Curcumol inhibits the proliferation and metastasis of melanoma via the miR-152-3p/PI3K/AKT and ERK/NF-κB signaling pathways

Melanoma is the most aggressive and treatment-resistant form of skin cancer. Curcumol is a Chinese medicinal herb traditionally used as a cancer remedy. However, the molecular mechanisms underlying the anticancer activity of curcumol in melanoma remains largely unknown. In the present study, we observed that Curcumol decreased mouse melanoma B16 cell proliferation and migration. The xenograft tumor assay showed that curcumol reduced melanoma volume and lung metastasis. Curcumol upregulated the expression of E-cadherin and downregulated the expression of N-cadherin, MMP2 and MMP9 in mouse melanoma B16 cell. Western blot analysis revealed that curcumol reduced the translocation of p65 to the nucleus and decreased p-ERK. Furthermore, curcumol attenuated c-MET, P13K and p-AKT protein expression and upregulated miR-152-3p gene expression. The dual-luciferase reporter assay indicated that c-MET was a target gene of miR-152-3p. Reduced expression of miR-152-3p partially attenuated the effect of curcumol on mouse melanoma B16 cell proliferation and migration. The decrease in c-MET, P13K and p-AKT protein expression following curcumol treatment in mouse melanoma B16 cells was notably attenuated by the miR-152-3p inhibitor. Taken together, our findings suggested that curcumol attenuated melanoma progression and concomitantly suppressed ERK/NF-κB signaling and promoted miR-152-3p expression to inactivate the c-MET/PI3K/AKT signaling pathway.


Introduction
Melanoma is the most prevalent, as well as the most aggressive and treatment-resistant malignant skin cancer 1,2 . Melanoma causes ~80% of skin cancer-associated mortalities worldwide, and its incidence has increased by 45% since the 20th century, with an annual percentage change being estimated at 3.1% 3,4 . The rising incidence and mortality of melanoma worldwide, as well as its high propensity for metastasis, highlights the urgency of exploring the molecular mechanism governing the progression of melanoma and developing effective strategies for melanoma.
Curcuma is traditionally used to treat various ailments, including cacochylia, traumatic hematoma,
MicroRNAs (miRNAs) are a class of small, noncoding RNA molecules containing approximately 19~22 nucleotides 17 . miRNAs mostly bind to the 3'-untranslated region (3'-UTR) of the target mRNAs, regulating gene expression at the posttranscriptional level 18 . miR-152 is a member of the miR-148/152 family 19 , which is expressed not only in normal tissues but also in numerous tumors, such as colorectal tumors 20 and breast 21 and human gastric tumors 22 . miR-152 inhibits cancer stemness, cell proliferation, and tumor angiogenesis 23 . Luan et al found that miR-152-3p was a target that decreased the expression of Cellular mesenchymal-epithelial transition factor (c-MET) and then suppressed melanoma progression and invasion via the PI3K/Akt/mTOR signaling pathway 24 . Therefore, these studies showed that miR-152-3p could be a novel therapeutic small molecule to suppress cancer.
In this study, we assessed the effect of curcumol on mouse melanoma B16 cells. We hypothesized that curcumol suppresses the ERK/NF-κB signaling pathway and upregulates miR-152-p to suppress the c-MET/PI3K/AKT pathway and inhibit melanoma cell growth and invasion. The study elucidated the possible molecular mechanisms responsible for curcumol anticancer activity.

Cell culture
Mouse B16 melanoma cells were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured according to the manufacturer's instructions. Briefly, we cultured these cells with Dulbecco's modified Eagle's medium (DMEM). Additionally, 10% FBS and 1% penicillin/streptomycin were added (Gibco, Rockville, MD, USA) to culture melanoma cell lines. Cell incubators in a humidified atmosphere containing 5% CO2 were employed for all cell cultures.

Cell viability assay
We assessed cell viability using a Cell Counting Kit-8 assay (CCK-8) following the protocol from the manufacturer (Dojindo, Tokyo, Japan). We seeded 2 × 10 3 cells in 96-well plates and incubated at 37 °C for 24 h. The cells were treated with the indicated concentrations (0, 25, 50, 100 and 200 µM) of curcumol for 24 h, 48 h or 72 h in a humidified chamber containing 5% CO 2 . Then, we added 10 μl CCK-8 reagent into each well and incubated for 1 h at 37 °C. A microplate reader (Bio-Tech, USA) was used to determine the absorbance of cells at 450 nm (OD450).

Colony formation assay
Mouse B16 melanoma cells were seeded into 60-mm dishes (200 cells/dish) for 24 h and then incubated with 0, 25, 50, 100 and 200 µM curcumol for 2 weeks to form colonies. Then, the colonies were washed with PBS three times. Next, the cells were fixed with methanol for 15 min, and they were stained with 0.1% crystal violet for 30 min at room temperature. Finally, we counted the number of colonies containing ≥50 cells under a microscope. Each assay was performed in triplicate.

Xenograft tumor assay
The xenograft tumor growth assay was performed in four-week-old male C57 mice (purchased from Shanghai SLAC Laboratory Animal Co., Ltd, China), and the animal experiments were approved by the Hunan Provincial People's Hospital. First, 2 × 10 6 mouse melanoma B16 cells were suspended in sterile saline (200 µl) or injected subcutaneously into the right lower paw and intravenously into the tail vein of mice. Curcumol (20 mg/kg) was injected intraperitoneally into mice 3 times per week. Tumor volumes were calculated according to the formula (0.5 × length × width) every 5 days. After 30 days, the mice were sacrificed, we tested the tumor volume in the right lower paw and harvested xenograft pulmonary tumors immediately.

Wound healing assay
We used a wound healing assay to evaluate the migration ability of mouse B16 melanoma cells. Mouse B16 melanoma cells were plated at a concentration of 1×10 5 cells/ml and incubated for 24 h, and a 200-μl pipette tip was used to scratch the cell layers to form wound gaps. The cells were incubated with 0, 50 and 100 µM curcumol for the indicated times (0, 24 and 48 h). The cells were photographed under a microscope to record the wound width. Wound healing was analyzed using ImageJ software.

Western blot assays
Cells were washed with ice-cold PBS and RIPA buffer with proteinase inhibitor was used to extract the total protein from tissues and cells, and protein concentrations were quantified with a BCA Protein Assay Kit (Beyotime, China). Briefly, equal amounts of protein (20 µg) were separated by 10% or 12% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Subsequently, the membranes were blocked in 5% nonfat milk in TBST for 1 h at room temperature and then incubated overnight with diluted primary antibodies at 4 °C followed by incubation with HRP-conjugated secondary antibody. GAPDH was used as a control. Proteins were visualized using the Luminescent Image Analyzer (ImageQuant LAS500, GE, USA), and densitometric analysis was performed using ImageJ software.

Transwell Migration and Invasion Assay
Cell migration was measured using a Transwell migration assay according to the manufacturer's instructions. The mouse B16 melanoma cells were mixed by the blowing method and added to the serum-free DMEM for incubator. After the cell concentration was adjusted to 5×10 5 cells/ml, 800 µl of DMEM containing 5% FBS was added to the lower chamber of the Transwell permeable support, and 200 µl of cell suspension was added to the upper chamber. Before the experiment, 10 μg/ml collagen was added into the Transwell at 50 µl/well. The cells were then cultured with the indicated concentrations (0, 50 and 100 µM) of curcumol in an incubator for 24 h followed by fixation with 4% paraformaldehyde and staining with crystal violet for 20 min. Microscopic images were used to observe the migration and invasion of cells, and visual fields were randomly selected and photographed. ImageJ (1.48 v) software (National Institutes of Health, Bethesda, MD, USA) was used to obtain an average cell count of the four stained membrane images.

Luciferase reporter assay
The wild-type and mutant 3'-untranslated region (3'-UTR) fragment of c-MET containing the putative binding sequences of miR-152-3p were cloned into the pGL2-basic vector (NEB) as wild-type (WT) or mutant (Mut). Mouse B16 melanoma cells were cotransfected with hsa-miR-152-3p mimic or NC and related reporter constructs. Luciferase activity was detected using the Dual Luciferase Reporter Assay System (Promega, USA) after transfection for 48 h according to the manufacturer's protocol.

Data analysis
The results of at least three independent datasets are presented as the mean ± standard deviation (S.D.). For statistical analysis, statistical significance was analyzed using Tukey's multiple comparison tests and one-way analysis of variance. Data analysis was performed with SPSS 16.0. Statistical significance was determined at p< 0.05.

Effect of curcumol on the proliferation of mouse melanoma B16 cells in vitro and in vivo
To determine whether curcumol influences the proliferation of mouse melanoma B16 cells, we first examined the effect of curcumol on the proliferation of the cells with the CCK-8 assay. Mouse melanoma B16 cells were treated with different concentrations of curcumol (0, 25, 50, 100 and 200 μM) for 0, 24, 48 and 72 h, respectively. As shown in Figure 1B, curcumol decreased cell viability in a dose-dependent and time-dependent manner, and compared to the control, 50-and 100-μM curcumol treatment for 48 h reduced the cell viability by 55%. Second, mouse melanoma B16 cells were incubated with 0, 50 and 100 μM for 2 weeks, and colony formation assays were performed. The results confirmed that curcumol decreased colony formation ( Figure 1C and 1D) in a dose-dependent manner, and when curcumol concentration reached 100 μM, the number of colonies decreased by 60% compared to the control.
To further test the function of curcumol in vivo, we established a melanoma xenograft model by subcutaneously injecting mouse melanoma B16 cells. As shown in Figure 1E and 1F, the average tumor volume in the DMSO group was significantly greater than that in the curcumol group after 30 days. These findings suggest that curcumol suppresses the proliferation of mouse melanoma B16 cells and melanoma growth in vivo.

Effect of curcumol on the migration and invasion of mouse melanoma B16 cells and mouse pulmonary metastasis model
Subsequently, we defined the role of curcumol in melanoma metastasis, and we examined the metastatic properties of the cells treated with 50 and 100 μM curcumol for 24 and 48 h in serum-free medium and examined the cells by wound-healing assay. Our data (Figure 2A and 2B) showed that there was a significantly lower migration rate for the cells in the curcumol group compared with the DMSO group. Then, we performed a migration assay to further detect the effect of curcumol on cell migration. As shown in Figure 2C and 2D, curcumol significantly inhibited cell migration in a dose-dependent manner, and 54% inhibition was observed at a concentration of 100 μM Tangeretin.
We observed that curcumol markedly suppressed cell migration in vitro and then examined the effect of curcumol on tumorigenicity and metastasis in vivo. To this end, mouse melanoma B16 cells were injected intravenously into C57 mice. As shown in Figure 2E and 2F, the intrapulmonary metastasis rate of mouse melanoma B16 cells was 80% in the DMSO group, and there was a lower 20% metastasis rate in the curcumol group. Taken together, these data support an important role for curcumol in the suppression of invasion and metastasis in vivo and in vitro.

Curcumol modulates the expression of MMP2/9 and E/N-cadherin in mouse melanoma B16 cells
Matrix metalloproteinase (MMPs) are the principal enzymes in extracellular matrix (ECM) degradation 25 . It has been shown that elevated expression of MMP2 and MMP9 is positively correlated with tumor progression, metastasis, and poor prognosis 26 . Curcumol has been shown to decrease the MMP to inhibit lung adenocarcinoma cancer cell migration and invasion 12,13 . Furthermore, our results indicated that curcumol decreased the expression of MMP2 and MMP9 in mouse melanoma B16 cells ( Figure 3A and 3B).
Epithelial to mesenchymal transition (EMT) refers to the loss of cell adhesion, repression of E-cadherin expression and increasing cell mobility 27 . The aberrant activation of EMT promotes tumor cell invasion and dissemination 28 . Yan et al found that curcumol altered the levels of E-cadherin and N-cadherin in a dose-dependent manner in a nasopharyngeal carcinoma cell xenograft model 29 .
Our results indicated that curcumol attenuated the expression of MMP2 and MMP9; therefore, we sought to determine whether curcumol was responsible for regulating epithelial-mesenchymal transition(EMT) in mouse melanoma B16 cells. Our data showed that curcumol increased the expression of E-cadherin and prominently decreased the expression of N-cadherin ( Figure 3C and 3D). Taken together, these data clearly demonstrate that curcumol limited the metastatic process of mouse melanoma B16 cells by regulating the ECM and EMT.

Effect of curcumol on the PI3K/Akt and ERK/NF-κB pathways in mouse melanoma B16 cells
Cellular c-MET acts as an oncogene in malignant melanoma, and c-MET expression is markedly increased in melanoma cells 24 . It has been demonstrated that c-MET enhanced melanoma cell proliferation and invasive capacity and protected cells from apoptosis via the PI3K/AKT signaling pathway 24,30 . Thus, based on our results showing that curcumol affected mouse melanoma B16 cell proliferation and invasion, we next investigated the effect of curcumol on c-MET, PI3K and Akt protein expression. Western blot analysis revealed that the c-MET and PI3K protein levels in the curcumol group were significantly lower than those in the control group. Additionally, curcumol attenuated Akt phosphorylation in mouse melanoma B16 cells ( Figure 4A and 4B).  Numerous studies have shown that MMP2/9 expression is regulated by NF-κB signaling pathways in melanoma 31-33 , and curcumol induced HSC-T6 cell death via this pathway. Therefore, we further observed the effects of curcumol on the NF-κB pathway in mouse melanoma B16 cells. We applied tumor necrosis factor alpha (TNF-α) as a positive control in experiments. Our results demonstrate that curcumol substantially decreased the phosphorylation of ERK and activation of p65 compared with the control group ( Figure 4C-E). Overall, these data indicate that curcumol mediates the inhibition of the PI3K/Akt and NF-κB signaling pathways to modulate migration and invasion in mouse melanoma B16 cells.

Curcumol inhibited mouse melanoma B16 cell growth and metastasis by upregulating miR-152-3p
miR-152-3p is a tumor suppressor gene that is expressed at low levels in melanoma 20-24 . Therefore, we performed miRNA microarray analysis to determine whether curcumol affects miRNA expression in mouse melanoma B16 cells. The heat map data revealed 69 differentially expressed miRNAs in the 100 μM curcumol group compared with the DMSO group ( Figure 5A). Treatment with 100 μM curcumol upregulated 38 miRNAs and downregulated 31 miRNAs. Notably, 100 μM curcumol significantly upregulated miR-152-3p expression, which was greater than a seven-fold change compared with the DMSO group (Table 1 and Figure 5B). To further confirm the data obtained from the miRNA microarray, we used qRT-PCR to examine miR-152-3p expression in mouse melanoma B16 cells. Our results showed that curcumol upregulated the level of miR-152-3p in a dose-dependent and time-dependent manner ( Figure 5C and 5D).
The results above clearly indicate that curcumol increases the level of miR-152-3p in mouse melanoma B16 cells. Subsequently, we used siRNA technology to inhibit miR-152-3p expression and investigated whether curcumol prevented mouse melanoma B16 cell proliferation and migration via miR-152-3p. Treatment with siRNA for miR-152-3p downregulated miR-152-3p expression by 85% ( Figure 6A), and curcumol led to a reduction in mouse melanoma B16 cell viability, which was significantly reversed by the addition of miR-152-3p inhibitor ( Figure 6B). As shown ( Figure 6C-F), 100 μM curcumol reduced the migration rate of the mouse melanoma B16 cells and was markedly attenuated by the miR-152-3p inhibitor. Taken together, our data suggest that curcumol prevents mouse melanoma B16 cell proliferation and migration by upregulating the level of miR-152-3p.

Curcumol-mediated suppression of the PI3K/AKT signaling pathway partially depends on miR-152-3p
miR-152-3p, through direct targeting of c-MET, suppresses oral squamous cell carcinoma 34 , and our results also demonstrated that miR-152-3p inhibitor reduced the c-MET gene transcription and protein expression ( Figure 7A and 7B). We predicted the potential miR-152-3p binding sites in c-MET using Starbase 2.0 (http://www.targetscan.org/vert_71/). Dual luciferase reporter assays showed that a miR-152-3p mimic reduced the luciferase activity of pMIR-c-MET-WT but not that of pMIR-c-MET-MUT in mouse melanoma B16 cells ( Figure 7C and 7D). Table 1. miR gene change in the 100 μM curcumol group compared with the DMSO group. Based on our results, we used a miR-152-3p inhibitor to further examine the effects of curcumol on MMP2/9 and E/N-cadherin in mouse melanoma B16 cells. As demonstrated in Figure 8A and 8B, the decrease in MMP2 and MMP9 following curcumol-treated mouse melanoma B16 cells was markedly attenuated by the miR-152-3p inhibitor. Additionally, the miR-152-3p inhibitor reversed the curcumol effect by decreasing N-cadherin and increasing E-cadherin ( Figure 8C and 8D). These results indicated that curcumol regulated ECM and EMT by miR-152-3p. In addition, the expression of c-MET and PI3K and the phosphorylated form of AKT were considerably upregulated in the curcumol treatment group. In contrast, the miR-152-3p inhibitor led to a marked decrease in c-MET, PI3K and p-AKT levels, and the miR-152-3p inhibitor attenuated the effect of curcumol ( Figure 8E-H). In summary, curcumol inhibited mouse melanoma proliferation and migration, largely by enhancing miR-152-3p and downregulating the c-MET/PI3K/AKT pathway.

Discussion
Melanoma is considered to be one of the most malignant cancers, and approximately 200,000 new cases are diagnosed worldwide each year 35 . Currently, the major challenges to conventional antitumor drugs include chemotherapy resistance and severe systemic adverse effects 36,37 . Therefore, interest has increasingly focused on finding novel and alternative medicines that are safe and effective against melanoma. Curcumol has been reported to possess antitumor and antiviral activities with low cytotoxicity 38 . In the present study, we demonstrated that curcumol decreased the expression of c-MET, PI3K and p-AKT by increasing the level of miR-152-3p. Simultaneously, curcumol decreased ERK phosphorylation and prevented the activity of NF-κB, thereby driving the c-MET/PI3K/AKTdependent and ERK/NF-κB-dependent pathways associated with mouse melanoma B16 cell death (Figure 9).
Curcumol is one of the major monomeric components of Rhizoma Curcumae and is widely used in the treatment of cancer in China 39 . Curcumol has a wide range of biological activities, including proliferation inhibition and apoptosis induction of a variety of cancer cells 9-16 . Consistent with the aforementioned reports, we found that curcumol decreased mouse melanoma B16 cell proliferation and migration. Curcumol is a multitarget natural anticancer substance, and the main targets involved in different cancers are also different, but the PTEN/P13K/AKT pathway and the ERK/NF-κB pathway are the main regulatory points for curcumol to inhibit cancer cell growth. Liu et al found that curcumol inhibited colorectal cancer proliferation via modulating PTEN/PI3K/AKT pathways 40 . Li et al indicated that curcumol induces cell cycle arrest and apoptosis by inhibiting the IGF-1R/PI3K/ Akt signaling pathway in human nasopharyngeal carcinoma CNE-2 cells 41 . Our current findings also revealed that the inhibitory effect of curcumol on the proliferation and migration of mouse melanoma B16 cells is related to the inhibition of the PI3K/AKT pathway. Consistent with previous reports 9, [31][32][33]42 , our results also showed that curcumol exerted an inhibitory effect on mouse melanoma B16 cells by inhibiting the ERK/NF-κB signaling pathway. Inhibition of the c-MET pathway is a potential therapeutic strategy against melanoma 24,30,43,44 . Overexpression of c-MET in melanoma cells enhances cell protection from cell death and correlates with a poor clinical outcome 45,46 , which was shown to be mediated by the activation of the PI3K/AKT pathways 47,48 . Furthermore, some synthetic cyclic compounds were designed and used to suppress MET kinase activity, such as cabozantinib, foretinib, BMS777607, MGCD-265 and pyrrolo[23-b]pyridine derivatives 49 . Our data also showed that curcumol decreased the expression of c-MET, which inhibited downstream PI3K/AKT pathways.
A number of studies have revealed that miRNA plays a critical role in health and diseases, including cell growth, migration, apoptosis and cancer cell resistance [50][51][52] . A decrease in miR-152 has been proved to be associated with the processes of cell proliferation, invasion and angiogenesis in different neoplasms [53][54][55] . In melanoma, miR-152-3p levels are relatively decreased compared to normal mammary tissues 24 . Then, we used miRNA microarrays to test the potential miRNAs that could be regulated by curcumol. miR-152-3p expression was significantly higher in the curcumol treatment group in mouse melanoma B16 cells, which was consistent with the qRT-PCR results. In addition, c-MET plays an essential role in miR-152-regulated PI3K/AKT signaling in osteosarcoma cells 56 . Based on these findings, we hypothesized that curcumol exerts anti-tumor activity, which may be associated with an increase in the level of miR-152-3p to decrease the c-MET in mouse melanoma B16 cells. To test this hypothesis, miR-152-3p that could interact with c-MET was predicted using Starbase 2.0. Western blotting and qRT-PCR results indicated that miR-152-3p reduced the level of c-MET. Moreover, dual luciferase reporter assays confirmed that miR-152-3p directly binds to c-MET. Finally, we found that curcumol inhibits c-MET expression and the downstream signaling pathway PI3k/Akt, and this inhibition effect of curcumol on mouse melanoma B16 cells can be reversed by miR-152-3p inhibitor. These results reveal that curcumol suppressed melanoma cell growth and metastasis by, at least in part, upregulating the level of miR-152-3p and promoting miR-152-3p to bind c-MET. Figure 9. Schematic illustrating the working principle of curcumol related signaling in mouse melanoma B16 cells. We propose that curcumol maintained the level of miR-152-p in mouse melanoma B16 cells, thereby inactivating c-MET/PI3K/AKT-dependent signaling pathway associated with mouse melanoma B16 cell death. In addition, our results also imply that ERK/NF-κB signaling pathway contributes partially in curcumol-mediated effects.
To summarize, this study is the first to demonstrate that curcumol, via the c-MET/PI3K/ AKT and ERK/NF-κB signaling pathways, deceases melanoma cell proliferation and migration. Moreover, our research has demonstrated a potential role for curcumol in the treatment of melanoma, and miR-152-3p was targeted by curcumol to treat melanoma. However, due to the unique five-membered and seven-membered ring chemical structure of curcumol, the drug solubility is poor, and the bioavailability is not high. Therefore, further studies are warranted to improve the physical properties and modify the structure of curcumol in a clinical context.

Ethics Committee Approval and Patient Consent
Investigations involving humans will have been performed in accordance with the principles of Declaration of Helsinki. And the use of animals in experiments will have observed the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Testing, and Education by the New York Academy of Sciences, Ad Hoc Animal Research Committee.