Antitumor activities of a newly synthesized shikonin derivative, 2-hyim-DMNQ-S-33

Cancer Letters 172 (2001) 171–175 www.elsevier.com/locate/canlet

Antitumor activities of a newly synthesized shikonin derivative, 2-hyim-DMNQ-S-33 Sung Hoon Kim a,1,*, In-Cheol Kang a,1, Taek Joon Yoon a, Young Mee Park b, Kyung-Sun Kang c, Gyu Yong Song d, Byung Zun Ahn d a

Department of Oncology, Graduate School of East-West Medical Science, KyungHee University, 1 Seochun-ri, Kiheung-eup, Yongin 449-701 South Korea b Department of Biology, University of Incheon, Incheon 402-749, South Korea c Department of Veterinary Public Health Colleage of Veterinary Medicine, Seoul National University, 103 Seodun-Dong, Kwonsun -Ku, Su-won 441-744, South Korea d College of Pharmacy, Chungnam National University, Taejon 305-764, South Korea Received 4 April 2001; received in revised form 18 June 2001; accepted 25 June 2001

Abstract 2- or 6-(1-hydroxyiminoalkyl)-5,8-dimethoxy-1, 4-naphthoquinone(2- or 6-hyim-DMNQ) derived from the roots of Lithospermum erythrorhizon was synthesized for the evaluation of antitumor activities. Among those derivatives, 2-hyim-DMNQS33 was found to be a potent anticancer agent. This compound suppressed the proliferation of Radiation Induced Fibrosarcoma (RIF) cells in a dose-dependent manner. 2-hyim-DMNQ-S33 significantly prolonged the survival time by 239% as compared with Sarcoma 180 tumor-bearing control mice in vivo. We found that the compound significantly suppressed phosphorylation of extracellular signal-regulated kinase (pERK) and activated c-jun-N-terminal kinase (JNK) and protein kinase C (PKC)-a following 4 h-treatment. These findings indicate that 2-hyim-DMSQ-S33 exerts antitumor activities by regulating pERK, JNK and PKC-a. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Shikonin; c-jun-N-terminal kinase; Extracellular signal-regulated kinase; Protein kinase C; Antitumor activity

1. Introduction The extract of the roots of Lithospermum erythrorhizon was reported to restore immunosuppression induced by the anti-tumor agent, cyclophosphamide [1]. The extract also significantly inhibited mutagenic effects of carcinogen N-butyl-N-butanolnitrosoamine (BBN) by suppression of chemotactic activity and production of IL-1 and TNF-alpha by macrophages [2]. * Corresponding author Tel.: 182-31-201-2179; fax: 182-31205-1074. E-mail address: [email protected] (S.H. Kim). 1 These authors contributed equally to this work.

Shikonin was known to be a main ingredient of the roots of Lithospermum erythrorhizon. Some shikonin derivatives have revealed an inhibitory effect on the proliferation of several tumor cell lines. Shikonin analogue 93/637 (SA) specifically suppressed the growth of PC-3 prostate cancer cells as well as vascular endothelial growth factor (VEGF) and insulin-like growth factors (IGFs) expression in the cells, suggesting that SA may have therapeutic potential for the treatment of prostate cancer [3]. Shikonin induced apoptosis in HL-60, a human premyelocytic leukemia cell line [4]. Beta-hydroxyisovalerylshikonin (beta-HIVS) induced apoptosis by activation of MAP kinases, such as ERK2, JNK and p38, which is different from the

0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00665-6

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apoptosis induced by Fas and anticancer drugs [5]. Alkannin and shikonin, naturally occurring naphthoquinones, and acylshikonin analogue were reported to be potent inhibitors of topoisomerase I [6,7]. Shikonin also inhibited tumor necrosis factor (TNF)-alpha- or B16 melanoma cell-induced angiogenesis in mice and normal developmental angiogenesis of endothelial cells in the yolk-sac membranes of chick embryos by blocking integrin alpha v beta 3 expression [8]. Clinical observations in later-stage lung cancer patients who were not candidates for surgery, radiotherapy or chemotherapy showed that the shikonin mixture inhibited the growth of lung tumors and improved the immune function of the body. Moreover, the shikonin mixture had no harmful effects on peripheral blood, cells, heart, kidney or liver [9]. We previously reported the inhibitory effect of 2- or 6-(1-oxyiminoalkyl)-5,8-dimethoxy-1,4-naphthoquinones(2- or 6-oxo-DMNQ) on DNA topoisomerasel and cytotoxicity [7,10]. In this study, 2-hyim-DMNQS33, a new shikonin derivative, was synthesized to improve the poor solubility of 2-oxo-DMNQ-S33 (Fig. 1). We also evaluated the effect of 2-hyimDMNQ-S33 on proliferation of RIF cells, expression of mitogen-activated protein (MAP) kinase and PKCa, and prolongation of life-span in tumor-bearing animals in vivo.

2. Materials and methods 2.1. Materials A 2-hyim-DMNQ-S33 derivative was synthesized at

Chungnam University, College of Pharmacy, Taejon, Korea. Tissue culture and biochemical reagents were purchased from Sigma–Aldrich (USA). 2.2. Animals Male ICR and C57BL/6 mice, 4 weeks old, were purchased from Korea Research Institute of Chemical Technology. They were maintained under standard vivarium conditions. Laboratory pellet chow (Samyang, Inc.) and water were administered ad libitum. 2.3. Tumor cells and culture condition S-180 was purchased from ATCC. RIF cells were derived from a radiation –induced fibrisancoma [17]. Cells were cultured in RPMI 1640 (GIBCO-BRL, Richmond,USA) supplemented with l-glutamine, 10% fetal bovine serum (FBS), 1% antibiotics (penicillin 100,000 units/ml, streptomycin 10 mg/ml), and 2 g sodium bicarbonate at 378C in 5% CO2, 95% air. Exponentially growing cultures were used for all experiments. 2.4. Cell proliferation assay Cell proliferation was evaluated by SRB (Sulforhodamine B) assay [11]. Harvested tumor cells (2 £ 10 4/ well) were seeded onto 96-well plates and cultured for 24 h. The cells were treated with 2-hyim-DMNQ-S33 for 4 h by replacing the media in the absence of FBS. Immediately after the drug treatment, cells were fixed by 100 ml of 10% trichloroacetic acid (TCA) in each well for 1 h at 48C. The plate was washed five times with distilled water and dried. Two hundred and fifty microlitres of 0.4% SRB in 1% acetic acid was added to each well for 30 min. The plate was washed 5–6 times with 1% acetic acid and dried. SRB stain in the cells was dissolved in 10 mM Tris–HCl and the color was measured at 520 nm. 2.5. Western immunoblot analysis of PKC, JNK and MAP kinase

Fig. 1. Chemical structure of 2-hyim-DMNQ-S33[2-(1-hydroxyiminoalkyl)-5,8-dimethoxy-1, 4-naphthoquinone-S33].

The subcellular fractionations of PKC and MAP kinase were carried out according to standard procedures with a slight modification [12]. In brief, the cells were exposed to 2-hyim-DMNQ-S33 for different time periods. Cells were washed, centrifuged, resuspended

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in 1 ml of homogenization buffer (20 mM Tris–HCl, 5 mM EGTA, 5 mM EDTA, 10 mM benzamidine, 0.3% mercaptoethanol, 50 mg/ml leupeptin and 50 mg/ml PMSF) and left for 20 min at 48C to swell. Cells were homogenized with a Dounce homogenizer until it was clear from microscopic observation that the cells were ruptured. Homogenates were then centrifuged at 100,000 £ g at 48C for 30 min and the supernatant was collected as the cytosol fraction. The pellet was resuspended in the homogenization buffer containing 1% Triton X-100 and left for 30 min at 48C. The suspension was centrifuged again at 100,000 £ g and the supernatant was taken as the membrane fraction. The expression of PKC and MAP kinase was determined by Western blot analysis. 2.6. Antitumor activity in ICR mice bearing sarcoma 180 cells Male ICR mice were inoculated intraperitoneally (injection volume; 0.2 ml per mouse) with sarcoma 180 cells suspended in saline (1 £ 10 7 cells/ml)[11]. The mice were divided into groups of eight, 24 h after the inoculation. The test compounds (adriamycin and 2-hyim-DMNQ-S33) were dissolved in a predetermined amount of 50% PEG200 and stored at 48C, Compounds were administered with the dose of 6 mg/kg by intraperitoneal injection over 7 consecutive days. The mice were kept for 51 days. The survival rate (T/C %) was calculated by following equation; T/C (%) ¼ [Average survival period in the test group/average survival period in the control group] £ 100. The survival ratio (T/C%), the ratio of the number of the surviving mice 50 days after sample treatment to the eight mice tested, is expressed as an important parameter for antitumor activity.

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survival was observed after 4 h treatment with 2hyim-DMNQ S-33.

3.2. Effect of 2-hyim-DMNQ S-33 on MAP kinases and protein kinase C (PKC) To examine the effect of 2-hyim-DMNQ S-33 on MAP kinases, we investigated the expression level and phosphorylation of ERK, p38, and JNK by Western blot analysis. 2-hyim-DMNQ S-33 (3.0 mM) significantly downregulated active pERK within 1 h without any change of inactive ERK. On the other hand, it remarkably upregulated active pJNK without any change of inactive JNK after a 4 h treatment. However, it had no effect on the expression or phosphorylation of p38 (Fig. 3). To further investigate the effect of 2-hyim-DMNQ S-33 on signal transduction pathways in RIF cells, the expression level of PKC-a in RIF cells treated with 2-hyim-DMNQ S-33 was measured by Western blot analysis. Several PKC isotypes (PKC- a, -G, -d, and -1) were constitutively expressed in RIF cells by RT-PCR analysis (data not shown). When we analyze the activation of PKC-a after 3.0 mM 2-hyim-DMNQ S-33 treatment, membrane translocation of PKC-a was observed (Fig. 3).

3. Results 3.1. Inhibition of tumor cell proliferation by 2-hyimDMNQ-S33 In order to assess the effect of 2-hyim-DMNQ S-33 on the proliferation of RIF cells, we employed an in vitro proliferation assay. As shown in Fig. 2, 2-hyimDMNQ S-33 inhibited RIF cell growth in a dosedependent manner. A remarkable inhibition of cell

Fig. 2. Inhibition of tumor cell proliferation by 2-hyim-DMNQ-S33. RIF cells were incubated with 2-hyim-DMNQ-S33 on the 96-well plates for 4 h. The wells were fixed and adherent cells were stained with SRB. The stain was dissolved in 10 mM Tris–HCl and the color was measured at 520 nm. The mean and SD of triplicate determinations are presented. Each bar represents the mean for n ¼ 3 determinations expressed as percent inhibition of cell proliferation.

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S.H. Kim et al. / Cancer Letters 172 (2001) 171–175 Table 1 Effect of 2-hyim-DMNQ-S33 on MST and T/C % in ICR mice bearing sarcoma 180 a Group

No. of Animals

MST (days)

T/C (%)

Vehicle Adriamycin 2-hyim-DMNQ-S33

8 8 8

17.6 42.3 42.1

100 239 239

a

Fig. 3. Effect of 2-hyim-DMNQ-S33 on the expression and activation of PKC and MAP kinases. The expression pattern of ERK (A), p38 (B), JNK (C) in 2-hyim-S33 (3.0 mM)-treated RIF cells was determined by Western blot analysis. For PKC-a (D), membrane fraction was analyzed to probe for its traslocation.

MST, mean survival time.

2- or 6-substituted naphthazarins. Recently, 6-(1hydroxyalkyl)- and 6-acyl-DMNQ derivatives showed improved potency in the inhibition of DNA topoisomerase-I and antiproliferative activities against L1210 leukemia cells by a sterically non-hindered quinonoid moiety. However, 2-hyim-DMNQ derivatives showed no inhibition of DNA topoisomerase-I activity [15,16]. In a previous report and this paper, 2-hyim-DMNQ derivatives revealed the suppression of L1210 leukemia cell [10] and RIF cell proliferation. These results strongly suggested that the inhibition of L1210 leukemia cell and RIF cell proliferation by 2-hyim-DMNQ derivatives might be caused by another growth inhibitory mechanism in the cells, not by inhibiting DNA topoisomerase-I activity. PKC and MAP kinase play a critical role in signal transduction pathways in cells [13,14]. MAP kinase is

3.3. Prolongation of life-span by 2-hyim-DMNQ S-33 The in vivo activity of 2-hyim-DMNQ S-33 was examined in tumor-bearing mice. The life span of 2hyim DMNQ-S-33 (6 mg/kg) was monitored as compared with vehicle and adriamyicn-treated groups. It was interesting that the life span in 2hyim DMNQ-S-33-treated mice was significantly prolonged (up to 239%) similar to the adriamycintreated group (Table 1, Fig. 4). Based on the results obtained in the present work, it is possible to explain that 2-hyim-DMNQ S-33 had antitumor effects by inhibiting tumor cell proliferation.

4. Discussion We have synthesized several shikonin derivatives,

Fig. 4. Effect of 2-hyim-DMNQ-S33 on the survival of sarcoma 180 tumor-bearing mice. Mice were injected intraperitoneally with sarcoma 180 cells (2 £ 10 6). After 24 h, vehicle (X), 2-hyimDMNQ-S33 (P), and adriamycin(W) were administered intraperitoneally into the mice for 7 days. The mice were monitored for 51 days and the T/C (%) was calculated.

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critically involved in the regulation of cell proliferation and differentiation as well as apoptosis. We demonstrated in this report that 2-hyim-DMNQ S-33 was able to prevent RIF cell proliferation and induce high expression of JNK and PKC-a in the cells. Based on these results, it is possible to postulate that 2-hyimDMNQ S-33 may inhibits RIF cell proliferation by inducing apoptosis via upregulation of JNK and PKC-a and downregulation of MAP kinase activation. The in vitro effects of 2-hyim-DMNQ S-33 on tumor cell proliferation were reflected in an increase in the life-span of S-180-bearing mice treated with 2hyim-DMNQ S-33. Further investigation will be necessary to demonstrate the cytotoxic mechanism of 2-hyim- DMNQ S-33 related to MAP kinase and the PKC signaling pathway in detail.

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