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BIOPHA-3372; No. of Pages 4 Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx
Available online at
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Original article
DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer Hanna Piotrowska a, Krzysztof Myszkowski a, Joanna Abraszek a, Eliza Kwiatkowska-Borowczyk b,c, Ryszard Amarowicz d, Marek Murias a, Marcin Wierzchowski e, Jadwiga Jodynis-Liebert a,* a
Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznan, Poland c Department of Cancer Diagnostics and Immunology, Greater Poland Cancer Centre, Poznan, Poland d Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland e Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Poznan, Poland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 August 2013 Accepted 28 February 2014
DMU-212 has been shown to evoke a mitochondrial apoptotic pathway in transformed fibroblasts and breast cancer. However, recently published data indicated the ability of DMU-212 to evoke apoptosis in both mitochondria- and receptor-mediated manner in two ovarian cancer cell lines, namely A-2780 and SKOV-3, which showed varied sensitivity to the compound tested. The pronounced cytotoxic effects of DMU-212 observed in A-2780 cells were related to the execution of extracellular apoptosis pathway and cell cycle arrest in G2/M phase. In view of the great anticancer potential of DMU-212 against A-2780 cell line, the aim of the current study was to assess antiproliferative activity of DMU-212 in xenograft model of ovarian cancer. To evaluate in vitro metabolic properties of cells that were to be injected into SCID mice, uptake and decline of DMU-212 in A-2780 ovarian cancer cell line was investigated. It was found that the concentration of the test compound in A-2780 cells was growing within first eight hours, and then the gradual decline was observed. A-2780 cells stably transfected with pcDNA3.1/Zeo(-)-Luc vector were subcutaneously inoculated into the right flanks of SCID mice. After seven days of the treatment with DMU-212 (50 mg/kg b.w), tumor growth appeared to be suppressed in the animals treated with the compound tested. At day 14 of the experiment, tumor burden in mice treated with DMU-212 was significantly lower, as compared to untreated controls. Our findings suggest that DMU-212 might be considered as a potential anticancer agent used in ovarian cancer therapy. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: 3,4,40 5-tetramethoxystilbene (DMU-212) Ovarian cancer Xenograft model
1. Introduction Resveratrol (3,40 ,5-trans-trihydroxystilbene) is a naturally occurring phytoalexin present in grapes, peanuts, cranberries and other dietary constituents. The highest concentration of resveratrol is found in the roots of Japanese knotweed (Polygonum cuspidatum), which have been used in traditional Asian medicine for centuries to treat inflammation [1,2]. An expanding body of preclinical evidence shows resveratrol to exert a wide variety of * Corresponding author. Tel.: +48 618470721; fax: +48 618470721. E-mail addresses: [email protected] (H. Piotrowska), [email protected] (K. Myszkowski), [email protected] (E. Kwiatkowska-Borowczyk), [email protected] (R. Amarowicz), [email protected] (M. Murias), [email protected] (M. Wierzchowski), [email protected] (J. Jodynis-Liebert).
pharmacological activities, including anti-inflammatory, antioxidative, anti-tumour, anti-platelet, anti-fungal and estrogenic ones [3,4]. However, its biological effects in vivo appear to be strongly limited because of a poor bioavailability and a rapid clearance from the circulation [5]. The trihydroxystilbene scaffold of resveratrol has been the subject of structural alternations to improve the pharmacokinetic properties of the compound. The structure-activity studies have shown that the introduction of methoxy groups in place of hydroxy moieties increases the molecule stability, making it less susceptible to phase II conjugation reactions in vivo. Additionally, the methoxy groups added to the stilbene backbone of resveratrol may enhance its cytotoxic activity [6]. It has been also revealed that the methoxy moieties at positions 3,5- and 3,4,5- of the trihydroxystilbene framework play a crucial role for pro-apoptotic activity of the molecule [7].
http://dx.doi.org/10.1016/j.biopha.2014.02.001 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Piotrowska H, et al. DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.02.001
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To date, several studies have shown the potent anti-cancer activities of the methylated analogue of resveratrol, 3,4,40 ,5tetramethoxystilbene (DMU-212). In vitro, DMU-212 inhibits the growth of transformed fibroblasts, prostate, cervical, ovarian, breast, liver and colon cancer cell lines. DMU-212 has been found to exert anti-proliferative effects through multiple mechanisms, including down-regulation of the expression of enzymes catalyzing carcinogens activation, induction of cell cycle arrest and initiation of apoptotic mechanisms [8–12]. It has been reported that DMU-212 triggered a mitochondrial apoptotic pathway in transformed fibroblasts and breast cancer cells [9,10]. However, our previously published results demonstrated the ability of DMU212 to evoke apoptosis in both mitochondria- and receptormediated manner in two ovarian cancer cell lines, namely A-2780 and SKOV-3, which showed varied sensitivity to the test compound. The pronounced cytotoxic effects of DMU-212 observed in A-2780 cells were related to the execution of extracellular apoptosis pathway and cell cycle arrest in G2/M phase. It has been also suggested that inhibition of CYP1B1 by DMU-212 may affect its cytotoxicity [12,13]. In view of the great anticancer potential of DMU-212 against A2780 cell line, the aim of the current study was to evaluate its inhibitory effect in vivo. We attempted to verify DMU-212 activity in xenograft model using severely compromised immunodeficient (SCID) mice injected with A-2780 human ovarian cancer cells.
SV40 late polyA signal, into pcDNA3.1/Zeo(-). A-2780 cells were stably transfected with pcDNA3.1/Zeo(-)-Luc vector using lipofectamine LTX with Plus Reagent (Invitrogen). Zeocin (Invitrogen) (300 mg/mL) was added 48 h later; clones were picked 16 days after transfection into 48-well plate and 10 days later were transferred into 24-well plate. Luciferine expression was measured using ONE-GloTM Luciferase Assay (Promega). Clone with highest expression level were chosen for further analysis.
2. Materials and methods
2.5. SCID mice xenograft model
2.1. Materials
Subcutaneous xenograft tumors derived from human ovarian cancer cell line A-2780 were generated in CB17-SCID mice. Briefly, 3 106 A-2780 cells in 0,2 mL PBS were subcutaneously inoculated into the right flanks of SCID mice. At day 8 after injection, mice with equal level of bioluminescence were divided into two groups (control and DMU-212-treated) of six animals each. The compound tested was administered in a dose of 50 mg/kg b.w. three times a week by gavage. DMU-212 was suspended in 50% bcyclodextrin to improve its bioavailability (Lin et al., 2010). The control group was given the vehicle. D-Luciferin was injected intraperitoneally at a dose of 150 mg/kg b.w., followed by inhaled isoflurane anesthesia and bioluminescence imaging (IVIS 200, Caliper Life Sciences, Hopkinton, MA). Images of tumor-bearing mice were captured three times a week and data were analyzed using the Living Images software package (Caliper Life Sciences). Data are presented as the mean of bioluminescence intensity (BLI). All animals were humanely sacrificed after 14 days of monitoring due to the excessive tumor burden.
DMU-212 (3,4,40 ,5-trans-tetramethoxystilbene) was synthesised as described elsewhere [14,15]. The identity and purity of the compound was confirmed by NMR and LCMS. Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), and trypsin-EDTA were purchased from Sigma-Aldrich St Louis, MO. Plasmid pcDNA3.1/Zeo(-) and plasmid pGL3-Basic were obtained from Invitrogen Carlsbad, CA and Promega, Madison, WI, respectively. Forane (isofluranum) was acquired from Abbott Laboratories Ltd (Illinois, USA). D-Luciferin (Potassium salt) was from Gold Biotechnology (St Louis, MO). 2.2. Animals SCID female mice (CB17/Icr-Prkdcscid/IcrCrl), 6-weeks-old and weighing 20–24 g were obtained from Charles Riever (Wilmington, MA, USA). Animals were housed (5/cage) in filter top cages equipped with HEPA filters (Tecniplast, Italy). Room climate was maintained at a temperature of 22 3 8C, 40–65% relative humidity, with 10 air changes per hour. Commercial diet (ISO 9001 certified laboratory feed, total pathogen free, Altromin, Germany) and drinking water were available ad libitum. The experiment was performed according to the guidelines of the Local Animal Ethics Committee for animal experimentation.
2.4. High performance liquid chromatography The determination of DMU-212 in cells and culture medium was performed using a Shimadzu System (Shimadzu, Kyoto, Japan) equipped with a L-C-10 AD VP pump, a SCL-10 A VP interface, and a UV-Vis SPD-M10 A VP detector (set at a wavelength 326 nm). For separation of DMU-212 a LUNA C18 column (4.60, 250 mm; 5 mm; Phenomenex, Torrence USA) was used. The mobile phases used in assay were: 30% acetonitrile containing 0.1% trifluoroacetic acid (mobile phase A), and 100% acetonitrile (mobile phase B), the flow rate was 1 mL/min. The linear gradient system ranged from 0 to 100% of mobile phase B over 50 min of analysis. Every analysis was followed by equilibration of the column with 100% phase A for 8 min before application of the next sample. The sample injection volumes were 100 mL. Calibration was accomplished using the external standard method at a concentration range of 0.1–75 mg/ mL.
2.6. Statistical analysis GraphPad Prism 5.0 version 5.00 for Windows, GraphPad Software, San Diego California USA was employed for the statistical evaluation of the data. Statistical significance was tested by twotailed Student’s t-test, with P-value of less than 0.05 considered statistically significant.
2.3. Cell culture and transfection A-2780 ovarian cancer cell line was purchased from the European Type Culture Collection (Sigma-Aldrich Co., St Louis, MO). A-2780 cells were maintained in phenol red-free DMEM medium supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/mL), and streptomycin (0.1 mg/mL). Cells were cultivated under standard conditions at 37 8C in a humidified atmosphere containing 5% CO2 and 95% air. Expression vector pcDNA3.1/Zeo(-)- Luc was constructed by cloning of XhoI – BamHI fragment of pGL3-Basic, encoding firefly luciferase and
3. Results and discussion DMU-212 and its prototype compound resveratrol have been reported to exert antiproliferative activity in the Apc(Min+) mice [11]. However, Ziegler et al. showed that resveratrol failed to decline tumor size in this experimental model [16]. Furthermore, according to more recent report, resveratrol promoted the growth of prostate cancer LAPC-4 xenografts in SCID mice [17]. Klink et al. demonstrated that resveratrol worsen survival in SCID mice with
Please cite this article in press as: Piotrowska H, et al. DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.02.001
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prostate cancer xenografts via up-regulating of oncogenic pathways. In conclusion, they found that resveratrol used in phase I clinical trials may be harmful for some patients with prostate cancer [17]. In view of findings mentioned above, there is a great need of structural alternation of resveratrol scaffold to generate its novel analogues with improved growth-inhibitory and apoptogenic properties. In the present study, we assessed the antiproliferative activity of the methylated analogue of resveratrol, 3,4,40 ,5-tetramethoxystilbene (DMU-212) in xenograft model of ovarian cancer. To evaluate in vitro metabolic properties of cells that were to be injected into SCID mice, uptake and decline of DMU-212 in A-2780 ovarian cancer cell line was investigated. It was found that the concentration of the test compound in A-2780 cells was growing within first eight hours, and then the gradual decline was observed (Fig. 1). In medium we noted time-dependent decrease in DMU-212 concentration. However, after 50 hours incubation a plateau has been established (Fig. 1). It can be suggested that the rapid absorption and slow elimination of the test compound may provide its effective growth inhibitory activity against ovarian cancer cells. Based on these data and our previously published results indicated potent antitumor activity of DMU-212 in A-2780 ovarian cancer cell line [12] we extended our studies to in vivo conditions by generating A-2780 ovarian cancer xenografts in SCID mice. In recent years, several new mouse models for cancer research have been developed. The discovery of T-cell deficient athymic nude mice, and later T-cell- and B-cell deficient SCID mice, allowed the inoculation and dissemination of human cancer cells in mice [18]. Bioluminescent imaging is one of the most common methods to assess tumor response to the test compound in mice. It allows non-invasive, longitudinal evaluation of tumor growth before, during and after treatment [19]. In our study, it has been initially observed that mice treated with vehicle and DMU-212 at dose 50 mg/kg b.w. exhibited similar tumor size as reflected by approximately equal bioluminescence intensity (Figs. 2 and 3). However, after seven days of treatment, tumor growth appeared to be suppressed in the animal group treated with the compound tested (Figs. 2 and 3). At day 14 of the experiment, tumor burden in mice treated with DMU-212 was significantly lower, as compared to untreated controls (Figs. 2 and 3). Our results are in agreement with the previous findings indicated antiproliferative activity of DMU-212 in the Apc(Min+) mice, a model of human intestinal
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3
1.2 10 6 1.0 10 6 8.0 10
5
6.0 10
5
4.0 10 5
control DMU-212
* *
2.0 10 5 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
treatment time (days) Fig. 2. Effect of DMU-212 on tumor burden in SCID mice with ovarian cancer xenografts. Tumor inhibition was evidenced by the bioluminescence intensity. A significant decrease in tumor burden in DMU-212-treated mice was first observed at 12 day of DMU-212 treatment. All data are presented as mean SD (n = 6 in each group, *P < 0.05).
carcinogenesis. Although an experimental model was different, decreased tumor size in mice treated with DMU-212 has been also observed [11]. Similarly, other methylated resveratrol derivatives 2,30 ,4,40 ,50 -pentamethoxystilbene (PMS) and 3,5,40 -trimethoxystilbene (MR-3) have been shown to exhibit potent antitumor activity in vivo [7,20,21]. Pan et al. (2008) showed that treatment with MR-3 three times a week at a dose of 50 mg/kg b.w. reduced tumor size in SCID mice injected with colon cancer cells [21]. While their experiment lasted 23 days, we shortened the treatment time to 14 days due to excessive tumor burden, which may be explained by differences in cell division between colorectal cancer cells and ovarian ones [22].
A2780 25
medium cells
20 15
DMU-212 [ M/mg protein]
10 0.03 0.02 0.01 0.00 0
10
20
30
40
50
60
70
time [h]
Fig. 1. Uptake of DMU-212 into cytoplasm of A-2780 cells ( ) and decrease in DMU212 levels in incubation medium ( ), cells were treated with DMU-212 for various time intervals, scrapped, centrifuged, lysed by freeze-thaw stress and centrifuged again. The supernatant and medium from the cells were analyzed using HPLC.
Fig. 3. The changes in tumor development in mice treated with DMU-212 (50 mg/kg b.w.) The figure shows one representative mouse from (A) DMU-212 treated group and (B) control group.
Please cite this article in press as: Piotrowska H, et al. DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.02.001
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To evaluate the potential toxic effects of DMU-212 treatments, clinical observations have been performed everyday as well as body weight, water and food intake were recorded. DMU-212 at a dose of 50 mg/kg b.w. was shown to be well tolerated without any adverse effects (data not shown). Our results are in agreement with the other authors’ findings indicated that DMU-212 was well tolerated and failed to affect body weight in Apc(Min+) mice [11]. Ovarian cancer is the fifth most common cause of cancerrelated deaths in women [23]. Although the incidence of ovarian cancer is lower than many other malignancies, the death rate is higher due to its early propagation and rapid development of chemotherapy resistance [23,24]. In our study, DMU-212, methylated analogue of resveratrol, displayed antitumor activity in an ovarian cancer model in vivo without systemic toxicity throughout the whole period of drug treatment. Our findings suggest that DMU-212 might be considered as a potential anticancer agent used in ovarian cancer therapy. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This study was supported by a research grant of the Polish Ministry of Science and Higher Education No. IP2012046072. References [1] Saiko P, Szakmary A, Jaeger W, Szekeres T. Resveratrol and its analogs: defense against cancer, coronary disease and neurodegenerative maladies or just a fad? Mutat Res 2008;658:68–94. [2] Stervbo U, Vang O, Bonnesen C. Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells. Cell Prolif 2006;39:479–93. [3] Shakibaei M, Harikumar KB, Aggarwal BB. Resveratrol addiction: to die or not to die. Mol Nutr Food Res 2009;53:115–28. [4] Whitlock NC, Baek SJ. The anticancer effects of resveratrol: modulation of transcription factors. Nutr Cancer 2012;64:493–502. [5] Walle T, Hsieh F, DeLegge MH, Oatis Jr JE, Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos 2004;32:1377–82. [6] Roberti M, Pizzirani D, Simoni D, Rondanin R, Baruchello R, Bonora C, et al. Synthesis and biological evaluation of resveratrol and analogues as apoptosisinducing agents. J Med Chem 2003;46:3546–54. [7] Li H, Wu WK, Li ZJ, Chan KM, Wong CC, Ye CG, et al. 2,3’,4,4’,5’-Pentamethoxytrans-stilbene, a resveratrol derivative, inhibits colitis-associated colorectal carcinogenesis in mice. Br J Pharmacol 2010;160:1352–61.
[8] Sale S, Verschoyle RD, Boocock D, Jones DJ, Wilsher N, Ruparelia KC, Potter GA, Farmer PB, Steward WP, Gescher AJ. Pharmacokinetics in mice and growthinhibitory properties of the putative cancer chemopreventive agent resveratrol and the synthetic analogue trans 3,4,5,4’-tetramethoxystilbene. Br J Cancer 2004;90:736–44. [9] Gosslau A, Chen M, Ho CT, Chen KY. A methoxy derivative of resveratrol analogue selectively induced activation of the mitochondrial apoptotic pathway in transformed fibroblasts. Br J Cancer 2005;92:513–21. [10] Ma Z, Molavi O, Haddadi A, Lai R, Gossage RA, Lavasanifar A. Resveratrol analog trans 3,4,5,4’-tetramethoxystilbene (DMU-212) mediates anti-tumor effects via mechanism different from that of resveratrol. Cancer Chemother Pharmacol 2008;63:27–35. [11] Sale S, Tunstall RG, Ruparelia KC, Potter GA, Steward WP, Gescher AJ. Comparison of the effects of the chemopreventive agent resveratrol and its synthetic analog trans 3,4,5,4’-tetramethoxystilbene (DMU-212) on adenoma development in the Apc(Min+) mouse and cyclooxygenase-2 in human-derived colon cancer cells. Int J Cancer 2005;115:194–201. [12] Piotrowska H, Myszkowski K, Ziolkowska A, Kulcenty K, Wierzchowski M, Kaczmarek M, et al. Resveratrol analogue 3,4,4’,5-tetramethoxystilbene inhibits growth, arrests cell cycle and induces apoptosis in ovarian SKOV-3 and A2780 cancer cells. Toxicol Appl Pharmacol 2012;263:53–60. [13] Piotrowska H, Kucinska M, Murias M. Biological activity of piceatannol: leaving the shadow of resveratrol. Mutat Res 2012;750:60–82. [14] Androutsopoulos VP, Ruparelia KC, Papakyriakou A, Filippakis H, Tsatsakis AM, Spandidos DA. Anticancer effects of the metabolic products of the resveratrol analogue, DMU-212: structural requirements for potency. Eur J Med Chem 2011;46:2586–95. [15] Murias M, Handler N, Erker T, Pleban K, Ecker G, Saiko P, et al. Resveratrol analogues as selective cyclooxygenase-2 inhibitors: synthesis and structureactivity relationship. Bioorg Med Chem 2004;12:5571–8. [16] Ziegler CC, Rainwater L, Whelan J, McEntee MF. Dietary resveratrol does not affect intestinal tumorigenesis in Apc(Min/+) mice. J Nutr 2004;134:5–10. [17] Klink JC, Tewari AK, Masko EM, Antonelli J, Febbo PG, Cohen P, et al. Resveratrol worsens survival in SCID mice with prostate cancer xenografts in a cell-line specific manner, through paradoxical effects on oncogenic pathways. Prostate 2013;73:754–62. [18] Richmond A, Su Y. Mouse xenograft models vs GEM models for human cancer therapeutics. Dis Model Mech 2008;1:78–82. [19] Zinn KR, Chaudhuri TR, Szafran AA, O’Quinn D, Weaver C, Dugger K, et al. Noninvasive bioluminescence imaging in small animals. ILAR J 2008;49(1):103–15. [20] Li H, Wu WK, Zheng Z, Che CT, Yu L, Li ZJ, et al. 2,3’,4,4’,5’-pentamethoxy-transstilbene, a resveratrol derivative, is a potent inducer of apoptosis in colon cancer cells via targeting microtubules. Biochem Pharmacol 2009;78: 1224–32. [21] Pan MH, Gao JH, Lai CS, Wang YJ, Chen WM, Lo CY, et al. Antitumor activity of 3,5,4’-trimethoxystilbene in COLO 205 cells and xenografts in SCID mice. Mol Carcinog 2008;47:184–96. [22] Pease JC, Brewer M, Tirnauer JS. Spontaneous spheroid budding from monolayers: a potential contribution to ovarian cancer dissemination. Biol Open 2012;1:622–8. [23] Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin 2010;60:277–300. [24] Jemal A, Center MM, Desantis C, Ward EM. Global patterns of cancer incidence and mortality rates and trends. Cancer Epidemiol Biomarkers Prev 2010;19:1893–907.
Please cite this article in press as: Piotrowska H, et al. DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.02.001
BIOPHA-3372; No. of Pages 4 Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx
Available online at
ScienceDirect www.sciencedirect.com
Original article
DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer Hanna Piotrowska a, Krzysztof Myszkowski a, Joanna Abraszek a, Eliza Kwiatkowska-Borowczyk b,c, Ryszard Amarowicz d, Marek Murias a, Marcin Wierzchowski e, Jadwiga Jodynis-Liebert a,* a
Department of Toxicology, Poznan University of Medical Sciences, Poznan, Poland Chair of Medical Biotechnology, Poznan University of Medical Sciences, Poznan, Poland c Department of Cancer Diagnostics and Immunology, Greater Poland Cancer Centre, Poznan, Poland d Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland e Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Poznan, Poland b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 1 August 2013 Accepted 28 February 2014
DMU-212 has been shown to evoke a mitochondrial apoptotic pathway in transformed fibroblasts and breast cancer. However, recently published data indicated the ability of DMU-212 to evoke apoptosis in both mitochondria- and receptor-mediated manner in two ovarian cancer cell lines, namely A-2780 and SKOV-3, which showed varied sensitivity to the compound tested. The pronounced cytotoxic effects of DMU-212 observed in A-2780 cells were related to the execution of extracellular apoptosis pathway and cell cycle arrest in G2/M phase. In view of the great anticancer potential of DMU-212 against A-2780 cell line, the aim of the current study was to assess antiproliferative activity of DMU-212 in xenograft model of ovarian cancer. To evaluate in vitro metabolic properties of cells that were to be injected into SCID mice, uptake and decline of DMU-212 in A-2780 ovarian cancer cell line was investigated. It was found that the concentration of the test compound in A-2780 cells was growing within first eight hours, and then the gradual decline was observed. A-2780 cells stably transfected with pcDNA3.1/Zeo(-)-Luc vector were subcutaneously inoculated into the right flanks of SCID mice. After seven days of the treatment with DMU-212 (50 mg/kg b.w), tumor growth appeared to be suppressed in the animals treated with the compound tested. At day 14 of the experiment, tumor burden in mice treated with DMU-212 was significantly lower, as compared to untreated controls. Our findings suggest that DMU-212 might be considered as a potential anticancer agent used in ovarian cancer therapy. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: 3,4,40 5-tetramethoxystilbene (DMU-212) Ovarian cancer Xenograft model
1. Introduction Resveratrol (3,40 ,5-trans-trihydroxystilbene) is a naturally occurring phytoalexin present in grapes, peanuts, cranberries and other dietary constituents. The highest concentration of resveratrol is found in the roots of Japanese knotweed (Polygonum cuspidatum), which have been used in traditional Asian medicine for centuries to treat inflammation [1,2]. An expanding body of preclinical evidence shows resveratrol to exert a wide variety of * Corresponding author. Tel.: +48 618470721; fax: +48 618470721. E-mail addresses: [email protected] (H. Piotrowska), [email protected] (K. Myszkowski), [email protected] (E. Kwiatkowska-Borowczyk), [email protected] (R. Amarowicz), [email protected] (M. Murias), [email protected] (M. Wierzchowski), [email protected] (J. Jodynis-Liebert).
pharmacological activities, including anti-inflammatory, antioxidative, anti-tumour, anti-platelet, anti-fungal and estrogenic ones [3,4]. However, its biological effects in vivo appear to be strongly limited because of a poor bioavailability and a rapid clearance from the circulation [5]. The trihydroxystilbene scaffold of resveratrol has been the subject of structural alternations to improve the pharmacokinetic properties of the compound. The structure-activity studies have shown that the introduction of methoxy groups in place of hydroxy moieties increases the molecule stability, making it less susceptible to phase II conjugation reactions in vivo. Additionally, the methoxy groups added to the stilbene backbone of resveratrol may enhance its cytotoxic activity [6]. It has been also revealed that the methoxy moieties at positions 3,5- and 3,4,5- of the trihydroxystilbene framework play a crucial role for pro-apoptotic activity of the molecule [7].
http://dx.doi.org/10.1016/j.biopha.2014.02.001 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Piotrowska H, et al. DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.02.001
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BIOPHA-3372; No. of Pages 4 2
H. Piotrowska et al. / Biomedicine & Pharmacotherapy xxx (2014) xxx–xxx
To date, several studies have shown the potent anti-cancer activities of the methylated analogue of resveratrol, 3,4,40 ,5tetramethoxystilbene (DMU-212). In vitro, DMU-212 inhibits the growth of transformed fibroblasts, prostate, cervical, ovarian, breast, liver and colon cancer cell lines. DMU-212 has been found to exert anti-proliferative effects through multiple mechanisms, including down-regulation of the expression of enzymes catalyzing carcinogens activation, induction of cell cycle arrest and initiation of apoptotic mechanisms [8–12]. It has been reported that DMU-212 triggered a mitochondrial apoptotic pathway in transformed fibroblasts and breast cancer cells [9,10]. However, our previously published results demonstrated the ability of DMU212 to evoke apoptosis in both mitochondria- and receptormediated manner in two ovarian cancer cell lines, namely A-2780 and SKOV-3, which showed varied sensitivity to the test compound. The pronounced cytotoxic effects of DMU-212 observed in A-2780 cells were related to the execution of extracellular apoptosis pathway and cell cycle arrest in G2/M phase. It has been also suggested that inhibition of CYP1B1 by DMU-212 may affect its cytotoxicity [12,13]. In view of the great anticancer potential of DMU-212 against A2780 cell line, the aim of the current study was to evaluate its inhibitory effect in vivo. We attempted to verify DMU-212 activity in xenograft model using severely compromised immunodeficient (SCID) mice injected with A-2780 human ovarian cancer cells.
SV40 late polyA signal, into pcDNA3.1/Zeo(-). A-2780 cells were stably transfected with pcDNA3.1/Zeo(-)-Luc vector using lipofectamine LTX with Plus Reagent (Invitrogen). Zeocin (Invitrogen) (300 mg/mL) was added 48 h later; clones were picked 16 days after transfection into 48-well plate and 10 days later were transferred into 24-well plate. Luciferine expression was measured using ONE-GloTM Luciferase Assay (Promega). Clone with highest expression level were chosen for further analysis.
2. Materials and methods
2.5. SCID mice xenograft model
2.1. Materials
Subcutaneous xenograft tumors derived from human ovarian cancer cell line A-2780 were generated in CB17-SCID mice. Briefly, 3 106 A-2780 cells in 0,2 mL PBS were subcutaneously inoculated into the right flanks of SCID mice. At day 8 after injection, mice with equal level of bioluminescence were divided into two groups (control and DMU-212-treated) of six animals each. The compound tested was administered in a dose of 50 mg/kg b.w. three times a week by gavage. DMU-212 was suspended in 50% bcyclodextrin to improve its bioavailability (Lin et al., 2010). The control group was given the vehicle. D-Luciferin was injected intraperitoneally at a dose of 150 mg/kg b.w., followed by inhaled isoflurane anesthesia and bioluminescence imaging (IVIS 200, Caliper Life Sciences, Hopkinton, MA). Images of tumor-bearing mice were captured three times a week and data were analyzed using the Living Images software package (Caliper Life Sciences). Data are presented as the mean of bioluminescence intensity (BLI). All animals were humanely sacrificed after 14 days of monitoring due to the excessive tumor burden.
DMU-212 (3,4,40 ,5-trans-tetramethoxystilbene) was synthesised as described elsewhere [14,15]. The identity and purity of the compound was confirmed by NMR and LCMS. Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS), and trypsin-EDTA were purchased from Sigma-Aldrich St Louis, MO. Plasmid pcDNA3.1/Zeo(-) and plasmid pGL3-Basic were obtained from Invitrogen Carlsbad, CA and Promega, Madison, WI, respectively. Forane (isofluranum) was acquired from Abbott Laboratories Ltd (Illinois, USA). D-Luciferin (Potassium salt) was from Gold Biotechnology (St Louis, MO). 2.2. Animals SCID female mice (CB17/Icr-Prkdcscid/IcrCrl), 6-weeks-old and weighing 20–24 g were obtained from Charles Riever (Wilmington, MA, USA). Animals were housed (5/cage) in filter top cages equipped with HEPA filters (Tecniplast, Italy). Room climate was maintained at a temperature of 22 3 8C, 40–65% relative humidity, with 10 air changes per hour. Commercial diet (ISO 9001 certified laboratory feed, total pathogen free, Altromin, Germany) and drinking water were available ad libitum. The experiment was performed according to the guidelines of the Local Animal Ethics Committee for animal experimentation.
2.4. High performance liquid chromatography The determination of DMU-212 in cells and culture medium was performed using a Shimadzu System (Shimadzu, Kyoto, Japan) equipped with a L-C-10 AD VP pump, a SCL-10 A VP interface, and a UV-Vis SPD-M10 A VP detector (set at a wavelength 326 nm). For separation of DMU-212 a LUNA C18 column (4.60, 250 mm; 5 mm; Phenomenex, Torrence USA) was used. The mobile phases used in assay were: 30% acetonitrile containing 0.1% trifluoroacetic acid (mobile phase A), and 100% acetonitrile (mobile phase B), the flow rate was 1 mL/min. The linear gradient system ranged from 0 to 100% of mobile phase B over 50 min of analysis. Every analysis was followed by equilibration of the column with 100% phase A for 8 min before application of the next sample. The sample injection volumes were 100 mL. Calibration was accomplished using the external standard method at a concentration range of 0.1–75 mg/ mL.
2.6. Statistical analysis GraphPad Prism 5.0 version 5.00 for Windows, GraphPad Software, San Diego California USA was employed for the statistical evaluation of the data. Statistical significance was tested by twotailed Student’s t-test, with P-value of less than 0.05 considered statistically significant.
2.3. Cell culture and transfection A-2780 ovarian cancer cell line was purchased from the European Type Culture Collection (Sigma-Aldrich Co., St Louis, MO). A-2780 cells were maintained in phenol red-free DMEM medium supplemented with 10% foetal bovine serum (FBS), 2 mM glutamine, penicillin (100 U/mL), and streptomycin (0.1 mg/mL). Cells were cultivated under standard conditions at 37 8C in a humidified atmosphere containing 5% CO2 and 95% air. Expression vector pcDNA3.1/Zeo(-)- Luc was constructed by cloning of XhoI – BamHI fragment of pGL3-Basic, encoding firefly luciferase and
3. Results and discussion DMU-212 and its prototype compound resveratrol have been reported to exert antiproliferative activity in the Apc(Min+) mice [11]. However, Ziegler et al. showed that resveratrol failed to decline tumor size in this experimental model [16]. Furthermore, according to more recent report, resveratrol promoted the growth of prostate cancer LAPC-4 xenografts in SCID mice [17]. Klink et al. demonstrated that resveratrol worsen survival in SCID mice with
Please cite this article in press as: Piotrowska H, et al. DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.02.001
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prostate cancer xenografts via up-regulating of oncogenic pathways. In conclusion, they found that resveratrol used in phase I clinical trials may be harmful for some patients with prostate cancer [17]. In view of findings mentioned above, there is a great need of structural alternation of resveratrol scaffold to generate its novel analogues with improved growth-inhibitory and apoptogenic properties. In the present study, we assessed the antiproliferative activity of the methylated analogue of resveratrol, 3,4,40 ,5-tetramethoxystilbene (DMU-212) in xenograft model of ovarian cancer. To evaluate in vitro metabolic properties of cells that were to be injected into SCID mice, uptake and decline of DMU-212 in A-2780 ovarian cancer cell line was investigated. It was found that the concentration of the test compound in A-2780 cells was growing within first eight hours, and then the gradual decline was observed (Fig. 1). In medium we noted time-dependent decrease in DMU-212 concentration. However, after 50 hours incubation a plateau has been established (Fig. 1). It can be suggested that the rapid absorption and slow elimination of the test compound may provide its effective growth inhibitory activity against ovarian cancer cells. Based on these data and our previously published results indicated potent antitumor activity of DMU-212 in A-2780 ovarian cancer cell line [12] we extended our studies to in vivo conditions by generating A-2780 ovarian cancer xenografts in SCID mice. In recent years, several new mouse models for cancer research have been developed. The discovery of T-cell deficient athymic nude mice, and later T-cell- and B-cell deficient SCID mice, allowed the inoculation and dissemination of human cancer cells in mice [18]. Bioluminescent imaging is one of the most common methods to assess tumor response to the test compound in mice. It allows non-invasive, longitudinal evaluation of tumor growth before, during and after treatment [19]. In our study, it has been initially observed that mice treated with vehicle and DMU-212 at dose 50 mg/kg b.w. exhibited similar tumor size as reflected by approximately equal bioluminescence intensity (Figs. 2 and 3). However, after seven days of treatment, tumor growth appeared to be suppressed in the animal group treated with the compound tested (Figs. 2 and 3). At day 14 of the experiment, tumor burden in mice treated with DMU-212 was significantly lower, as compared to untreated controls (Figs. 2 and 3). Our results are in agreement with the previous findings indicated antiproliferative activity of DMU-212 in the Apc(Min+) mice, a model of human intestinal
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carcinogenesis. Although an experimental model was different, decreased tumor size in mice treated with DMU-212 has been also observed [11]. Similarly, other methylated resveratrol derivatives 2,30 ,4,40 ,50 -pentamethoxystilbene (PMS) and 3,5,40 -trimethoxystilbene (MR-3) have been shown to exhibit potent antitumor activity in vivo [7,20,21]. Pan et al. (2008) showed that treatment with MR-3 three times a week at a dose of 50 mg/kg b.w. reduced tumor size in SCID mice injected with colon cancer cells [21]. While their experiment lasted 23 days, we shortened the treatment time to 14 days due to excessive tumor burden, which may be explained by differences in cell division between colorectal cancer cells and ovarian ones [22].
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Fig. 1. Uptake of DMU-212 into cytoplasm of A-2780 cells ( ) and decrease in DMU212 levels in incubation medium ( ), cells were treated with DMU-212 for various time intervals, scrapped, centrifuged, lysed by freeze-thaw stress and centrifuged again. The supernatant and medium from the cells were analyzed using HPLC.
Fig. 3. The changes in tumor development in mice treated with DMU-212 (50 mg/kg b.w.) The figure shows one representative mouse from (A) DMU-212 treated group and (B) control group.
Please cite this article in press as: Piotrowska H, et al. DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.02.001
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To evaluate the potential toxic effects of DMU-212 treatments, clinical observations have been performed everyday as well as body weight, water and food intake were recorded. DMU-212 at a dose of 50 mg/kg b.w. was shown to be well tolerated without any adverse effects (data not shown). Our results are in agreement with the other authors’ findings indicated that DMU-212 was well tolerated and failed to affect body weight in Apc(Min+) mice [11]. Ovarian cancer is the fifth most common cause of cancerrelated deaths in women [23]. Although the incidence of ovarian cancer is lower than many other malignancies, the death rate is higher due to its early propagation and rapid development of chemotherapy resistance [23,24]. In our study, DMU-212, methylated analogue of resveratrol, displayed antitumor activity in an ovarian cancer model in vivo without systemic toxicity throughout the whole period of drug treatment. Our findings suggest that DMU-212 might be considered as a potential anticancer agent used in ovarian cancer therapy. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgments This study was supported by a research grant of the Polish Ministry of Science and Higher Education No. IP2012046072. References [1] Saiko P, Szakmary A, Jaeger W, Szekeres T. Resveratrol and its analogs: defense against cancer, coronary disease and neurodegenerative maladies or just a fad? Mutat Res 2008;658:68–94. [2] Stervbo U, Vang O, Bonnesen C. Time- and concentration-dependent effects of resveratrol in HL-60 and HepG2 cells. Cell Prolif 2006;39:479–93. [3] Shakibaei M, Harikumar KB, Aggarwal BB. Resveratrol addiction: to die or not to die. Mol Nutr Food Res 2009;53:115–28. [4] Whitlock NC, Baek SJ. The anticancer effects of resveratrol: modulation of transcription factors. Nutr Cancer 2012;64:493–502. [5] Walle T, Hsieh F, DeLegge MH, Oatis Jr JE, Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos 2004;32:1377–82. [6] Roberti M, Pizzirani D, Simoni D, Rondanin R, Baruchello R, Bonora C, et al. Synthesis and biological evaluation of resveratrol and analogues as apoptosisinducing agents. J Med Chem 2003;46:3546–54. [7] Li H, Wu WK, Li ZJ, Chan KM, Wong CC, Ye CG, et al. 2,3’,4,4’,5’-Pentamethoxytrans-stilbene, a resveratrol derivative, inhibits colitis-associated colorectal carcinogenesis in mice. Br J Pharmacol 2010;160:1352–61.
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Please cite this article in press as: Piotrowska H, et al. DMU-212 inhibits tumor growth in xenograft model of human ovarian cancer. Biomed Pharmacother (2014), http://dx.doi.org/10.1016/j.biopha.2014.02.001
