Choroidal Melanoma Cells Research Articles

Expression of the human poliovirus receptor (CD155) as an unfavorable prognostic factor for the choroid melanoma

Aim: to characterize CD155 expression in primary choroidal melanoma cells and determine its prognostic value for survival.Material and methods: 68 patients with primary choroidal melanoma were included. The levels and patterns of CD155 expression in tumor tissues were studied using immunohistochemical method. Regression analysis was performed to identify independent factors predicting the course of the disease.Results: in 51 % of cases, membrane expression of CD155 was registered in choroidal melanoma cells. In the group of tumors with any type of membrane expression of CD155, the signs of poor prognosis were statistically significantly more common. Membrane expression of CD155 was found to be an independent prognostic factor for the development of distant metastases according to multivariate regression analysis (RR 3.7 95 % CI 1.3–10.4, p = 0.005), as well as for death from choroidal melanoma (RR 3.2 95 %CI 1.2–8.3, p = 0.009).Conclusion: the expression patterns of CD155 in primary choroidal melanoma were studied. It was found that the presence of membrane expression of CD155 was associated with an increased risk of choroid melanoma progression by 3.7 times and death by 3.2 times.

Simultaneous choroidal melanoma and renal cell carcinoma in middle-aged patient

Simultaneous choroidal melanoma and renal cell carcinoma in middle-aged patient

HDAC7/c-Myc signaling pathway promotes the proliferation and metastasis of choroidal melanoma cells

Choroidal melanoma (CM) is the most common type of diagnosed uveal melanoma (UM), which is prone to metastasis and exhibits a poor prognosis. The molecular mechanisms underlying CM progression need further elucidation to research effective therapeutic strategies. Histone deacetylase 7 (HDAC7) is very important in regulating cancer progression, but the significance and effect of HDAC7 on CM progression are unclear. In the present study, we found that HDAC7 is overexpressed in CM tissues versus normal tissues. We built HDAC7 overexpressing CM cell lines to study the functions of HDAC7 in CM progression and verified that upregulation of HDAC7 promoted the proliferation and metastasis of CM cells, while pharmacological inhibition of HDAC7 suppressed both the proliferation and metastasis of CM cells. Furthermore, we found that the aforementioned cancer-promoting effect of HDAC7 was mediated by c-Myc. Targeted inhibition of c-Myc inhibited CM progression by interfering with the HDAC7/c-Myc signaling pathway. Our study highlighted the function of targeting the HDAC7/c-Myc signaling pathway to intervene in the pathological process of CM, which provides potential therapeutic strategies for CM treatment.

METTL14 promotes migration and invasion of choroidal melanoma by targeting RUNX2 mRNA via m6A modification.

The modification of N6-methyladenosine is involved in the progression of various cancers. This study aimed to clarify its regulatory mechanism in the pathogenesis of choroidal melanoma. Expression of methyltransferase-like 14 in choroidal melanoma or normal choroidal tissues was determined by Western blot and immunohistochemistry. The impacts of methyltransferase-like 14 on invasion and migration of choroidal melanoma cells were determined using functional and animal experiments. The interaction between methyltransferase-like 14 and its downstream target was identified by methylated RNA immunoprecipitation and a dual-luciferase reporter assay. Additionally, Wnt/β-catenin signalling pathway was evaluated by Western blot. Methyltransferase-like 14 was upregulated in choroidal melanoma compared to the normal choroidal tissues. Overexpression or knockdown of methyltransferase-like 14 enhanced or inhibited the invasion and migration of choroidal melanoma cells, respectively, both in vivo and in vitro. Methyltransferase-like 14 directly targeted downstream runt-related transcription factor 2 mRNA, depending on N6-methyladenosine. Additionally, the Wnt/β-catenin signalling pathway was activated by methyltransferase-like 14 in choroidal melanoma cells. Our study identified a novel RNA regulatory mechanism in which runt-related transcription factor 2 was upregulated by enhanced expression of methyltransferase-like 14 via N6-methyladenosine modification, thus facilitating migration and invasion of choroidal melanoma cells.

The Prognostic Relevance of PMCA4 Expression in Melanoma: Gender Specificity and Implications for Immune Checkpoint Inhibition.

PMCA4 is a critical regulator of Ca2+ homeostasis in mammalian cells. While its biological and prognostic relevance in several cancer types has already been demonstrated, only preclinical investigations suggested a metastasis suppressor function in melanoma. Therefore, we studied the expression pattern of PMCA4 in human skin, nevus, as well as in primary and metastatic melanoma using immunohistochemistry. Furthermore, we analyzed the prognostic power of PMCA4 mRNA levels in cutaneous melanoma both at the non-metastatic stage as well as after PD-1 blockade in advanced disease. PMCA4 localizes to the plasma membrane in a differentiation dependent manner in human skin and mucosa, while nevus cells showed no plasma membrane staining. In contrast, primary cutaneous, choroidal and conjunctival melanoma cells showed specific plasma membrane localization of PMCA4 with a wide range of intensities. Analyzing the TCGA cohort, PMCA4 mRNA levels showed a gender specific prognostic impact in stage I–III melanoma. Female patients with high transcript levels had a significantly longer progression-free survival. Melanoma cell specific PMCA4 protein expression is associated with anaplasticity in melanoma lung metastasis but had no impact on survival after lung metastasectomy. Importantly, high PMCA4 transcript levels derived from RNA-seq of cutaneous melanoma are associated with significantly longer overall survival after PD-1 blockade. In summary, we demonstrated that human melanoma cells express PMCA4 and PMCA4 transcript levels carry prognostic information in a gender specific manner.

Dual VEGF/PDGF knockdown suppresses vasculogenic mimicry formation in choroidal melanoma cells via the Wnt5a/β-catenin/AKT signaling pathway

This study aimed to explore the effects of knocking down both vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF) on vasculogenic mimicry (VM) formation in choroidal melanoma (CM) cells. Cell counting Kit (CCK)-8, monoclonal formation, wound healing, transwell and flow cytometry assays were used to observe the cell effects in CM cell line, ocular choroidal melanoma-1 cells (OCM-1) with respect to proliferation, migration, invasion and apoptosis. Three-dimensional (3D) cultures were also used to characterize VM tube structural effects in OCM-1 cells and western blotting was used to characterize protein expression changes in VM-related markers. Dual VEGF/PDGF knockdown suppressed cell proliferation, migration and invasion, but promoted cell apoptosis. It also reduced VM tube structures in OCM-1 cells. VM associated markers including, VE-cadherin, EphA2 and MT1-MMP were also down-regulated in OCM-1 cells. Similarly, Wnt5a, β-catenin and phosphorylated-AKT levels were also down-regulated. Western blotting and 3D cultures further demonstrated that combined Wnt5a silencing with dual VEGF/PDGF knockdown significantly decreased VE-cadherin and EphA2 levels and reduced VM tube structures in OCM-1 cells. Dual VEGF/PDGF knockdown suppressed cell growth and metastasis in OCM-1 cells, and blocked the Wnt5a/β-catenin/AKT signaling pathway thereby inhibiting VM formation.

Combination of oridonin and TRAIL induces apoptosis in uveal melanoma cells by upregulating DR5.

To investigate the inhibitory effect of the combined use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and oridonin on choroidal melanoma cell lines, and to explore its underlying mechanism. MUM-2B and C918 cells were treated with different concentrations of TRAIL and oridonin, and MTT assay used to evaluate the inhibition rate of the two compounds on cells. Then, the cell cycle distribution and apoptosis were detected by flow cytometry, and changes in apoptosis-related proteins such as death receptor 5 (DR5), a-caspase-3, and x-linked inhibitor of apoptosis protein (XIAP) were detected by Western blot. MUM-2B cells were transfected with si-DR5, which interfered with the expression of the DR5 gene. MTT and Western blot assay were used to detect cell activity and apoptosis-related proteins. When TRAIL and oridonin were simultaneously administered to the MUM-2B cells, the apoptosis rate was significantly higher than that by the two drugs individually. However, the effect of combined use of TRAIL and oridonin on C918 cells was not significantly different from that used alone. Cell cycle analysis showed that TRAIL and oridonin could induce G2/M arrest in MUM-2B cells. The Western blot results showed that the protein expression levels of the DR5, a-caspase-3, and BAX increased, while the expression levels of the anti-apoptosis-related proteins XIAP and BCL-2 were suppressed when TRAIL and oridonin simultaneously administered to MUM-2B cells. Interfering the expression of DR5 gene in MUM-2B cells could reverse the inhibitory effect of oridonin and TRAIL on the proliferation and apoptosis induction of MUM-2B cells. The inhibitory effects of oridonin and TRAIL on MUM-2B cells are significantly enhanced when they were administered as a combined treatment, which may ascribe to up-regulation of DR5.

Carbazole-based chemosensor for highly sensitive and selective bioimaging identification of hydrazine in multiple model systems via ratiometric and colorimetric

Hydrazine (N2H4) is a widely used propellent in spacecraft propulsion, a chemical catalyst, a common precursor to several pharmaceuticals, and a big threat to human and animal health. Overexposure to it can cause a series of diseases, which underlies the importance of developing an effective probe for monitoring its levels. Herein, a carbazole-based novel ratiometric and colorimetric chemosensor, PCC-EC, was successfully developed and well-characterized. PCC-EC has demonstrated ratiometric blue-shifted fluorescent response to hydrazine, which induced naked-eye color and fluorescent color change, respectively. Besides, PCC-EC realized a highly sensitive and selective response to hydrazine in multiple model systems ranging from aqueous solutions, water samples, zebrafish, and mung bean sprouts, even imaging in choroidal melanoma cells of the human eye cells with a rapid response, good cell/tissue permeability, low cytotoxicity, and a wide pH-working range. We also created a simple paper strip system to facilitate the quick detection of hydrazine. The sensing mechanism of PCC-EC has been explored by DFT calculations and mass spectrometry. Overall, PCC-EC employs a rarely-reported strategy in sensing hydrazine and its successful application in multiple biological and environmental systems highlights its good application potential in the future.

Luteolin inhibits the proliferation, adhesion, migration and invasion of choroidal melanoma cells in vitro

Choroidal melanoma is a devastating disease that causes visual loss and a high mortality rate due to metastasis. Luteolin, a potential anticancer compound, is widely found in natural plants. The aim of this study was to evaluate the antiproliferative, antiadhesive, antimigratory and anti-invasive effects of luteolin on choroidal melanoma cells invitro and to explore its potential mechanism. Cell counting kit-8 (CCK-8) assays, 5-ethynyl-2'-deoxyuridine (EdU) assays, Cell adhesion, migration, and invasion assays were performed to examine the inhibitory effects of luteolin on cell cell viability, proliferation, adhesion, migration and invasion capacities, respectively. Considering the correlation between Matrix metalloenzymes and tumor metastasis, Enzyme-linked immunosorbent assays (ELISAs) were used to assess matrix metalloproteases MMP-2 and MMP-9 secretion. Western blotting was performed to detect p-PI3K P85, Akt, and p-Akt protein expression. The cytoskeletal proteins vimentin were observed with cell immunofluorescence staining. Luteolin can inhibit OCM-1 cell proliferation, migration, invasion and adhesion and C918 cell proliferation, migration, and invasion through the PI3K/Akt signaling pathway. Therefore, Luteolin may have potential as a therapeutic medication for Choroidal melanoma.

Identification of Precise Therapeutic Targets and Characteristic Prognostic Genes Based on Immune Gene Characteristics in Uveal Melanoma.

The tumor microenvironment is an important factor for the immunotherapy of tumor patients. The sequenced transcriptome data can be used to describe the tumor microenvironment and various immune subtypes. We exploited published data on patients with uveal melanoma (UVM) to identify immune subtypes. Based on the immune-related gene sets of 80 patients with UVM in the TCGA database, we used consensus clustering to identify two immune subgroups. In the two immune subtypes, we analyzed clinical characteristics and immune infiltration. Class1 has low immune infiltration, contains memory B cells, Th2 cells, Th17 cells, eosinophils, natural killer cells, and has a better prognosis. Class2 has higher immune infiltration. CD8+ T cells, Th1 cells, MDSCs, and Dendritic cells are enriched in class2, which has strong cytolytic activity, high expression of immune checkpoint genes, and poor outcome. Moreover, we have developed and verified an immune characteristic model that can predict the prognosis of patients well. Through this model, we screened prostaglandin-endoperoxide synthase 2 (PTGS2) as the therapeutic target of UVM. Treatment of choroidal melanoma cell line (OCM1) cells with celecoxib (an inhibitor of PTGS2) effectively inhibits cell growth, proliferation, and promotes apoptosis. Our results show the immunological heterogeneity of UVM patients and also provide an ideal therapeutic target for the future treatment design of patients.

Efficient Anticancer Effect on Choroidal Melanoma Cells Induced by Tanshinone IIA Photosensitization

Tanshinone IIA (TanIIA) has multiple biological functions and already been clinically used to treat many cardiovascular diseases. TanIIA is a photoactive molecule and can be excited by light to generate 3 TanIIA*. Generation of 3 TanIIA* by TanIIA photosensitization indicates that TanIIA may serve as a photosensitizer to bring photodynamic damage to organisms. Therefore, human choroidal melanoma MUM-2B cell was chosen as a superficial tumor model and the photodynamic effect of TanIIA on tumor cells was evaluated in this study. The results showed that TanIIA photosensitization could generate singlet oxygen in noncellular system. MTT, clone formation and wound-healing assays showed that the survival and migration of MUM-2B cells could be efficiently inhibited by TanIIA photosensitization. And then, laser confocal microscope and flow cytometry were used to try to elucidate related mechanism. It was found that TanIIA could pass through cellular membrane and preferably accumulate in nucleus. TanIIA photosensitization could efficiently induce cell apoptosis and necrosis, increase intracellular ROS levels, decrease mitochondria membrane potential, and lead to cell cycle arrest in G2/M phase. Our findings indicate that TanIIA photosensitization can exert remarkable toxicity on choroidal melanoma cells.

Effect of luteolin on apoptosis and vascular endothelial growth factor in human choroidal melanoma cells.

To investigate the effects of luteolin on apoptosis, the cell cycle, and the expression and secretion of vascular endothelial growth factor (VEGF) in human choroidal melanoma cells (C918 and OCM-1). C918 and OCM-1 cells cultured in vitro were treated with various concentrations of luteolin (0, 5, 10, 15 µmol/L). Cell growth was observed with an inverted microscope, and cell cycle arrest was detected by propidium iodide (PI) staining using flow cytometry. Apoptosis was detected by Hoechst33342 staining, and apoptosis rate was determined by Annexin V-FITC/PI experiments using flow cytometry. The expression of apoptosis-related proteins Bcl-2, Bax and VEGF was analyzed using Western blots. The levels of VEGF secreted by the cells into the supernatant was analyzed using ELISA. After treating with 5 to 15 µmol/L luteolin for 48h, the fusion degree of C918 and OCM-1 cells decreased, and more floating apoptotic cells appeared. Luteolin treatment increased the G0-G1 phase ratio of the C918 and OCM-1 cells, blocked cell cycle progression, and increased the apoptosis rate of the C918 and OCM-1 cells. Western blot showed that luteolin decreased the expression of Bcl-2 and VEGF in the C918 and OCM-1 cells and increased the expression of Bax protein. The ELISA results showed that 10 to 15 µmol/L luteolin decreased the cell secretion of VEGF. Luteolin may induce apoptosis by regulating the levels of apoptosis-related proteins in C918 and OCM-1 cells. Luteolin can induce cell cycle arrest, decrease the expression of VEGF.

LiCl induces apoptosis via CHOP/NOXA/Mcl-1 axis in human choroidal melanoma cells

BackgroundChoroidal melanoma is the most common primary intraocular malignancy that occurs in adults. Lithium Chloride Promotes Apoptosis in Human Leukemia NB4 Cells by Inhibiting Glycogen Synthase Kinase-3 Beta. In this study, we aimed to understand whether LiCl exerts anticancer effects on choroidal melanoma cells and elucidate the underlying molecular mechanisms.MethodsHuman choroidal melanoma cells were treated with LiCl, and cell survival was assessed with MTT assays. Cell reproductive viability was measured by plate colony formation assays. Cell apoptosis was evaluated using flow cytometry, and proteins were detected using western blotting. A human choroidal melanoma xenograft model was established to demonstrate the effect of LiCl on human choroidal melanoma in vivo.ResultsWe found that LiCl inhibited cell survival and clonogenic potential and induced apoptosis in human choroidal melanoma cells. LiCl also reduced the proliferation of choroidal melanoma cells in vivo. Moreover, the upregulation of NOXA and downregulation of Mcl-1 were responsible for LiCl-induced apoptosis. Mcl-1 overexpression obviously impaired LiCl-induced apoptosis and cleavage of caspase8, caspase9, caspase3 and PARP. Moreover, the protein expression of endoplasmic reticulum stress markers, including IRE1α, Bip, p-eIF2α, ATF4 and CHOP, were upregulated following treatment with LiCl. When CHOP expression was knocked down and cells were treated with LiCl, the protein level of NOXA was partially increased, and Mcl-1 expression was increased, while the cleavage of caspase8, caspase9, caspase3 and PARP that was induced by the LiCl was reduced compared with the vehicle treated group. Prolonged ER stress results in the activation of the apoptotic pathway.ConclusionsIn summary, LiCl induced an endoplasmic reticulum stress response while activating intrinsic apoptosis. Furthermore, the CHOP/NOXA/Mcl-1 axis contributed to LiCl-induced apoptosis both in vitro and in vivo. The present study provides important mechanistic insight into potential cancer treatments involving LiCl and enhances the understanding of human choroidal melanoma.

Orbital exenteration and conjunctival melanoma: a 14-year study at the Jules Gonin Eye Hospital.

PurposeTo report our 14-year experience with orbital exenteration and assess risk factors for poor prognosis by focusing on conjunctival melanoma.Patients and methodA retrospective study was conducted in our tertiary care centre (Jules Gonin Eye Hospital, Lausanne, Switzerland) between 2003 and 2017. Inclusion criteria were patients aged ≥18 years with a follow-up >12 months, without metastatic spread at the time of surgery. Data recorded were age, gender, tumour histology, surgical technique, postoperative complications, surgical margin status, local recurrence, postoperative radiation beam therapy and metastatic status.ResultsTwenty-five patients with a mean age of 63.2 years (38–92) were included. Conjunctival melanoma was the most frequently identified tumour (n = 14, 56%) followed by conjunctival squamous cell carcinoma (n = 4, 16%), sebaceous carcinoma (n = 3, 12%), choroidal melanoma (n = 2, 8%) and basal cell carcinoma (n = 2, 8%). Eighteen tumours (72%) originated from the conjunctival tissue. Clear surgical margins were achieved in 21 (84%) patients. Fourteen (56%) patients experienced distant metastases and died from metastatic spread after a mean follow-up of 52.3 months (6–120). The 1-, 3- and 5-year overall survival (OS) was 96%, 72% and 60%, respectively. In the univariate analysis, positive surgical margins, local recurrence and metachronous metastases were associated with a decreased OS (p = 0.002, p = 0.005 and p = 0.007, respectively). In the multivariate analysis, positive surgical margins and metachronous metastases were also associated with a decreased OS (p = 0.02 and p = 0.042, respectively). Conjunctival melanoma was not associated with a poorer prognosis (p = 0.280).ConclusionFree surgical margins are needed to increase OS. To achieve clearer surgical margins, neoadjuvant targeted therapies/immunotherapies may be considered.

MiR-9-5p Inhibits the Proliferation, Migration and Invasion of Choroidal Melanoma by Targeting BRAF.

Objective:According to different reports, miR-9-5p either facilitates or suppresses the occurrence of tumors. BRAF is a serine/threonine kinase involved in the MAPK pathway and is a proto-oncogene promoting the progression of many tumors, especially melanoma. The present study aimed to reveal the mechanism of action of miR-9-5p and BRAF in choroidal melanoma (CM).Methods:RT-qPCR was used to detect the expression of miR-9-5p in CM cells after transfection with miR-9-5p mimics and inhibitor. EdU assay and Transwell assay, respectively, showed the proliferation, migration and invasion of CM cells after transfection with miR-9-5p mimics and inhibitor. A bioinformatics website was used for target prediction and the dual luciferase reporter assay was used to verify the interaction between miR-9-5p and BRAF. RT-qPCR and Western blot were performed to examine the expression of BRAF mRNA and protein, respectively. The BRAF protein was knocked down by siRNAs and then examined by Western blot. The effects of BRAF in CM cells were investigated by EdU assay and Transwell assay. Overexpressing BRAF and transfecting miR-9-5p mimics into choroidal melanoma cells confirmed the interaction between miR-9-5p and BRAF.Results:miR-9-5p could bind to the BRAF mRNA 3’UTR and inhibit the transcription and translation of BRAF, thereby suppressing the proliferation, migration and invasion of CM cell lines. Moreover, silencing BRAF inhibited the progression of CM cells.Conclusions:In conclusion, this study is the first to investigate the association among BRAF, miR-9-5p and the progression of CM cells. In addition, the interaction between BRAF and miR-9-5p was explored for the first time in CM. Thus, our study suggests that miR-9-5p, BRAF and their interaction may act as potential therapeutic targets for CM.

The Use of TAT Peptide-Functionalized Graphene as a Highly Nuclear-Targeting Carrier System for Suppression of Choroidal Melanoma.

Tumorous metastasis is a difficult challenge to resolve for researchers and for clinicians. Targeted delivery of antitumor drugs towards tumor cells’ nuclei can be a practical approach to resolving this issue. This work describes an efficient nuclear-targeting delivery system prepared from trans-activating transcriptional activator (TAT) peptide-functionalized graphene nanocarriers. The TAT peptide, originally observed in a human immunodeficiency virus 1 (HIV-1), was incorporated with graphene via an edge-functionalized ball-milling method developed by the author’s research group. High tumor-targeting capability of the resulting nanocarrier was realized by the strong affinity between TAT and the nuclei of cancer cells, along with the enhanced permeability and retention (EPR) effect of two-dimensional graphene nanosheets. Subsequently, a common antitumor drug, mitomycin C (MMC), was covalently linked to the TAT-functionalized graphene (TG) to form a nuclear-targeted nanodrug MMC-TG. The presence of nanomaterials inside the nuclei of ocular choroidal melanoma (OCM-1) cells was shown using transmission electron microscopy (TEM) and confocal laser scanning microscopy. In vitro results from a Transwell co-culture system showed that most of the MMC-TG nanodrugs were delivered in a targeted manner to the tumorous OCM-1 cells, while a very small amount of MMC-TG was delivered in a non-targeted manner to normal human retinal pigment epithelial (ARPE-19) cells. TEM results further confirmed that apoptosis of OCM-1 cells was started from the lysis of nuclear substances, followed by the disappearance of nuclear membrane and cytoplasm. This suggests that the as-synthesized MMC-TG is a promising nuclear-target nanodrugfor resolution of tumorous metastasis issues at the headstream.

Fabrication of chitosan based nanocomposite with legumain sensitive properties using charge driven self-assembly strategy.

Chitosan (CS) based nanoparticles (NPs) have several advantages in delivering drugs. They are usually prepared in a micro-emulsion solvent system but this route can leave significant levels of potentially harmful organic solvent residue in the NPs. In this study, we prepared CS based nanocomposites using charge driven self-assembly in an aqueous buffer, thus avoiding the use of organic solvents. Doxorubicin (DOX) was covalently attached to positive charged CS with a legumain substrate peptide to confer targeted drug release property, since legumain is often overexpressed in tumors or tumor associated micro environments. This DOX prodrug solution interacted with negative charged methoxyl poly (ethylene glycol)-block-poly (glutamic acid) copolymer (PEG-PGA) in an aqueous buffer forming nanocomposite with a regular morphology. The particle size and zeta potential of these NPs was regulated by the addition of different PEG-PGA concentrations into the DOX prodrug solution. Due to its potential for legumain triggered release, this DOX NP exhibited enhanced cytotoxicity against choroidal melanoma cell line (Mum-2C) and reduced cytotoxicity on normal human corneal epithelial cells (HCEC), suggesting a good potential for enhanced targeted delivery of chemotherapeutic agents. A chitosan based nanocomposite with legumain sensitive properties are rapidly controllable prepared in aqueous buffer by charge driven self-assembly strategy, without using micro-emulsion solvent system and cross-linking agents.

Gamma irradiation of ocular melanoma and lymphoma cells in the presence of gold nanoparticles: invitro study.

The aim of this work was to determine whether conjugation of cultivated choroidal melanoma and Burkitt's lymphoma cells with gold nanoparticles (GNPs) is beneficial for these series of ocular cancer patients. GNPs are radiosensitizers and can sensitize tumors to radiotherapy.This application has been examined in several tumor types, but not in choroidal melanoma. This study shows the results of in vitro study on the choroidal melanoma and also Burkitt's lymphoma cells in the presence of GNPs during continuous gamma irradiation. Cytotoxicity of GNPs were assessed for five different concentrations then cultured melanoma and Burkitt's lymphoma cells were irradiated with a Gamma source in the presence and absence of NPs. Incubation of melanoma cells with GNP concentrations below 100 μg/ml, accompanied by gamma irradiation, increased cell death (P value = 0.016) . In the absence of irradiation, GNPs at these concentrations did not affect cultured melanoma cell metabolism. Reduced cell viability resulted from a significant increase in absorbed energy by the tumor. Moreover, GNP concentrations higher than 200 μg/ml induced cytotoxicity in melanoma cells. Cytotoxicity assay in GNPs‐loaded Burkitt's lymphoma cells showed a slight decrease in cell viability at 50 μg/ml and clear cytotoxicity at concentrations higher than 100 μg/ml (P value = 0.035). Concentration and proper injection doses of GNPs in sensitive tissues such as the human eye are important variables yet to be determined.This is the first report of choroidal melanoma dosimetry performed in the presence of GNPs and provides valuable insights into future therapeutic approaches. Further in vitro study with more different sizes and concentrations is needed to determine the optimum size and concentration before any clinical research in this regard.

Quantitative Evaluation of Vascular Microcirculation Using Contrast-Enhanced Ultrasound Imaging In Rabbit Models of Choroidal Melanoma.

We investigated the application of contrast-enhanced ultrasound (CEUS) to quantify vascular perfusion in two animal models of choroidal melanoma. B16F10 and choroidal melanoma cell line (OCM1) melanoma xenografts were implanted into the cyclodialysis cleft of rabbits' right eyes. Fundus photography, indocyanine green angiography (ICGA), B-mode ultrasound, and CEUS were used to examine the tumors. Standard vascular parameters of CEUS imaging were evaluated, including the area under the receiver operating characteristic curve (AUC), peak intensity (IMAX), mean transit time (mTT), rise time, and time to peak. Kruskal-Wallis tests followed by Nemenyi tests and Bonferroni corrections were used to analyze the findings. OCM1 and B16F10 animal models of choroidal melanoma were established. Tumor size was larger in the animals receiving B16F10 xenografts compared with the OCM1. Choroidal melanoma received a more abundant blood supply and had higher blood flow velocities than normal eye tissue. The animals receiving B16F10 xenografts had larger blood volume but smaller blood flow velocity compared with the OCM1 models. Vascular mimicry was present in seven of eight OCM1 tumors (87.5%). Growth and blood circulation patterns differed between OCM1 and B16F10 melanomas. CEUS could be used to noninvasively dynamically, and safely detect the microcirculation patterns, quantify blood circulation volume, and velocity in ocular melanoma in animal models. The blood circulation patterns of two models were different in both qualitatively and quantitative analysis under CEUS and ICGA.

C-FLIP and the NOXA/Mcl-1 axis participate in the synergistic effect of pemetrexed plus cisplatin in human choroidal melanoma cells.

Choroidal melanoma is the most common primary malignant intraocular tumor, and very few effective therapies are available to treat it. Our study aimed to understand whether pemetrexed plus cisplatin exerts a beneficial synergistic effect in human choroidal melanoma cells and to delineate the underlying molecular mechanism. To accomplish these aims, we treated choroidal melanoma cells with pemetrexed and cisplatin and assessed cell survival with SRB and MTT assays. Proteins were detected using western blotting analysis. NOXA and CHOP were knocked down with siRNA. We found that pemetrexed or cisplatin alone inhibited survival and induced apoptosis in human choroidal melanoma cells. Furthermore, the expression levels of c-FLIP, an anti-apoptotic protein in the extrinsic apoptosis pathway, and Mcl-1, an anti-apoptotic protein in the intrinsic apoptosis pathway, were decreased by pemetrexed or cisplatin respectively, while the expression of a pro-apoptotic protein in the intrinsic apoptosis pathway, NOXA, was up-regulated. Moreover, pemetrexed or cisplatin alone increased the protein expression of the endoplasmic reticulum stress markers IRE1α, Bip and CHOP. Silencing CHOP expression reduced NOXA expression. These findings suggest that the pemetrexed or cisplatin induced intrinsic apoptosis via activation of the ER stress response. Importantly, combining the two compounds more strongly induced apoptosis. Following the cotreatment, CHOP and NOXA expression increased, while c-FLIP and Mcl-1 expression decreased, and these effects were more pronounced than when using either compound alone. This result suggests that pemetrexed and cisplatin synergistically activate ER stress response-induced apoptosis in choroidal melanoma cells. To summarize, the c-FLIP and NOXA/Mcl-1 axis participated in the synergistic effect of pemetrexed plus cisplatin in human choroidal melanoma cells. Intrinsic apoptosis was induced via activation of the ER stress response. Our study provides important mechanistic insights into potential cancer treatment with pemetrexed plus cisplatin and enriches our understanding of human choroidal melanoma.