Melatonin regulates cancer migration and stemness and enhances the anti‐tumour effect of cisplatin

Abstract Melatonin, a lipophilic hormone released from the pineal gland, has oncostatic effects on various types of cancers. However, its cancer treatment potential needs to be improved by deciphering its corresponding mechanisms of action and optimising therapeutic strategy. In the present study, melatonin inhibited gastric cancer cell migration and soft agar colony formation. Magnetic‐activated cell sorting was applied to isolate CD133+ cancer stem cells. Gene expression analysis showed that melatonin lowered the upregulation of LC3‐II expression in CD133+ cells compared to CD133− cells. Several long non‐coding RNAs and many components in the canonical Wnt signalling pathway were altered in melatonin‐treated cells. In addition, knockdown of long non‐coding RNA H19 enhanced the expression of pro‐apoptotic genes, Bax and Bak, induced by melatonin treatment. Combinatorial treatment with melatonin and cisplatin was investigated to improve the applicability of melatonin as an anticancer therapy. Combinatorial treatment increased the apoptosis rate and induced G0/G1 cell cycle arrest. Melatonin can regulate migration and stemness in gastric cancer cells by modifying many signalling pathways. Combinatorial treatment with melatonin and cisplatin has the potential to improve the therapeutic efficacy of both.


| INTRODUC TI ON
Cancer cells can spread directly from the primary lesion to distal sites, or metastasize, by invading the lymphatic system or the bloodstream, thereby forming metastatic cancer. Metastasis makes surgical and locoregional therapy for cancer extremely difficult. In addition, metastasis is often associated with cancer stem cells identified with high global and confidence biomarker scores, 1,2 and cancer stem cells are considered as the leading factor of chemotherapeutic resistance. 2 Therefore, the development of advanced anticancer drugs and treatment strategies is necessary to inhibit cancer cell migration. Some chemicals secreted by the body may exhibit excellent cancer-inhibiting effects. Melatonin, whose secretion is controlled by circadian rhythms, is a hormone synthesized and secreted mainly by the pineal gland. 3 Many studies have shown that melatonin can effectively inhibit all stages of cancer development and enhance the sensitivity of cancer cells to various anticancer drugs. [4][5][6] Melatonin is a pleiotropic anticancer molecule that regulates malignant cells via multiple mechanisms, including enhancement of mitochondrial reactive oxygen species, 7 targeting of Wnt/beta-catenin signalling, 8 regulation of histone modification, 9,10 etc. In our previous study, melatonin was shown to suppress gastric cancer by modulating endoplasmic reticulum (ER) stress, autophagy and the Ras-Raf-ERK signalling pathway. 11 However, a greater effort is needed to improve the utility of melatonin as an anticancer treatment. It is especially important to further uncover the role of melatonin in metastasis and discover suitable strategies to promote its application in cancer treatment by minimising the potential therapeutic risks.
Various studies have suggested that single-drug chemotherapy could cause cancer cells to develop resistance, thus greatly reducing the effectiveness of cancer treatment. Combination therapies could reduce the likelihood of acquired resistance, shorten the duration of treatment and speed up recovery. 12,13 Cisplatin, a platinumcontaining anticancer drug, is a non-specific modulator of the cell cycle and exhibits a strong anticancer effect by interacting with N 7guanine and N 3 -adenine on cancer cells, damaging the structures on the cell membrane, and inhibiting replication of cancer cells, ultimately leading to cancer cell necroptosis. [14][15][16][17] However, cisplatin also causes severe toxicities, including nephrotoxicity, peripheral neuropathy and ototoxicity. Long-term exposure to cisplatin could cause cancer cells to develop drug resistance. 16,18 We conducted an in-depth study of the anticancer effects of melatonin and cisplatin combination therapy with a view to identifying new diagnostic markers and developing appropriate therapeutic approaches for cancer patients, as well as to broadening the application of both melatonin and cisplatin in cancer therapy.

| Cell culture
Human gastric cancer cells, SGC-7901, were cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 1% glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in 5% CO 2 atmosphere. Melatonin (Sigma-Aldrich) was prepared in dimethyl sulfoxide (DMSO) at 5.0 mol/L and diluted with medium to create different concentrations.

| Flow cytometry analysis of apoptosis
After incubation with melatonin (2.5 mmol/L) and/or cisplatin (0.25, 0.5, 2.5 or 5.0 mmol/L) for 24 h, cells were digested with 0.25% trypsin, collected after centrifugation by gentle blowing and washed twice with cold phosphate buffer. The Annexin V-FITC/PI apoptosis detection kit was then applied. In brief, 100 μL of 1× binding buffer was added to dilute the precipitate, followed by addition of 5 μL of Annexin V-FITC and 5 μL of PI. After incubation at room temperature (in the dark) for 15 min, 400 μL of 1× binding buffer was added and the solution was immediately analysed using flow cytometry (Fortessa, BD Biosciences).

| Cell cycle distribution analysis
Gastric cancer cells were treated with melatonin and/or cisplatin for 24 h, collected in 0.1% trypsin and 2.5 mmol/L EDTA, and washed using cold phosphate buffer. The cells were fixed using cold 70% ethanol and refrigerated overnight at −20°C. They were then washed with phosphate buffer, and 500 μL of PI/RNase staining buffer was added to them. The cells were incubated for 30 min at room temperature then analysed using a flow cytometer (Fortessa, BD Biosciences).

| Real-time quantitative PCR
Real-time quantitative PCR (RT-qPCR) was used to determine gene expression at transcriptional level in gastric cancer cells. RNA was isolated using TRIzol reagent, and the purity of RNA was determined according to the instructions. Briefly, 2 μg of RNA was reversetranscribed into complementary cDNA using the All-in-One cDNA Synthesis SuperMix kit (Bimake). RT-qPCR was performed with 2× SYBR Green qPCR Master Mix (Bimake) on a CFX96 real-time PCR detection system. Relative expression of target genes was calculated using the 2 −ΔΔCt method based on analysis of amplification and melting curves. All experiments were repeated three times.

| Western blotting
After being washed with PBS twice, the cells were lysed with phenylmethane sulfonyl fluoride lysis buffer. Total protein was collected by centrifugation and concentration was measured using a bicinchoninic acid protein assay kit (Beyotime). Next, 20 μg of the extracted protein samples was separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk and incubated at room temperature for 1 h. The primary antibody was added and incubated overnight at 4°C. After being washed with TBST three times, the membranes were incubated for 1 h with the appropriate amount of secondary antibody.
Protein bands were visualized using an enhanced chemiluminescence detection system.

| Soft agar colony formation assay
To each well of a 6-well plate, 1.5 mL of 0.6% agar containing 10% FBS, which served as the agar base, was added and allowed to solidify. Then, 500 μL of 0.6% agar solution was mixed with 500 μL of DMEM containing 20% FBS and 3000 gastric cancer cells treated with 0, 1.0, 2.5 or 5.0 mM melatonin. These mixtures were seeded on the top of the agar base and allowed to solidify. Cells were then incubated in a 37°C, humidified, 95% air and 5% CO 2 atmosphere to form colonies. Next, 100 μL of DMEM containing 10% FBS was added to the wells every 3 days. The colony assay was terminated at Day 21 and colonies were stained with 0.1% crystal violet solution.
Colonies with a diameter larger than 50 μm were counted and visualized under a microscope.

| Cell migration assay
The cell migration assay was performed using 24-well-plate transwell chambers (8 μm pore size). In brief, 2 × 10 4 gastric cancer cells in The migrated cells were then observed using an optical microscope.

| Isolation of CD133 + cancer stem cells with magnetic-activated cell sorting system
Treated gastric cancer cells were harvested, and CD133 + cells were isolated using the MACS indirect CD133 MicroBead kit (MiltenyiBiotec) according to standard enrichment procedures.
In brief, cells were labelled with CD133 micro-magnetic beads and incubated at 4°C for 30 min in the dark. CD133 + and CD133 − cells were isolated separately using the sorting column. Cell sheet precipitates were obtained by centrifugation and prepared as cell suspensions using PBS buffer.

| Statistical analysis
Data are expressed as mean ± standard error of the mean (SEM).
Statistical analyses were performed using SPSS software, and statistically significant differences were determined using one-way analysis of variance (anova). p < 0.05 was considered statistically significant. Experiments were conducted in triplicate.

| Melatonin-silenced cell migration and remodelled cytoskeleton
Gastric cancer cells were treated with different concentrations of melatonin (0, 1.0, 2.5 and 5.0 mM) and evaluated using the transwell assay to investigate the effects of melatonin on their migration ability.
Cell migration was reduced in the presence of melatonin ( Figure 1A) and was dramatically inhibited in cells treated with 5.0 mM melatonin.

| Melatonin treatment participated in the regulation of cancer stemness
Cancer stem cells show extreme tumorigenic capacity in inducing tumour proliferation, recurrence and metastasis. Considering the significant role played by cancer stem cells in cancer development, it is meaningful to explore the effects of melatonin on cancer stem cells. CD133 is a potential marker of gastric cancer stem cells. Therefore, we examined gene expression in CD133 + cells treated with melatonin. E-cadherin expression in CD133 + cells was reduced compared to that in CD133 − cells ( Figure 3A). In addition, after being treated with 2.5 mM melatonin, CD133 + cells showed further downregulation of E-cadherin expression, indicating that cancer stem cell metastasis might be exacerbated after melatonin treatment. PTEN is a widely studied tumour suppressor. In the present study, PTEN expression in CD133 + cells was lower than that in CD133 − cells. PTEN expression could be promoted by melatonin treatment, although its expression in melatonin-treated CD133 + cells was lower than in melatonin-treated CD133 − cells. In general, anti-tumour mechanisms were likely activated by melatonin.
Tumorigenesis is believed to be associated with dysregulation of cellular autophagy. Autophagy has both tumour promotion and suppression functions depending on context. Therefore, the expression of autophagy-related genes, ATG3, ATG5, ATG7, Beclin1, LC3 and phosphorylated Akt (p-Akt), was examined using western blotting. ATG3, ATG5, ATG7 and Beclin1 expression in CD133 − cells was enhanced by melatonin treatment (Figure 3). In CD133 + cells, ATG5 and ATG7 expression was promoted by melatonin treatment.
As expected, LC3-II expression increased in melatonin-treated cells, but its expression in melatonin-treated CD133 − cells was lower than that in melatonin-treated CD133 − cells. In addition, p-Akt expression in both melatonin-treated CD133 + and CD133 − cells was lower than that in non-treated cells. These results indicate that autophagy might play a regulatory role in the stemness of melatonin-treated cancer cells.
Our previous study showed that melatonin altered the expression of lncRNAs. 11 Therefore, the present study attempted to further uncover the role of melatonin treatment in lncRNA expression. As shown in Figure S2  The above findings indicate that knockdown of lncRNA H19 assisted melatonin in inducing cancer cell death.

| Melatonin in combination with cisplatin enhanced the induction of cancer cell apoptosis
The above results suggest that melatonin treatment could be effective in cancer cell inhibition. However, a large number of residual cells still remained after melatonin treatment. Therefore, combinatorial treatment might be an appropriate strategy to strengthen the effects of melatonin on cancer cells. Cisplatin is a classic platinum-based anticancer drug widely used to treat various types of cancers. In the present study, the therapeutic outcomes of combining melatonin with cisplatin were investigated. Apoptosis was assessed first via the Annexin V-FITC/PI assay in cells that underwent combinatorial treatment with melatonin (2.5 mM) and cisplatin (0.25, 0.5, 2.5 or 5.0 μg/mL). Results showed that the rate of apoptosis of gastric cancer cells under melatonin and cisplatin combinatorial treatment was higher than that of the control, melatonin-alone group and cisplatin-alone group ( Figure 6). In addition, PI staining-based flow cytometry showed that the combination of melatonin and cisplatin increased cell distribution in the G0/G1 phase ( Figure 7A). IRE1α and Bip expression in the cells under combinatorial treatment was higher than that in the control ( Figure 7B). In the case of autophagy, melatonin and cisplatin alone, as well as in combination, increased Beclin1 and LC3-II expression. Furthermore, Beclin1 and LC3-II expression in cells receiving combinatorial treatment was the highest among the groups examined. Western blotting was used to determine MAPK signalling pathway expression; the results showed that combinatorial treatment triggered higher expression of phospho-ERK and phospho-p38 compared to the control ( Figure 7C). Taken together, these findings suggest that combining melatonin with cisplatin might be a potential strategy to enhance the effectiveness of therapy for gastric cancer. EMT, invasion and migration. 26 However, pretreatment with melatonin has also been shown to increase vimentin expression in porcine trophectoderm cells. 27 That tumour cells originate from stem cells has been a longstanding proposition. Cancer stem cells, are a small heterogeneous population within the tumour that exhibits self-renewal and unlimited proliferative capacity and functions in cancer recurrence, metastasis and drug resistance. 1,28 Many studies have shown that melatonin can disrupt cancer stem cells. 29,30 To further evaluate the impact of melatonin treatment on cancer metastasis, CD133 + cells were isolated in this study. CD133 is highly expressed in various tumours and is often used as a cancer stem cell marker in stemness studies of gastric cancer. 31,32 Gene expression analysis showed that the CD133 + subpopulation of melatonin-treated cells had the lowest E-cadherin expression. In addition, autophagy induction by melatonin is weakened in the CD133 + subpopulation compared to the CD133 − subpopulation. Melatonin appears to have an unsatisfactory therapeutic outcome against gastric cancer stem cells. In fact, melatonin was found to increase c-Myc S62 phosphorylation in this study.
Enhancement of c-Myc S62 phosphorylation is well known to promote c-Myc stabilisation to increase tumorigenic potential. 33,34 However, there are unresolved controversies about CD133 as a marker for cancer stem cells. [35][36][37] Prolonged culture of a pure CD133 − population can lead to the re-emergence of CD133 + cells. 37 Therefore, melatonin-promoted ATG3 and Beclin 1 expression in CD133 − cells might be associated with other cancer stem cell subpopulations. This suggests that cancer treatment with melatonin alone may not be the optimal strategy.
LncRNA, a type of non-coding regulatory RNA, is closely associated with tumour invasion and metastasis. 38,39 In previous studies, regulation of lncRNA expression together with drug treatment showed a promising combinatorial effect as a cancer therapy. [40][41][42] The present study found that LINC01121, lncRNA COX2, H19, TUG1, HOTAIR, MALAT1, SAMMSON and UCA1 expression was altered after melatonin exposure. Furthermore, knockdown of H19 in melatonin-treated cells could further enhance the expression of pro-apoptotic genes Bax and Bak, suggesting that modulation of H19 expression may contribute to the enhancement of the inhibitory effect of melatonin on cancer progress. However, melatonin has been shown to both downregulate 43  In the case of melatonin based-cancer treatment, several previous reports focused on combinatorial therapy. For example, in head and neck cancer therapy, the combination of melatonin and rapamycin enhanced apoptosis via regulation of the mitochondrial function. 45 In colon cancer, melatonin synergized the chemotherapeutic effect of 5-fluorouracial via inhibition of the AKT and iNOS signalling pathways. 46 Therefore, to strengthen the impact of melatonin on cancer, the synergized effect of melatonin in a combinatorial treatment was also determined. Cisplatin is a well-known chemotherapeutic drug used for the treatment of various human cancers. 47 As expected, the combinatorial treatment of melatonin and cisplatin could induce large-scale apoptosis in gastric cancer cells by triggering G0/G1 phase arrest. These results suggest that co-treatment with melatonin and cisplatin might be an appropriate strategy for cancer treatment.