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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 G₁ Arrest and Differentiation¹

Center for Cancer Causation and Prevention, AMC Cancer Research Center, Denver, Colorado 80214 [C. A., Y. S., J. Z., R. A.], and University of Colorado Cancer Center, University of Colorado Health Sciences Center, Denver, Colorado 80262 [R. A.]

In recent years, significant emphasis is being placed on identifying naturally 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 constituted with procyanidins that are strong antioxidants. Breast cancer is the most common invasive malignancy and the second leading cause of cancer-related deaths in United States women. Accordingly, here we investigated the effect of GSP on mitogenic signaling and regulators of cell cycle and apoptosis as molecular targets for the growth arrest, apoptotic death, and/or differentiation of estrogen-independent MDA-MB468 human breast carcinoma cells. Treatment of cells with GSP (at 25-, 50-, and 75-µg/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)/extracellular signal-regulated protein kinase1/2 in a dosedependent manner after 72 h of treatment. Whereas GSP treatment of cells did not show a conclusive effect on MAPK/JNK1 activation, a moderate to highly significant inhibition (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-treated cells resulted in their accumulation in G₁ 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 µg/ml GSP-treated cells in a time-dependent manner. Additional studies assessing the biological fate of GSP-treated cells showed that they do not undergo apoptotic death, as evidenced by a lack of DNA fragmentation, poly (ADP ribose) polymerase cleavage, and apoptotic morphology of the cells. A morphological change suggestive of differentiation 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 time-dependent manner, in cytokeratin 8 protein level, a marker of differentiation. 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 developed as preventive and/or interventive agents against breast cancer in humans.

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