Normal cells have higher extracellular pH (pHe) of 7.4 and lower intracellular pH (pHi) of 7.2. However, like other cancer cells, breast cancer cells has a reversed pH gradient. Cancer cells have lower pHe of ~6.7 - 7.1 and higher pHi of 7.4. MDA-MB-231 cells experience different cell growth, morphology, and cell metabolism in different exogenous pHs. The acidic pHe or increased pHi is necessary for directed cell migration.26 In this study, MDA-MB-231 cells, breast cancer cells, were incubated with various organic acids, and we monitored the cytotoxicity of organic acids with concentrations, and migration of MDA-MB-231 cells.
In Figure 1, ten organic acids were selected and tested for the cytotoxicity to MDA-MB-231 cells. Organic acids include valproic acid, aminocaproic acid, acetylsalicylic acid, maleic aid, dithiobutyric acid, dithiopropionic acid, succinic acid (SA), fumaric acid (FA), tartaric acid (TA), and citric acid (CA). The most common organic acids is carboxylic acid, which has the molecular formula R–COOH, where H is the acidic proton and R is an aliphatic or aromatic group. Based on the number of carboxylic (-COOH) group attached to an R group, organic acid can be classified into aliphatic monocarboxylic acid (valproic acid and aminocaproic acid), aliphatic dicarboxylic acid (succinic acid, fumaric acid, maleic acid, tartaric acid, dithiobutyric acid, and dithiopropionic acid), aliphatic tricarboxylic acid (citric acid), and aromatic acid (acetylsalicylic acid). Next, ten organic acids were tested for the cytotoxicity to MDA-MB-231 cells.
For the cytotoxicity, MDA-MB-231 cell, a metastatic human breast cancer cell line, were cultured in medium with or without organic acids. Cells (1x105 cells/well, 96 well plate) were treated with 20 mM organic acids (final concentration of organic acid in medium) for 24 hours. Based on the guideline of the International Organization for Standardization, ISO 10993-5 regarding the in vitro cytotoxicity tests, organic acids were considered toxic to cells if the percentage cell vitality is <70%, and non-cytotoxic if the percentage cell vitality is >70%. At this concentration (20 mM) and incubation time (24 hours), two organic acids (valproic acid, aminocaproic acid) were not toxic to MDA-MB-231 cell. There was no significant difference statistically between organic acid treatment and negative control groups. Other four organic acids (acetylsalicylic acid, maleic aid, dithiobutyric acid, and dithiopropionic acid) were relatively toxic to MDA-MB-231 cell at this condition. Cell viabilities of MDA-MB-231 cell treated by these four organic acids were between 20 % and 60 %, and cell viabilities in these organic acid groups was significantly lower than in the negative control group. The other four organic acids (succinic acid, fumaric acid, tartaric acid, and citric acid) are the most toxic groups and cell viabilities are less than 10 %. Based on in vitro cytotoxicity in Figure 2, the last four organic acids (succinic acid (SA), fumaric acid (FA), tartaric acid (TA), and citric acid (CA)) could be a new class of anticancer drug candidate, and the four organic acids were used for further experiments.
Organic acids have been examined to see if there is any role of organic acids in cancers. The citric acid cycle or tricarboxylic acid (TCA) cycle is a metabolic pathway to produce energy in mitochondria. The metabolic pathway is derived from the citric acid, and both succinate and fumarate are an intermediate in the citric acid cycle. Tartaric acid is a white, crystalline organic acid that also exists naturally in many fruits, and it is a dihydroxyl derivative of succinic acid. Increased level of organic acid can be found in cancer and it can promote cancer migration. Fumaric acid is found in high level around tumors,27 and cancer cell migration or cancer metastasis can be enhanced by succinic acid secreted by cancer.28 To the contrary, some other reports demonstrated that organic acid could interfere the tumor growth. Fumaric acid inhibits the solid growth of mouse tumor cells. 29 Citrate suppresses tumor growth in vivo using genetic tumor models of A549 cells, adenocarcinomic human alveolar basal epithelial cells.30 Different concentrations of organic acids could explain the contradictory effect on cancers.
Next, we investigated the effect of concentrations of four organic acids (SA, FA, TA, and CA) on the cell viability of MDA-MB-231. Cells were incubated with 3, 5, 10, 20, 30 and 40 mM of each organic acid for 24 hours, and the cell viability was measured by MTT assay.
Figure 3 demonstrated that the cell viabilities of all four organic acids to MDA-MB-231 cells were over 70 % at low concentrations below or equal to 5 mM. However, all four organic acids at higher concentrations than 10 mM caused a significant decrease in cell viability of MDA-MB-231 cells. For example, 10 mM of all four organic acids reduced the viability of MDA-MB-231 to lower than 50 % compared to the cell viability of control group (no treatment of organic acids).
MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) is widely used for the cell viability. Yellow MTT is reduced by succinate dehydrogenase system of active or live mitochondria to form an insoluble purple formazan. Some chemicals such as ascorbic acid (AA), retinol, or α-tocopherol can reduce the MTT in the absence of cells to change the color to purple.31, 32 Therefore, they could affect the cell viability of MTT assay (false positive). In Figure S1, organic acids were mixed with MTT solution, and the result demonstrated that only ascorbic acid (MTT + AA) changed the color of MTT to purple, which would affect the absorbance of MTT in the cytotoxicity assay. Fortunately, other four organic acids (SA, FA, TA, and CA) didn’t change the color of MTT, and the MTT assay is still a useful method to measure the cytotoxicity of four organic acids.
In order to understand the concentration or pH effect of organic acids on the cell viability of MDA-MB-231, we measured pH of medium when organic acids were dissolved in the medium. A pH meter was used to measure the acidity of medium with organic acids. The medium, DMEM, also included phenol red as a pH indicator. The color of phenol red becomes yellow below pH 6.8, and it is orange-red around physiological pH, pH 7.4, and it turns bright pink over pH 8.2.
In Figure 4A and 4C, MDA-MB-231 cells were treated with of four organic acids (SA, FA, TA, and CA). In Figure 4B and 4D, acidic medium was prepared by adding HCl to the medium, and cells were cultured in various pHs. Then, pH of each medium was measured by a pH meter, and images of cell morphology and cell density were captured using a phase-contrast microscope.
Figure 4A demonstrated that high concentrations of organic acids reduced pH, and increased the cytotoxicity. In Figure 4B, when pH was reduced by simply adding H+ to the medium, pH changes decreased the cell viability. The cells were stable in neutral pH ranges; however, cells were dead via apoptosis or necrosis at acidic pH ranges. The cell viability profiles of Figure 4A and 4B were very similar to each other. The result suggested that pH of organic acids was the major effect on cell viability rather than other chemical properties of organic acids. In Figure 4C and 4D, cell density and morphological changes of cells depended on pH rather than other chemical properties of organic acids.
In order to confirm the effect of pH on cell viability, organic acid medium was prepared by adding either 10 mM or 20 mM organic acids to medium. One set of medium (10 mM or 20 mM organic acid medium) was used without modification. In the other set of media (10 mM or 20 mM organic acid media), pH was adjusted to the physiological pH, pH 7.4 by adding 1 M NaOH to the medium. As shown in Figure 5, cytotoxicity of organic acid was increased at higher concentration (20 mM); however, the toxicity was alleviated when pH of 10 mM or 20 mM organic acid medium was adjusted to the physiological pH, pH 7.4. This result also supports that pH of organic acids is a dominant effect on cell toxicity rather than concentration.
Cell motility is one of key features of live cells. Cell migration is involved in many pathological processes of cancer such as metastasis and inflammation. Among the numerous ways to study cell migration, the transwell migration is one of the simplest. In the transwell cell migration assay, 24-well inserts from Corning were used. MDA-MB-231 cells with or without organic acids were placed in the upper chamber. After incubation for 24 hours at 37 °C, cells were fixed with formaldehyde and methanol, and they were stained with 10 % Giemsa stain. In Figure 6A, images of migrated cells on the upper chamber were taken.
Giemsa stain is a mixture of methylene blue, azure B, and eosin. Methylene blue is a cationic dye that binds the negatively charged components of the cells such as DNA in the nucleus, or RNA present in the cytoplasm. Azure B is a cationic dye and the major metabolite of Methylene blue. Eosin is anionic and negatively charged dye. It can react with positively charged amino acids in proteins nonspecifically. In a typical tissue, nuclei are stained blue, whereas the cytoplasm and extracellular matrix have varying degrees of pink staining. In order to enhance the contrast of cells (black spots) compared to the background, the images were converted to black and white, and the contrast and brightness were adjusted. The number of migrated cells in 6 randomly selected microscope fields were counted as shown in Figure 6B. Some cells were migrated and reached to the lower chamber, and they were also imaged in Figure 6C. Salicylic acid slightly reduced the migration of MDA-MB-231; however, there were no significant changes statistically in cell migration between control group and organic acid-treated groups.
We investigated the effect of organic acids on the spheroid growth of metastatic MDA-MB-231 breast cancer cells. 3D-cultured spheroid of MDA-MB-231 breast cancer cells were formed in low-cell-attachment Nunclon Sphera 96-well round-bottom plates. Dead cells were labeled with a red fluorescent dye, propidium Iodide (PI) (1 μg/mL) that was impermeable to the membrane of live cells. Morphology of cells and PI fluorescence (red color) of dead cells were displayed in fluorescence images. In Figure 7, MDA-MB-231 spheroid in medium (Figure 7a) was smaller than other MDA-MB-231 spheroids in medium with organic acids (Figure 7b-7e); however, cell density of MDA-MB-231 spheroid in medium was much higher. MDA-MB-231 spheroids in medium with organic acids were bulky, and dead cells were spread all over the spheroid. The result suggested that organic acids enhanced the cell toxicity and they inhibit cell growth and cell cycle.
In summary, we investigated how organic acids affected breast cancer cells. Ten organic acids including carboxylic acid (R–COOH) were selected and tested for the cytotoxicity to MDA-MB-231 cells. Among ten organic acids, four organic acids, succinic acid (SA), fumaric acid (FA), tartaric acid (TA), and citric acid (CA), showed more cytotoxicity to MDA-MB-231 cells, and four organic acids were used for further experiment.
Higher concentrations of four organic acids reduced pH, and increased the cytotoxicity. pH of organic acids was the major effect on cell toxicity, cell density, and morphology rather than other chemical properties of organic acids. The organic acids also affected the formation of MDA-MB-231 spheroids and toxicity of cells in the spheroid. The results in this research enhanced the understanding of pH effect of organic acids on MDA-MB-231 cancer cells.