Melatonin enhances sensitivity to fluorouracil in oesophageal squamous cell carcinoma through inhibition of Erk and Akt pathway - PubMed (original) (raw)

. 2016 Oct 27;7(10):e2432.

doi: 10.1038/cddis.2016.330.

Dong-Liang Chen 1 2, De-Shen Wang 1 2, Le-Zong Chen 1 2, Hai-Yu Mo 1, Hui Sheng 1, Long Bai 1 2, Qi-Nian Wu 1, Hong-En Yu 1 2, Dan Xie 1, Jing-Ping Yun 1 3, Zhao-Lei Zeng 1, Feng Wang 1 2, Huai-Qiang Ju 1, Rui-Hua Xu 1 2

Affiliations

Melatonin enhances sensitivity to fluorouracil in oesophageal squamous cell carcinoma through inhibition of Erk and Akt pathway

Yun-Xin Lu et al. Cell Death Dis. 2016.

Abstract

Oesophageal squamous cell carcinoma (ESCC) is the sixth most common cause of cancer-associated death in the world and novel therapeutic alternatives are urgently warranted. In this study, we investigated the anti-tumour activity and underlying mechanisms of melatonin, an indoleamine compound secreted by the pineal gland as well as naturally occurring plant products, in ESCC cells and revealed that melatonin inhibited proliferation, migration, invasion and induced mitochondria-dependent apoptosis of ESCC cells in vitro and suppressed tumour growth in the subcutaneous mice model in vivo. Furthermore, after treatment with melatonin, the expressions of pMEK, pErk, pGSK3β and pAkt were significantly suppressed. In contrast, treatment of the conventional chemotherapeutic drug fluorouracil (5-Fu) resulted in activation of Erk and Akt, which could be reversed by co-treatment with melatonin. Importantly, melatonin effectively enhanced cytotoxicity of 5-Fu to ESCC in vitro and in vivo. Together, these results suggested that inhibition of Erk and Akt pathway by melatonin have an important role in sensitization of ESCC cells to 5-Fu. Combined 5-Fu and melatonin treatment may be appreciated as a useful approach for ESCC therapy that warrants further investigation.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Melatonin inhibits proliferation, colony formation, migration and invasion of ESCC cells. (a) Viability of the indicated cells exposed to melatonin at different concentrations (72 h) was detected with MTS kit. (b) MTS assay of Eca109 (left panel) and KYSE150 (right panel) cells treated with melatonin (Control, 1 mM or 2 mM) at indicated time points. (c) Representative images (upper panel) and quantification (lower panel) of colony formation of the indicated cells cultured with melatonin at different concentrations for 14 days. (d) Representative images (left panel) and quantification (right panel) of colony formation of NE1 and NE3 cells cultured with melatonin at different concentrations for 14 days. Representative images and quantification of migration (e) and invasion (f) assay of the indicated cells treated with melatonin (Control, 1 mM or 2 mM) for 24 h. (g) Immunoblotting of CCND1, PCNA of cell extracts from Eca109 and KYSE150 cells after treated with indicated concentrations of melatonin for 24 h. _β_-Actin was used as loading control. Data in (a), (b), (c), (d), (e) and (f) are presented as mean±S.E. derived from three individual experiments with triplicate wells. **P<0.01 versus corresponding control. ns, no significant. Error bars, S.E.

Figure 2

Figure 2

Melatonin induces mitochondria-dependent apoptosis of ESCC cells. (a) Representative images of mitochondrial transmembrane potential (left panel) and quantification (right panel) of cells negative for rhodamine staining in Eca109, KYSE150, KYSE510 cells treated with melatonin (Control, 4 mM, 6 mM) for 24 h. (b) Representative images of Annexin-V/PI assays (left panel) and quantification (right panel) of dual negative percentage in Eca109, KYSE150, KYSE510 cells treated with melatonin (Control, 4 mM, 6 mM, 8 mM) for 24 h. (c) Relative caspase 3/7 activity of Eca109, KYSE150 and KYSE510 cells treated with melatonin (Control, 4 mM, 6 mM, 8 mM) for 24 h. (d) Immunoblotting of PARP in the indicated cells treated with melatonin (Control, 4 mM, 8 mM) for 24 h. _β_-Actin was used as a loading control. Data in (a), (b) and (c) are presented as mean±S.E. derived from three individual experiments with triplicate wells. **P<0.01 versus corresponding control. Error bars, S.E.

Figure 3

Figure 3

Melatonin inhibits MEK/Erk and GSK3β/Akt pathway in ESCC cells. (a) Immunoblotting of pMEK, MEK, pErk, Erk of cell extracts from Eca109 and KYSE150 cells after treated with indicated concentrations of melatonin for 24 h. GAPDH was used as a loading control. (b) Immunoblotting of pGSK3β, GSK3β, pAkt, Akt of cell extracts from Eca109 and KYSE150 cells after treated with indicated concentrations of melatonin for 24 h. GAPDH was used as a loading control. (c) Quantification of migration assays in Eca109 and KYSE150 cells treated with melatonin (1 mM), MEK inhibitor selumetinib (10 nM) or GSK3β inhibitor BIO (1 nM) for 24 h. Data in (c) are presented as mean±S.E. derived from three individual experiments with triplicate wells. *P<0.05 and **P<0.01 versus corresponding control. Error bars, S.E.

Figure 4

Figure 4

Melatonin inhibitis 5-Fu induced Erk and Akt phosphorylation. (a) Eca109 and KYSE150 cells were treated with 5-Fu (Control, 5 μM, 10 μM, 20 μM) for 24 h. Expression of pErk, Erk, pAkt, Akt was detected by western blot. GAPDH was used as a loading control. (b) Immunobloting of p-Erk, Erk, pAkt, Akt in Eca109 and KYSE150 cells treated with DMSO, melatonin (5 mM), 5-Fu (10 μM) or both agents for 24 h. GAPDH was used as a loading control. (c) Quantification analysis of pErk and pAkt expression in Eca109 and KYSE150 cells treated with DMSO, melatonin (5 mM), 5-Fu (10 μM) or both agents. Data in (c) are presented as mean±S.E. derived from three individual experiments with triplicate wells. **P<0.01 versus corresponding control. Error bars, S.E.

Figure 5

Figure 5

Synergistic effects between melatonin and 5-Fu in ESCC cells in vitro. (a) Cell viability of Eca109 (upper panel) and KYSE150 (lower panel) cells treated with 5-Fu alone or combined with melatonin (0.5 mM) at indicated concentrations was detected by MTS. (b) Representative images (left panel) and quantification (right panel) of migration assays in Eca109 and KYSE150 cells treated with 5-Fu (1 μM) and melatonin (1 mM) for 24 h. (c) Representative images (left panel) and quantification (right panel) of invasion assays in the indicated cells treated with 5-Fu (1 μM) and melatonin (1 mM) for 24 h. (d) Representative images (upper panel) and quantification (lower panel) of colony formation in Eca109 and KYSE150 cells treated with 5-Fu (0.5 μM) and melatonin (1 mM) for 14 days. (e) Representative images (upper panel) and quantification (lower panel) of Annexin-V/PI assays in the indicated cells treated with 5-Fu (10 μM) and melatonin (6 mM) for 24 h. (f) Immunoblotting of PARP in Eca109 and KYSE150 cells treated with 5-Fu (10 μM) and melatonin (6 mM) for 24 h. _β_-Actin was used as a loading control. Data in (a), (b), (c), (d) and (e) are presented as mean±S.E. derived from three individual experiments with triplicate wells. *P<0.05 and **P<0.01 versus corresponding control. Error bars, S.E.

Figure 6

Figure 6

Melatonin enhance sensitivity to 5-Fu in ESCC cells in vivo. (a) Eca109 (1 × 106/mouse) cells were subcutaneously inoculated into the dorsal flank of nude mice before they were treated with PBS, 5-Fu (20 mg/kg, twice per week), melatonin (20 mg/kg, once per day), or 5-Fu combined with melatonin. Tumour volumes were measured at indicated days. Data are shown as mean±S.E. of six mice in each group. (b) Excised tumour weight from the four separate groups was recorded. (c) Weight of the mice was recorded. (d) Left panel: representative hematein-eosin (H&E) and immunohistochemistry staining of pErk, pAkt, and Ki67 from tumour sections. Scale bar: 50 μm. Right panel: quantification of pErk, pAkt and Ki67 immunoreactivity in tumour sections. Data in (a), (b), (c) and (d) are presented as mean±S.E. (_n_=6). *P<0.05 and **P<0.01 versus corresponding control. Error bars, S.E.

Similar articles

Cited by

References

    1. Rustgi AK, El-Serag HB. Esophageal carcinoma. N Engl J Med 2014; 371: 2499–2509. - PubMed
    1. Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol: Official Journal of the American Society of Clinical Oncology 2006; 24: 2137–2150. - PubMed
    1. Ohashi S, Miyamoto S, Kikuchi O, Goto T, Amanuma Y, Muto M. Recent advances from basic and clinical studies of esophageal squamous cell carcinoma. Gastroenterology 2015; 149: 1700–1715. - PubMed
    1. Chen W, Zheng R, Zeng H, Zhang S. The updated incidences and mortalities of major cancers in China, 2011. Chin J Cancer 2015; 34: 502–507. - PMC - PubMed
    1. Pennathur A, Gibson MK, Jobe BA, Luketich JD. Oesophageal carcinoma. Lancet 2013; 381: 400–412. - PubMed

MeSH terms

Substances

LinkOut - more resources