Evaluating medicinal plants for anticancer properties: testing plant extracts for cytotoxicity

Evaluating medicinal plants for anticancer properties: testing plant extracts for cytotoxicity. Vadym Trokhymchuk, Carlos Planchart, Andrea Peterson, Adriana Reytor, Dora Pilar Maul, Maria Pina, Luis C. Fernandez-Torres, Alexis Tapanes-Castillo (E-mail: atapanes-castillo@stu.edu) a School of Science, St. Thomas University, 16401 NW 37 Avenue, Miami Gardens, FL 33054 b InterAmerican Campus, Miami-Dade College, 627 SW 27 Avenue, Miami, FL 33135 . Graphical Abstract


Introduction
Treating cancers requires an integrative approach, utilizing multiple therapies that complement one another. Cancer development and progression has been described as exhibiting the following characteristics: (1) maintained proliferative signaling, (2) evasion of growth suppressors, (3) genomic instability and mutation, (4) replicative immortality, (5) avoidance of cell death, (6) tumor-promoting inflammation, (7) activation of invasion and metastatic pathways, (8) induction of angiogenesis, (9) reprogramming of cellular metabolism, and (10) escape from immune destruction [1]. Plants traditionally used for medicinal purposes contain a rich repository of bioactive compounds, which have the potential to therapeutically target these features of cancer biology [2].  [3][4][5][6][7][8][9][10]. Dimethyl sulfoxide (DMSO) was added to extracts at the concentrations listed for the control (Table 1) to improve solubility and cellular internalization. Extracts were then filter-sterilized and diluted in media as described in Table 1. Concentrations varied between extracts because the aim was to maximize extract concentration, not to test uniform extract concentrations.

Materials and Methods
Cells were first cultured for 48 hours and then treated with plant extracts for 72 hours. Methylthiazol tetrazolium (MTT) assays were conducted to evaluate cytotoxicity. Culture media was replaced with RPMI1640 (without phenol red), 10% fetal bovine serum, and 0.5 mg/mL MTT. Formazan crystals were solubilized in 0.1 N HCl (diluted in isopropanol). Samples were read on a Synergy H1 (BioTek) plate reader set to 570 nm with a 630 nm reference background subtraction.
Each extract was tested using 5-16 replicates in one or two independent experiments. Data from extracttreated cells were compared to that obtained from untreated controls with the same DMSO concentration. Absorbance values were averaged and normalized to controls. Standard deviations of normalized values were calculated. Two-sample, two-tail t-tests assuming unequal variances were utilized to calculate p-values. In the case of multiple experiments, the largest p-value was reported. Figure 1 summarizes preliminary data regarding the effect of plant extracts on MCF7 breast cancer cells. Extracts were not cytotoxic at the concentrations tested. The greater the absorbance, the higher the concentration of formazan, a purple product generated in live cells by mitochondrial succinate dehydrogenase (SDH) enzyme reduction of MTT. Variability in the data, especially evident in the larger error bars observed at higher values, are primarily attributed to the challenge of consistently solubilizing higher formazan concentrations with manual trituration.

Results and Discussion
Wells treated with extracts from each type of plant had significantly higher SDH activity during the MTT assay than untreated wells (Fig. 1). This activity is proportional to the number of live cells in a well, which is related to cell survival and cellular proliferation. Hence, overall the extracts reduced cell death. These results are not surprising given that extracts from these plants have been reported to have medicinal effects [reviewed in [3][4][5][6][7][8]. Moreover, recent studies suggest several of the extracts demonstrate antioxidant activity in chemical reactions [3][4][5][6][7][8]. In general, antioxidants improve cell viability, including that of cancer cells, which tend to exhibit elevated levels of reactive oxygen species due to metabolic and signal transduction aberrations related to tumorigenesis [11]. Antioxidants provided by the plant extracts could reduce electron leakage during mitochondrial respiration and superoxide formation, increasing the number of live cells in treated samples.

Conclusions
In summary, plant extracts derived from muscadine, scarlet bush, Brazilian pepper tree, anamú, moringa, guanábana, oyster plant, and Okinawa spinach were not cytotoxic to MCF7 breast cancer cells. These findings are relevant, as they indicate the listed concentrations can be used in other assays to study potential anticancer properties present in the plant extracts. Future experiments will test how the plant extracts affect invasion-associated processes, such as cell migration, cell adhesion, and cell aggregation in breast cancer cells, as well as other cancer cell types.