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. 2021 May 25:14:2253-2265.
doi: 10.2147/JIR.S305450. eCollection 2021.

Melatonin Inhibits Migration and Invasion in LPS-Stimulated and -Unstimulated Prostate Cancer Cells Through Blocking Multiple EMT-Relative Pathways

Qi-Xing Tian #  1 , Zhi-Hui Zhang #  1 , Qing-Lin Ye  1 , Shen Xu  1 , Qian Hong  1 , Wei-Yang Xing  1 , Lei Chen  1 , De-Xin Yu  1 , De-Xiang Xu  2   3 , Dong-Dong Xie  1
Affiliations

Melatonin Inhibits Migration and Invasion in LPS-Stimulated and -Unstimulated Prostate Cancer Cells Through Blocking Multiple EMT-Relative Pathways

Qi-Xing Tian et al. J Inflamm Res. .

Retraction in

Abstract

Purpose: Gram-negative bacteria are usually found in prostate cancer (PCa) tissues. This study aims to investigate the role of lipopolysaccharide (LPS), a glycolipid compound found in the outer membrane of gram-negative bacteria, on the migration and invasion of PCa cells, and to evaluate the protective effect of melatonin.

Materials and methods: DU145, PC-3 and LNCaP cells were incubated with LPS in the presence or absence of melatonin. Wound healing and Transwell assays were used to analyze migration and invasion of PCa cells. RT-PCR and Western blotting were used to assess the mRNA and protein levels, respectively. Co-IP was used to analyze β-catenin ubiquitination.

Results: Our results showed that LPS promoted migration and invasion of PCa cells. In addition, LPS stimulated inflammatory reaction and induced epithelial-mesenchymal transition (EMT) in PCa cells by activating several TLR4 downstream pathways. Specifically, LPS promoted NF-κB/IL-6/STAT3 signal transduction. In addition, LPS upregulated phosphorylation levels of cytoplasmic AKTSer473 and GSK-3βSer9. Moreover, LPS induced phosphorylation of GSK-3βSer9 in the "disruption complex", and then inhibited phosphorylation and ubiquitination of cytoplasmic β-catenin, leading to β-catenin nuclear translocation. Interestingly, melatonin inhibited invasion and migration not only in LPS-stimulated but also in LPS-unstimulated PCa cells. Melatonin suppressed PCa cells migration and invasion by blocking EMT mediated by IL-6/STAT3, AKT/GSK-3β and β-catenin pathways.

Conclusion: This study provides evidence that melatonin inhibits migration and invasion through blocking multiple TLR4 downstream EMT-associated pathways both in LPS-stimulated and -unstimulated PCa cells. Our results provide new insights into the role of bacterial infection in PCa metastasis and a potential therapeutic agent.

Keywords: EMT; lipopolysaccharide; melatonin; prostate cancer; β-catenin.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
Melatonin inhibits migration and invasion in LPS-stimulated and -unstimulated DU145 and PC-3 cells. After pretreated with 2 mM melatonin for 30 min, DU145 and PC-3 cells were incubated with LPS (2.0 μg/mL). (A) The migration of PC-3 cells was measured using wound healing assay. After 12 and 24 h migration, the scratches were photographed (left panel) and wounded areas were calculated (right panel). (B) The migration of DU145 and PC-3 cells were measured using Transwell migration assay. (C) The invasion of DU145 and PC-3 cells were measured using Transwell invasion assay. All experiments were repeated three times. Data were expressed as means ± S.E.M., **P<0.01 versus control group, ##P<0.01 versus LPS group.
Figure 2
Figure 2
Melatonin inhibits EMT in LPS-stimulated and -unstimulated DU145 cells. (A) DU145 cells were incubated with LPS (2 μg/mL) for indicated time. N-cadherin, E-cadherin and Vimentin were measured using Western blotting. (B) DU145 cells were incubated with LPS (2 μg/mL) with/without melatonin (2 mM) for 24 h. E-cadherin, N-cadherin, and Vimentin were measured. (C) DU145 cells were incubated with melatonin at an indicated dose for 24 h. E-cadherin, N-cadherin, and Vimentin were measured. (D) DU145 cells were incubated with LPS (2 μg/mL) for indicated time. Nuclear Zeb1, Snail, Slug and Twist were measured using Western blotting. (E) DU145 cells were incubated with LPS (2 μg/mL) with/without melatonin (2 mM) for 12 h. Nuclear Zeb1, Snail, Slug and Twist were measured. (F) DU145 cells were incubated with melatonin at indicated dose for 12 h. Nuclear Zeb1, Snail, Slug and Twist were measured. All experiments were repeated three times. Data were expressed as means ± S.E.M., *P<0.05, **P<0.01 versus control group, #P<0.05, ##P<0.01 versus LPS group.
Figure 3
Figure 3
Melatonin inhibits activation of NF-κB mediated inflammatory signal in LPS-stimulated and -unstimulated DU145 and PC-3 cells. (A) DU145 cells were incubated with LPS (2 μg/mL) for indicated time. Phosphorylated I-κB was measured using Western blotting. (B) DU145 cells were incubated with LPS (2 μg/mL) in absence or presence of melatonin (2 mM) for 2 h. Phosphorylated I-κB was measured. (C) DU145 cells were incubated with melatonin for indicated dose. Phosphorylated I-κB was measured. (D–I) PC-3 cells were incubated with LPS (2 μg/mL) in absence or presence of melatonin (2 mM) for 6 h. (D) IL-1α; (E) IL-1β; (F) TNF-α; (G) COX-2; (H) IL-8; (I) MCP-1 mRNAs were detected using RT-PCR. All experiments were repeated three times. Data were expressed as means ± S.E.M., *P<0.05, **P<0.01 versus control group, ##P<0.01 versus LPS group.
Figure 4
Figure 4
Melatonin inhibits STAT3 signal transduction in LPS-stimulated and -unstimulated DU145 and PC-3 cells. (A and B) PC-3 cells were incubated with LPS (2 μg/mL) with/without melatonin (2 mM) for 6 h. (A) IL-6, mRNAs were detected using RT-PCR. (B) IL-6 levels in the supernatant were detected by ELISA. (C and D) PC-3 and DU145 cells were incubated with LPS (2 μg/mL) in absence or presence of melatonin (2 mM) for 6 h. STAT3 phosphorylation was measured. (E) DU145 cells were incubated with melatonin at indicated dose for 6 h. STAT3 phosphorylation was measured. All experiments were repeated three times. Data were expressed as means ± S.E.M., *P<0.05, **P<0.01 versus control group, ##P<0.01 versus LPS group.
Figure 5
Figure 5
Effect of melatonin on AKT/GSK-3β pathway in LPS-stimulated and -unstimulated DU145 cells. (A) DU145 cells were incubated with LPS (2 μg/mL) for indicated time. Phosphorylation of AKT on serine 473 (p-AKTSer473) in cytoplasm was measured using Western blotting. (B) DU145 cells were incubated with LPS (2 μg/mL) with/without melatonin (2 mM) for 2 h. Cytoplasmic p-AKTSer473 was measured. (C) DU145 cells were incubated with melatonin for indicated dose. Cytoplasmic p-AKTSer473 was measured. (D) DU145 cells were incubated with LPS (2 μg/mL) for indicated time. Cytoplasmic p-GSK3βSer9 was measured. (E) DU145 cells were incubated with LPS (2 μg/mL) with/without melatonin (2 mM) for 2 h. Cytoplasmic p-GSK3βSer9 was measured. (F) DU145 cells were incubated with melatonin for indicated dose. Cytoplasmic p-GSK3βSer9 was measured. All experiments were repeated three times. Data were expressed as means ± S.E.M., **P<0.01 versus control group, ##P<0.01 versus LPS group.
Figure 6
Figure 6
Melatonin inhibits β-catenin nuclear translocation in LPS-stimulated and -unstimulated DU145 cells. (A and B) DU145 cells were incubated with LPS (2 μg/mL) for indicated time. (A) Phosphorylated β-catenin in cytoplasm was measured. (B) Nuclear β-catenin was measured. (C) DU145 cells were incubated with LPS (2 μg/mL) with/without melatonin (2 mM) for 2 h. Phosphorylated β-catenin in cytoplasm was measured. (D) DU145 cells were incubated with LPS (2 μg/mL) in absence or presence of melatonin (2 mM) for 6 h. Nuclear β-catenin was measured. (E and F) DU145 cells were incubated with melatonin for indicated dose. The levels of β-catenin in (E) cytoplasm and (F) nuclear were measured. (G and H) DU145 cells were incubated with LPS (2 μg/mL) in absence or presence of melatonin (2 mM) for 2 h. (G) p-GSK3βSer9 in “destruction complex” was determined using Co-IP. (H) Ubiquitination of cytoplasm β-catenin was determined using Co-IP. All experiments were repeated three times. Data were expressed as means ± S.E.M., *P<0.05, **P<0.01 versus control group, ##P<0.01 versus LPS group.
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Grants and funding

This research was funded by the Key Project of the National Natural Science Foundation of China (Grant no. 81630084).