The Effect And Mechanism Of Zeaxanthin Induces Apoptosis And Inhibit Cell Migration, Invasion In Human Uveal Melanoma Cells

Uveal melanoma is the most common primary intraocular tumor in the adult population, with an incidence of 6-7 cases per million per year in the United States. Uveal melanoma has a poor prognosis, with half of uveal melanoma patients dying from their disease within 25 years. The high mortality rate is related to the development of metastasis, which has a strong preference for the liver. Most uveal melanoma patients with liver metastasis die within 6 months.Because of the poor prognosis of metastatic uveal melanoma, new therapies are urgently required.Zeaxanthin is a carotenoid pigment, which belongs to the xanthophyll subclass.It is found at high levels in various foods(e.g., egg yolk, corn and many vegetables and fruits), herbs and traditional Chinese medications(Hippophae rhamnoides, Lycium barbarum, Lycium chinese, Lilium hansonii, Cycas revolute and Crocus sativus).The relationship between the incidence of various malignant tumors and zeaxanthin in the diet or blood has been studied previously. Epidemiological studies showed that high intake and high blood levels of zeaxanthin(alone or combined with lutein) might be associated with a lower risk of occurrence of various malignant tumors, including non-hodgkin lymphoma, cervical, esophagus,stomach, lung, breast, kidney, head and neck, colon and pancreas cancers. There were only a few reports on the effects of zeaxanthin on various cancer cells in vitro and these studies were tested only in few cancer cell lines. It has been reported that zeaxanthin might inhibit cell growth or cause apoptosis of lymphoma, breast cancer, colon cancer and neuroblastoma cells in vitro. To our best knowledge, the relationship between the incidence of uveal melanoma andzeaxanthin in the diet or blood, the effect and mechanism of zeaxanthin induces apoptosis and inhibit cell migration, invasion in human uveal melanoma cells has not been reported previously.Purpose: The purpose of the present study was to investigate the effect and mechanism of zeaxanthin induces apoptosis and inhibit metastatic uveal melanoma cell migration and invasion, to suggest that zeaxanthin may be a promising agent worth exploring for the treatment of uveal melanoma and other malignant tumors.Methods: Three different normal cell lines, human normal uveal melanocytes,fibroblasts and retinal pigment epithelial(RPE) cells, and two human choroidal melanoma cell lines(SP6.5 and C918) were treated with zeaxanthin at different dosages(10,30,100 and 300 μM). MTT Assay for cell viability. Using flow cytometric detection of apoptotic cells to analyze the cells. Western Blot analysis for Bcl-2 family proteins. MTP assessment(JC-1). The cytochrome c level in the cytosol and the activities of caspase-8, caspase-9 and caspase-3 were measured using enzyme-linked immunosorbent assay(ELISA) kit. The effect of zeaxanthin on the cell migration of cultured C918 cell line as determined by wound healing assay and Boydon chamber assay. Matrigel invasion assay showed the cell invasion. Secretion of MMP-2 and the NF-κB levels were measured by ELISA kit.Results: Zeaxanthin at 10, 30, 100, and 300 μM levels did not affect the cell viability of cultured human uveal melanocytes, RPE cells, or fibroblasts, but significantly decreased the cell viability of uveal melanoma cells(SP6.5 and C918) in a dose-dependent manner. Cells cultured with zeaxanthin for 6 and 24 h showed a significant increase in total apoptotic rate(P < 0.05, as compared with cells cultured without zeaxanthin). Western blot analysis revealed that 30 μM zeaxanthin significantly decreased the expression of Bcl-x L and Bcl-2,anti-apoptotic proteins, and increased the expression of Bak, a pro-apoptotic protein, in SP6.5 cells(P < 0.05). Zeaxanthin significantly decreased the expression of Bcl-x L, an anti-apoptotic protein, and increased the expression ofBax, a pro-apoptotic protein, in C918 cells(P < 0.05). Treatment with zeaxanthin(30 μM) caused a diffuse green staining pattern, representative of damaged MTP.Zeaxanthin increased cytosol cytochrome c levels, the Caspase-3 and-9 activities in melanoma cells in a dose-dependent manner, but it did not affect Caspase-8activities. Zeaxanthin significantly inhibited the C918 cell migration and invasion.Secretion of MMP-2 and the NF-κB levels by melanoma cells were significantly inhibited by zeaxanthin(P <0.05).Conclusions: In the present study, zeaxanthin significantly decreased cell viability of two different human melanoma cell lines at concentration of 10 – 100μM in a dose-dependent manner, but the cell viability of normal cells was not affected at even the highest tested levels of zeaxanthin(300 μM, which is 30-fold of the minimal toxic levels in melanoma cells), suggesting that zeaxanthin has specific anticancer activity in uveal melanoma cells. C918 is more sensitive to cytotoxic effect of zeaxanthin as compared with the non-metastatic melanoma cell line(SP6.5), indicating that zeaxanthin may be a promising candidate for prevention and treatment of metastatic uveal melanoma. Among cells treated with zeaxanthin, annexin-stained cells(indicating apoptotic changes) significantly increased compared to controls. The percentage of apoptotic cells also increased with time, suggesting that zeaxanthin can induce apoptosis of melanoma cells.The results suggest that the zeaxanthin induced apoptosis effect occurs via the intrinsic cell death pathway and is regulated by Bcl-2 family proteins.Accumulation of pro-apoptotic proteins on the mitochondrial outer membrane results in increased mitochondrial membrane permeability, causing the release of cytochrome c into the cytoplasm. Cytochrome c promotes activation of caspase-9,which in turn promotes activation of caspase-3, leading to apoptosis of the tumor cell, this effect is mainly through the regulation of intrinsic mitochondrial pathway. Zeaxanthin can also inhibit the cell migration and invasion of cultured human UM cells by the decrease of secretion of MMP-2. This effect is attributed to the inhibition of NF-κB pathway in UM cells by zeaxanthin. These results suggest that zeaxanthin might be a potentially therapeutic approach in theprevention of metastasis in uveal melanoma.