Introduction
Melanoma is one of the most threatening malignancies and has high metastatic potential. Although in the recent years, significant progresses have been made in melanoma treatment with the appearance and widespread application of the combinational immunotherapy [
1‐
4], it is still necessary to explore other treatment options to get better clinical output because the response rates to immunotherapy are not 100%. This might be mainly due to that the antigens selected for these approaches do not cover the full spectrum of melanoma cells present in a tumor [
5,
6]. The studies on cancer stem cells in melanoma raise the possibility that this long-lived tumor subpopulation is resistant to clinical therapy [
7]. Normal stem cells are thought to achieve their longevity by several mechanisms among which are slow divisions, anti-apoptotic mechanisms, and expression of efflux pumps that provide protection from toxins [
7,
8], and the design of more effective therapeutic strategies targeting melanoma stem cells and associated molecular pathways and their application hold promise for melanoma treatment. Inflammation is an important feature of the tumor microenvironment in melanoma, and previous studies showed that inducible nitric oxide synthase (INOS), one of the most common inflammation factors, is an important inducer of melanoma tumorigenesis, tumor growth, invasion and metastasis [
9,
10], and INOS abrogation has been proved to contribute to melanoma treatment.
BRAF mutations have been found in melanoma [
11,
12], and V600E is the most common mutation in BRAF leading to constitutive activation of the MAPK signaling pathway in malignant melanomas [
13]. The MAPK signaling pathway is involved in activation of BRAF which phosphorylates and activates MEK, and in turn phosphorylates and activates ERK [
14]. These reactions result in the activation of transcription factors that regulate cell survival, proliferation and differentiation. Vemurafenib, a small molecule inhibitor of serine/threonine protein kinase BRAF, shows initial good clinical responses [
15]. Unfortunately, the relative initial success of vemurafenib has been dampened by the development of acquired resistance to the drug [
16]. Moreover, the patients received vemurafenib treatment easily present a severe anterior uveitis secondary to this drug. In general, the reactivation of MAPK signaling pathway, the bypass of oncogenic pathway via activation of alternative signaling pathways, and other uncharacterized mechanisms are considered to be the cause of therapeutic resistance in kinase-driven cancers [
17‐
19].
Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous physiological mediator secreted by the pineal gland. In mammals, the pineal gland [
20,
21] secretes melatonin into the blood circulation to exert a range of well-documented physiological functions [
22]. It is well-known melatonin is an important endogenous synchronizer of the circadian day–night rhythm and seasonal biorhythms on a variety of target organs [
23‐
25]. Functionally, melatonin has been widely documented because of its significant antitumor effects on the ovarian carcinoma [
26], human melanoma [
27] and breast cancer [
28] et.al. In addition, melatonin can induce cancer cell apoptosis and suppress tumor metastasis, angiogenesis and inflammatory reaction, which indicate its potential clinical applications [
29‐
31]. Melatonin has been shown to function as a potent combination therapeutic agent in human cancer cells by enhancing the efficacy of conventional anticancer agents and meanwhile reducing their side effects [
32‐
34]. Notably, melatonin reduces cancer cell proliferation and decreases self-renewal and clonogenic capability through the decreased expression of stem cell markers [
35]. However, the mechanisms associated with the melatonin-regulated gene expression remain unclear. Therefore, it is important to clarify the underlying molecular mechanisms involved in the combination and to discover newly potential therapeutic targets.
In this study, we assessed the role of melatonin in the enhancement of the vemurafenib-mediated antitumor effect and identified the underlying mechanism of the combination treatment in melanoma.
Methods
Cell lines and cell culture
Human melanoma cell lines SK-Mel-28, A375, A431 and G361 were all obtained from the American Type Culture Collection (ATCC). All the cells were grown in Dulbecco,s Modified Eagle Medium (HyClone, Thermo Scientific) supplemented with 10% heat-inactivated fetal bovine serum (Gibco).
Establishment of melanoma cell line with relative vemurafenib resistances
Briefly, we cultivated A375 cells under the treatment of increasing amounts of vemurafenib (VE), firstly 0.5 μM VE was used for 2 weeks, and then 1μΜ VE was added as a part of the culture medium. Cells that survive the conditions were selected and amplified.
Western blot
Proteins from melanoma cell lysate were quantified using a BCA protein assay kit and were loaded onto a 10% polyacrylamide gel (SDS-PAGE), then then transferred onto a polyvinylidene fluoride (PVDF, Millipore, USA) membrane. Western blots were incubated with the specific primary antibodies. Finally immunoreactivity were detected by enhanced chemiluminescence.
Reagents and antibodies
Melatonin was purchased from J&K, Chemical Ltd. Vemurafenib was obtained from Selleck (PLX4032) and dissolved in dimethyl sulphoxide (DMSO) before addition to the complete cell culture medium. For the experiment, the solution of melatonin (1 M) and vemurafenib (VE) (10 mM) in DMSO was prepared and kept at 4 °C for further dilution in culture medium to maintain stability of used drugs. The InSolution™ NF-κB Activation Inhibitor controlling the biological activity of NF-κB (481407) was bought from Merck Millipore. Antibodies against β-catenin, MMP-1, MMP-9, Apaf-1 were purchased from Santa Cruz (USA). The antibodies against β-actin, IKKα, IKKβ, p-IKKα/β, IκB-α, p-IκB-α, cleaved caspase-3, cleaved caspase-9, cleaved PARP, Bcl-2, NF-κB p50, p65, p-PDK1, p-PTEN, p-AKT and AKT were purchased from Cell Signaling Technology (USA). The anti-TFIIB, E-cadherin antibodies were purchased from Proteintech group (USA), and anti-iNOS antibody was purchased from Wanlei Biotechnology (China). The anti-BRAF V600E antibody was purchased from Omnimabs (USA).
Plasmid vectors
The transfection was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA). Recombinant plasmid vectors pGL3-hTERT-438 expressing luciferase driven by hTERT promoter (− 378 to + 60) were produced in our lab.
Tumorsphere culture
Cells with indicated treatment were digested into single cell with trypsin-EDTA and were respectively seeded in 35 mm non-treated cell culture dishes (BIOFIL, 2000 cells/dish) with continuous culture in DMEM/F12 medium (HyClone) containing B27 supplement (Gibco), N2 supplement (Gibco), bFGF (20 ng/ml), and EGF (20 ng/ml) for two weeks. Then the pictures of the formed tumorspheres were taken by inverted microscope (Leica) and the number of the spheres with diameter larger than 50um was counted.
Flow cytometry assay of CD44
Expression of stemness-associated marker, CD44, was detected by flow cytometer. A375 and SK-MEL-28 cells with indicated treatment were digested with trypsin-EDTA and washed twice in PBS containing 2% BSA and centrifuged at 300×g for 3 min. Cells were divided into two groups and resuspended in 100 ul PBS with 2% BSA on ice. Then the antibody APC-IgG and APC-CD44 (BD Pharmingen) were respectively added into single tube of each group on ice to incubate for 30 min. The fluorescence value was detected finally by FACS Accuri C6 (Genetimes Technology Inc.).
Cell viability assay
Cell viability was assessed using the MTT assay (Roche Diagnosis, Indianapolis, IN). Briefly, melanoma cell lines were seeded onto 96 well plates for following overnight incubation. Five replicated wells were put up for each group. Then cells were treated with melatonin or vemurafenib by indicated dose. Finally, the effect for cell viability was assessed by the absorbance of the supernatant at a wavelength of 450 nm comparing to the vehicle-treated control group. The drug concentration required to cause 50% cell growth inhibition (IC50) was determined by interpolation from dose-response curves.
Melanoma cells were seeded into six well plate (2 × 103 per well) and were incubated for 12 h. Then, the medium was removed and cells were exposed to various drugs. After 24 h, cells were changed into fresh medium containing 10% FBS and incubated in a 37 °C, 5% CO2 incubator for 14 days until cells grew into macroscopic colonies. Finally, the medium was removed, and the colonies were stained by 0.1% crystal violet and counted.
Scratch assay
A375 and SK-mel-28 cells treated with indicated doses of vemurafenib or melatonin. The cells were seeded into six-well plates and incubated in the medium with 2.5% FBS until grown to full confluency, then scraped by a sterile 200 μl pipette tip. After 36 h, medium was replaced with PBS buffer and the wound gap was photographed by inverted microscope (Leica DM 14000B microscope fixed with digital camera) at 0 h and 48.
Transwell assay
For the transwell assay, 4 × 104 cells were treated with indicated doses of vemurafenib or melatonin per chamber were plated. Cells were allowed to invade through the matrigel-coated inserts. After the cells that remained in the gel or attached to the upper side of the filter were removed with cotton swabs, the invaded cells were stained with 0.1% crystal violet solution and then was photographed using a Leica DM 14000B microscope fixed with digital camera. Finally, we used image-Pro plus software for counting the invaded cells. The concrete steps are as following: First, load the file to be analyzed from the folder, and then select the rectangular AOI tool, define the AOI to cover the image, and click on select colors in the Count/Size dialog box, and select Histogram Based button to set the range. Next, from the Measure menu in the Count/Size dialog box, select the Select Measurements command. Add the Area measurement in the Filter Ranges, and set the threshold, then click on Measure, and click the count in the Count/Size dialog box. From the View menu in the Count/Size dialog box, select the Statistics command.
Cell cycle and apoptosis assay
In brief, A375 and SK-mel-28 cells (105 cells) seeded in 6-well plates were treated with indicated doses of vemurafenib or melatonin. Cell cycle assay: after 48 h, cells were collected and stained DNA with PI, finally sorted by FACS Accuri C6 (Genetimes Technology Inc.) and analyzed by using FlowJo 7.6 software; Apoptosis assay: after 48 h, cells were collected subsequently stained simultaneously with FITC-labeled annexin V and PI. Stained cells were analyzed using FACS Accuri C6 (Genetimes Technology Inc.).
Acridine orange /ethidium bromide fluorescence staining
A375 or SK-mel-28 cells were grown on chamber slides and treated with indicated doses of vemurafenib or melatonin. After 48 h, cells were washed by PBS, and then fixed by 95% ethanol for 15 min. After slightly drying cells, 5 ul AO/EB (50 μg/ml) were added with gently pipetting to mix before photographing by Leica DM 14000B microscope fitted with digital camera.
Pulldown assay
The biotin-labeled double-stranded oligonucleotide probes, which correspond to hTERT promoter sequence or iNOS promoter sequence, were synthesized by PCR using biotin-labeled primers from TAKARA Company. The nucleus proteins (400 μg) were mixed with double-strand biotinylated hTERT or iNOS promoter probe (4 μg), streptavidin agarose beads (50 ml) in 500 ml PBSI buffer (0.5 mM PMSF, 10 mM NaF, 25 mM β-glycerophosphate) and rotated for 4 h at RT. The beads were centrifuged, washed with PBSI buffer for two times, and then were resuspended by loading buffer and boiled at 100 °C for 10 min. The supernatant was analyzed by Western blot.
Chromatin immunoprecipitation assay (ChIP)
Briefly, the A375 and SK-mel-28 cells were fixed with 1% formaldehyde, sonicated on ice to shear the DNA into the fragments from 200 bp to 500 bp. The lysate were subjected to immunoprecipitations with anti-p65 or non-specific rabbit IgG. The immunoprecipitated DNA was subjected to PCR to amplify a 220 bp fragment of hTERT promoter or a 510 bp fragment of iNOS promoter. The PCR products were run electrophoretically on a 1% agarose gel and visualized by ethidium bromide staining.
Confocal immunofluorescence assay
A375 and SK-mel-28 cells were incubated on chamber slides in 6-well plates with indicated treatment, cells fixed for 10 min at room temperature (RT) with 4% paraformaldehyde, and then permeabilized with PBST (PBS with 0.2%Triton X-100), blocked with bovine serum albumin (BSA) 30 min and incubated with Cytochrome-c, or p65 or p50 antibodies (1:200 dilution) for overnight at 4 °C. Following 10-min washes for three times with PBS, cells were incubated with the fluorescein isothiocyanate- and rhodamineconjugated secondary antibodies for 30 min. Subsequently, the nuclei of stained samples were mounted with Vectashield solution containing 4′6-diamidino-2-phenylindole (DAPI). After five additional 10-min washes, the results were visualized by Leica DM 14000B confocal laser scanning microscope.
Xenograft tumor models of human melanoma
Male nude mice (4-5 weeks old) were obtained. All animal maintenance and procedures were carried in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Animal Research Committee of Dalian Medical University. A375 melanoma cells (2 × 106) were inoculated subcutaneously into the flank of the nude mice. Mice were randomly divided into 4 groups (5 mice per group): DMSO, melatonin (25 mg/kg), vemurafenib (20 mg/kg) and melatonin + vemurafenib, which were started 5 days after injection and co-treatment every other day for 2 weeks. The tumor volume in mm3 was calculated as V = (width2 × length)/2 using digital calipers and the tumor weight was recorded after the mice were sacrificed. Partial tumor was lysed for protein expression analysis through western blot analysis, and partial was sliced and fixed in formalin and embedded in paraffin for protein expression analysis through the immunohistochemical staining.
Immunohistochemistry staining
Briefly, Tumors were dissected and fixed in 10% formalin overnight, embedded in paraffin, and incised to 4 um thick. Immunohistochemistry (IHC) staining was performed using following the DAB Kit (Origene, China). The primary antibodies iNOS, hTERT, p-p65, Epcam, CD44, PCDNA were used with indicated dilution ratio. Sections were stained with hematoxylin to recognize nucleus.
Statistical analysis
Data are represented as mean ± standard deviation (SD). Analysis of variance and Student’s t test were used to compare the values of the test and control samples. P < 0.05 was considered to be a statistically significant difference. SPSS17.0 software was used for statistical analysis, and all the experiments were done three times.