OCM-1 Cells Research Articles

The BET inhibitor JQ1 suppresses tumor survival by ABCB5-mediated autophagy in uveal melanoma

Uveal melanoma (UM), the most common adult ocular tumor, is aggressive and resistant to treatment, posing threat to patients' lives. The novel, effective therapies and the exploration of chemosensitizer for UM are imperative. The anticancer efficacy was evaluated with and without JQ1 treatment or ABCB5 gene silencing or overexpression. RNA sequencing identified downstream effectors in JQ1-treated cells. Integrated analysis of The Cancer Genome Atlas data (TCGA) and immunohistochemistry (IHC) revealed the oncogenic role of ABCB5. Functional analyses of JQ1 and defective ABCB5 were conducted using flow cytometry, transmission electron microscopy (TEM), IHC and western blot. The effects of JQ1 were validated in a heterotopic tumor model derived from OCM-1 cells. JQ1 inhibited cell proliferation, migration and invasion, induced cell cycle arrest and promoted apoptosis. JQ1 also suppressed the survival of UM in heterotopic tumor model. RNA sequencing indicated that JQ1 down-regulated the expressions of ABCB5 and autophagy-related genes, which was confirmed in vitro and in vivo by western blot. ABCB5, a marker associated with cancer stem cells and chemo-resistance, exhibited heightened expression in UM tissues, linked to immune infiltration. Notably, disrupting ABCB5 expression impeded UM cell proliferation and interfered with autophagy. Moreover, the overexpression of ABCB5 promoted cell proliferation, migration and invasion, and rescued autophagy related gene expression. Of note, JQ1 enhanced the sensitivity of OCM-1 cells to chemotherapy. Thus JQ1 inhibits UM survival via ABCB5-mediated autophagy and enhances chemo-sensitivity, suggesting potential for BET-based approaches in UM clinical management.

Single cell sequencing analysis constructed the N7-methylguanosine (m7G)-related prognostic signature in uveal melanoma.

Uveal melanoma is a highly malignant tumor in the eye. Its recurrence and metastasis are common, and the prognosis is poor. The transcriptome data of UVM were downloaded from TCGA database, and the single cell sequencing dataset GSE139829 was downloaded from GEO database. Weighted co-expression network analysis was used to explore the modules associated with m7G. Lasso regression was used to construct M7G-related prognostic signature. Immune infiltration analysis was used to explore the significance of the model in the tumor immune microenvironment. Finally, cell assays were used to explore the function of key genes in the MUM-2B and OCM-1 cell lines of UVM. The prognostic signature was constructed by Cox regression and Lasso regression. Patients could be divided into high-risk group and low-risk group by this signature, and the high-risk group had worse prognosis (P<0.05). Cell experiments showed that the proliferation, invasion and migration ability of UVM cell lines were significantly decreased after the knockdown of PAG1, a key gene in signature, which proved that PAG1 might be a potential target of UVM. Our study explored the significance of m7G in UVM, provided biomarkers for its diagnosis and treatment.

Quercetin Mediated TET1 Expression Through MicroRNA-17 Induced Cell Apoptosis in Melanoma Cells.

A previous report suggested that the expression of ten-eleven translocation (TET) proteins is abnormal in certain cancers. Quercetin has been demonstrated as anti-cancer role in cancer development. In order to explore the inhibitory effect and mechanism of quercetin on uveal melanoma cells, the expression of TET proteins was analyzed in the present study. Our results suggest that the expression of TET1 was increased following treatment with quercetin in OCM-1, SK-MEL-1, and B16 cells. In addition, quercetin treatment induced apoptosis and inhibited migration and invasion. To further investigate the association of the expression of TET1 with cell growth, apoptosis, migration, and invasion, cell lines in which TET1 was knocked-down or overexpressed were constructed. The results showed that the increased expression of TET1-induced apoptosis, increased 5-hydroxymethylcytosine (5hmC). and inhibited invasion. Our bioinformatics studies indicated that TET1 is a target gene of microRNA-17 (miR-17) Our results showed that inhibition of the expression of miR-17 resulted in increased TET1 expression in OCM-1 cells. Furthermore, our results indicated that quercetin treatment increased TET1 expression and inhibited melanoma growth in nude mice. Taken together, our results suggest that quercetin can regulate cell proliferation and apoptosis through TET1 via miR-17 in melanoma cells.

Sclerostin Suppression Facilitates Uveal Melanoma Progression Through Activating Wnt/β-Catenin Signaling Via Binding to Membrane Receptors LRP5/LRP6.

ObjectiveUveal melanoma (UM) is the most frequent primary eye cancer in adults with a 50% mortality rate. Characterizing the fundamental signaling pathways that drive UM is of importance for the development of targeted therapy. This study aims to probe the impact of sclerostin (SOST) on malignant progression of UM and regulation of Wnt/β-catenin signaling.MethodsEpithelial-type (n=20) and spindle-type (n=16) UM tissues were collected for immunohistochemical staining of SOST, Wnt-1, and β-catenin expressions. SOST was silenced in three UM cell lines (primary spindle-type OCM-1 cells, metastatic epithelial Mum-2B cells, and metastatic spindle-type Mum-2C cells) through transfecting specific siRNA. RT-qPCR and Western blot were presented for examining the levels of SOST, and markers in Wnt/β-catenin signaling. Flow cytometry, MTT, EdU, transwell, and tube formation assays were conducted, respectively. By implanting BALB/c nude murine models in situ, the function of SOST on tumor growth was investigated, followed by immunofluorescence double staining of SOST and LRP5/6.ResultsLow SOST expression as well as high Wnt-1 and β-catenin expressions were found in epithelial-type (high malignancy) than spindle-type (low malignancy) UM tissues. Silencing SOST activated the markers in Wnt/β-catenin signaling as well as accelerated cell cycle progression, migration, invasion, angiogenesis, and reduced apoptosis in UM cells. In situ tumor formation in murine eyes showed that SOST knockdown promoted tumor growth. Moreover, SOST interacted with LRP5/LRP6.ConclusionSOST silencing may facilitate the malignant progression of UM cells through activating Wnt/β-catenin signaling. Mechanistically, SOST may exert this function by interacting with LRP5/LRP6 membrane receptors.

Spheroid-induced heterogeneity and plasticity of uveal melanoma cells

PurposeThe mechanism underlying cancer heterogeneity and plasticity remains elusive, in spite of the fact that multiple hypotheses have been put forward. We intended to clarify this heterogeneity in uveal melanoma (UM) by looking for evidence of cancer stem cell involvement and a potential role of ZEB1 in cancer cell plasticity.MethodsSpheroids derived from human UM cells as well as xenograft tumors in nude mice were dissected for signs of heterogeneity and plasticity. Two human UM cell lines were studied: the epithelioid type C918 cell line and the spindle type OCM1 cell line. We knocked down ZEB1 in both cell lines to investigate its involvement in the regulation of stem-like cell formation and vascularization by qRT-PCR, immunohistochemistry, flow cytometry, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays.ResultsWe found that a small side population (SP) in OCM1 showed stem cell-like properties such as heterogeneity, remote dissemination and nuclear dye exclusion after spheroid formation in vitro. ZEB1 regulated UM stem cell generation indirectly by promoting cell proliferation to form large size tumors in vivo and spheroid in vitro, and directly by binding to stemness genes such as TERT and ABCB1. In addition, we found that ZEB1 participates in vasculogenic mimicry system formation through the regulation of CD34 and VE-cadherin expression.ConclusionsFrom our data we conclude that cancer stem cells may contribute to UM heterogeneity and plasticity and that ZEB1 may play a regulatory role in it.

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

ObjectiveThis 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. MethodsCell 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. ResultsDual 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. ConclusionsDual 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.

Artesunate Suppresses Choroidal Melanoma Vasculogenic Mimicry Formation and Angiogenesis via the Wnt/CaMKII Signaling Axis.

Angiogenesis and vasculogenic mimicry (VM) are considered to be the main processes to ensure tumor blood supply during the proliferation and metastasis of choroidal melanoma (CM). The traditional antimalarial drug artesunate (ART) has some potential anti-CM effects; however, the underlying mechanisms remain unclarified. Recent studies have shown that the Wnt5a/calmodulin-dependent kinase II (CaMKII) signaling pathway has a close correlation with angiogenesis and VM formation. This study demonstrated that ART eliminated VM formation by inhibiting the aforementioned signaling pathway in CM cells. The microvessel sprouting of the mouse aortic rings and the microvessel density of chicken chorioallantoic membrane (CAM) decreased significantly after ART treatment. VM formation assay and periodic acid schiff (PAS) staining revealed that ART inhibited VM formation in CM. Moreover, ART downregulated the expression levels of the angiogenesis-related proteins vascular endothelial growth factor receptor (VEGFR) 2, platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor (VEGF) A, and VM-related proteins ephrin type-A receptor (EphA) 2 and vascular endothelial (VE)-cadherin. The expression of hypoxia-inducible factor (HIF)-1α, Wnt5a, and phosphorylated CaMKII was also downregulated after ART treatment. In addition, we further demonstrated that ART inhibited the proliferation, migration, and invasion of OCM-1 and C918 cells. Collectively, our results suggested that ART inhibited angiogenesis and VM formation of choroidal melanoma likely by regulating the Wnt5a/CaMKII signaling pathway. These findings further supported the feasibility of ART for cancer therapy.

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 in vitro 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.

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.

MicroRNA-145 suppresses uveal melanoma angiogenesis and growth by targeting neuroblastoma RAS viral oncogene homolog and vascular endothelial growth factor.

BackgroundUveal melanoma (UM) is the most common primary intraocular malignancy in adults. It has been demonstrated that microRNA-145 (miR-145) is correlated with the progression of various cancers by regulating the expression of multiple target genes, especially a number of genes that regulate angiogenesis and proliferation. However, the underlying mechanisms of miR-145 in tumor angiogenesis of UM are still not well illustrated. Thus, we aimed to explore the potential target genes or pathways regulated by miR-145 in UM and the effect of miR-145 on invasion and angiogenesis.MethodsTotally, 24 choroid samples were collected in our study, including 12 UM samples and 12 normal uveal tissues. The expression of neuroblastoma RAS viral oncogene homolog (N-RAS), phosphorylated protein kinase B (p-AKT), and vascular endothelial growth factor (VEGF) in UM tissues and normal uveal tissues was analyzed using Western blotting analysis. Lentivirus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Transwell and endothelial cell tube formation assay were used to measure the effects of miR-145 on the invasion and angiogenesis of UM in vitro. The downstream target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase assay. BALB/c nude mice models were established to investigate the mechanisms of miR-145 on tumor growth and angiogenesis in vivo. Group data comparisons were performed using analysis of Student's t test. A two-tailed P < 0.05 was considered as statistically significant.ResultsThe results of Western blotting analysis indicated that the expressions of N-RAS (1.10 ± 0.35 vs. 0.41 ± 0.36, t = 3.997, P = 0.012), p-AKT (1.16 ± 0.22 vs. 0.57 ± 0.03, t = 7.05, P = 0.001), and VEGF (0.97 ± 0.32 vs. 0.45 ± 0.21, t = 3.314, P = 0.008) in UM tumor tissues were significantly higher than those in normal uveal tissue. Luciferase assay demonstrated N-RAS and VEGF as downstream targets of miR-145. Moreover, tube formation assay revealed that miR-145-transfected human microvascular endothelial cell line formed shorter tube length (36.10 ± 1.51 mm vs. 42.91 ± 0.94 mm, t = 6.603, P = 0.003) and less branch points (350.00 ± 19.97 vs. 406.67 ± 17.62, t = 3.685, P = 0.021) as compared with controls. In addition, the numbers of invaded MUM-2B and OCM-1 cells with miR-145 overexpression were significantly lower than the controls (35.7 ± 3.3 vs. 279.1 ± 4.9, t = 273.75, P < 0.001 and 69.5 ± 4.4 vs. 95.6 ± 4.7, t = 21.27, P < 0.001, respectively). In vivo, xenografts expressing miR-145 had smaller sizes (miR-145 vs. miR-scr, 717.41 ± 502.62 mm3vs. 1694.80 ± 904.33 mm3, t = 2.314, P = 0.045) and lower weights (miR-145 vs. miR-scr, 0.74 ± 0.46 g vs. 1.65 ± 0.85 g, t = 2.295, P = 0.045).ConclusionOur results indicated that miR-145 is an important tumor suppressor and the inhibitory strategies against N-RAS/VEGF signaling pathway might be potential therapeutic applications for UM in the future.

Study of Cinobufagin as a Promising Anticancer Agent in Uveal Melanoma Through Intrinsic Apoptosis Pathway.

Uveal melanoma (UM) is the most common primary intraocular carcinoma in adults. Cinobufagin, secreted by the Asiatic toad Bufo gargarizans, is a traditional Chinese medicine, widely used in tumor treatment. Here, we explored the potential antitumor function of cinobufagin and investigated its biochemical mechanisms in UM cells. The antitumor potential of cinobufagin was determined via cell viability, cell cycle, and apoptosis assays. Colony formation assays confirmed that cinobufagin exerted potent antitumor activity in a dose-dependent manner. We found that cinobufagin could induce cell apoptosis and upregulate the expression of cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), and cleaved caspase-9 in vivo and in vitro. In addition, after treatment with increased concentrations of cinobufagin, the intrinsic mitochondrial apoptosis pathway was also activated, which was demonstrated by increased cell apoptosis with increased expression of Bad and Bax, decreased expression of Bcl-2 and Bcl-xl, and reduced mitochondrial membrane potential (MMP) in OCM1 cells. Taken together, the results of this preclinical study suggest that cinobufagin can both inhibit cell survival and induce cell apoptosis in a dose-dependent manner in UM cells, which provides new insights into the biochemical mechanism of cinobufagin and its potential as a future chemotherapeutic agent for UM.

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.

KIF15 plays a role in promoting the tumorigenicity of melanoma

Kinesins are a superfamily of motor proteins and are often dysregulated in many cancers. KIF15, which belongs to the kinesin-12 family, has been shown to function in many different cellular processes, including proliferation, apoptosis, differentiation and development. However, the role of KIF15 in melanoma, remains unknown. In this study, the expression levels of KIF15 in melanoma cells lines and tissues were determined via real-time PCR, immunohistochemical staining and western blot. The effect of KIF15 on tumorigenesis was evaluated by using MTT and colony information. The function of KIF15 on cell survival was detected through flow cytometry assay. Microarray assay and bioinformatics analysis were used to find the potential target of KIF15. We show that KIF15 was significantly upregulated in melanoma cells and tissues. The suppression of KIF15 in tumors significantly reduced tumor growth and increased apoptosis in A375 and OCM1 cells. Findings based on the subcutaneous xenograft model were further consistent with the in vitro results that KIF15 knockdown inhibited melanoma tumor growth in vivo. Microarray assay and bioinformatics indicated that BIRC5, CDK4 and WNT5A were three potential targets of KIF15. Taken together, our results suggest that KIF15 plays a positive role in the tumorigenicity of melanoma and it may serve as a novel diagnostic and therapeutic target for melanoma, especially uveal melanoma.

The oncolytic virus H101 combined with GNAQ siRNA-mediated knockdown reduces uveal melanoma cell viability.

Uveal melanoma (UM) is a severe human malignancy with a high mortality rate, as well as high metastasis and recurrence potential. The active mutation of G protein subunit alpha q (GNAQ) or G protein subunit alpha 11 (GNA11) is a major trigger for UM. Oncolytic adenovirus H101 (H101) is the first oncolytic virus approved for clinical applications in cancer therapy by the China Food and Drug Administration. We investigated whether combining H101 with the downregulation of GNAQ expression would act synergistically in UM therapy. Three UM cell lines OMM2.3 and 92.1, harboring GNAQ mutation, and OCM1, harboring B-Raf proto-oncogene mutation, were chosen for our research. The cellular toxicity of adenoviral infection and the cell growth rate were measured with a Cell Counting Kit-8. Western blot analysis was used to detect GNAQ, p-MEK1/2, YAP, and p-YAP expression. The apoptosis and cell-cycle distribution of cells were evaluated with annexin-V and propidium iodide staining. Our results revealed that OMM2.3 and 92.1 cells were more sensitive to H101 infection than OCM1 cells. GNAQ expression was markedly reduced by small interfering RNA, siGNAQ. Combined treatment of siGNAQ and H101 inhibited the proliferation and activated the apoptosis of OMM2.3 and 92.1 cells by blocking the phosphorylation of MEK1/2 and increasing the phosphorylation of YAP. In summary, a therapy combining H101 and siGNAQ is feasible, with potential utility as a novel targeted molecular therapy for UM, especially those carrying a GNAQ mutation.

Role of microRNA-21 in uveal melanoma cell invasion and metastasis by regulating p53 and its downstream protein.

To reveal the insight mechanism of liver metastasis in uveal melanoma, we investigated cell functions of microRNA-21 in three different uveal melanoma cell lines and analyze the relationship of target gene p53 and its downstream targets. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect microRNA-21 expression in normal uveal tissue and uveal melanoma cell lines. Lenti-virus expression system was used to construct OCM-1, MuM-2B and M619 cell line with stable overexpression and inhibition of microRNA-21. In vitro cell function tests such as cell proliferation, cell apoptosis, cell circle and abilities of migration and invasion were examined by MTT, BrdU assay, flow cytometry, transwell assay and Matrigel invasion assay respectively. The target gene was predicted by bioinformatics and confirmed by using a dual luciferase reporter assay. The expression of p53 and its suspected downstream targets LIM and SH3 protein 1 (LASP1) and glutathione S transferase pi (GST-Pi) were determined by qRT-PCR in mRNA level and Western blotting analysis in protein level. Finally, the effect of microRNA-21 in a xenograft tumor model was assessed in four-week-old BALB/c nude mice. Compared to normal uveal melanoma, expressions of microRNA-21 were significantly higher in uveal melanoma cell lines. Overexpression of microRNA-21 promoted proliferation, migration, and invasion of OCM-1, M619 and MuM-2B cells, while inhibition of microRNA-21 reveal opposite effects. Wild type p53 was identified as a target gene of microRNA-21-3p, and proved by dual luciferase reporter assay. Up-regulated microRNA-21 inhibited the expression of wild type p53 gene, and the increased expression of LASP1 in mRNA level and protein level, while down-regulated microRNA-21 presented opposite way. However, GST-pi showed the potential pattern as expected, but relative mRNA level showed no statistically significant difference in OCM-1 cells. Furthermore, the mRNA expression of GST-pi was decreased in microRNA-21 overexpressing MuM-2B, and increased in M619 cells with inhibition of microRNA-21. In vivo, inhibition of microRNA-21 reduced tumor growth with statistically significant difference. These findings provide novel insight into molecular etiology of microRNA-21 in uveal melanoma cell lines, and suggest that microRNA-21 might be a potential candidate for the diagnosis and prognostic factor of human uveal melanoma.

Somatostatin Receptors as Molecular Targets in Human Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, with an incidence of 4–5 cases per million. The prognosis of UM is very poor. In the present study, our aim was to investigate the expression of mRNA and protein for somatostatin receptor types-1, -2, -3, -4, -5 (SSTR-1–5) in human UM tissue samples and in OCM-1 and OCM-3 human UM cell lines by qRT-PCR, western blot and ligand competition assay. The mRNA for SSTR-2 showed markedly higher expression in UM tissues than SSTR-5. The presence of SSTRs was demonstrated in 70% of UM specimens using ligand competition assay and both human UM models displayed specific high affinity SSTRs. Among the five SSTRs, the mRNA investigated for SSTR-2 and SSTR-5 receptors was strongly expressed in both human UM cell lines, SSTR-5 showing the highest expression. The presence of the SSTR-2 and SSTR-5 receptor proteins was confirmed in both cell lines by western blot. In summary, the expression of somatostatin receptors in human UM specimens and in OCM-1 and OCM-3 human UM cell lines suggests that they could serve as a potential molecular target for therapy of UM using modern powerful cytotoxic SST analogs targeting SSTR-2 and SSTR-5 receptors.

Role of miR-23a/Zeb1 negative feedback loop in regulating epithelial-mesenchymal transition and tumorigenicity of intraocular tumors.

Role of the two-way negative feedback regulation channel formed by miR-23a and Zeb1 in epithelial-mesenchymal transition (EMT), tumorigenic ability, and migration and metastasis capacity of the intraocular malignant tumor cells was investigated. Molecular biological methods such as real time-quantitative PCR (RT-qPCR), immunoblotting method, and immunofluorescence were used to detect the expression levels of mRNA and protein in the Zeb1 factor in OCM-1, WERI-RB1, and Y79 cells before and after miR-23a transfection. Transwell cells were used to detect the in vitro membrane permeation and migration ability in OCM-1, WERI-RB1, and Y79 cells (non-transfection group, blank control transfection group, mimic transfection group, inhibitor transfection group). The results revealed that the relative expression of miR-23a in the cells in the miR-23a mimic transfection group increased significantly compared with that in the control group (p<0.05). There were significant differences in the relative expression of mRNA between the mimic transfection and control group (p<0.05). RT-qPCR detection showed that the relative expression of mRNA of the epithelial-labeled factor E-cadherin increased significantly in the miR-23a mimics group (p<0.05). Expression of the protein E-cadherin increased while the expression of the mesenchyme-labeled proteins of vimentin and N-cadherin decreased in the mimics group. Zeb1 has a negative feedback effect on miR-23a. They can form a negative feedback loop. The results showed that miR-23a and Zeb1 form a bidirectional inhibitory negative feedback loop, which plays an important role in regulating EMT. In conclusion, the significant changes in the mesenchymal phenotype of the stable strains with Zeb1 overexpressed in the OCM-1 cells cannot be completely explained with the changes in cytoskeleton caused by EMT.

Antitumor efficacy of VP22-CD/5-FC suicide gene system mediated by lentivirus in a murine uveal melanoma model

Uveal melanoma (UM) is the most common primary intraocular tumor in adults, which has high frequency of metastasis to the liver, typically causing a fatal outcome. Chemo-resistance remains a major obstacle in the therapeutic approach to UM, leaving limited choice for treating UM. Other possible treatments have been explored but the results are yet to be evident. To improve therapy for UM, transcriptional suicide genes were transfected into the OCM-1 cell line. In the current study, OCM-1 cells transfected with lentiviral-meditated EGFP, cytosine deaminase (CD)/EGFP, and VP22-CD/EGFP were established. Of the three groups, we examined the cell growth in vitro and in vivo by using the MTT method with cell culture media and MRI in murine UM models. According to our results, the cell proliferation in the transfected CD/EGFP group was slower than the non-suicide gene group. The VP22-CD/EGFP group manifested superior cell cytotoxicity than the CD/EGFP group. Further analysis of MRI and fluorescent imaging of the murine UM model identified significant differences in tumor volume among the three groups. Collectively, our study demonstrated that CD/5-FC is a potent therapeutic approach for UM. With the efficacy of VP22, suicide gene-induced cytotoxicity was superior to applying CD alone. Taken together, we concluded that novel therapy with the VP22-CD suicide gene may further contribute to treatment of UM.

Characterization of luteinizing hormone-releasing hormone receptor type I (LH-RH-I) as a potential molecular target in OCM-1 and OCM-3 human uveal melanoma cell lines.

IntroductionUveal melanoma (UM) is the most common primary intraocular malignancy with very poor prognosis. Conventional chemotherapy only rarely prolongs the survival, therefore patients require novel treatment modalities. The discovery of specific receptors for hypothalamic hormones on cancer cells has led to the development of radiolabeled and cytotoxic hormone analogs.Materials and methodsIn the present study, our aim was to investigate the expression of mRNA for receptors of luteinizing hormone-releasing hormone type I (LH-RH-I) and LH-RH ligand in OCM-1 and OCM-3 human uveal melanoma cell lines. The presence and binding characteristics of LH-RH-I receptor protein was further studied by Western blot, immunocytochemistry and ligand competition assay. The expression of mRNA and protein for LH-RH-I receptors has been also studied using tumor samples originating from nude mice xenografted with OCM-1 or OCM-3 cells.ResultsThe mRNA for LH-RH-I receptor has been detected in OCM-1 and OCM-3 cell lines and was found markedly higher in OCM-3 cells. The mRNA for LH-RH-I receptors was also observed in both UM xenograft models in vivo with higher levels in OCM-3. The presence of LH-RH-I receptor protein was found in both cell lines in vitro by immunocytochemistry and Western blot, and also in tumor tissue samples grown in nude mice by Western blot. Both human uveal melanoma models investigated showed specific high affinity receptors for LH-RH-I using ligand competition assay. The mRNA for LH-RH ligand has also been detected in OCM-1 and OCM-3 cell lines and cancer tissues.ConclusionThe demonstration of the expression of LH-RH-I receptors in OCM-1 and OCM-3 human UM cell lines suggests that they could serve as potential molecular target for therapy. Our findings support the development of new therapeutic approaches based on cytotoxic LH-RH analogs or modern powerful antagonistic analogs of LH-RH targeting LH-RH-I receptors in UM.

Gambogenic acid induces cell growth inhibition, cell cycle arrest and metastasis inhibition in choroidal melanoma in a dose-dependent manner.

The aim of the present study was to explore the effects of gambogenic acid (GNA) on the malignant behaviors of choroidal melanoma cells, including cell viability, cell cycle, migration and invasion, and to elucidate the underlying regulatory mechanism. The human choroidal melanoma cell line OCM-1 was treated with different concentrations of GNA and cell viability, colony formation ability, cell cycle, migration and invasion were analyzed. Additionally, cells were incubated with or without LY294002, a specific inhibitor of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, for 24 h. Levels of cell cycle-associated proteins (cyclin D1, cyclin E, cyclin-dependent kinase 2 and P21), epithelial-mesenchymal transition (EMT)-associated molecules (epithelial-cadherin, α-smooth muscle actin and vimentin) and phosphorylated (p)-AKT/AKT were determined using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The results demonstrated that GNA significantly inhibited cell viability and induced cell cycle arrest at the G0/G1 phase in a dose-dependent manner (P<0.01). Furthermore, GNA administration significantly suppressed cell migration and invasion in a dose-dependent manner (P<0.01). Treatment with GNA or LY294002 induced a marked decrease in the expression of p-AKT/AKT, a significant downregulation in cell cycle-associated molecules (P<0.01), and a significant decrease in cell viability (P<0.01). Co-treatment with LY294002 and GNA had an additive effect on the growth of OCM-1 cells. In conclusion, the results of the present study suggest that treatment with GNA may inhibit cell viability and induce G0/G1 arrest. Furthermore, GNA may also inhibit cell metastasis via regulating EMT-associated molecules. The PI3K/Akt signaling pathway may be a key mechanism involved in the progression of choroidal melanoma, and GNA may serve as a potential therapeutic reagent for the treatment of this disease.