The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells

Abstract

There is an urgent need to identify targeting molecules to control invasion and metastasis in cancer patients. We first isolated cancer stem cells (CSCs) from SKOV3 ovarian cancer cells and then investigated the role of melatonin in invasiveness and migration of CSCs compared to SKOV3 cells. The proportion of CSCs in SKOV3 cells was as low as 1.28% with overexpression of both CD133 and CD44. The ability of spheroid formation along with SOX2 overexpression revealed a high self-renewal potential in isolated cells. Melatonin (3.4 mM) inhibited proliferation of CSCs by 23% which was confirmed by a marked decrease in protein expression of Ki67, as a proliferation marker. Applying luzindole, a melatonin receptor 1, 2 inhibitor, partially abolished anti-proliferative effect of melatonin. Melatonin also decreased Epithelial mesenchymal transition (EMT) related gene expressions including ZEB1, ZEB2, snail and vimentin with increase in E-cadherin as a negative EMT regulator. Incubation of CSCs with melatonin showed a marked decrease in matrix metalloproteinase 9 (MMP9) expression and activity. Melatonin also inhibited CSCs migration in a partially receptor dependent and PI3k and MAPK independent manner. Melatonin can be considered as an important adjuvant to control invasion and metastasis especially in patients with high melatonin receptor expression.

Figure 1

Cancer stem cells (CSCs) isolation and characterization from human ovarian SKOV3 cell line. (a) SKOV3 cells were labeled with FITC-conjugated anti-CD133 and PE-conjugated anti-CD44 antibodies, and analyzed by a flow cytometer as described in Materials and methods. (b) Immunofluorescence staining of CD133 and CD44 in CSCs and SKOV3 cell line. Cells were directly labeled using FITC-conjugated anti-CD133 (green) and PE-conjugated anti-CD44 (red) antibodies; nuclei were stained blue with 4′,6-diamidino-2-phenylindole (DAPI). (c) SOX2 protein expression was stained using FITC-conjugated anti-SOX2 and analyzed by Flow cytometry in SKOV3 cells, immediately after isolation and after 48 h incubation in medium supplemented by 2% fetal bovine serum (FBS). (d) SOX2 mRNA expression in CSCs was carried out by real-time RT-PCR and compared with that expression in SKOV3 cells. Data are expressed as mean ± SD of three independent experiments. Statistically significant differences are indicated as *p < 0.05. (e) Cells (3 × 104) were cultured in serum free media supplemented with 10 µM epidermal growth factor (EGF) and 10 µM basic fibroblast growth factor (bFGF) in non-treated six-well plates for seven days and spheroid formation was visualized by light microscopy in both CSCs and SKOV3 cells.

Figure 2

Melatonin effects on proliferation and stemness properties of cancer stem cells (CSCs). (a) Inhibitory effects of melatonin on viability of SKOV3 cells. (b) The effects of melatonin (IC50 value = 3.4 mM) on viability of CSCs and SKOV3 cells were determined after 48 h incubation. (c) The protein expression of Ki67 proliferation marker was determined after incubation of the cells with melatonin (3.4 mM) for 48 h in both CSCs and SKOV3 cells by applying FITC conjugated anti-Ki67 antibody and analyzed by flow cytometry. (d) Nanog mRNA expression in CSCs was carried out by real-time RT-PCR and compared with that expression in SKOV3 cells after incubation of the cells with melatonin (3.4 mM) for 48 h. (e) The role of melatonin in SOX2 stemness marker expression was evaluated after incubation of the cells with melatonin (3.4 mM) for 48 h using real-time RT PCR analysis Results are presented as mean ± SD of three independent experiments. Statistically significant differences are indicated as *p < 0.05 and **p < 0.01.

Figure 3

Effects of melatonin on MT1 and MT2 melatonin receptors in cancer stem cells (CSCs) and SKOV3 cells. MT1 (a) and MT2 (b) mRNA expression levels were determined by real-time RT PCR after incubation of both CSCs and SKOV3 cells after with and without melatonin (3.4 mM) for 48 h. Relative changes in gene expression levels were determined using the Pfaffl method. Data, normalized against GAPDH, are presented as fold change from the control. (c) SKOV3 and isolated CSCs were treated with the 3.4 mM melatonin for 48 hr. The cell lysates were prepared and used for Western blot with MT1, MT2 and b-actin antibodies. (d) The role of melatonin receptors on melatonin–induced changing in cell viability was evaluated after pretreatment of the cells with a pan melatonin receptor inhibitor (luzindole) and then incubation of the cells with melatonin (3.4 mM) for 48 h using MTT assay. All experiments were performed in triplicate and data were expressed as mean ± SD. *p < 0.05 and **p < 0.01.

Figure 4

The role of melatonin in invasion activity of cancer stem cells (CSCs). (a) MMP-2 mRNA expression levels were determined by real-time RT PCR after 48 h incubation of both CSCs and SKOV3 cells with melatonin (3.4 mM). (b) Equal amounts of extracted protein following incubation with and without melatonin (3.4 mM) for 48 h were subjected to gelatin zymography to determine MMP-2 activity. (c) The role of melatonin in MMP-9 expression level was evaluated after incubation of the cells with melatonin (3.4 mM) for 48 h using real-time RT PCR analysis. (d) MMP-9 activity was determined by gelatin zymography after incubation with or without 3.4 mM melatonin for 48 h. (e–i) mRNA levels of ZEB1, ZEB2, Snail, vimentin and E-cadherin quantified by real-time RT PCR analysis. Relative changes in gene expression levels were determined using the Pfaffl method. Data, normalized against GAPDH, were presented as fold change from the control. All experiments were performed in triplicate and data were expressed as mean ± SD. *p < 0.05 and **p < 0.01.

Figure 5

Effects of melatonin on migration ability in ovarian cancer stem cells (CSCs) and SKOV3 cells. (a) SKOV3 and isolated CSCs were treated with the 3.4 mM melatonin for 48 hr. The cell lysates were prepared and used for Western blot with p-ERK1/2, total ERK, p-Akt, total Akt and b-actin antibodies. (b) Cells were pretreated with MAPK inhibitor (PD98059) (40 µM), PI3K inhibitor (LY294002) (20 µM) and Melatonin receptor antagonist (luzindole) (10 µM) for 1 h and then incubated with melatonin (3.4 mM) for 48 h. Migrated cells in basal media (RPMI 1640 with 2% FBS) was compared to migration in the presence of melatonin with or without inhibitors. All experiments were performed in triplicate and data were expressed as mean ± SD. *p < 0.05 and **p < 0.01.