Hydrogen phosphate selectively induces MDA MB 231 triple negative breast cancer cell death in vitro

Phosphate ions are the most abundant anions inside the cells, and they are increasingly gaining attention as key modulators of cellular function and gene expression. However, little is known about the effect of inorganic phosphate ions on cancer cells, particularly breast cancer cells. Here, we investigated the toxicity of different phosphate compounds to triple-negative human breast cancer cells, particularly, MDA-MB-231, and compared it to that of human monocytes, THP-1. We found that, unlike dihydrogen phosphate (H2PO4−), hydrogen phosphate (HPO42−) at 20 mM or lower concentrations induced breast cancer cell death more than immune cell death, mainly via apoptosis. We correlate this effect to the fact that phosphate in the form of HPO42− raises pH levels to alkaline levels which are not optimum for transport of phosphate into cancer cells. The results in this study highlight the importance of further exploring hydrogen phosphate (HPO42−) as a potential therapeutic for the treatment of breast cancer.

particularly via apoptosis. We attribute this effect to the fact that phosphate in the form of HPO 4 2− raises pH levels to alkaline levels which are not optimum for phosphate transport into cancer cells. Taken together, these results indicate the significance of further exploring HPO 4 2− as potential therapeutic for the treatment of breast cancer.

Results
Cytotoxicity of different phosphate compounds. In order to assess the effect of different phosphate compounds on the cell viability of MDA-MB-231 and THP-1 cells, three inorganic phosphates (NaH 2 PO 4 , Na 2 HPO 4 , and KH 2 PO 4 ) and three organic phosphates (adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP)) were tested. Both MDA-MB-231 and THP-1 cells were incubated with 20 mM of NaH 2 PO 4 , Na 2 HPO 4 , KH 2 PO 4 , ATP, ADP and AMP for 48 h, then assessed for their viability via MTT assay. As shown in Fig. 1, almost all phosphate-containing compounds were toxic (viability < 80%) to both cell types and their toxicity was more pronounced on THP-1 cells than it was on MDA-MB-231 cells. Interestingly, only Na 2 HPO 4 was more toxic to MDA-MB-231 cells than it was to THP-1 cells. In particular, the viability of MDA-MB-231 cells incubated with 20 mM Na 2 HPO 4 was 37.5% lower than that of THP-1 (relative viability of MDA-MB-231 was 54.1% while that of THP-1 was 86.6%). This indicates that the toxicity of Na 2 HPO 4 is selective to the cell type.
In order to understand the difference in the effect of the various phosphate compounds on the two cell types, the pH of the different phosphate solutions was measured. In Fig. 2, all phosphate compounds produced acidic solutions except Na 2 HPO 4 which produced a basic one. In supporting Fig. 1, the cytotoxicity of the different phosphate compounds was compared to controls at suitable pH (i.e. to cells incubated in media with no phosphate compound but having their media pH adjusted by either HCl or NaOH to a level nearly equal to that containing phosphate compound). The figure shows that pH is a factor contributing to the toxicity of Na 2 HPO 4 to MDA-MB-231 cells but is not the only one. This, as well as the fact that Na 2 HPO 4 is the only compound containing phosphate in the form of HPO 4 2− , were a drive to further explore Na 2 HPO 4 compound. In the medium, inorganic phosphate compounds are dissociated into ions. All phosphate-containing compounds reduced viability of both cell types significantly except Na 2 HPO 4 , which only reduced the viability of MDA-MB-231 cells but not that of THP-1 cells. Experiment was done in triplicate and the sample number for each replicate is 3. ) on viability of MDA-MB-231 and THP-1 cells, cells were incubated with different concentrations of Na 2 HPO 4 for 48 h then assessed for their viability via MTT or live/dead assay. Figure 3 (both MTT and live/dead) demonstrates that, at low concentrations, HPO 4 2− was more toxic to MDA-MB-231 cells than it was to THP-1 cells. Interestingly, for MDA-MB-231, even low concentrations (< 20 mM) caused a significant decrease in cell viability. For example, 5 mM Na 2 HPO 4 reduced the viability of MDA-MB-231 by almost 20%. However, for THP-1 cells, there was no significant decrease in the viability of cells incubated with 5 mM, 10 mM and 20 mM Na 2 HPO 4 compared to control cells. Only concentrations above 20 mM produced a significant decrease in viability. In order to examine the type of death induced by Na 2 HPO 4 on MDA-MB-231 cells, cells were treated with various concentrations of Na 2 HPO 4 for 48 h then stained by apoptosis/necrosis dyes. As seen in Fig. 4, results indicate that Na 2 HPO 4 induces both necrosis and apoptosis, with apoptosis being more pronounced (particularly at concentrations of 20 mM and 40 mM).

Scientific Reports
In order to confirm that the observed toxicity of Na 2 HPO 4 on MDA-MB-231 was induced by phosphate and not by sodium in Na 2 HPO 4 or by simple osmotic pressure, the toxicity of NaCl, sucrose and sodium bicarbonate (NaHCO 3 ) on MDA-MB-231 was assessed. In Fig. 5, none of the compounds caused a significant decrease in the viability of cells which indicates that the phosphate is the probable cause for the induced toxicity observed.
In order to explore the possible mechanisms of action in which HPO 4 2− induces its toxicity, the effect of HPO 4 2− on pH of the media was assessed. For that, phosphate solutions at different concentrations of Na 2 HPO 4 (5, 10, 20, 40, 80, 100 and 120 mM) were prepared in media then their pH measured. As seen in Fig. 6a, results indicate that as the concentration of HPO 4 2− increases, the pH of the solutions increases. These results are in contrast to those of H 2 PO 4 − , Fig. 6b, in which the pH decreases with increasing NaH 2 PO 4 concentration.
Tolerance of MDA-MB-231 and THP-1 cells to different pH levels. As HPO 4 2− was found to alter the pH of the media, the difference in the tolerance of MDA-MB-231 and THP-1 cells to different pH levels was examined. For this, cells were incubated in media solutions at pH values equal to 5, 6, 7, 8 and 9 for 48 h then their viability determined using MTT assay. As seen in Fig. 7, results demonstrated that both cell types were more tolerant to basic conditions than they were to acidic conditions, and that MDA-MB-231 cells were slightly more tolerant to pH fluctuations than THP-1 cells were.    ), which exists at alkaline pH 1 . Since triple negative breast cancer cells are known to require elevated amounts of phosphate to meet their metabolic demands, failure to efficiently transport phosphate into cancer cells alters their functioning and probably leads to cellular death 30,31 . This study demonstrates that phosphate in its monoprotic form (HPO 4 2− ) causes significant death in MDA-MB-231 cells but not in THP-1 cells, unlike phosphate in its diprotic form.
Since phosphate in general is a tryptotic acid and, thus, has different physiological forms according to the pH range in which it is found, the effect of adding Na 2 HPO 4 to MDA-MB-231 and THP-1 cells and then adjusting the pH to a physiological one (pH = 7.4) was also explored 32 . As seen in Fig. 8, results demonstrate that adjusting the pH to physiological range after adding HPO 4 2− slightly enhanced viability of MDA-MB-231 cells to near significant values but had no effect on viability of THP-1 cells. The level of enhancement in viability was also pH dependent. These results support the hypothesis that cancer cells seem to favor diprotic form of phosphate than monoprotic one.

Discussion
In this study, we demonstrated the potential of HPO 4 2− to contribute to the treatment of triple negative breast cancer cells. This is quite significant as there is no effective therapy for triple-negative breast cancer to date and it continues to acclaim the lives of its patients 33,34 . P i is emerging as a crucial signaling molecule capable of altering signal transduction pathways, gene expression, and protein abundance in many cell types 9,35-37 . Interestingly, the effect of phosphate on cells is not universal; it varies according to the cellular type and the cellular background. In this study, we showed via MTT and live/dead assays that phosphate, in the form of HPO 4 2− , induces cell death in a specific type of triple negative breast cancer cells, namely MDA-MB-231 cells, but not in monocytes (THP-1 cells). This is done via both apoptosis and necrosis with apoptosis being more pronounced. In other studies, P i was shown to increase cell proliferation in some cell types, such as preosteoblastic cells, lung cells, and epidermal JB6 cells while decreasing proliferation in other cell types such as human osteosarcoma U2OS cells and MDA-MB-231 cells 3,19,23,23,35,[38][39][40][41] . In addition, P i was shown to induce apoptosis in MO6-G3 odontoblast-like cells 42 . Interestingly, Spina et.al showed that Pi inhibits proliferation in MDA-MB-231 cells but not in MCF-7 breast cancer cells, which are not "triple negative" and express estrogen and progesterone receptors 14 . Taken together, these results strongly suggest that Pi has a discrete effect on cells depending on their type and their background. www.nature.com/scientificreports/ In this study, we also show that the specific type of phosphate available to the cells has a great influence on its response to it. In particular, hydrogen phosphate causes significant death in MDA-MB-231 cells but not in THP-1 cells, unlike dihydrogen phosphate. Figure 9 is a schematic showing the observed effect of hydrogen phosphate and dihydrogen phosphate on the viability of both MDA-231 cells and THP-1 cells. We hypothesize that this effect is related to the fact that MDA-MB-231 cells require high amounts of phosphate to meet their metabolic needs but are unable to attain it due to the inability of phosphate transporters to carry phosphate into the cells when it is in the form of HPO 4 2−43 . Studies show that cancer cells depend on several transporters to transport phosphate into the cells, but the key ones are sodium dependent transporters and hydrogen dependent transporters, both of which are highly overexpressed in MDA-MB-231 cells 1,2,28,29 . Interestingly, sodium dependent phosphate transporters have higher affinity for dihydrogen phosphate (H 2 PO 4 − ) than they do to hydrogen phosphate (HPO 4 2− ) 1 . In addition, hydrogen dependent transporters tend to transport phosphate more efficiently in acidic conditions than they do in basic conditions due to the greater availability of protons (H+) in acidic environments compared to basic environments 2 . These findings are, in fact, in consistence with the results obtained from this study in which hydrogen phosphate was more toxic to MDA-MB-231 cells than dihydrogen phosphate. www.nature.com/scientificreports/ Finally, we suggest that hydrogen phosphate is a suitable candidate for further investigation as a therapeutic for the treatment of triple negative breast cancer, due to its selective ability to induce cell death in cancer cells but not in immune cells. This is quite crucial as effective cancer therapeutics require high antitumor activity and minimal toxicity to normal tissues. We emphasize here that the effect of phosphate on cells is dependent on their type. In this study, phosphate, in the form of HPO 4 2− was shown to induce cell death in MDA-MB-231 cells but may not have the same effect in other breast cancer cell types. Therefore, future work would include examining the effect of hydrogen phosphate on various cell types (non-cancerous cells and non-triple negative breast cancer cells) and at various concentrations and studying the effect of hydrogen phosphate using in vivo models.
All cells were incubated at 37 °C and 5% CO 2 .  www.nature.com/scientificreports/ Cytotoxicity of sodium phosphate dibasic. To further examine the effect of phosphate in the form of HPO 4 2− on MDA-MB-231 cells and on THP-1 cells, cells were incubated with different concentrations of Na 2 HPO 4 for 48 h then their viability assessed via both MTT and live/dead assays. In brief, cells were seeded in 96 well plates at a concentration of 6 × 10 5 cells/mL and volume of 100 µL. Cells were then incubated for 24 h at 37 °C and 5% CO 2 . After 24 h of incubation, Na 2 HPO 4 was added to cells at variable concentrations (5,10,20,40,80, 100 and 120 mM) while maintaining a total volume of 200 µL for each sample. Cells with 200 µL media and no Na 2 HPO 4 were kept as a control. Cells were then incubated for an additional 48 h. After 48 h, MTT or live/dead (Thermo Fisher Scientific, Waltham, Massachusetts, USA) were added to cells. Cells treated with MTT were subsequently incubated for extra 4 h. After 4 h, the absorbance of each sample was measured and subsequent cellular viability determined. For cells treated with live/dead, cells were incubated for 15 min and then imaged under a fluorescence microscope (Zeiss AxioCam HRm, Carl Zeiss, Oberkochen, Germany) at 10× magnification where live cells were stained green by Calcein AM and dead cells were stained red by ethidium homodimer-1. Percent cell viability was calculated as: Percentage cell viability = (number of live cells (green))/ (number of live cells (green) + number of dead cells (red)) × 100. Cell counting was performed via ImageJ analysis software 44,45 .

Cytotoxicity of different phosphate compounds.
To confirm that any observed effects are due to phosphate and not to osmotic pressure or to sodium in Na 2 HPO 4 , MTT assay was performed to quantify the toxicity of sodium and the effect of osmotic pressure. In particular, cells were incubated with 20 mM, 40 mM, 60 mM sucrose to examine the effect of osmotic pressure and with 20 mM, 40 mM NaCl and 20 mM sodium bicarbonate to examine effect of sodium.
To determine the mechanism of cell death induced by Na 2 HPO 4 on MDA-MB-231 cells, cells were incubated with Na 2 HPO 4 for 48 h and then treated with Apoptosis/Necrosis dyes (abcam, Cambridge, UK) following the manufacturer's recommendations. Cells were then imaged using a fluorescence microscope. The images of fluorescent cells were taken at a specific z-plane and the background noise was removed during image analysis via "Noise Tolerance" option in ImageJ.

Measurement of pH.
In order to investigate the possible ways in which phosphate influences MDA-MB-231 and THP-1 cell survival, the pH of the resultant solutions (after adding different phosphate compounds and after adding different concentrations of Na 2 HPO 4 and NaH 2 PO 4 ) was measured using a pH meter (ORION STAR A211, Thermo Scientific).

Tolerance of MDA-MB-231 and THP-1 cells to different pH levels.
To study the tolerance of MDA-MB-231 and THP-1 cells to different pH levels, cells were incubated for 48 h in media having pH levels ranging from 5 to 9 then their viability was assessed using MTT assay. In brief, MDA-MB-231 and THP-1 cells were seeded in 96 well plates at a concentration of 6 × 10 5 cells/mL and incubated for 24 h at 37 °C and 5% CO 2 . After 24 h of incubation, the old media was replaced by new media having its pH adjusted by HCl (Sigma-Aldrich) or NaOH (Sigma-Aldrich) to obtain a specific pH; either 5, 6, 8 or 9. Media without any addition of acid or base was kept as a control (pH 7.2). The cells were incubated for 48 h. After 48 h, MTT was added to cells and their viability was quantified.
Cytotoxicity of sodium phosphate dibasic at physiological pH. To study the toxicity of Na 2 HPO 4 on MDA-MB-231 and THP-1 cells at physiological pH, cells were incubated for 48 h with 20 mM sodium phosphate dibasic (Na 2 HPO 4 ) solutions having their pH adjusted to 7.4 then their viability was assessed via MTT assay. In brief, cells were seeded in 96 well plates at a concentration of 6 × 10 5 cells/mL and volume of 100 µL. Cells were then incubated for 24 h at 37 °C and 5% CO 2 . After 24 h of incubation, 20 mM Na 2 HPO 4 at pH 8.1 and at pH 7.4 were added to the cells while maintaining a total volume of 200 µL for each sample. Two controls were maintained for this experiment: cells kept in 200 µL media at pH 7.4 (negative control) and cells kept in 200 µL media having its pH adjusted to 8 or 8.5 by NaOH (Sigma-Aldrich) (positive control). The cells were incubated for 48 h. After 48 h, MTT was added to cells and their viability was quantified.

Data analysis.
All experiments were done in triplicates with the sample number in each replicate being 3.
The data is presented as mean ± standard error. Student t-test was performed to determine significance. P < 0.05 was regarded as significant (*), P < 0.01 was regarded as highly significant (**) and P < 0.001 was regarded as very highly significant (***). www.nature.com/scientificreports/