Lipotropes (methyl nutrients) inhibit growth of feline lymphoma in vitro

Research in Veterinary Science 93 (2012) 259–263

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Lipotropes (methyl nutrients) inhibit growth of feline lymphoma in vitro L. Mabasa, K. Cho, S. Bae, D.M. Walsh, P. Asija, C.S. Park ⇑ Department of Animal Sciences, North Dakota State University, Fargo, ND 58108, USA

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Article history: Received 4 November 2010 Accepted 18 May 2011

Keywords: Lipotropes Methyl nutrients Feline lymphoma DNA methylation p53 Caspase 3

a b s t r a c t Feline lymphoma is one of the most frequently diagnosed tumors in cats. Lipotropes are dietary methyl donors that may modulate DNA methylation status and the expression of genes involved in growth and apoptosis of feline lymphoma cells. The specific objective of the study was to determine if lipotropes affect the growth of feline lymphoma cells, which entailed examining a correlation between lymphoma cell proliferation and apoptosis. F1B and FeLV-3281 cells were cultured and treated with 20 times the level of lipotropes contained in the basal culture medium. Cell growth and death and caspase 3 and tumor protein p53 activity were measured. Lipotropes were found to significantly reduce cell growth; increased cell death and caspase 3 and p53 activity was seen in F1B cells after 72 h, but the effect was minimal on FeLV-3281. These results could be useful in the development of dietary strategies for treating and preventing feline lymphoma. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Lymphoma is the malignant transformation of lymphocytes, which are cells of the immune system. It is the most commonly diagnosed neoplasm in cats, accounting for approximately 30% of all diagnosed tumors (Twomey and Alleman, 2005; Tzannes et al., 2008). Feline lymphoma may be classified as retrovirus-associated or non-viral in origin (Louwerens et al., 2005). The most lymphomagenic of the viruses is feline leukemia virus (FeLV), with 70% of feline lymphomas causally associated with persistent productive FeLV infection (Louwerens et al., 2005; Rojko et al., 1989). FeLV is a contagiously transmitted retrovirus of cats which suppresses the immune system (Hoover and Mullins, 1991). The mechanisms of the FeLV-induced tumors are not completely understood, and little information exists in relation to the involvement of apoptosis-related proteins (Suntz et al., 2010). Most recently, a study on feline lymphomas showed a high expression of Bcl-2, a member of the rapidly expanding Bcl-2 family of genes that regulate apoptosis (Kano et al., 2008). This evidence suggests that apoptosis-related proteins may play a critical role as target components of the genomic pool in prevention and treatment of feline lymphoma. Lipotropes are essential nutrients (methionine, choline, folate, and vitamin B12) containing methyl groups (CH3). They are important methyl donors and cofactors that play key roles in one-carbon metabolism. One-carbon metabolism provides methyl groups for ⇑ Corresponding author. Address: Department of Animal Sciences, North Dakota State University, North University Drive, Fargo, ND 58108, USA. Tel.: +1 701 231 7670; fax: +1 701 231 7590. E-mail address: [email protected] (C.S. Park). 0034-5288/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2011.05.015

all biological methylation pathways, and is highly dependent on methyl donors and cofactors (Newberne and Rogers, 1986; Riggs et al., 1996; Van den Veyver, 2002). Dietary supplementation of lipotropes may increase methyl metabolism (i.e., genomic DNA methylation and nucleic acid synthesis), which is required for cell proliferation and maintenance of tissue integrity (Ross, 2003; Van den Veyver, 2002). Until now, investigation of the involvement of methyl donors in health has been limited to deficiency in individual nutrients in human health. This study is the first to look at the role of methyl donor supplementation in feline lymphoma growth. The specific objective of the study was to determine if lipotropes affect the growth of feline lymphoma cells, which entailed examining a correlation between lymphoma cell proliferation and apoptosis.

2. Materials and methods 2.1. Cell culture F1B (adherent fibroblast, submandibular lymph node origin) and FeLV-3281 (non-adherent lymphoblast, subgroup A feline leukemia virus origin) feline lymphoma cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in a 5% CO2-humidified atmosphere at 37 °C and maintained in basal medium (Roswell Park Memorial Institute for FeLV-3281, and Dulbecco’s Modified Eagle Medium for F1B, Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco Invitrogen) and 1% antibiotic–antimycotic (Gibco Invitrogen) as recommended by the supplier.

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2.2. Cell proliferation assay F1B and FeLV-3281 feline lymphoma cells were seeded at a density of 5  104 cells/mL in flat–bottomed 96-well plates with either basal control medium [normal levels of lipotropes in basal culture medium (1): 17 mg/L L-methionine, 9 mg/L choline, 3 mg/L folic acid and 2 mg/L vitamin B12] or medium supplemented with 10, 20, 30, or 40 times the above mentioned methyl nutrients contained in basal culture medium for dose response experiment. After 0, 24, 48, 72, and 96 h of incubation, cell proliferation was measured using colorimetric MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (CellTiter 96 AQueous One Solution Reagent; Promega, Madison, WI, USA). MTS was added to each well (at 10%) and the plates incubated for a further 2 h at 37 °C in a 5% CO2 incubator. The absorbance was measured at 490 nm with a plate reader (Molecular Devices, Sunnyvale, CA, USA). The observed optimal dose of lipotropes for maximum cell growth inhibition from the above experiment was used for further cell proliferation assays and subsequent experiments. 2.3. Flow cytometric analysis Cell death was determined by double-staining with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) [Sigma–Aldrich, St Louis, MO, USA] as described previously (Cho et al., 2010). Cells treated with the 20 times lipotropes level were harvested after a 72 h incubation and washed with PBS containing 0.5% bovine serum albumin (Sigma–Aldrich) and 0.1% sodium azide (Sigma–Aldrich). They were then incubated with FITC-conjugated Annexin V and PI. Cells were washed and then analyzed using an Accuri C6 cytometer and Cflow software (Accuri Cytometers, Ann Arbor, MI, USA). 2.4. Caspase 3 apoptosis assay F1B and FeLV-3281 feline lymphoma cells were cultured at a density of 5  104 cells/mL in flat-bottomed 24-well plates in media containing the optimal 20 times lipotropes’ level and incubated for 72 h based on data collected from the cell proliferation experiment. The activity of caspase 3 was measured by colorimetric assay with CaspACE assay system (Promega, Madison, WI, USA). Cultured cells were washed twice with ice–cold PBS and resuspended in cell lysis RIPA buffer (Promega). Cell lysates were incubated with the colorimetric substrate N-acetyl-Asp-Glu-Val-Asp-amino-p-nitroanilide (Ac-DEVD-pNA). After a 4 h incubation at 37 °C, the release of p-nitroaniline from Ac-DEVD-pNA was measured at 405 nm. 2.5. Tumor protein p53 assay Tumor protein p53 activity was determined by using an enzyme immunometric assay kit (TiterZyme EIA Kit, Assay Designs, Ann Arbor, MI, USA). Cells were treated with the 20 times lipotropes level at a density of 5  104 cells/mL and incubated for 72 h. Following incubation, cells were washed twice with PBS and re-suspended in cell lysis RIPA buffer (Sigma–Aldrich). The cell lysates were centrifuged. The supernatant was incubated on a plate pre-immobilized with p53 polyclonal antibody and it then reacted with the labeled antibody. The absorbance was measured at 450 nm. 2.6. Statistical analyses A minimum of four replications of three independent culture experiments were required to detect a statistical difference. Statistical analysis was performed using Student’s t test and one-way ANOVA followed by Tukey’s test (SAS 9.1, Statistical Analysis Sys-

tem Institute, Cary, NC, USA). Differences were considered significant at P < 0.05. 3. Results 3.1. Effect of lipotropes on the proliferation of F1B and FeLV-3281 lymphoma cells To investigate the effect of lipotropes supplementation on cancer cell growth and apoptosis, feline lymphoma F1B and FeLV-3281 cell lines were cultured in medium supplemented with 10, 20, 30, or 40 times the level of lipotropes present in basal culture medium to determine the optimal dose of lipotropes for maximal cell growth inhibition. The 20 times lipotropes level was identified as the optimal dose for maximal inhibition of growth of the F1B cell line after 72 h of incubation. However, the proliferation of the FeLV-3281 cell line was not affected by lipotropes (data not shown). The observed optimal level was then used for further individual cell proliferation assays and other experiments. As shown in Fig. 1A, lipotropes (20) significantly reduced growth of F1B cells by 45.4% after 72 h (P < 0.05). However, the growth of FeLV-3281 lymphoma cells was not affected by lipotropes at 72 h (Fig. 1B). At 96 h, growth of FeLV-3281 tended to be lower with lipotropes treatment as compared to the control (P > 0.05). 3.2. Analysis of F1B and FeLV-3281 lymphoma cell death by flow cytometry Populations of viable cells were gated for analysis of apoptosis (Fig. 2A, B, E, F). F1B cells treated with lipotropes showed 2.2% more apoptosis (Fig. 2D) than F1B control cells (Fig. 2C). Also, late apoptotic cells were 3.2% higher in the lipotropes treated cells (Fig. 2D) as compared to the control (Fig. 2C). There was no difference between the control (Fig. 2G) and the lipotropes-treated FeLV-3281 cells (Fig. 2H). Consistent with the cell proliferation data, we showed that the 20 times lipotropes level increased the death of F1B cells as compared to the control at 72 h (2.3% vs. 0.1%, respectively; Fig. 2D and C). Death of FeLV-3281 was not affected by the lipotropes treatment as compared to the control (0% vs. 0.1%, respectively; Fig. 2H and G). 3.3. Expression of caspase 3 enzyme Effect of lipotropes on the expression of caspase 3 on both F1B and FeLV-3281 cells was studied. Lipotropes significantly increased caspase 3 activity of F1B cells after 72 h as compared to the control (93.0 ± 0.9 vs. 88.7 ± 1.0%; P < 0.05; Fig. 3A). However, caspase 3 activity of FeLV-3281 was not affected by the lipotropes treatment (Fig. 3A). 3.4. Tumor protein p53 expression Results of p53 protein show that the concentration of p53 in F1B lymphoma cells tended to be higher with lipotropes treatment as compared to the control after a 72 h incubation as shown in Table 1 (P > 0.05). Fig. 3B shows that there was a 52% increase in p53 protein. These results are also consistent with the cell proliferation and cell death data. In FeLV-3281, the lipotropes treatment did not affect the concentration of p53 as compared to the control (Fig. 3B). 4. Discussion Lipotropes have been shown to reduce tumor incidence in humans and rodents (Moon et al., 1998; Park et al., 2008). Here,

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Fig. 1. Effect of lipotropes on time-dependent feline lymphoma cell growth. (A) F1B and (B) FeLV-3281 cells were treated with 20 the level of lipotropes present in basal culture medium. Data in A and B are means ± SEM (n = 12) and expressed as percentages of cell proliferation ([treated cell absorbance - initial (seeding) cell absorbance]/ initial (seeding) cell absorbance)  100. ⁄Different from control, P < 0.05.

Fig. 2. Gating, cell death and apoptosis of 20 lipotropes-supplemented F1B and FeLV-3281 cells after a 72 h incubation. Boxes A, B, E and F show gating of the population. C represents the control for the F1B cell line and D is the treatment; G is the control for the FeLV cell line and H is the treatment. Cells were stained with Annexin V-FITC (FL1, X axis) and PI (FL2, Y axis). Lower left quadrant represents living cells, upper left quadrant represents dead cells, lower-right quadrant represents early apoptotic cells, while upper-right quadrant represents late apoptotic cells.

we attempted to investigate the effect of supplementation with lipotropes on the proliferation of feline lymphoma cells, F1B and FeLV-3281. We have demonstrated a significant reduction in growth of the F1B cell line due to lipotropes treatment after 72 h of incubation, while the effect was minimal on the FeLV-3281 cell line. To determine if the reduction in growth of feline lymphoma by methyl nutrients was apoptosis-related, flow cytometry was used to evaluate the extent to which methyl donors affect death of both F1B and FeLV-3281 lymphoma cells. A good population of viable cells was found for both cell lines and appropriate gates were made. 2.3% of the F1B lipotrope-treated population stained single-positive for Annexin V, whereas only 0.1% of the F1B control population stained this way. There was no significant difference

between the populations of lipotrope-treated and control FeLV cells. This indicates that treatment with lipotropic nutrients slightly increased apoptosis after 72 h in the F1B cell line, supporting the cell proliferation data. This suggests that methyl donors may be involved in initiating death of F1B lymphoma cells but not FeLV-3281 cells. The activities of proteins that regulate the cell death pathway were also investigated. p53 is a tumor suppressor gene which is mutated in about 50% of human tumors. While investigating whether mutational inactivation of p53 protein occurred in feline lymphomas, Miki et al. (2004) discovered that, of the three feline lymphoma cell lines studied, one had an aberration of the p53-amino acid sequence, while the others did not. p53 is greatly involved

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Fig. 3. (A) Caspase 3 and (B) p53 activities of 20 lipotropes-supplemented F1B and FeLV-3281 cells after a 72 h incubation. Data in A and B are means ± SEM (n = 9 for A and n = 4 for B) and expressed as percentages of control cell caspase 3 or p53 activities (treated cell absorbance/control cell absorbance)  100. ⁄Different from control, P < 0.05.

Table 1 Differences in the concentration of p53 tumor suppressor gene in feline lymphoma cells grown in culture medium supplemented with or without 20 times the level of lipotropes in basal culture medium.a

a b c

Cell lines

Control

Lipotropes

SEMb

Pc

p53 Expression of F1B, pg/mL p53 Expression of FeLV, pg/mL

0.73 1.14

1.11 1.24

0.20 0.09

0.18 0.43

Values are means ± SEM and also log scaled. SEM = standard error of the mean. P (probability) = significance level of F-test for equality.

in the feline lymphoma cell death pathway; p53 either directly or indirectly transcriptionally down-regulates the expression of the Bcl-2 gene involved in the regulation of programmed cell death (Marin et al., 1994; Miyashita et al., 1994). p53 may in turn be inactivated by damage to the genome through mutation, chromosomal rearrangement, gene conversion, over-expression of the MDM2 gene (which is a dominant p53 inhibitor), and imprinting (Harris, 1996; Miyashita et al., 1994; Miki et al., 2004). Methyl nutrients have been shown to regulate the activity of p53 tumor suppressor gene and inhibition of several human cancers’ development (Kim et al., 1997). We show that methyl nutrients increased the expression of p53 protein in F1B lymphoma cells as compared to the control, while the effect was minimal in FeLV-3281 cells. These findings suggest that methyl nutrients may be directly or indirectly involved in restoration of p53 protein, although it is not clear as to how. Apoptosis is also mediated by a frequently activated terminal protease, caspase 3; caspase 3 is responsible for catalyzing specific cleavage of key proteins (Porter and Janicke, 1999). We investigated the effect of lipotropes on caspase 3 activity. Statistically, caspase 3 activity was significantly increased in the F1B lymphoma cells, while the expression in FeLV-3281 was not affected by lipotropes treatment. This information suggests that lipotropes induced caspase 3-dependent F1B cell death. Although signs of apoptosis were reflected in the cell death analysis, the difference between the treatments was minimal, as reported earlier. This could be due to the timing of the analyses. For instance, Sundquist et al. (2006) have noted that monitoring apoptosis-related events after the optimal time period could result in little to no signal, leading to the erroneous conclusion of no occurrence of apoptosis. Also, they indicated that cultured cells induced to undergo apoptosis exhibit signs of apoptotic events within the first 10 h. However, detection of apoptosis-related events at 72 h in this study was based on the period of maximal cell growth inhibition on the cell proliferation experiment. In addition, induction of apoptosis by nutritional components may not be as prompt as other specific apoptosis-inducing chemicals; hence, we chose to detect at 72 h. Future studies would need to determine the optimal time in a 24 h interval. Moreover, different cell lines respond differently to identical conditions, so the same chemical may induce

cell death in one cell line but not in another (Sundquist et al., 2006). Although the cell lines used in our experiment are of feline lymphoma disease, they are of different origins and characteristics, which may explain why lipotropes only reduced growth of F1B and not that of the FeLV-3281 cell line. FeLV-3281 is a suspension subgroup A feline leukemia virus, while F1B is an adherent lymphoma cell line of submandibular lymph node origin. Chromosomal instability enables tumor cells to acquire genetic alterations responsible for initiating and maintaining oncogenesis (Luke et al., 2003). DNA methylation has been shown to regulate chromosomal stability, p53 tumor suppressor gene activity, and inhibit lymphoma development (Luke et al., 2003; Eden et al., 2003). As methyl donors, lipotropes are involved in one-carbon metabolism, a process responsible for regulating the DNA methylation pathway and nucleic acid production (Mason, 2003). From these findings, we can postulate that lipotropes may alter DNA methylation status, which then induces genomic stability, leading to p53 tumor suppressor gene restoration, and in turn, inhibits lymphoma development and growth through apoptosis-related processes. Thus, an in-depth investigation of these processes is warranted. 5. Conclusion This study is the first demonstration of the effect of lipotropes on the growth of feline lymphoma cells. We showed that lipotropes reduce the growth of F1B lymphoma cells in association with enhanced expression of cell death-related proteins. Inhibition of apoptosis is a strategy employed by cells with genetic modifications to survive death-inducing agents. Hence, this data may serve as the basis for future studies aimed at establishing a correlation between dietary lipotropes, DNA and histone modifications (e.g., histone deacetylation), chromatin structure, and expressions of tumor suppressor p53 and lymphoma-related genes. This information may be particularly important given the emergence of histone deacetylase inhibitors in lymphoma treatment. Conflict of interest There were no conflicts of interest regarding this article. Acknowledgment This research was supported by the Morris Animal Foundation. References Cho, K., Mabasa, L., Fowler, A.W., Walsh, D.M., Park, C.S., 2010. Canola oil inhibits breast cancer cell growth in cultures and in vivo and acts synergistically with chemotherapeutic drugs. Lipids 45 (9), 777–784. Eden, A., Gaudet, F., Waghmare, A., Jaenisch, R., 2003. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 300, 455.

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