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Melatonin potentiates cisplatin-induced apoptosis and cell cycle arrest in human lung adenocarcinoma cells - PubMed

Melatonin potentiates cisplatin-induced apoptosis and cell cycle arrest in human lung adenocarcinoma cells

P Plaimee et al. Cell Prolif. 2015 Feb.

Abstract

Objectives: Melatonin produces anti-cancer effects via several mechanisms, including by induction of apoptosis. In this way, it has been shown to be of use, in combination with chemotherapeutic drugs, for cancer treatment. The study described here has evaluated effects of melatonin on cytotoxicity, apoptosis and cell cycle arrest induced with the chemotherapeutic agent cisplatin, in human lung adenocarcinoma cisplatin-sensitive cell line (SK-LU-1), which previously had only limit data.

Materials and methods: Cells of the SK-LU-1 line were treated with melatonin alone at 1-5 mM concentration or cisplatin alone 10-200 μM, for 48 h in culture. Cytotoxicity was measured by MTT reduction assay. Apoptosis induction was detected by annexin V/PI staining using flow cytometric analysis and DAPI nuclear staining. Change in mitochondrial membrane potential (ΔΨm) was quantified using DiOC6(3) reagent and activities of caspases-3/7 were also investigated. DNA fractions were measured using propidium iodide (PI) staining.

Results: Melatonin or cisplatin alone had 50% (IC50 ) cytotoxicity at 5 mM or 34 μM concentrations respectively. Combination of 1 or 2 mM melatonin and cisplatin significantly augmented cytotoxicity of cisplatin by reducing its IC50 to 11 and 4 μM, respectively. Consistently, combined treatment increased population of apoptotic cells by elevating mitochondrial membrane depolarization, activating caspases-3/7 and inducing cell cycle arrest in the S phase, compared to treatment with cisplatin alone.

Conclusion: These data demonstrate that melatonin enhanced cisplatin-induced cytotoxicity and apoptosis in SK-LU-1 lung cancer cells. SK-LU-1 cell population growth inhibition was mediated by cell cycle arrest in the S phase. These findings suggest that melatonin has the potential to be used for NSCLC treatment in combination with a chemotherapeutic agent such as cisplatin.

© 2015 John Wiley & Sons Ltd.

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Figures

Figure 1
Figure 1

Effect of melatonin (a), cisplatin (b) or the combination of melatonin with cisplatin (c) on cytotoxicity of

SK

‐

LU

‐1 for 48 h, evaluated by

MTT

reduction assay. Chemical structure and cytotoxicity of melatonin (C13H16N2O2, molecular weight (MW) = 232.28) and cisplatin (Cl2(NH3)2Pt, MW = 300.04) are shown in dose dependent manner in (a) and (b), respectively. (c) The cytotoxicity of the combination of 1 or 2 m

m

melatonin and cisplatin (10–200 μ

m

) are also shown in

SK

‐

LU

‐1 cells. (d) Morphological changes of

SK

‐

LU

‐1 cells were observed under inverted microscopy (20× magnification) after treatment with melatonin or cisplatin (50 μ

m

) or combination treatment for 48 h. Cytotoxicity is expressed as a percentage versus controls, cultured without any treatment. Five samples were averaged for each group. Error bars show

SD

of five samples. *P < 0.05 versus untreated cells, a P < 0.05 versus cisplatin treated alone.

Figure 2
Figure 2

Effect of melatonin, cisplatin and the combination of melatonin with cisplatin on the induction of apoptosis of

SK

‐

LU

‐1. Flow cytometry analyses of

SK

‐

LU

‐1 cells using double staining with annexin V (Annexin V, horizontal line) and propidium iodide (

PI

, vertical line); (a) untreated group; melatonin treated group at (b) 1 m

m

and (c) 2 m

m

; (d) cisplatin treated group (50 μ

m

); (e) combination treatment of 1 m

m

melatonin and cisplatin (50 μ

m

) and (f) 2 m

m

melatonin and cisplatin (50 μ

m

). Flow cytometry apoptotic results are shown in four subpopulations which indicate: early apoptotic cells (lower right), late apoptotic cells (upper right), normal cells (lower left) and necrotic cells (upper left). (g) The level of

SK

‐

LU

‐1 cell apoptosis expressed as the percentage of apoptotic cells are depicted in the graph (mean ± SD of at least three samples). (h) Correlation graphs of apoptotic cells and cytotoxicity of cisplatin treated

SK

‐

LU

‐1 alone and combination treatment are shown. Error bars show

SD

of three samples. a P < 0.05 versus cisplatin treated alone.

Figure 3
Figure 3

Apoptotic bodies of

SK

‐

LU

‐1 after treatment with melatonin, cisplatin and the combination of melatonin and cisplatin detected by

DAPI

staining; (a) untreated group; melatonin treated group at (b) 1 m

m

and (c) 2 m

m

; (d) cisplatin treated group (50 μ

m

); (e) combination treatment of 1 m

m

melatonin and cisplatin (50 μ

m

) and (f) 2 m

m

melatonin and cisplatin (50 μ

m

). Apoptotic bodies were observed under inverted fluorescence microscopy (20× magnification).

Figure 4
Figure 4

Effect of melatonin, cisplatin and the combination of melatonin with cisplatin on the mitochondrial membrane potential (ΔΨm) of

SK

‐

LU

‐1. Flow cytometry analyses of loss of ΔΨm

SK

‐

LU

‐1 cells using Di

OC

6(3) staining. (a) The level of loss of ΔΨm

SK

‐

LU

‐1 cells expressed as a bar graph (mean ± SD of duplicate experiments). (b) Correlation graphs of the loss of ΔΨm cells and apoptotic cells or cytotoxicity of cisplatin treated

SK

‐

LU

‐1 alone or the combination treatment with melatonin (1 or 2 m

m

) are shown. a P < 0.05 versus cisplatin treated alone.

Figure 5
Figure 5

The activity of caspase‐3/7 in SK‐LU‐1 cells after treatment with melatonin, cisplatin and the combination of melatonin with cisplatin. Caspases activity was assessed using caspase‐Glo assay. The incremental increase of caspases activity is expressed as a percent versus control, untreated group. Error bars show SD of at least two experiments (n = 6). *P < 0.05 versus untreated cells, a P < 0.05 versus cisplatin treated alone.

Figure 6
Figure 6

Alterations of the cell cycle measured in the

SK

‐

LU

‐1 cells after treatment with melatonin, cisplatin and the combination of melatonin with cisplatin; duplicate experiments were performed while the

DNA

fraction profile from one experiment is shown; (a) untreated group; melatonin treated group at (b) 1 m

m

and (c) 2 m

m

; (d) cisplatin treated group (50 μ

m

); (e) combination treatment of 1 m

m

melatonin and cisplatin (50 μ

m

) and (f) 2 m

m

melatonin and cisplatin (50 μ

m

). (g) Percentage of cell numbers of each cell cycle phase in each group of treatment are shown as a bar graph (mean ± SD of duplicate experiments). *P < 0.05 versus untreated cells, a P < 0.05 versus cisplatin treated alone.

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