Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Mar 23;12(6):5423-5438.
doi: 10.18632/aging.102968. Epub 2020 Mar 23.

Melatonin reverses nasopharyngeal carcinoma cisplatin chemoresistance by inhibiting the Wnt/β-catenin signaling pathway

Jian Zhang  1 , Tao Xie  1 , Xi Zhong  2 , Hua-Li Jiang  3 , Rong Li  1 , Bai-Yao Wang  1 , Xiao-Ting Huang  1 , Bo-Hong Cen  1 , Ya-Wei Yuan  1
Affiliations

Melatonin reverses nasopharyngeal carcinoma cisplatin chemoresistance by inhibiting the Wnt/β-catenin signaling pathway

Jian Zhang et al. Aging (Albany NY). .

Abstract

Cisplatin (DDP)-based concurrent chemo-radiotherapy is a standard approach to treat locoregionally advanced nasopharyngeal carcinoma (NPC). However, many patients eventually develop recurrence and/or distant metastasis due to chemoresistance. In this study, we aimed to elucidate the effects of melatonin on DDP chemoresistance in NPC cell lines in vitro and vivo, and we explored potential chemoresistance mechanisms. We found that DDP chemoresistance in NPC cells is mediated through the Wnt/β-catenin signaling pathway. Melatonin not only reversed DDP chemoresistance, but also enhanced DDP antitumor activity by suppressing the nuclear translocation of β-catenin, and reducing expression of Wnt/β-catenin response genes in NPC cells. In vivo, combined treatment with DDP and melatonin reduced tumor burden to a greater extent than single drug-treatments in an orthotopic xenograft mouse model. Our findings provide novel evidence that melatonin inhibits the Wnt/β-catenin pathway in NPC, and suggest that melatonin could be applied in combination with DDP to treat NPC.

Keywords: Wnt/β-catenin; chemoresistance; cisplatin; melatonin; nasopharyngeal carcinoma.

PubMed Disclaimer

Conflict of interest statement

CONFLICTS OF INTEREST: The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
DDP chemoresistance in NPC cells is mediated through activation of the Wnt/β-catenin pathway. (A) Heatmap of differentially expressed genes in 5-8F and 5-8F/DDP cell lines by RNA sequencing. (B) Differentially enriched Wnt/β-catenin pathway-related signatures between 5-8F and 5-8F/DDP cell lines, determined by gene set enrichment analysis (GSEA). (C) Protein expression of Wnt/β-catenin in the CNE2 and CNE2/DDP, 5-8F and 5-8F/DDP cell lines, as determined by western blot. (D, E) mRNA expression of the Wnt/β-catenin downstream genes (AXIN2, SOX9, CD44 and CCND2) in CNE2 and CNE2/DDP cell lines (D) and 5-8F and 5-8F/DDP cell lines (E) as determined by qPCR. All of the experiments were performed at least three times. Data presented are the mean ± SD; **P < 0.01 compared with using the Student t-test.
Figure 2
Figure 2
Melatonin reverses DDP chemoresistance by inhibiting β-catenin nuclear translocation. (A, B) Protein expression of β-catenin, DKK1, c-Myc and CylincD1 in the CNE2 and CNE2/DDP (A), 5-8F and 5-8F/DDP cell lines (B) treated with melatonin (2 mM) for 48 hr, as determined by western blot. (C) mRNA expression of Wnt/β-catenin downstream genes (AXIN2, SOX9, CD44 and CCND2) in CNE2 and CNE2/DDP, 5-8F and 5-8F/DDP cell lines treated with melatonin (2 mM) for 48 hr, as determined by qPCR. (D, E) Representative images of immunofluorescent staining for β-catenin in CNE2 and CNE2/DDP (D), 5-8F and 5-8F/DDP (E) treated with or without melatonin (2 mM) for 48 hr. (F) Relative luciferase activity of NPC cells transfected with the TOP/FOPFlash vector and pRL-TK vector. Data presented are the mean ± SD; **P < 0.01 compared with control using Student t-test.
Figure 3
Figure 3
Melatonin inhibits malignant properties of NPC cells. (A) Representative images (left panel) and quantification (right upper panel) of cell apoptosis in the indicated cells treated with melatonin (Mel, 48 hr), as determined by AnnexinV/propidium iodide (PI) assay. (B) The cell viability of the indicated cells incubated with melatonin (72 hr) was determined by CCK-8 assay. (C, D) Images and quantification of migrated (C) and invaded (D) NPC cells treated with melatonin (2 mM) for 24 hr were analyzed in transwell assays. Data presented are the mean ± SD; **P < 0.01 compared with control using Student t-test.
Figure 4
Figure 4
Melatonin reverses DDP chemoresistance in NPC DDP-resistant cells. (A, B) The cell viability of the CNE2/DDP (A) and 5-8F/DDP (B) cells treated with melatonin (2 mM) was determined by CCK-8 assay. (C, D) Images (C) and quantification (D) of transwell migration and invasion assays. (E) Images (up) and quantification (down) clonogenic colony formation assays of the CNE2/DDP and 5-8F/DDP cells treated with melatonin for 2 weeks. All of the experiments were performed at least three times. Data presented are the mean ± SD; **P < 0.01, *P < 0.05 compared with control using Student t-test.
Figure 5
Figure 5
Effects of melatonin and DDP combination on malignant properties of nasopharyngeal carcinoma (NPC) cells. (A–D) The cell viability of the CNE2 (A), 5-8F (B), HONE1 (C) and SUNE1 (D) cells incubated with melatonin (72 hr) (green line) alone and DDP alone (blue line) or melatonin with DDP combination (red line) was determined by CCK-8 assay. (E, F) Images (E) and quantification (F) clonogenic colony formation assays of the indicated cells treated with melatonin for 2 weeks. All of the experiments were performed at least three times. Data presented are the mean ± SD; **P < 0.01, *P < 0.05 compared with control using Student t-test.
Figure 6
Figure 6
Antitumor activity of melatonin plus DDP in vivo in a lung metastasis model. (A) Tumor progression timeline with experimental treatment time points. (B) Tumor cell luminescence from each mouse in each group was acquired on treatment day 28. (C, D) Representative front images (C) and back images (D) of macroscopic lung metastases, arrowheads indicate the metastatic nodes. (E) Quantification of the average number of macroscopic metastatic nodes formed on the lung surface. (F) Paraffin-embedded tumor sections were stained with H&E, anti-Ki67, and β-catenin antibodies (Scar bar: 100 μm). Right panel: quantification of the proliferation index and β-catenin index in tumor sections. These animal experiments were repeated once (n = 3 mice per treatment group). Data presented are the mean ± SD; **P < 0.01 compared with control using Student t-test.
Figure 7
Figure 7
Proposed model of the combinatorial effect of DDP and melatonin on NPC cells.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Chen W, Zheng R, Baade PD, Zhang S, Zeng H, Bray F, Jemal A, Yu XQ, He J. Cancer statistics in China, 2015. CA Cancer J Clin. 2016; 66:115–32. 10.3322/caac.21338 - DOI - PubMed
    1. Chua ML, Wee JT, Hui EP, Chan AT. Nasopharyngeal carcinoma. Lancet. 2016; 387:1012–24. 10.1016/S0140-6736(15)00055-0 - DOI - PubMed
    1. Lai SZ, Li WF, Chen L, Luo W, Chen YY, Liu LZ, Sun Y, Lin AH, Liu MZ, Ma J. How does intensity-modulated radiotherapy versus conventional two-dimensional radiotherapy influence the treatment results in nasopharyngeal carcinoma patients? Int J Radiat Oncol Biol Phys. 2011; 80:661–68. 10.1016/j.ijrobp.2010.03.024 - DOI - PubMed
    1. Amable L. Cisplatin resistance and opportunities for precision medicine. Pharmacol Res. 2016; 106:27–36. 10.1016/j.phrs.2016.01.001 - DOI - PubMed
    1. Chen SH, Kuo CC, Li CF, Cheung CH, Tsou TC, Chiang HC, Yang YN, Chang SL, Lin LC, Pan HY, Chang KY, Chang JY. O(6) -methylguanine DNA methyltransferase repairs platinum-DNA adducts following cisplatin treatment and predicts prognoses of nasopharyngeal carcinoma. Int J Cancer. 2015; 137:1291–305. 10.1002/ijc.29486 - DOI - PubMed

Publication types