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.

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.

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.

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.

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.

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.

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.

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.

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.

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.