Delphinidin induces apoptosis and growth inhibition of prostate cancer PC-3 cells via inhibition of Notch-1 and NF-   κβ/PI3κ    pathways

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Prostate cancer (PCa) is one of the most common invasive malignancies and the second leading cause of cancer related deaths in males in the United States. At present, treatment options available for the management of this disease are inadequate. One approach to control this malignancy is to slow its progression through the use of natural agents present in the diet consumed by humans. Delphinidin, a major anthocyanidin present in many pigmented fruits and vegetables possesses anti-oxidant, anti-inflammatory and anti-angiogenic properties. We first observed that delphinidin treatment (60-240 µM; 48 hrs) results in dose-dependent growth inhibition of human PCa PC-3 cells as assessed by an MTT assay. Using TUNEL assay and western blotting, we also observed induction of apoptosis as suggested by increase in TUNEL positive cells and PARP cleavage. Since Notch and NF-kB pathways are involved in cell proliferation, differentiation and apoptosis of various types of cancer cells, we hypothesized that delphinidin induced cell growth inhibition and apoptosis is mediated through inhibition of Notch-1 and/or NF-κB/PI3K pathways. We found that delphinidin treatment to PC-3 cells resulted in inhibition of constitutive expression of Notch-1 alongwith inhibition of its active cleaved form (notch intracellular domain). Over expression of Hes-1, a down stream target of Notch-1 that promotes cell proliferation by repressing cell cycle inhibitory proteins is observed in various cancer cells including prostate cancer. We observed that delphinidin treatment to PC-3 cells results in inhibition of protein expression of Hes-1 suggesting deregulation of cell cycle by delphinidin. Since it is well documented that anti-apoptotic effects of Notch-1 proteins are regulated through increased NF-κB and PI3K signaling, we next assessed the effect of delphinidin on these pathways. Our data showed that delphinidin treatment to PC-3 cells resulted in dose dependent inhibition of (i) phosphorylation of upstream kinases Iκκα, and IKKγ (NEMO) that regulate NF-κB activity, (ii) decrease in NF-κB inhibitory protein IκBα, (iii) NF-κB/p50 and NF-κB/p65 protein expression, (iv) phosphorylation of NF-κB/p65 at Ser⁵³⁶ and NF-κB/p50 at Ser⁵²⁹ and (v) NF-κB/p50 and NF-κB/p65 DNA binding activities. Delphinidin treatment to cells also resulted in inhibition of NF-κB luciferase activity suggesting inhibition of NF-κB/p65 at transcription level. Delphinidin treatment to PC-3 cells significantly inhibited p110 (catalytic) and p85 (regulatory) subunits of PI3K, an essential downstream regulator of Notch-1/NF-κB signaling. Taken together, our data suggest that delphinidin is involved in inducing apoptosis and cell growth inhibition of PC-3 cells via inhibition of Notch-1/NF-κB/PI3K signaling. It is tempting to suggest that delphinidin could be a novel therapeutic agent against PCa.