Fig 1.
Melatonin proliferation in PCa cell lines and RNA-seq data.
(A, B) The cell viability assay in PCa cells. (C) Melatonin treated transcriptome changes in Du145 cell lines in vitro, distribution of GO categories (Biological process, cellular component, and molecular functions) of top 30 terms in Du145 cell line. (D) Heatmap of global differentially expressed genes in Du145 cell lines treated with 0.5 mM melatonin: sample T1, T2, T3 represent the melatonin treated group, Sample C1, C2, C3 represent the control group. (E) KEGG enrichment for top 20 pathways in DU145 cell lines. (F) Differentially expressed genes in DU145 after administration with melatonin: up-regulation signalled by 97 genes (p<0.05) and 174 genes signalled down-regulation (p<0.05).
Table 1.
Primers used in real-time RT-PCR.
Table 2.
RNA oligonucleotide sequences.
Fig 2.
Validation of RNA-seq data using differentially expressed genes (DEGs): (A) DU145 and PC3 cell lines detected by qPCR (B) IL2Rβ, NGFR, IL6, IGBP3 genes explored in PCa cell lines were measured by Western blotting (C) Densitometry analysis of gene intensity was carried out using ImageJ software. Data were normalized with loading control GAPDH. Each bar represents the mean ± SD of the three independent experiments (D) Cell cycle analysis in PCa cells treated with melatonin. PC3 and DU145 PCa with 0.1% DMSO or with melatonin (0, 0.5mM, 0.8mM) for 48h, stained with PI, as indicated in the materials and methods, and then subjected to flow cytometry analysis (E) The bar graphs show the percentages of cells in the sub-G1 region and G0/G1, S, and G2/M phases. Data represents three independent experiments (F) Apoptotic cell determinations in PCa cells treated with melatonin (0, 0.5mM, 0.8mM) for 48h. Cancer cells were treated with 0.1% DMSO dissolved in culture medium or with melatonin diluted in 0.1% DMSO. After 48h cells were simultaneously stained with Alexa Fluor-488-Annexin V and propidium iodide, and analyzed by flow cytometry to determine apoptosis described in the materials and methods. One representative experiment of three performed. For each panel, the cytograms represent viable (Annexin V-negative/PI-negative), early apoptotic (Annexin V-positive/PI-negative), late apoptotic (Annexin V-positive/PI-positive) cells. (G) The bar graphs represent the percentage of early and late apoptotic cells described above.
Fig 3.
Melatonin suppresses PCa proliferation.
(A) DU145 cell line (B) PC3 cell line were seeded in 6-well plates, and a wound line was produced between the cells. Transfected cells were treated with melatonin. (C, D) Mobility rate DU145 and PC3 cell lines: The migration rate was plotted in a graph (n = 3, p*<0.05) (E) Transwell assay showing the effects of melatonin on DU145 and PC3 cell lines’ migration. Control and melatonin-treated cells were seeded in the transwell upper chamber for 24h. The migrated cells were fixed using 4% paraformaldehyde and subjected for imaging (100x). (F) The number of migrated cells was counted from 5 random places and plotted in a bar diagram. Data are represented as mean ± SD (n = 3, p*<0.05).
Fig 4.
Melatonin suppressed PCa cell migration by IL2Rβ.
(A) Knockdown of IL2Rβ inhibited PCa cell migration. DU145 cells were transiently transfected with either scrambled or SiIL2Rβ and wound healing assay was performed in the presence or absence of melatonin. (B) PC3 cells were also performed like before. The wound closure was quantified from the difference between wound arrest at the beginning and incubation period of experiments. (C, D) Bar diagrams were generated for the migration rate from three independent experiments (p*<0.005) on the PCa cell lines for 24h respectively (E) Knockdown of IL2Rβ inhibited PCa cell migration. Cells transfected with the scramble and SiIL2Rβ in the presence or absence of melatonin for 48h treatment were subjected for transwell and migration rates were calculated. (F) Data were normalized with loading control GAPDH. Each bar represents the mean ± SD of the three independent experiments (n = 3, p*<0.05).
Fig 5.
Inhibition of IL2Rβ by melatonin reduces prostate cancer cell proliferation.
(A) MTT cell viability assay; Transient transfection with siRNA (siIL2Rβ) of PCa (DU145 and PC3) cells (B) Cell proliferation assay; melatonin treated concentration (0; 0.5; 0.8mM).
Fig 6.
Melatonin-mediated IL2Rβ inhibits the NF-κB pathway: (A) PCa cells were transiently transfected with scrambled siRNA or empty vector and SiIL2Rβ followed by treated with or without melatonin for 48 hours. (B) Data were normalized with loading control GAPDH. Each bar represents the mean ± SD of the three independent experiments. (C) PCa cells were treated with melatonin for 48h and total collected protein was subjected to Immunoblot analysis for the effect of the melatonin on NF-κB pathway proteins. PCa cells were treated melatonin (0, 0.5, 0.8 mM). Indicated antibodies (IKBα, P- IKBα, p65, P-p65, P50) were used to observe their expression. (D) Densitometry analysis of gene intensity was carried out using ImageJ software. (E) PCa cancer cells were transient with scramble or SiIL2Rβ and later treated with 0.5 mM of melatonin for 48h. (F) Densitometry analysis of gene intensity was carried out using ImageJ software. All Data were normalized with loading control GAPDH and each bar represents the mean ± SD of the three independent experiments.