A polyphenolic fraction from grape seeds causes irreversible growth inhibition of breast carcinoma MDA-MB468 cells by inhibiting mitogen-activated protein kinases activation and inducing G(1) arrest and differentiation

In recent years, significant emphasis is being placed on identifying natura lly occurring cancer preventive and interventive agents. In this regard, a polyphenolic fraction isolated from grape seeds (hereafter referred as GSP) has recently been shown by us and others to prevent tumorigenesis in mouse skin models. Chemical analysis of GSP has shown that it is largely constit uted with procyanidins that are strong antioxidants. Breast cancer is the m ost common invasive malignancy and the second leading cause of cancer-relat ed deaths in United States women, Accordingly, here we investigated the eff ect of GSP on mitogenic signaling and regulators of cell cycle and apoptosi s as molecular targets for the growth arrest, apoptotic death, and/or diffe rentiation of estrogen-independent MDA-MB468 human breast carcinoma cells. Treatment of cells with GSP (at 25-, 50-, and 75-mu g/ml doses for 1-3 days ) resulted in a highly significant inhibition (90% to complete, P < 0.001) of constitutive activation of mitogen-activated protein kinase (MAPK)/extra cellular signal-regulated protein kinase1/2 in a dose-dependent manner afte r 72 h of treatment. Whereas GSP treatment of cells did not show a conclusi ve effect on MAPK/JNK1 activation, a moderate to highly significant inhibit ion (15-70%, P < 0.1-0.001) of constitutive activation of MAPK/p38 was also observed in a dose-dependent manner as early as 24 h of GSP treatment. GSP -treated cells also showed a strong induction (1.7-2.7 fold, P < 0.001) of cyclin-dependent kinase inhibitor Cip1/p21 and a decrease (10-50%, P < 0.1- 0.001) in cyclin-dependent kinase 4, Consistent with these findings, GSP-tr eated cells resulted in their accumulation in G(1) phase of the cell cycle in a dose-dependent manner, An irreversible growth inhibition (44-88%, P < 0.001) was also observed in 50 and 75 mu g/ml GSP-treated cells in a time-d ependent manner. Additional studies assessing the biological fate of GSP-tr eated cells showed that they do not undergo apoptotic death, as evidenced b y a lack of DNA fragmentation, poly (ADP ribose) polymerase cleavage, and a poptotic morphology of the cells. A morphological change suggestive of diff erentiation was observed in GSP-treated cells that was further confirmed by a significant induction (1.7-2.6 fold, P < 0.001), in both a dose- and tim e-dependent manner, in cytokeratin 8 protein level, a marker of differentia tion. An irreversible growth-inhibitory effect of GSP possibly via terminal differentiation of human breast carcinoma cells suggests that GSP and the procyanidins present therein should be studied more extensively to be devel oped as preventive and/or interventive agents against breast cancer in huma ns.