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The underlying mechanism of colorectal cells developing into cancer cells has been extensively investigated, yet is still not fully delineated, resulting in the treatment of advanced colorectal cancer (CRC) remains regrettably an unmet need. Zinc Finger Protein 746/Parkin-interacting substrate (ZNF746/PARIS) has previously been identified to play a fundamental role on bladder cancer cell proliferation and metastasis that were effectively inhibited by melatonin (Mel). In this study, we utilized ex vivo/in vivo studies to verify whether the ZNF746 signaling was also crucial in CRC growth/invasion/migration. Tissue-bank specimens showed that the protein expression of ZNF746 was significantly increased in CRC than that of healthy colorectal tissues (p < 0.001). Additionally, in vitro study demonstrated that excessive expression of ZNF746 significantly inhibited mitochondrial activity via (1) interfering with the dynamic balance of mitochondrial fusion/fission and (2) inhibiting the protein expression of MFN1/MFN2/PGC1a (all p < 0.001). Furthermore, we identified that inhibition of ZNF746 protein expression significantly reduced the resistance of CRC cell lines to the anticancer drug of 5-FU (p < 0.001), whereas overexpression of ZNF746 significantly augmented resistance of CRC cells to 5-FU (all p < 0.001). Finally, using the cell culture method, we found that combined Mel and 5-FU was superior to merely one on promoting the CRC cell apoptosis (p < 0.001). Our results confirmed that ZNF746 signaling played a cardinal role of CRC cell proliferation/survival and combined Mel and 5-FU treatment attenuated the resistance of CRC cells to the drug mainly through suppressing this signaling., (© 2023 Wiley Periodicals LLC.)

This study investigated whether melatonin (Mel) would promote cisplatin to suppress the proliferation and growth of bladder cancer (BC) cells by inhibiting cellular prion protein (PrP C )-mediated cell stress and cell proliferation signaling. An immunohistochemical staining of tissue arrays from BC patients demonstrated that the PrP C expression was significantly upregulated from stage I to III BC ( p < 0.0001). The BC cellline of T24 was categorized into G1 (T24), G2 (T24 + Mel/100 μM), G3 (T24+cisplatin/6 μM), G4 (PrP C overexpression in T24 (i.e., PrP C-OE -T24)), G5 (PrP C-OE -T24+Mel), and G6 (PrP C-OE -T24+cisplatin). When compared with a human uroepithelial cell line (SV-HUC-1), the cellular viability/wound healing ability/migration rate were significantly increased in T24 cells (G1) and further significantly increased in PrP C-OE -T24 cells (G4); and they were suppressed in Mel (G2/G5) or cisplatin (G3/G6) treatment (all p < 0.0001). Additionally, the protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrP C ), cell cycle/mitochondrial functional integrity (cyclin-D1/clyclin-E1/ckd2/ckd4/mitochondrial-cytochrome-C/PINK1), and cell stress (RAS/c-RAF/p-MEK1/2, p-ERK1/2) markers showed a similar pattern of cell viability among the groups (all p < 0.001). After the BC cell line of UMUC3 was implanted into nude mouse backs, by day 28 mthe BC weight/volume and the cellular levels of PrP C /MMP-2/MMP-9 were significantly, gradually reduced from groups one to four (all p < 0.0001). The protein expressions of cell proliferation (PI3K/p-Akt/p-m-TOR/MMP-9/PrP C ), cell cycle/mitophagy (cyclin-D1/clyclin-E1/ckd2/ckd4/PINK1), and cell stress (RAS/c-RAF/p-MEK1,2/p-ERK1,2) signaling were significantly, progressively reduced from groups one to four, whereas the protein expressions of apoptotic (Mit-Bax/cleaved-caspase-3/cleaved-PARP) and oxidative stress/mitochondrial damaged (NOX-1/NOX-2/cytosolic-cytochrome-C/p-DRP1) markers expressed an opposite pattern of cell proliferation signaling among the groups (all p < 0.0001). Mel-cisplatin suppressed BC cell growth/proliferation via inhibiting the PrP C in upregulating the cell proliferation/cell stress/cell cycle signaling.

Background: Cerebral ischemic events, comprising of excitotoxicity, reactive oxygen production, and inflammation, adversely impact the metabolic-redox circuit in highly active neuronal metabolic profile which maintains energy-dependent brain activities. Therefore, we investigated neuro-regenerative potential of melatonin (Mel), a natural biomaterial secreted by pineal gland., Methods: We specifically determined whether Mel could influence tunneling nanotubes (TNTs)-mediated transfer of functional mitochondria (Mito) which in turn may alter membrane potential, oxidative stress and apoptotic factors. In vitro studies assessed the effects of Mito on levels of cytochrome C, mitochondrial transfer, reactive oxygen species, membrane potential and mass, which were all further enhanced by Mel pre-treatment, whereas in vivo studies examined brain infarct area (BIA), neurological function, inflammation, brain edema and integrity of neurons and myelin sheath in control, ischemia stroke (IS), IS + Mito and IS + Mel-Mito group rats., Results: Results showed that Mel pre-treatment significantly increased mitochondrial transfer and antioxidants, and inhibited apoptosis. Mel-pretreated Mito also significantly reduced BIA with improved neurological function. Apoptotic, oxidative-stress, autophagic, mitochondrial/DNA-damaged biomarkers indices were also improved., Conclusion: Conclusively, Mel is a potent biomaterial which could potentially impart neurogenesis through repairing impaired metabolic-redox circuit via enhanced TNT-mediated mitochondrial transfer, anti-oxidation, and anti-apoptotic activities in ischemia., (Copyright © 2021. Published by Elsevier Masson SAS.)

Background: This study tested the hypothesis that combined melatonin (Mel) and adipose-derived mesenchymal stem cells (ADMSCs) treatment was superior to either one alone on protecting the testis against acute testicular torsion-induced ischemia-reperfusion (TTIR) injury., Methods and Results: Male adult SD rats (n = 30) were equally categorized into group 1 (sham-operated control), group 2 [TTIR/by torsion of right/left testis (i.e., ischemia) with rotated 720° counterclockwise for 2 h, then detorsion (i.e., reperfusion) to the original position for 72 h], group 3 (TTIR + Mel/intraperitoneal administration/50 mg/kg at 30 min after ischemia, followed by 20 mg at 3 h and days 1/2/3 after TTIR), group 4 (TTIR + ADMSCs/1.2 × 10 6 cells/by tail-vein administration at 30 min after ischemia, followed by days 1/2 TTIR), and group 5 (TTIR + Mel + ADMSCs/tail-vein administration). The result showed that the protein expressions of oxidative-stress (NOX-1/NOX-2/oxidized-protein), apoptotic/mitochondrial-damaged (mitochondrial-Bax/cleaved-caspase3/cleaved-PARP/cytosolic-cytochrome C), and fibrotic (TGF-ß/Smad3) biomarkers as well as testicular damage scores were lowest in group 1, highest in group 2, and significantly higher in groups 3/4 than in group 5, but they showed no difference between groups 3/4, whereas the protein expressions of androgen receptor (AR) and vimentin showed an opposite pattern of oxidative stress (all p < 0.0001). The cellular levels of inflammation (MMP-9/MPO/CD68) exhibited an identical pattern, whereas the numbers of Sertoli cells, α-tubulin, AR and vimentin as well as thickness of seminiferous tubule exhibited an opposite pattern of oxidative stress among the groups (all p < 0.0001)., Conclusion: Mel-ADMSCs effectively protected the testis against TTIR injury.

Background: This study tested whether combined cyclosporin-A (CsA) and melatonin (Mel) was superior to either one on protecting the brain against ischemia (occluded left-middle-cerebral-artery for 90-min)-reperfusion (for 14 days) injury., Methods and Results: Neuro-2a cells (N2a) were categorized into groups 1 (N2a), 2 (N2a-IR), 3 (N2a-IR-Mel), 4 (N2a-IR-CsA) and 5 (N2a-IR-CsA-Mel). in vitro results showed the protein expressions of cytosolic-cytochrome-C/mitochondrial-Bax/cleaved-capase-3/NOX-1/NOX-2 and flow-cytometric results of ROS (DCFDA/Mito-SOX) were highest in group 2, lowest in group 1, significantly lower in group 5 than in groups 3/4, but they showed no difference in groups 3/4 (all p < 0.001). Male-adult-SD rats (50) were equally categorized into groups 1 (sham-operated-control), 2 (IR), 3 (IR-CsA/20.0 mg/kg at 0.5/24/48 h intraperitoneally after IR), 4 (IR-Mel/50.0 mg/kg intraperitoneally at 30 min and 30 mg/kg at 6/24/48 h after IR) and 5 (IR-CsA-Mel). The brain-infarct-area (BIA) (at day-3 by TTC-stain) was lowest in group 1, highest in group 2, significantly lower in group 5 than groups 3/4, but it showed no difference between groups 3/4 whereas the brain-infarct-volume (at day 14 by MRI) was similar as BIA except for significantly lower in group 4 than in group 3 (all p < 0.0001). By day 14, microscopic finding showed the numbers of glial+/GFAP+/AQP + cells expressed an identical trend whereas the number of NeuN + cells exhibited an opposite pattern of BIA among the groups (all p < 0.0001). The protein expressions of oxidative-stress (NOX-1/NOX-2/p22phox/oxidized-protein), inflammatory (TNF-α/p-NF-κB/MMP-9), apoptotic (mitochondrial-Bax/caspase-3/PARP) and mitochondrial-damaged (Cyclophilin-D/DRP1/cytosolic-cytochrome-C) biomarkers displayed an identical pattern of BIA among the five groups (all p < 0.0001)., Conclusion: Combined CsA-Mel was superior to either CsA or Mel on protecting the brain against IR injury., (Copyright © 2021 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Acute liver ischaemia-reperfusion injury (IRI), commonly encountered during liver resection and transplantation surgery, is strongly associated with unfavourable clinical outcome. However, a prompt and accurate diagnosis and the treatment of this entity remain formidable challenges. This study tested the hypothesis that 31 P-magnetic resonance spectroscopy ( 31 P-MRS) findings could provide reliable living images to accurately identify the degree of acute liver IRI and melatonin-pretreated mitochondria was an innovative treatment for protecting the liver from IRI in rat. Adult male SD rats were categorized into group 1 (sham-operated control), group 2 (IRI only) and group 3 (IRI + melatonin [ie mitochondrial donor rat received intraperitoneal administration of melatonin] pretreated mitochondria [10 mg/per rat by portal vein]). By the end of study period at 72 hours, 31 P-MRS showed that, as compared with group 1, the hepatic levels of ATP and NADH were significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1. The liver protein expressions of mitochondrial-electron-transport-chain complexes and mitochondrial integrity exhibited an identical pattern to 31 P-MRS finding. The protein expressions of oxidative stress, inflammatory, cellular stress signalling and mitochondrial-damaged biomarkers displayed an opposite finding of 31 P-MRS, whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. Microscopic findings showed that the fibrotic area/liver injury score and inflammatory and DNA-damaged biomarkers exhibited an identical pattern of cellular stress signalling. Melatonin-pretreated mitochondria effectively protected liver against IRI and 31 P-MRS was a reliable tool for measuring the mitochondrial/ATP consumption in living animals., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: This study assessed the expression of Jagged2 in human bladder cancer (BC) tested the hypothesis that melatonin (Mel) inhibited the tumorigenesis of BC cells mainly through downregulating the Notch/Jagged2 and PI3K/AKT/mTOR/MMPs (2&9) signaling pathways. Methods and Results: Tissue array from BC patients showed that the gene and protein expressions of JAG2/Jagged2 were significantly upregulated from T1 to T3 (primary tumor size) and from stage I to III (all p<0.001). In vitro study showed that in BC cell line of UMUC3, the cellular and protein expressions of Jagged2 were significantly attenuated in Mel-treated UMUC3 and further attenuated in UMUC3 shRNA silenced Notch/JAG2 (UMUC3 KD ) than in UMUC3 only (all p<0.0001). The protein expressions of Notch/Jagged2/MMPs (2&9) /PI3K/p-AKT/mTOR/p53/ratio of LC3BII/LC3B-I were significantly progressively reduced from UMUC3 to UMUC3+Mel/1.0mM, further to UMUC3+Mel/2.0mM and furthermore to UMUC3 KD (all p<0.0001). The cell proliferation/invasion/colony formation/healing-process were significantly inhibited in Mel-treated/2.0mM UMUC3 and further significantly inhibited in UMUC3 KD regardless of Mel treatment as compared with UMUC3 only (all p<0.0001). By day 28 after UMUC3 implanted into nude mouse back, the BC weight/volume were significantly reduced in UMUC3+Mel (100 mg/kg/day) and furthermore reduced in UMUC3 KD (all p<0.0001) as compared with UMUC3 only (all p<0.0001). The cellular (MMPs (2&9) /Notch/Jagged2) and protein (Notch/Jagged2/PI3K/p-AKT/mTOR/MMPs (2&9) ) exhibited a similar trend, whereas the PTEN protein level exhibited an opposite pattern of PI3K among three groups (all p<0.0001). Conclusion: Notch/Jagged-PI3K/p-AKT/mTOR/MMPs is one essential signaling pathway for BC survival, proliferation and invasion that were remarkably suppressed by Mel treatment., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

This study tested the hypothesis that exposure to ambient fine particulate matter (PM 2.5 ) pollution increased susceptibility of rat lung to damage from acute ischemia-reperfusion (IR) injury that was reversed by melatonin (Mel) treatment. Male-adult SD rats (n = 30) were categorized into group 1 (normal control), group 2 (PM 2.5 only), group 3 (IR only at day 8 after PM 2.5 exposure), group 4 (PM 2.5 + IR) and group 5 (PM 2.5 + IR + Mel), and all animals were sacrificed by day 10 after PM 2.5 exposure. Oxygen saturation (%) was significantly higher in group 1 than in other groups and significantly lower in group 4 than in groups 2, 3 and 5 but it did not differ among the latter three groups (p < 0.01). Pulmonary protein expressions of inflammation (MMP-9/TNF-α/NF-kB), oxidative stress (NOX-1/NOX-2/oxidized protein), apoptosis (mitochondrial-Bax/caspase-3/PARP) and fibrosis were lowest in group 1, highest in group 4, significantly higher in group 5 than in groups 2 and 3 (all p < 0.0001), but they did not differ between groups 2 and 3. Inflammatory cell infiltration in lung parenchyma, specific inflammatory cell surface markers (CD14+, F4/88+), allergic inflammatory cells (IgE+, eosinophil+), number of goblet cells, thickness of tracheal epithelial layer and fibrotic area exhibited an identical pattern of protein expressions to inflammation among the five groups (all p < 0.0001). In conclusion, lung parenchymal damage and a rigorous inflammatory response were identified in rodent even with short-term PM 2.5 exposure., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

We evaluated the ability of extracorporeal shock wave (ECSW)-assisted melatonin (Mel) therapy to offer an additional benefit for alleviating the neuropathic pain (NP) in rats. Left sciatic nerve was subjected to chronic constriction injury (CCI) to induce NP. Animals (n = 30) were randomized into group 1 (sham-operated control), group 2 (CCI only), group 3 (CCI + ECSW), group 4 (CCI + Mel) and group 5 (CCI + ECSW + Mel). By days 15, 22 and 29 after CCI, the thermal paw withdrawal latency (TPWL) and mechanical paw withdrawal threshold (MPWT) were highest in group 1, lowest in group 2, significantly higher in group 5 than in groups 3 and 4, but they showed no difference between the later two groups (all p < 0.0001). The protein expressions of inflammatory (TNF-α, NF-κB, MMP-9, IL-1ß), oxidative-stress (NOXs-1, -2, -4, oxidized protein), apoptotic (cleaved-caspase3, cleaved-PARP), DNA/mitochondrial-damaged (γ-H2AX/cytosolic-cytochrome C), microglia/astrocyte activation (ox42/GFAP), and MAPKs [phosphorylated (p)-p38, p-JNK, p-ERK] biomarkers in dorsal root ganglia neurons (DRGs) and in spinal dorsal horn were exhibited an opposite pattern of TPWL among the five groups (all p < 0.0001). Additionally, protein expressions of Nav.1.3, Nav.1.8 and Nav.1.9 in sciatic nerve exhibited an identical pattern to inflammation among the five groups (all p < 0.0001). The numbers of cellular expressions of MAPKs (p-ERK1/2+/peripherin + cells, p-ERK1/2+/NF200 + cells and p-JNK+/peripherin + cells, p-JNK+/NF200 + cells) and voltage-gated sodium channels (Nav.1.8+/peripherin + cells, Nav.1.8+/NF200 + cells, Nav.1.9+/peripherin + cells, Nav.1.9+/NF200 + cells) in small and large DRGs displayed an identical pattern to inflammation among the five groups (all p < 0.0001). In conclusion, the synergistic effect of combined ECSW-Mel therapy is superior to either one alone for long-term improvement of mononeuropathic pain-induced by CCI in rats.

There still lacking effective treatment for bladder cancer. This study investigated whether melatonin (Mel) can suppress the growth and invasion of bladder cancer cells. Male C57B/L6 mice were categorized into control group (ie, subcutaneous injection of HT1197 bladder cancer cell line at the back] and treatment group [subcutaneous HT1197 cells + intraperitoneal Mel (100 mg/kg/d) from day 8 to day 21 after tumor cell injection]. In vitro Mel suppressed cell growth of four bladder cancer cell lines (ie, T24, RT4, HT1197, HT1376), cell migration in HT1197/HT1376, mitochondrial membrane potential (MMP) in T24 and colony formation in RT4 cells as well as arrested the cell cycle at G0 phase and inhibited the mitotic phase of T24 cells (all P < 0.0001). Protein expression of ZNF746 in RT4/T24 cells and protein expression phosphorylated (p)-AKT/MMP-2/MMP-9 in HT1197/HT1376 cells were reduced following Mel treatment (all P < 0.001). Transfection of T24 cells with plasmid-based shRNA (ie, ZNF746-silencing) downregulated the protein expression of MMP-9, cell growth, and invasion and attachment to endothelial cells but upregulated the colony formation (all P < 0.001). Mel suppressed oxidative stress and MMP but upregulated mitochondria mass in ZNF746-silenced T24 cells, whereas these parameters exhibited a similar patter to Mel treatment in ZNF746-silenced T24 cells (all P < 0.0001). In vivo study demonstrated that Mel treatment significantly suppressed cellular expressions of MMP-9/MMP-2, protein expressions of ZNF746/p-AKT, and tumor size (all P < 0.001). Mel treatment suppressed the growth, migration, and invasion of bladder carcinoma cells through downregulating ZNF746-regulated MMP-9/MMP-2 signaling., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

We tested the hypothesis that daily melatonin treatment protects endothelial lineage and functional integrity against the aging process, oxidative stress/endothelial denudation (ED), and toxic environment and restored blood flow in murine critical limb ischemia (CLI). In vitro study using HUVECs, in vivo models (ie, CLI through left femoral artery ligation and ED through carotid artery wire injury), and model of lipopolysaccharide-induced aortic injury in young (3 months old) and aged (8 months old) mice were used to elucidate effects of melatonin treatment on vascular endothelial integrity. In vitro study showed that menadione-induced oxidative stress (NOX-1/NOX-2), inflammation (TNF-α/NF-kB), apoptosis (cleaved caspase-3/PARP), and mitochondrial damage (cytosolic cytochrome c) in HUVECs were suppressed by melatonin but reversed by SIRT3-siRNA (all P < .001). In vivo, reduced numbers of circulating endothelial progenitor cells (EPCs) (C-kit/CD31+/Sca-1/KDR+/CXCR4/CD34+), and angiogenesis (Matrigel assay of bone marrow-derived EPC and ex vivo aortic ring cultures) in older (compared with younger) mice were significantly reversed through daily melatonin administration (20 mg/kg/d, ip) (all P < .001). Aortic vasorelaxation and nitric oxide release were impaired in older mice and reversed in age-match mice receiving melatonin (all P < .01). ED-induced intimal/medial hyperplasia, reduced blood flow to ischemic limb, and angiogenesis (reduced CD31+/vWF+ cells/small vessel number) were improved after daily melatonin treatment (all P < .0001). Lipopolysaccharide-induced aortic endothelial cell detachment, which was more severe in aged mice, was also alleviated after daily melatonin treatment (P < .0001). Daily melatonin treatment protected both structural and functional integrity of vascular endothelium against aging-, oxidative stress-, lipopolysaccharide-, and ischemia-induced damage probably through upregulating the SIRT signaling pathway., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)