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Article

Effects of Polybrominated Diphenol Ethers from a Marine Sponge Phyllospongia dendyi on IL-8 Production in a PMAstimulated Promyelocytic Leukemia Cell Line

1
Department of Basic Biological Sciences, Kyoritsu College of Pharmacy, Shibakoen 1-chome, 5-30, Minato-ku, Tokyo, 105-8512, Japan
2
Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo, 108-8477, Japan
*
Author to whom correspondence should be addressed.
Mar. Drugs 2005, 3(4), 119-125; https://doi.org/10.3390/md304119
Submission received: 28 August 2005 / Accepted: 3 November 2005 / Published: 3 November 2005

Abstract

:
The effects of five polybrominated diphenol ethers from a Marine Sponge, Phyllospongia dendyi, on the relative plating efficiencies in V79 cells and the cell proliferation and IL-8 production in PMA-stimulated HL-60 cells were examined. Three compounds, possessing two phenol units, were reported to inhibit the assembly of microtubule proteins and showed the inhibition of colony formation of V79 cells and increase in IL-8 production. Interestingly, a monomethoxy compound exhibited the stronger activity than the three active compounds in both bioassays. This compound did not inhibit the assembly of microtubule proteins. A dimethoxy compound was not active in either bioassays. In these experimental conditions, the biological activities were not high, but in future, these compounds will be expected to be made stronger by structural modifications.

Introduction

Marine sponges are prolific sources of biologically active agents [1,2]. In the course of our studies on biologically active natural products from marine organisms, we isolated eleven polybrominated diphenol ethers from the ethanol extract of a marine sponge, Phyllospongia dendyi, and reported the structures and the inhibitory activity on the assembly of microtubules in the purified porcine brain microtubule proteins of these compounds [3]. Three compounds (13) showed weak inhibition of the assembly of microtubules in the in vitro system.
In this study, we attempted to show the effects of these compounds on colony formation in Chinese hamster V79 cells, because the inhibition of microtubules correlates to cell proliferation. Moreover, the effects of these compounds on inflammatory cytokine, interleukin-8 (IL-8), production were determined. IL-8 is a member of the superfamily of C-X-C chemokines and a chemotactic factor for T cells, neutrophils and basophils [4]. The expression of IL-8 has been detected in a variety of human cancers and is suggested to be a factor in tumor progression and metastasis [514]. Therefore, the regulation of IL-8 production is an important medical problem.
Compounds 13 and 5 showed weak inhibitory activity on colony formation in V79 cells. The IL-8 production was stimulated by compounds 13 and 5 at higher concentrations, but 4 was not active at 50 μM. Interestingly, compound 5, a monomethoxy derivative of 2, exhibited the stronger activity than 2 and the other compounds.

Materials and Methods

Materials

2-(3′,5′-Dibromo-2′-hydroxyphenoxy)-3,4,5,6-tetrabromophenol (1) [15,16], 2-(3′,5′-dibromo-2′-hydroxyphenoxy)-3,5,6-tribromophenol (2) [15,16], 2-(4′,6′-dibromo-2′-hydroxyphenoxy)-3,4,5-tribromophenol (3) [16], 2-(3′,5′-dibromo-2′-methoxyphenoxy)-3,4,5,6-tetrabromoanisole (4) [15] and 4,6-dibromo-2-(3′,4′,6′-tri-bromo-2′-meyhoxyphenoxy)phenol (5) [16] (Figure 1) were prepared as described previously [3]. The structures of five compounds are shown in Figure 1. Dimethylsulfoxide (DMSO) was purchased from Pierce Chemical Co. (Rockfield, IL) and fetal bovine serum (FBS) was obtained from GIBCO after checking the lot. All other reagents and chemicals used were of the highest grade available commercially.

Cell lines and culture conditions

Cell culture for Chinese hamster V79 cells were grown in monolayer culture in Eagle’s minimum essential medium (MEM, Nissui Seiyaku Co., Ltd., Tokyo, Japan) with 10(v/v)% heat-inactivated FBS.
The human promyelocytic cell line, HL-60, was obtained from the Japanese Cancer Research Resources Bank (JCRB, Kamiyoga, Tokyo, Japan). This cell line was maintained in tissue culture dishes in RPMI 1640 medium (Nissui Seiyaku Co., Ltd., Tokyo, Japan), supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100 U/ml of penicillin G and 100 μg/ml of streptomycin.

Relative Plating Efficiency

The method was essentially the same as that described in our previous paper [17]. The relative plating efficiencies in the presence of different concentrations of drugs were determined as the ratio of the number of colonies at a given drug concentration to that obtained in the control culture in the absence of any drug. Two hundred cells were seeded onto 60/15-mm Petri dishes in 4 ml MEM with 10(v/v)% FBS and incubated overnight at 37°C, after which the samples dissolved in DMSO (4 μl) were added. The relative plating efficiencies were determined as the ratio of the number of colonies in the presence of different sample concentrations to that obtained in the control culture.

Detection of human IL-8 by ELISA

The IL-8 concentrations of the culture supernatants under control and various test conditions were measured by ELISA using a combination of monoclonal and polyclonal antibodies [18]. All samples were assayed at least in duplicate. Data are presented as the mean ± SE of three independent experiments.

Determination of cell proliferation

The cell proliferation was evaluated by enumerating the viable cells using the MTT formazan production method [19]. HL-60 cells (1 x 106 cells/ml) were treated with PMA (with or without test compounds) and then transferred to 96-well microtiter plates. After a 24 h incubation, 20 μl of MTT reagent (5 mg/ml in PBS) was then added to each well. After incubation for 3 h, formazan production was assessed by measuring the optical density (OD570 nm).

Results and Discussion

The effects of polybrominated diphenol ethers 15 on relative plating efficiencies of Chinese hamster V79 cells

Compounds 13 showed inhibitory activities to the assembly of microtubule proteins (IC50: 29.6, 33.5, and 20.9 μM, respectively) and to the meiotic maturation of starfish oocytes (IC50: 3.6, 4.2, and 4.2 μM, respectively) [3]. On the other hand, we showed that the inhibition of microtubule polymerization correlated with the inhibition of colony formation in Chinese hamster V79 cells. However, it will be expected that the biological activities are generally different between in vitro and in vivo systems. Then, we examined the effects of these polybrominated diphenol ethers on colony formation in Chinese hamster V79 cells. Two doses (1 and 10 μM) of each compound were examined, and the results shown in Figure 2.
Compounds 2 and 5 showed stronger inhibition than those of 1 and 3 on colony formation. On the other hand, compound 4 had no effect at either concentration. These results indicated that the monomethoxy derivative (5) displayed the strongest inhibition compared with those of the two OH derivatives (13), and the dimethoxy derivative (4) did not inhibit its activity. This result shows that the inhibited activities of microtubule polymerization in vitro systems were not correlated with the inhibition of cell growth.

The effects of polybrominated diphenol ethers 15 on IL-8 production by PMA-stimulated HL-60 cells

To further examine the effects of compounds 15, we used PMA-stimulated HL-60 cells because of their IL-8 production. Comparison of the results of relative plating efficiencies and IL-8 production were very interesting, because IL-8 production correlated to the stoppage of cell growth. We examined the effects of compounds 15 on IL-8 production by the PMA (2 or 20 nM)-stimulated HL-60 cells, and the results are shown in Figure 3. The high concentration of compounds 1, 2, 3 and 5 showed the increases in the IL-8 production under the additional two doses of PMA; on the other hand, compound 4 did not increase the IL-8 production under the same experimental conditions. Moreover, the cell proliferations of each IL-8 production condition were determined by MTT methods (data not shown). The results, these proliferations at 10 and 50 μM of compounds 1, 2, 3 and 5 were inhibited strongly (20–80%). Thus, IL-8 production correlated with cell proliferation in this case.
On the other hand, the monomethoxy derivative (5) showed the strongest inhibition compared with those of the two OH derivatives (13), and the dimethoxy derivative (4) did not inhibit the activity. As compound 2 showed greater inhibition than 1, it was suggested that the 4-Br in the ring A has a negative effect on IL-8 production. Moreover, the relative inhibited activities of all compounds for IL-8 production agreed with these relative plating efficiencies, but did not agree with in vitro microtubule polymerization system, as shown in the previous report [3].
In this study, we elucidated the effects of polybrominated diphenol ethers from a Marine Sponge, Phyllospongia dendyi, on the relative plating efficiencies in V79 cells and the cell proliferation and IL-8 production in PMA-stimulated HL-60 cells. The monomethoxy compound (5) has greater activity than those of the compounds possessing two phenol units. One of the reasons suggested was that the monomethoxy derivative incorporates more easily into cells than the two OH derivatives. In this experimental data, the biological activities were so fine what weak, but after this, these compounds will be expect to have their activity enhanced by any structure modifications.
Figure 1. Structures of compounds 1–5.
Figure 1. Structures of compounds 1–5.
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Figure 2. Relative plating efficiencies of Chinese hamster V79 cells treated with compounds 1–5 for 48 h with 1 and 10 μM concentrations of drugs. The experimental conditions were as described in the Materials and Methods. The data are the mean values of three independent experiments.
Figure 2. Relative plating efficiencies of Chinese hamster V79 cells treated with compounds 1–5 for 48 h with 1 and 10 μM concentrations of drugs. The experimental conditions were as described in the Materials and Methods. The data are the mean values of three independent experiments.
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Figure 3. Effects of compounds 1–5 on IL-8 production and cell proliferation in PMA-stimulated HL-60 cells. A and B show the effects of compounds 15 on IL-8 production by 2 and 20 nM PMA added, respectively. HL-60 cells (1 x 106 cells/ml) were treated with PMA (2 or 20 nM) and the indicated concentration of each compound for 24 h. The IL-8 concentrations in the culture supernatant of the PMA-stimulated cells were determined to be ca. 14 and 40 ng/ml by ELISA, as described in the Materials and Methods. The data are shown as values relative (%) to the PMA-stimulated each IL-8 productions (14 and 40 ng/ml). The data are the mean values of three independent experiments.
Figure 3. Effects of compounds 1–5 on IL-8 production and cell proliferation in PMA-stimulated HL-60 cells. A and B show the effects of compounds 15 on IL-8 production by 2 and 20 nM PMA added, respectively. HL-60 cells (1 x 106 cells/ml) were treated with PMA (2 or 20 nM) and the indicated concentration of each compound for 24 h. The IL-8 concentrations in the culture supernatant of the PMA-stimulated cells were determined to be ca. 14 and 40 ng/ml by ELISA, as described in the Materials and Methods. The data are shown as values relative (%) to the PMA-stimulated each IL-8 productions (14 and 40 ng/ml). The data are the mean values of three independent experiments.
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Acknowledgments

We thank Prof. Tadashi Kasahara for providing IL-8 monoclonal and polyclonal antibodies.
  • Sample availability: Not available.

References and Notes

  1. Faulkner, D.J. Marine natural products. Nat. Prod. Rep 19, 1–48.2000, 18, 1–49.1999, 17, 7–55.1998, 16, 155–198.1997, 15, 113–158.1996, 14, 259–302.1995, 13, 75–125.1994, 12, 223–269.1993, 11, 355–394.1992, 10, 497–539.1991, 9, 323–364.1990, 8, 97–147.1988, 7, 269–309.1987, 5, 613–576.1986, 4, 539–576.1984, 3, 1–33.1.
  2. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Marine natural products. Nat. Prod. Rep 22, 15–61.2003, 21, 1–49.20, 1–48.
  3. Liu, H.; Namikoshi, M.; Meguro, S.; Nagai, S.; Kobayashi, H.; Yao, X. Isolation and Characterization of Polybrominated Diphenyl Ethers as Inhibitors of Microtubule Assembly from a Marine Sponge Phyllospongia dendyi Collected at Palau. J. Nat. Prod 2004, 67, 472–474. [Google Scholar]
  4. Graves, D.T.; Jiang, Y. Molecular cloning and functional analysis of the promoter of the human squalene synthase gene. Crit. Rev. Oral Biol. Med 1995, 6, 109–118. [Google Scholar]
  5. Zachariae, C.O.; Thestrup-Pedersen, K.; Matsushima, K. Expression and secretion of leukocyte chemotactic cytokines by normal human melanocytes and melanoma cells. J. Invest. Dermatol 1991, 97, 593–599. [Google Scholar]
  6. Di Celle, P.F.; Carbone, A.; Marchis, D.; Zhou, D.; Sozzani, S.; Zupo, S.; Pini, M.; Mantovani, A.; Foa, R. Cytokine gene expression in B-cell chronic lymphocytic leukemia: evidence of constitutive interleukin-8 (IL-8) mRNA expression and secretion of biologically active IL-8 protein. Blood 1994, 84, 220–228. [Google Scholar]
  7. Morita, M.; Kasahara, T.; Mukaida, N.; Matsushima, K.; Nagashima, T.; Nishizawa, M.; Yoshida, M. Induction and regulation of IL-8 and MCAF production in human brain tumor cell lines and brain tumor tissues. Eur. Cytokine. Netw 1993, 4, 351–358. [Google Scholar]
  8. Green, A.R.; Green, V.L.; White, M.C.; Speirs, V. Expression of Cytokine messenger RNA in normal and neoplastic human breast tissue: identification of interleukin-8 as a potential regulatory factor in breast tumours. Int. J. Cancer 1997, 72, 937–941. [Google Scholar]
  9. Mizuno, K.; Sone, S.; Orino, E.; Mukaida, N.; Matsushima, K.; Ogura, T. Spontaneous production of interleukin-8 by human lung cancer cells and its augmentation by tumor necrosis factor alpha and interleukin-1 at protein and mRNA levels. Oncology 1994, 51, 467–471. [Google Scholar]
  10. Suliman, M.E.; Royds, J.A.; Baxter, L.; Timperley, W.R.; Cullen, D.R.; Jones, T.H. IL-8 mRNA expression by in situ hybridisation in human pituitary adenomas. Eur. J. Endocrinol 1999, 140, 155–158. [Google Scholar]
  11. Konig, B.; Steinbach, F.; Janocha, B.; Drynda, A.; Stumm, M.; Philipp, C.; Allhoff, E.P.; Konig, W. The differential expression of proinflammatory cytokines IL-6, IL-8 and TNF-alpha in renal cell carcinoma. Anticancer Res 1999, 19, 1519–1524. [Google Scholar]
  12. Yoshida, M.; Matsuzaki, H.; Sakata, K.; Takeya, M.; Kato, K.; Mizushima, S.; Kawakita, M.; Takatsuki, K. Neutrophil chemotactic factors produced by a cell line from thyroid carcinoma. Cancer Res 1992, 52, 464–469. [Google Scholar]
  13. Brew, R.; Erikson, J.S.; West, D.C.; Flanagan, B.F.; Christmas, S.E. Interleukin-8 as a growth factor for human colorectal carcinoma cells in vitro. Biochem. Soc. Trans 1997, 25, 264S–268S. [Google Scholar]
  14. Galffy, G.; Mohammed, K.A.; Dowling, P.A.; Nasreen, N.; Ward, M.J. Antony, V.B. Interleukin 8: an autocrine growth factor for malignant mesothelioma. Cancer Res 1999, 59, 367–371. [Google Scholar]
  15. Carté, B.; Faulkner, D. J. Polybrominated diphenyl ethers from Dysidia herbaces, Dysidia chlorea and Phyllospongia foliascens. Tetrahedron 1981, 37, 2335–2339. [Google Scholar]
  16. Fu, X.; Schmitz, F. J.; Govindan, M.; Abbas, S. A.; Hanson, K. M.; Horton, P. A.; Crews, P.; Laney, M.; Schatzman, R. C. Enzyme inhibitors: new and known polybrominated phenols and diphenyl ethers from four Indo-Pacific Dysidia sponges. J. Nat. Prod 1995, 58, 1384–1391. [Google Scholar]
  17. Sato, Y.; Sakakibara, Y.; Oda, T.; Aizu-yokota, E.; Ichinoseki, I. Effects of estradiol and ethynylestradiol on microtubule distribution in Chinese hamster V79 cells. Chem. Pharm. Bull 1992, 40, 182–184. [Google Scholar]
  18. Kasahara, T.; Oda, T.; Hatake, K.; Akiyama, M.; Mukaida, N.; Matsushima, K. Interleukin-8 and monocyte chemotactic protein-1 production by a human glioblastoma cell line, T98G in coculture with monocytes: involvement of monocyte-derived interleukin-1alpha. Eur. Cytokine Netw 1998, 9, 47–55. [Google Scholar]
  19. Carmichael, J.; DeGraff, W.G.; Gazdar, A.F.; Minna, J.D.; Mitchell, J.B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res 1987, 47, 939–942. [Google Scholar]

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MDPI and ACS Style

Oda, T.; Liu, H.; Namikoshi, M. Effects of Polybrominated Diphenol Ethers from a Marine Sponge Phyllospongia dendyi on IL-8 Production in a PMAstimulated Promyelocytic Leukemia Cell Line. Mar. Drugs 2005, 3, 119-125. https://doi.org/10.3390/md304119

AMA Style

Oda T, Liu H, Namikoshi M. Effects of Polybrominated Diphenol Ethers from a Marine Sponge Phyllospongia dendyi on IL-8 Production in a PMAstimulated Promyelocytic Leukemia Cell Line. Marine Drugs. 2005; 3(4):119-125. https://doi.org/10.3390/md304119

Chicago/Turabian Style

Oda, Taiko, Hongwei Liu, and Michio Namikoshi. 2005. "Effects of Polybrominated Diphenol Ethers from a Marine Sponge Phyllospongia dendyi on IL-8 Production in a PMAstimulated Promyelocytic Leukemia Cell Line" Marine Drugs 3, no. 4: 119-125. https://doi.org/10.3390/md304119

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