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• W2015560109 abstract "Anthocyanins are a group of naturally occurring phenolic compounds widely available in fruits and vegetables in human diets. They have broad biological activities including anti-mutagenesis and anticarcinogenesis, which are generally attributed to their antioxidant activities. We studied the effects and the mechanisms of the most common type of anthocyanins, cyanidin-3-rutinoside, in several leukemia and lymphoma cell lines. We found that cyanidin-3-rutinoside extracted and purified from the black raspberry cultivar Jewel induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Paradoxically, this compound induced the accumulation of peroxides, which are involved in the induction of apoptosis in HL-60 cells. In addition, cyanidin-3-rutinoside treatment resulted in reactive oxygen species (ROS)-dependent activation of p38 MAPK and JNK, which contributed to cell death by activating the mitochondrial pathway mediated by Bim. Down-regulation of Bim or overexpression of Bcl-2 or Bcl-xL considerably blocked apoptosis. Notably, cyanidin-3-rutinoside treatment did not lead to increased ROS accumulation in normal human peripheral blood mononuclear cells and had no cytotoxic effects on these cells. These results indicate that cyanidin-3-rutinoside has the potential to be used in leukemia therapy with the advantages of being widely available and selective against tumors. Anthocyanins are a group of naturally occurring phenolic compounds widely available in fruits and vegetables in human diets. They have broad biological activities including anti-mutagenesis and anticarcinogenesis, which are generally attributed to their antioxidant activities. We studied the effects and the mechanisms of the most common type of anthocyanins, cyanidin-3-rutinoside, in several leukemia and lymphoma cell lines. We found that cyanidin-3-rutinoside extracted and purified from the black raspberry cultivar Jewel induced apoptosis in HL-60 cells in a dose- and time-dependent manner. Paradoxically, this compound induced the accumulation of peroxides, which are involved in the induction of apoptosis in HL-60 cells. In addition, cyanidin-3-rutinoside treatment resulted in reactive oxygen species (ROS)-dependent activation of p38 MAPK and JNK, which contributed to cell death by activating the mitochondrial pathway mediated by Bim. Down-regulation of Bim or overexpression of Bcl-2 or Bcl-xL considerably blocked apoptosis. Notably, cyanidin-3-rutinoside treatment did not lead to increased ROS accumulation in normal human peripheral blood mononuclear cells and had no cytotoxic effects on these cells. These results indicate that cyanidin-3-rutinoside has the potential to be used in leukemia therapy with the advantages of being widely available and selective against tumors. Natural products derived from plants have recently received much attention as potential chemopreventive and chemotherapeutic agents. Among them great attention has been given to natural products with established antioxidant activities and less toxicity in normal cells, such as tea polyphenols and resveratrol (1Surh Y.J. Nat. Rev. Cancer. 2003; 3: 768-780Crossref PubMed Scopus (2511) Google Scholar, 2Feng R. Lu Y. Bowman L.L. Qian Y. Castranova V. Ding M. J. Biol. Chem. 2005; 280: 27888-27895Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 3Lopez-Lazaro M. Curr. Med. Chem. Anticancer Agents. 2002; 2: 691-714Crossref PubMed Scopus (237) Google Scholar). These substances appear very promising for cancer prevention and treatment in preclinical models and clinical trials (1Surh Y.J. Nat. Rev. Cancer. 2003; 3: 768-780Crossref PubMed Scopus (2511) Google Scholar, 2Feng R. Lu Y. Bowman L.L. Qian Y. Castranova V. Ding M. J. Biol. Chem. 2005; 280: 27888-27895Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 3Lopez-Lazaro M. Curr. Med. Chem. Anticancer Agents. 2002; 2: 691-714Crossref PubMed Scopus (237) Google Scholar, 4Jang M. Cai L. Udeani G.O. Slowing K.V. Thomas C.F. Beecher C.W. Fong H.H. Farnsworth N.R. Kinghorn A.D. Mehta R.G. Moon R.C. Pezzuto J.M. Science. 1997; 275: 218-220Crossref PubMed Scopus (4551) Google Scholar). Perhaps among the most common type of plant polyphenols is flavonoids, which provide much of the flavor and color to fruits and vegetables. More than 5000 different flavonoids have been described (5Ross J.A. Kasum C.M. Annu. Rev. Nutr. 2002; 22: 19-34Crossref PubMed Scopus (1805) Google Scholar). There are six major subclasses of flavonoids (5Ross J.A. Kasum C.M. Annu. Rev. Nutr. 2002; 22: 19-34Crossref PubMed Scopus (1805) Google Scholar): anthocyanins, flavones (e.g. apigenin), flavonols (e.g. quercetin), flavanones (e.g. naringenin), catechins of flavanols (e.g. epicatechin), and isoflavones (e.g. genistein). Anthocyanins probably deserve the most attention, as the daily uptake of anthocyanins in the human diet is remarkable, estimated at 180–215 mg/day in the United States (6Hertog M.G. Hollman P.C. Katan M.B. Kromhout D. Nutr. Cancer. 1993; 20: 21-29Crossref PubMed Scopus (1155) Google Scholar), which is much higher than the total intake estimated for other flavonoids (23 mg/day). Anthocyanins are the glycosylated form of anthocyanidins, which are polyhydroxyl and polymethoxy derivatives of 2-phenylbenzopyrylium or flavylium salts (7Mazza G. Miniati E. Anthocyanins in Fruits, Vegetables, and Grains. CRC Press, Boca Raton, FL1993Google Scholar). Structurally, there are more than a dozen different anthocyanidins in plants, but cyanidin is the most frequently found, naturally occurring anthocyanidin. Cyanidin is widely available in the human diet through crops such as beans, fruits, vegetables, and red wine (7Mazza G. Miniati E. Anthocyanins in Fruits, Vegetables, and Grains. CRC Press, Boca Raton, FL1993Google Scholar), and cyanidin-3-rutinoside (C-3-R) 2The abbreviations used are: C-3-R, cyanidin-3-rutinoside; DCF, 2′,7′-dichlorofluorescein; H2DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; NAC, N-acetyl-cysteine; PBMC, peripheral blood mononuclear cells; PEITC, β-phenylethyl isothiocyanate; PI, propidium iodide; shRNA, short hairpin RNA; JNK, c-Jun NH2-terminal kinase; MAP, mitogen-activated protein; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AFC, 7-amino-4-trifluoromethyl coumarin; Z, benzyloxycarbonyl. is a common glycosylated form (8Galvano F. La Fauci L. Lazzarino G. Fogliano V. Ritieni A. Ciappellano S. Battistini N.C. Tavazzi B. Galvano G. J. Nutr. Biochem. 2004; 15: 2-11Crossref PubMed Scopus (281) Google Scholar). Anthocyanins demonstrate strong antioxidant activities in a variety of in vitro assays (7Mazza G. Miniati E. Anthocyanins in Fruits, Vegetables, and Grains. CRC Press, Boca Raton, FL1993Google Scholar, 8Galvano F. La Fauci L. Lazzarino G. Fogliano V. Ritieni A. Ciappellano S. Battistini N.C. Tavazzi B. Galvano G. J. Nutr. Biochem. 2004; 15: 2-11Crossref PubMed Scopus (281) Google Scholar, 9Wang H. Nair M.G. Strasburg G.M. Chang Y.C. Booren A.M. Gray J.I. DeWitt D.L. J. Nat. Prod. 1999; 62: 294-296Crossref PubMed Scopus (620) Google Scholar, 10Zheng W. Wang S.Y. J. Agric. Food Chem. 2003; 51: 502-509Crossref PubMed Scopus (660) Google Scholar). These natural compounds can react with reactive oxygen species (ROS) and thus interrupt the propagation of new free radical species. The double bonds present in the phenolic ring, the hydroxyl side chains, and even the glycosylation contribute to the scavenging activity. Anthocyanins, particularly cyanidin glycosides, have been found to possess a broad spectrum of biological activities, including scavenging effects on activated carcinogens and mutagens and effects on cell cycle regulation (7Mazza G. Miniati E. Anthocyanins in Fruits, Vegetables, and Grains. CRC Press, Boca Raton, FL1993Google Scholar, 8Galvano F. La Fauci L. Lazzarino G. Fogliano V. Ritieni A. Ciappellano S. Battistini N.C. Tavazzi B. Galvano G. J. Nutr. Biochem. 2004; 15: 2-11Crossref PubMed Scopus (281) Google Scholar, 11Hagiwara A. Miyashita K. Nakanishi T. Sano M. Tamano S. Kadota T. Koda T. Nakamura M. Imaida K. Ito N. Shirai T. Cancer Lett. 2001; 171: 17-25Crossref PubMed Scopus (165) Google Scholar, 12Serafino A. Sinibaldi Vallebona P. Lazzarino G. Tavazzi B. Rasi G. Pierimarchi P. Andreola F. Moroni G. Galvano G. Galvano F. Garaci E. FASEB J. 2004; 18: 1940-1942Crossref PubMed Scopus (52) Google Scholar, 13Lazze M.C. Savio M. Pizzala R. Cazzalini O. Perucca P. Scovassi A.I. Stivala L.A. Bianchi L. Carcinogenesis. 2004; 25: 1427-1433Crossref PubMed Scopus (168) Google Scholar). Previous work has indicated that cyanidin-rich berry or pomegranate extracts or purified cyanidins possess remarkable chemopreventive activities in cell culture and animal models (11Hagiwara A. Miyashita K. Nakanishi T. Sano M. Tamano S. Kadota T. Koda T. Nakamura M. Imaida K. Ito N. Shirai T. Cancer Lett. 2001; 171: 17-25Crossref PubMed Scopus (165) Google Scholar, 14Feng R. Bowman L.L. Lu Y. Leonard S.S. Shi X. Jiang B.H. Castranova V. Vallyathan V. Ding M. Nutr. Cancer. 2004; 50: 80-89Crossref PubMed Scopus (30) Google Scholar, 15Ding M. Feng R. Wang S.Y. Bowman L. Lu Y. Qian Y. Castranova V. Jiang B.H. Shi X. J. Biol. Chem. 2006; 281: 17359-17368Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 16Malik A. Afaq F. Sarfaraz S. Adhami V.M. Syed D.N. Mukhtar H. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 14813-14818Crossref PubMed Scopus (377) Google Scholar, 17Fimognari C. Berti F. Nusse M. Cantelli-Forti G. Hrelia P. Chemotherapy. 2005; 51: 332-335Crossref PubMed Scopus (21) Google Scholar). Furthermore, cyanidin glycosides purified from berry extracts possess proapoptotic properties in human cancer cells including leukemia cells (13Lazze M.C. Savio M. Pizzala R. Cazzalini O. Perucca P. Scovassi A.I. Stivala L.A. Bianchi L. Carcinogenesis. 2004; 25: 1427-1433Crossref PubMed Scopus (168) Google Scholar, 18Wenzel U. Kuntz S. Brendel M.D. Daniel H. Cancer Res. 2000; 60: 3823-3831PubMed Google Scholar, 19Katsuzaki H. Hibasami H. Ohwaki S. Ishikawa K. Imai K. Date K. Kimura Y. Komiya T. Oncol. Rep. 2003; 10: 297-300PubMed Google Scholar, 20Fimognari C. Berti F. Nusse M. Cantelli-Forti G. Hrelia P. Biochem. Pharmacol. 2004; 67: 2047-2056Crossref PubMed Scopus (76) Google Scholar). These studies suggest that the antitumor activities of cyanidin glycosides could be related to their ability to induce apoptosis and that these products may have potential as agents for cancer treatment as well. All of these activities are thought to be related to the antioxidant properties of the cyanidins (8Galvano F. La Fauci L. Lazzarino G. Fogliano V. Ritieni A. Ciappellano S. Battistini N.C. Tavazzi B. Galvano G. J. Nutr. Biochem. 2004; 15: 2-11Crossref PubMed Scopus (281) Google Scholar, 21Kahkonen M.P. Hopia A.I. Vuorela H.J. Rauha J.P. Pihlaja K. Kujala T.S. Heinonen M. J. Agric. Food Chem. 1999; 47: 3954-3962Crossref PubMed Scopus (2917) Google Scholar, 22Zheng Y. Wang C.Y. Wang S.Y. Zheng W. J. Agric Food Chem. 2003; 51: 7162-7169Crossref PubMed Scopus (152) Google Scholar, 23Wang S.Y. Feng R. Bowman L. Penhallegon R. Ding M. Lu Y. J. Agric Food Chem. 2005; 53: 3156-3166Crossref PubMed Scopus (68) Google Scholar). However, this notion has not been carefully examined, particularly for the apoptotic activity. Furthermore, the molecular mechanisms and signaling pathways initiated by cyanidin glycosides in cell death are also poorly understood. In the present study, we have found that cyanidin-3-rutinoside paradoxically increases the level of peroxides in the human leukemia cells, and this increase is responsible for its apoptotic effects. Cyanidin-3-rutinoside subsequently activates the p38 MAP kinase and the pro-death Bcl-2 family proteins, leading to mitochondrial release of apoptogenic factors and cell death. In contrast, cyanidin-3-rutinoside caused little ROS generation in the normal human peripheral blood mononuclear cells (PBMC) and had very low toxicity toward these cells. Our study thus indicates that natural antioxidative products such as cyanidin 3-rutinoside may exhibit pro-oxidant activities selectively in leukemic cells, an effect that could be exploited in the development of antitumor agents having a low toxicity toward normal cells. Reagents—C-3-R (purity >99%) was purified from black raspberry cultivar Jewel extract as described previously (10Zheng W. Wang S.Y. J. Agric. Food Chem. 2003; 51: 502-509Crossref PubMed Scopus (660) Google Scholar). Purified C-3-R was stored at –80 °C, and the stock solution (150 mm) was prepared fresh before use by dissolving the powder in Me2SO. The following primary or secondary antibodies were used: anti-Bak (NT) (Upstate Biotechnology, Lake Placid, NY); anti-Bak (Ab-1) (Oncogene, Cambridge, MA); anti-Bax (N-20) (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Bax (clone 6A7), anti-Bcl-2, anti-JNK (clone 666), anti-cytochrome c, and anti-Smac (all from BD Biosciences, San Jose, CA); anti-Bcl-xL, anti-Bim, antiphosphorylated JNK and p38 MAP kinase (all from Cell Signaling, Beverly, MA); anti-β-actin (Sigma); anti-COX IV subunit II (Invitrogen-Molecular Probes); and horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Cell Lines and Cell Cultures—The human leukemia and lymphoma cell lines HL-60 (myeloblastic), MOLT-4 (lymphoblastic), and Daudi (lymphoblastic) were obtained from American Type Culture Collection (Manassas, VA). Human leukemia cells HL-60/neo, HL-60/Bcl-2, and HL-60/Bcl-xL were derived from the laboratory of Dr. Kapil N. Bhalla (Medical College of Georgia) and cultured as described previously (24Amarante-Mendes G.P. Naekyung Kim C. Liu L. Huang Y. Perkins C.L. Green D.R. Bhalla K. Blood. 1998; 91: 1700-1705Crossref PubMed Google Scholar). The T cell acute lymphoblastic leukemia cell line, CCRF-CEM, and its subline stably expressing shRNA against Bim were cultured as described previously (25Lu J. Quearry B. Harada H. FEBS Lett. 2006; 580: 3539-3544Crossref PubMed Scopus (89) Google Scholar). Human PBMC were prepared from healthy donors (Pittsburgh Blood Bank) using Ficoll-Hypaque centrifugation. The leukemia/lymphoma cell lines and PBMC were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Analysis of Cell Death—Apoptotic and necrotic cell death were determined as described previously, with modifications (26Ding W.X. Ni H.M. DiFrancesca D. Stolz D.B. Yin X.M. Hepatology. 2004; 40: 403-413Crossref PubMed Scopus (104) Google Scholar). Briefly, cells were double-stained with 10 μmol/liter bisbenzimide Hoechst 33258 and propidium iodide (PI, 1 μg/ml) (Invitrogen-Molecular Probes) for 30 min and analyzed by fluorescence microscopy. Approximately 300–500 cells/condition were randomly selected and assessed. Hoechst 33258 positive cells with apoptotic (condensed and/or fragmented) nuclei were considered as apoptotic cells regardless of whether they were PI positive or not, whereas PI-stained cells without apoptotic nuclear changes were considered as necrotic cells. Caspase activities were measured using 15 μg (caspase-3) or 20 μg (caspase-8 and -9) of proteins and 20 μm fluorescent substrates (Ac-DEVD-AFC, Ac-IETD-AFC, and Ac-LEHD-AFC for caspase-3, -8, and -9, respectively) (Biomol, Plymouth Meeting, PA) (26Ding W.X. Ni H.M. DiFrancesca D. Stolz D.B. Yin X.M. Hepatology. 2004; 40: 403-413Crossref PubMed Scopus (104) Google Scholar). The fluorescence signals were detected using a fluorometer (Tecan GENios, Durham, NC) at excitation and emission wavelengths of 400 and 510 nm, respectively. Cytochrome c release and Bax translocation to the mitochondria were analyzed by immunoblot assay using the cytosolic and membrane fractions prepared from treated cells. Subcellular fractionation was conducted using limited plasma membrane permeabilization with 0.05% digitonin as described previously (27Mikhailov V. Mikhailova M. Pulkrabek D.J. Dong Z. Venkatachalam M.A. Saikumar P. J. Biol. Chem. 2001; 276: 18361-18374Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar). Measurement of ROS—For the in vitro study, superoxide radicals were quantified spectrophotometrically in a xanthine/xanthine oxidase system as described previously (28Richmond R. Halliwell B. Chauhan J. Darbre A. Anal. Biochem. 1981; 118: 328-335Crossref PubMed Scopus (207) Google Scholar). The final results were expressed as percent inhibition of superoxide radical production in the presence of an antioxidant (C-3-R, ascorbic acid, or Trolox). The assay for hydrogen peroxide (H2O2) levels was carried out following procedures described previously (29Patterson B.D. MacRae E.A. Ferguson I.B. Anal. Biochem. 1984; 139: 487-492Crossref PubMed Scopus (985) Google Scholar). The final results were expressed as percent inhibition of H2O2 levels in a Ti(IV)-H2O2 complex system in the presence of antioxidants. For determination of the cellular ROS level, two different methods were used. First, at various times (15 min to 4 h) after C-3-R treatment, HL-60 cells were incubated for another 15 min with either 2 μm dihydroethidium bromide or 2 μm 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). These ROS-dependent fluorescent probes were converted to ethidium or 2′,7′-dichlorofluorescein (DCF) in the presence of superoxide radicals or H2O2, respectively, which can be detected by flow cytometry (26Ding W.X. Ni H.M. DiFrancesca D. Stolz D.B. Yin X.M. Hepatology. 2004; 40: 403-413Crossref PubMed Scopus (104) Google Scholar). To measure the intracellular accumulation of C-3-R-induced H2O2 production, cells were treated simultaneously with C-3-R and H2DCFDA for 1.5–3 h and then harvested for flow cytometry analysis. In another set of experiments, intracellular H2O2 levels were determined by a method based on horseradish peroxidase-dependent oxidation of phenol red, which is assessed by the increased absorbance at 610 nm (30Pick E. Keisari Y. J. Immunol. Methods. 1980; 38: 161-170Crossref PubMed Scopus (1005) Google Scholar). Immunofluorescence and Immunoblot Analysis—Immunofluorescence staining was carried out as described previously (26Ding W.X. Ni H.M. DiFrancesca D. Stolz D.B. Yin X.M. Hepatology. 2004; 40: 403-413Crossref PubMed Scopus (104) Google Scholar). Treated cells were harvested and pelleted to glass slides by Cytospin® (Thermo Shandon Inc., Pittsburgh, PA). Cells were fixed in 4% paraformaldehyde/phosphate-buffered saline (pH 7.4) for 1 h at 4°C and permeabilized in 0.5% Triton X-100 for another 1 h. Following incubation of the primary antibodies, the molecules of interest were detected using Alexa Fluor-conjugated secondary antibodies and fluorescence microscopy. For the immunoblot assay, treated cells were lysed with radioimmune precipitation assay buffer supplemented with 1 μm EDTA and a mixture of protease inhibitors. Proteins in the lysates were quantified and separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with the relevant antibodies. C-3-R Selectively Induces Apoptosis in Human Leukemic Cells—Treatment of the human leukemia cell line HL-60 with C-3-R induced a significant amount of apoptosis in a time- and dose-dependent manner (Fig. 1A) based on the morphology of the nucleus. About 50% of the cells became apoptotic within 18 h when 50 μm C-3-R was applied, and virtually all cells were apoptotic in the presence of 120 μm or greater C-3-R. There was very little necrosis as measured by PI staining. Consistently, a significant amount of caspase-3 and caspase-9 activity could be detected in a dose-dependent manner, which peaked around 16 h (Fig. 1, B and C). The detected activity was reduced thereafter, likely because of the increased cell death. Consequently, C-3-R-induced apoptosis and caspase activation in HL-60 cells could be suppressed by the effector caspase inhibitor DEVD-CHO (data not shown) and the pan-caspase inhibitor Z-VAD-fluoromethyl ketone (Fig. 1D). The mitochondrial pathway could be an important mechanism contributing to C-3-R-induced apoptosis, as we found that cytochrome c (Fig. 1E) and Smac (see below) was released upon C-3-R treatment. C-3-R also induced apoptosis in other human leukemia/lymphoma cell lines including MOLT-4, Daudi, and CCRF-CEM (Fig. 1F). It had little toxicity against the normal human PBMC as measured by nuclear staining with Hoechst 33258 and by MTT assay (Fig. 2, A and B). This probably is not a surprising finding, as C-3-R is a common constituent in human diets (5Ross J.A. Kasum C.M. Annu. Rev. Nutr. 2002; 22: 19-34Crossref PubMed Scopus (1805) Google Scholar, 6Hertog M.G. Hollman P.C. Katan M.B. Kromhout D. Nutr. Cancer. 1993; 20: 21-29Crossref PubMed Scopus (1155) Google Scholar). This property is similar to resveratrol, a natural product widely studied for its chemopreventive benefits (4Jang M. Cai L. Udeani G.O. Slowing K.V. Thomas C.F. Beecher C.W. Fong H.H. Farnsworth N.R. Kinghorn A.D. Mehta R.G. Moon R.C. Pezzuto J.M. Science. 1997; 275: 218-220Crossref PubMed Scopus (4551) Google Scholar). C-3-R had some toxicity against proliferating PBMC stimulated with phytohemagglutinin based on Hoechst staining (Fig. 2A), although this was not as obvious in the MTT assay. This toxicity, however, was much lower than that found in proliferating HL-60 cells (Fig. 1A). We also observed a similar level of toxicity of resveratrol in the proliferating PBMC using the Hoechst assay (data not shown). These observations suggest that proliferating PBMC may be, in general, more susceptible to these chemicals. C-3-R Selectively Alters the Intracellular Level of Peroxides in HL-60 Cells—Structurally C-3-R possesses the phenolic rings typical of the anthocyanidins (Fig. 3A), suggesting that it has antioxidant activity similar to that of other members of the family. Indeed, when using the standard in vitro assays, we found that C-3-R demonstrated concentration-dependent inhibition of H2O2 production in the Ti(IV)-H2O2 complex system (Fig. 3B). C-3-R could also effectively inhibit superoxide and peroxyl radicals (ROO·) production (data not shown). This activity might be related to its chemopreventive activity in the normal cells (8Galvano F. La Fauci L. Lazzarino G. Fogliano V. Ritieni A. Ciappellano S. Battistini N.C. Tavazzi B. Galvano G. J. Nutr. Biochem. 2004; 15: 2-11Crossref PubMed Scopus (281) Google Scholar, 21Kahkonen M.P. Hopia A.I. Vuorela H.J. Rauha J.P. Pihlaja K. Kujala T.S. Heinonen M. J. Agric. Food Chem. 1999; 47: 3954-3962Crossref PubMed Scopus (2917) Google Scholar, 22Zheng Y. Wang C.Y. Wang S.Y. Zheng W. J. Agric Food Chem. 2003; 51: 7162-7169Crossref PubMed Scopus (152) Google Scholar). Consistently, we have found that treatment of normal human PBMC with C-3-R led to reduced intracellular peroxide levels in the time frame of 15 min to 4 h based on the use of the cell-permeable nonfluorescent H2DCFDA, which is oxidized to generate the fluorescent product DCF by the peroxides (Fig. 2C). In addition, the increased reduction of MTT in the C-3-R-treated cells could also suggest an overall less oxidative environment in these cells (Fig. 2B). In contrast, when the leukemic HL-60 cells were treated with C-3-R, there was an increased level of H2O2 accumulated in the cells. In the first assay, we added H2DCFDA only for the last 15 min of the culture, at each given time point, to detect the intracellular level of H2O2 at that particular moment. We found that although similar to what was observed in the normal PBMC, C-3-R treatment in HL-60 cells led to the reduction of intracellular peroxides in the first 15 min of the treatment. The peroxide level began to reverse within 30 min and was significantly above the control level by 1 h of culture (Fig. 3C). The accumulated level of peroxide was gradually reduced thereafter to the control level by 4 h after treatment. To examine the overall impact of C-3-R treatment on the intracellular accumulation of H2O2, we added the H2DCFDA at the beginning of the culture through the first 1.5 and 3 h. This allowed the level of DCF to accumulate as the production of H2O2 was increased. The result indicated that despite the initial reduction of H2O2 in the first 15 min after treatment, the net level of H2O2 was increased during the first 3 h and peaked at the first 1.5 h (Fig. 3D). We confirmed this result using an enzyme-based assay that measures the concentration of H2O2 based on the horseradish peroxidase-mediated oxidation of phenol red (30Pick E. Keisari Y. J. Immunol. Methods. 1980; 38: 161-170Crossref PubMed Scopus (1005) Google Scholar) (Fig. 3E). In contrast to the observed change in H2O2 levels, there were no significant changes in the intracellular superoxide (O–2) levels using dihydroethidium staining (data not shown). These results suggest that C-3-R led to a biphasic change in the intracellular redox status, with the net impact being an increase in the intracellular H2O2 level. C-3-R-induced Apoptosis Could be Mediated by ROS, p38 MAP Kinase, and JNK—Based on the above observations, it was possible that C-3-R-induced apoptosis could be related to its activity in enhancing cellular peroxide level. To investigate this possibility, we first determined whether a preincubation of a nontoxic low dose of H2O2 would inhibit C-3-R-induced apoptosis. Such treatment is well known to induce an adaptive response from the cells, which leads to cellular resistance to the subsequent challenge of a larger dose of H2O2 due to the induction of antioxidative gene expression (31Davies K.J. IUBMB Life. 1999; 48: 41-47Crossref PubMed Google Scholar). Indeed, this pretreatment with 0.5–4 μm H2O2 for a period of 3 h dramatically suppressed C-3-R-induced apoptosis in HL-60 cells (Fig. 4A). Consistently, C-3-R-induced apoptosis, caspase activity, and mitochondrial release of apoptogenic factors also could be suppressed by the general antioxidant N-acetyl-cysteine (NAC) or the H2O2 scavenger catalase (Fig. 4, B and C). These observations suggest that C-3-R-induced oxidative stress can activate the mitochondrial apoptosis pathway in HL-60 cells. To explore the signaling events upstream the mitochondria, we examined the redox-sensitive stress kinase pathways, which are involved in apoptosis (32Matsuzawa A. Ichijo H. Antioxid. Redox Signal. 2005; 7: 472-481Crossref PubMed Scopus (243) Google Scholar). In untreated HL-60 cells, neither p38 MAP kinase nor JNK was phosphorylated (Fig. 4D). Upon C-3-R treatment, there was a rapid increase in the phosphorylation of both p38 MAP kinase and JNK, correlating with the increase of intracellular peroxides levels (Fig. 3, C–E). Indeed, treatment of cells with the antioxidant NAC and the H2O2 scavenger catalase suppressed the activation of p38 MAP kinase and JNK at the early time point (Fig. 4E). Although JNK activation seemed to be relatively transient, p38 MAP kinase phosphorylation was sustained throughout the time course. However, both the p38 MAP kinase inhibitor SB203580 and the JNK inhibitor SP600125 could significantly inhibit mitochondrial release of cytochrome c and Smac, caspase activation, and apoptosis induced by C-3-R (Fig. 4, B and C), indicating that both stress kinases were involved in the C-3-R-activated mitochondrial apoptosis pathway following C-3-R-induced oxidative stress. Activation of the Mitochondrial Pathway by C-3-R—To understand how C-3-R-mediated oxidative stress and stress kinase activated the mitochondrial apoptosis pathway, we examined activation of the pro-death Bcl-2 family proteins, which are perhaps the most important molecules promoting this pathway (33Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2831) Google Scholar). The multidomain pro-death molecules, Bax and Bak, are responsible for the mitochondrial release of apoptogenic factors. When activated, they become oligomerized, which can be detected by conformation-sensitive antibodies. Indeed, the mitochondria localized Bak could be activated by C-3-R treatment as shown by immunostaining with a conformation-sensitive antibody (Ab-1) (Fig. 5A). Immunostaining with a conformation-sensitive anti-Bax antibody (clone 6A7) also detected Bax in a distinctive punctated pattern following C-3-R treatment, suggesting that the normally cytosol-located Bax was translocated to the mitochondria and was in an activated status (Fig. 5A). Fractionation studies confirmed Bax translocation upon C-3-R treatment (Fig. 5B). Interestingly, catalase, SB203580, or SP600125 had a much stronger effect on Bax conformational change (Fig. 5C) than on Bax translocation (Fig. 5B). These data suggest that ROS and stress kinases mainly mediate the second step of Bax activation, the oligomerization. This activation step of Bax and Bak (oligomerization and conformation change) was most likely caused by the BH3-only Bcl-2 family proteins (33Green D.R. Kroemer G. Science. 2004; 305: 626-629Crossref PubMed Scopus (2831) Google Scholar). Among the possible BH3-only molecules that could be activated under the current experimental system, we focused on Bim, as it is reported to be activated by stress kinases, mostly via direct phosphorylation of protein (34Ley R. Ewings K.E. Hadfield K. Cook S.J. Cell Death Differ. 2005; 12: 1008-1014Crossref PubMed Scopus (258) Google Scholar, 35Lei K. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2432-2437Crossref PubMed Scopus (904) Google Scholar, 36Putcha G.V. Le S. Frank S. Besirli C.G. Clark K. Chu B. Alix S. Youle R.J. LaMarche A. Maroney A.C. Johnson E.M. Neuron. 2003; 38: 899-914Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, 37Cai B. Chang S.H. Becker E.B. Bonni A. Xia Z. J. Biol. Chem. 2006; 281: 25215-25222Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). We found that although the general level of BimEL did not change significantly following C-3-R treatment, the migrati" @default.
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