Inositol hexakisphosphate blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting the Akt/NF
B-mediated cell survival pathway
Sandra Ferry1,
Miho Matsuda1,
Hiroki Yoshida2 and
Masato Hirata1,3
1 Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science and Station for Collaborative Research, Kyushu University, Fukuoka 812-8582, Japan and
2 Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
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Abstract
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It has been reported that inositol hexakisphosphate (InsP6, phytic acid), a natural product, has an anticancer role. However, there is inadequate information regarding the mechanism by which InsP6 exerts anticancer actions, and the effect requires relatively high concentration of the agent, both of which hinders the usage of InsP6 as an anticancer drug. In the present study, we investigated the mechanism by which InsP6 acts as an anticancer agent, and tried to reduce the concentration of effective InsP6. Treatment of HeLa cells with InsP6 at 1 mM induced apoptosis, as assessed by counting the cell number, and by Hoechst and TUNEL staining. This is probably mediated by intracellular InsP6 itself and/or the dephosphorylated forms of metabolized InsP6, because incubation of HeLa cells with [3H]InsP6 produces dephosphorylated forms such as InsP4 and InsP5. Induction of apoptosis by InsP6 was examined in two ways: inhibition of cell survival signaling and direct induction of apoptosis. Treatment of HeLa cells with tumor necrosis factor (TNF) or insulin stimulated the Akt-nuclear factor
B (NF
B) pathway, a cell survival signal, which involves the phosphorylation of Akt and I
B, nuclear translocation of NF
B and NF
B-luciferase transcription activity. InsP6 blocked all these cellular events, but phosphatidylinositol 3-kinase activity was not affected. As well as inhibiting the Akt-NF
B pathway, InsP6 itself caused mitochondrial permeabilization, followed by cytochrome c release, which later caused activation of the apoptotic machinery, caspase 9, caspase 3 and poly (ADP-ribose) polymerase. When InsP6 was applied together with histone, the effective concentration to induce apoptosis was ~10-fold lower. These results revealed that extracellularly applied InsP6 directly activates the apoptotic machinery as well as inhibits the cell survival signaling, probably by the intracellular delivery followed by a dephosphorylation.
Abbreviations: 
m, change in mitochondrial membrane potential; I
B, inhibitor of NF
B; NF
B, nuclear factor
B; PARP, poly (ADP-ribose) polymerase; PH domain, pleckstrin homology domain; PI3K, phosphatidylinositol 3-kinase; PtdInsPx and InsPx, phosphatidylinositol phosphate and inositol phosphate, respectively (position of phosphates is indicated in the parentheses and `x' indicates the number of phosphates); TNF, tumor necrosis factor.
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Introduction
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Inositol hexakisphosphate (InsP6) is present in plants, particularly in cereals and legumes, at concentrations ranging from 0.4 to 6.4%. In plants, InsP6 exists as a salt with monovalent and divalent cations such as Ca2+, Mg2+ and K+ (1,2). Inositol, InsP6 and lower inositol phosphates (InsP15) are all present as intracellular molecules in mammalian cells as well (1,2). InsP5 and InsP6 are present in virtually all mammalian cells at substantial amounts, between 10 and 100 µM, much higher than any other InsPs (1,2). There are several reports that extracellularly applied InsP6 exhibits an anticancer effect on several cancers in the blood, colon, liver, lung, mammary and prostate (38). Comparison of synthetic compounds and natural products, such as InsP6, suggests that the latter may be more suitable for chemoprevention of carcinogenesis, because their actions are milder (9). It has also been reported that InsP6 is involved in cell apoptosis and differentiation (10). As to the mechanisms by which InsP6 exerts anticancer effects, there have been several reports that InsP6 inhibits the transforming growth factor
-stimulated-phosphatidylinositol 3-kinase (PI3K)-Akt pathway in prostate carcinoma cells; InsP6 inhibited PI3K activity, followed by the activation of Akt which is involved in cell survival through the phosphorylation of Bad, a pro-apoptotic molecule, of caspase 9 and/or of I
B kinase
(IKK
) (11). However, another report is conflicting; InsP6 had no effect on PI3K activation, but was effective in the inhibition of nuclear factor
B (NF
B) activation (12). Since the AktNF
B pathway is known to be a strong cell survival pathway (13,14), it is important to determine which step is impaired by InsP6.
The dephosphorylation of InsP6 to InsP15 could enhance its anticancer function (15). Razzini et al. (16) reported that Ins(1,3,4,5,6)P5 and Ins(1,4,5,6)P4, at relatively low concentrations, exhibited anticancer activity in human breast cancer and human ovarian cancer cells to the same extent as InsP6 at millimolar concentrations. This effect is a consequence of the competition between InsPs and PtdIns(3,4,5)P3 for proteins bearing the pleckstrin homology (PH) domain favoring PtdIns(3,4,5)P3, a product of PI3K activity such as that of Akt.
In the present study, we investigated the mechanism of InsP6 internalization and its competence to inhibit cell proliferation and to induce apoptosis. We also looked into the mechanisms by which InsP6 inhibits the tumor necrosis factor (TNF)-induced PI3KNF
B survival pathway on which we reported previously (14) and whether InsP6 itself causes activation of the apoptotic pathway. Furthermore, we examined the effects of histone to reduce the effective concentration of InsP6.
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Materials and methods
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Cell culture and growth assays
HeLa cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum and were treated with InsP6 (Sigma, St Louis, MO) at various concentrations. The number of viable and dead cells was counted on the first, second, third and fourth day after InsP6 treatment. Cells were stained with trypan blue to distinguish the viable and dead cells.
Soft agar colony formation assay
This assay was performed using 6 well plates. The bottom layer contained 2 ml of 0.5% agar in medium. The middle layer contained 1 ml of 0.38% agar in medium with 103 HeLa cells. The top layer contained 1 ml of 0.38% agar in medium with various concentrations of InsP6. Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2.
Hoechst and TUNEL staining
This experiment was performed according to a method described by Negri et al. (17). Briefly, HeLa cells were seeded onto coverslips, stimulated, rinsed in PBS, fixed with ice-cold 10% trichloroacetic acid, and washed in cold 70, 80, 90% and absolute ethanol. DNA apoptotic fragments were end-labeled using the Apoptosis Detection System, Fluorescein (Promega, Madison, WI). After washing, the TUNEL-processed samples were incubated with Hoechst 33258 (Sigma).
Flow cytometry
Flow cytometry detection of apoptotic cells was performed as described by Yoshida et al. (18). Briefly, pretreated HeLa cells were harvested and suspended in 1 ml of hypotonic fluorochrome solution (50 µg/ml propidium iodide in 0.1% sodium citrate containing 0.1% Triton X-100). Cell suspensions were placed at 4°C in the dark overnight before flow cytometric analysis. The propidium iodide fluorescence of individual nuclei was measured using an EPICS XL flow cytometer.
Immunoblotting
Stimulated cells were incubated with lysis buffer [20 mM HEPES buffer pH 7.2, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 50 mM sodium fluoride, 40 mM sodium ß-glycerophosphate, 2 mM sodium orthovanadate, 30 mM sodium pyrophosphate and a cocktail of protease inhibitors (see below)] and scraped. Samples were separated by SDSPAGE and transferred to a polyvinylidene difluoride sheet. After blocking with 5% skimmed milk in Tris-buffered saline (150 mM NaCl and 20 mM TrisHCl, pH 7.2), the sheets were incubated with the first antibody. The secondary antibody was horseradish peroxidase-conjugated donkey anti-rabbit or anti-mouse IgG antibody (Amersham, Buckinghamshire, UK). The bound antibodies were visualized using the ECL system (Amersham). The cocktail of protease inhibitors contained 10 µM aprotinin, 10 µM pepstatin A, 10 µM leupeptin and 1 mM p-amidinophenyl methanesulfonyl fluoride (PMSF).
Nuclear extract
After washing with PBS, the cells were incubated in 400 µl buffer A (10 mM HEPES buffer pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol and the cocktail of protease inhibitors) on ice for 15 min. Nonidet P-40 at 10% (25 µl) was then added and the cells vortexed for 10 s. After centrifugation for 1 min at 12 000 r.p.m., the resulting pellet was collected, suspended in 50 µl of buffer B (20 mM HEPES buffer pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol and the cocktail of protease inhibitors) and incubated on ice for 15 min with occasional vortex mixing. After a final centrifugation of the sample for 10 min at 12 000 r.p.m., the supernatant was taken as the nuclear extract.
Luciferase assay
HeLa cells were transfected with NF
B-luc (Clontech, Palo Alto, CA). Luciferase assay was performed using the luciferase assay system (Promega). Briefly, after washing with PBS, 400 µl of lysis buffer was added to the cells, followed by a single freezethaw to ensure complete lysis. The culture dish was rocked several times, scraped and the contents transferred to a microcentrifuge tube. The tube was vortexed for 15 s and centrifuged at 12 000 g for 2 min. The resulting supernatant was collected and added to a luminometer tube containing luciferase assay reagent. The tube was then read for 10 s to measure luciferase activity.
PI3K assay
Stimulated HeLa cells were lysed in a buffer (20 mM TrisHCl buffer pH 8.0, 137 mM NaCl, 1 mM MgCl2, 10% Nonidet P-40, 1 mM dithiothreitol, 0.4 mM sodium orthovanadate and 1 mM PMSF). The cell lysate supernatant was collected by centrifugation and incubated with monoclonal anti-PI3K (p85
) antibody immobilized with protein GSepharose. The beads were washed with PBS containing 1% Nonidet P-40 and 1 mM dithiothreitol, with a solution consisting of 0.1 M TrisHCl buffer (pH 7.6), 0.5 M LiCl and 1 mM dithiothreitol, and finally with a solution containing 10 mM TrisHCl buffer (pH 7.6), 0.1 M NaCl and 1 mM dithiothreitol. This was followed by incubation with 0.5 mg/ml phosphoinositide fraction containing PtdIns(4,5)P2. Reaction buffer (50 mM MgCl2, 100 mM HEPES buffer pH 7.6, 250 µM ATP containing 5 µCi of [
-32P]ATP) was added and incubated at 30°C for 10 min. The assay was continued as described by Chen et al. (12).
PtdIns(3,4,5)P3 production assay
[32P]Orthophosphate-labeled HeLa cells were pre-treated with buffer A (30 mM HEPES buffer pH 7.4, 110 mM NaCl, 1 mM MgCl2 and 10 mM glucose) containing 25 µM LY294002 or 1 mM InsP6 for 2 h at 37°C, and incubated in buffer A containing 100 ng/ml TNF or insulin for 5 min. This assay was continued as described by Hidaka et al. (19).
Immunofluorescence
The stimulated HeLa cells were fixed using a solution of 4% paraformaldehyde in PBS. After washing in PBS, the fixed HeLa cells were treated with 100 mM glycine and 0.2% Triton X-100 in PBS. Samples were incubated in 0.1% bovine serum albumin in PBS to block non-specific binding. Polyclonal anti-Akt antibody (New England BioLabs, Beverly, MA) was applied as the first antibody, followed by FITC-conjugated goat F(ab')2 anti-rabbit IgG (Biosource, Camarillo, CA) as the second antibody.

m analysis

m analysis was carried out as described by Yoshida et al. (20). Briefly, the cell pellet was suspended in 250 µl of 20 nM DiOC6(3) in PBS and incubated in an incubator for 15 min. 
m was measured using an EPICS XL flow cytometer.
Mitochondrial fraction
This assay was carried out according to the MBL cytochrome c kit protocol. Briefly, cells were homogenized in 200 µl of ice-cold solution containing 10 mM TrisHCl (pH 7.5), 0.3 M sucrose, and the cocktail of protease inhibitors, followed by centrifugation at 100 000 g at 4°C for 60 min. The supernatant was collected as the cytosol fraction. The precipitate was resuspended in 200 µl ice-cold solution containing 10 mM TrisHCl (pH 7.5), 1% Triton X-100, 150 mM NaCl and protease inhibitor mixture, sonicated with an ultra sonicator, and centrifuged at 10 000 g, 4°C for 30 min. The supernatant was collected as the mitochondrial fraction.
InsP6 internalization
Pretreated cells were labeled with 0.5 µCi of [3H]InsP6 for 6 h. After washing with PBS, they were incubated with 0.8 ml of 2 M perchloric acid containing 1 mg/ml InsP6 for 15 min on ice. Then 0.4 ml of 1 M potassium carbonate and 5 mM EDTA was added and a freezethaw cycle was carried out twice. The mixture was kept at 4°C for 2 h, then centrifuged at 15 000 r.p.m. for 15 min. The resulting supernatant was applied to a strong anion exchange column (Whatman, Kent, UK), mounted on a high performance liquid chromatography system at a flow rate of 1 ml/min. Fractions were collected at 1.1 min intervals, followed by liquid scintillation counting after mixing with 4 ml of ultima-flo (Packard, Grönigen, The Netherlands).
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Results
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InsP6 inhibited the growth and induced apoptosis of HeLa cells
HeLa cells were cultured at an initial density of 2x104 cells in each well of a 6 well culture dish for 2 days. Various concentrations of InsP6 were added to the well, followed by further cultivation for up to 4 days (6 days after the initiation of culture). The growth of HeLa cells was inhibited by InsP6, and cell death was induced as well (Figure 1A and B
). The increase in dead cells was evident at concentrations as minimal as 0.25 mM InsP6, and increased concentrations increased the effect concomitantly. The inhibition of the proliferation of HeLa cells by InsP6 was also seen in an anchorage-free colony formation assay in soft agar (Figure 1C
). Such an effect was not observed with inositol hexakissulfate (data not shown), indicating the specific effect of InsP6.

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Fig. 1. Growth inhibition and apoptosis induction by InsP6. HeLa cells (2x104) were starved for 12 h and treated with InsP6 at various concentrations for 2 h. Dulbecco's modified Eagle medium containing 10% fetal bovine serum and InsP6 at various concentrations was then added. Pre-incubation with InsP6 in the absence of serum for 2 h is to facilitate the entry into cells, preventing the complex formation with serum protein. The number of living cells (A) and the percentage of dead cells (B) were counted on the first, second, third and fourth day after InsP6 treatment. Soft agar colony formation assay was performed as described in the Materials and methods. The number of 15-cell-colonies was counted under a microscope at x100 magnification (C). Each panel shows the mean ± SEM of five independent determinations.
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Cell death caused by InsP6 appeared to be apoptosis, since Hoechst 33258 staining of cells treated with InsP6 showed nuclear fragmentation in a manner similar to cells treated with Fas, which was used as a positive control. Also, the cells stained with TUNEL (Figure 2A
). Furthermore, flow cytometric analysis of cells (both dish-attached and floating cells) stained with propidium iodide confirmed the presence of nuclear fragmentation in cells treated with InsP6, as shown in Figure 2B
. This also showed that lower concentrations of InsP4 and InsP5 were effective at causing apoptosis. Chelation of divalent cations such as Ca2+ and Mg2+ by millimolar concentrations of InsP6 was not a cause of apoptosis, because supplementing with Ca2+ and Mg2+ did not change the results (data not shown). Furthermore, addition of EDTA at millimolar concentrations for up to 4 days to chelate the cations did not induce cell death (data not shown).

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Fig. 2. (A) Hoechst and TUNEL staining. HeLa cells (1x106) were seeded on coverslips, starved for 12 h and treated with 1 mM InsP6 (c and d) or 5 µg/ml Fas (e and f) for 6 h. Hoechst and TUNEL staining were carried out as described in the Materials and methods. The experiment was repeated three times. (B) Flow cytometry detection of apoptotic cells. HeLa cells (1x106) were starved for 12 h and then stimulated with 50 µM Ins(1,4,5,6)P4 (Cell Signals, Lexington, KY) (b), or 50 µM Ins(1,3,4,5,6)P5 (Cell Signals) (c), 0.5 mM or 1 mM InsP6 (d and e) for 48 h. The procedures were described in the Materials and methods. This experiment was repeated five times.
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InsP6 inhibited cell survival signaling through the AktNF
B pathway
Apoptosis can be the result of either or both of inhibition of the cell survival signal and the direct induction of apoptosis. Thus, we first examined whether InsP6 inhibits cell survival signaling. For this purpose, we examined whether InsP6 inhibits the TNFPI3KAktNF
B pathway, which has been shown to stimulate cell survival in SAS cells by us (14) and in 293 and HeLa cells by Ozes et al. (13).
Phosphorylation of Akt at both Ser473 and Thr308 was seen within 1 min of stimulation with TNF at 100 ng/ml, and reached maximal levels within 10 min. Stimulation with insulin at 100 ng/ml for 5 min provided similar results (Figure 3A and B
). Pre-incubation with 1 mM InsP6 prevented the Akt phosphorylation at both sites (Figure 3A and B
). In vitro Akt activity was assayed using the phosphorylation of histone 2B (H2B) as the substrate. The results were apparently parallel to those of the phosphorylation of Akt; almost maximal Akt activity was seen at 10 min, and pre-incubation with 1 mM InsP6 abolished the Akt activity (Figure 3C
).

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Fig. 3. Phosphorylation and activity of Akt. HeLa cells (1x106) were starved for 12 h and treated with or without 1 mM InsP6 for 2 h, followed by 100 ng/ml TNF stimulation for 1, 3, 5, 10, 30 min or 100 ng/ml insulin for 5 min. Cell lysates were immunoprecipitated by polyclonal anti-Akt antibody (New England BioLabs), coupled to protein GSepharose (Amersham) for detecting Akt phosphorylation (A and B). The precipitates were analyzed by immunoblotting using polyclonal anti-phospho-Akt (Ser473) (New England BioLabs) (A) or polyclonal anti-phospho-Akt (Thr308) (New England BioLabs) (B). (C) The immunoprecipitates were assayed for kinase activity using H2B and [ -32P]ATP as substrates (14). The membranes were reprobed for Akt using polyclonal anti-Akt (New England BioLabs). To detect the phosphorylation and degradation of I B, cell lysates were analyzed by immunoblotting using polyclonal anti-phospho-specific-I B (New England BioLabs) (D) or polyclonal anti-I B (Santa Cruz Biotechnology, Santa Cruz, CA) (E). To detect NF B nuclear translocation, nuclear extracts, prepared as described in the Materials and methods, were analyzed by immunoblotting using polyclonal anti-NF B p50 (Santa Cruz Biotechnology) (F) or polyclonal anti-NF B p65 (Santa Cruz Biotechnology) (G). To detect NF B nuclear translocation and NF B-luciferase activity, luciferase assay was performed as described in the Materials and methods (H). Each panel shows the typical results and the mean ± SEM of five independent determinations.
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It has been reported that Akt activated by phosphorylation can then induce the phosphorylation of IKKs, mainly IKKß, which later induces the phosphorylation and degradation of I
B (21). Considering that Akt is upstream of this pathway, we needed to check whether this pathway was also inhibited. Stimulation with 100 ng/ml TNF showed I
B phosphorylation in 3 min, which reached maximal levels in 10 min, whilst I
B degradation was seen in 3 min (Figure 3D and E
). Similar results were seen in cells stimulated with 100 ng/ml insulin. I
B phosphorylation and degradation by 100 ng/ml TNF or insulin was abolished by pre-incubation with 1 mM InsP6 (Figure 3D and E
).
Following I
B phosphorylation and degradation, NF
B is free to translocate to the nucleus (21,22). We also noticed this phenomenon in the present results, in that the amount of NF
B (p65 and p50) was increased in the nucleus after 100 ng/ml TNF stimulation. This translocation was totally inhibited in the cells pre-incubated with 1 mM InsP6 (Figure 3F and G
). Results from the NF
B-luciferase transfected HeLa cells also agreed with this result (Figure 3H
). Treatment with TNF increased the NF
B transcriptional activity gradually, but the 1 mM InsP6-pre-incubated sample showed only a slight rise in NF
B activity. InsP6 also had the same capacity of inhibiting insulin-induced NF
B nuclear translocation and activity (Figure 3H
).
Cell survival is also promoted by activation of the mitogen-activated protein kinase (MAPK) pathway (23,24). InsP6 had little effect on this pathway as assessed by the phosphorylation of MAPK (data not shown), indicating that the inhibition by InsP6 is specific to the Akt cell survival pathway.
InsP6 did not affect the activity of PI3K and PtdIns(3,4,5)P3 production
An investigation as to whether InsP6 affects the activity of PI3K, responsible for producing PtdIns(3,4,5)P3, was performed. Tyrosine phosphorylation of p85, a regulatory subunit of type 1 PI3K, was increased by stimulation with 100 ng/ml TNF or insulin. Pre-incubation with 25 µM LY294002, a PI3K inhibitor, inhibited the phosphorylation of p85, but pre-incubation with InsP6 was ineffective (Figure 4A
). The ability of PI3K to convert PtdIns(4,5)P2 to PtdIns(3,4,5)P3 was enhanced by immunoprecipitates of the anti-p85 subunit prepared from cells stimulated with TNF or insulin. Pre-incubation with 25 µM LY294002 diminished this effect, but pre-incubation with 1 mM InsP6 did not cause any reduction in the activity (Figure 4B
). [32P]Orthophosphate-labeled HeLa cells also showed the same result; PtdIns(3,4,5)P3 production was upregulated by stimulation with 100 ng/ml of TNF or insulin. Pre-incubation with 25 µM LY294002 decreased this production but pre-incubation with 1 mM InsP6 had no effect (Figure 4C
).

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Fig. 4. Phosphorylation and activity of PI3K and PtdIns(3,4,5)P3 production. HeLa cells (1x106) were starved for 12 h and pretreated with or without 1 mM InsP6 or 25 µM LY294002 for 2 h, followed by 100 ng/ml TNF or 100 ng/ml insulin for 5 min. (A) Cell extracts were immunoprecipitated by monoclonal anti-PI3K (p85 ) (PharMingen, San Diego, CA), coupled to protein GSepharose (Amersham). The precipitates were analyzed by immunoblotting using monoclonal anti-phosphotyrosine (Upstate Biotech, NY). Each panel shows the typical results and the mean ± SEM of five independent determinations. (B and C) PI3K and PtdIns(3,4,5)P3 production assays were performed as described in the Materials and methods. The experiments were repeated three times.
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InsP6 caused the inhibition of Akt membrane translocation
When PtdIns(3,4,5)P3 is produced in the cell membrane, Akt, present mainly in the cytosol, moves to the cell membrane by binding to PtdIns(3,4,5)P3 via its PH domain. This translocation was seen after stimulation with 100 ng/ml TNF or insulin, and was prevented by pre-incubation with 25 µM LY294002, a potent PI3K inhibitor. Interestingly, the same results were obtained in HeLa cells pre-incubated with 1 mM InsP6 (Figure 5
), in spite of little inhibition of PtdIns(3,4,5)P3 production.

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Fig. 5. Akt membrane translocation. HeLa cells (1x106) were seeded on coverslips, starved for 12 h and pretreated with or without 1 mM InsP6 or 25 µM LY294002 for 2 h, followed by 100 ng/ml TNF or 100 ng/ml insulin for 5 min. Immunofluorescence was carried out as described in the Materials and Methods. This experiment was repeated three times.
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Direct effects of InsP6 on events relating to cell apoptosis
We then examined whether InsP6 directly activates events relating to apoptosis. We first analyzed 
m in cells treated with InsP6. We found that 0.5 mM InsP6 lowered 
m by ~50%. A more dramatic change was seen when 1 mM InsP6 was used (Figure 6A
). Cytochrome c release was observed after 6 h treatment with 0.5 mM InsP6 and 12 h treatment caused more cytochrome c release. More cytochrome c release was detected after treatment with higher concentrations of InsP6 (Figure 6B
).
To examine whether InsP6 activates the apoptotic machinery, we studied the cleavage of caspase 9 (caspase recruiting domain), caspase 3 (downstream caspase), and poly (ADP ribose) polymerase (death substrate, abbreviated as PARP). Degradation and cleavage of caspase 9, 3 and PARP were detected slightly after treatment with 0.5 mM InsP6 (Figure 7
). Much more degradation and cleavage was seen after 6 h treatment with 1 mM InsP6. The degradation and cleavage were increased by treatment for 12 h.

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Fig. 7. Caspase 9, caspase 3 and PARP. HeLa cells (1x106) were starved for 12 h and then stimulated with 0.5 or 1 mM InsP6 for 2, 6 or 12 h. Cell extracts were analyzed by immunoblotting using polyclonal anti-cleaved-caspase-9 (Asp330) (New England BioLabs) (A), polyclonal anti-cleaved-caspase-3 (Asp175) (New England BioLabs) (B), polyclonal anti-cleaved-PARP (Asp214) (New England BioLabs) (C). Cytc-Jurkat and eto-Jurkat were included in the apoptosis sampler kit as positive controls. Each panel shows the typical results and the mean ± SEM of five independent determinations.
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InsP6 internalization and dephosphorylation
To determine how extracellularly applied InsP6 affects the events observed above, HeLa cells were cultured with 1 mM InsP6 (containing 0.5 µCi [3H]InsP6) for 6 h. The extract prepared from perchloric acid treated cells was analyzed by applying it to a strong anion exchange column, followed by scintillation counting. As shown in Figure 8
, [3H]InsP6 was internalized and dephosphorylated into InsP3, InsP4 and InsP5. However, pre-incubation with colchicine, a pinocytosis inhibitor, did not produce the dephosphorylated forms of InsP6 (Figure 8
). [3H]InsP6 was incubated for 6 h with culture medium prepared just in the same way as for the analysis of [3H]inositol phosphates, but with no addition of an isotope InsP6, followed by the analysis of [3H]inositol phosphates. There was little production of lower [3H]inositol phosphates in the presence or absence of colchicines, indicating that they were the products inside cells.

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Fig. 8. InsP6 internalization and dephosphorylation. HeLa cells (1x106) were starved for 12 h and then incubated with or without 10 µg/ml colchicine for 2 h. The cells were then stimulated with 1 mM InsP6 for 2 h, followed by the addition of 0.5 µCi of [3H]InsP6 for 6 h. The procedures used were as described in the Materials and methods. The fractions of InsPx indicated in the figure were examined by applying an authentic [3H]InsPx to the same column in advance. Most of the radioactivity corresponding to InsP6 appears to be originated from the extracellular space even after extensive washing. Open squares indicate the results obtained from the cells pretreated with colchicines. The figure represents a typical analysis and the experiment was repeated three times.
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Effect of histone to lower the effective concentration of InsP6
Ozaki et al. (25) successfully used histone for intracellular delivery of chemically synthesized analogs of phosphoinositides and inositol phosphates. Therefore, we investigated the effect of histone on InsP6-induced apoptosis, as assessed by nuclear staining with propidium iodide followed by a flow cytometry. As shown in Figure 9
, InsP6 at concentration of 100 µM exhibited a partial induction of apoptosis, but the inclusion of histone enhanced the InsP6 action to the similar extent as that observed at 1 mM (see also Figure 2B
).

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Fig. 9. Flow cytometry detection of apoptotic cells. HeLa cells (1x106) were starved for 12 h and then stimulated with none (A), 100 µM InsP6 (B) or 100 µM InsP6 plus 50 µM histone (C) for 48 h. Histone alone exhibited the same pattern as that seen in (A). The procedures were described in the Materials and methods. This experiment was repeated five times.
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Discussion
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There have been many efforts to induce apoptosis in cancer cells, by activating Fas (26), Apaf-1 (20), apoptosis-inducing factor (27), and inhibiting the survival pathway via mechanisms independent of (28), or dependent on (14,29), Akt activation. It has been reported that inositol phosphates, which are recognized as second messengers inside cells, have the potential to inhibit cell growth, especially when InsP4, InsP5 and InsP6 are extracellularly applied (16).
The present results show that, besides having the potential to inhibit cell growth in both anchorage-dependent culture and anchorage-independent soft agar assays, InsPxs also have the potential to induce cell death. The ability of InsP6 to cause cell death has already been described by another group (11) but the mechanisms underlying this are not clearly understood. It has been shown that the Akt pathway is a major pathway that can induce cell survival (13,21). Therefore, we first investigated the effect of InsP6 on the Akt pathway.
Akt phosphorylated at Ser473 and Thr308, an active form, was upregulated by TNF stimulation, whilst pre-incubation with InsP6 inhibited the phosphorylation. This effect of InsP6 was also shown in an in vitro model assay using H2B as a substrate. Akt activated by phosphorylation has many downstream effects (30), one of which is the phosphorylation of I
B and consequent release of NF
B. Released NF
B is translocated to the nucleus, which is an important stage in the continuation of this survival pathway (31,32). We also noticed that pre-incubation with InsP6 prior to TNF stimulation inhibited all of the activation signals mentioned above; the phosphorylation and degradation of I
B and NF
B translocation were abolished. We further pursued this investigation on NF
B-luciferase activity, and again we found that after pre-incubation with InsP6, the NF
B-luciferase activity was inhibited.
It was clear that InsP6 could inhibit the AktNF
B pathway, but it was necessary to investigate the effect of InsP6 on PI3K. This is a key regulator upstream of Akt (33), involved in a complex series of events, including genetic alterations, leading to aberrant cell cycle, progression and inhibition of apoptosis (34,35). In the present study, we found that InsP6 did not inhibit the phosphorylation of p85, a regulatory subunit of type1 PI3K. We also found that InsP6 did not decrease PI3K activity, as assessed by both an in vitro kinase assay using immunoprecipitates of anti-p85 antibody as an enzyme source and PtdIns(3,4,5)P3 production in intact cells labeled with [32P]orthophosphate. However, Akt membrane translocation, probably caused by the binding of the Akt PH domain to PtdIns(3,4,5)P3 produced in the plasma membrane, was inhibited by pre-incubation with InsP6. Another group reported the inhibition of this process by Ins(1,4,5,6)P4 (16), probably because of the binding of Ins(1,4,5,6)P4 to Akt so that the PH domain of Akt cannot bind to PtdIns(3,4,5)P3. We reported previously the binding affinity between a variety of InsPxs and the PH domain of Akt (36,37), showing that the PH domain of Akt binds preferably to Ins(1,4,5,6)P4, interfering with the binding of Akt to PtdIns(3,4,5)P3.
Extracellularly applied InsP6 is effective in the events observed in the present study, entering cells pinocytotically and then being further dephosphorylated into the appropriate InsPs. This was proved by analyzing [3H]inositol-labeled InsPxs in extracts of intact cells incubated with [3H]InsP6. [3H]InsP3, [3H]InsP4 and [3H]InsP5 metabolized from [3H]InsP6 were found to be accumulated inside cells, but we have not yet analyzed which isomers are dominant. Morrison et al. (38) have suggested that InsP6 kinase 2, which phosphorylates InsP6 to InsP5-PP, might be important in apoptosis. We also found a small radioactive peak, corresponding to InsP5-PP (InsP7) in HeLa cells incubated with [3H]InsP6 (data not shown), indicating that this inositol polyphosphate might also be involved in the apoptosis. These metabolites from [3H]InsP6 would be produced inside cells, because the treatment with colchicine, an inhibitor of pinocytosis, did not produce the metabolites. Furthermore, little metabolites were produced by incubating [3H]InsP6 with the culture medium prepared in the same way as for the assay of [3H]inostol phosphates. Since the radioactivities of [3H]InsP6 were much the same between the cell extracts treated with or without colchicine, the majority of the radioactivity would be due to the extracellular binding.
In response to death stimuli, mitochondrial membranes are permeabilized (39,40) and cytochrome c is released from the mitochondria (41,42). To find out whether InsP6 is a death stimulator, we investigated the effect of InsP6 on 
m without any stimulants, because the presence of stimulants, including serum, always enhances cell survival signals. The results showed that InsP6 itself could cause a decrease in 
m, indicating that the mitochondria were permeabilized. Although there are reports that Akt is involved in maintaining the integrity of the mitochondrial membrane (43,44), in the present experimental system InsP6 was applied without any stimulation, so that the Akt pathway was not upregulated. The results suggest that InsP6 itself could directly cause the mitochondrial defect. We realize that inhibition of the Akt pathway by InsP6 might play a role in affecting the mitochondria membrane potential as well. We also found increased amounts of cytochrome c in the cytosol and reduced cytochrome c levels in the mitochondria, indicating that cytochrome c was released from the mitochondria. Released cytochrome c induces the cleavage of caspase 9, a caspase recruiting domain, which induces the activation of caspase 3, and consequently the activation of apoptotic substrates (45). Our present results showed that, after treatment with InsP6, caspase 9, caspase 3 and PARP, an apoptotic substrate, were cleaved in a time- and InsP6 concentration-dependent manner. Treatment of HeLa cells with 1 mM InsP6 for 6 h, which was sufficient to cause mitochondrial permeabilization, cytochrome c release, activation of caspase 9, caspase 3 and PARP, was carried out to observe DNA fragmentation by TUNEL.
As mentioned in the introduction, the dephosphorylation of InsP6 to InsP15 can possibly enhance its anticancer function (16). We tested the potential of the lower forms to induce apoptosis. We treated HeLa cells with Ins(1,4,5,6)P4 or Ins(1,3,4,5,6)P5, and detected apoptosis by flow cytometry. We found that these lower metabolites are more active than InsP6. This result agreed with the report by Razzini et al. (16). It suggested that InsP6 enters the cell and is dephosphorylated to lower forms, which are more potent at inducing apoptosis. The lower phosphorylated forms, especially Ins(1,4,5,6)P4, which has the highest affinity for the Akt PH domain (36,37), also inhibit the Akt pathway (16), indicating that lower concentration of these agents could be used for anticancer action. However, InsP4, especially an isomer whose position of the phosphates is defined is expensive. So the trial to reduce the effective concentration of InsP6 was achieved by using histone, the principle to use it is to neutralize the negative charges of the phosphates of InsP6 to facilitate the transport through the plasma membrane (25).
In conclusion, we suggest that InsP6, which is probably pinocytotically transported into cells and subsequently dephosphorylated to InsP4 and InsP5, can inhibit the Akt-NF
B cell survival pathway by inhibiting the translocation of Akt to the plasma membrane and, thus, its activation. It can also induce the apoptotic machinery directly. The next step is to clarify the mechanism by which extracellularly applied InsP6 induces mitochondrial permeabilization.
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Notes
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3 To whom correspondence should be addressed Email: hirata1{at}dent.kyushu-u.ac.jp 
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Acknowledgments
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We would like to thank Drs S.Shears and K.Hidaka (NIEHS, USA) for help with the analysis of inositol phosphates. This work was supported by the Japan Society for Promotion of Science (JSPS) (to M.H.) and a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology of Japan (to M.H.), by the Kyushu University Interdisciplinary Programs in Education and Projects in Research Development (to M.H.).
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Received June 14, 2002;
revised August 20, 2002;
accepted August 30, 2002.