Edited by: Jerry Edward Chipuk, Mount Sinai School of Medicine, USA
Reviewed by: Gavin Mc Stay, Columbia University, USA; Paulo J. Oliveira, Center for Neuroscience and Cell Biology, Portugal
*Correspondence: Fred Saad, Department of Surgery, Hôpital Notre-Dame, 1560 Sherbrooke East, Montreal, QC, Canada H2L 4M1. e-mail:
†Blandine Betton and Philippe O. Gannon have contributed equally to this work.
This article was submitted to Frontiers in Molecular and Cellular Oncology, a specialty of Oncology.
This is an open-access article subject to a non-exclusive license between the authors and Frontiers Media SA, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and other Frontiers conditions are complied with.
Inositol hexakisphosphate (IP6) is a phytochemical that exhibits promising anti-tumoral properties against several mouse, rat, and human cancer cell lines including prostate cancer cells (Vucenik and Shamsuddin,
Effects on tumor cell proliferation, survival, and angiogenesis, were documented as being key anti-tumoral properties of IP6 against prostate cancer cells. Initial studies in PC-3 cells demonstrated that IP6 inhibits the growth and promotes the differentiation of PC-3 cells (Shamsuddin and Yang,
In prostate cancer mice models, IP6 has documented
In terms of intracellular signaling, IP6 affects several pathways. IP6 decreases phospho-AKT (S473) in DU145 cells (Jagadeesh and Banerjee,
As IP6 is generally administered orally through the diet, typically in drinking water, IP6 must be buffered to a neutral pH before ingestion. IP6 salts are commercially available in several forms, which when reconstituted in water result in widely different pH. Typical formulations of the IP6 salt are basic and when reconstituted in water result in a solution at pH 12, which is not apt for consumption. The goal of this study was thus to validate whether the previously documented anti-tumoral properties of IP6 were modulated by the pH. Our results demonstrate that IP6 at pH 12 had different effects than IP6 solutions at pH 7 or pH 5. We conclude that the effect of pH should be carefully monitored when evaluating the cytotoxic and anti-cancer properties of IP6 and possibly other phytochemicals.
PC-3 and LNCaP cells were obtained from ATCC (Rockville, MD, USA). Myo-Inositol hexakisphosphate dodecasodium salt (IP6; US Biological, Swampscott, MA, USA) was diluted in water as a 100-mM stock solution. The IP6 stock solution was at pH 12. The pH was adjusted to pH 5 or pH 7 by addition of 1 N hydrochloric acid (HCl). It required 6.4 and 10.9 ml of 1 N HCl to buffer 100 ml of 100 mM IP6 pH 12 to pH 7 and pH 5, respectively. Antibodies recognizing phospho-AKT (S473; cat# 9271), phospho-PDK1 (S241; 3438) and phospho-ERK (T202/Y204; cat# 9106L) were obtained from Cell Signaling (Danvers, MA, USA). Antibodies detecting PARP full-length (sc7150), PARP 85 kDa (cat# 9541), PARP 25 kDa (cat# 32064) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Cell Signaling and Abcam (Cambridge, MA, USA), respectively. The anti-Ran antibody (sc1146) and the horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibodies recognizing actin (ab-6278) and GAPDH (ab-9485) were obtained from Abcam (Cambridge, MA, USA).
The WST-1 metabolic assay was done as previously described by our group (Diallo et al.,
PC-3 cells were plated at 15,000 cells/ml in 6-well plates, allowed to adhere overnight and then treated with 2 mM IP6 at pH 5, pH 7, or pH 12 or the equivalent volumes of pH-adjusted H2O controls over a period of 72 h. At indicated times cells were trypsinized, combined with non-adherent cells and counted using a cell counter (Casy, Roche Innovatis, Germany).
PC-3 cells were plated at 200,000 cells/ml in 6-well plates. PC-3 cells were exposed to H2O or 2 mM IP6 at the various pH for 8, 24, 48, and 72 h. Adherent cells were trypsinized, combined with non-adherent cells, stained with propidium iodide (PI), and analyzed by flow cytometry. All experiments were repeated at least three times.
Western blots were performed as previously described by our group (Diallo et al.,
The mitochondrial assay was done as previously described by our group (Diallo et al.,
Data are expressed as mean ± SD. Comparisons between the IP6 or H2O controls at the various pH were performed using one-way ANOVA followed by
We first evaluated the effect of the pH of IP6 on the metabolic activity of PC-3 cells. The cells were treated with either increasing doses of IP6 at pH 5, pH 7, and pH 12 or the corresponding volumes of H2O also at pH 5, pH 7, and pH 12. IP6 at pH 5 (2.5, 4, and 5 mM) and pH 12 (4 and 5 mM) induced a statistically significant reduction in the metabolic activity of PC-3 cells (Figure
We then evaluated the IC50 and IC75 for the various IP6 solutions. Due to the absence of a significant reduction in the metabolic activity following treatment with IP6 at pH 7 no IC50 could be calculated. The IC50 for IP6 at pH 5 was 2.39 ± 0.19 mM and 2.97 ± 0.45 for IP6 at pH 12 (Table
[] at |
[] at |
|
---|---|---|
IP6 pH 5 | 2.39 ± 0.19 | 3.96 ± 0.60 |
IP6 pH 7 | N/A | N/A |
IP6 pH 12 | 2.97 ± 0.45 | 4.04 ± 0.64 |
Finally, we inquired whether the addition of small volumes (60 μl) of IP6 or buffered H2O solutions could significantly change the pH of the culture media (Figure
We next evaluated the impact of pH on the anti-proliferation action of IP6. As previously described, PC-3 cells were treated with 2 mM IP6 solutions or with the corresponding volumes of buffered H2O. We chose the 2-mM IP6 dose in line with other studies by our group (Diallo et al.,
We then assessed whether the reduction in metabolic activity and in cellular proliferation (Figure
G1 | S | G2/M | Sub-G1 | |
---|---|---|---|---|
Ctrl | 53.56 ± 2.01 | 9.23 ± 1.43 | 25.96 ± 0.86 | 0.51 ± 0.09 |
MG132 | 54.10 ± 2.55 | 7.86 ± 1.13 | 27.76 ± 4.33 | 0.59 ± 0.08 |
H2O pH 5 | 53.18 ± 2.15 | 11.00 ± 2.60 | 25.67 ± 1.33 | 0.45 ± 0.09 |
H2O pH 7 | 53.53 ± 1.91 | 11.23 ± 2.78 | 25.72 ± 1.39 | 0.41 ± 0.08 |
H2O pH 12 | 53.15 ± 2.53 | 10.10 ± 1.75 | 25.72 ± 1.31 | 0.71 ± 0.25 |
IP6 pH 5 | 53.95 ± 1.85 | 10.88 ± 2.70 | 25.80 ± 0.86 | 0.61 ± 0.10 |
IP6 pH 7 | 53.63 ± 2.62 | 10.41 ± 1.48 | 26.03 ± 0.58 | 0.68 ± 0.13 |
50.24 ± 3.25 | 8.85 ± 1.80 |
By Western blot, we then studied whether Poly (ADP-ribose) polymerase 1 (PARP-1), was cleaved following IP6 treatment, as an indication of apoptosis induction. Following a 24-h stimulation with 2 mM IP6 at pH 12, we observed a most complete disappearance of full-length PARP-1 (Figure
We also noticed that the effects observed by IP6 at different pH varied according to the cell line used in the experiment. Using the hormone-sensitive LNCaP cell line treated with the various IP6 solutions, we observed an elevated expression of the cleaved-PARP-1 25 kDa fragment following exposure to IP6, something that was not observed with the PC-3 cell line (Figure
The reduction in metabolic activity, cellular proliferation combined with the abundance of sub-G1 PC-3 cells lead us to investigate mitochondrial depolarization or mitochondrial outer membrane permeation (MOMP), another event associate with apoptotic cell death. Similar to PARP cleavage (Kaufmann et al.,
Finally, we then evaluated whether the pH of the IP6 solution could differentially affect the phosphorylation status of intracellular effectors documented to be modulated by IP6. We detected a clear decrease in the phosphorylation status of AKT (S473) and PDK1 (S241) following treatment with IP6 at pH 12 (Figure
Numerous publications discuss the anti-cancer properties of IP6 in
The results presented in this study confirmed that, compared to pH-adjusted control H2O solutions, IP6 at any pH reduced the metabolic rate and the proliferation of hormone-refractory PC-3 cells. However, it was the IP6 solutions at pH 5 and pH 12 that offered the most significant inhibitory potential on the metabolic rate of PC-3 cells and the IP6 at pH 12 that most significantly
The intriguing results observed in our study concerns accumulation of sub-G1 PC-3 cells following treatment with IP6 at pH 12. Whereas short-lived and low level MOMP is observed following treatment with both water pH 12 and IP6 pH 12, only IP6 pH 12 leads to increased cytotoxicity, acute sub-G1 cell accumulation, and reduced cell growth. We must therefore conclude that the MOMP observed in both cases is not linked with apoptosis but may instead be an artifact induced by basic pH. The observation of sub-G1 cells only in the IP6 pH 12-treated cells (and not at neutral or acidic pH) is nonetheless intriguing. To explain this phenomenon, we need to consider the fact that the pI/cell cycle assay sub-G1 peak actually measures cells in which DNA has been fragmented and lost from the cells due to the fixation procedure. While DNA fragmentation occurs at late stages of apoptosis (e.g., after 24 h with MG132) it can also occur following extensive DNA-damage via double-strand (DS) breaks. Note that the IP6 sub-G1 is maximal at 8 h then progressively decreases. This could suggest that DNA DS breaks are being induced by IP6 pH 12 early on then progressively repaired. We would further predict that such DS breaks would occur preferentially in the G2/M phase of the cell cycle, as the fraction of cells in this phase inversely correlates with the sub-G1 peak. This idea is particularly interesting considering that in the literature, endogenous IP6 normally found in cells (which should be at ∼pH 7 as opposed to pH 12) has been found to stimulate non-homologous end-joining (NHEJ) by binding Ku70/80. It is tempting to speculate that IP6 at pH 12 inhibits rather that stimulates repair via NHEJ, leading to accumulation of DS breaks in cells undergoing G2/M transition. As the pH/ionic balance eventually re-equilibrates following IP6 pH 12 treatment (see Figures
One mechanism that has been proposed to mediate the chemopreventive abilities of IP6 activity is through its role as an anti-oxidant. The negatively charged phosphates in position 1, 2, and 3 constitute a unique (axial–equatorialaxial) conformation that confers anti-oxidant properties to IP6 by chelating Fe3+ and preventing Fe3+-catalyzed hydroxyl radical formation (Graf et al.,
Since the pH can change drastically as the molecule travels through the gastro-intestinal tract, our findings have implications for
In conclusion, our work demonstrates that the pH of the IP6 solution must be taken in consideration when evaluating the anti-tumoral properties of this phytochemical. We demonstrate significant differences in the activity of IP6 depending on its pH on the metabolic activity, cell proliferation, and cell death of PC-3 cells.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
G1 | G2/M | |||||||
---|---|---|---|---|---|---|---|---|
8 h | 24 h | 48 h | 72 h | 8 h | 24 h | 48 h | 72 h | |
Ctrl | 53.56 ± 2.0 | 55.11 ± 2.3 | 58.15 ± 4.8 | 56.07 ± 3.4 | 25.96 ± 0.9 | 25.28 ± 4.0 | 24.80 ± 4.0 | 27.14 ± 4.0 |
MG132 | 54.10 ± 2.6 | 63.22 ± 3.5 | 55.33 ± 6.6 | 33.12 ± 3.5 | 27.76 ± 4.3 | 14.15 ± 2.6 | 10.70 ± 3.2 | 11.62 ± 3.8 |
H2O pH 5 | 53.18 ± 2.2 | 55.06 ± 2.3 | 59.76 ± 4.6 | 57.35 ± 2.9 | 25.67 ± 1.3 | 24.17 ± 2.9 | 24.31 ± 3.1 | 27.04 ± 2.2 |
H2O pH 7 | 53.53 ± 1.9 | 54.42 ± 2.1 | 58.29 ± 4.8 | 55.01 ± 3.0 | 25.72 ± 1.4 | 25.18 ± 3.8 | 23.78 ± 4.0 | 28.46 ± 3.5 |
H2O pH 12 | 53.15 ± 2.5 | 52.22 ± 2.0 | 56.64 ± 5.3 | 55.73 ± 2.8 | 25.72 ± 1.3 | 26.66 ± 3.9 | 24.83 ± 3.6 | 27.35 ± 3.7 |
IP6 pH 5 | 53.95 ± 1.8 | 56.71 ± 2.2 | 58.13 ± 4.0 | 56.89 ± 3.1 | 25.80 ± 0.9 | 23.94 ± 3.1 | 24.33 ± 2.7 | 28.43 ± 2.1 |
IP6 pH 7 | 53.63 ± 2.6 | 57.72 ± 2.8 | 58.70 ± 5.1 | 56.14 ± 2.6 | 26.03 ± 0.6 | 24.03 ± 3.5 | 25.11 ± 3.7 | 26.03 ± 2.5 |
IP6 pH 12 | 50.24 ± 3.2 | 55.29 ± 2.8 | 56.60 ± 3.5 | 52.48 ± 2.7 | 22.29 ± 1.9 | 26.38 ± 3.3 | 26.69 ± 3.1 |
S | Sub-G1 | |||||||
---|---|---|---|---|---|---|---|---|
8 h | 24 h | 48 h | 72 h | 8 h | 24 h | 48 h | 72 h | |
Ctrl | 9.23 ± 1.4 | 7.79 ± 0.8 | 6.73 ± 0.7 | 7.89 ± 1.7 | 0.51 ± 0.1 | 0.56 ± 0.1 | 0.71 ± 0.2 | 0.52 ± 0.1 |
MG132 | 7.86 ± 1.1 | 6.00 ± 0.6 | 5.19 ± 0.9 | 5.82 ± 1.0 | 0.59 ± 0.1 | 2.37 ± 0.7 | 20.28 ± 3.4 | 40.13 ± 7.5 |
H2O pH 5 | 11.00 ± 2.6 | 8.15 ± 0.3 | 6.99 ± 0.8 | 8.23 ± 1.7 | 0.45 ± 0.1 | 0.74 ± 0.2 | 0.58 ± 0.1 | 0.34 ± 0.1 |
H2O pH 7 | 11.23 ± 2.8 | 8.30 ± 0.7 | 7.10 ± 1.6 | 8.44 ± 2.1 | 0.41 ± 0.1 | 0.78 ± 0.1 | 0.65 ± 0.1 | 0.37 ± 0.0 |
H2O pH 12 | 10.10 ± 1.7 | 8.70 ± 0.8 | 6.72 ± 1.0 | 7.24 ± 1.8 | 0.71 ± 0.2 | 0.59 ± 0.2 | 0.73 ± 0.1 | 0.35 ± 0.0 |
IP6 pH 5 | 10.88 ± 2.7 | 7.64 ± 1.0 | 8.10 ± 1.3 | 8.81 ± 2.4 | 0.61 ± 0.1 | 1.33 ± 0.2 | 0.87 ± 0.2 | 0.53 ± 0.2 |
IP6 pH 7 | 10.41 ± 1.5 | 7.92 ± 0.9 | 7.51 ± 1.0 | 8.36 ± 1.8 | 0.68 ± 0.1 | 1.41 ± 0.3 | 0.92 ± 0.1 | |
IP6 pH 12 | 8.85 ± 1.8 | 8.29 ± 1.0 | 7.71 ± 1.1 | 8.32 ± 1.8 |
The authors would like to thank laboratory members for helpful discussions. We are grateful to Nathalie Delvoye for her technical support. This work is supported by the René Malo Initiative of the Institut du cancer de Montréal and the Fondation Sybila Hesse. Fred Saad holds the Université de Montréal Chair in Prostate Cancer Research. Philippe O. Gannon, Ismaël Hervé Koumakpayi, and Jean-Simon Diallo receive support from the Fonds de la Recherche en Santé du Québec and Defi Canderel.