Inositol Hexaphosphate Inhibits Growth and Induces G1 Arrest and Apoptotic Death of Androgen-Dependent Human Prostate Carcinoma LNCaP Cells

Abstract

Prostate cancer (PCA) is the most common invasive malignancy and the second leading cause of cancer-related deaths in the US male population. One approach to control this malignancy is its preventive intervention by dietary agents. Inositol hexaphosphate (IP6), a dietary constituent, has shown promising efficacy against various cancers; however, limited studies have been performed with IP6 against PCA. Here, we investigated the growth-inhibitory effect and associated mechanisms of IP6 in androgen-dependent human prostate carcinoma LNCaP cells. IP6 treatment of cells resulted in a strong growth inhibition and an increase in G1 cell population. In mechanistic studies, IP6 resulted in an increase in cyclin-dependent kinase inhibitors (CDKIs) Cip1/p21 and Kip1/p27 levels, together with a decrease in cyclin-dependent kinase (CDK) 4 and cyclin D1 protein levels. An increase in CDKI levels by IP6 also led to a concomitant increase in their interactions with CDK2 and CDK4, together with a strong decrease in the kinase activity of both CDKs. Downstream in CDKI-CDK-cyclin cascade, consistent with its inhibitory effect on CDK kinase activity, IP6 treatment of cells increased hypophosphorylated levels of retinoblastoma (Rb) with a decrease in Rb phosphorylation at serine 780, 807, and 811 sites, and caused a moderate to strong decrease in the levels of transcription factors E2F1, E2F4, and E2F5. In other studies, IP6 caused a dose- and a time-dependent apoptotic death of LNCaP cells, and a decrease in Bcl2 levels, causing a strong increase in Bax versus Bcl2 ratio, as well as an inhibition of constitutively active AKT phosphorylation. Taken together, these molecular alterations provide an insight into IP6-caused growth inhibition, G1 arrest, and apoptotic death of human prostate carcinoma LNCaP cells. Because early clinical PCA growth is an androgen-dependent response, the results of the present study employing androgen-dependent LNCaP cells suggest that IP6 has promise and potential to be effective against PCA.

Introduction

Statistics of prostate cancer (PCA) show that it is the second most common male malignancy in the United States and European countries [1]. It is estimated that 220,900 new cases and 28,900 associated deaths would have occurred from PCA in 2003 [1]. In the genesis of PCA, a variety of pathogenetic pathways/factors exist, and the rates of PCA differ up to 90-fold between different populations [2–4]. Both epidemiological and laboratory studies suggest that diet and androgen increase PCA risk through a common etiologic pathway [3–6]. Importantly, several studies in recent years have also shown that diet and nutrition play a significant role in PCA control, prevention, and/or intervention, and, accordingly, extensive efforts are being made for the prevention/intervention of PCA by altering dietary choices or the use of dietary supplements [7–9].

Inositol hexaphosphate (IP6), also known as phytic acid, is one such dietary agent, which is described as “natural cancer fighter,” being an important constituent of natural diets. It is found in abundance in high-fiber-content diets [10]. Most cereals, legumes, nuts, oil seeds, and soybean contain 0.5% to 6.4% (wt/wt) or even higher levels of IP6 [10]. In an animal study, it has been reported that physiological levels of IP6 are more dependent on consumption of IP6-rich diet compared to the levels of its lower phytate derivatives, IP5 and IP4 [11]. Another study suggests that humans become deficient in IP6 if they consume IP6-poor diet for as little as 2 weeks [12].

Almost three decades of research with IP6 have revealed its broad-spectrum antineoplastic activities in different cancer models (reviewed in Refs. [10,13]). In cell culture, IP6 has been shown to inhibit: 1) growth of human breast, colon, and liver cancer cells, and rhabdomyosarcoma and erythroleukemia cells; and 2) cell transformation in mouse epidermal JB6 cells [14–19]. IP6 has also been shown to impair epidermal growth factor (EGF) or phorbol ester-induced ERK1/2-AP1 activation and phosphotidylinositol-3 kinase (PI-3K) activity in JB6 cells as its anti-tumor promotion mechanism [19]. However, there are only a few studies showing the anticancer potential of IP6 against PCA cells; these reports employed advanced and androgen-independent human PCA cell lines PC3 and DU145. The first study reported that IP6 inhibits growth and induces differentiation of human PCA PC-3 cells [20]. More recently, we showed that IP6 impairs erbB1 receptor-associated mitogenic signaling [21], and induces G1 arrest in cell cycle progression and modulates cell cycle regulatory proteins in DU145 cells [22]. Most of the abovementioned in vitro studies have used up to 5 mM IP6.

With regard to in vivo anticancer efficacy of IP6, it has been shown that 1% IP6 in drinking water 1 week before or 2 weeks after the administration of azoxymethane inhibits the development of large intestinal cancer in F344 rats [23]. Later, it was reported that in same animal model, treatment with 2% IP6 in drinking water, even after 5 months of carcinogen induction, significantly inhibits both number and size of tumors in large intestines [24]. IP6 was also found to suppress dimethylhydrazine-induced large intestinal cancer in CD-1 mice [25]. Furthermore, IP6 has been shown to: 1) inhibit DMBA-induced rat mammary cancer growth [26]; 2) regress liver cancer xenotransplant [27]; 3) prevent pulmonary adenomas in mice [28]; 4) prevent DMBA-induced skin tumorigenesis [29]; 5) inhibit the growth of rhabdomyosarcoma tumor xenograft growth [16]; 6) inhibit the growth of mouse fibrosarcoma FSA-1 tumor xenografts in nude mice [30]; and 7) inhibit colon carcinogenesis [31,32]. In another study, it is reported that IP6 treatment reveres the colon carcinogen (dimethylhydrazine)-induced suppression of natural killer cells activity, as well as enhances the baseline natural killer cells' cytotoxicity in mice [33]. With regard to PCA, only very recently did we report, for the first time, that 1% and 2% (wt/vol) IP6 feeding in drinking water inhibits DU145 tumor xenograft growth in athymic nude mice, which was associated with anti-proliferative, pro-apoptotic, and anti-angiogenic effects of IP6 in the tumor xenograft [34]. Various animal studies reported above, including ours, have also shown that IP6 does not cause any noticeable harmful side effects or toxicity even at higher doses (up to 2% wt/vol or 15 mM in drinking water) and chronic administration [24,34,35]. With regard to epidemiological studies with IP6, consumption of IP6-rich cereals and legumes has been suggested to be associated with a reduction in prostate, breast, and colon, cancers [12].

The induction of PCA in humans has been viewed as a multistage process involving progression from low-grade small latent carcinoma to higher-grade large metastasizing carcinoma [36]. PCA growth and progression involve different molecular events converging into an aberrant (deregulated) cell cycle progression and loss of apoptosis of transformed and cancerous prostate cells [36]. The defects in cell cycle progression in cancer cells, including PCA, also involve altered expression, interaction, and/or function of different cell cycle regulators [36–39]. A controlled cell cycle progression is regulated by cyclin-dependent kinases (CDKs), the activities of which are positively regulated by interactions with their regulatory subunits—cyclins [36–39]. There are numerous evidences, however, showing an overexpression of cyclins and/or CDKs in many human cancers and derived cell lines, including PCA [36–39]. The other important regulators in a controlled cell cycle progression are cyclin-dependent kinase inhibitors (CDKIs), such as Cip1/p21, Kip1/p27, and Kip2/p57, which control CDK kinase activity in response to both mitogenic and growth-inhibitory signals [36–43]. Frequent mutations in CDKIs or a lack of their expression have been shown to be another common feature in the majority of human cancers including PCA [36–39]. Taken together, these studies clearly suggest that a controlled regulation of cell cycle progression by an increase in expression and function of CDKIs, causing a decrease in CDK kinase activity and associated downstream events, could be an effective approach to control the growth and proliferation of PCA cells and facilitate their apoptotic death [44].

Because clinical PCA is initially androgen-dependent for its growth [36], employing androgen-dependent human prostate carcinoma LNCaP cells as a model system, in the present study, we assessed the efficacy of IP6 on cell growth, cell cycle progression, and apoptotic death, and associated mechanisms. The results obtained demonstrate that IP6 inhibits LNCaP cell growth, together with G1 arrest, through an induction in Cip1/p21 and Kip1/p27 levels, causing their increased interaction with CDKs and leading to a decrease in kinase activity toward retinoblastoma (Rb) phosphorylation. IP6 also showed the apoptotic death of LNCaP cells that was associated with a decrease in Bcl2, concomitant with an increase in Bax versus Bcl2 levels and an inhibition in constitutively active survival factor AKT favoring apoptotic response.

Materials and Methods

Cell Lines and Reagents

The human prostate carcinoma cell line LNCaP was obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 with 10% fetal bovine serum (Hyclone, Logan, UT) and 1% penicillin-streptomycin under standard culture conditions (37°C, 95% humidified air, and 5% CO₂). IP6 and anti-actin antibody were from Sigma-Aldrich Chemical Co. (St. Louis, MO). RPMI 1640 and other culture materials were from Life Technologies, Inc. (Gaithersburg, MD). Anti-Cip1/p21 antibody was from Calbiochem (Cambridge, MA), and anti-Kip1/p27 antibody was from Neomarkers, Inc. (Fremont, CA). Antibodies to CDK2 and CDK4; cyclins D1 and E; E2F1, E2F2, E2F3, E2F4, and E2F5; and Rb (recognizes both hyperphosphorylated and hypophosphorylated Rb forms as slow-migrating and fast-migrating bands, respectively) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Histone H1 was from Boehringer Mannheim Corp. (Indianapolis, IN). [γ-³²P] ATP (specific activity, 3000 Ci/mmol); anti-mouse horseradish peroxidase-conjugated secondary antibody and ECL detection system were from Amersham Corp. (Arlington Heights, IL). The phospho-specific Rb antibodies, phospho-AKT and total AKT antibodies, Rb-C fusion protein, and anti-goat anti-rabbit immunoglobulin horseradish peroxidase-conjugated secondary antibody were from Cell Signaling Technology (Beverly, MA). Cell Death Detection ELISA^plus kit was from Roche Diagnostics (Indianapolis, IN). Other chemicals were obtained in their commercially available highest purity grade.

Cell Growth Assay

LNCaP cells were plated at 5000 cells/cm² in 60-mm plates under standard culture conditions detailed above for 24 hours. Thereafter, cells were fed with fresh medium and treated with various doses (0.5–4 mM, final concentration in the medium) of IP6 dissolved in distilled water and pH-adjusted (7.4). The doses of IP6 selected in this study are those used by others and us in several studies [19–22]. After 24 and 48 hours, cells were collected by a brief trypsinization, and counted in duplicate with a hemocytometer. Each treatment and time point had three independent plates. The representative data shown in this study were reproducible in an additional independent experiment.

Fluorescence-Activated Cell Sorter (FACS) Analysis for Cell Cycle Phase Distribution

LNCaP cells were grown under 10% serum conditions in RPMI 1640 medium as detailed above. In the first experiment, cells were treated with 0.5 to 4 mM IP6 for 24 and 48 hours and, in the second experiment, with 1 and 2 mM IP6 for 6, 12, and 24 hours. At the end of each treatment, cells were collected after a brief incubation with trypsin-EDTA followed by processing for cell cycle analysis. Briefly, 0.5 x 10⁶ cells were suspended in 0.5 ml of saponin/propidium iodide (PI) solution [0.3% saponin (wt/vol), 25 µg/ml PI (wt/vol), 0.1 mM EDTA, and 10 mg/ml RNase (wt/vol) in PBS] and incubated overnight at 4°C in the dark. Cell cycle distribution was then analyzed by flow cytometry using the FACS analysis core facility of the University of Colorado Cancer Center (Denver, CO). Finally, the percentage of cells in different phases of cell cycle was determined by ModFit LT cell cycle analysis software. In both experiments, each treatment and time point had three independent plates. The representative data shown in this study were reproducible in an additional independent experiment.

Cell Culture Treatments for Molecular Analyses

LNCaP cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin under standard culture conditions. At 60% confluency, cultures were treated with different doses of IP6 (1, 2, and 4 mM) for desired time periods. After these treatments, cell lysates were prepared in nondenaturing lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.3 mM phenyl methyl sulfonyl fluoride, 0.2 mM sodium orthovanadate, 0.5% NP-40, and 5 U/ml aprotinin). Briefly, the medium was aspirated and cells were washed with ice-cold PBS for two times followed by incubation in lysis buffer for 10 minutes on ice. Then cells were scrapped and kept on ice for 30 minutes, and, finally, cell lysates were cleared by centrifugation at 4°C for 30 minutes at 14,000 rpm. Protein concentrations in lysates were determined using Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA).

Immunoprecipitation and Immunoblotting

Cell lysates (200 µg of proteins per sample) were diluted to 1 ml with lysis buffer, precleared with protein A/G plus agarose for 1 hour, then incubated overnight with primary antibody against Cip1/p21 or Kip1/p27 plus protein A/G plus agarose beads; and immunocomplexes were collected and washed three times with lysis buffer. For immunoblotting, immunocomplexes or total cell lysates were denatured with 2x sample buffer. Samples were subjected to SDS-PAGE on 12% or 16% gel and separated proteins were transferred onto membrane by Western blot analysis. Membranes were blocked with blocking buffer for 1 hour at room temperature and, as desired, probed with primary antibody against Cip1/p21, Kip1/p27, CDK2, CDK4, cyclin E, cyclin D1, total Rb, phospho-specific Rb, E2F1, E2F2, E2F3, E2F4, E2F5, Bax, Bcl2, total AKT, phospho-AKT, and β-actin overnight at 4°C followed by peroxidase-conjugated appropriate secondary antibody and ECL detection.

Kinase Assays

To assess CDK4 kinase activity, 200 µg of protein lysate from each sample was precleared with protein A/G plus agarose beads and CDK4 protein was immunoprecipitated using anti-CDK4 antibody and protein A/G plus agarose beads. After overnight incubation at 4°C, beads conjugated with antibody and protein were washed three times with Rb lysis buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, 80 mM β-glycerophosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, 0.1% Tween 20, 10% glycerol, 1 mM PMSF, and 10 µg/ml aprotinin and leupeptin) and twice with Rb kinase assay buffer (50 mM HEPES-KOH, pH 7.5, 2.5 mM EGTA, 1 mM DTT, 10 mM β-glycerophosphate, 10 mM MgCl₂, 1 mM NaF, and 0.1 mM sodium orthovanadate). Phosphorylation of Rb was measured by incubating the beads with 30 µl of Rb kinase solution (1 µg of Rb fusion protein and 0.1 mM ATP in Rb kinase buffer) for 30 minutes at 37°C. Reaction was stopped by boiling the samples in 5x SDS sample buffer for 5 minutes. Samples were analyzed by SDS-PAGE and Western blot analysis followed by detection of Rb fusion protein phosphorylation employing phospho-specific Rb antibodies.

Similarly, to determine the CDK2-associated H1 histone kinase activity, CDK2 protein was immunoprecipitated using anti-CDK2 antibody and protein A/G plus agarose beads. Beads were washed two times with lysis buffer and, finally, once with kinase assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, and 1 mM DTT). Phosphorylation of histone H1 was measured by incubating the beads with 30 µl of hot kinase solution [2.5 µg of histone H1, 0.5 µl (5 mCi) of [γ-³²P] ATP, 0.5 µl of 0.1 mM ATP, and 28.75 µl of kinase buffer] for 30 minutes at 37°C. The samples were then boiled in SDS sample buffer for 5 minutes to stop the reaction and subjected to 12% SDS-PAGE, and the gel was dried and analyzed by autoradiography.

Quantitative Apoptotic Cell Death Assay

To quantify IP6-induced apoptotic death of LNCaP cells, cells were cultured and treated with IP6 at 1, 2, or 4 mM dose for various time points (1–48 hours) as detailed above. After these treatments, cells were harvested and processed for the analysis of apoptotic DNA fragments using Cell Death Detection ELISA^plus kit and following the step-by-step protocol provided by the vendor. This method detects the mononucleosomal and oligonucleosomal DNA fragments released into the cytoplasm using anti-DNA-POD and anti-histone-biotin antibodies. Quantitative ELISA at OD 405 to 490 nm then measures subsequent color formation after the addition of ABTS substrate. Enrichment factor (EF) was calculated using the formula: EF = absorbance of treated sample/absorbance of corresponding untreated sample. In other experiments, LNCaP cells were cultured under standard culture conditions and, at ∼80% confluency, were treated with PI-3K inhibitor LY294002, EGF, and IP6 either alone or LY294002 plus EGF and IP6 plus EGF for 12 or 24 hours in serum-free medium. After these treatments, cells were harvested and processed for the analysis of apoptotic DNA fragments using Cell Death Detection ELISA^plus kit, as detailed above.

Densitometry and Statistical Analysis

Autoradiograms and immunoblots were scanned with Adobe Photoshop 6.0 (Adobe Systems, Inc., San Jose, CA). The density for each band in immunoblotting, binding, and kinase activity assays was analyzed using the Scion Image program, National Institutes of Health (Bethesda, MD). The numerical data discussed in the Results section for an observed change in a specific molecule are based on the densitometric analyses of bands in different studies. In each case, these numbers and associated discussions are based on the observations from two to three independent experiments. Statistical significance of differences between control and treated samples was calculated by Student's t test (SigmaStat 2.0; Jandel Scientific, San Rafeal, CA). P values of less than .05 were considered statistically significant.

Results

Effect of IP6 on Growth and Cell Cycle Progression of LNCaP Cells

First, studies were performed to assess the effects of IP6 on LNCaP cell growth. The cells were seeded and treated 24 hours later with varying doses of IP6 for 24 or 48 hours, and the total number of cells was counted manually by hemocytometer. As shown in Figure 1, treatment of cells with IP6 resulted in a moderate to strong cell growth inhibition at both 24 and 48 hours of treatment. Overall, 0.5 to 4 mM IP6 doses resulted in a 23%, 19%, 29%, and 58% decrease (P < .01 to P < .001) in LNCaP cell growth following 24 hours of treatment, and 44%, 50%, 38%, and 63% growth inhibition (P < .01 to P < .001) after 48 hours of treatment, respectively (Figure 1).

Figure 1.

Effect of IP6 on LNCaP cell growth. Cells were plated at 5000 cells/cm² in 60-mm plates under standard culture conditions and, 24 hours later, fed with fresh medium and treated with various doses of IP6 as detailed in Materials and Methods section. After 24 and 48 hours, cells were collected by a brief trypsinization and counted in duplicate with a hemocytometer. The data shown are mean ± SE of three independent plates, which were reproducible in an additional independent experiment.

Because cell cycle progression is an essential event for cellular growth, based on our results showing that IP6 causes cell growth inhibition, we next examined whether this effect of IP6 is mediated through an alteration in a specific phase of the cell cycle progression. For this experiment, identical IP6 treatments were performed as for cell growth study, and cell cycle progression was studied by FACS analysis following saponin/PI staining. As shown in Figure 2, IP6 treatment for 24 hours resulted in a moderate to strong increase in G1 cell population in a dose-dependent manner; however, it showed only moderate response following 48 hours of similar treatment. Compared to controls showing 66.5% cells in G1 phase at 24 hours, IP6 treatment at 0.5, 1, 2, or 4 mM doses resulted in 69.2 (P < .05), 70.9 (P < .01), 74.9 (P < .01), and 81.9% (P < .001) cells in the G1 phase of the cell cycle, respectively (Figure 2A). The observed accumulation of the cells in G1 phase by IP6 was accompanied by a decrease in both S phase (Figure 2B) and G2/M phase (Figure 2C) in a dose-dependent manner. Because we did not observe any affect of IP6 on cell cycle progression following 48 hours of its treatment, an additional experiment was next conducted at early time points to seek a time kinetics of G1 arrest by IP6. As shown in Figure 2D, IP6 treatment of cells at 1 and 2 mM doses, which showed a strong G1 arrest in 24 hours of treatment (Figure 2A), resulted in no changes following 6 hours of treatment, but caused a G1 arrest in cell cycle progression at 12 and 24 hours of its treatment, which was consistent with a decrease in both S and G2/M phase cell populations (Figure 2, E and F), as observed in the first experiment (Figure 2, B and C). Together, these results suggested that IP6 causes a strong G1 arrest in the cell cycle progression of LNCaP cells, and that such an affect is evident as early as 12 hours of its treatment. These observations also suggested that IP6-caused G1 arrest might possibly be involved in its growth-inhibitory efficacy in LNCaP cells.

Figure 2.

Effect of IP6 on cell cycle progression of LNCaP cells. Cells were cultured under standard conditions and, in the first experiment (A–C), were treated with 0.5 to 4 mM IP6 for 24 and 48 hours. In the second experiment (D–F), under similar conditions, cells were treated with 1 and 2 mM IP6 for 6, 12, and 24 hours. At the end of each treatment, cells were collected after a brief incubation with trypsin-EDTA followed by processing for cell cycle phase distribution, as detailed in Materials and Methods section. In both experiments, the data shown are mean ± SE of three independent plates, which were reproducible in an additional independent experiment.

Effect of IP6 on G1 Cell Cycle Regulators in LNCaP Cells

The levels, interactions, and kinase activities of CDK cyclins drive an overall cell cycle progression, where G1 to S phase transition is mediated by CDK4/cyclin D1 at early G1 and by CDK2/cyclin E during late G1 to S phase [36–40,44]. The other major regulator of cell cycle transitions is CDKI, whose level and interaction with CDK cyclin govern a controlled (in normal cell) or an uncontrolled (in transformed and cancerous cells) cell cycle progression [36–40,44]. The two important CDKIs are Cip1/p21, which regulates all phases of the cell cycle, and Kip1/p27, which specifically controls G1 phase [40–44]. Based on our findings showing a moderate to strong increase in G1 phase cell population following IP6 treatment of cells as early as 12 hours and sustained up to 24 hours, we next assessed the mechanism of this affect of IP6 under similar treatment conditions. First, studies were performed to assess whether IP6 causes an alteration in CDKIs Cip1/p21 and Kip1/p27. As shown in Figure 3, compared to controls, IP6 treatment of cells resulted in a moderate to strong increase in the protein levels of both Cip1/p21 (Figure 3A) and Kip1/p27 (Figure 3B) in a dose-dependent manner, where 12 hours of treatment time showed more induction in these CDKIs compared to 24 hours of exposure. In other studies assessing assessing the effect of IP6 on CDK and cyclin levels, as shown in Figure 3, C and D, IP6 treatment of LNCaP cells showed a moderate decrease in CDK2 protein levels (∼20–30% decrease compared to control), but caused a stronger decrease in CDK4 levels (∼40–50% decrease compared to control) at all doses and time points studied. A similar trend was also observed for cyclins, where IP6 treatment of cells resulted in a moderate decrease in cyclin E levels (∼20–30%; Figure 3E) but a relatively stronger decrease in cyclin D1 levels (∼20–40%; Figure 3F) at all doses and time points studied; actin blot (Figure 3G) confirmed protein loading.

Figure 3.

Effect of IP6 on G1 cell cycle regulators in LNCaP cells. Cells were cultured under standard conditions and, at 60% confluency, treated with different doses of IP6 (1, 2, and 4 mM) for 12 and 24 hours. After these treatments, cell lysates were prepared in nondenaturing lysis buffer and subjected to SDS-PAGE followed by Western blot analysis, as detailed in Materials and Methods section. Membranes were probed with anti-Cip1/p21 (A), anti-Kip1/p27 (B), anti-CDK2 (C), anti-CDK4 (D), anti-cyclin E (E), anti-cyclin D1 (F), or anti-actin (G) antibody followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. Different treatments are as labeled in the figure.

Effect of IP6 on CDKI-CDK Binding and CDK Kinase Activity in LNCaP Cells

Based on the findings showing that IP6 strongly induces CDKIs Cip1/p21 and Kip1/p27 protein expression in LNCaP cells, and the reports showing that such an induction in CDKIs results in an increase in their interaction with CDKs leading to a decrease in CDK kinase activity [36,44], we next assessed whether IP6-caused increase in both Cip1/p21 and 2Kip1/p27 also leads to an increase in their interactions with CDK2 and/or CDK4. To assess the effect of IP6 on these bindings, cell extracts prepared under nondenaturing conditions and employed above for Western immunoblotting of CDKIs, CDKs, and cyclins were subjected to immunoprecipitation using Cip1/p21 or Kip1/p27 antibody, and immunoprecipitates were run on SDS-PAGE followed by blotting. In the case of both Cip1/p21 and Kip1/p27, membranes were probed with anti-CDK2 and anti-CDK4 antibodies. As shown in Figure 4A, compared to controls, IP6 treatment of cells resulted in an increased binding of Cip1/p21 with both CDK2 and CDK4; similar results were also evident for Kip1/p27 showing an increased binding with both CDK2 and CDK4 following IP6 treatments (Figure 4A). Together, these results suggest that an increased interaction between induced levels of CDKIs (by IP6) with CDKs plays an important regulatory role in possibly inhibiting CDK kinase activity, leading to a G1 arrest in the cell cycle progression of LNCaP cells.

Figure 4.

Effect of IP6 on CDKI-CDK binding and CDK kinase activity in LNCaP cells. Cells at 60% confluency under standard culture conditions were treated with different doses of IP6 for 12 or 24 hours, and cell lysates were prepared in nondenaturing lysis buffer as detailed in Materials and Methods section. For CDKI-CDK bindings (A), Cip1/p21 or Kip1/p27 was immunoprecipitated from the total cell lysates and subjected to SDS-PAGE followed by immunoblotting. The membranes in both cases were probed with anti-CDK2 and anti-CDK4 antibodies followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. For CDK2 kinase activity (B), CDK2 was immunoprecipitated from the total cell lysates and subjected to kinase assay in the presence of [γ-³²P] ATP and histone H1 as substrate, as detailed in Materials and Methods section. Samples were then subjected to SDS-PAGE followed by gel drying and autoradiography. For CDK4 kinase activity (C), CDK4 was immunoprecipitated from the total cell lysates and subjected to kinase assay in the presence of ATP and Rb fusion protein as substrate, as detailed in Materials and Methods section. Samples were then subjected to SDS-PAGE followed by immunoblotting. The membranes were probed with phosphoserine-specific Rb antibodies followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. Different treatments are as labeled in the figure.

To support the above suggestion, cell lysates prepared from control and IP6-treated cells were subjected to immunoprecipitation with anti-CDK2 or anti-CDK4 antibody followed by kinase assays. As shown in Figure 4B, compared to control, IP6 treatment of cells resulted in a strong decrease (∼80%) in CDK2 kinase activity as evidenced by a reduction in phosphorylation of histone H1 used as substrate. Similarly, IP6 also showed an inhibition in CDK4 kinase activity as observed by a reduction in Ser780 and Ser807/811 phosphorylation of Rb fusion protein used as substrate (Figure 4C).

IP6 Increases Hypophosphorylated Rb Levels by Decreasing Rb Phosphorylation at Serine Residues in LNCaP Cells

An active CDK phosphorylates Rb at five different serine residues and a threonine residue, releasing an E2F family of transcription factors from the Rb-E2F complex that induces expression of a number of genes required for S phase transition [45–47]. Hyperphosphorylated Rb (ppRb) exerts most of its cell cycle transition effects in a specific time frame during the first two thirds of the G1 phase, where cells make most decisions for proliferation versus quiescence [45–47]. Based on our results showing that IP6 strongly induces CDKI levels and their increased interaction with CDKs, causing a strong decrease in the kinase activity of the latter, we next assessed the effect of IP6 treatment on total Rb protein levels, as well as any change in ppRb versus hypophosphorylated Rb (pRb). As shown in Figure 5A, IP6 treatment of LNCaP cells resulted in an increase in the amount of total Rb (ppRb plus pRb) at both 1 and 2 mM doses following both 12 and 24 hours of treatment; however, this increase in total Rb levels was due to an increase only in pRb levels (fast-migrating lower band in the immunoblots) following IP6 treatment of the cells (Figure 5A). Based on these results showing a strong increase in pRb levels by IP6 in LNCaP cells, we next assessed whether this effect is due to a change in Rb phosphorylation at specific Ser phosphorylation sites. Employing phospho-specific Rb antibodies in immunoblotting, we observed that IP6 strongly decreases the levels of Rb phosphorylated at Ser780 (Figure 5B) and Ser807/811 (Figure 5C); the equal protein loading in these blots was conformed by actin immunoblotting (blot shown just below Figure 5C).

Figure 5.

Effect of IP6 on total Rb phosphorylation, serine residue-specific Rb phosphorylation, and different E2F levels in LNCaP cells. Cells were cultured under standard conditions and, at 60% confluency, treated with different doses of IP6 (1 and 2 mM) for 12 and 24 hours. After these treatments, cell lysates were prepared in nondenaturing lysis buffer and subjected to SDS-PAGE followed by Western blot analysis, as detailed in Materials and Methods section. Membranes were probed with anti-total Rb (A), anti-phosphoserine780-specific Rb (B), anti-phosphoserine807/811-specific Rb (C), anti-E2F1 (D), anti-E2F2 (E), anti-E2F3 (F), anti-E2F4 (G), anti-E2F5 (H), or anti-actin antibody followed by peroxidaseconjugated appropriate secondary antibody and visualization by ECL detection system. Different treatments are as labeled in the figure.

IP6 Decreases Transcription Factor E2F Levels in LNCaP Cells

Transcription factor E2F family members (E2F1–E2F5) play an important role in cell cycle progression following a growth signal that phosphorylates Rb, releasing E2Fs free from Rb-bound form [45,48–51]. Furthermore, it is the levels of E2Fs that play a detrimental role in driving cell cycle progression even in the free form [48–51]. Recently, it has also been shown that knocking down the levels of cell cycle regulators upstream of E2Fs is sufficient to inhibit E2F expression as well as cell growth [52]. Based on these reports and our findings that IP6 strongly modulates CDKI-CDK-cyclin levels, CDKI-CDK interaction, and the kinase activity of CDKs together with a strong increase in hypoRb levels, we next assessed the effect of IP6 on E2F protein levels. As shown in Figure 5, compared to controls, IP6 treatment of LNCaP cells resulted in a moderate to strong decrease in the protein levels of different E2Fs. In case of E2F1, IP6 showed a ∼50% decrease following 24 hours of treatment (Figure 5D); however, almost no change was observed in E2F2 (Figure 5E) and E2F3 (Figure 5F) protein levels following IP6 treatments of cells. In case of E2F4, IP6 treatment for 24 hours resulted in ∼50% decrease only at higher dose used in the study, but did not show any observable changes at 12 hours of treatment (Figure 5G). Similarly, higher IP6 dose (2 mM) and longer treatment time (24 hours) also resulted in ∼70% decrease in E2F5 protein levels (Figure 5H). When the same blots were reprobed with anti-actin antibody, they confirmed equal protein loading (lower panel in Figure 5H).

Effect of IP6 on Apoptosis Induction in LNCaP Cells

In addition to constitutively active mitogenic signaling as well as alterations in cell cycle regulators' expression and function causing deregulated cell cycle progression, a loss of apoptotic response due to constitutively active cell survival and anti-apoptotic cascades are major hallmarks of various human malignancies including PCA [36]. In this regard, human prostate carcinoma LNCaP cells are an excellent example as well as a model system where constitutively active survival factor Akt plays an important role in their survival even in this absence of growth factors [53,54]. These observations suggest that the agents that could induce apoptotic death of cancer cells could be effective in both cancer prevention and intervention. Based on our studies showing strong efficacy of IP6 in LNCaP cells, we next assessed whether this phytonutrient also induces apoptotic death of LNCaP cells. As shown in Figure 6, treatment of cells with IP6 at varying doses and time periods resulted in a dose-dependent and time-dependent apoptotic death at 12, 24, and 48 hours. The time points earlier (1, 3, and 6 hours) than 12 hours were also studied in these experiments, but were not effective in causing apoptosis induction (data not shown). Overall, IP6 treatment at 1 mM dose for 12 to 48 hours resulted in 1.6- to 2.2-fold increase (P < .05 to P < .01) in EF, a measure of apoptosis, compared to controls (Figure 6). Comparable effects of IP6 were also evident at 2 mM dose; however, its treatment at 4 mM dose for 12, 24, and 48 hours resulted in a 2.4-, 5.0-, and 7.0-fold increase in apoptosis over control, respectively (Figure 6).

Figure 6.

Effect of IP6 on apoptotic induction in LNCaP cells. Cells were cultured under standard conditions and treated with IP6 at 1, 2, or 4 mM dose for various time points (12–48 hours). After these treatments, cells were harvested and processed for the analysis of apoptotic DNA fragments using Cell Death Detection ELISA^plus kit and following the step-by-step protocol provided by the manufacturer, as detailed in Materials and Methods section. The data shown are mean ± SE of three independent samples, which were reproducible in an additional independent experiment.

Effect of IP6 on Bax and Bcl2 Levels and Their Ratio in LNCaP Cells

The Bcl2 family includes both anti-apoptotic (e.g., Bcl2 itself) and pro-apoptotic (e.g., Bax) proteins, and the levels, interaction, ratio, and translocation of different members in this family determine the overall pro-apoptotic or anti-apoptotic fate of the cell following an agent's treatment [55,56]. Based on our data showing that IP6 causes apoptotic death of LNCaP cells, we next assessed whether it is associated with alterations in Bax and Bcl2 levels as well as Bax versus Bcl2 ratio where an increase in this ratio favors apoptosis [55,56]. As shown in Figure 7A, IP6 treatment of cells at various doses (1–4 mM) for 12, 24, and 48 hours did not show a noticeable alteration in Bax protein levels, but resulted in a moderate to strong decrease in Bcl2 levels (Figure 7B). The IP6 doses used in the study caused ∼60% to 70%, 20% to 80%, and 60% to 70% decrease in Bcl2 levels following 12, 24, and 48 hours of treatments, respectively (Figure 7B). Actin blotting confirmed the equal protein loading (Figure 7C). Densitometric analysis of Bax and Bcl2 immunoblots, followed by a calculation of Bax/Bcl2 ratio, showed that compared to controls, IP6 treatments for 12, 24, and 48 hours result in 2.2- to 3.3-fold, 1.5- to 5-fold, and 2.4- to 3.3-fold increase in this ratio, respectively (Figure 7D), which possibly is one of the mechanisms of the strong apoptotic effects of IP6 in LNCaP cells observed in the present study (Figure 6).

Figure 7.

Effect of IP6 on Bax and Bcl2 levels and their ratio on LNCaP cells. Cells were cultured under standard conditions and, at 60% confluency, treated with different doses of IP6 (1, 2, and 4 mM) for 12, 24, and 48 hours. After these treatments, cell lysates were prepared in nondenaturing lysis buffer and subjected to SDS-PAGE followed by Western blot analysis, as detailed in Materials and Methods section. Membranes were probed with anti-Bax (A), anti-Bcl2 (B), or anti-actin (C) antibody followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. Different treatments are as labeled in the figure. For determining Bax/Bcl2 ratio (D), immunoblots were scanned and arbitrary densitometric values for Bax and Bcl2 were employed in the final calculations after correction for actin.

Effect of IP6 on Constitutively Active Survival Factor Akt and Associated Apoptosis in LNCaP cells

As mentioned earlier, survival factor Akt is constitutively active in LNCaP cells, which make them survive even in the absence of growth factors [53,54]. A couple of studies in recent years have shown that a selective inhibition of PI3K causes inhibition of constitutively active Akt in LNCaP cells and induces their apoptotic death; however, a cotreatment with growth factor partially reverses the apoptotic response of PI3K inhibitors by activating other survival pathways [53,54]. Based on these studies and our results showing that IP6 causes apoptotic death of LNCaP cells, we next assessed whether this agent also inhibits constitutively active survival factor Akt and that such an affect is associated with apoptosis response. The LNCaP cells were treated with a PI3K inhibitor LY294002 or IP6 in serum-free medium as a function of time, and total cell lysates were analyzed for phospho-Akt and total Akt levels. Compared to LY294002 showing a complete inhibition in Akt phosphorylation as early as 3 hours after treatment, IP6 at 2 and 4 mM doses did not show any noticeable response at this early time point (data not shown). However, similar treatments for 6 and 12 hours resulted in a strong decrease in phospho-Akt levels at 4 mM IP6 dose without any effect at 2 mM dose; the LY294002 PI3K inhibitor showed complete inhibition at these time points, too (Figure 8A). The observed decrease in phospho-Akt by IP6 and LY294002 was not due to an overall decrease in total Akt, except in the case of LY294002 treatment for 12 hours (Figure 8B).

Figure 8.

Effect of IP6 on constitutively active Akt and associated apoptosis in LNCaP cells. Cells were cultured under standard conditions, and treated with PI3K inhibitor LY294002 at 25 µM dose, or different doses of IP6 (2 and 4 mM) for 6 and 12 hours under serum-free medium condition. After these treatments, cell lysates were prepared in nondenaturing lysis buffer and subjected to SDS-PAGE followed by Western blot analysis, as detailed in Materials and Methods section. Membranes were probed with anti-phospho-Akt (A) or anti-total Akt (B) antibody followed by peroxidase-conjugated appropriate secondary antibody and visualization by ECL detection system. Different treatments are as labeled in the figure. In other experiments assessing apoptosis induction in LNCaP cells (C), LNCaP cells were cultured under standard culture conditions and treated with PI3K inhibitor LY294002 (25 µM), EGF (100 ng/ml), and IP6 (4 mM) either alone or LY294002 plus EGF and IP6 plus EGF for 12 or 24 hours in serum-free medium. After these treatments, cells were harvested and processed for the analysis of apoptotic DNA fragments using Cell Death Detection ELISA^plus kit, as detailed in Materials and Methods section. The data shown are mean ± SE of three independent samples, which were reproducible in an additional independent experiment.

Based on the observation that IP6 at higher dose reduces phospho-Akt levels similar to PI3K inhibitor, we next assessed its association with apoptosis induction. In case of PI3K inhibitors, because cotreatment with growth factors such as EGF partially reverses their apoptotic response, we also employed similar experimental design to assess whether IP6 acts through similar a pathway(s). As shown in Figure 8C, compared to controls, LY294002 treatment of cells for 12 and 24 hours resulted in four-fold increase (P < .001) in EF; however, a cotreatment with EGF reduced (P < .01 at 12 hours and P < .05 at 24 hours, for LY294002 versus EGF plus LY294002) the apoptotic response of LY294002 at both time points studied. In case of IP6 compared to controls, it showed a ∼2.2- to 2.5-fold increase (P < .05) in EF, which, unlike LY294002, was not reduced by similar cotreatments with EGF (Figure 8C). In fact, EGF plus IP6 treatment showed a further increase in EF (2.4- to 3.8-fold, P < .05 compared to controls), although it was not statistically significant when compared to IP6 alone (Figure 8C). Together, these results suggest that IP6 inhibits multiple components of mitogenic and survival pathways compared to selective PI-3K inhibitors such as LY294002 used in the present study.

Discussion

The major findings in the present study are that IP6 inhibits androgen-dependent human prostate carcinoma LNCaP cell growth possibly through a G1 arrest in cell cycle progression, and causes their apoptotic death. In this regard, it is important to emphasize here that whereas IP6 showed a significant cell growth inhibition at all the doses (0.5–4 mM) and time points (24 and 48 hours) employed in the present study, its effect on a G1 arrest was not evident earlier than 12 hours of treatment and was not sustained at 48 hours. These observations suggest that, in part, a G1 arrest by IP6 following 12 hours of its treatment drives the LNCaP cells to a resultant growth inhibition and apoptotic death at both early and late time points, and that other molecular mechanisms such as an increased Bax/Bcl2 ratio and inhibition in constitutively active Akt contribute additionally to its strong efficacy in causing both growth inhibition and apoptotic death of LNCaP cells at later time points. The doses of IP6 (0.5–4 mM) showing strong efficacy in the present study are those employed in the earlier cell culture studies (up to 5 mM IP6) by the present and other authors [10,19–22]. The physiological and/or pharmacological significance of these levels of IP6 in cell culture studies needs to be studied and established clinically in the future; however, when these cell culture concentrations of IP6 (with only single treatment) are compared with published literature showing its anti-cancer activity in animal models by using up to 15 mM dose in continuous feeding in drinking water (reviewed in Refs. [10,26,34]), the concentrations in cell culture studies are much lower, clearly establishing their relevance in defining the molecular mechanism of IP6 efficacy at pharmacological doses at least in preclinical anti-cancer studies.

The regulation of cell cycle progression in eukaryotes is controlled by CDKs, the activities of which are regulated by interactions with their regulatory subunits (cyclins) and CDKIs [36–40,44]. Cyclins and their cognate CDK catalytic subunits noncovalently form complexes to produce the CDK holoenzyme, which is activated by phosphorylation of catalytic subunits through CDK-activating kinase [36–40,44]. CDK activities are specific to the distinct phases of the cell cycle, where CDK4 complexed with D-type cyclins is responsible for G1 phase progression; a complex of CDK2-cyclin E is responsible for G1-S phase transition; CDK2-cyclin A is required for S phase progression; and a complex of CDK1 (or cdc2)-cyclin B is needed for G2-M phase transition [36–40,44]. CDK4-cyclin D1 and CDK2-cyclin E regulate the phosphorylation of Rb protein and thus the level of free E2Fs, thereby controlling the progression from G1 to S phases of cell cycle [36–40,44]. The controlled cell cycle progression is regulated, in part, by CDKIs (Cip1/p21, Kip1/p27, and Kip2/p57) in response to both growth-stimulating and growth-inhibitory signals [36–44]. CDKIs have been shown to inhibit the kinase activity of CDK-cyclin complexes that modulates Rb phosphorylation events, which are essential for the transition at various cell cycle checkpoints [36–44]. The unphosphorylated forms of Rb and its related proteins have been shown to inhibit cell proliferation by sequestering the E2F family of transcription factors [44–47]. The results obtained in our present study are consistent with these reports related to the biologic role of CDKIs, CDKs, and cyclins in driving cell cycle progression. For example, we observed that IP6 causes a strong increase in Cip1/p21 and Kip1/p27 levels, together with their increased interaction (binding) with CDK2 and CDK4, and a strong inhibition in CDK2 and CDK4 kinase activity. The IP6 also caused a decrease in CDK4, cyclin D1, and different E2Fs levels as well as Rb phosphorylation. All these alterations in cell cycle regulators by IP6 involve those molecules that govern a G1 to S phase transition and, therefore, the net biologic outcome observed was indeed G1 arrest and growth inhibition of LNCaP cells by IP6.

In human PCA patients, a decreased p27 expression has been shown to be associated with aggressive phenotype of prostatic carcinomas [57]. It has also been observed that the loss of p21 function results into the failure of irradiation response in PCA patients [57]. Mechanistic studies have shown that a decrease in the expression and/or function of these CDKIs could be mediated by DNA methylation, posttranscriptional modification, and, most commonly, posttranslational modification [58,59]. At posttranslational level, altered subcellular localization of p27 and degradation by the ubiquitin-proteosome pathway are observed in a number of human cancers [60–62]. An important role of CDKI expression and function in human malignancies including PCA is further supported by recent studies showing that overexpression of Kip1/p27 or Cip1/p21 using adenoviral approaches inhibits the growth of different types of tumors [63–68]. Together, these findings suggest that Cip1/p21 and Kip1/p27 are important molecular targets in the etiology of various malignancies including PCA, and that the agents that could induce their functional levels should be effective in controlling human malignancies including PCA. In accord with these suggestions, the results of our present study showing a strong increase in Cip1/p21 and Kip1/p27 protein levels by IP6 in LNCaP cells might be of high significance for PCA control. More studies, however, are needed to define the mechanisms of these CDKI induction by IP6 and to establish a cause-versus-effector role of their induction in G1 arrest and growth inhibition of LNCaP cells by IP6.

Several evidences suggest that apoptosis frequently occurs in cells at the G1 phase of the cell cycle, and that an arrest in late G1 or S phase could accelerate apoptosis [69–71]. Accordingly, the proteins responsible for the G1 or G1/S phase arrest, such as Kip1/p27 and Cip1/p21, could be the logical targets in apoptotic events. In this regard, overexpression of these CDKIs in cancer cells through adenoviral vectors is shown to block all cyclin/CDK activities and to induce both cell cycle arrest and apoptosis [71–77]. Consistent with these reports, we also observed that IP6 causes an induction in Cip1/p21 and Kip1/p27, and inhibits CDK kinase activity together with cell cycle arrest and apoptosis induction, suggesting a possible role of IP6-caused CDKI induction in its apoptotic activity.

The role of Bcl2 family members and their interplay are well studied and established in apoptosis induction [55]. With regard to Bcl2/Bax in apoptosis process, Bcl2 is an anti-apoptotic molecule whereas Bax supports apoptosis. A homodimerization of Bcl2 is an anti-apoptotic effect; however, recent studies have shown that serine/threonine phosphorylation of Bcl2 inactivates it, which favors apoptosis response [55,56]. When it heterodimerizes with Bax, Bcl2 loses its anti-apoptotic function and causes apoptosis; the better scenario for apoptosis is the homodimerization of Bax [55,56]. Consistent with these reports, we observed that IP6 treatment of LNCaP cells results in a decrease in the levels of Bcl2 without any noticeable changes in Bax levels; however, Bcl2 decrease was sufficient for a substantial increase in Bax/Bcl2 ratio, a situation that favors apoptosis [55,56]. Recent studies have also established the role of Bcl2 in mitochondrial damage causing apoptotic death [55,56]. Regarding Bax, published studies demonstrate that the pro-apoptotic effects of Bax are tightly linked to its caspase activation-dependent redistribution from the cytosol to the mitochondrial membrane, which promotes dissipation of mitochondrial membrane potential, cytochrome C release, and subsequent caspase activation [55,56,78]. Based on these studies and our data showing the effect of IP6 on Bcl2 decrease and Bax/Bcl2 ratio increase, more studies are needed in the future to assess the effect of IP6 on Bcl2 family members and their interplay and subcellular distribution in IP6-induced apoptosis. Additional studies are also needed in the future to define whether IP6-mediated alterations in Bcl2 levels and Bax/Bcl2 ratio activate mitochondrial damage, leading to cytochrome C release and caspase activation in its overall apoptotic response in LNCaP cells.

The requirement for membrane-associated cell survival, and mitogenic and/or anti-apoptotic signaling is a universal characteristic of cells [79–82]; however, both genetic and epigenetic alterations lead to a persistent autocrine stimulation of cells by secreted growth factors, which has been implicated in a wide variety of human malignancies including PCA [36,44,83]. PI3K is a ubiquitous lipid kinase composed of a regulatory (p85) and a catalytic (p110) subunit, and is involved in receptor signaling through tyrosine kinase receptors regulating various important cellular processes such as cell survival, growth, transformation, and so on [79–81]. Phosphatase and tensin homologue (PTEN), a tumor-suppressor gene, antagonizes PI3K activity by dephosphorylating the D3 phosphate group of lipid second messenger. Therefore, PTEN functions as a negative regulator of PI3K/Akt pathway [79,82]; however, it is mutated or nonfunctional in many cancer cells including human PCA LNCaP cells [53,54]. PI3K catalyzes the phosphorylation of phosphoinositol-4 phosphate and phosphoinositol-4,5 phosphate at D3 position, and activates various downstream elements including Akt/protein kinase B; phosphorylation at Ser473 and Thr308 is inducible and essential for Akt activation [84,85]. Inhibition of pro-apoptotic signals and induction of survival signals lead to Akt activation, which may also contribute to malignant transformation [84,85]. Both Cip1/p21 and Kip1/p27 have been shown as targets of Akt for their phosphorylation and subsequent degradation, leading to progression of cell cycle [86]. Bad and caspase 9 are other downstream targets of Akt, the phosphorylation of which mediates anti-apoptotic effect mainly through inhibition of mitochondrial apoptosis through Akt signaling [85,87].

Together, the above studies suggest that inhibition of constitutively active Akt signaling could be a potential target to control PCA cell growth and induce apoptotic death. In this regard, recent studies have shown that PI3K pathway is a dominant growth factor-activated cell survival pathway in human PCA LNCaP cells, where treatment of LNCaP cells with PI3K inhibitors under serum-free conditions results in apoptotic death, and that serum, several growth factors, or androgen treatment, together with PI3K inhibitors, blocks the apoptotic response of PI3K inhibitors [53,54]. In these studies, an inhibition of MEK1 activation by PD98059 did not cause apoptotic death [53,54]. Together, these studies suggested that inhibition of PI3K activation, followed by AKT activation, is sufficient for apoptotic death of LNCaP cells only if other mitogenic responses are not activated. Furthermore, these studies also emphasize that inhibiting one signaling pathway is possibly not enough to control the growth of PCA cells and to induce their apoptotic death, and that agents that exert diversified inhibitory effects on both mitogenic and cell survival signaling in PCA cells, leading to their growth inhibition and apoptotic death, are needed. Consistent with these reports, in our studies, we found that IP6 inhibits constitutively active Akt in LNCaP cells together with their apoptotic death, and that cotreatment with EGF does not reverse IP6-caused apoptotic death, in contrast to PI3K inhibitor where apoptosis is partially reversed by EGF cotreatment. These observations suggest that IP6 has diversified inhibitory effects on different mitogenic and survival signalings, causing both cell cycle arrest and apoptotic death of LNCaP cells. Because Akt activation also targets CDKIs phosphorylation and degradation leading to cell cycle transition, it is plausible that IP6 inhibits Akt activation, thereby causing CDKI accumulation followed by cell cycle arrest in LNCaP cells as one of its mechanisms of efficacy, other than its apoptotic effect through inhibition of Akt activation causing caspases activation and molecular alterations. In the future, more studies are needed in these directions to further define the mechanisms of IP6 efficacy in PCA cells.

Overall, the findings in the present study revealed the anticancer efficacy of IP6 against hormone-dependent human PCA LNCaP cells, which involves inhibition of cell cycle progression in G1 phase and an induction of apoptosis. IP6 upregulates CDKIs accompanied by its increased binding with CDKs, causing a strong decrease in their kinase activity (Figure 9). This further leads to a decrease in the hyperphosphorylated form of Rb with a concomitant increase in its hypophosphorylated form, causing an increased binding of E2Fs with pRb in the cytoplasm, thereby reducing their transcriptional activity for growth-responsive genes. This effect is also augmented by a decrease in some E2F levels by IP6. Furthermore, IP6 inhibits Akt activation and showed an overall increase in Bax/Bcl2 ratio, which might be prominent mechanisms for the apoptotic death of PCA cells (Figure 9). However, it would be interesting to investigate in the future whether inhibition of Akt signaling by IP6 has any role in the upregulation of CDKIs in LNCaP cells. Moreover, the translation of mechanism-based in vitro anticancer efficacy of IP6 into in vivo condition in future studies would be helpful in reducing the risk, as well as management and control, of PCA in humans.

Figure 9.

Proposed mechanisms of anticancer efficacy of IP6 associated with a G1 arrest in cell cycle progression and apoptosis induction in human prostate carcinoma LNCaP cells.

Abbreviations

PCA

prostate cancer

Rb

retinoblastoma protein

pRb

hypophosphorylated Rb

ppRb

hyperphosphorylated Rb

CDKI

cyclin-dependent kinase inhibitor

CDK

cyclindependent kinase

IP6

inositol hexaphosphate