Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

RETRACTED: Molecular mechanism of inhibitory effects of melatonin on prostate cancer cell proliferation, migration and invasion

  • Altannavch Nyamsambuu,

    Roles Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Visualization, Writing – original draft, Writing – review & editing

    Affiliation Department of Biochemistry and Molecular Biology, School of Life Sciences, Central South University, Changsha, Hunan, China

    ⨯
  • Md. Asaduzzaman Khan,

    Roles Project administration, Writing – review & editing

    Affiliations Department of Biochemistry and Molecular Biology, School of Life Sciences, Central South University, Changsha, Hunan, China, Key Laboratory of Epigenetics and Oncology, The Research Center for Preclinical Medicine, Southwest Medical University, Luzhou, Sichuan, China

    ⨯
  • Xi Zhou,

    Roles Conceptualization, Data curation, Formal analysis, Methodology

    Affiliation Department of Biochemistry and Molecular Biology, School of Life Sciences, Central South University, Changsha, Hunan, China

    ⨯
  • Han-Chun Chen

    Roles Conceptualization, Funding acquisition, Investigation, Project administration, Supervision, Writing – review & editing

    chenhanchun@csu.edu.cn

    Affiliation Department of Biochemistry and Molecular Biology, School of Life Sciences, Central South University, Changsha, Hunan, China

    ⨯

Retraction

After this article [1] was published, concerns were raised about Figs 2-4 and 6, and the underlying data provided in the Supporting Information. Specifically,

  • The underlying image data published in the Supporting Information of this article are incomplete and some images do not appear to match the corresponding panels presented in Figs 2B, 6A, 6C, or 6E. Upon editorial follow-up, first author AN indicated that these differences in appearance arose owing to loss of quality in the stored raw data files.
  • In the p50 panel of Fig 6E there appears to be a vertical discontinuity between lanes 4 and 5. Upon editorial follow-up, first author AN stated this arose owing to a natural boundary between adjacent areas of the same membrane, citing uneven exposure and scanner shading during image acquisition. They stated that all lanes were run on the same gel and belong to the same continuous membrane, and that no lanes were added, removed, or rearranged. In the absence of the raw data, this issue could not be fully resolved.
  • The following microscopy images in this article [1] appear to fully or partially overlap:
    • The Fig 3A 0.5 mM and 0.8 mM 0 h panels
    • The Fig 3A 0 mM, 0.5 mM, and 0.8 mM 24 h panels
    • The Fig 3B 0.5 mM 24 h panel and the Fig 4B Scramble + melatonin 24 h panel
    • The Fig 3B 0.5 mM 48 h panel and the Fig 4B Scramble + melatonin 48 h panel
    • The Fig 3E 0.5 mM Du145 panel and the Fig 4E Scramble (-) Du145 and SiIL2RB (-) Du145 panels
    • The Fig 3E 0.8 mM Du145 panel, and the Fig 4E SiIL2RB (+) Du145 and SiIL2RB (+) PC3 panels
    • The Fig 3E 0mM PC3 panel and the Fig 4E Scramble(-) PC3 panel
  • Editorial assessment of the individual-level data provided in the S1 File of [1] raised concerns about the normalization procedure of these data. In addition, PLOS identified discrepancies between the data provided in the Supporting Information files and their corresponding panels in the published figures.

First author AN acknowledged the panel overlaps observed in Figs 3 and 4 and provided replacement data and underlying microscopy image files, along with a quantitative analysis file for the western blot quantification data. Editorial assessment of the underlying and replacement data identified additional concerns about the reliability of the image data.

In light of the above concerns, which question the reliability and integrity of reported results and conclusions, the PLOS One Editors retract this article.

AN and MAK did not agree with the retraction. XZ and HCC either did not respond directly or could not be reached.

12 Feb 2026: The PLOS One Editors (2026) Retraction: Molecular mechanism of inhibitory effects of melatonin on prostate cancer cell proliferation, migration and invasion. PLOS ONE 21(2): e0342720. https://doi.org/10.1371/journal.pone.0342720 View retraction

Abstract

The increasing incidence of prostate cancer (PCa) indicates an urgent need for the development of new effective drug therapy. There are limited options to treat the PCa, this study tried to determine a new therapy option for this acute cancer. Androgen-independent PCa cell lines PC3 and DU145 were treated with different melatonin concentrations (0.1~3.5 mM) for 1~3 days and assessed cell migration, cell invasion, cycle arrest in G0/G1 phase as well as apoptosis. We utilized RNA-seq technology to analyze the transcriptional misregulation pathways in DU145 prostate cancer cell line with melatonin (0.5 mM) treatment. Data revealed 20031 genes were up and down-regulated, there were 271 genes that differentially expressed: 97 up-regulated (P<0.05) and 174 down-regulated (P<0.05) genes. Furthermore, RNA-seq results manifested that the melatonin treatment led to a significant increase in the expression levels of HPGD, IL2Rβ, NGFR, however, IGFBP3 and IL6 (P <0.05) had decreased expression levels. The immunoblot assay revealed the expression of IL2Rβ and NGFR genes was up-regulated, qPCR confirmed the gene expression of HPGD and IL2RB were also up-regulated in Du145 cells. Consequently, we probed mechanisms that generate kinetic patterns of NF-κB-dependent gene expression in PCa cells responding to a NF-κB-activation, the significant results were indicated by the inhibition of the NF-kB pathway via IL2Rβ actions. Based on our investigation, it could be concluded that melatonin is a chemotherapeutic molecule against PCa and provides a new idea for clinical therapy of PCa.

Citation: Nyamsambuu A, Khan MA, Zhou X, Chen H-C (2022) RETRACTED: Molecular mechanism of inhibitory effects of melatonin on prostate cancer cell proliferation, migration and invasion. PLoS ONE 17(1): e0261341. https://doi.org/10.1371/journal.pone.0261341

Editor: Antimo Migliaccio, Universita degli Studi della Campania Luigi Vanvitelli, ITALY

Received: July 5, 2021; Accepted: November 30, 2021; Published: January 21, 2022

Copyright: © 2022 Nyamsambuu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the Supporting information files.

Funding: The work was supported by the Department of Biochemistry and Molecular Biology, School of Life Sciences, Central South University. And solely funded by the National Basic Research Program of China (Grant No. 2011CB910700-704), the funding body helped to interpret the data and guided in preparing the manuscript.

Competing interests: The authors declare that they have no competing interests.

Introduction

The hallmarks of cancer assist to understand the cancer, one of the hallmarks of cancer is to avoid the cell mortality [1, 2]. The developed countries indicate it as one of the major public health problems among their elderly people so, there is a high demand for new clinical therapies. The incidence of PCa is directly proportional to the demographic changes of population [3, 4]. Prostate cancer (PCa) attains the fourth position being as common cancer, second in male population and contributes 3.8% to deaths due to cancer malignancy [5, 6]. At the time of diagnosis it is noted that the growing age >65 years is associated with the incidence and mortality of PCa globally, Of note, the incidence rates are higher for African-American men relative to white men, with 158.3 new cases diagnosed per 100,000 men and their mortality is around twice as high as for white men [7]. However, it is predicted that more than two million new cases will occur by 2040, with a minor increase difference in mortality rate of 1.05% [8], Social, environmental, and genetic variables have been speculated as explanation for this difference. Rarely the PCa is androgen-dependent, to treat metastatic androgen-deprivation therapy together with radiotherapy and chemotherapy is used. On the other hand, primary and acquired resistance to androgen-deprivation therapy is also frequent. Androgen deprivation therapy is less effective alone perhaps than the androgen signal block therapy with beta-blockers combined with abiraterone and enzalutamide drugs [9]. The melatonin was first time reported as a free radical scavenger and potential antioxidant in 1993 [10, 11] and proven to be twice as vitamin E an effective lipophilic antioxidant that occurs with mitochondrial fluid and exceeds the melatonin plasma concentration [12–14]. Melatonin (N-acetyl-5-methoxytryptamine, Fig 1) is an indolic compound secreted primarily by the pineal gland of human and mammals in response to darkness [15, 16]. On the one hand, melatonin is effective in suppressing CRC development and progression [17]. Natural melatonin has been reported in foods such as tart cherries [18], bananas, grapes, rice, cereals, herbs, plums [19], olive oil, wine, and beer [20].

thumbnail
Download:
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).

https://doi.org/10.1371/journal.pone.0261341.g001

In 1958, Aron Lerner and colleagues first isolated and characterized melatonin (N-acetyl-5-methoxytryptamine) having molecular formula/mass C13H16N2O2 / 232.121178 g/mol [21] from bovine pineal gland and intestinal tract [22], and since, has been found across kingdoms including bacteria, fungi [23], and plants [24]. The pineal gland produces melatonin is an indole-amine that commands circadian rhythms and plays a role as a cytokine, biological response modifier, and neuromodulator, however various actions are mediated by G-protein coupled melatonin receptors in cellular membranes, and indole seems to be involved in interactions with orphan nuclear receptors calmodulin in the cytosol [25]. Epidemiological and clinical studies showed the melatonin as a potential prostate tumor oncostatin hormone, neuro-hormone, directs circadian rhythms to environmental factors and simultaneously involved in diverse physiological processes, such as the regulation of blood pressure, body temperature, oncogenesis, and immune function [26]. It is also helpful in promoting apoptosis, anti-proliferation, pro-oxidation, metabolic shifting, inhibiting neovasculogenesis, controlling inflammation, and restoration of chemosensitivity [27]. MT1 and MT2 are high-affinity membrane receptors through which melatonin exert physiological effects [28]. To the best of our literature review, previously it has not been shown the inhibitory role of melatonin in PCa. To exploit this deep insight present study investigates the therapeutic activity of melatonin, on PCa cells by treating them in small doses of melatonin and its effects were analyzed by detecting differentially expressed genes, antiproliferative, migration and invasion activities in PCa cells.

Materials and methods

Ethics statement

This research work was approved by the ethical committee of School of Life Science, Central South University (Changsha, Hunan 410013, China). No human or animal subject was used in this study.

Cell culture and reagents

Human Prostate cancer (PCa) cell lines: PC3 and DU145 were obtained from ATCC: The global Bioresource center (https://www.atcc.org). These cell lines were maintained in RPMI 1640 (Gibco, MA, USA) medium supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, MA, USA), 100μl/ml streptomycin at 37°C, and 5% CO2 humid atmosphere. Melatonin (MLT) was acquired from Sigma-Aldrich China and the stock solution of melatonin (99.5%) was prepped in DMSO (Sigma Chemical Co., St. Louis, MO, USA). For further use, it is diluted to different levels of concentrations during cell culture experiments.

Cell viability assay

Prostate cancer cells (PC3 and DU145) were seeded in 96-well plates (at a ratio of 1×104cells/well with 200 μL of RPMI 1640 medium) and incubated at 37°C with a 5% CO2 humid atmosphere for 24h. Cells were treated with different diluted melatonin doses (0; 0.5; 1.0; 1.5; 2.0; 2.5; 3.0; 3.5mM) always freshly prepared from stock (99.5% DMSO) for 24, 48 and 72 hours. The cell viability and proliferation were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma Aldrich, USA), after incubation time 150μl (0.5mg/ml) of MTT reagent was served to each well plate at 37°C, 5% humid atmosphere [29]. This action revealed formation of purple formazan crystal in metabolically reduced active cells, subsequently at a wavelength of 490nm the absorbance of cell was assessed for each cell group of well plate (Fig 1). The experiments were performed in quadruplicate with three times revision.

RNA sequencing and cluster analysis of DU145

The DU145 cancer cells were divided into 6 groups, of which three were treated with melatonin (0.5mM) in complete medium added with serum for 48h while the other three cell groups were kept as control. After 48h of incubation, cells were collected and lysed by triazole solution (Carlsbad CA 92008, USA). Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s protocol. Transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd. (Shanghai, China). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number ≥7 were subjected to the subsequent analysis [30]. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions then these libraries were sequenced on the Illumina sequencing platform (Illumina HiSeq X Ten) and 125bp to 150bp paired-end reads were generated.

Differentially expressed genes selection and functional analysis / Bioinformatic analysis for DEGs/

Differentially expressed genes (DEGs) were identified performing a t-test. Genes with P-value ≤0.05, fold change <0.05 or >2, were set as threshold significant for the gene’s differential expression and we also applied Gene Ontology (GO) and KEGG pathway analysis to explore the differentially expressed genes as well as transcript level.

Gene-level quantification: FPKM [30] value of each gene was calculated using cufflinks [31], and the read counts of each gene were obtained through hiseq-count [32]. DEGs were identified using the DESeq [33] (2012) R package functions estimateSizeFactors and nbinomTest.

Transcript-level quantification: Transcript-level quantification, FPKM [34] and reads count value of each transcriptome sequencing and analysis were performed by OE Biotech Co., Ltd. (Shanghai, China) [35] and eXpress (version1.0) [36]. DEGs were identified using the DESeq [33] (2012) functions estimateSizeFactors and nbinomTest.

Hierarchical cluster analysis of DEGs was performed to explore the gene expression and transcripts expression patterns. GO enrichment and KEGG [37] pathway enrichment analysis of DEGs was performed using R based on the hypergeometric distribution respectively.

Cell migration assay

PCa DU145 and PC3 were digested with 1ml (0.25%) trypsin at a cell density of 5.0 to 10×104 cells/mL and mixed with 2mL of completed cell suspensions containing 10% FBS incubated at 37°C, 5% CO2. When the growth of the cells reached at a confluence of 90% in each well/plate then the cells were used to prepare three uniforms equidistantly cuts within 6-well plates using 200μl tips and incubated at 37°C, 5% CO2 with saturated humidity for 24, 48, 72 hours, and photographed by selecting 3 fixed points in each plate using a 10x optical microscope, and measures were taken to avoid cell contamination such as sterilization.

Cell invasion assay

The cells were suspended and adjusted to a cell density of 5.0 to 10×104 cells/mL in a 6-well plate, incubated at 37°C, 5% humidity under observation. The cells were treated with melatonin (0.5 mM and 0.8 mM). After 48h cells were digested with 1ml of 0.25% trypsin, and 600ml of medium containing 10% FBS was added to the lower chamber of the transwell plate, and 100μl of the prepared cell suspension was added to the upper chamber and shaken slightly to avoid air bubbles so that the lower chamber medium evenly contacts the bottom of the upper chamber by even distribution and incubated for 24h at 37°C, in 5% CO2 saturated humidity. After that the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 minutes. The fixative was removed, by washing twice with PBS again, and then stained with 0.1% crystal violet (CV) dye for 20 minutes. The cells in the upper chamber were wiped with a wet cotton swab gently, then the chamber was dried approximately for 1 day at room temperature and then the Boyden chamber was placed under the microscope, photographs were captured at 10x randomly. An addition, we used a modified Boyden chamber assay to evaluate the invasiveness of PC3 cancer cells following siIL2RB knockdown (S1 Fig in S1 File).

Isolation of total RNA and quantitative real-time PCR

Total RNA was extracted from cancer cell samples using an RNA isolator (Nanjing Vazyme BioTech Co., Ltd, China) and was immediately reverse-transcribed using the Prime Script RT Reagent Kit with cDNA Eraser (Nanjing Vazyme Biotech Co., Ltd, China). The mRNA Levels of HPGD, IL6, IGBF3, IL2RB, NGFR genes as well as GAPDH control gene were investigated by qPCR using Bio-Rad CFX96 Real-time System (Bio-Legend Biotech Co., Ltd, China). Reaction mixtures consisted of 16 μl of ChamQ Universal SYBR qPCR master mix (Nanjing Vazyme Biotech Co., Ltd, China), 1μmol/L forward and reverse primers (Table 1: Primers used in real-time RT-PCR), 1μl cDNA, 3 μl ddH2O, further ddH2O added up to 10μl to complete the total volume of 16μl in every tube. The thermocycler was set to an initial temperature of 95°C for 3 minutes, followed by 40 cycles of 65°C for 5 seconds, 95°C for 10 seconds, and 55°C for 30 seconds. A melting curve was obtained from 65°C to 95°C, increasing in increments of 0.5°C every 5 seconds. Expression levels of target genes were calculated as relative values using 2−ΔΔCT method. The primers were synthesized (Table 1: Primers used in real time-PCR) by Sangon Biotech, Shanghai, China.

thumbnail
Download:
Table 1. Primers used in real-time RT-PCR.

https://doi.org/10.1371/journal.pone.0261341.t001

Cell cycle analysis by flow cytometry

Cell cycle phase (G0/G1, S, and G2/M) distribution was analyzed by flow cytometry based on DNA content. PC3 and DU145 cells were seeded in 60-mm plates for 24h and then treated with melatonin (0.5mM and 0.8mM). After 48h of treatment cells were collected, washed with PBS three times, and stained for 30 min with 1 mL of DNA-staining solution in Triton X-100 (Sigma-Aldrich, Milan, Italy), 5 μg/mL RNAse A (type IIIA, Sigma-Aldrich), and 1 μg/mL PI (propidium iodide) (Biotechnology Co., Ltd, Shanghai, China) at 37°C. For staining with PI, cells were kept in dark at room temperature for 30 min and then washed with chilled PBS, centrifuged for 5 minutes, at 3500 rpm, 4°C. The cell samples were transferred to glass tubes (1.5 mL Falcon tubes) and by keeping on ice the cellular DNA content was analyzed through Cyto-Flex (Youcheng Co., Ltd, Changsha, China).

Detection of apoptosis by annexin V-FITC/propidium iodide assay

The PCa cells PC3 and DU145 were seeded in 60-mm plates and incubated at 37°C with a 5% CO2 humidified atmosphere. Left to grow for 24 and 48 h later, to reach maximum confluency, later the cells were treated with melatonin (0.5 mM and 0.8 mM) freshly prepped (99.5% DMSO). After 24h, and 48h MLT (melatonin) treatment cells were stained with 1×Binding Buffer conjugated Annexin V-FITC and PI (Absin A biochemical company, China) simultaneously, using the Apoptosis Assay kit (Absin, A Biochemical Company, China), according to the manufacturer’s instructions. Samples were analyzed by FSC/SSC flow cytometer (Becton Dickinson). In each analysis, 10000 events were recorded and the percentage of apoptotic cells was estimated through the Modfit (Becton Dickinson). The simultaneous staining of cells with Annexin-V and PI allowed the resolution of viable cells, early apoptotic cells, and late apoptotic cells (Annexin V-positive/PI-positive), respectively.

Study of NF-κB signaling pathway by immunoblot analysis

DU145 and PC3 cells were cultured in 6 well plates at a density of 5×105 cells/mL and incubated at 37°C with 5% CO2 humid atmosphere, harvested for 24h, later treated with melatonin (0.5 mM and 0.8 mM) for 48h and then washed with pre-cooled PBS (phosphate-buffered saline) 3 times, lysate RIPA and PMSF were mixed at a ratio (1:100) on the ice after 30 minutes collected by centrifugation at 12000 rpm for 14 min. Equivalent amounts of proteins were analyzed by BCA protein quantification kit (Nanjing Vazyme BioTech Co., Ltd, China), and then SDS–polyacrylamide gel (15%) was run with electrophoresis for approximately 1hour 30 minutes, 24-30W of voltage. After electrophoretic separation, the proteins were transferred onto Immobilon-PSQ transfer membrane (Merck KGaA, Darmstadt, Germany) and incubated for 2h in blocking buffer solution (5% milk in TBST), filters were incubated for whole night with the appropriate antibodies: Anti-NGFR antibody (ratio 1:500; D261027), Anti-IL2Rβ (ratio 1:330; 13602-1-AP) (IBBI, Changsha, China); Anti-IGFBP3 Rabbit pAb (1:1000; WL01195), Anti-IL6 Rabbit pAb (1:1500; WL02715) (Wanleibo, Changsha, China) as well as HPGD Mouse Monoclonal antibody (1:5000; 66798-1-lg). Proteins were visualized after incubation in secondary antibody HRP goat anti-rabbit LgG (ratio 1:50000; D110058) as well as HRP mouse anti-mouse LgG (H+L) (1:4000; D20691) using enhanced chemiluminescence (ECL) for detection (GE Healthcare). Densitometry was performed on scanned immunoblot images using the SageCapture version 2.17.12.170316) (Sagecreation Co., Ltd, Beijing, China).

Small interfering RNA

RNA oligonucleotides for human IL2Rβ (Gene Pharma Co., Ltd, Shanghai, China) synthesized (Table 2: RNA oligonucleotide sequences).

thumbnail
Download:
Table 2. RNA oligonucleotide sequences.

https://doi.org/10.1371/journal.pone.0261341.t002

The well-grown cells were selected one day before the transfection and inoculated into a six-well culture plate containing an antibiotic-free medium to make the cells proliferative to 80% during transfection. Lipofectamine 2000 (3μL) was diluted in 250 μL serum and antibiotic with RPMI 1640 medium, incubated for 5 minutes at room temperature. 100 pmol of siRNA was diluted in 250 μL serum and antibiotic with RPMI 1640 medium gently mixed, and then both solutions were mixed gently with the pipette. Note that the diluted Lipofectamine 2000 was slowly added to the siRNA solution and the solution was let stand at room temperature for 20 minutes. Using a pipette, 500 μL of the above mixture was added to each well and shaked gently, and incubated at 37°C for 6 hours. The medium mixture in the six-well plate was removed by pipette, and 2 mL of the complete RPMI 1640 medium containing 10% FBS with antibodies was added to continue the culture, and gene expression was examined after 48 h. siRNAs targeting IL2Rβ were purchased from Gene Pharma (Shanghai, China). DU145 and PC3 cells were transfected with siRNAs by Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Knockdown efficiency was determined by Western blot analysis. Three independent transfection experiments were performed.

Statistical analysis

Data are mean standard deviation of at least three independent experiments and were evaluated by Student t-test. The statistical significant difference was considered as p-value < 0.01.

Results and discussion

Analysis for suppression of cell proliferation in PCa cells

Melatonin selectively inhibits PCa cell proliferation.

Earlier study have shown that melatonin has very high antioxidant activity [11], however, its biological functions in PCa cells are still unclear. The present study examined two PCa cell lines DU145 and PC3 (androgen-independent) treated with a range of melatonin concentrations (0.1–3.5mM) for 24, 48, and 72h. Both cell lines revealed sensitivity to melatonin in millimolar range in the dose and time-dependent manner investigated by cell proliferation assay, the IC50 value for DU145 is 1.1mM, and for PC3 = 1.0mM treated for 48h (Fig 1A and 1B). The DU145 and PC3 cells treated with low doses of melatonin 0.5mM and 0.8mM respectively for 48h demonstrated the morphological differences and gradually widened their morphology (S1 Fig in S1 File).

GO and KEGG pathway analysis of differentially expressed genes in DU145 cell line.

Based on the significant differences top 30 GO terms were obtained and genes were grouped into three categories: biological process, cellular component, and molecular function (Fig 1D). KEGG enrichment analysis indicated top 20 signaling pathways predicted to involve the targeted genes. GO enrichment and KEGG [37] pathway enrichment analysis of DEGs was performed using R based on the hypergeometric distribution. The pathway relevant to cancer signaling includes transcriptional misregulation in cancer (Fig 1F).

Melatonin changes transcriptome sequencing in DU145 cell lines.

After 48h of treatment with melatonin (0.5mM), we performed RNA-seq analysis to elucidate the function of melatonin in controlling genome-wide gene expression. Since melatonin can act at fewer concentrations as a hormone and at high concentrations as an antioxidant [38], to analyze the mechanism of PCa suppression with melatonin, RNA-seq was performed to detect the global gene expression profiles. We used screen differential expression levels of mRNAs following the criteria: P≤0.05; log2FC >0.58 and detected differential gene expression levels by RNA-seq to confirm their expression levels. RNA-seq data revealed 20031 genes were up and down-regulated in melatonin (0.5mM) treated cells (Fig 1C). Especially, we determined top 271 total genes with differential expression (P<0.05): 97 up-regulated (P<0.05), and 174 down-regulated genes (P<0.05) (Fig 1E).

Effect of melatonin on gene expression of transcriptional misregulation in cancer

Effect of melatonin on gene expression in PCa cells.

To determine the reliability of the RNA-seq data, we selected 5 genes (HPGD, NGFR, IL2Rβ, IGFBP3, and IL6) from RNA-seq data for transcriptional misregulation in cancer. Our RNA-seq data analysis showed HPGD, NGFR, and IL2Rβ were upregulated and IL6, IGFBP3 were downregulated for gene expression in DU145 cells. To examine their expression levels, DU145 and PC3 cells were treated with melatonin (0 mM, 0.5 mM, and 0.8 mM) and tested with the Bio-Rad CFX96 Real-time System. We found that qPCR results were not completely consistent with RNA-seq data analysis, but we have identified that HPGD and IL2Rβ as up-regulated genes consistent with RNA- seq data, while NGFR, IL6, and IGFBP3 were downregulated in DU145 cancer cells (Fig 2A). On the other hand, in PC3 cells the IL2Rβ, IL6, and IGFBP3 were upregulated, and HPGD and NGFR genes were downregulated (Fig 2A). These differences may be because of various biological signaling networks or physiological changes of the cells. In both cell lines, IL2Rβ was up-regulated consistent with RNA- seq data that would be of interest in future investigations of PCa cells.

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0261341.g002

Melatonin activates NF-κB pathway in PCa cells.

Several genes have been predicted to function as novel upstream or downstream NF-κB regulatory targets in PCa. Many anticancer compounds activate the NF-κB signaling pathway [39–41] and ultimately cause apoptosis in PCa cells [40]. In our study, we have shown that melatonin could inhibit the proliferation of human PCa cells and activates the NF-κB pathway. Especially, transcriptional misregulation in cancer pathway (S2 Fig in S1 File) included 4 genes (IL2Rβ, NGFR, IL6, IGBP3) differentially expressed in PCa and extremely functionally related to NF-κB. To investigate the effect of melatonin on protein levels, activators, and inhibitors of NF-κB, both DU145 and PC3 cell lines were treated with 0mM, 0.5mM, and 0.8mM. The effect of melatonin on the protein expression levels of IL2Rβ, we assessed interestingly decreased in DU145 PCa cells (Fig 2B). The relative value of proteins plotted against increasing concentrations of melatonin in DU145 and PC3 cell lines, protein levels are expressed as relative ratios to GAPDH levels (n = 3) (Fig 2C).

Melatonin induced cell cycle arrest in G0/G1 phase.

Growth inhibition by melatonin altered the percentage of cells in G0-G1 and G2/M phases indicating cell cycle arrest in the G2/M phase [42]. Through the flow cytometry, the cell cycle was assessed in PCa cells treated with melatonin to examine the possible causal relationship between cell proliferation and cell cycle arrest induced. The results indicated that melatonin increased cell number in G0/G1 phase, and the percentage of the arrested cells decreased with 0.5 mM conc., increased with 0.8 mM conc: after 48h of melatonin treatment in DU145 cell lines. On the other hand, In PC3 the percentage of the arrested cells slowly increased in G0/G1 phase but as decrease in DU145 cells (Fig 2D). The bar graphs show the percentages of cells in the sub-G1 region and G1, S, and G2/M phases. Data are representative of three independent experiments (Fig 2E). Apoptosis is a well-known mechanism of programmed cell death other than normal cells [43], and is triggered by chemotherapeutic substances [44], adjacent cells, and decreased local inflammation [45] (Fig 2F) The gradient concentration of melatonin increases apoptosis in both cancer cells, while cell necrosis was rarely seen. The proportion of apoptotic cells increased with increased drug concentrations. Similar results were found in both of the cancer cells. The bar graphs represent the percentage of early and late apoptotic cells described above (n = 3, p<0.05) (Fig 2G).

The potential therapeutic effect of melatonin on human PCa by inhibition of invasion and migration of prostate cancer cells

Melatonin suppresses cell proliferation in prostate cancer.

In our study, we first performed a wound-healing assay to examine PCa cells mobility. With melatonin treatment (0.5 and 0.8mM) for 24h and 48h, the cells showed a significant delay in wound closure compared to controls in DU145 cells (Fig 3A) and PC3 cells (Fig 3B). The mobility rate was calculated using ImageJ software and plotted in a graph (n = 3, p<0.05) (Fig 3C and 3D) respectively. Cell migration or mobility is related to treated melatonin concentration and time. In the case of DU145, 0.5mM treated cells showed higher mobility than 0.8mM treated cells, while in PC3 cells the cell mobility was found nearly equal in both treated concentrations. Subsequently, cell migration was examined by transwell migration assay. The results appeared similar to the wound-healing assay showing a decreased number of migrated cells in the lower chamber in compliance with melatonin treatment (Fig 3E). The transwell migration rate was calculated using ImageJ software and plotted in a graph (n = 3, p<0.05) (Fig 3F).

thumbnail
Download:
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).

https://doi.org/10.1371/journal.pone.0261341.g003

Melatonin suppressed PCa cell migration by IL2Rβ.

Afterward, we performed gene knockdown to assess the possible role of IL2Rβ in mediating melatonin function. Treatment of PCa with 0.5mM and 0.8mM melatonin concentration knockdowns the IL2Rβ levels that eventually inhibited PCa cell growth and proliferation (Fig 4A and 4B). The knockdown of IL2Rβ reduced the rate of wound closure and migration rate in PCa cells. The mobility rate was calculated using ImageJ software and plotted in a graph (n = 3, p<0.05) (Fig 4C and 4D). The knockdown of IL2Rβ reduced the rate of wound closure and migration rate of DU145 cancer cells (Fig 4E). The transwell migration rate was calculated using ImageJ software and plotted in a graph (n = 3, p<0.05) (Fig 4F).

thumbnail
Download:
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).

https://doi.org/10.1371/journal.pone.0261341.g004

Molecular mechanism underlying melatonin function

IL2Rβ acts as a mediator of melatonin mediated inhibition of PCa cell lines proliferation.

Transient transfection of siIL2Rβ resulted in a significant knockdown of IL2Rβ protein both in the presence or absence of melatonin in the cell line tested (Fig 5A). Besides, the silencing of IL2Rβ significantly inhibited PCa cell growth and proliferation when treated with melatonin 0.5 and 0.8 mM (Fig 5B).

thumbnail
Download:
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).

https://doi.org/10.1371/journal.pone.0261341.g005

Melatonin mediated degradation of IL2Rβ inhibits NF-κB pathway.

Aberrant regulation of the NF-κB pathway is believed to be a major event contributing to the malignant transformation and progression of PCa [46–48]. Studies have shown that NF-κB plays an important role in PCa growth, survival, angiogenesis, tumor genesis, and metastatic progression [46]. Abundant data support a key role for the NF-κB signaling pathway in controlling the initiation and progression of human cancer [49, 50]. In the current study, the biological effect of melatonin on the loss of function of IL2Rβ was assessed through siRNA. Significant results indicated the specific SiIL2Rβ (RNA oligonucleotides for human IL2Rβ (Gene Pharma Co., Ltd, Shanghai, China)) in transiently transfected cells knockdown IL2Rβ protein in both cell lines (Fig 6A). Data were normalized with loading control GAPDH. Each bar represents the mean ± SD of the three independent experiments (Fig 6B).

thumbnail
Download:
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.

https://doi.org/10.1371/journal.pone.0261341.g006

We aimed to evaluate the modulatory effect of melatonin on this pathway in both cells. We found that treatment with melatonin decreased the phosphorylation of IkBα and P-65. Besides, the IkBα and p65 were increased at protein levels in independent cancer cells. Furthermore, the level of P50 was decreased in DU145 cell lines, on the contrary, to increase the level in PC3 cancer cells (Fig 6C). Data were normalized with loading control GAPDH. Each bar represents the mean ± SD of the three independent experiments (Fig 6D).

To examine whether IL2Rβ could be a possible mediator of the melatonin affecting the NF-κB signaling pathway, we utilized the silencing of IL2Rβ. The knockdown of IL2Rβ triggered decreased phosphorylation of IkBα, p65 and increased the nuclear translocation of IkBα and p65 in both cancer cells. On the other hand, the P50 level increased in DU145 and, decreased in PC3 cancer cells compared to scrambled siRNA-transfected cells (Fig 6E). GAPDH used as loading control. Each bar represents the mean ± SD of the three independent experiments (Fig 6F). Furthermore, the effect of IL2Rβ silencing was enhanced remarkably in the presence of melatonin. Altogether, the derived results allude that IL2Rβ performs a key role in melatonin-mediated suppression of the NF-κB pathway.

Finally, Co-localization of IL2RB proteins was visualized by immunofluorescence assay. We used a modified Boyden chamber assay to evaluate the invasiveness of PC3 cancer cells following siIL2RB knockdown. Nuclei were counter-stained with DAPI (blue color) and imaged were captures using a fluorescence microscope with 100x magnification (S3 Fig in S1 File).

Discussion

Chemotherapy plays an important role in the treatment of PCa. In recent years, new chemotherapeutic drugs have been developed focusing mostly on targeting the androgen receptors. However, the development of other chemotherapeutic approaches for the treatment of PCa has been limited by problems associated with specificity and high systemic toxicity. The multidrug resistance shown in PCa became the main cause of tumor recurrence, metastasis, and even treatment failure. It is the most common and most difficult problem to overcome in the treatment of PCa. Previous studies considered only as a pharmaceutical product for rhythm regulation and sleep-aiding has shown melatonin potency as a co-adjuvant treatment in intestinal diseases [51, 52]. Melatonin has multiple effects for example antioxidative properties found in various edible and medicinal plants. In accordance with its multiple effects, it might resist growing pathological conditions such as carcinogenesis and evidences gathered to date strongly indicates that melatonin is a powerful free radical scavenger [53–55]. It plays critical roles in the suppression of lung injury [56], breast cancer risk [57, 58], myeloid leukemia cells [59]. The current common chemotherapy drugs are not sufficiently effective for anti-PCa, and there is an urgent need to screen for active ingredients of natural chemical compounds in the application for the new anti-PCa drugs. Although, melatonin cancer mechanisms are still unclear in prostate cancer cells.

To the best of our knowledge by reviewing literature, we could not find a single study on the anti-proliferation effect of melatonin in vitro. In the current study by utilizing RNA-seq data, we found a total of 20031 genes were upregulated and downregulated in the melatonin (0.5mM) treated cells. Of these we selected the top determining differentially expressed genes (n = 271) especially those having p < 0.05, these included 97 up-regulated (P<0.05), 174 down-regulated genes (P<0.05). We selected GO and KEGG signaling pathway to analyze the transcriptional misregulation in DU145 prostate cancer cell line. The gene ontology (GO) consortium consists of a number of databases working together to define standardized ontologies and provide annotations. GO can describe gene, gene product function and it can provide that similar gene often have conserved functions in different organisms [60, 61]. GO and KEGG enrichment analysis revealed that differentially expressed genes are playing important roles in the biological processes, cellular components, molecular functions and cell death before and after treatment with melatonin.

The GO analysis of differentially expressed genes indicates that melatonin plays an important role in cancer cell proliferation, migration, invasion, and cell cycle. By KEGG pathway analysis, it was found that significant differences existed in transcriptional misregulation of the cancer cells. qPCR and Immunoblotting analysis revealed that the highest differential expression in RNA-seq results revealed melatonin treatment led to a significant increase in the expression levels of HPGD, IL2Rβ, NGFR and decreased the expression level of IGFBP3 and IL6 genes (P <0.05). On the other hand, HPGD and IL2Rβ upregulated, NGFR, IGFBP3, and IL6 downregulated genes were confirmed by the qPCR assay as well as IL2Rβ, NGFR were upregulated, IL6 and IGFBP3 were downregulated in DU145 cell lines investigated by immunoblot assay. Furthermore, IL6 and IGFBP3 were upregulated, HPGD, NGFR, and IL2Rβ were downregulated in PC3 cell lines confirmed by qPCR results and IGFBP3 and IL6 genes had upregulation, NGFR and IL2Rβ genes had downregulation in PC3 cell lines confirmed by immunoblot analysis. According to RNA-seq results, the HPGD gene is an overexpressed gene and so, we planned to explore HPGD by qPCR and immunoblot analysis. We show that RNA-seq, qPCR, and immunoblot all results were demonstrated IL2Rβ (Interleukin 2 Receptor Subunit Beta) gene upregulation level.

PCa growth is initially androgen-dependent and thus androgen ablation is the standard therapeutic option. Nevertheless, the malignant prostate tumor eventually relapses after treatment, becoming hormone-independent and resistant to conventional therapies [62]. An inverse relationship between melatonin blood levels and tumor growth has been reported as well [63]. The results from the wound healing assay and transwell migration assay show that melatonin reduces the migration of PCa cells. The anti-inflammatory effects of melatonin were mediated by the inhibition of P38 phosphorylation and NF-κB P65 activation, and the anti-proliferative effects of melatonin were mediated by the regulation of cell cycle-related regulatory proteins and by the inhibition of mTOR phosphorylation [64]. Aberrant regulation of the NF-κB pathway is believed to be a major event contributing to the malignant transformation and progression of PCa [46–48]. Studies have shown that NF-κB plays an important role in PCa growth, survival, angiogenesis, tumor genesis, and metastatic progression [46]. For further we investigated the relationship between NF-κB signaling pathway (used antibodies: Anti-NGFR antibody (ratio 1:500), Anti-IL2Rβ (ratio 1:330) (IBBI, Changsha, China); Anti-IGFBP3 Rabbit pAb (1:1000), Anti-IL6 Rabbit pAb (1:1500) (Wanleibo, Changsha, China); HPGD Mouse Monoclonal antibody (1:5000) as well as HPGD Rabbit Polyclonal antibody (Proteintech, Changsha, China) and IL2Rβ gene expression in melatonin’s anti-proliferative actions as well as melatonin two receptors serves as a common regulators of NF-κB pathway in independent prostate cancer cell lines, the results revealed that melatonin can be used as a potential therapeutic and clinical agent for curing prostate diseases.

Conclusions

Our data confirmed that melatonin can efficaciously suppress human prostate cancer cell proliferation in vitro. In this study, we used various techniques such as RNA sequencing analysis, quantitative real-time PCR, flow cytometry, cell growth assay, cell migration, invasion assay as well as immunoblot analysis. The results indicated that melatonin can be considered as a potential novel chemotherapeutic for the treatment of PCa, and provide new ideas for the application of in-clinic therapy and research of PCa. The role of IL2Rβ in the immune system, molecular mechanisms have not yet been clearly defined, we planned to explore the molecular mechanism MT1 and MT2 of melatonin in near future studies.

Acknowledgments

We acknowledge and appreciate our lab fellows and related staff for their hard work and continuous support to continuously schedule our experiments.

References

  1. 1. Fouad YA, Aanei C. Revisiting the hallmarks of cancer. Am J Cancer Res. 2017;7(5):1016–36. pmid:28560055
  2. 2. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. 2011;144(5):646–74. pmid:21376230
  3. 3. Miller KD, Nogueira L, Mariotto AB, Rowland JH, Yabroff KR, Alfano CM, et al. Cancer treatment and survivorship statistics, 2019. CA Cancer J Clin. 2019. pmid:31184787
  4. 4. Kim TH, Park JM, Kim MY, Ahn YH. The role of CREB3L4 in the proliferation of prostate cancer cells. Scientific reports. 2017;7:45300. pmid:28338058
  5. 5. Fidler MM, Gupta S, Soerjomataram I, Ferlay J, Steliarova-Foucher E, Bray F. Cancer incidence and mortality among young adults aged 20–39 years worldwide in 2012: a population-based study. The Lancet Oncology. 2017;18(12):1579–89. pmid:29111259
  6. 6. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 2018;68(6):394–424.
  7. 7. Panigrahi GK, Praharaj PP, Kittaka H, Mridha AR, Black OM, Singh R, et al. Exosome proteomic analyses identify inflammatory phenotype and novel biomarkers in African American prostate cancer patients. Cancer Med. 2019;8(3):1110–23. pmid:30623593
  8. 8. Rawla P. Epidemiology of Prostate Cancer. World J Oncol. 2019;10(2):63–89. pmid:31068988
  9. 9. Chen D, Ayala GE. Innervating Prostate Cancer. The New England journal of medicine. 2018;378(7):675–7. pmid:29443663
  10. 10. Arnao MB, Hernández-Ruiz J. The physiological function of melatonin in plants. Plant signaling & behavior. 2006;1(3):89–95. pmid:19521488
  11. 11. Gobbo MG, Dizeyi N, Abrahamsson PA, Bertilsson PA, Masitéli VS, Pytlowanciv EZ, et al. Influence of Melatonin on the Proliferative and Apoptotic Responses of the Prostate under Normal and Hyperglycemic Conditions. Journal of diabetes research. 2015;2015:538529. pmid:26295055
  12. 12. Manchester LC, Coto-Montes A, Boga JA, Andersen LP, Zhou Z, Galano A, et al. Melatonin: an ancient molecule that makes oxygen metabolically tolerable. Journal of pineal research. 2015;59(4):403–19. pmid:26272235
  13. 13. Reiter RJ, Mayo JC, Tan DX, Sainz RM, Alatorre-Jimenez M, Qin L. Melatonin as an antioxidant: under promises but over delivers. J Pineal Res. 2016;61(3):253–78. pmid:27500468
  14. 14. Reiter RJ, Rosales-Corral S, Tan DX, Jou MJ, Galano A, Xu B. Melatonin as a mitochondria-targeted antioxidant: one of evolution’s best ideas. Cell Mol Life Sci. 2017;74(21):3863–81. pmid:28864909
  15. 15. Grant SG, Melan MA, Latimer JJ, Witt-Enderby PA. Melatonin and breast cancer: cellular mechanisms, clinical studies and future perspectives. Expert Rev Mol Med. 2009;11:e5. pmid:19193248
  16. 16. Li Y, Li S, Zhou Y, Meng X, Zhang JJ, Xu DP, et al. Melatonin for the prevention and treatment of cancer. Oncotarget. 2017;8(24):39896–921. pmid:28415828
  17. 17. Wu H, Liu J, Yin Y, Zhang D, Xia P, Zhu G. Therapeutic Opportunities in Colorectal Cancer: Focus on Melatonin Antioncogenic Action. Biomed Res Int. 2019;2019:9740568. pmid:31637261
  18. 18. Burkhardt S, Tan DX, Manchester LC, Hardeland R, Reiter RJ. Detection and quantification of the antioxidant melatonin in Montmorency and Balaton tart cherries (Prunus cerasus). Journal of agricultural and food chemistry. 2001;49(10):4898–902. pmid:11600041
  19. 19. González-Flores D, Belén V, Garrido M, Gonzalez-Gomez D, Lozano M, Concepción Ayuso M, et al. Ingestion of Japanese plums (Prunus salicina Lindl. cv. Crimson Globe) increases the urinary 6 sulfatoxymelatonin and total antioxidant capacity levels in young, middle-aged and elderly humans: Nutritional and functional characterization of their content. Journal of food and nutrition research. 2011;50:229–36.
  20. 20. Lamont KT, Somers S, Lacerda L, Opie LH, Lecour S. Is red wine a SAFE sip away from cardioprotection? Mechanisms involved in resveratrol- and melatonin-induced cardioprotection. Journal of pineal research. 2011;50(4):374–80. pmid:21342247
  21. 21. Chowdhury I, Sengupta A, Maitra SK. Melatonin: fifty years of scientific journey from the discovery in bovine pineal gland to delineation of functions in human. Indian J Biochem Biophys. 2008;45(5):289–304. pmid:19069840
  22. 22. Zhao F, Ma C, Zhao G, Wang G, Li X, Yang K. Rumen-Protected 5-Hydroxytryptophan Improves Sheep Melatonin Synthesis in the Pineal Gland and Intestinal Tract. Med Sci Monit. 2019;25:3605–16. pmid:31091223
  23. 23. Kim SW, Kim S, Son M, Cheon JH, Park YS. Melatonin controls microbiota in colitis by goblet cell differentiation and antimicrobial peptide production through Toll-like receptor 4 signalling. Sci Rep. 2020;10(1):2232. pmid:32042047
  24. 24. Zhan H, Nie X, Zhang T, Li S, Wang X, Du X, et al. Melatonin: A Small Molecule but Important for Salt Stress Tolerance in Plants. Int J Mol Sci. 2019;20(3). pmid:30736409
  25. 25. Reiter RJ, Tan DX, Fuentes-Broto L. Melatonin: a multitasking molecule. Progress in brain research. 2010;181:127–51. pmid:20478436
  26. 26. Shiu SY, Leung WY, Tam CW, Liu VW, Yao KM. Melatonin MT1 receptor-induced transcriptional up-regulation of p27(Kip1) in prostate cancer antiproliferation is mediated via inhibition of constitutively active nuclear factor kappa B (NF-kappaB): potential implications on prostate cancer chemoprevention and therapy. J Pineal Res. 2013;54(1):69–79. pmid:22856547
  27. 27. de Almeida Chuffa LG, Seiva FRF, Cucielo MS, Silveira HS, Reiter RJ, Lupi LA. Mitochondrial functions and melatonin: a tour of the reproductive cancers. Cellular and molecular life sciences: CMLS. 2019;76(5):837–63. pmid:30430198
  28. 28. Liu VWS, Yau WL, Tam CW, Yao KM, Shiu SYW. Melatonin Inhibits Androgen Receptor Splice Variant-7 (AR-V7)-Induced Nuclear Factor-Kappa B (NF-kappaB) Activation and NF-kappaB Activator-Induced AR-V7 Expression in Prostate Cancer Cells: Potential Implications for the Use of Melatonin in Castration-Resistant Prostate Cancer (CRPC) Therapy. Int J Mol Sci. 2017;18(6). pmid:28561752
  29. 29. Altannavch N, Zhou X, Khan MA, Ahmed A, Naranmandakh S, Fu JJ, et al. Anti-Oxidant and Anticancerous Effect of Fomitopsis officinalis (Vill. ex Fr. Bond. et Sing) Mushroom on Hepatocellular Carcinoma Cells In Vitro through NF-kB Pathway. Anti-cancer agents in medicinal chemistry. 2021.
  30. 30. Roberts A, Trapnell C, Donaghey J, Rinn JL, Pachter L. Improving RNA-Seq expression estimates by correcting for fragment bias. Genome biology. 2011;12(3):R22. pmid:21410973
  31. 31. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature biotechnology. 2010;28(5):511–5. pmid:20436464
  32. 32. Anders S, Pyl PT, Huber W. HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics (Oxford, England). 2015;31(2):166–9. pmid:25260700
  33. 33. Love MI, Soneson C, Patro R. Swimming downstream: statistical analysis of differential transcript usage following Salmon quantification. F1000Research. 2018;7:952. pmid:30356428
  34. 34. Kim D, Langmead B, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nature methods. 2015;12(4):357–60. pmid:25751142
  35. 35. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature methods. 2012;9(4):357–9. pmid:22388286
  36. 36. Roberts A, Pachter L. Streaming fragment assignment for real-time analysis of sequencing experiments. Nature methods. 2013;10(1):71–3. pmid:23160280
  37. 37. Kanehisa M, Araki M, Goto S, Hattori M, Hirakawa M, Itoh M, et al. KEGG for linking genomes to life and the environment. Nucleic acids research. 2008;36(Database issue):D480–4. pmid:18077471
  38. 38. Park S, Lee DE, Jang H, Byeon Y, Kim YS, Back K. Melatonin-rich transgenic rice plants exhibit resistance to herbicide-induced oxidative stress. J Pineal Res. 2013;54(3):258–63. pmid:22856683
  39. 39. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer. 2015;136(5):E359–86. pmid:25220842
  40. 40. Kumar C, Rasool RU, Iqra Z, Nalli Y, Dutt P, Satti NK, et al. Alkyne-azide cycloaddition analogues of dehydrozingerone as potential anti-prostate cancer inhibitors via the PI3K/Akt/NF-kB pathway. MedChemComm. 2017;8(11):2115–24. pmid:30108729
  41. 41. Hazafa A, Rehman KU, Jahan N, Jabeen Z. The Role of Polyphenol (Flavonoids) Compounds in the Treatment of Cancer Cells. Nutrition and cancer. 2020;72(3):386–97. pmid:31287738
  42. 42. Martin-Renedo J, Mauriz JL, Jorquera F, Ruiz-Andres O, Gonzalez P, Gonzalez-Gallego J. Melatonin induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cell line. J Pineal Res. 2008;45(4):532–40. pmid:19012662
  43. 43. Hassan M, Watari H, AbuAlmaaty A, Ohba Y, Sakuragi N. Apoptosis and molecular targeting therapy in cancer. Biomed Res Int. 2014;2014:150845. pmid:25013758
  44. 44. Plaimee P, Weerapreeyakul N, Barusrux S, Johns NP. Melatonin potentiates cisplatin-induced apoptosis and cell cycle arrest in human lung adenocarcinoma cells. Cell Prolif. 2015;48(1):67–77. pmid:25580987
  45. 45. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000;21(3):485–95. pmid:10688869
  46. 46. Jin R, Yi Y, Yull FE, Blackwell TS, Clark PE, Koyama T, et al. NF-kappaB gene signature predicts prostate cancer progression. Cancer Res. 2014;74(10):2763–72. pmid:24686169
  47. 47. Shukla S, MacLennan GT, Fu P, Patel J, Marengo SR, Resnick MI, et al. Nuclear factor-kappaB/p65 (Rel A) is constitutively activated in human prostate adenocarcinoma and correlates with disease progression. Neoplasia (New York, NY). 2004;6(4):390–400. pmid:15256061
  48. 48. Staal J, Beyaert R. Inflammation and NF-kappaB Signaling in Prostate Cancer: Mechanisms and Clinical Implications. Cells. 2018;7(9). pmid:30158439
  49. 49. Hoesel B, Schmid JA. The complexity of NF-kappaB signaling in inflammation and cancer. Mol Cancer. 2013;12:86. pmid:23915189
  50. 50. Inoue J, Gohda J, Akiyama T, Semba K. NF-kappaB activation in development and progression of cancer. Cancer Sci. 2007;98(3):268–74. pmid:17270016
  51. 51. Ma N, Zhang J, Reiter RJ, Ma X. Melatonin mediates mucosal immune cells, microbial metabolism, and rhythm crosstalk: A therapeutic target to reduce intestinal inflammation. Med Res Rev. 2019.
  52. 52. Auld F, Maschauer EL, Morrison I, Skene DJ, Riha RL. Evidence for the efficacy of melatonin in the treatment of primary adult sleep disorders. Sleep medicine reviews. 2017;34:10–22. pmid:28648359
  53. 53. Favero G, Franceschetti L, Buffoli B, Moghadasian MH, Reiter RJ, Rodella LF, et al. Melatonin: Protection against age-related cardiac pathology. Ageing research reviews. 2017;35:336–49. pmid:27884595
  54. 54. Bonomini F, Borsani E, Favero G, Rodella LF, Rezzani R. Dietary Melatonin Supplementation Could Be a Promising Preventing/Therapeutic Approach for a Variety of Liver Diseases. Nutrients. 2018;10(9). pmid:30134592
  55. 55. Salehi B, Sharopov F, Fokou PVT, Kobylinska A, Jonge L, Tadio K, et al. Melatonin in Medicinal and Food Plants: Occurrence, Bioavailability, and Health Potential for Humans. Cells. 2019;8(7).
  56. 56. Han B, Li S, Lv Y, Yang D, Li J, Yang Q, et al. Dietary melatonin attenuates chromium-induced lung injury via activating the Sirt1/Pgc-1alpha/Nrf2 pathway. Food Funct. 2019. pmid:31429458
  57. 57. Dodda BR, Bondi CD, Hasan M, Clafshenkel WP, Gallagher KM, Kotlarczyk MP, et al. Co-administering Melatonin With an Estradiol-Progesterone Menopausal Hormone Therapy Represses Mammary Cancer Development in a Mouse Model of HER2-Positive Breast Cancer. Frontiers in oncology. 2019;9:525. pmid:31355130
  58. 58. Menéndez-Menéndez J, Hermida-Prado F, Granda-Díaz R, González A, García-Pedrero JM, Del-Río-Ibisate N, et al. Deciphering the Molecular Basis of Melatonin Protective Effects on Breast Cells Treated with Doxorubicin: TWIST1 a Transcription Factor Involved in EMT and Metastasis, a Novel Target of Melatonin. Cancers. 2019;11(7). pmid:31331001
  59. 59. Tian T, Li J, Li Y, Lu YX, Tang YL, Wang H, et al. Melatonin enhances sorafenib-induced cytotoxicity in FLT3-ITD acute myeloid leukemia cells by redox modification. Theranostics. 2019;9(13):3768–79. pmid:31281512
  60. 60. du Plessis L, Skunca N, Dessimoz C. The what, where, how and why of gene ontology—a primer for bioinformaticians. Briefings in bioinformatics. 2011;12(6):723–35. pmid:21330331
  61. 61. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nature genetics. 2000;25(1):25–9. pmid:10802651
  62. 62. Rodriguez-Garcia A, Hevia D, Mayo JC, Gonzalez-Menendez P, Coppo L, Lu J, et al. Thioredoxin 1 modulates apoptosis induced by bioactive compounds in prostate cancer cells. Redox biology. 2017;12:634–47. pmid:28391184
  63. 63. Blask DE, Brainard GC, Dauchy RT, Hanifin JP, Davidson LK, Krause JA, et al. Melatonin-depleted blood from premenopausal women exposed to light at night stimulates growth of human breast cancer xenografts in nude rats. Cancer research. 2005;65(23):11174–84. pmid:16322268
  64. 64. Li HY, Leu YL, Wu YC, Wang SH. Melatonin Inhibits in Vitro Smooth Muscle Cell Inflammation and Proliferation and Atherosclerosis in Apolipoprotein E-Deficient Mice. J Agric Food Chem. 2019;67(7):1889–901. pmid:30661353