©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

[Cell Cycle 7:11, 1648-1657; 1 June 2008]; ©2008 Landes Bioscience

1648 Cell Cycle 2008; Vol. 7 Issue 10

Cocoa-derived flavanols and procyanidins have been previously reported to exhibit anti-oxidant and anti-tumor properties. In this study, we have investigated the cellular growth inhibitory effect of chemically-synthesized procyanidin [3-O-galloyl]-(-)-epicatechin-(4β,8)-(+)-catechin-3-O-gallate (GECGC) on a variety of human cancer cell lines. Among 16 human cancer cell lines tested, GECGC selectively inhibited proliferation of a subset of human cancer cell lines, especially those of short doubling time. In contrast, all 6 normal cell lines tested including human mammary epithelial cells and skin fibroblast were resistant to GECGC’s cytotoxicity. Cell cycle analysis and apoptosis assay showed that GECGC increased sub-G1 population and increased the population of propidium iodide and Annexin V staining cells in GECGC-sensitive cell lines, suggesting that cell growth inhibition by GECGC may be mediated through both apoptotic and non-apoptotic mechanisms. Further characterization of GECGC cytotoxicity on 30 genetically modified cell lines with overexpression or depletion of key proteins involved in cell cycle regulation and signal transduction pathways suggested that GECGC-mediated cell death involves IKKα and IKKγ. Collectively, our observations indicate that synthesized GECGC has selective anti-proliferative effect on human cancer cells and warrant further evaluation as a preventive and chemo-therapeutic reagent to human malignancies.

Introduction

Polyphenols found naturally in vegetables and fruits have been reported to possess a wide range of biological properties that may help to explain the health benefits associated with diets rich in these food.1-3 For example, a grape seed proanthocyanidin extract was shown to exhibit potent anti-oxidant effects as well as anti-proliferative

properties in breast cancer and colon cancer cell lines.4 Cocoa derived flavanols and procyanidins have also been shown to possess potent antioxidant, antitumor properties and immunomodulatory functions in vitro.5-7 The flavanols found in cocoa exist as both the monomeric flavanols (-)-epicatechin and to a much lesser extent (+)-catechin, and they are structurally related oligomers known as procyanidins.8 Previous studies of cocoa flavanols and procyanidins showed 70% growth inhibition with a blockade of the cell cycle of Caco-2 cells at the G2/M phase.5 Synthetic trimeric, tetrameric, pentameric and higher oligomeric epicatechin-derived procyanidins inhibited breast cancer cell growth and induced cell death possibly through non-apoptotic mechanisms.9,10 Pentameric procyanidin has been shown to selectively inhibit growth of human breast cancer cells through regulation of site-specific phosphorylation of several cell cycle regulatory proteins including p53 and pRb.11

The molecular mechanisms underlying the anti-proliferative properties of the cocoa derived flavanols and procyanidins are largely unknown. It has been suggested that the degree of oligomerization of procyanidin correlates with its protective effects from oxidative stress.12 However, there are contradictory reports9,13 and a systematic screening of the anti-proliferative activities of the lower molecular weight oligomers is lacking. In the current study, we used synthesized procyanidin, [3-O-galloyl]-(-)-epicatechin-(4β,8)-(+)-catechin-3-O-gallate (GECGC) from Mars Inc., and its cytotoxicity was examined in 6 normal cell lines and in 16 human cancer cell lines including breast cancer, colon cancer, lung cancer, cervical cancer, ovary cancer, prostate cancer, leukemia, astrocytoma and renal adenocar-cinoma. Furthermore, the molecular signaling pathways involved in GECGC-mediated cytotoxicity were also examined.

Results

Selective growth inhibition effect of GECGC on human cancer cells. A detail description of the cells lines tested is listed in Table 1. The panel of the cell lines used in the current study repre-sent the most common human cancer types including breast cancer (MCF-7, MDA-MB-231, MDA-MB-435, T-47D and BT-20), lung cancer (NCI-H28, H520 and H596), colon cancer (HCT-116 and SW480), cervical cancer (Hela), ovary cancer (OVCAR), prostate cancer (PC-3), leukemia (K562), renal carcinoma (ACHN) and a tumor derived from central nervous system (U373MG). The doubling time of these cell lines ranges from 17.4 hr to 50 hrs

*Correspondence to: Maofu Fu; Department of Cancer Biology; Kimmel Cancer Center; Thomas Jefferson University; 233 S. 10th Street; Bluemle Life Sciences Building; Room 1050A; Philadelphia, Pennsylvania 19107 USA; Email: Annie.mathies@kimmelcancercenter.org/ Richard G. Pestell; Department of Cancer Biology; Kimmel Cancer Center; Thomas Jefferson University; 233 S. 10th Street; Bluemle Life Sciences Building; Room 1050A; Philadelphia, Pennsylvania 19107 USA; Email: Annie.mathies@kimmelcancercenter.org

Submitted: 03/19/08; Accepted: 03/21/08

Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5980

Report

Selective cytotoxicity of synthesized procyanidin 3-O-galloylepicatechin-4b, 8-3-O-galloylcatechin to human cancer cellsMin Kim,1 Xiaofang Wu,1 Insun Song,1 Maofu Fu,2,* Seo-Hee Chang,2 Michael P. Lisanti2 and Richard Pestell2,*

1Department of Oncology; Lombardi Comprehensive Cancer Center; Georgetown University School of Medicine; Washington, USA; 2Department of Cancer Biology; Kimmel Cancer Center; Thomas Jefferson University; Philadelphia, Pennsylvania USA

Key words: Cocoa flavonol, cocoa procyanidin, cytotoxicity, cancer, cell cycle, apoptosis

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

www.landesbioscience.com Cell Cycle 1649

(mean = 28.98 hrs). 5,000 to 20,000 cells per well were seeded in 96-well culture plate to ensure the cells were in exponential growth at the time of GECGC treatment. A wide range of dose titration (0, 1.6, 3.1, 6.2, 12.5, 25, 50, 75, 100 and 200 μM) of GECGC was applied in the study and the cells were incubated with or without GECGC for 12, 24 and 48 hrs. MTT assay and Cell Titer-Glo assay were used to evaluate the effects of GECGC on cellular growth. As shown in Figure 1A by MTT assay, among the 5 breast cancer cell lines tested, MDA-MB-231 showed greatest sensitivity to GECGC. About 30% growth inhibition was observed at 25 μM and at 50 μM, only 32% of cells were viable (32% viable, N = 4; p < 0.05). About 80% of MDA-MB-231 cells were killed by GECGC at 100 μM. T-47D cells showed modest sensitivity to GECGC with cell viability being 60% at 75 μM. The other 3 lines, MCF-7, MDA-MB-435 and BT-20 were more resistant to GECGC at 75 μm concentration. Colon cancer cell lines SW480 and HCT-116 were more sensitive than breast cancer cell lines to GECGC cytotoxicity (Fig. 1B). For SW480, 50% growth inhibition was seen at 12.5 μM and the cell viability was less than 20% at 50 μM. 50% growth inhibition of HCT-116 was seen at 50 μM GECGC (0.55% viable, N = 4; p < 0.05). Hela cell line and astrocytoma cell line U373MG were also sensitive to GECGC treatment. However, the ovary cancer cell line OVCAR was resistant to GECGC except at the highest concentra-tion (Fig. 1B). The lung cancer cell NCI-H520 and the leukemia cell line K562 were sensitive to GECGC treatment with growth inhibi-tion of ~80% at 50 μM GECGC. The renal adenocarcinoma ACHN cell line showed a modest response to GECGC whereas NCI-H596, NCI-H28 (lung cancer) and PC3 (prostate cancer) were relatively resistant with some reduction in cell viability at highest concentra-tion (Fig. 1C). The IC50 of GECGC in different cancer cell lines is listed in Table 2. The IC50 of GECGC was less than 50 μM in 5 out of 16 cell lines tested in this studies (Table 3), and these cell lines were therefore considered as GECGC sensitive cell lines. Most of the GECGC sensitive cell lines have a relative short doubling time (17.4–38 hr, mean = 23.6 hr). The results of 24 hrs were similar to 48 hrs. (data not shown).

Effect of GECGC on fibroblast and normal epithelial cells. In order to test the selectivity of GECGC for cancer cells, the cyto-toxicity of GECGC was examined in 6 normal noncancerous cells lines including 3 fibroblast and 3 epithelial cells derived from either lung or mammary gland (Table 1). In Figure 2, the normal cell lines tested were relatively resistant to GECGC as demonstrated by both MTT assay and Cell Titer-Glo (LUC) assay although the H568 and 184B5 showed ~20% inhibition at 100% μM. The IC50 values of GECGC in 6 normal cell lines with 5% of FBS were mostly above 100 μM (Table 4).

Tumor cytotoxicity of GECGC is not mediated by produc-tion of H2O2. Previous studies suggested that the green tea-derived catechins (-)-epigallocatechin-3-gallate (EGCG) and (-)-epigallocat-echin (EGC) may mediate apoptosis through production of H2O2.14 As research suggests that the gallate structure may contribute to the production of hydrogen peroxide in vitro, we chose to examine if in fact hydrogen peroxide was formed and if its production affected cell viability. Catalase (250 unit/ml) was added into two GECGC-sensitive cell lines, Hela and HCT-116 cells, in addition to GECGC treatment and incubated for 24 hrs. As a control, the cells was first treated with H2O2 at different concentration as indicated in

Figure 3A and C, and the cell viability was examined by MTT assay. Treatment of Hela and HCT-116 cells with H2O2 at 2 mM result in total cell death which was prevented by addition of exogenous catalase (Fig. 3A and C). The coincubation of GECGC with exog-enous catalase resulted in partial protection of Hela and HCT-116 cells from GECGC-mediated cell death (Fig. 3B snd D), suggesting that GECGC cytotoxicity might be partially mediated through auto-oxidation of GECGC leading to the generation of H2O2 in vitro. To further clarify if GECGC produces hydrogen peroxide in the cell culture medium, H2O2 levels were measured by the FOX and Amplex assays. After adding GECGC (0.1–400 μM) into water (FOX assay) or DMEM + 10% FCS (Amplex) with or without cells, the H2O2 content was analyzed within 15 min based on the detection protocol. We could not detect any significant production of H2O2 from GECGC treated medium up to 400 μM whereas the addition of exogenous H2O2 to the culture medium at different concentra-tions produced an obvious color reaction (data not shown).

Effect of GECGC on cell cycle profile. To investigate the mecha-nism of cell growth inhibition by GECGC, cell cycle analysis was performed on the GECGC-sensitive cell lines Hela and HCT-116, and the GECGC-resistant breast cancer MDA-MB-435 and skin fibroblast HS-68 cell lines. Cells were treated with GECGC at 50 μM and 200 μM for 24 hrs. Compared to the untreated cells, treat-ment of GECGC at 50 μM and 200 μM in both HCT-116 (Fig. 4A) and Hela cells (Fig. 4B) for 24 hrs resulted in an altered cell cycle profile. GECGC increased the sub-G1 population in GECGC sensi-tive cell lines and decreased the proportion of cells in the G1 phase.

Table 1 Human cancer cell lines tested

Cell lines Origin Doubling time (h)MCF-7 Breast cancer 25.4MDA-MB-231 Breast cancer 41.9T-47D Breast cancer 44.5MDA-MB-435 Breast cancer 25.8BT-20 Breast cancer 21.0Hela Cervical cancer 19.0HCT-116 Colon cancer 17.4SW480 Colon cancer 24.0U373MG Astrocytoma 28.8OVCAR Ovary cancer 34.7PC-3 Prostate cancer 27.1K562 Leukemia 19.6ACHN Kidney Adenocarcinoma 27.5NCI-H596 Lung cancer 50.0NCI-H520 Lung cancer 38.0NCI-H28 Lung cancer 19.0HUVS112D Umbilical vein fibroblast NDHS-68 Skin fibroblast 105.6184B5 Mammary Epithelial cell 38.6A-1N4 Mammary Epithelial cell NDCCD-27SK Skin fibroblast 93.6Beas-2B Lung epithelial cell ND

ND, not determined.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

1650 Cell Cycle 2008; Vol. 7 Issue 10

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

Cell debris was increased with GECGC treatment, suggesting that GECGC may induce both apoptotic and non-apoptotic cell death. However, GECGC did not significantly alter the cell cycle profile in MDA-MB-435 (Fig. 4C) or HS-68 cells (Fig. 4D), consistent with the cell proliferation studies.

Effect of GECGC on apoptosis and cytotoxicity. To further examine the mechanisms involved in GECGC-mediated cell death,

the GECGC-sensitive cell line HCT-116 and the resistant cell line MDA-MB-435 were selected for apoptosis studies by propidium iodide (PI)/Annexin-V staining and flow cytometry analysis. PI(-)/Annexin-V(+) staining (lower right quadrant) represents early apop-totic events; PI(+)/Annexin-V(+) staining (upper right quadrant) represent late apoptotic events; double negative cells (lower left quad-rant) are viable cells, whereas PI(+)/Annexin-V(-) fraction (upper left quadrant) are nonviable cells. Compared to the untreated cells, GECGC treatment of HCT-116 cells resulted in a significant increase of the late apoptotic cell fraction in a dose-dependent manner (from 13.38% in control, untreated cells to 24.7% in cells treated with 50 μM GECGC, and to 50.24% among cells treated with 200 μM GECGC for 24 hrs), with only a slight increase of early apoptotic cells (from 2.48% to 4.51% at 50 μM) (representative example of 3 experiments shown in Fig. 5A (p > 0.05)). Nonviable cells were increased from 7.74% among untreated, control cells to 11.22% and 16.30% among cells treated with 50 uM and 200 μM GECGC, respectively (N = 3; p > 0.05). In MDA-MB-435 cells, GECGC treatment at 50 μM and 200 μM did not significantly change either apoptotic cell death indices (Fig. 5B). This result suggests that both apoptotic and non-apoptotic mechanisms are involved in GECGC-mediated cytotoxicity.

An early transient burst of poly-ADP-ribosylation of nuclear proteins is required for apoptosis to proceed in various cell lines followed by cleavage of poly-ADP-ribosepolymerase (PARP).15 PARP cleavage followed by caspase-3 activation plays an active and complex role in apoptosis.16 In order to examine the mechanisms governing GECGC-mediated cell death, we examined the effect of GECGC treatment on PARP cleavage and caspase-3 activation. In

Figure 1. Cytotoxicity of GECGC in different cancer cell lines. (A–C). Five to twenty thousand cells (dependent on cell type) were seeded into 100 ul DMEM + 5% FBS in 96-well plates and incubated for 24 hr at 37°C, 5% CO2. The cells were then treated with either DMSO or GECGC at concentrations indicated for 48 hrs. Viability of the cells was tested by MTT assay or Cell Titer-Glo assay. Data are expressed as the mean ± SEM for N ≥ 12 separate experiments. (D). Phase contrast image of Hela and MDA-MB-435 cells treated with or without GECGC as indicated for 48 hrs.

Table 2 IC50 (μM) of GECGC in different cancer cell lines

Cell lines IC50, MTT 5% FBS IC50, LUC 5% FBSMCF-7 200 150MDA-MB-231 60 75T-47D 150 120MDA-MB-435 150 75BT-20 >200 >200Hela 40 40HCT-116 60 70SW480 12.5 12.5U373MG 60 50OVCAR >200 150PC-3 200 150K562 35 35ACHN 50 40NCI-H596 150 45NCI-H520 35 25NCI-H28 >200 150

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

www.landesbioscience.com Cell Cycle 1651

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

MDA-MB-435, HCT-116 cells and in Hela cells. GECGC (200 uM) treatment induced PARP cleavage and Caspase-3 activation were Hela, HCT 116 cells and MDA-MB-435 cells (Fig. 5C).

Effects of GECGC on membrane potential. Cellular energy produced during mitochondrial respiration is stored as an elec-trochemical gradient across the mitochondrial membrane.17 This membrane potential enables the cell to drive the synthesis of ATP and its disruption is associated with a variety of cellular phenomena, including apoptosis.18 We examined the effect of GECGC treatment on mitochondrial membrane potential in two GECGC-sensitive cell lines (Hela and HCT-116) compared with the GECGC-resistant MDA-MB-435 cell line. The mitochondrial membrane potential was not altered by GECGC treatment in MDA-MB-435 cells (Fig. 6A). Treatment of Hela and HCT-116 cells with GECGC (50 μM for 24 hrs) decreased mitochondrial potential in both cell types, evidenced by the left-shift of the red-fluorescence curve (Fig. 6B and C) suggesting GECGC may disrupt mitochondrial membrane integ-rity in sensitive cells.

Signal transduction pathways involved in GECGC-mediated cytotoxicity. Alterations of signaling pathways within cells have been shown to play important roles in the initiation and progression of cancer.19,20 To identify the molec-ular pathways that may be involved in GECGC-mediated cytotoxicity, we tested the effect of GECGC on 30 cell lines with different compositions of key cell cycle regulated genes (p53, p21, p27, p300, cyclin D1), key oncogenic signaling path-ways (P/TEN, NeuT, ErbB2, β-Catenin, Ras, Raf, c-Myc, IκBSR) and kinases (IKK-α, IKK-β, IKK-γ). The cells were treated with 3.2–200 μM GECGC for 48 hrs and cell viability was analysed by MTT assay. The IKK (IKK-α, IKK-β, IKK-γ) genes form a complex that activates NFκB signaling.21 Deletion of the IKK genes in fibroblasts, especially IKK-α, and γ and to a lesser degress IKKβ reduced sensitivity to GECGC (Fig. 7A and B). Overexpression of c-Myc in mammary epithelial MCF-10A cells also conferred GECGC sensitivity as MCF10A-c-Myc cells showed a greater reduction in viability with GECGC treatment compared with parental MCF10A cells (Fig. 7C). Deletion of the p53 gene in fibroblasts did not affect the cell viability (Fig. 7D). These results indicated that the IKK-α, and γ signaling pathways contribute to GECGC-induced growth inhibition.

Apoptosis and cell cycle gene analysis by multiplex PCR. In order to determine the molecular mechanisms governing GECGC apoptosis, we characterized expression of 66 apoptosis and cell cycle regulatory genes upon treatment with GECGC in sensitive (HCT-116) and resistant (Hs68) cell types. Cells were treated with GECGC for 24 hrs and total RNA were collected for multiplex PCR. We found that 7 genes (Caspase10, Bid, Bcl3, BcLx, Apc, Akt1 and

cyclin D3) were upregulated in the GECGC sensitive HCT-116 cell line and 1 gene (CDC25A) was downregulated (Fig. 8).

Discussion

Consumption of polyphenol-rich food or beverages has been implicated in prevention of a number of human diseases including

Table 3 Doubling time of GECGC sensitive cell lines

Cell lines Doubling time (h)Hela (Cervical Cancer) 19.0HCT116 (Colon Cancer) 17.4SW480 (Colon Cancer) 24.0K562 (Leukemia) 19.6NIC H520 (Lung Cancer) 38.0

Figure 2. Non-cancer normal cells are resistant to GECGC toxicity. Skin fibroblast (A and B), or non-cancer normal mammary epithelial cells (C and D), were treated with either with DMSO or GECGC at different concentrations as indicated for 48 hrs. The viability of the cells was evaluated using the MTT assay (MTT) or the Cell Titer-Glo (LUC) assay. Data are expressed as the mean ± SEM for N ≥ 12 separate experiments.

Table 4 IC50 (μM) of GECGC in normal cell lines (5% FBS)

Cell lines IC50, MTT IC50, LUCHUVS112D >200 200HS-68 >200 150184B5 150 120A-1N4 200 >200CCD-27SK >200 200Beas-2B 75 ND

*Not determined.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

1652 Cell Cycle 2008; Vol. 7 Issue 10

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

cardiovascular and neurodegenerative disorders as well as cancer.22 The most abundant flavanoids in the diet are flavanols (cate-chins and proanthocyanidins), anthocyanins and their oxidation products. Composition analysis of procyanidin-enriched cocoa extracts demonstrated that the major constituents of cocoa procya-dins are low molecular weight oligomers.8 In the previous study with different cocoa procyanidins, the cytotoxic effects of cocoa

procyanidins were observed only in high molecular weight oligomers (pentamers and higher) at the 100 μg/ml level in human breast cancer cells lines; however, cytotoxicity was not observed for the dimeric, trimeric or tetrameric epicatechin-derived procyanidins.9 However, procyanidin dimers and epicatechin trimers purified from bark tannins have been shown to inhibit the biochemical effects of the potent tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) in mouse epidermis in vivo.23 These monomeric, dimeric, and trimeric procyani-dins did not differ significantly in their abilities to inhibit TPA-stimulated DNA synthesis.23

In the current study, the anti-proliferative effect of the chemically synthesized procyanidin dimer, [3-O-galloyl]-(-)-epicatechin-(4β,8)-(+)-catechin-3-O-gallate (GECGC) was examined in 6 normal cell lines and in 16 human cancer cell lines (including 5 breast cancer, 2 colon cancer, 3 lung cancer, and 1 of each for cervical cancer, ovary cancer, prostate cancer, leukemia, astrocytoma and renal adenocarcinoma cell lines). To our knowl-edge, this is first extensive cytotoxicity screening of this chemically synthesized procyanidin dimer in human cancer cell lines.

Within tested concentrations, most breast cancer cell lines were resistant to the growth inhibi-tory effect of GECGC. However, one of the breast

cancer cell lines, MDA-MB-231 was sensitive to GECGC treatment. 30% growth inhibition was observed at 25 μM, and 80% of MDA-MB-231 cells were nonviable at 100 μM. In the presence of 100 μM GECGC, both the SW480 and HCT-116 colon cancer cell lines were sensitive to the GECGC. The cervical cancer line Hela, the leukemia cell line K562, and the lung cancer cell line NCI-H520, together with the colon cancer cell lines SW480 and HCT-116 were among the most GECGC sensi-tive cell lines. The common characteristic of these GECGC’s sensitive cell lines is that they are all fast growing cells with short doubling times (mean = 23.6 hrs). Population doubling time could be an important factor determining sensitivity of cells to treatment with drugs that interfere with DNA/RNA synthesis.24 Although the differences in cellular toxicity may primarily depend on the proliferation rate, it remains unclear whether other factors, such as tissue origin, histology type, GECGC uptake and bioavailability, or pattern of metabolism may play a role in determining an individual cell line’s sensi-

tivity to treatment with GECGC.The current studies demonstrated that the cytotoxicity of GECGC

is selective for tumorous compared with non-tumorous lines. Of the 22 cell lines tested, only cancer cells responded to GECGC treat-ment. None of the normal epithelial cells were sensitive to GECGC’s toxicity. The high sensitivity of colon cancer cell lines raises the possibility that GECGC could serve as a promising therapeutic for

Figure 3. Tumor cytotoxicity of GECGC is partillay blocked with catalase pre treatment. Hela (A) or HCT-116 (C) cells were first treated with H2O2 at different concentration as indicated with or without addition of catalase (250 unit/ml), and then the cell viability was then examined by using the MTT assay. Hela (B) or HCT-116 (D) cells were treated with GECGC at the concentra-tions indicated, with or without addition of catalase (250 unit/ml), and the cell viability was examined using the MTT assay after 24 hrs. Data are expressed as mean ± SEM for N ≥ 12 separate experiments.

Figure 4. See figure legend, page 6

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

www.landesbioscience.com Cell Cycle 1653

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

Figure 5. Effect of GECGC on cell apop-tosis. HCT-116 (A) and MDA-MB-435 (B) cells were treated with control (DMSO) or GECGC (50 μM or 200 μM) for 24 Hrs. Cellular apoptosis were examined using an Annexin V-FITC Apoptosis Detection Kit. PI(-)/Annexin-V(+) staining (Lower right quadrant) represent early apoptotic events; PI(+)/Annexin-V(+) staining (upper right quadrant) represent late apoptotic events; double negative cells (lower left quadrant) are viable cells whereas PI(+)/Annexin-V(-) fraction (upper left quadrant) are nonviable cells. (C) PARP expression and caspase-3 activation in GECGC-sensitive (HCT-116 and Hela) and GECGC-resistant cell lines (MDA-MB-435) following treatment with 0, 50 and 200 μM GECGC for 24 hrs. PARP expression (intact and cleaved) and caspase-3 expression were analyzed by Western blot.

colon cancer. In the current study, hydrogen peroxide production was not detected with addition of GECGC. However, GECGC cytotoxicity was partially blocked by catalase pretreat-ment, suggesting that catalase might interact with GECGC independently of hydrogen peroxide production. We conclude that the cytotoxicity of GECGC is largely due to peroxide-independent mechanisms.

Previous studies of cocoa flavanols and procyanidins in Caco-2 cells suggested that the PE extracts (procy-anidin-enriched extracts) caused non-apoptotic cell death.5 In the current study, GECGC incubation increased the sub-G1 population and increased cellular debris in the sensitive cell lines (N = 3; p < 0.05). Consistent with the cell cycle analysis, PI and annexin V staining demonstrated a significant increase in the population of nonvi-able and late apoptotic cells. PARP, a sensitive apoptosis marker, as well as caspase-3 cleavage, was induced by GECGC in HCT-116 cells. GECGC treatment in Hela and HCT-116 cells also induced depolarization of the mitochondrial membrane, an early event that triggers cell death. In the resistant cell line, MDA-MB-435, GECGC did not induce membrane depolarization. Collectively, the data suggest that GECGC treatment can cause both apoptotic and non-apop-totic cell death in sensitive cancer cell lines. The fact that PARP and

Figure 4. Effect of GECGC on cell cycle profile. Hela (A), HCT-116 (B), MDA-MB-435 (C) or normal fibroblast cell line HS-68 (D) cells were treated with or without GECGC at the concentrations indicated for 24 hrs. Cell cycle profile was analyzed using a fluorescence-activated sorter with a 360 nm Argon-Iron laser. 10,000 events per sample were collected and analyzed using the Cell Fit cell analysis program (Becton Dickinson).

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

1654 Cell Cycle 2008; Vol. 7 Issue 10

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

caspase-3 cleavage patterns between HCT-116 cells line and Hela cell line suggests that different molecular mechanisms are involved in GECGC-mediated cell killing in different cell lines.

Overall, the molecular targets for GECGC in cancer cells remain largely elusive. Flavanols and procyanidins of cocoa and chocolate have been shown to inhibit cell growth through inhib-iting cell cycle progression in Caco-2 cells, suggesting this class of compounds has anti-proliferative effects in vitro.5 The compound examined in this study is not found in cocoa and there was no G1

phase accumulation upon treatment of GECGC in either Hela or HCT-116 cells. Furthermore, deletion of the cell cycle regulatory genes cyclin D1, p21, p27 and p53 did not alter GECGC-medi-ated toxicity. Interestingly, c-Myc overexpression and competent IKK signaling enhanced sensitivity to GECGC. Multiplex PCR screening assay data suggest that the expression of several genes was induced by GECGC treatment. Further functional studies using RNAi will be necessary to verify the role of these genes in GECGC sensitivity.

Figure 6. Effect of GECGC on mitochondrial membrane potential. MDA-MB-435 (A), HCT-116 (B) or Hela (C) cells were treated with or without GECGC (50 μM) for 24 hrs. The mitochondrial membrane potential was evaluated using the JC-1 mitochondrial membrane sensor kit. (Molecular Probe). ΔΨm was measured by flow cytometry. The effect of GECGC on the mitochondrial potential was shown in red.

Figure 7. Signal transduction pathways and GECGC cytotoxicity. Fibroblasts with deletions of IKKα, IKKβ or IKKγ (A and B) or p53 (D), and mammary epi-thelial cell MCF-10A cells overexpressing c-Myc (C) were tested for GECGC cytotoxicity. The cells were treated with either with DMSO or GECGC at the con-centrations indicated for 48 hrs. Viability of the cells was tested by MTT assay. Data are expressed as the mean ± SEM for N ≥ 12 separate experiments.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

www.landesbioscience.com Cell Cycle 1655

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

In conclusion, our studies demonstrated that chemically synth- esized dimer [3-O-galloyl]-(-)-epicatechin-(4β,8)-(+)-catechin-3-O-gallate (GECGC) has selective anti-proliferative effects on a subset of human cancer cell lines. The cytotoxicity associated with GECGC was greater in cells with shorter doubling times. Further studies are

required to determine the toxicity, efficacy and bioavailability of GECGC in animal models. The observation that GECGC cytocidal effects are relatively selective for colon cancer cells in vitro, raises an interesting possibility that oral intake of GECGC, could be beneficial for the prevention and treatment of colorectal malignancies.

Figure 8. (A) Expression of apoptosis genes in GECGC treated human cancer cell lines. After reverse transcription, a multiplex polymerase chain reaction with primers for the 30 genes was performed. After 40 cycles of amplification, the samples were run by electrophoresis and stained with ethidium bromide. (B) Expression of cell cycle regulator genes in GECGC treated human cancer cell lines. After reverse transcription, a multiplex polymerase chain reaction with primers for the 36 genes was performed. After 40 cycles of amplification, the samples were run by electrophoresis and stained with ethidium bromide. P: Positive size marker, Lane 1: HS68, Lane 2: HS68 + 50 μM GECGC, Lane 3: HCT-116, Lane 4: HCT-116 + 50 μM GECGC. * cDNAs were normalized by using House Keeping Gene (HPRT1:hypoxanthine guanine phosphoribosyltransferase).

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

1656 Cell Cycle 2008; Vol. 7 Issue 10

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

Materials and Methods

Preparation of GECGC. [3-O-galloyl]-(-)-epicatechin-(4β,8)-(+)-catechin-3-O-gallate (GECGC) was chemically-synthesized and provided by Mars Inc (Hackettstown, NJ, USA). The molecular weight of GECGC is 882.8 g/mol. Briefly 10 mg of GECGC was freshly dissolved in 50 μl of DMSO and diluted with 28.3 ml DMEM to a final concentration of 400 μM. Then serial dilution was performed with tissue culture media. The highest concentration of DMSO was 0.17%. Cytotoxicity of DMSO up to 0.5% has been tested. DMSO alone did not reveal any cytotoxic effects within the tested range (data not shown).

Cell culture. All the cell lines, including the 16 human cancer cell lines and 6 normal epithelial cell or fibroblasts (Table 1) were obtained from the Tissue Culture Shared Resource at the Lombardi Comprehensive Cancer, and grown in DMEM supplemented with FBS (Gibco, Gaithersburg, MD), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were incubated at 37°C in a humidi-fied atmosphere of 5% CO2. 5,000 to 20,000 cells (dependent on the doubling time of the specific cell type) were seeded into 100 μl DMEM + 5% FBS or 10% FBS in 96-well plates and incubated for 24 hr at 37°C, 5% CO2. The cells were then treated with either with DMSO (control) or GECGC for 12, 24 hrs or 48 hrs. Cell conflu-ence was adjusted to 70% at the beginning of GECGC treatment using a coulter counter. The media was changed every day.

Cell viability assay. Viability of the cells was tested by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay according to the manufacturer’s instructions (Promega, Madison, WI). 5,000 to 20,000 cells per well were plated in 96-well culture plate and incubated with or without GECGC at the indicated concentrations and time. To avoid the possible binding of MTT with GECGC, culture medium containing GECGC was replaced with fresh media before addition of the MTT reagent. 10 μl of stock solution of MTT (5 mg/ml) was added to each well with 90 μl of DMEM and incubated for 3 hrs at 37°C. The incubation medium was removed and the formazan crystals were dissolved in 100 μl of solution of DMSO. MTT reduction was quantified by measuring the light absorbance with a microplate counter (VICTOR2-Wallac) at 570 nM. Viability of the cells was also tested by Cell Titer-Glo assay (Promega Catalog # G7571) according to the instructions provided by the manufacturer (Promega, Madison, WI). Statistical analysis was conducted using the Student’s t-test.

Measurements of hydrogen peroxide production. After adding various concentrations of GECGC (0.1–400 μM) into DMEM + 10% FBS with or without cells, the H2O2 content was analyzed within 15 min by the ferrous ion oxidation-xylenol orange method (FOX assay) using the FOX Quantitative Peroxide Assay Kit according to the manufacturer’s protocol (R&D systems, Minneapolis, MN). As the manufacturer had reported that binding of Fox reagent and DMEM results in abnormal reactions in the Fox assay, water was used instead of DMEM to test H2O2 production by GECGC. The Amplex kit was used to avoid the binding of Fox reagents and DMEM (Invitrogen, Carlsbad, CA). Samples were assayed in a 96-well microplate and absorbance was measured in a plate reader at 595 nm. Catalase was added 5 minutes before addition of GECGC.

Cell cycle analysis. Cell cycle analysis was performed as described previously.25 Cells were harvested and single cell suspension was prepared in PBS/2% FBS. The cells were washed twice with PBS

and resuspended at 1–2 x 106 cells/ml. The cells were then fixed in 70% ethanol at 4°C for 1 hr. After 5 min incubation in citrate buffer (750 mM Na2HPO4, 750 μM sodium citrate, pH 7.8), the cells were resuspended in 1 mL PBS containing 20 μg/ml propidium iodide (Sigma-Aldrich, Saint Louis, MO), 5 units of RNase A (Sigma-Aldrich, Saint Louis, MO) and were incubated for 30 min at room temperature. Flow cytometric analyses were carried out in a fluores-cence-activated sorter (FACStar plus; Becton Dickinson) with a 360 nm Argon-Iron laser. 10,000 events per sample were collected and analyzed using the Cell Fit cell analysis program (Becton Dickison, Franklin Lakes, NJ).

Cell apoptosis analysis. Cell apoptosis were examined using an Annexin V-FITC Apoptosis Detection Kit which measures phosphatidylserine membrane externalization (Sigma-Aldrich, Saint Louis, MO). Briefly, cells were washed in cold PBS and resuspended in 1x binding buffer at a concentration of 1 x 106 cells/ml. 500 μl of the apoptotic cell suspension was transferred to a 12 x 75 mm test tube which contains 5 μl of annexin V-FITC and 10 μl of propidium iodide and allowed to incubate at RT in the dark for 10 min. Each sample was diluted with binding 1x buffer to obtain a final volume appropriate for flow cytometry.

Analysis of mitochondrial membrane potential. Mitochondrial membrane potential was evaluated using the JC-1 mitochondrial membrane sensor kit (Molecular Probe, Eugene, OR). Cells were seeded and grown to 60–70% confluence in 25 cm2 culture dishes. The medium was replaced 24 hrs later with fresh medium containing GECGC, DMSO or medium only at the indicated concentration and time. Both floating and adherent cells were harvested and resus-pended. 1 x 106 cells were incubated with JC-1 (J-aggregate-forming lipophilic cation, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimida-zolocarbocyanine iodide) at a final concentration of 3 μM at 37°C in the dark for 30 min. ΔΨm was measured immediately by flow cytometry.

Western blot analysis. Total cellular lysates were prepared with RIPA buffer and resolved by 4–20 % SDS-PAGE gels. The gels were then transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk in PBS containing 0.05% Tween 20 (TPBS), the membranes were probed with the primary antibody at room temper-ature for 2 hrs. The membrane was washed with 0.1% TPBS 3 times and then incubated with appropriate HRP-conjugated secondary antibody. The proteins were visualized by the enhanced chemilumi-nescence (ECL) system (Amersham Biosciences, Piscataway, NJ). The following antibodies were used with 1:1000 dilution: SA249-0100, mouse monoclonal antibodies to PARP (Biomol, Plymouth Meeting, PA); sc-1224, goat polyclonal antibodies to caspase 3 (Santa Cruz Biotechnology, Santa Cruz, CA); sc-1616 goat polyclonal antibody to actin (Santa Cruz Biotechnology, Santa Cruz, CA).

Gene expression assay by multiplex-PCR. The GECGC sensi-tive cell line (HCT-116) and resistant cell line (MDA-MB-435) were treated with 50 μM GECGC for 24 hrs. RNA was extracted by Trizol and apoptosis and cell cycle related genes were screened through Multiplex-PCR assay which was performed by Seegene system (Rockville, MD. USA 20850). RNA optical densities (OD) were adjusted and 40 cycles of PCR were performed. RNA quality and genomic DNA contamination was determined by gel electro-phoresis using 2.5% agarose gel. PCR products were assessed by gel electrophoresis using a 2.5% agarose gel.

©2008 L

ANDES BIOSCI

ENCE.

DO NOT DIST

RIBUTE.

www.landesbioscience.com Cell Cycle 1657

Synthesized procyanidin GECGC selectively inhibits human cancer cell growth

Acknowledgements

We are thankful to the cell culture core facility in Lombardi Cancer Center, Georgetown University for providing the fibroblast and cancer cell lines. GECGC was provided as a gift by Mars, Incorporated. Grant support for these studies (R.G.P) was from Mars, Incorporated, McLean, VA. This work was supported in part by R01CA70896, R01CA75503, R01CA86072, R01CA107382 (R.G.P.). The Kimmel Cancer Center was supported by the NIH Cancer Center Core grant P30CA56036 (R.G.P).

Financial disclosure

This work was supported in part by awards from the NIH R01CA70896, R01CA75503, R01CA107382. This project is funded, in part, under a grant with the Pennsylvania Department of Health and Dr. Ralph and Marian C. Falk Medical Research Trust (R.G.P.). The Department specifically disclaims responsibility for any analyses, interpretations or conclusions (R.G.P.). Work conducted at the Kimmel Cancer Center was supported by the NIH Cancer Center Core Grant P30CA56036 (R.G.P.).

References 1. Thompson LU, Yoon JH, Jenkins DJ, Wolever TM and Jenkins AL. Relationship between

polyphenol intake and blood glucose response of normal and diabetic individuals. Am J Clin Nutr 1984; 39:745-51.

2. Selvendran RR. Developments in the chemistry and biochemistry of pectic and hemicel-lulosic polymers. J Cell Sci 1985; 2:51-88.

3. Rice-Evans CA, Miller NJ, Bolwell PG, Bramley PM and Pridham JB. The relative antioxi-dant activities of plant-derived polyphenolic flavonoids. Free Radic Res 1995; 22:375-83.

4. Ye X, et al. The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells. Mol Cell Biochem 1999; 196:99-108.

5. Carnesecchi S, et al. Flavanols and procyanidins of cocoa and chocolate inhibit growth and polyamine biosynthesis of human colonic cancer cells. Cancer Lett 2002; 175:147-55.

6. Li QL, et al. Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 2002; 109:113-24.

7. Selmi C, Mao TK, Keen CL, Schmitz HH and Eric Gershwin M. The anti-inflammatory properties of cocoa flavanols. J Cardiovasc Pharmacol 2006; 47:163-71.

8. Hammerstone JF, Lazarus SA, Mitchell AE, Rucker R and Schmitz HH. Identification of procyanidins in cocoa (Theobroma cacao) and chocolate using high-performance liquid chromatography/mass spectrometry. J Agric Food Chem 1999; 47:490-6.

9. Kozikowski AP, Tuckmantel W, Bottcher G and Romanczyk LJ Jr. Studies in polyphenol chemistry and bioactivity. 4.(1) Synthesis of trimeric, tetrameric, pentameric, and higher oligo-meric epicatechin-derived procyanidins having all-4beta,8-interflavan connectivity and their inhibition of cancer cell growth through cell cycle arrest. J Org Chem 2003; 68:1641-58.

10. Kozikowski AP, Tuckmantel W and Hu Y. Studies in polyphenol chemistry and bioactivity. 3.(1,2) stereocontrolled synthesis of epicatechin-4alpha,8-epicatechin, an unnatural isomer of the B-type procyanidins. J Org Chem 2001; 66:1287-96.

11. Ramljak D, et al. Pentameric procyanidin from Theobroma cacao selectively inhibits growth of human breast cancer cells. Mol Cancer Ther 2005; 4:537-46.

12. Fujii H, Yokozawa T, Kim YA, Tohda C and Nonaka G. Protective effect of grape seed polyphenols against high glucose-induced oxidative stress. Biosci Biotechnol Biochem 2006; 70:2104-11.

13. Elbling L, et al. Green tea extract and (-)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities. Faseb J 2005; 19:807-9.

14. Yang GY, Liao J, Kim K, Yurkow EJ and Yang CS. Inhibition of growth and induction of apoptosis in human cancer cell lines by tea polyphenols. Carcinogenesis 1998; 19:611-6.

15. Kim M, et al. Induction of apoptosis in p16INK4A mutant cell lines by adenovirus-mediated overexpression of p16INK4A protein. Cell Death Differ 2000; 7:706-11.

16. Boulares AH, et al. Role of poly(ADP-ribose) polymerase (PARP) cleavage in apoptosis. Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells. J Biol Chem 1999; 274:22932-40.

17. Darzynkiewicz Z, et al. Features of apoptotic cells measured by flow cytometry. Cytometry 1992; 13:795-808.

18. Richter C, Schweizer M, Cossarizza A and Franceschi C. Control of apoptosis by the cellular ATP level. FEBS Lett 1996; 378:107-10.

19. Hanahan D and Weinberg R. The Hallmarks of Cancer. Cell 2000; 100:57-70. 20. Nguyen DX and Massague J. Genetic determinants of cancer metastasis. Nat Rev Genet

2007; 8:341-52. 21. Joyce DE, Gelbert L, Ciaccia A, DeHoff B and Grinnell BW. Gene expression profile of

antithrombotic protein c defines new mechanisms modulating inflammation and apoptosis. J Biol Chem 2001; 276:11199-203.

22. Khan N, Afaq F, Saleem M, Ahmad N and Mukhtar H. Targeting multiple signaling path-ways by green tea polyphenol (-)-epigallocatechin-3-gallate. Cancer Res 2006; 66:2500-5.

23. Galien R, Ravier-Emanoil R and Mercier G. Differential effects of c-jun and CREB on cAMP response element activation by Ha-ras. Oncogene 1994; 9:1101-8.

24. Bykov VJ, Issaeva N, Selivanova G and Wiman KG. Mutant p53-dependent growth sup-pression distinguishes PRIMA-1 from known anticancer drugs: a statistical analysis of information in the National Cancer Institute database. Carcinogenesis 2002; 23:2011-8.

25. Wu K, et al. Flavopiridol and trastuzumab synergistically inhibit proliferation of breast cancer cells: association with selective cooperative inhibition of cyclin D1-dependent kinase and Akt signaling pathways. Mol Cancer Ther 2002; 1:695-706.