Cinnamic Aldehyde Inhibits Proliferation And Invasion Of Human Cutaneous Melanoma Cells In Two-Dimensional And Three-Dimensional Culture

Melanoma usually originates from skin and is one of the most aggressive canceramong human tumors with a high mortality. The immortalization and invasive growth ofmalignant melanoma is the key factor in determining its clinical prognosis. In order tosearch for a safe and effective treatment，it is requisite to understand its biologicalcharacteristics and invasional ability.Two-dimensional (2D) cell culture is a major tool in melanoma cell biology research.2D culture of A375cells in vitro reproduces some of the biological characteristics ofmelanoma cells in vivo; however, it cannot be used to observe the processes involved in thecell-cell connection, the signal transduction between the cell and extracellular matrix(ECM). Three-dimensional (3D) cell culture mimics the melanoma environment in vivo andhas the advantage to be used to observe cell-cell interaction and cell-ECM communication.Using tissue-engineered skin as a3D model of invasion of malignant melanoma in vitro hasgreat potential for studying the biological characteristics of malignant melanoma andevaluating the efficacy of drugs. Up to date, a variety of melanoma cell lines have beenisolated, cultured and used in medical research. A375, an invasive melanoma cell line, hasbeen used in the present study.Cinnamic aldehyde (CA) is an α, β-unsaturated aldehyde that has been approved byFDA for use in foods. CA is the active constitute extracted from cinnamon and possessesversatile biological functions, such as anti-fungus, anti-virus and anti-cancer. The effect ofCA on proliferation, apoptosis and invasion of A375melanoma cells in2D culture wasobserved in this research. In order to establish a3D culture model of skin melanomainvasion in vitro, A375melanoma cells were inoculated onto the surface oftissue-engineered skin and cultured at the air-liquid interface. The effect of CA on proliferation and invasion of A375melanoma cells in3D culture was also detected.Methods:1. Different concentration of CA (0,10,20and40μM) was added to the culturemedium when A375melanoma cells reached about60%confluence. The cell proliferationwas tested with MTT methods. The cell cycle and apoptosis was tested by flow cytometry.The VEGF protein expression level was detected with ELISA and Western blot. The levelof MMP-9secretion was detected by gelatin zymography assay. Melanoma A375cellmigration was measured by In Vitro Wound Closure assay. Melanoma A375cellinvasiveness was assessed by transwell assay.2. Different concentration of CA (0,10,20and40μM) was added to the culturemedium when A375melanoma cells grew in3D scaffold. The cell proliferation was testedwith MTT methods. The VEGF protein expression level was detected with ELISA andWestern blot. The level of MMP-9secretion was detected by gelatin zymography assay.3. A375melanoma cells were inoculated onto the surface or into the matrix gel oftissue-engineered skin and cultured at the air-liquid interface or submerged in liquid. Thegrowth pattern of A375cells was detected with hematoxylin and eosin (HE) staining inorder to select a better inoculation and culture methods.4. Keratinocytes (KCs) and fibroblasts (FBs) were obtained from forskins of healthchildren. KC and FB were cultured and passaged in vitro. A375melanoma cells wereinoculated onto the surface of tissue-engineered skin and cultured at the air-liquid interface.Skin samples cultured for five, ten and fifteen days were stained with HE and observedunder transmission electron microscopy (TEM). The immunohistochemical staining ofE-cadherin, proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase(MMP)-9was carried out separately.5. From day5, CA at a concentration of20μΜ was added to the culture medium.Onday10and day15, the CA-treated skin samples were also fixed and stained with H.E. Theimmunohistochemical staining of PCNA and MMP-9was also carried out separately.6. After the human A375melanoma cells were treated with CA under2D and3Dculture condition, oxidative stress response genes of A375cells were detected with genechip.The NF-κB activation in A375cells was detected with ELISA. Results:1. Under2D culture condition, CA showed to promote A375cell apoptosis and inhibitcell proliferation after24hours. The cell proliferation showed significant differencesamong cells treated with different concentration of CA (P＜0.01). The cell proliferationwas most remarkably inhibited when CA was at40μM for72hours. The highest inhibitionrate was88.91±4.5%. The proliferation index was also showed the lowest when contrastedwith the control group (35.13±3.24%vs53.38±4.25%) while the ratio of apoptotic cell wasmost remarkably increased (10.50±0.37%vs4.37±0.25%). The cell apoptosis exhibitedsignificant differences among cells treated with different concentration of CA (P＜0.05).CA also inhibited the proliferation of A375cells in3D culture. The cell proliferation alsoshowed significant differences among cells treated with different concentration of CA (P＜0.01).2. CA also inhibited A375cells migration to scratch wounds (P＜0.05). CA reducedthe invasion of A375cells substantially in a concentration-dependent manner (P＜0.05).3. CA inhibited VEGF expression of A375cells in2D and3D culture condition whencompared with the control group (2D:0.204±0.017vs1.193±0.195；3D:0.392±0.019vs1.549±0.214). The VEGF secretion levels were significantly different among cells treatedwith different concentration of CA (P＜0.01). CA also decreased MMP-9secretion of A375cells in2D and3D culture when compared with the control group (2D:14150.77±555.63vs24885.83±865.80；3D:15121.43±497.95vs29408.50±817.62). The MMP-9expressionlevels were significantly different among cells treated with different concentration of CA (P＜0.05).4. HE staining showed that the melanoma cells formed clumps in the dermis and fromthere invaded downward. The invasion became deeper with the extension of culture time.After five days of culture, the tumor cells were distributed in the superficial layer of thedermis. A large number of A375cell clumps had invaded the deep dermis on day15. Themelanoma cells grew well within the dermis, and presented atypically shaped nuclei andabundant organelles. While the expression of E-cadherin was lost in the tumor cells,expression of MMP-9and PCNA increased with the increase of invasion depth. Theexpression levels were also significantly different among cells after five, ten, and fifteendays of culture (P＜0.05). The rates of PCNA-positive cells also exhibited significantdifferences among the five-, ten-, and fifteen-day cultures (P＜0.05). 5. CA inhibited the proliferation and invasion of melanoma cells in the3D culturemodel. Melanoma cell clumps became smaller and closer to the epidermal-dermal junctionin CA-treated skin equivalents and melanoma cells faintly expressed PCNA and MMP-9.The expression levels were significantly different between control and CA-treated skinequivalents (P＜0.01).6. CA suppressed NF-κB activation and induced oxidative stress responses in humanA375melanoma cells in2D and3D culture.Conclusion:In conclusion, these data demonstrate that the3D culture model of skin malignantmelanoma has been successfully established in the present study and exhibits good stabilityand controllability. It exhibits the biological characteristics of proliferation and invasion ofmalignant melanoma cells and hence can be used as a model system to study its biologicalcharacteristics and to evaluate the efficacy of therapeutic drugs. We found that CAsignificantly reduces the growth of melanoma and prevents its invasion in2D culture and3D model. On the basis of this model, we have preliminary evidence to suggest that CA haspotential anti-melanoma activity.