NecroX-5 Can Suppress Melanoma Metastasis by Reducing the Expression of Rho-Family GTPases

NecroX-5 (NX-5) is a cell-permeable necrosis inhibitor with cytoprotective effects. Although it has been reported to inhibit lung and breast cancer metastasis by modulating migration, its therapeutic effect on melanoma metastasis is still unknown. In this study, we examined the anti-metastatic effect of NX-5 on melanoma cell lines and its related therapeutic mechanism. The anti-metastatic effect of NX-5 on melanoma cell lines was determined using a transwell migration assay. We performed a quantitative real-time polymerase chain reaction and western blot analysis to measure changes in the expression of mRNA and protein, respectively, for major mediators of Rho-family GTPases after NX-5 treatment in melanoma cells. In addition, after constructing the 3D melanoma model, the expression of Rho-family GTPases was measured by immunohistochemistry. NX-5 (10 μM and 20 μM) treatment significantly reduced melanoma cell migration (p < 0.01). Additionally, NX-5 (20 μM) treatment significantly decreased the mRNA and protein expression levels of Cdc42, Rac1, and RhoA in melanoma cells compared with the untreated group (p < 0.001 and p < 0.05, respectively). Immunohistochemistry for our 3D melanoma model showed that Cdc42, Rac1, and RhoA were constitutively expressed in the nuclei of melanoma cells of the untreated group, and NX-5 treatment decreased their expression. These results demonstrate that NX-5 can suppress melanoma metastasis by reducing the expression of Rho-family GTPases.


Introduction
Melanoma is a fatal disease that accounts for 90% of skin cancer-related deaths, and metastasis easily occurs with a poor prognosis [1,2]. Most melanomas metastasize to distant organs such as the liver, bones, and brain; skin, subcutaneous tissue, and lymph nodes are the primary sites of metastasis [3]. Although several anti-cancer drugs and immunotherapeutic agents have been tried, thus far, their therapeutic efficacy against invasion and metastasis is still limited [4,5].
Cancer metastasis is a process of cell migration, epithelial-mesenchymal transition (EMT), intravascular invasion, the potential circulation of tumor cells, and deposition to secondary organs. In cancer cells, EMT increases mobility and invasiveness and gives cancer cells the ability to degrade the extracellular matrix (ECM) [6,7].
Rho-family GTPases have Cdc42, Rac1, and RhoA as the main subfamily and play important roles in various cell functions including angiogenesis and invasion, cytoskeletal structure formation, cell proliferation, apoptosis, and cancer metastasis [8][9][10]. In 2 of 10 melanoma, Cdc42 contributes both amoeboid and mesenchymal movement and overall tumor cell invasion. [11]. Rac1 is known to enhance the elongated protruding movement of melanoma cells by promoting nuclear alterations through PAK1 and the tubulin cytoskeleton [12]. Additionally, RhoA is activated by the chemokine CXCL12, which triggers a cellular polar response, leading to rounded, amoeboid movement [13,14]. When Rhofamily GTPases move to the cell membrane and are activated, they induce cancer cell transplantation and metastasis through downstream target molecules [15]. Mediation of Rho-protein activity can advance tumor cell metastasis by disrupting epithelial-sheet organization, increasing cell motility, and promoting ECM degradation. Rho-family GTPase overexpression is associated with the progression of some cancers, including breast and lung cancers [16].
NecroX-5 (NX-5) is a derivative of the NecroX series of compounds and has been introduced as a drug for inhibiting cancer metastasis [17]. NX-5 has been reported to reduce the calcium concentration in cells, thereby reducing the invasion ability of breast cancer cells [18]. However, the anti-metastatic effect of NX-5 on melanomas has not yet been fully elucidated. In this study, we examined whether NX-5 has the potential to inhibit the metastatic response of melanoma cells.

Cell Viability Assay
Melanoma cells (1 × 10 4 /mL) were seeded onto 96-well plates with 100 µL/well culture medium. After 24 h, the medium was replaced with serum-free medium, then treated with NX-5 (1-50 µM) for 24 h, when cell viability was examined using MTT. MTT reagent (20 µL, 5 mg/mL PBS) was added to each well. The cells were further incubated at 37 • C for 4 h. After careful removal of the medium, 100 µL of buffered DMSO was added to each well, and the plates were shaken. Cellular metabolism was determined by recording the absorbance of samples at 540 nm in a VersaMax Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The effect of each compound on cell growth was assessed as a percentage of cell viability where vehicle-treated cells were taken as 100% viable.

Cell Migration Assay
Melanoma cell migration was evaluated using 24-well Transwell plates (Costar, Corning Inc., Corning, NY, USA). The appropriate number of A375P and A375SM cells were added to the upper chamber of transwell plates with 8µm pores with or without NecroX-5 at 10 µM or 20 µM and incubated for 24 h. The upper surface of the membrane was wiped with a cotton-tipped applicator to remove residual cells. Cells in the bottom compartment were fixed and stained with hematoxylin, and a photographic image of the bottom of the transwell membrane was taken. Migration of the human melanoma cell lines was determined by counting three fields randomly selected at ×100-200 magnification from the upper chamber to the bottom side of the membrane.

Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)
Melanoma cells were seeded at a density of 1 × 10 4 mL/well in plates. After being cultured for 24 h, the medium was replaced with serum-free medium. After starvation, the cells were treated with NX-5 (10, 20 µM), ZCL278 (10 µM), NCS23766 (10 µM), or CCG-1423 (100 nM). The negative control cells were incubated with sterile distilled water. Total RNA from tissues was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and reverse-transcribed to cDNA using a cDNA synthesis kit (Doctor Protein, Seoul, Korea). RT-qPCR was performed using a CFX96™ Real-Time PCR Detection System ® (Bio-Rad, Hercules, CA, USA) with MG 2X qPCR MasterMix II (SYBR Green) (MGmed, Seoul, Korea) and specific primers. Conditions of the RT-qPCR reaction consisted of an initialization step for 10 s at 95 • C followed by two-step PCR for 45 cycles of 95 • C for 5 s (denaturation) and 58-60 • C for 30 s (annealing/extension). Results were normalized to the level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression. The analysis of relative gene expression data was conducted using the 2 −∆∆CT method.

Fabrication of Nanofibrous Membrane
Electrospinning was used to fabricate a nanofibrous membrane [19][20][21]. Briefly, the polycaprolactone (PCL) with a number-average molecular weight of 80,000 (440,744, Sigma-Aldrich, St Louis, MO, USA) was dissolved in a 9:1 mixture of chloroform (C0584, 99.5%, Samchun Pure Chemical Co., Ltd., Seoul, Korea) and dimethylformamide (Sigma-Aldrich) at a concentration of 17% by weight. During electrospinning, the temperature and relative humidity were 20-21 • C and 50-55%, respectively. The flow rate of the solution, voltage, and tip-to-collector-distance were maintained at 0.2 mL/h, 18.0-18.1 kV, and 18 cm. A rotating drum collector was used to collect continuously electrospun nanofibers. The rotating speed (5 rpm) of the collector drum was set low enough to prevent fiber alignment. The fabricated nanofibrous membrane was 35 ± 5 µm-thick. To evaporate the remaining chloroform, the membrane was placed in a fume hood at room temperature for 24 h. Then the nanofibrous membrane was soaked in 70% ethanol overnight for sterilization under an ultraviolet lamp. Finally, the nanofibrous membrane was washed twice using PBS (pH 7.4, Gibco, Rockville, MD, USA).

Printing Melanoma Cells and Aggregation
A375P cells were printed in a drop-on-demand manner on the nanofibrous membrane using a micro dispensing system (CT-M3-04 Micro Dispensing, Microfab, Inc., Plano, TX, USA). The cell suspensions in culture media (1 × 10 6 cells/mL) were loaded in a 3 mL syringe. The syringe is assembled with a piezoelectric printer head and three-dimensional stage. The printer head has an inner diameter of 120 µm (MJ-ABL-01-120-8MX, Microfab, Inc.). Approximately 900 microdroplets, generated by the piezoelectric print-head, were dropped on the surface of the nanofibrous membrane. The printing frequency was set at 3 Hz, and the distance between the end tip of the printer head and the nanofibrous membrane surface is approximately 2 mm. The dropped cells formed an aggregation on the surface of the membrane because of highly porous and randomly intertwined nanofiber structures [19]. Immediately after the printing, the printed cell aggregates on the nanofibrous membrane were cultured with DMEM in 5% CO 2 at 37 • C for 1 day to ensure stable formation of cell aggregates ( Figure 4).

Development of Epidermis
The melanoma printed nanofibrous membrane was coated with collagen (2.5 mg/mL, collagen type I, Thermo Fisher Scientific) to enhance normal human epidermal keratinocytes (NHEK, Lonza Walkersville, MD, USA) adhesion, and the NHEK cells (2 × 10 5 cell/cm 2 ) were seeded with keratinocytes basal medium (KBM, Lonza) on the melanoma printed side of the membrane and incubated for 2 h in 5% CO 2 at 37 • C for initial cell adhesion. After that, they were air-liquid interface (ALI) cultured in 6-well plates with keratinocytes differentiation medium: a 1:1 mixture of DMEM and KBM containing calcium chloride (Sigma-Aldrich) at 1.8 mM (Figure 4) for more than 7 days. The medium was changed every two or three days.

Statistical Analysis
All data are representative data from at least three independent experiments. Statistical analysis was performed via one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test using GraphPad Prism, version 5.02, GraphPad Software (La Jolla, CA, USA). Unpaired t-tests were used for comparisons of the two groups. All data are expressed as the mean ± standard error of the mean (SEM). All graphs were generated using GraphPad Prism 5 (La Jolla, CA, USA). Statistical significance was considered when a p-value was less than 0.05 (* p < 0.05; ** p < 0.01; and *** p < 0.001).

Viability of Human Melanoma Cells after Treatment with NecroX-5
A375P showed a significant decrease in cell viability at concentrations of 50 µM of NX-5 (35.7%, p < 0.001), whereas A375SM showed little to no change in cell viability at all concentration levels, with the exception of a significant decrease at the 50 µM concentration (37.4%, p < 0.001). Based on these results, the NX-5 concentrations of 10 and 20 µM with relatively high cell viability of A375P and A375SM were chosen for further study (Figure 1). Cells were treated with various concentrations of NecroX-5 for 24 h. Cell viability was measured using the MTT assay. Results are expressed as the mean ± SEM from the three independent experiments. * p < 0.05, *** p < 0.001.
These results indicate that NX-5 can inhibit melanoma cell migration by reducing the expression of Rho-family GTPases such as Cdc42, Rac1, and RhoA, which differs depending on NX-5 concentration and the melanoma cell line.

Necrox-5 Treatment Down-Regulated the Expression of Rho-Family GTPase in 3D Melanoma Model
To confirm the anti-metastatic effect of NX-5 on melanoma cells, we constructed a 3D melanoma model ( Figure 4A-E) and then measured the expression of Rho-family GT-Pases after NX-5 (20 µM) treatment ( Figure 5). Immunohistochemistry showed that Cdc42, Rac1, and RhoA were constitutively expressed in the nuclei of melanoma cells of the untreated group, and NX-5 treatment decreased their expression. These results demonstrated that NX-5 treatment can down-regulate the expression of Rho-family GTPases in a 3D melanoma model. and RhoA were measured by qRT-PCR (A-C,G-I). The protein levels of Cdc42, Rac1, and RhoA were measured using western blot analysis (D-F,J-L). GAPDH was used as an internal control for mRNA and protein normalization (ZCL278 (10 μM), Cdc42 GTPase inhibitor; NCS23766 (10 μM), Rac GTPase inhibitor; CCG-1423 (100 nM), RhoA GTPase inhibitor). Results are expressed as the mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Necrox-5 Treatment Down-Regulated the Expression of Rho-Family GTPase in 3D Melanoma Model
To confirm the anti-metastatic effect of NX-5 on melanoma cells, we constructed a 3D melanoma model ( Figure 4A-E) and then measured the expression of Rho-family GTPases after NX-5 (20 μM) treatment ( Figure 5). Immunohistochemistry showed that Cdc42, Rac1, and RhoA were constitutively expressed in the nuclei of melanoma cells of the untreated group, and NX-5 treatment decreased their expression. These results demonstrated that NX-5 treatment can down-regulate the expression of Rho-family GTPases in a 3D melanoma model.   and RhoA were measured by qRT-PCR (A-C,G-I). The protein levels of Cdc42, Rac1, and RhoA were measured using western blot analysis (D-F,J-L). GAPDH was used as an internal control for mRNA and protein normalization (ZCL278 (10 μM), Cdc42 GTPase inhibitor; NCS23766 (10 μM), Rac GTPase inhibitor; CCG-1423 (100 nM), RhoA GTPase inhibitor). Results are expressed as the mean ± SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Necrox-5 Treatment Down-Regulated the Expression of Rho-Family GTPase in 3D Melanoma Model
To confirm the anti-metastatic effect of NX-5 on melanoma cells, we constructed a 3D melanoma model ( Figure 4A-E) and then measured the expression of Rho-family GTPases after NX-5 (20 μM) treatment ( Figure 5). Immunohistochemistry showed that Cdc42, Rac1, and RhoA were constitutively expressed in the nuclei of melanoma cells of the untreated group, and NX-5 treatment decreased their expression. These results demonstrated that NX-5 treatment can down-regulate the expression of Rho-family GTPases in a 3D melanoma model.

Discussion
In this study, NX-5 treatment decreased A375P and A375SM cell migration. This result was elucidated by reducing the expression of Rho-family GTPases (Cdc42/Rac1/RhoA) in human melanoma cell lines after NX-5 treatment. NX-5 treatment has been shown to down-regulate the expression of Rho-family GTPases in the 3D melanoma model as well.
NX-5 is one of the derivatives of the NecroX series of compounds that effectively inhibit mitochondrial ROS generation and oxidize low-density lipoprotein cholesterol generation [22,23]. Nam et al. reported that regulation of intracellular ROS through NX-5 attenuated p-glycoprotein expression via inhibiting STAT1 phosphorylation [24]. Park et al. showed that NX-5 prevents metastasis by inhibiting the migration of human cancer cells including breast and lung cancers. They also found that NX-5 inhibits metastasis-related cell migration by down-regulation of AKT through inhibiting Ca 2+ influx [18]. In this study, NX-5 treatment significantly reduced melanoma cell migration in the highly metastatic A375SM cell line as well as in the low metastatic A375P cell line.
In cancer, metastasis occurs when tumor cells migrate from their original site and invade other tissues in the 3D environment. There are two types of movement in tumor cell migration, mesenchymal and amoeboid modes, and both are involved in melanoma metastasis [25]. The Rho-family GTPases Cdc42, Rac1, and RhoA are key substances involved in the mesenchymal movement as well as amoeboid movement including actin dynamics and cell movements [26][27][28]. In addition, they are linked with MAPK and PI3K pathways involved in melanoma progression [16]. Overexpression of P29S mutation after UV exposure is reportedly involved in cancer proliferation and migration through Cdc42 and Rac1 [29,30]. RhoA has also been found to be involved in cell mobility in B16F10 melanoma cells [31]. ZCL278, a known Cdc42 inhibitor, directly blocks guanine nucleotide exchange factor (GEF) binding, and NSC23766, a Rac1 inhibitor, suppresses Rac-GTP loading by blocking GEF-mediated activation [32,33]. CCG-1423, a RhoA inhibitor, reduces RhoA-mediated transcription [34]. Our current study demonstrates that NX-5 significantly down-regulates mRNA and protein expression of Rho-family GTPases, which are important for metastasis in a 3D environment. In addition, NX-5 has the potential to have similar inhibitory effects as Cdc42 inhibitor, Rac1 inhibitor, and RhoA inhibitor in human melanoma cell lines. Taken together, our results suggest that NX-5 can replace known Rho-family GTPase inhibitors.
One of the strengths of this study is that a new melanoma model using 3D printing was applied. A 3D model of melanoma better represents the tumor environment found in patients than classical cell cultures. Especially, it is well known that the three-dimensional microenvironment influences cell functionality, phenotype, and morphology. We successfully demonstrate the fabrication of a 3D melanoma model using a drop-on-demand inkjet-based 3D printer and nanofibrous membrane which is advantageous in terms of maintaining the shape of cell aggregates after printing. Moreover, we make our model more clinically relevant by forming an epidermis layer on the top of the melanoma aggregates. The model described herein is highly reproducible and can be formed in a shorter period of time than existing 3D culture melanoma models [35] making it easier to access the experiment. Using the 3D melanoma model prepared in this way, the reduction in the expression of Rho-family GTPase (Cdc42/Rac1/RhoA) protein by NX-5 treatment was confirmed by immunohistochemical staining. We believe that this model could be used for further drug research on melanoma.
In conclusion, in this study, we showed that NX-5 treatment on human melanoma cell lines inhibited cell migration by suppressing the activity of Rho-family GTPases. This suggests the potential efficacy of NX-5 as a treatment modality for melanoma metastasis.

Informed Consent Statement:
In accordance with Article 2 of the Supplementary Regulations of the Bioethics and Safety Act, which has been in force since February 2013 in Korea, patient consent was waived because human materials used in this study were already collected prior to the enforcement of the law.

Data Availability Statement:
The data presented in this study are available in the article.