Anti-alopecia mechanisms of soymetide-4, an immunostimulating peptide derived from soy β-conglycinin

Peptides 26 (2005) 707–711

Anti-alopecia mechanisms of soymetide-4, an immunostimulating peptide derived from soy ␤-conglycinin Takahiro Tsurukia , Kyoya Takahatab , Masaaki Yoshikawaa,∗ a

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan b Faculty of Agriculture, Okayama University, Okayama 700-0082, Japan

Received 25 October 2004; received in revised form 29 December 2004; accepted 4 January 2005 Available online 12 February 2005

Abstract Previously, we found that orally administered soymetide-4 (MITL), an immunostimulating peptide derived from soybean ␤-conglycinin ␣ subunit, suppressed alopecia induced by the anti-cancer drug etoposide in neonatal rats. Soymetide-4 has weak affinity for N-formylmethionyl-leucyl-phenylalanine (fMLP) receptor. fMLP showed an anti-alopecia effect after intraperitoneal administration, though it was inactive after oral administration. Anti-alopecia effect of fMLP was blocked by pyrilamine or cimetidine, antagonists for histamine H1 or H2 receptor, respectively. However, the anti-alopecia effect of soymetide-4 was not inhibited by the histamine antagonists but by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP4 receptor for PGE2 . Anti-alopecia effect of soymetide-4 was also blocked by pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-␬B (NF-␬B). These results suggest that PGE2 , which is produced after activation of COX by soymetide-4, might suppress apoptosis of hair matrix cells and etoposide-induced alopecia by activating NF-␬B. © 2005 Elsevier Inc. All rights reserved. Keywords: Alopecia; Etoposide; Peptide; Prostaglandin; Soybean

1. Introduction Soymetide-13 (MITLAIPVNKPGR) is an immunostimulating peptide isolated from trypsin digest of soy protein based on stimulatory activity for phagocytosis by human polymorphonuclear leukocytes (PMN) in vitro [11]. Soymetide-13 is derived from residues 173–185 of the soybean ␤-conglycinin ␣ subunit. Soymetide-4 (MITL) is the minimum structure required for immunostimulating activity and has weak affinity for the chemotactic peptide Abbreviations: fMLP, N-formyl-methionyl-leucyl-phenylalanine; PG, prostaglandin; COX, cyclooxygenase; NF-␬B, nuclear factor-␬B ∗ Corresponding author. Tel.: +81 774 38 3725; fax: +81 774 38 3774. E-mail address: [email protected] (M. Yoshikawa). 0196-9781/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2005.01.010

N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor [3,8,11]. After oral administration, soymetide-4 (300 mg/kg for 8 days) suppressed alopecia (hair loss) induced by the anticancer drug etoposide in a neonatal rat model [12,13]. Etoposide, effective against various leukemia and lung cancer, is recently regarded as a useful anti-cancer drug since its application has been extended for treatment of advanced skin Kaposi sarcoma. Anti-alopecia agent is valuable to improve quality of life (QOL) in medical treatment using this anticancer drug. fMLP (30 mg/kg for 4 days) also induced the antialopecia effect after intraperitoneal injection, but it was ineffective after oral administration. Histamine seems to be involved in the anti-alopecia effect by fMLP since it

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was blocked by antagonists for both the histamine H1 and H2 receptors, pyrilamine and cimetidine, respectively [14]. In the present study, we found that the anti-alopecia effect of soymetide-4 was not inhibited by the histamine antagonists. We investigated the mechanism of the anti-alopecia effect of soymetide-4 after oral administration.

2. Materials and methods 2.1. Animals Experimental protocols involving laboratory animals were approved by the ethical committee of the Graduate School of Agriculture, Kyoto University. Lactating Sprague–Dawley (SD) rats were purchased from Japan SLC Inc. Ten neonatal male and female rats nursed from one lactating mother rat. Six-day-old rats were used for experiments.

2.2. Reagents Etoposide (VP-16) was purchased from Nippon Kayaku Co. Ltd. (Tokyo, Japan). Indomethacin, a COX inhibitor, was from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Pyrilamine a histamine H1 antagonist, was bought from Sigma Chemical Co. (St. Louis, MO, USA). Cimetidine, an H2 antagonist, was from Sumitomo Pharmaceuticals Co. Ltd. (Osaka, Japan). AH23848B, an antagonist of the PGE2 receptor EP4 , was a kind gift from Glaxo Smith Cline (Stevenage, UK). Pyrrolidine dithiocarbamate ammonium (PDTC), an inhibitor of nuclear factor-␬B (NF-␬B) activation, was purchased from Nacalai Tesque Inc. (Kyoto, Japan). 2.3. Peptide synthesis Soymetide-4 (MITL) was prepared using a solid phase peptide synthesizer (PS3, Protein Technologies Inc., Tuscon, AZ, USA) according to the 9-fluoroenyl methoxy-

Fig. 1. Effect of histamine antagonists on the anti-alopecia effect of orally administered soymetide-4. Etoposide was injected at a dose of 1.2 mg/kg intraperitoneally for 3 days to 11-day-old rats. Soymetide-4 (300 mg/kg) was administered orally for 8 consecutive days beginning 5 days before the first etoposide injection. (A) Pyrilamine (10 mg/kg i.p. for 8 days) or (B) cimetidine (10 mg/kg i.p. for 8 days) was injected alone or simultaneously with soymetide-4. Pictures were taken 7 days after the last etoposide injection. Hair area was analyzed as described in Section 2. Values are expressed as mean ± S.E.M., (*** P < 0.001 vs. etoposide-injected group).

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carbonyl (Fmoc) method. Peptides were purified with reversed-phase high performance liquid chromatography (HPLC) on octadecyl silica (ODS) column (Cosmosil 5C18-AR-II, 20 mm × 250 mm, Nacalai Tesque Inc., Kyoto, Japan). 2.4. Evaluation of anti-alopecia effect In this study, reagents were dissolved in saline and injected in a volume of 10 ml/kg. Etoposide, an anti-cancer agent that characteristically induces alopecia as a side effect, was injected daily in 11-day-old SD rats at 1.2 mg/kg i.p. for 3 consecutive days. Alopecia was induced by 7 days after the last injection of etoposide. Beginning 5 days before the first injection of etoposide, soymetide-4 was administered orally for 8 consecutive days. Indomethacin, AH23848B, pyrilamine, cimetidine, and PDTC were administered intraperitoneally concomitantly with administration of soymetide-4, as indicated above. Degrees of alopecia were evaluated and pictures were taken 7 days after the last etoposide injection. Percentage of hair area on total back skin area was analyzed by NIH image.

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3. Results 3.1. Effect of histamine antagonists on anti-alopecia effect of orally administered soymetide-4 In our previous study, the anti-alopecia effect of fMLP was blocked by pyrilamine and cimetidine [14]. We investigated whether histamine receptors might also be involved in the anti-alopecia effect of orally administered soymetide4. The protective effect of orally administered soymetide-4 (300 mg/kg for 8 days) against etoposide (1.2 mg/kg i.p. for 3 days)-induced alopecia was not inhibited by either pyrilamine (10 mg/kg i.p. for 8 days) or cimetidine (10 mg/kg i.p. for 8 days) (Fig. 1A and B), suggesting that histamine is not involved in the anti-alopecia effect of orally administered soymetide-4. 3.2. Effects of indomethacin and an EP antagonist We investigated whether cyclooxygenase (COX) is involved in the anti-alopecia effect of orally administered soymetide-4, since fMLP agonists are known to induce

Fig. 2. Effect of a COX inhibitor and a PGE2 antagonist on the anti-alopecia effect of orally administered soymetide-4. Etoposide was injected at a dose of 1.2 mg/kg intraperitoneally for 3 days to 11-day-old rats. (A) Indomethacin (5 mg/kg i.p. for 8 days) or (B) AH-23848B (10 mg/kg i.p. for 8 days) was injected alone or simultaneously with soymetide-4 (300 mg/kg p.o. for 8 days). Pictures were taken 7 days after the last etoposide injection. Values are expressed as mean ± S.E.M., (*** P < 0.001, ** P < 0.01 vs. etoposide-injected group, ### P < 0.001, ## P < 0.01 vs. etoposide + soymetide-4-injected group).

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COX-2 in neutrophils [7]. The COX inhibitor indomethacin (5 mg/kg i.p. for 8 days) abolished the protective effect of orally administered soymetide-4 against etoposide-induced alopecia (Fig. 2A) though it did not induce alopecia by itself under these conditions. These suggest that COX metabolites are involved in the anti-alopecia effect of orally administered soymetide-4. PGE2 is a prostaglandin involved in hair growth [4,9] and fMLP agonists are reported to stimulate PGE2 release [7]. Then, we tested whether PGE2 is associated with the antialopecia effect of orally administered soymetide-4. There are four subtypes of PGE2 receptors: EP1 , EP2 , EP3 , and EP4 [1]. AH-23848B (10 mg/kg i.p. for 8 days), an antagonist of the EP4 receptor subtype, blocked the anti-alopecia effect of soymetide-4 (Fig. 2B) though it did not induce alopecia when

given alone. On the other hand, AH-6809 (10 mg/kg i.p. for 8 days), an antagonist of the EP1 , EP2 , and EP3 receptor subtypes [5], did not inhibit the anti-alopecia effect (data not shown). These results suggest that anti-alopecia effects of soymetide-4 are mediated via EP4 . 3.3. Effect of an NF-κB inhibitor on the anti-alopecia effect of orally administered soymetide-4 Etoposide-induced alopecia is caused by apoptosis of hair follicle cells. The anti-alopecia effect of orally administered soymetide-4 might be associated with suppression of apoptosis. NF-␬B is a nuclear factor which prevents apoptosis and is activated by various factors including PGE2 [2]. Thus, we investigated the effect of PDTC, an inhibitor of NF-␬B

Fig. 3. Effect of an NF-␬B inhibitor on the anti-alopecia effect of orally administered soymetide-4. Etoposide was injected at a dose of 1.2 mg/kg intraperitoneally for 3 days to 11-day-old rats. Pyrrolidine dithiocarbamate (PDTC, 100 mg/kg i.p. for 8 days) was injected alone or simultaneously with soymetide-4 (300 mg/kg p.o. for 8 days). Pictures were taken 7 days after the last etoposide injection. Values are expressed as mean ± S.E.M., (*** P < 0.001 vs. etoposide-injected group, ## P < 0.01 vs. etoposide + soymetide-4-injected group).

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The next problem is whether the anti-cancer effect of etoposide is not inhibited by soymetide-4. We have confirmed that etoposide-induced apoptosis of human leukemia cells HL-60 was not inhibited by soymetide-4 in vitro (data not shown).

Acknowledgments This work was supported in part by Grant-in-Aid for Scientific Research from the Japanese Society for the Promotion of Science and the PROBRAIN grant.

References Fig. 4. Hypothetical mechanism of anti-alopecia effect of orally administrated soymetide-4.

activation, on the soymetide-4-induced anti-alopecia effect. PDTC (100 mg/kg i.p. for 8 days) abolished the anti-alopecia effect of soymetide-4 (Fig. 3) though it did not induce alopecia by itself. These suggest that activation of NF-␬B is involved in the anti-alopecia effect of orally administered soymetide-4.

4. Discussion The anti-alopecia effect of orally administered soymetide4 was not inhibited by pyrilamine or cimetidine while the anti-alopecia effect of intraperitoneally injected fMLP was blocked by the antagonists [14]. On the other hand, the anti-alopecia effect of orally administered soymetide-4 was blocked by indomethacin. COX metabolites are associated with hair growth; COX inhibitors, such as indomethacin or aspirin-like drugs, induce alopecia [6,10] and PGE2 are known to be involved in hair growth [4]. EP3 and EP4 receptor subtypes expresses on hair follicle cells and are considered to be involved in hair development and re-growth [9]. Next, we examined the effect of PGE2 antagonists on the anti-alopecia effect of soymetide-4. The EP4 antagonist AH23848B blocked the effect, while AH-6809, a nonselective antagonist which binds to EP1 , EP2 , and EP3 , did not, suggesting that only the EP4 receptor subtype mediates the anti-alopecia effect of soymetide-4. Thus, we found for the first time that PGE2 mediates protection against etoposide-induced alopecia. The anti-alopecia effect of orally administered soymetide4 was blocked by PDTC an inhibitor of NF-␬B. NF-␬B is known to be activated by PGE2 [2]. These results suggest that PGE2 , which is produced after activation of COX by soymetide-4, might suppress apoptosis of hair follicle cells and etoposide-induced alopecia by activating NF-␬B (Fig. 4).

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