Pomegranate extract induces apoptosis in human prostate cancer cells by modulation of the IGF-IGFBP axis

Abstract

The IGF axis is critical for the regulation of apoptosis in many human cancer cell lines. Recently, potent anti-tumorigenic effects of pomegranate juice and extracts have been reported. Consequently, pomegranate has potential not only as a treatment but also as a preventative measure against certain types of cancer, including prostate. In this study, we investigated the relationship between pomegranate-induced apoptosis in human prostate cancer cells and the IGF/IGFBP system. Treatment of LAPC4 prostate cancer cells with 10microg/ml POMx, a highly potent pomegranate extract prepared from skin and arils minus seeds and standardized to ellagitannin content (37% punicalagins by HPLC), resulted in inhibition of cell proliferation and induction of apoptosis. Interestingly, co-treatment with POMx and IGFBP-3 revealed synergistic stimulation of apoptosis and additive inhibition of cell growth. Western blot analysis revealed that treatment with POMx or POMx/IGFBP-3 combination resulted in increased JNK phosphorylation, and decreased Akt and mTOR activation, consistent with a growth inhibitory, pro-apoptotic function. We also investigated the relationship between IGF-1 and pomegranate-induced apoptosis in 22RV1 prostate cancer cells. Co-treatment with 100ng/ml IGF-1 completely blocked apoptosis induction by POMx. In contrast, IGF-I failed to inhibit POMx-induced apoptosis in R(-) cells, suggesting the importance of IGF-IR. POMx-treatment decreased Igf1 mRNA expression in a dose-dependent manner indicating that its actions also involve tumor-specific suppression of IGF-1. These studies revealed novel interactions between the IGF system and pomegranate-induced apoptosis.

Figure 1. IGFBP-3 and POMx additively inhibit the growth of LAPC4 cells

LAPC4 prostate cancer cells were incubated in the presence and absence of 10 μg/ml POMx and/or 1 μg/ml IGFBP-3 for 72 h in SF media. Cell proliferation was assessed by enzymatic reduction of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), to a formazan product. n=3; significance that mean is difference from untreated control: *P<0.05, **P<0.01, ***P<0.001.

Figure 2. Synergistic apoptosis induction by IGFBP-3 and POMx

LAPC4 cells were incubated in the presence and absence of 10 μg/ml POMx and/or 1 μg/ml IGFBP-3 for 24 h in SF media. Apoptosis was assessed by ELISA for cytoplasmic histone-associated–DNA fragments. n=3; significance that mean is different from 1 (untreated control): *P<0.05, **P<0.01

Figure 3. Dose-dependent increase in JNK phosphorylation by POMx

A, LAPC4 cells were incubated with increasing concentrations of POMx in SF media for 24 h. Levels of total and phospho-JNK were determined by immunoblotting, n=3. B, LAPC4 cells were treated in the presence or absence of 10 μg/ml POMx and/or 1 μg/ml IGFBP-3 for 24 h in SF media. Levels of total and phospho-JNK were determined by immunoblotting, n=3 (upper panel).

Figure 4. Modulation of PI3 kinase/Akt/mTOR signaling by POMx

LAPC4 cells were treated in the presence or absence of 10 μg/ml POMx and/or 1 μg/ml IGFBP-3 for 24 h in SF media. A, Total and phospho-Akt (Thr308 and Ser473) were assessed by immunoblotting, n=3. B, Total and phospho-mTOR (Ser2448 and Ser2481) were assessed by immunoblotting, n=3.

Figure 5. IGF-I antagonizes POMx-induced apoptosis

A, 22RV1 human prostate cancer cells were incubated in the presence or absence of 10 μg/ml POMx and/or 100 ng/ml IGF-I for 24 h in SF media. Apoptosis was assessed by ELISA for histone-associated DNA fragments. n=3; significance that mean is different from 1 (untreated control): *P<0.05, **P<0.01. B, Apoptosis was assessed by ELISA for histone-associated –DNA-fragments in R^- cells (IGF-IR null MEFs) incubated in the presence and absence of 10 μg/ml POMx and/or 100 ng/ml IGF-I for 24 h in SF media. n=3; significance that mean is different from 1 (untreated control): *P<0.05, **P<0.01.

Figure 6. Synergistic activation of JNK phosphorylation by IGF-I and POMx

A, 22RV1 cells were incubated with increasing concentrations POMx in SF media for 24 h. Levels of total and phospho-JNK were determined by immunoblotting, Blots are representative of 3 independent experiments. B, 22RV1 cells were treated in the presence and absence of 10 μg/ml POMx and/or 100 ng/ml IGF-I for 24 h in SF media. Levels of total and phospho-JNK were determined by immunoblotting (upper panel). Blots are representative of 3 independent experiments.

Figure 7. POMx decreases Igf1 mRNA expression in a dose-dependent manner

A, 22RV1 cells were incubated with increasing concentrations POMx in SF media for 24 h. Total RNA was prepared using TRIzol Reagent. Levels of Igf1 and β-actin mRNA were determined by RT-PCR. B, 22RV1 cells were incubated with and without 1 μg/ml IGFBP-3, 100 ng/ml IGF-I and/or 10 μg/ml POMx for 24 h in SF media. Total RNA was prepared using TRIzol Reagent. Levels of Igf1 and β-actin mRNA were determined by RT-PCR.