| Uveal melanoma is the most common primary intraocular tumor in adults. Theincidence of uveal melanoma is approximately6–7cases per million populations peryear in the USA. Despite advances in diagnosis and treatment of uveal melanoma inthe last decades, uveal melanoma still has a poor prognosis.Uveal melanoma-relatedmortality is31%after5years following diagnosis, and approximately50%after25years. Systemic metastasis is common in uveal melanoma and the liver is the mostfrequent organ for metastasis. Ten-year cumulative metastasis rate in uveal melanomapatients is34%. Various procedures have been reported for the treatment of metastaticuveal melanoma, but the response rate is very poor. Most uveal melanoma patientswith liver metastasis die within6month and the median survival time after diagnosisof metastasis is only3.6month. Because of the poor prognosis of metastaticmelanoma, new therapies are urgently required.Butein is a polyphenol extracted from stembark of cashews (Semecarpusanacardium), the heartwood of Dalbergia odorifera, caragana jubata, and Rhusverniciflua Stokes. It has been used as a traditional Chinese and Korean herbalmedicine to treat various diseases, including stomach cancer. Previous studies havesuggested that Butein has different pharmacological effects, including antioxidant,anti-inflammatory, and anti-angiogenic activities. Recently, Butein has beendemonstrated to possess anticancer effects in vitro and in vivo. It can suppress theproliferation and induce cell apoptosis in different human malignant tumor cellsincluding leukemia and various solid tumors. Butein has recently received a great dealof attention as useful chemopreventive and chemotherapeutic agents with onlyminimal toxicity in normal cells. studied the anticancer activity in an experimentalanimal model, found that Butein inhibit the tumor growth of hepatocellular cancer. Apreliminary clinical trail on the effects of flavonoids (mainly containing Butein) ingastric cancer patient indicated that this drug was well-tolerated and showed a markeddecrease in the size of tumor.The effect of Butein on melanoma has only been tested in mouse cutaneous melanoma cells, which showed that Butein inhibited cellproliferation and induced apoptosis in B16mouse cutaneous melanoma cell line.Theeffect of Butein on uveal melanoma cells has not been reported previously. Uvealmelanoma and cutaneous melanoma differ not only on the epidemiologic andetiologic aspects, but also on their molecular biological patterns. Therefore, these twomelanomas must be considered as different disease entities, independent studies arerequired for each type of melanoma to develop new treatments. The purpose of thepresent study was to explore the possibility of using Butein in the prevention ortreatment of uveal melanoma by the investigation on the cytotoxic effects of Buteinon human uveal melanoma cells in vitro with comparison to normal human scleralfibroblasts and retinal pigment epithelial (RPE) cells. Butein-induced biologicalchanges in melanoma cells, such as changes of mitochondrial transmembranepotential (MTP), release of cytochrome c from mitochondria, and the activation ofcaspase-9and caspase-3, were also studied to elucidate the signaling pathwayinvolved in Butein-induced apoptosis of melanoma cells.Methods: Three human uveal melanoma cell lines (M17, SP6.5, and C918),retinal pigment epithelial (RPE) cells and scleral fibroblasts were treated with Buteinat different dosages. The effects of Butein on cell viability were assessed by using theMTT assay. Cell apoptosis was determined using annexin V-FITC/ethidiumhomodimer III flow cytometry. Mitochondrial transmembrane potential changes wereassessed by using the JC-1fluorescent reader, cytosol cytochrome c levels, and theactivities of caspase-3,-8, and-9were measured by using an enzyme-linkedimmunosorbent assay or colorimetric assay.Results: Butein reduced the cell viability of cultured human uveal melanomacells in a dose-dependent manner (10,30, and100μM), with IC50at13.3μM and15.8μM in SP6.5and M17cell lines, respectively. Similar effects were also found ina highly aggressive and metastatic C918cell line (IC5016.7μM). Butein at lowerconcentrations (10–30μM) selectively reduced the cell viability of uveal melanomacells, without affecting cell viability of RPE cells and fibroblasts. Butein-inducedapoptosis of melanoma cells, increased mitochondrial permeability and the level ofcytosol cytochrome c, caspase-9and-3activities (but not caspase-8) in adosedependent manner.Conclusions: In our study, Butein significantly induced apoptosis of cultured human uveal melanoma cells at30μM. Therefore, although Butein-induces apoptosisin these different melanoma cell lines through the same pathway, human uvealmelanoma cells might be more sensitive to Butein than murine cutaneous melanomacells. In the present study, Butein significantly decreased cell viability of threedifferent human melanoma cell lines (M17, SP6.5, and C918) at concentration of10–300μM. Butein significantly decrease the cell viability of C918(anaggressive cellline) at a concentration comparatively to the other two melanoma cell lines, indicatingthat cultured human uveal melanoma cells are sensitive to the treatment of Butein,even the highly aggressive melanoma cell line. The effects of Butein on human uvealmelanoma cells were compared with scleral fibroblasts and RPE cells. Butein at10and30μM levels induces apoptosis of uveal melanoma cells, whereas the viability offibroblasts and RPE cells is not affected, suggesting that anticancer specificitypresents in Butein for uveal melanoma cells. In cells treated with low concentrationsof Butein, part of the cells were stained by annexin but not EtD-III, indicating earlyapoptotic changes. In cells treated with Butein at high concentrations, numerous cellswere stained by annexin and part of these cells also stained positively by EtD-III(apoptosis at advanced stage with damage of cell membrane). Butein significantlydamaged the MTP, increased the levels of cytosol cytochrome c and the activities ofcaspase-9and-3in a dose-dependent manner. Caspase-8, which plays an importantrole in the activation of extrinsic apoptosis pathway, did not increase after thestimulation of melanoma cells by Butein in this study. These observations areconsistent with many previous studies and suggest that Butein induces apoptosis inuveal melanoma cells mainly via the intrinsic mitochondrial pathway. In summary,Butein at a relatively low concentration significantly decreases cell viability andinduces apoptosis of human uveal melanoma cells without affecting the cell viabilityof RPE cells and fibroblasts, indicating that Butein has a selective and potentpro-apoptotic effect on human uveal melanoma cells in vitro. Since metastaticmelanoma is strongly resistant to conventional chemotherapy, the potent and selectivecytotoxic effects of Butein on C918cell line (a highly aggressive and metastatichuman melanoma cell line) indicate that Butein may be a promising agent to beexplored for prevention of uveal melanoma and/or treatment of metastatic uvealmelanoma. Determination of the bioavailability and plasma levels of Butein after oraladministration is currently underway. If the bioavailability is relatively poor as in several other phytochemicals with anticancer activities, further studies may berequired to enhance the bioavailability of Butein by modulating its metabolism,devising an appropriate formulation, identifying possible interaction with otheranticancer drugs, and developing more bioavailable analogues of the compound. |
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