Anti-Tumor Effects of Vitamin B2, B6 and B9 in Promonocytic Lymphoma Cells

Abstract

Chronic inflammation can lead to tumour initiation and progression. Vitamin B complex has the ability to regulate the immune response and, therefore, inflammation but many of the mechanistic and molecular processes involved in this regulation are still not fully understood. This study sought to determine some of these processes by studying the effects of vitamin B2 (riboflavin) B6 (pyridoxine) and B9 (folic acid) on un-differentiated pro-monocytic lymphoma cells in regard to their ability to alter the proliferation, migration, apoptosis, cytokines and expression levels of programmed death ligand 1. We show that vitamin B2, B6 and B9, on pro-monocytic lymphoma cells exerted an anti-tumorigenic effect. This data could form the basis for future studies in using vitamin B supplementation to reduce cancer cell growth in vivo.

Keywords: U937 cell line; folate; pro-monocytes; pyridoxine; riboflavin; vitamin B complex; vitamin B2; vitamin B6; vitamin B9.

Figure 1

(A,B), Effect of vitamin B2 (riboflavin) (C,D), vitamin B6 (pyridoxine) (E,F), vitamin B9 (folic acid) (G,H), NaOH control on U937 cell proliferation. Cells were incubated with increasing doses of vitamin B for 6 days in 96 well U bottom plates and analyzed by MTT assay. Absorbance readings were taken at 540 nm to assess for cellular proliferation compared to control well (0 μg/mL). Significance was established at p, 0.05, two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test and marked with asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Cells were viewed under an IX81 Olympus microscope at 4x magnification and photos taken at each concentration and control NaOH on day 6 of culture.

Figure 2

Annexin V-FITC/propridium iodide (PI) staining of undifferentiated U937 cells incubated with vitamin B. 1 × 10⁶ of U937 cells treated with 0.25 μg/mL of B2 and 250 μg/mL of vitamin B6 and B9 for 72 h were used for analysis. Resuspended cells were incubated with Annexin V-FITC at 1:1000 for 15 min in the dark. PI at 0.5 μg/mL was used as a counterstain to differentiate necrotic/dead cells from apoptotic cells. Shown in the figure are (A) controls, (B) vitamin B samples.

Figure 3

Effect on cell migration of pro-monocytic cells in the presence of (A) vitamin B2 (riboflavin), (B) vitamin B6 (pyridoxine) and (C) vitamin B9 (folic acid) using Boyden chamber assay. Data presented as mean +/− standard error of the mean of duplicate wells. Excel and Prism excel (Graph Pad Software, La Jolla, CA, USA) were used to aid in the statistical analysis using students t-Test and * p < 0.05 was considered significant.

Figure 4

Expression of PD-L1 as assessed by confocal imaging on U937 cells in the presence of (A) vitamin B2 at 0.125 μg/mL and vitamin B2 vehicle control (NaOH) and untreated control. (B) Vitamin B6 at 125 μg/mL and untreated control, and, (C) vitamin B9 at 125 μg/mL and untreated control. (* p < 0.05, *** p < 0.001, **** p < 0.0001

Figure 5

U937 cells were treated with (A) 0.125 μg/mL of vitamin B2 and NaOH vehicle control, (B) 125 μg/mL and 250 μg/mL of vitamin B6 and (C) 125 μg/mL of vitamin B9 and NaOH vehicle control for 3 days. Media and fresh vitamin B were replaced and cultured for a further 3 days. Supernatants were collected and cytokines secretion analyzed by bioplex. Significance in relation to untreated control is indicated by Asterisks (* p < 0.05, ** p < 0.01, **** p < 0.0001). Bioplex data for IL-1β, IL-2, Il-4, IL-6, Il-8, Il-10, IFNγ, TNFα, and GM-CSF are shown.