracil. After a treatment period of 72 h, cell viability was estimated with the MTT assay or the resazurin assay [2,15]. Due to the fact that plant extracts contain compounds (e.g., polyphenols) that may interfere with these assays, cell culture medium was removed before conducting these assays in all adherent cell lines.
The MTT assay is based on the ability of viable cells to convert the MTT compound (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into an insoluble and purple formazan product. After an incubation period of the cells with the MTT and a solubilization step, the quantity of the colored product was measured with a plate reading spectropho-tometer. Dead cells are metabolically inactive and cannot produce the colored product. Briefly, after the 72 h treatment period, the medium was removed, the cells were washed with PBS, and 125 µL MTT (1 mg/mL in medium) was added to each well. The plates were incubated for 4 h to allow viable cells to transform the MTT compound into an insoluble formazan product. This insoluble compound was solubilized by adding 80 µL 20% SDS in 0.02M HCl to each well and by incubating the plates overnight at 37 • C. Finally, optical densities were measured at 540 nm on a multi-well plate spectrophotometer reader (Multiskan EX Labsystems, Vantaa, Finland).
The resazurin assay is a redox-based colorimetric or fluorometric assay based on the capacity of viable cells to reduce the blue compound resazurin into the pink, fluorescent and soluble product resorufin. The quantity of resorufin produced is proportional to the number of viable cells. After treatment period, medium was removed and 150 µL of resazurin (20 µg/mL in medium) was added to each well for 5-6 h. The absorbance was measured at 540 nm and 620 nm on a multi-well plate spectrophotometer reader (Multiskan EX Labsystems, Vantaa, Finland).
In both assays, cell viability was calculated as a percentage in relation to untreated cells from at least two independent experiments. IC50 values were calculated by linear regression analysis after selecting the two concentrations leading to cell viability values immediately above and below 50%. With this approach, IC50 values always are within the experimentally observed concentration range [15,[34][35][36][37]. After estimating cell viability and calculating IC50 values, results were expressed as mean ± standard error of the mean (SEM), and a t-test (paired, two tailed) was used for statistical analysis. A p-value > 0.05 was not considered statistically significant and was not represented by any symbol. A p-value ≤ 0.05 was considered statistically significant and was represented with an asterisk, two asterisks (p ≤ 0.01), or three asterisks (p ≤ 0.001). Since selectivity is the most relevant parameter to detect anticancer potential in vitro [3,4], selectivity indices were used to quantify this parameter. The selectivity index (S.I.) was calculated as the average of the IC50 value in the normal cell line (MRC-5) divided by the IC50 value in the cancer cell line (A549) obtained in each independent experiment [3].
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/plants10102193/s1. Figure S1: Comparison of cytotoxicity of Tetraclinis articulata (Vahl) Mast. extract (58) assessed by MTT and resazurin assays. A549 and MRC-5 cells were treated for 72 h.  Acknowledgments: Authors would like to thank to the biology and herbarium services of CITIUS-University of Seville for maintaining the cell lines and preserving the vouchers of the plants. They are especially grateful for the financial support from V Plan Propio (University of Seville). The graphical abstract was created with BioRender.

Conflicts of Interest:
The authors declare no conflict of interest.