Fig 1.
In vitro supplementation of PCa cells with sarcosine.
(A) Viability (MTT) assay showing the trend in (Aa) PC-3 and (Ab) LNCaP cells growth under 24 h supplementation with sarcosine (0–10 μM). Values are expressed as the means ± standard deviations of six independent replicates (n = 6). (B) Micrographs of densities of (Ba) PC-3 cultivated in standard Ham’s F12 medium (left) and in Ham’s F12 medium enriched for 1 μM sarcosine (right) and (Bb) LNCaP cultivated in RPMI-1640 medium (left) and in RPMI-1640 medium enriched for 1 μM sarcosine (right). The length of scale bar is 500 μm.
Fig 2.
The murine PC-3 and LNCaP xenografts were treated with sarcosine i.p.
(A) Schematic depiction of experimental workflow beginning with the PCa cells (5×106) s.c. inoculation. (B) Average weight of mice determined after the termination of experiment. (C) Selected photographs of excised ectopic prostate tumors treated with sarcosine (left) and non-treated after termination of the experiment (right). (D) Average tumor weight at the endpoint of the experiment. Values are expressed as the means ± standard deviations of three independent replicates (n = 3). Vertical bars indicate standard error. Asterisks indicate significant differences (p < 0.05) compared to the untreated group. (E) H&E-stained tissue sections of ectopic prostate tumors after treatment with sarcosine (left) and non-treated (right). The length of scale bar is 100 μm.
Fig 3.
Effect of sarcosine treatment on the tumor tissue concentrations of sarcosine pathway-related amino acids.
(A) Schematic depiction showing the sarcosine pathway ongoing in mitochondrion. Red rectangles highlight the amino acids, whose tissue levels were increased due to sarcosine treatment. Dmg-Dimethylglycine, Gly-Glycine, Ser-Serine, DMGHD-Dimethylglycine dehydrogenase, SARDH-Sarcosine dehydrogenase, GNMT-Glycine-N-methyltransferase, GDC-Glycine decarboxylase, SHMT-Serine hydroxymethyltransferase, THF-Tetrahydrofolate, 10f-THF-10-Formyltetrahydrofolate, CH2-THF-Methylenetetrahydro-folate, FDH-Formyltetrahydrofolate dehydrogenase, FTS-Formyltetrahydrofolate synthase. Bar graphs showing the differences in tumor tissue Sar, Gly, Dmg and Ser levels in sarcosine treated and non-treated (B) PC-3 and (C) LNCaP mice. The levels of GNMT and SARDH were estimated in (D) PC-3 and (E) LNCaP mice by ELISA. Values are means ± standard deviations of three independent replicates (n = 3). Asterisks indicate significant differences (*, p < 0.05) or (**, p < 0.01) compared to the untreated group.
Table 1.
List of genes up-regulated in PC-3 xenografts in a response to sarcosine treatment (genes with the median fold ratio ≥ 1.5 are shown).
Table 2.
List of genes up-regulated in LNCaP xenografts in a response to sarcosine treatment (genes with the median fold ratio ≥ 1.5 are shown).
Fig 4.
Comparison of gene expression in PC-3 and LNCaP xenografts.
(A) Venn diagram showing the number of overlapping genes up-regulated after treatment of PC-3 and LNCaP cells with sarcosine. (B) SQ-RT-PCR validation of 10 selected genes up-regulated in murine PC-3 and LNCaP xenografts after sarcosine treatment (up-regulation ≥ 2.5). Expression of β-actin constituted as loading control. Lanes 1–3—RNA isolated from sarcosine treated mice, lane 4–6—RNA isolated from non-treated mice.
Table 3.
Percentage of genes up-regulated in ectopic prostate xenografts treated with sarcosine classified with respect to their biological functions.
Fig 5.
Interactome network showing the genes, which were up-regulated after sarcosine treatment and which are fundamental for apoptosis and cell cycle (PC-3 on the left, LNCaP on the right).
The red nodes highlight the genes, which were up-regulated and are involved in negative regulation of a cell death. The interior of the circle represents the structure of proteins. The color of the line provides evidence of the different interactions among proteins. A red line indicates the presence of fusion evidence; a green line, neighborhood evidence; a blue line, concurrence evidence; a purple line, experimental evidence; a light blue line, database evidence; a black line, coexpression evidence. The genes were analyzed using STRING software (version 10.0).
Fig 6.
KEGG PCa-specific metabolism diagram for PC-3 cells.
Gene expression shifts are projected as comparison of sarcosine treated and non-treated prostate tumors. The genes highlighted in red were found up-regulated after the sarcosine treatment in both PCa xenografts. The pathway map is species-specific (Homo sapiens).
Fig 7.
KEGG PCa-specific metabolism diagram for LNCaP cells.
Gene expression shifts are projected as comparison of sarcosine treated and non-treated prostate tumors. The genes highlighted in red were found up-regulated after the sarcosine treatment in both PCa xenografts. The pathway map is species-specific (Homo sapiens).
Fig 8.
Schematic depiction of cell cycle and illustration of genes, which induce progression of cell cycle, performed using the Reactome software.
Black frames indicate the genes, which were up-regulated after sarcosine treatment.