Antitumor activity of tributyrin in murine melanoma model

Cancer Letters 164 (2001) 143±148

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Antitumor activity of tributyrin in murine melanoma model Adam Giermasz a, Dominika Nowis a, Ahmad Jalili a, Grzegorz Basak a, Maria Marczak c, Marcin Makowski a, Anna Czajka a, Izabela Møynarczuk a,b, GrazÇyna Hoser d, Tomasz Stok osa a, Sebastian Lewandowski e, Marek JakoÂbisiak a,* a

Department of Immunology, Centre of Biostructure, The Medical University of Warsaw, ul. Chalubinskiego 5, 02-004 Warsaw, Poland b Department of Histology and Embriology, Centre of Biostructure, The Medical University of Warsaw, Warsaw, Poland c Department of Dermatology, The Medical University of Warsaw, Warsaw, Poland d Department of Clinical Cytology, Postgraduate Center for Medical Study, Warsaw, Poland e Oncology Clinic, Central Clinical Hospital, Military School of Medicine, Warsaw, Poland Received 11 July 2000; accepted 11 September 2000

Abstract Butyric acid has been known to inhibit growth and to induce differentiation of a variety of tumor cells. Butyrate-treated tumor cells have also been observed to undergo apoptosis. Although butyrate compounds have demonstrated antitumor activity in murine tumor models and have already been admitted to clinical trials in tumor patients, the exact mechanism of their antitumor effects has not been elucidated. The results of our study showed antitumor activity of tributyrin, a butyric acid prodrug, in murine melanoma model and are strongly suggestive that antiangiogenic effects could participate in antitumor effects of butyrate compounds in vivo. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tributyrin; Butyrate; Melanoma; Mouse; Tumor therapy

1. Introduction Butyric acid belongs to short-chain fatty acids produced by anareobic fermentation of dietary ®bers in the human colon [1]. It has been demonstrated to stimulate differentiation [2,3] and to inhibit growth [4±6] of a number of tumor cells. Butyrate has also been shown to induce apoptosis in both leukemia and solid tumor lines [5±8]. Due to the above-mentioned properties butyrate compounds have received attention as potential cancer therapeutics and have been shown to produce antitumor effects in murine tumor models [9±11]. Although administration of butyrate * Corresponding author. Tel./fax: 148-22-622-6306. E-mail address: [email protected] (M. JakoÂbisiak).

compounds to cancer patients has already been reported [12,13] the exact mechanism responsible for their antitumor effects remains to be elucidated. In our present study we decided to examine potential antitumor effects of tributyrin in murine melanoma model and to shed some light on the mechanism of its antitumor activity. 2. Materials and methods 2.1. Mice (C57BL/6 £ DBA/2)F1, called hereafter B6D2F1 and BALB/c mice, 8±12 weeks old, were used in the experiments. Breeding pairs were obtained from Inbred Mice Breeding Center of the Institute of Immu-

0304-3835/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(01)00375-5

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nology and Experimental Medicine, Worcøaw, Poland. Mice were bred and kept in local animal facility. All experiments with animals were performed in accordance with the guidelines approved by the Ethical Committee of the Medical University of Warsaw. 2.2. Tumor cells B16F10 melanoma cells were obtained from Dr M. Kubin, Immunex Corp., Seattle, WA. Tumor cells were cultured in Dulbecco's modi®ed Eagle medium (Gibco BRL Life technologies Ltd, Paisley, UK) supplemented with 10% heat-inactivated FBS, standard antibiotics, 2-mercaptoethanol (50 mM), l-glutamine (2 mM), all from Gibco BRL. Cells were maintained in a humidi®ed 5% CO2 atmosphere at 378C. 2.3. Drugs and reagents Butyric acid sodium salt (NaBut, butyrate) and tributyrin were purchased from Sigma Chemicals, St. Louis, MO. 2.4. In vitro experiments Cytostatic/cytotoxic effect of NaBut on B16F10 cells were studied in a standard MTT assay as described elsewhere [14]. Brie¯y, tumor cells were dispensed in a 96-well ¯at bottom microtiter plate (Nunc, Roskilde, Denmark) at concentration of 5 £ 103 cells in 100 ml per well. After 1 day of culture to allow the cells to attach they were treated with serial dilutions of NaBut for 3 days. Four hours before the end of incubation 25 ml of 3-(4,5-dimethylthiazol-2-yl)diphenyltetrazolium bromide (MTT; Sigma) solution (2.5 mg/ml) was added to each well. After 4 h resulting formazon crystals were dissolved in acidic DMSO. The absorbance was read at 540 nm on an ELISA reader (SLT Labinstrument GmBH, Salzburg, Austria). The cytostatic/cytotoxic effect was expressed as relative viability and calculated as follows: Relative viability ˆ

experimental absorbance 2 background absorbance £ 100% untreated control absorbance 2 background absorbance

2.5. Flow cytometric analysis of the cell cycle Cells were plated in 6-well plates (Nunc, Roskilde, Denmark) and sodium butyrate was added (the ®nal

concentration was 10 mM) for 12, 24, 36 and 48 h prior to harvesting. After washing in phosphatebuffered saline (PBS) twice, the cells were trypsinized and ®xed in 75% ethanol in 50 mM PBS, pH 7.3 and kept at 2208C till analysis (up to 1 week). Prior to staining cells were spun at 300 £ g for 10 min and washed twice with cold 50 mM PBS (pH 7.3) supplemented with 2% fetal-bovine serum (FBS). The cells were resuspended in 10 mg/ml propidium iodide in PBS with 0.1% Nonidet P40 and RNAse free from DNAse (100 mg/ml), and were incubated for 10 min at 378C. Then the cells were incubated with propidium iodide (5 mg/ml) for 4 h at 48C in darkness. The cells were analyzed by FACSCalibur ¯ow cytometer (Beckton Dickinson, San Diego, CA) and CellQuest software. Argon laser excitation wavelength was 488 nm, while emission data were acquired at wavelength 580 nm [15]. 2.6. In vivo experiments B6D2F1 mice were inoculated with exponentially growing B16F10 melanoma cells (1 £ 106 ) into the footpad of the right hind-limb. Tumor-bearing mice (6±7 mice in each group) were treated intraperitoneally with doses of 5 mg/kg of tributyrin in different time schedules. Control mice received mock injections with saline. Local tumor growth was determined by measuring footpad diameter in two dimensions (anterior±posterior and side-to-side) with caliper every 2 days. Tumor volume was estimated as in previous experiments [16] by the formula: Tumor volume …mm3 † ˆ …longer diameter† £ …shorter diameter†2

Relative tumor volume was calculated as follows: Relative tumor volume ˆ

tumor volume £ 100 initial tumor volume

where the initial tumor volume was that measured at the ®rst day of treatment. 2.7. Angiogenesis assay The tumor induced angiogenesis assay was performed as previously described [17]. Brie¯y, BALB/c mice were irradiated and anaesthetized with chloral hydrate and shaved on both ¯anks. To induce angiogenesis, each mouse was given a B16F10 tumor cell suspension intradermally

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(1 £ 10 cells in 0.1 ml saline with trypan blue for better visualization of the injection site). Tributyrin (5 mg/kg) was administered intraperitoneally for 3 consecutive days starting from the day of tumor cell inoculation; control mice were treated with saline. Mice were killed on day 4 and the inner surface of the skin was assessed blindly for the number of newly formed blood vessels. 2.8. Western blotting B16F10 cells were cultured with different concentrations of butyrate and for different times. Cells were washed with PBS and lysed in sample buffer containing 2% SDS with protease inhibitor. Equal amounts of protein were separated on 7.5% SDS±polyacrylamide gel, transferred onto PVDF membranes, blocked with TBST (Tris-buffered saline (pH 7.4), 0.05% Tween20) with 5% non-fat dry milk and 5% FBS. After washing, membranes were incubated with a polyclonal primary antibody anti poly-(ADP-ribose)-polymerase (PARP) (Boeringer Mannheim GmbH, Mannheim, Germany) for 4 h. After next washing, membranes were incubated for 45 min with alkaline phosphatase-coupled secondary antibody (Jackson Immuno Research Inc., West Grove, PA). The color reaction was developed using NBT (p-nitro-blue tetrazolium chloride) and BCIP (5-bromo-4-cholro-3indolyl phosphate). 2.9. Statistical analysis

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shown in Fig. 1. Dose-dependent cytostatic/cytotoxic effects were observed. Only higher doses of butyrate (0.625, 2.5 and 10 mM) produced signi®cant cytostatic/cytotoxic effects (P , 0:05, in comparison with control). For the cell cycle analysis 10 mM concentration of sodium butyrate was chosen. Significant accumulation in G1 phase was observed in cells incubated for 12, 24, 36 and 48 h with sodium butyrate. Transient increase in percentage of cells accumulated in G2/M phase was observed after 12 h incubation (Fig. 2). The highest number of apoptotic cells (counted as those with DNA content of less than 2 N) was observed after 36 h incubation; however, it never exceeded 3% of all counted cells (data not shown). To con®rm whether butyrate is inducing apoptosis in B16F10 cultures, Western blot analysis was performed. As shown in Fig. 3, a poly-(ADPribose)-polymerase (PARP) degradation was observed only after 48 h incubation with higher concentrations of butyrate (5 and 10 mM). 3.2. Effect of systemic treatment with tributyrin on growth of B16F10 cells in mice Mice bearing B16F10 melanoma in the footpad were treated intraperitoneally with 5 mg/kg of tributyrin or corresponding volume of saline every second day starting on day 7 after tumor inoculation. As presented in Fig. 4, relative tumor volume of mice treated with tributyrin was reduced in comparison

Data are presented as a mean ^ standard deviation (SD). Differences in in vitro experiments and angiogenesis assay were analyzed by Student's t-test (twotailed). Differences in data from animal experiments were analyzed for signi®cance with the Mann±Whitney U-test. Cell cycle data were analyzed by the chisquare test. 3. Results 3.1. Response of B16F10 cells to sodium butyrate in vitro To assess the in¯uence of butyrate on cell proliferation, attached cells were incubated for 3 days with serial dilutions of sodium butyrate. Results are

Fig. 1. The effect of butyrate on B16F10 melanoma cell growth in vitro. Cells were incubated with indicated concentrations of butyrate for 72 h. Signi®cant differences (Student's t-test, P , 0:05) with controls were observed for cells treated with 0.625, 2.5 and 10 mM concentrations.

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Fig. 2. Cell cycle analysis of B10F10 cells treated with 10 mM of butyrate for indicated times. Cells were stained with propidium iodide and analyzed for DNA content (as described in Section 2). Cell cycle phases of untreated and treated groups were counted as a percentage of all living cells. A signi®cant difference (P , 0:05, chi-square test) was observed for G1/G0 phase between all treated groups and control, and also between 12- and 24-h groups and between 36- and 48-h groups. For G2/M phase the difference was signi®cant between control and the 12-h group, and also between the 12- and 24-h groups and between the 36- and 48-h groups. A signi®cant decrease in the amount of cells in S phase as compared with control was observed in all treated groups.

Fig. 4. The effect of systemic administration of tributyrin (5 mg/kg) given every second day starting from day 7 after tumor inoculation on B16F10 melanoma growth in B6D2F1 mice. A signi®cant difference (P , 0:05, Mann±Whitney U-test) between treated groups and control was observed from day 13 and persisted until day 21.

with that of controls. A signi®cant difference was observed starting from day 13 after inoculation of tumor cells. Mice from both groups died within 36 days, but no difference in survival between groups was observed. The experiment was performed twice and similar results were obtained. 3.3. Antiangiogenic effects of tributyrin To determine whether the antitumor effect observed in vivo could be caused by antiangiogenic mechanisms, we used the tumor cell-induced angiogenesis model. As shown in Fig. 5, a short intraperitoneal treatment with tributyrin (5 mg/kg) for 3 consecutive days starting from the day of tumor cell inoculation resulted in signi®cant inhibition of the blood vessel formation at the site of B16F10 cells injection (P , 0:0001). 4. Discussion

Fig. 3. Western blot of B16F10 cells treated with indicated concentrations of butyrate for 6, 24 and 48 h. Arrows indicate poly-(ADPribose)-polymerase (PARP, 118 kDa) and degraded PARP (DPARP, 85 kDa). For positive control U937 apoptotic cells were used.

Butyrate was previously shown to induce apoptosis in a variety of tumor cells [7,8,18,19]. Although in our experiments we have shown that butyrate exerts concentration-dependent cytostatic/cytotoxic effects in B16F10 melanoma cells, ¯ow cytometric analysis revealed that only up to 3% of cells have undergone apoptosis. In Western blot analysis it was also shown that PARP degradation occurs only in long-time-

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References

Fig. 5. The effect of systemic administration of tributyrin (5 mg/kg) given for 3 consecutive days starting from the day of tumor cell inoculation on new vessel formation in BALB/c mice (as described in Section 2). Difference between groups was signi®cant for P , 0:0001 (Student's t-test).

incubated cells (48 h) and with higher doses of butyrate (5 and 10 mM). These results suggest that butyrate-induced cytostatic effects result in the induction of apoptotic events after prolonged period of time. Accumulation of butyrate-treated cells in G1 phase of the cell cycle seems to be responsible for these cytostatic effects. Our results are in accordance with earlier observations showing G1 arrest in cells treated with butyrate [4], although accumulation in G2 phase was also observed after incubation of these cells with this compound [6]. Our in vivo results show that systemic administration of a butyric acid prodrug, tributyrin, results in signi®cant retardation of B16F10 melanoma growth. The effect might be due to its cytostatic activity against B16F10 (as suggested by in vitro experiments). To look for other mechanisms of antitumor activity of tributyrin we performed a tumor cell-induced angiogenesis assay. Our results are the ®rst to suggest that antiangiogenic effects could participate in antitumor activity of butyrate compounds.

Acknowledgements This research was supported by Grant M19/S1/00 from The Medical University of Warsaw. The ®rst author is a recipient of The Award for Young Scientist of The Foundation of Polish Science.

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