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Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport
Cancer Letters 119 (1997) 207-2 12
Phloretin-induced apoptosis in B 16 melanoma 4A5 cells by inhibition of glucose transmembrane transport Masuko Kobori”, Hiroshi Shinmoto, Tojiro Tsushida, Kazuki Shinohara’ iVationa1 Food Research Institute, Ministry of Agriculture, Forestp and Fisheries, 2-1-2 Kannondai, Tsukuba, ltmruki +Oj. Jqxm Received 24 March 1997; received in revised form 8 May 1997: accepted 15 May 1997
Abstract Phloretin, a naturally occurring dihydrochalcone, is known to inhibit tumor cell growth in vitro and in viva. To clarify the anti-tumor effects of phloretin, its apoptosis-inducing effects in B 16 melanoma 4A.5 cells were examined. Phloretin induced the internucleosomal DNA fragmentation typical of apoptosis in B16 melanoma cells. The addition of extracelluIar glucose remarkably inhibited the phloretin-induced apoptosis in the cells. When apoptosis was strongly induced in the B16 cells by phloretin, protein kinase C activity was inhibited in the cells. Our results suggest that phloretin induced apoptosis in B 16 melanoma 4A5 cells mainly through the inhibition of glucose transmembrane transport. Inhibition of protein kinase C activity by phloretin probably promotes the ratio of apoptotic cells in the cells. 0 1997 Elsevier Science Ireland Ltd Keywords:
Phloretin; Apoptosis; Melanoma cells; Glucose transport; Protein kinase C
1. IntFodwtin The dihydrochalcone phloretin is the aglucon of phlorizin occurring in apple, pear and other Roseaceae. This compound, which is related to flavonoids, is known to inhibit glucose transmembrane transport [l] and protein kinase C (PKC) [2], and is one of the naturally occurring non-steroid estrogens [3]. Phloretin has been reported to inhibit the growth of Molt-4 human leukemic cells in vitro [4], and Fisher bladder
*Corresponding author. Tel.: +81 298 388055; fax: +81 298 387996. ’ Current address: National Research Institute of Fisheries Sciences, Ministry of Agriculture, Forestry and Fisheries, 2-12-4 Fukuura, Yokohama, Kanagawa 236, Japan.
carcinoma and rat mammary adenocarcinoma cells in vivo [5]. However, the mechanism of tumor cell death induced by phloretin has not been reported, whereas the flavonoids, quercetin and genistein, has been shown to induce apoptosis in some tumor cells. Many kinds of chemotherapeutic agents induce apoptosis in tumor cells via diverse pathways, such as inhibition of topoisomerase (topo) I or 11 [6], activation or inhibition of PKC [7,8] and inhibition of DNA replication [9]. Quercetin, which is a bioflavonoid found in most edible fruits and vegetables, induces apoptosis of K562 human leukemia and some tumor cells. The induction of apoptosis by quercetin is thought to proceed via inhibition of heat shock protein synthesis and expression [IO]. The isoflavone genistein, which is contained in soybeans and other plant foods, was shown to induce apoptosis in HL60
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human leukemia cells, immature human thymocytes and other cells [9,11]. Genistein is thought to induce apoptosis not by inhibition of tyrosine kinase but by inhibition of topo II [ 111. Since protein kinase C inhibitors induce apoptosis in HL60 and some other cells, phloretin might induce apoptosis in tumor cells by inhibition of PKC. Otherwise, phloretin may induce apoptosis in tumor cells through inhibition of glucose transport or stimulation of estrogen receptors, because inhibition of glucose uptake and the ligand-activated estrogen receptor were reported to induce apoptosis in some cell lines [12,13]. In this study, we demonstrated that phloretin induced apoptosis in B16 mouse melanoma 4A5 cells. Our results suggested that phloretin induced apoptosis mainly through inhibition of glucose transmembrane transport. Inhibition of PKC activity by phloretin probably promotes the ratio of apoptotic cells in B16 melanoma 4A5 cells.
2.3. DNA extraction and agarose gel electrophoresis Cells (1 x lo6 cells) were lysed in 1 ml lysis buffer (50 mM Tris-HCl (pH 8.0), 0.1 M EDTA and 0.5% SDS), incubated with 0.05 mg/ml RNase at 37°C for 30 min, and then incubated with 0.1 mg/ml proteinase K at 37°C for 30 min. DNA was extracted in phenol, precipitated in ethanol and resuspended in a buffer (10 mM Tris-HCl (pH 8.0) 1 mM EDTA). The samples were electrophoresed in Tris-borate buffer (pH 8.0) on a 2% agarose gel and the DNA was visualized with ethidium bromide. 2.4. Flocytometric analysis
2. Materials and methods
Flowcytometric analysis was performed to determine hypodiploid cells, The cells were stained with propidium iodide using a cycle TEST PLUS DNA Reagent kit (Becton Dickinson, USA), then analysed by FACScan (Becton Dickinson, USA) with Cell Fit software. Cells with DNA content less than Gl in the cell cycle distribution were counted as hypodiploid cells.
2.1. Chemicals
2.5. PKC activity in cells
Phloretin (2’,4’,6’-trihydroxy-3-(p-hydroxyphenyl)propiophenone) was purchased from Funakoshi Chemicals. /3-Estradiol and H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) were purchased from Sigma (St. Louis, MO, USA). D-(+)-glucose was purchased from WAKO Pure Chemical Industries (Osaka, Japan). Phloretin and H7 were dissolved in dimethyl sulfoxide, fi-estradiol was dissolved in ethanol, then these chemicals were added into a culture medium of B16 mouse melanoma 4A5 cell line. 2.2. Cells and cell culture
Cells were suspended in homogenization buffer (50 A
B
ABCDE
5x10-5 ~4x16 3 Z3xld f :8
2x16 1Xld
0
10
20
30
40
50
Incubation time (hr)
B16 mouse melanoma 4A5 cells (RCB557) were provided by RIKEN Cell Bank (RCB, Ibaraki, Japan). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nissui) supplemented with 10% heat-inactivated fetal calf serum (FCS; Bioserum, Australia) at 37°C in the presence of 5% C02. Viable cells were counted with a hemocytometer by Trypan Blue exclusion.
Fig. 1. (A) Effect of phloretin on the cell growth of B16 melanoma cells. B 16 melanoma cells (1.4 x 10’ cells/ml) were cultured for 24 h prior to phloretin treatment. 0, none; A, 0.1 mM; n , 0.2 mh4. (B) Analysis of DNA fragmentation patterns by agarose gel electrophoresis. B16 melanoma cells (1 x 10’ cells/ml) were cultured for 24 h prior to phloretin treatment. DNA was extracted from B16 melanoma cells treated for 24 h with or without phloretin. A, marker: B, control cells; C, 0.1 mM phloretin; D, 0.15 mM phloretin; E, 0.2 n&l phloretin.
M. Kobori et al. /Cancer
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A
B
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Fig. 2. Effect of /3-estradiol on the induction of hypodiploid cells by phloretin. B16 melanoma 4A5 cells (5 x lo4 cells/ml) were cultured for 24 h prior to fl-estradiol-treatment for 24 h and then the cells were treated with 0.1 mM phloretin for 24 h. The ratio of hypodiploid cells was calculated from a DNA fluorescence histogram. A. control cells; B, 10m5M @-estradiol; C, 0.1 mM phloretin; D, IO4 M P-estradiol, 0.1 mM phloretin; E, lo-’ M @-estradiol, 0.1 mM phloretin.
mM Tris-HCl (pH 7.5) 0.3% (w/v) 2-mercaptoethanol, 5 mM EDTA, 10 mM EGTA, 0.05 mg/ml phenylmethylsulfonyl fluoride (PMSF) and 10 mM benzamidine). The suspension (2 x lo7 cells/ml) was lysed on ice by sonication and centrifuged at 10000 x g for 1 h at 4°C. The supernatant (25 ~1) was incubated with 25 ~1 of the reaction buffer (50 mM Tris-HCl (pH 7.5), 12 mM calcium acetate, 30 mM dithiothreitol, 0.3 mg/ml a-phosphatidyl-L-serine, 24 pg/ml phorbol 12-myristate 13-acetate and 900 PM substrate peptide) and 5 ~1 of 1.2 mM [y-32P]ATP (0.4 &i) for 15 min at 37°C. The reaction was stopped by the addition of 300 mM orthophospheric acid. 32P-Phosphorylated peptides in the reaction mixture were bound to peptide binding paper and counted using 10 ml of Aqueous Counting Scintillant (ACS II, Amersham) and Tricarb 1900TP liquid scintillation analysis (Packard). The chemicals and reagents were purchased from Amersham (England) as a protein kinase C enzyme assay system.
3. Results When 0.1 or 0.2 mM phloretin was added to the culture medium 24 h after the B 16 melanoma 4A5
Letters 119 (1997) 207.-212
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cells (1.4 x lo5 cells/ml) were plated, the cell growth was markedly inhibited during the subsequent 6-48 h of cultivation (Fig. 1A). Because the intemucleosomal DNA fragmentation typical of apoptosis was observed by agarose gel electrophoresis of the phioretin-treated B 16 cells, phloretin was demonstrated to induce apoptosis in B16 melanoma 4A5 cells (Fig. III). To determine the trigger of the phloretin-induced apoptosis in the B16 cells, the cells were treated with lo-” or low6 M P-estradiol for 24 h prior to phloretin treatment. The percentage of apoptotic cells was assessed as the ratio of hypodiploid cells to total cells using flowcytometry after @-estradiol and phloretin treatment. /3-Estradiol did not affect the phloretininduced apoptosis in the B16 cells (Fig. 2). &Zstradiol did not inhibit or induce the phloretin-induced apoptosis in B16 cells when it was added to the cells at the same time as phloretin (data not shown). It was thought that inhibition of PKC could induce an apoptosis pathway in B16 melanoma 4A5 cells. To investigate this possibility, the PKC activity in phloretin-treated B 16 melanoma 4A5 cells was determined. After 24 h of treatment, 0.1 mM phloretin reduced the cell number to 26% of the control, however, the PKC activity in the phloretin-treated cells was no different than that in the control cells. The higher concentration of phloretin (0.15 mM) inhibited both cell growth and PKC activity (Fig. 3)
100
20 0
0.1 0.15 0 Concentration of phloretin (mM)
Fig. 3. Effect of phloretin on the cell growth (f&d circle) and protein kinase C activity (hatched bar) in B 16 metanoma cells. B16 melanoma 4A5 cells (1.0 x IO5 cells/ml) were cultured for 24 h prior to phloretin-treatment for 24 h.
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0
A
B
C
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I
Fig. 4. Effect of extracellular glucose on the induction of hypodiploid cells by phlotetin and H7. B16 melanoma 4A5 cells (1 .O x lo5 cells/ml) were cultured for 24 h prior to 0.15 mM phloretin- or 0.2 mM H7-treatment for 24 h. Different concentrations of glucose were added into the culture medium at the same time with phloretin or H7. The ratio of hypodiploid cells was calculated from a DNA fluorescence histogram. A, control cells; B, 0.15 mM phloretin; C, 0.15 mM phloretin, 10 mM glucose; D, 0.15 mM phloretin, 20 mM glucose; E, 0.15 mM phloretin, 100 mh4 glucose; F, 0.2 mM H7; G, 0.2 mM H7, 10 mM glucose; H, 0.2 mM H7, 20 mM glucose; I, 0.2 mM H7, 100 mM glucose.
Subsequently the effect of extracellular glucose on the phloretin-induced apoptosis was examined. When 100 mM glucose was added into the culture medium of B16 melanoma cells with 0.15 mM phloretin, the induction of hypodiploid cells was remarkably inhibited (Fig. 4). The phloretin-induced hypodiploid cell population was reduced from 46 to 16% of the total cells by the addition of 100 mM glucose. On the other hand, 100 mM glucose did not inhibit the induction of hypodiploid cells by a protein kinase C inhibitor, H7 (Fig. 4). Because phloretin was reported to be a competitive inhibitor of glucose transport into the cells, this result suggested that phloretin induced the apoptosis in B 16 melanoma 4A5 cells through the inhibition of the transmembrane transport of glucose.
4. Discussion The data presented in this report indicate that phloretin induces apoptosis in B16 melanoma 4A5 cells. Phloretin is a naturally occurring non-steroidal estrogen, however, /3-estradiol does not affect phloretininduced apoptosis in B16 melanoma cells. The effect of phloretin on the estrogen receptor is supposed to be unrelated to the phloretin-induced apoptosis in B16
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melanoma cells. Phloretin is also known to be a non-specific PKC inhibitor. H7, staurosporine and other PKC inhibitors induced apoptosis in HL60 human leukemia, Molt-4 human lymphocytic leukemia and other cell lines [8,9,14,15]. Apoptosis in B16 melanoma cells was reported to be induced by downregulation of PKC activity [ 161; nevertheless 0.1 mM phloretin did not inhibit PKC activity but still inhibited cell growth. This result shows that phloretin is able to induce apoptosis without inhibiting PKC activity in B16 melanoma cells. In contrast, 0.15 mM phloretin strongly inhibited cell growth and PKC activity of B 16 melanoma cells. Inhibition of protein kinase C activity by phloretin probably promotes the ratio of apoptotic cells in B16 melanoma cells. We revealed that phloretin was more effective than a potent protein kinase C inhibitor, H7, in inducing apoptosis in B16 mouse melanoma 4A5 cells (Fig. 4). Inhibition of glucose transmembrane transport is one of the important physiological effects of phloretin. Phloretin competitively inhibits glucose transport into erythrocytes, intestine and brain [ 17- 191. Therefore, if the inhibition of glucose transport into the B 16 cells is the trigger of phloretin-induced apoptosis, the number of apoptotic cells will be reduced by the addition of excess glucose into the medium. Actually, since the induction of hypodiploid cells was remarkably inhibited by 100 mM glucose, phloretin was suggested to induce apoptosis mainly through inhibition of glucose transmembrane transport. Cytochalasin B, which inhibits glucose transport and polymerization of actin, induced apoptosis in the B16 cells at the concentration of 0.05 mM, while 2-deoxy-D-glucose, which inhibits glucose metabolism but not glucose transport, did not induce apoptosis in the B16 cells (unpublished observations). Cytochalasin B might induce apoptosis through the inhibition of actin polymerization. However, these observations support our conclusion that phloretin induces apoptosis in the B 16 cells through the inhibition of glucose transport in the cells. Phloretin was reported to inhibit glucose transport into tumor cells in vitro and in vivo [20]. The inhibition of glucose transport must be the most significant effect of phloretin for tumor suppression. The structure of phloretin is closely related to that of quercetin and genistein. Some of the physiological effects of phloretin are in common with these compounds. The bioflavonoid, quercetin, which induces
M. Kobori ef al. /Cancer Lerrers 119 (1997) 207-212
apoptosis in tumor cells, inhibited the activity of protein kinase C similar to phloretin, but only quercetin inhibited lipoxygenase and cyclooxygenase activities [2]. Quercetin was also reported to be a topo I and topo II inhibitor, whereas phloretin only inhibited topo II activity [21]. In spite of the many reported physiological functions of quercetin, only heat shock protein was suggested to play an important role in inducing apoptosis in tumor cells by quercetin [9]. The isoflavone, genistein, which has also been demonstrated to induce apoptosis in thymocytes and tumor cells, was suggested to induce apoptosis by topo II inhibition, however, phloretin is a less potent inhibitor of topo II [22]. Furthermore, genistein is a specific and potent inhibitor of protein tyrosine kinase activity, having little effect on protein kinase C [23]. Of these three inhibitors, only phloretin is known to inhibit glucose transport. Since apoptosis is induced by various pharmacological triggers, these naturally occurring flavonoids were supposed to promote apoptosis in tumor cells through different physiological effect(sj. Our results suggest that phloretin has chemotherapeutic effects on some tumor cells by inducing apoptosis through inhibition of glucose transport and inhibition of PKC activity.
References [I] D.W. Salter, S. Custead-Jones, J.S. Cook, Quercetin inhibits hexose transport in a human diploid fibroblast, J. Membr. Biol. 40 (1978) 67-76. [2] M. Gschwendt, F. Horn, W. Kittstein, G. Furstenberger, E. Besenfelder, F. Marks, Calcium and phospholipid-dependent protein kinase activity in mouse epidermis cytosol. Stimulation by complete and incomplete tumor promoters and inhibition by various compounds, B&hem. Biophys. Res. Commun. 124 (1984) 63-68. [3] R.J. Miksicek, Interaction of naturally occurring nonsteroidal estrogens with expressed recombinant human estrogen receptor, J. Steroid Biochem. Mol. Biol. 49 (1994) 153160. [4] M.A. Devi, N.P. Das, In vitro effects of natural plant polyphenols on the proliferation of normal and abnormal human lymphocytes and their secretions of interleukin-2, Cancer Lett. 69 (1993) 191-196. [5] J.A.S. Nelson, R.E. Falk, The efficacy of phloridzin and phloretin on tumor cell growth, Anticancer Res. 13 (1993) 2287-2292. 161 R. Bertrand, E. Solary, J. Jenkins, Y. Pommier, Apoptosis and its modulation in human promyelocytic HL-60 cells treated
‘il
with DNA topoisomerase I and II inhibitors, Exp. Cell Res. 207 (1993) 388-397. [7] D.E. Macfarlane, P.S. O’Donnel, Phorbol ester induces apoptosis in HL-60 promyelocytic leukemia cells but not in HI.-60 PET mutant, Leukemia 7 (1993) 1846- 185 1. [8] W.D. Jarvis, A.J. Turner, L.F. Povirk, R.S. Traylor, S. Grant, Induction of apoptotic DNA fragmentation and cell deatb in HL-60 human promyelccytic leukemia cells by pharmacoiogical inhibitors of protein kinase C. Cancer Res. 54 (1994) 1707-1714. [9] W. Gorczyca, J. Gong, B. Ardelt, F. Traganos, %. Darzynkiewicz, The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res. 53 (1993) 3186-3192. [lo] Y. Wei, X. Zhao, Y. Kariya, H. Fukata. K. I’eshiganara, A. Uchida. Induction of apoptosis by quercetin: involvement of heat shock protein, Cancer Res. 54 ( 19941 49.524957. [l I] M.J. McCabe Jr., S. Orrenius, Genistein induces apoptosis in immature human thymocytes by inhibiting topoisomerase-11, B&hem. Biophys. Res. Commun. 194 : 1993‘1944950. [12] 0. Kan, S.A. Baldwin, A.D. Whetton, Apoptos1a is regulated by the rate of glucose transport in a interleukin 3 dependent cell line, J. Exp. Med. 180 (1994) 917-923. [ 131 G.A. Blobel, S.H. Orkin, Estrogen-induced apoprosis by inhibition of the erythroid transcription factor GATA-1, ~Mol. Cell. Biol. 16 (1996) 1687-1694. [14] E. Falcieri, A.M. Martelli, R. Bareggi, A. Cataldi. I.. Cocco. The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in Mort-4 cells without concomitant DNA fragmentation, B&hem. Blophys. Res. Commun. 193 (1993) 19-25. [15] R. Bertrand, E. Solary, P. O’Connor, K.W. Kohn, Y. Pommier, Apoptosis and its modulation in human promyelocytic HL-60 cells treated with DNA topoisomerase I and II inhibitors, Exp. Cell Res. 21 1 : 1994) ?14-321. [ 161 Z. Zong, K. Fujikawa-Yamamoto, M. Tan&o, K. Teraoka. H. Yamagishi, S. Odashima, Saikosaponin b-induced apoptosis of cultured B 16 melanoma cell line through down-regulation of PKC activity, B&hem. Biophys. Res. (‘ommun. 219 (1996) 480-485. [17] R.M. Krupka, Asymmetrical binding of phlorerm to the glucose transport system of human erythrocytes. .I. Membr. Biol. 83 (1985) 71-80. [18] A.L. Betz, L.R. Drewes, D.D. Gilboe. Inhibition of glucose transport into brain by phlorizin, phloratin and glucose analogues, Biochim. Biophys. Acta 406 !1975) SO5-515. [19] B.M. Sahagian, Active glucose uptake by strip:~ of guinea pig intestine; competitive inhibition by phlorizio and phloretin. Can. J. B&hem. 43 (1965) 851-858. [20] J.A.S. Nelson, R.E. Falk, The efficacy of phioridzin and phloretin on tumor cell growth, Anticancer Res. 13 i 1993) 2293-2300. [21] A. Constantinous, R. Mehta, C. Runya. K. Rae. .r\ Vaughan.
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R. Moon, Flavonoids as DNA topoisomerase antagonists and poisons: structure-activity relationships, J. Nat. Prod. 58 (1995) 217-225. [22] D.S. Grove, A.M. Ma&o, Prevention of the TPA-mediated down-regulation of protein kinase C, Biochem. Biophys. Res. Commun. 151 (1988) 94-99.
[23] T. Akiyama, J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, Y. Fukami, Genistein, a specific inhibitor of tyrosine-specific protein kinases, J. Biol. Chem. 262 (1987) 5592-5595.
Phloretin-induced apoptosis in B 16 melanoma 4A5 cells by inhibition of glucose transmembrane transport Masuko Kobori”, Hiroshi Shinmoto, Tojiro Tsushida, Kazuki Shinohara’ iVationa1 Food Research Institute, Ministry of Agriculture, Forestp and Fisheries, 2-1-2 Kannondai, Tsukuba, ltmruki +Oj. Jqxm Received 24 March 1997; received in revised form 8 May 1997: accepted 15 May 1997
Abstract Phloretin, a naturally occurring dihydrochalcone, is known to inhibit tumor cell growth in vitro and in viva. To clarify the anti-tumor effects of phloretin, its apoptosis-inducing effects in B 16 melanoma 4A.5 cells were examined. Phloretin induced the internucleosomal DNA fragmentation typical of apoptosis in B16 melanoma cells. The addition of extracelluIar glucose remarkably inhibited the phloretin-induced apoptosis in the cells. When apoptosis was strongly induced in the B16 cells by phloretin, protein kinase C activity was inhibited in the cells. Our results suggest that phloretin induced apoptosis in B 16 melanoma 4A5 cells mainly through the inhibition of glucose transmembrane transport. Inhibition of protein kinase C activity by phloretin probably promotes the ratio of apoptotic cells in the cells. 0 1997 Elsevier Science Ireland Ltd Keywords:
Phloretin; Apoptosis; Melanoma cells; Glucose transport; Protein kinase C
1. IntFodwtin The dihydrochalcone phloretin is the aglucon of phlorizin occurring in apple, pear and other Roseaceae. This compound, which is related to flavonoids, is known to inhibit glucose transmembrane transport [l] and protein kinase C (PKC) [2], and is one of the naturally occurring non-steroid estrogens [3]. Phloretin has been reported to inhibit the growth of Molt-4 human leukemic cells in vitro [4], and Fisher bladder
*Corresponding author. Tel.: +81 298 388055; fax: +81 298 387996. ’ Current address: National Research Institute of Fisheries Sciences, Ministry of Agriculture, Forestry and Fisheries, 2-12-4 Fukuura, Yokohama, Kanagawa 236, Japan.
carcinoma and rat mammary adenocarcinoma cells in vivo [5]. However, the mechanism of tumor cell death induced by phloretin has not been reported, whereas the flavonoids, quercetin and genistein, has been shown to induce apoptosis in some tumor cells. Many kinds of chemotherapeutic agents induce apoptosis in tumor cells via diverse pathways, such as inhibition of topoisomerase (topo) I or 11 [6], activation or inhibition of PKC [7,8] and inhibition of DNA replication [9]. Quercetin, which is a bioflavonoid found in most edible fruits and vegetables, induces apoptosis of K562 human leukemia and some tumor cells. The induction of apoptosis by quercetin is thought to proceed via inhibition of heat shock protein synthesis and expression [IO]. The isoflavone genistein, which is contained in soybeans and other plant foods, was shown to induce apoptosis in HL60
03t.W3835/97/$17.00 0 1997 Elsevier Science lreland Ltd. All rights reserved PII SO304-3835(97)00271-l
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M. Kobori et al. /Cancer Letters 119 (1997) 207-212
human leukemia cells, immature human thymocytes and other cells [9,11]. Genistein is thought to induce apoptosis not by inhibition of tyrosine kinase but by inhibition of topo II [ 111. Since protein kinase C inhibitors induce apoptosis in HL60 and some other cells, phloretin might induce apoptosis in tumor cells by inhibition of PKC. Otherwise, phloretin may induce apoptosis in tumor cells through inhibition of glucose transport or stimulation of estrogen receptors, because inhibition of glucose uptake and the ligand-activated estrogen receptor were reported to induce apoptosis in some cell lines [12,13]. In this study, we demonstrated that phloretin induced apoptosis in B16 mouse melanoma 4A5 cells. Our results suggested that phloretin induced apoptosis mainly through inhibition of glucose transmembrane transport. Inhibition of PKC activity by phloretin probably promotes the ratio of apoptotic cells in B16 melanoma 4A5 cells.
2.3. DNA extraction and agarose gel electrophoresis Cells (1 x lo6 cells) were lysed in 1 ml lysis buffer (50 mM Tris-HCl (pH 8.0), 0.1 M EDTA and 0.5% SDS), incubated with 0.05 mg/ml RNase at 37°C for 30 min, and then incubated with 0.1 mg/ml proteinase K at 37°C for 30 min. DNA was extracted in phenol, precipitated in ethanol and resuspended in a buffer (10 mM Tris-HCl (pH 8.0) 1 mM EDTA). The samples were electrophoresed in Tris-borate buffer (pH 8.0) on a 2% agarose gel and the DNA was visualized with ethidium bromide. 2.4. Flocytometric analysis
2. Materials and methods
Flowcytometric analysis was performed to determine hypodiploid cells, The cells were stained with propidium iodide using a cycle TEST PLUS DNA Reagent kit (Becton Dickinson, USA), then analysed by FACScan (Becton Dickinson, USA) with Cell Fit software. Cells with DNA content less than Gl in the cell cycle distribution were counted as hypodiploid cells.
2.1. Chemicals
2.5. PKC activity in cells
Phloretin (2’,4’,6’-trihydroxy-3-(p-hydroxyphenyl)propiophenone) was purchased from Funakoshi Chemicals. /3-Estradiol and H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) were purchased from Sigma (St. Louis, MO, USA). D-(+)-glucose was purchased from WAKO Pure Chemical Industries (Osaka, Japan). Phloretin and H7 were dissolved in dimethyl sulfoxide, fi-estradiol was dissolved in ethanol, then these chemicals were added into a culture medium of B16 mouse melanoma 4A5 cell line. 2.2. Cells and cell culture
Cells were suspended in homogenization buffer (50 A
B
ABCDE
5x10-5 ~4x16 3 Z3xld f :8
2x16 1Xld
0
10
20
30
40
50
Incubation time (hr)
B16 mouse melanoma 4A5 cells (RCB557) were provided by RIKEN Cell Bank (RCB, Ibaraki, Japan). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Nissui) supplemented with 10% heat-inactivated fetal calf serum (FCS; Bioserum, Australia) at 37°C in the presence of 5% C02. Viable cells were counted with a hemocytometer by Trypan Blue exclusion.
Fig. 1. (A) Effect of phloretin on the cell growth of B16 melanoma cells. B 16 melanoma cells (1.4 x 10’ cells/ml) were cultured for 24 h prior to phloretin treatment. 0, none; A, 0.1 mM; n , 0.2 mh4. (B) Analysis of DNA fragmentation patterns by agarose gel electrophoresis. B16 melanoma cells (1 x 10’ cells/ml) were cultured for 24 h prior to phloretin treatment. DNA was extracted from B16 melanoma cells treated for 24 h with or without phloretin. A, marker: B, control cells; C, 0.1 mM phloretin; D, 0.15 mM phloretin; E, 0.2 n&l phloretin.
M. Kobori et al. /Cancer
T
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A
B
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E
Fig. 2. Effect of /3-estradiol on the induction of hypodiploid cells by phloretin. B16 melanoma 4A5 cells (5 x lo4 cells/ml) were cultured for 24 h prior to fl-estradiol-treatment for 24 h and then the cells were treated with 0.1 mM phloretin for 24 h. The ratio of hypodiploid cells was calculated from a DNA fluorescence histogram. A. control cells; B, 10m5M @-estradiol; C, 0.1 mM phloretin; D, IO4 M P-estradiol, 0.1 mM phloretin; E, lo-’ M @-estradiol, 0.1 mM phloretin.
mM Tris-HCl (pH 7.5) 0.3% (w/v) 2-mercaptoethanol, 5 mM EDTA, 10 mM EGTA, 0.05 mg/ml phenylmethylsulfonyl fluoride (PMSF) and 10 mM benzamidine). The suspension (2 x lo7 cells/ml) was lysed on ice by sonication and centrifuged at 10000 x g for 1 h at 4°C. The supernatant (25 ~1) was incubated with 25 ~1 of the reaction buffer (50 mM Tris-HCl (pH 7.5), 12 mM calcium acetate, 30 mM dithiothreitol, 0.3 mg/ml a-phosphatidyl-L-serine, 24 pg/ml phorbol 12-myristate 13-acetate and 900 PM substrate peptide) and 5 ~1 of 1.2 mM [y-32P]ATP (0.4 &i) for 15 min at 37°C. The reaction was stopped by the addition of 300 mM orthophospheric acid. 32P-Phosphorylated peptides in the reaction mixture were bound to peptide binding paper and counted using 10 ml of Aqueous Counting Scintillant (ACS II, Amersham) and Tricarb 1900TP liquid scintillation analysis (Packard). The chemicals and reagents were purchased from Amersham (England) as a protein kinase C enzyme assay system.
3. Results When 0.1 or 0.2 mM phloretin was added to the culture medium 24 h after the B 16 melanoma 4A5
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cells (1.4 x lo5 cells/ml) were plated, the cell growth was markedly inhibited during the subsequent 6-48 h of cultivation (Fig. 1A). Because the intemucleosomal DNA fragmentation typical of apoptosis was observed by agarose gel electrophoresis of the phioretin-treated B 16 cells, phloretin was demonstrated to induce apoptosis in B16 melanoma 4A5 cells (Fig. III). To determine the trigger of the phloretin-induced apoptosis in the B16 cells, the cells were treated with lo-” or low6 M P-estradiol for 24 h prior to phloretin treatment. The percentage of apoptotic cells was assessed as the ratio of hypodiploid cells to total cells using flowcytometry after @-estradiol and phloretin treatment. /3-Estradiol did not affect the phloretininduced apoptosis in the B16 cells (Fig. 2). &Zstradiol did not inhibit or induce the phloretin-induced apoptosis in B16 cells when it was added to the cells at the same time as phloretin (data not shown). It was thought that inhibition of PKC could induce an apoptosis pathway in B16 melanoma 4A5 cells. To investigate this possibility, the PKC activity in phloretin-treated B 16 melanoma 4A5 cells was determined. After 24 h of treatment, 0.1 mM phloretin reduced the cell number to 26% of the control, however, the PKC activity in the phloretin-treated cells was no different than that in the control cells. The higher concentration of phloretin (0.15 mM) inhibited both cell growth and PKC activity (Fig. 3)
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Fig. 3. Effect of phloretin on the cell growth (f&d circle) and protein kinase C activity (hatched bar) in B 16 metanoma cells. B16 melanoma 4A5 cells (1.0 x IO5 cells/ml) were cultured for 24 h prior to phloretin-treatment for 24 h.
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Fig. 4. Effect of extracellular glucose on the induction of hypodiploid cells by phlotetin and H7. B16 melanoma 4A5 cells (1 .O x lo5 cells/ml) were cultured for 24 h prior to 0.15 mM phloretin- or 0.2 mM H7-treatment for 24 h. Different concentrations of glucose were added into the culture medium at the same time with phloretin or H7. The ratio of hypodiploid cells was calculated from a DNA fluorescence histogram. A, control cells; B, 0.15 mM phloretin; C, 0.15 mM phloretin, 10 mM glucose; D, 0.15 mM phloretin, 20 mM glucose; E, 0.15 mM phloretin, 100 mh4 glucose; F, 0.2 mM H7; G, 0.2 mM H7, 10 mM glucose; H, 0.2 mM H7, 20 mM glucose; I, 0.2 mM H7, 100 mM glucose.
Subsequently the effect of extracellular glucose on the phloretin-induced apoptosis was examined. When 100 mM glucose was added into the culture medium of B16 melanoma cells with 0.15 mM phloretin, the induction of hypodiploid cells was remarkably inhibited (Fig. 4). The phloretin-induced hypodiploid cell population was reduced from 46 to 16% of the total cells by the addition of 100 mM glucose. On the other hand, 100 mM glucose did not inhibit the induction of hypodiploid cells by a protein kinase C inhibitor, H7 (Fig. 4). Because phloretin was reported to be a competitive inhibitor of glucose transport into the cells, this result suggested that phloretin induced the apoptosis in B 16 melanoma 4A5 cells through the inhibition of the transmembrane transport of glucose.
4. Discussion The data presented in this report indicate that phloretin induces apoptosis in B16 melanoma 4A5 cells. Phloretin is a naturally occurring non-steroidal estrogen, however, /3-estradiol does not affect phloretininduced apoptosis in B16 melanoma cells. The effect of phloretin on the estrogen receptor is supposed to be unrelated to the phloretin-induced apoptosis in B16
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melanoma cells. Phloretin is also known to be a non-specific PKC inhibitor. H7, staurosporine and other PKC inhibitors induced apoptosis in HL60 human leukemia, Molt-4 human lymphocytic leukemia and other cell lines [8,9,14,15]. Apoptosis in B16 melanoma cells was reported to be induced by downregulation of PKC activity [ 161; nevertheless 0.1 mM phloretin did not inhibit PKC activity but still inhibited cell growth. This result shows that phloretin is able to induce apoptosis without inhibiting PKC activity in B16 melanoma cells. In contrast, 0.15 mM phloretin strongly inhibited cell growth and PKC activity of B 16 melanoma cells. Inhibition of protein kinase C activity by phloretin probably promotes the ratio of apoptotic cells in B16 melanoma cells. We revealed that phloretin was more effective than a potent protein kinase C inhibitor, H7, in inducing apoptosis in B16 mouse melanoma 4A5 cells (Fig. 4). Inhibition of glucose transmembrane transport is one of the important physiological effects of phloretin. Phloretin competitively inhibits glucose transport into erythrocytes, intestine and brain [ 17- 191. Therefore, if the inhibition of glucose transport into the B 16 cells is the trigger of phloretin-induced apoptosis, the number of apoptotic cells will be reduced by the addition of excess glucose into the medium. Actually, since the induction of hypodiploid cells was remarkably inhibited by 100 mM glucose, phloretin was suggested to induce apoptosis mainly through inhibition of glucose transmembrane transport. Cytochalasin B, which inhibits glucose transport and polymerization of actin, induced apoptosis in the B16 cells at the concentration of 0.05 mM, while 2-deoxy-D-glucose, which inhibits glucose metabolism but not glucose transport, did not induce apoptosis in the B16 cells (unpublished observations). Cytochalasin B might induce apoptosis through the inhibition of actin polymerization. However, these observations support our conclusion that phloretin induces apoptosis in the B 16 cells through the inhibition of glucose transport in the cells. Phloretin was reported to inhibit glucose transport into tumor cells in vitro and in vivo [20]. The inhibition of glucose transport must be the most significant effect of phloretin for tumor suppression. The structure of phloretin is closely related to that of quercetin and genistein. Some of the physiological effects of phloretin are in common with these compounds. The bioflavonoid, quercetin, which induces
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apoptosis in tumor cells, inhibited the activity of protein kinase C similar to phloretin, but only quercetin inhibited lipoxygenase and cyclooxygenase activities [2]. Quercetin was also reported to be a topo I and topo II inhibitor, whereas phloretin only inhibited topo II activity [21]. In spite of the many reported physiological functions of quercetin, only heat shock protein was suggested to play an important role in inducing apoptosis in tumor cells by quercetin [9]. The isoflavone, genistein, which has also been demonstrated to induce apoptosis in thymocytes and tumor cells, was suggested to induce apoptosis by topo II inhibition, however, phloretin is a less potent inhibitor of topo II [22]. Furthermore, genistein is a specific and potent inhibitor of protein tyrosine kinase activity, having little effect on protein kinase C [23]. Of these three inhibitors, only phloretin is known to inhibit glucose transport. Since apoptosis is induced by various pharmacological triggers, these naturally occurring flavonoids were supposed to promote apoptosis in tumor cells through different physiological effect(sj. Our results suggest that phloretin has chemotherapeutic effects on some tumor cells by inducing apoptosis through inhibition of glucose transport and inhibition of PKC activity.
References [I] D.W. Salter, S. Custead-Jones, J.S. Cook, Quercetin inhibits hexose transport in a human diploid fibroblast, J. Membr. Biol. 40 (1978) 67-76. [2] M. Gschwendt, F. Horn, W. Kittstein, G. Furstenberger, E. Besenfelder, F. Marks, Calcium and phospholipid-dependent protein kinase activity in mouse epidermis cytosol. Stimulation by complete and incomplete tumor promoters and inhibition by various compounds, B&hem. Biophys. Res. Commun. 124 (1984) 63-68. [3] R.J. Miksicek, Interaction of naturally occurring nonsteroidal estrogens with expressed recombinant human estrogen receptor, J. Steroid Biochem. Mol. Biol. 49 (1994) 153160. [4] M.A. Devi, N.P. Das, In vitro effects of natural plant polyphenols on the proliferation of normal and abnormal human lymphocytes and their secretions of interleukin-2, Cancer Lett. 69 (1993) 191-196. [5] J.A.S. Nelson, R.E. Falk, The efficacy of phloridzin and phloretin on tumor cell growth, Anticancer Res. 13 (1993) 2287-2292. 161 R. Bertrand, E. Solary, J. Jenkins, Y. Pommier, Apoptosis and its modulation in human promyelocytic HL-60 cells treated
‘il
with DNA topoisomerase I and II inhibitors, Exp. Cell Res. 207 (1993) 388-397. [7] D.E. Macfarlane, P.S. O’Donnel, Phorbol ester induces apoptosis in HL-60 promyelocytic leukemia cells but not in HI.-60 PET mutant, Leukemia 7 (1993) 1846- 185 1. [8] W.D. Jarvis, A.J. Turner, L.F. Povirk, R.S. Traylor, S. Grant, Induction of apoptotic DNA fragmentation and cell deatb in HL-60 human promyelccytic leukemia cells by pharmacoiogical inhibitors of protein kinase C. Cancer Res. 54 (1994) 1707-1714. [9] W. Gorczyca, J. Gong, B. Ardelt, F. Traganos, %. Darzynkiewicz, The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res. 53 (1993) 3186-3192. [lo] Y. Wei, X. Zhao, Y. Kariya, H. Fukata. K. I’eshiganara, A. Uchida. Induction of apoptosis by quercetin: involvement of heat shock protein, Cancer Res. 54 ( 19941 49.524957. [l I] M.J. McCabe Jr., S. Orrenius, Genistein induces apoptosis in immature human thymocytes by inhibiting topoisomerase-11, B&hem. Biophys. Res. Commun. 194 : 1993‘1944950. [12] 0. Kan, S.A. Baldwin, A.D. Whetton, Apoptos1a is regulated by the rate of glucose transport in a interleukin 3 dependent cell line, J. Exp. Med. 180 (1994) 917-923. [ 131 G.A. Blobel, S.H. Orkin, Estrogen-induced apoprosis by inhibition of the erythroid transcription factor GATA-1, ~Mol. Cell. Biol. 16 (1996) 1687-1694. [14] E. Falcieri, A.M. Martelli, R. Bareggi, A. Cataldi. I.. Cocco. The protein kinase inhibitor staurosporine induces morphological changes typical of apoptosis in Mort-4 cells without concomitant DNA fragmentation, B&hem. Blophys. Res. Commun. 193 (1993) 19-25. [15] R. Bertrand, E. Solary, P. O’Connor, K.W. Kohn, Y. Pommier, Apoptosis and its modulation in human promyelocytic HL-60 cells treated with DNA topoisomerase I and II inhibitors, Exp. Cell Res. 21 1 : 1994) ?14-321. [ 161 Z. Zong, K. Fujikawa-Yamamoto, M. Tan&o, K. Teraoka. H. Yamagishi, S. Odashima, Saikosaponin b-induced apoptosis of cultured B 16 melanoma cell line through down-regulation of PKC activity, B&hem. Biophys. Res. (‘ommun. 219 (1996) 480-485. [17] R.M. Krupka, Asymmetrical binding of phlorerm to the glucose transport system of human erythrocytes. .I. Membr. Biol. 83 (1985) 71-80. [18] A.L. Betz, L.R. Drewes, D.D. Gilboe. Inhibition of glucose transport into brain by phlorizin, phloratin and glucose analogues, Biochim. Biophys. Acta 406 !1975) SO5-515. [19] B.M. Sahagian, Active glucose uptake by strip:~ of guinea pig intestine; competitive inhibition by phlorizio and phloretin. Can. J. B&hem. 43 (1965) 851-858. [20] J.A.S. Nelson, R.E. Falk, The efficacy of phioridzin and phloretin on tumor cell growth, Anticancer Res. 13 i 1993) 2293-2300. [21] A. Constantinous, R. Mehta, C. Runya. K. Rae. .r\ Vaughan.
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R. Moon, Flavonoids as DNA topoisomerase antagonists and poisons: structure-activity relationships, J. Nat. Prod. 58 (1995) 217-225. [22] D.S. Grove, A.M. Ma&o, Prevention of the TPA-mediated down-regulation of protein kinase C, Biochem. Biophys. Res. Commun. 151 (1988) 94-99.
[23] T. Akiyama, J. Ishida, S. Nakagawa, H. Ogawara, S. Watanabe, N. Itoh, M. Shibuya, Y. Fukami, Genistein, a specific inhibitor of tyrosine-specific protein kinases, J. Biol. Chem. 262 (1987) 5592-5595.