Human Uveal Melanoma Cells Research Articles

Expression of BRCA1-associated protein 1 in primary tumors and metastasis in an animal model of uveal melanoma

Background: Uveal melanoma (UM) is the most common primary intraocular tumor in adults. Despite successful local treatment, approximately 50% of these patients develop lethal metastatic disease. BRCA1-associated protein 1 (BAP1) encodes BAP1, which functions as a tumor suppressor. Inactivation by a somatic mutation in BAP1 results in a loss of protein expression, which is associated with increased risk of metastasis.Aim: To analyze the expression of BAP1 protein in primary uveal tumors and their corresponding metastasis in an animal model of UM and to determine whether BAP1 expression is lost during the metastatic process.Subjects and Methods: Postmortem eyes and lungs with metastasis from nine rabbits previously inoculated with human UM cells expressing BAP1 (92.1 strain) were analyzed. Immunohistochemistry was performed on formalin-fixed paraffin-embedded samples using a monoclonal BAP1 (C-4) antibody. Expression was evaluated by the extent and intensity of staining. All primary tumors and metastasis were positive for BAP1. Results: The staining was diffuse and intense in the primary tumors of four rabbits (67%), focal-intense in one rabbit and focal-mild in one rabbit. All lung metastasis showed focal staining. All animals with intense BAP1 staining in the primary tumor showed mild BAP1 in the metastasis. Discussion: A predominant pattern observed between primary and metastatic lesion shows a transition from moderate into mild intensity and diffuse into the focal extent, suggesting a decrease in BAP1 expression. Future studies, including the human tissue of primary UM and their corresponding metastasis, should be performed to corroborate these findings.

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Parthenolide inhibits the proliferation and induces the apoptosis of human uveal melanoma cells.

To explore the effect of parthenolide (PTL) on human uveal melanoma (UM) cells (C918 and SP6.5 cells) and its molecular mechanism. Carboxyfluorescein succinimidyl amino ester (CFSE) assays and cell counting kit-8 (CCK-8) were performed to detect the cell viability. Flow cytometry was used to analyze cell cycle and apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot assays were performed to measure proliferation-related and apoptosis-related factors. Firstly, PTL decreased the viability of C918 and SP6.5 cells in a dose-dependent manner, and the effect of PTL on C918 cells was stronger than on SP6.5; however, it did not affect normal cells. Secondly, PTL increased the proportion of cell number at cell cycle G1 phase in C918 cells, and decreased the proportion of cell number at S phase, but the proportion did not change at G2 phase. In addition, PTL induced the apoptosis of C918 cells, and decreased the expressions of Cyclin D1, B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-XL). Also, PTL increased Cyclin inhibition protein 1 (P21), Bcl-2-associated X protein (Bax), Cysteinyl aspartate specific proteinas-3 (Caspase-3) and Caspase-9 expression. However, the expression of Caspase-8 was not changed. PTL inhibites proliferation and induces apoptosis in UM cells by arresting G1 phase and regulating mitochondrial pathway, however, it does not affect normal cells.

LncRNA PVT1 knockdown affects proliferation and apoptosis of uveal melanoma cells by inhibiting EZH2.

To detect the expression of long non-coding ribonucleic acid (lncRNA) plasmacytoma variant translocation gene 1 (PVT1) in uveal melanoma (UM) tissues, and to investigate its influence on the proliferation and apoptosis of UM cells as well as its mechanism. 40 cases of UM tissues and 40 cases of adjacent tissues surgically resected in our hospital from October 2015 to April 2018 were collected. The expression level of lncRNA PVT1 in these tissues was determined by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Stable knockdown of lncRNA PVT1 was constructed in human UM cell line OCM-1 using small interfering RNA (siRNA). The impact of lncRNA PVT1 on UM cell proliferation was detected by Cell Counting kit-8 (CCK-8) and colony formation assay. Flow cytometry was applied to measure the apoptotic level of UM cells in the blank control group and lncRNA PVT1 knockdown group. Meanwhile, the expression level of enhancer of zeste homologue 2 (EZH2) was determined by Western blotting. The expression level of lncRNA PVT1 in UM tissues was remarkably higher than that in the adjacent tissues (p<0.05). UM cell proliferation was notably repressed after lncRNA PVT1 knockdown by siRNA. Flow cytometry results indicated that the number of apoptotic UM cells in lncRNA PVT1 knockdown group significantly increased compared with that in the blank control group (p<0.05). The protein expression of EZH2 was suppressed after lncRNA PVT1 knockdown (p<0.05). LncRNA PVT1 knockdown in UM cells can repress the proliferation of UM cells and promote their apoptosis by regulating EZH2 expression.

TRPM8 Activation via 3-Iodothyronamine Blunts VEGF-Induced Transactivation of TRPV1 in Human Uveal Melanoma Cells.

In human uveal melanoma (UM), tumor enlargement is associated with increases in aqueous humor vascular endothelial growth factor-A (VEGF-A) content that induce neovascularization. 3-Iodothyronamine (3-T1AM), an endogenous thyroid hormone metabolite, activates TRP melastatin 8 (TRPM8), which blunts TRP vanilloid 1 (TRPV1) activation by capsaicin (CAP) in human corneal, conjunctival epithelial cells, and stromal cells. We compare here the effects of TRPM8 activation on VEGF-induced transactivation of TRPV1 in an UM cell line (92.1) with those in normal primary porcine melanocytes (PM) since TRPM8 is upregulated in melanoma. Fluorescence Ca2+-imaging and planar patch-clamping characterized functional channel activities. CAP (20 μM) induced Ca2+ transients and increased whole-cell currents in both the UM cell line and PM whereas TRPM8 agonists, 100 μM menthol and 20 μM icilin, blunted such responses in the UM cells. VEGF (10 ng/ml) elicited Ca2+ transients and augmented whole-cell currents, which were blocked by capsazepine (CPZ; 20 μM) but not by a highly selective TRPM8 blocker, AMTB (20 μM). The VEGF-induced current increases were not augmented by CAP. Both 3-T1AM (1 μM) and menthol (100 μM) increased the whole-cell currents, whereas 20 μM AMTB blocked them. 3-T1AM exposure suppressed both VEGF-induced Ca2+ transients and increases in underlying whole-cell currents. Taken together, functional TRPM8 upregulation in UM 92.1 cells suggests that TRPM8 is a potential drug target for suppressing VEGF induced increases in neovascularization and UM tumor growth since TRPM8 activation blocked VEGF transactivation of TRPV1.

Herpes Simplex Virus 1 Infection Promotes the Growth of a Subpopulation of Tumor Cells in Three-Dimensional Uveal Melanoma Cultures.

Herpes simplex virus 1 (HSV-1)-mediated oncolytic therapy is an emerging cancer treatment modality with potential effectiveness against a variety of malignancies. To better understand the interaction of HSV-1 with neoplastic cells, we inoculated three-dimensional (3D) cultures of human uveal melanoma cells with HSV-1. 3D melanoma cultures were established by placing tumor cells on the surface of a Matrigel matrix, which was followed by the growth of tumor cells on the matrix surface and invasion of the Matrigel matrix by some tumor cells to form multicellular tumor spheroids within the matrix. When established 3D melanoma cultures were inoculated with HSV-1 by placing virus on the surface of cultures, virus infection caused extensive death of melanoma cells growing on the surface of the 3D matrix and significantly decreased the number of tumor cell spheroids within the matrix. However, HSV-1 infection did not lead to a complete destruction of tumor cells in the 3D cultures during a 17-day observation period and, surprisingly, HSV-1 infection promoted the growth of some melanoma cells within the matrix as determined by the significantly increased size of residual viable multicellular tumor spheroids in virus-inoculated 3D cultures at 17 days after virus inoculation. Acyclovir treatment inhibited HSV-1-induced tumor cell killing but did not block the virus infection-induced increase in spheroid size. These findings suggest that although HSV-1 oncolytic virotherapy may cause extensive tumor cell killing, it may also be associated with the unintended promotion of the growth of some tumor cells.IMPORTANCE Cancer cells are exposed to HSV-1 during oncolytic virotherapy with the intention of killing tumor cells. Our observations reported here suggest that potential dangers of HSV-1 oncolytic therapy include promotion of growth of some tumor cells. Furthermore, our findings raise the possibility that HSV-1 infection of neoplastic cells during natural infections or vaccinations may promote the growth of tumors. Our study indicates that HSV-1 infection of 3D tumor cell cultures provides an experimental platform in which mechanisms of HSV-1-mediated promotion of tumor cell growth can be effectively studied.

Experimental therapy of doxorubicin resistant human uveal melanoma with targeted cytotoxic luteinizing hormone-releasing hormone analog (AN-152)

BackgroundCytotoxic analogs of LHRH (luteinizing hormone-releasing hormone) can be successfully used for the treatment of hormone-dependent cancers such as prostatic, ovarian, endometrial, but our knowledge about their effect on hormone-independent cancers such as human uveal melanoma (UM) is limited. Previously, we have demonstrated that 46% of UM express full-length LHRH receptors. This finding has led us to further examine the mechanism of action of LHRH receptor based targeted therapies in this malignancy. AimsIn the present study we investigated the cellular uptake of doxorubicin (DOX) and cytotoxic LHRH analog AN-152 (AEZS-108, zoptarelin doxorubicin) on human UM cell lines (OCM3) and its DOX resistant form OCM3DOX320 by confocal laser scanning microscopy. The LHRH receptor expression was characterized by RT-PCR and immunocytochemistry. ResultsWe were able to establish a new, stable and DOX resistant human UM cell line OCM3DOX320. Our results demonstrated the expression of splice variants and isoforms of receptor for LHRH in OCM3 UM cell line and its doxorubicin resistant form OCM3DOX320.It has been revealed by MTT assay that AN-152 inhibited cell proliferation in a dose dependent manner in OCM3DOX320 cells. Furthermore, receptor-mediated uptake of AN-152 was demonstrated using confocal laser scanning microscopy in both cell line. ConclusionsOur results suggest that the antiproliferative effect of AN-152 can be detected even if only LHRH receptor isoforms are expressed. Our study also demonstrates the LHRH receptor-mediated uptake of AN-152 in DOX resistant OCM3DOX320 cells. Our experiments provide new insights into a potential targeted therapy of UM and give further details about the accumulation of AN-152 in hormone-independent DOX-resistant cells expressing splice variants of the LHRH receptors.

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.

Zeaxanthin from Porphyridium purpureum induces apoptosis in human melanoma cells expressing the oncogenic BRAF V600E mutation and sensitizes them to the BRAF inhibitor vemurafenib

Zeaxanthin, an abundant carotenoid present in fruits, vegetables and algae was reported to exert antiproliferative activity and induce apoptosis in human uveal melanoma cells. It also inhibited uveal melanoma tumor growth and cell migration in nude mice xenograft models. Here we report that zeaxanthin purified from the rhodophyte Porphyridium purpureum (Bory) K.M.Drew & R.Ross, Porphyridiaceae, promotes apoptosis in the A2058 human melanoma cell line expressing the oncogenic BRAF V600E mutation. Zeaxanthin 40μM (IC50) induced chromatin condensation, nuclear blebbing, hypodiploidy, accumulation of cells in sub-G1 phase, DNA internucleosomal fragmentation and activation of caspase-3. Western blot analysis revealed that zeaxanthin induced up-regulation of the pro-apoptotic factors Bim and Bid and inhibition of NF-κB transactivation. Additionally, zeaxanthin sensitized A2058 melanoma cells in vitro to the cytotoxic activity of vemurafenib, a BRAF inhibitor widely used for the clinical management of melanoma, suggesting its potential interest as dietary adjuvant increasing melanoma cells sensitivity to chemotherapy.

Melanosome-Targeting Near-Infrared Fluorescent Probe with Large Stokes Shift for in Situ Quantification of Tyrosinase Activity and Assessing Drug Effects on Differently Invasive Melanoma Cells.

Tyrosinase (TYR) plays a vital role in melanin biosynthesis and is widely regarded as a relatively specific marker for melanocytic lesions which involve vitiligo, malignant cutaneous melanoma, Parkinson's disease (PD), etc. However, the detection of TYR in living cells with fluorescent probes is usually interfered by diverse endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS). Herein, we synthesized a melanosome-targeting near-infrared (NIR) fluorescent probe (HB-NP) with a large Stokes shift (195 nm), achieving a highly sensitive and selective in situ detection for intracellular TYR, by incorporating a m-hydroxybenzyl moiety that recognizes TYR specifically and the morpholine unit which facilitates the probe accumulating in the melanosome into a salicyladazine skeleton. When treated with TYR, the probe itself with weak fluorescence is lit up via an inhibited photoinduced electron-transfer (PET) effect and HB-NP shows a strong fluorescence signal (nearly 48-fold enhancement) with a low detection limit of 0.5 U mL-1. HB-NP has been successfully applied in visualizing and in situ quantification of the intracellular TYR activity. Moreover, owing to the different expression levels of TYR, two human uveal melanoma cells with different invasive behaviors are distinguished by means of bioimaging and the effects of the inhibitor, kojic acid, and the up-regulating treatment, psoralen/ultraviolet A, on TYR activity of the two melanoma cells are evaluated. HB-NP is expected to be a useful tool to monitor diseases associated with the abnormal level of melanin and screen medicines for TYR disorder more effectively.

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.

The miR-181 family promotes cell cycle by targeting CTDSPL, a phosphatase-like tumor suppressor in uveal melanoma

BackgroundMicroRNAs (miRNAs) have been shown to function in many different cellular processes, including proliferation, apoptosis, differentiation and development. miR-181a, -181b, -181c and -181d are miR-181 members of the family, which has been rarely studied, especially uveal melanoma.MethodsThe expression level of miR-181 family in human uveal melanoma cell lines was measured via real-time PCR (RT-PCR). The function of miR-181 on cell cycle was detected through Flow Cytometry assay. Microarray assay and Bioinformatics analysis were used to find the potential target of miR-181b, and dual-luciferase reporter assays further identified the target gene.ResultsMiR-181 family members were found to be highly homologous across different species and their upregulation significantly induces UM cell cycle progression. Of the family members, miR-181b was significantly overexpressed in UM tissues and most UM cells. Bioinformatics and dual luciferase reporter assay confirmed CTDSPL as a target of miR-181b. miR-181b over-expression inhibited CTDSPL expression, which in turn led to the phosphorylation of RB and an accumulation of the downstream cell cycle effector E2F1, promoting cell cycle progression in UM cells. Knockdown CTDSPL using siRNAs showing the same effect, including increase of E2F1 and the progression of cell cycle.ConclusionsMiR-181 family members are key negative regulators of CTDSPL-mediated cell cycle progression. These results highlight that miR-181 family members, especially miR-181b, may be useful in the development of miRNA-based therapies and may serve as novel diagnostic and therapeutic candidate for UM.

Electrochemotherapy with bleomycin and cisplatin enhances cytotoxicity in primary and metastatic uveal melanoma cell lines in vitro.

Electrochemotherapy (ECT) enhances responsiveness to cytotoxic drugs in numerous cell lines in vitro. Clinically ECT is widely applied for skin tumor ablation and has shown efficacy in treating non-resectable colorectal liver metastases. There is limited experience of ECT for ocular tumor therapy. We investigated the cytotoxic effect of bleomycin and cisplatin in combination with electroporation on chemoresistant human uveal melanoma (UM) cell lines in vitro. Four UM cell lines (Mel 270, 92-1, OMM-1, OMM-2.5) were treated with electroporation (pulse amplitude 300-1000 V/cm, 8-80 pulses, 100 μs, 5 Hz) and increasing concentrations of bleomycin and cisplatin (0-7.5 μg/ml). Cell survival was analyzed by MTT viability assay after 36 hours. UM cell lines were resistant to both bleomycin and cisplatin. In combination with electro- poration, the effects of bleomycin and cisplatin were increased 8-70 fold and 3-15 fold, respectively, in all UM cell lines. At the lowest concentration of bleomycin tested (1 μg/ml), viability was maximally reduced in all UM cell lines by ≥69% with electroporation conditions of 750 V/cm and 20 pulses. All UM cell lines were more resistant to cisplatin; however, electro- poration of 1000 V/cm and 8 pulses resulted in similar reductions in cell viability of 92-1, Mel270 with 2.5 μg/ml cisplatin, OMM2-5 cells with 5 μg/ml cisplatin and OMM1 cells with 1 μg/ml cisplatin. In vitro ECT with bleomycin or cisplatin is more effective than the highest concentration of the antineoplastic drug or electroporation alone, opening new perspectives in primary and metastatic UM treatment.

Forkhead box protein O3 suppresses uveal melanoma development by increasing the expression of Bcl‑2‑like protein 11 and cyclin‑dependent kinase inhibitor 1B.

Forkhead box protein O3 (FoxO3a) is a forkhead box family transcription factor which serves an important role in a number of biological functions, including tumor growth. A previous study indicated that FoxO3a serves a role in insulin like growth factor‑induced growth, migration and invasion of uveal melanoma (UM) cells; however, whether FoxO3a is associated with the development and formation of UM remains unknown. In the present study, the role of FoxO3a in UM development and formation was investigated by modulating the expression of FoxO3a in a human UM cell line. The results of the present study demonstrated thatFoxO3a overexpression in UM cells inhibited cell proliferation and promoted cellular apoptosis, leading to an accumulation of cells at the G1 cell cycle phase. Western blot analysis demonstrated that FoxO3a overexpression increased thetranscription and protein expression of Bcl‑2‑like protein 11 and cyclin‑dependent kinase inhibitor 1B, and inhibited cyclin D1 transcription and expression. The opposite effects were observed when FoxO3a was knocked down in UM cells. The results of the present study indicated that FoxO3a may exhibit a negative role in UM development and formation, which is consistent with its role as a tumor suppressor.

Expression of Antimicrobial Peptides by Uveal and Cutaneous Melanoma Cells and Investigation of Their Role in Tumor Cell Migration and Vasculogenic Mimicry

ABSTRACTAims: Antimicrobial peptides (AMPs) have been implicated in the pathogenesis of several cancers, although there is also evidence suggesting potential for novel, AMP-based antitumor therapies. Discerning potential roles of AMPs in tumor pathogenesis may provide valuable insight into the mechanisms of novel AMP-based antitumor therapy.Methods: mRNA expression of the AMPs α defensin (HNP-1); cathelicidin (LL-37); and β defensins (hBD-1, hBD-2, hBD-3, hBD-4) in human uveal and cutaneous melanoma cell lines, primary human uveal melanocytes, and primary human uveal melanoma cells was determined by reverse transcriptase polymerase chain reaction. An in vitro scratch assay and custom Matlab analysis were used to determine the AMP effects on melanoma cell migration. Last, the effect of specific AMPs on vasculogenic mimicry was determined by three-dimensional (3D) culture and light and fluorescence microscopy.Results: Low-to-moderate AMP transcript levels were detected, and these varied across the cells tested. Overall, LL-37 expression was increased while hBD-4 was decreased in most melanoma cell lines, compared to primary cultured uveal melanocytes. There was no observable influence of HNP-1 and LL-37 on tumor cell migration. Additionally, aggressive cutaneous melanoma cells grown in 3D cultures exhibited vasculogenic mimicry, although AMP exposure did not alter this process.Conclusions: Collectively, our data show that although AMP mRNA expression is variable between uveal and cutaneous melanoma cells, these peptides have little influence on major characteristics that contribute to tumor aggressiveness and progression.

Motile activity and cytoskeleton changes in uveal melanoma after proton beam radiation

PurposeProton beam irradiation is known to invoke different biological response than photons.We compared the effect of proton beam irradiation and X‐rays on the survival and migratory properties of melanoma cells.MethodsMel270, human uveal melanoma cell line was treated with physical doses of 1‐5 Gy of proton beam (58 MeV) from Proteus C‐235 cyclotron or X‐rays (300 kV Phillips) at the dose rate of 1 Gy/min were given to suspended cells.ResultsInvasive potential of cells was slightly diminished after both types of radiation at 20 days post‐radiation and then returned to normal at 40 days. Wound healing ability was decreased after X‐rays. The beta‐1‐integrin level was decreased after both types of radiation in Mel270 cells, and vimentin, a marker of EMT was not changed. Less stress fibers were seen in cytoskeleton of Mel270 cells after radiation.ConclusionsLow doses of both types of radiation influenced the motile activity and cytoskeleton in Mel270 cells.

Melanogenesis in uveal melanoma cells: Effect of argan oil.

The mechanisms underlying cutaneous melanogenesis have been widely studied; however, very little is known about uveal melanogenesis. Melanin is normally produced by uveal melanocytes and gives the color to the iris. A derangement from this normal production may occur, for instance, by iatrogenic events, such as glaucoma therapy with prostaglandins that may enhance cutaneous and iris pigmentation. In this study, we investigated the mechanisms that regulate uveal melanogenesis in human uveal melanoma cells(92.1) and murine cutaneous melanoma cells(B16-F1). In the first part of the study, we compared the effects of known cutaneous pigmenting agents on the B16-F1 and 92.1 cells, showing an opposite response of the two cell lines. Subsequently, using argan oil, a known depigmenting agent for murine cutaneous melanoma cells, on 92.1 cells, we found that in these cells, it also functioned as an inhibitor of melanogenesis and tyrosinase expression. From a molecular perspective, treatment of the 92.1cells with argan oil decreased melanogenesis-associated transcription factor(MITF) gene expression by inducing MITF phosphorylation at Ser73, thus leading to MITF ubiquitination and disposal. It also led to the downregulation of the extracellular signal-regulated kinase(ERK)1/2 and Akt pathways, also known to be involved in cutaneous melanogenesis, although with an opposing function. Taken together, our data indicate that: ⅰ)some differences exist in the regulation of melanogenesis between cutaneous and uveal melanoma cells; and ⅱ)argan oil exerts a depigmenting effect on 92.1cells through its action on the ERK1/2 and Akt pathways.

Sigma-1 and Sigma-2 receptor ligands induce apoptosis and autophagy but have opposite effect on cell proliferation in uveal melanoma.

Uveal melanoma is the most common primary intraocular tumor in adults, with about 1200–1500 new cases occurring per year in the United States. Metastasis is a frequent occurrence in uveal melanoma, and outcomes are poor once distant spread occurs and no clinically significant chemotherapeutic protocol is so far available. The aim of the present study was to test the effect of various σ1 and σ2 receptor ligands as a possible pharmacological strategy for this rare tumor. Human uveal melanoma cells (92.1) were treated with various concentrations of different σ2 ligands (haloperidol and haloperidol metabolite II) and σ1 ligand ((+)-pentazocine) at various concentrations (1, 10 and 25 μM) and time points (0, 4 h, 8 h, 24 h and 48 h). Cell proliferation and migration were evaluated respectively by continuous cell monitoring by xCELLigence analysis, clonogenic assay and wound healing. Apoptosis and autophagy were also measured by cytofluorimetric and microscopy analysis. Our results showed that σ2 receptor ligands significantly reduced cell proliferation whereas (+)-pentazocine exhibited opposite results. All tested ligands showed significant decrease in cell migration. Interestingly, both σ1 and σ2 receptor ligands showed significant increase of autophagy and apoptosis at all concentrations. Taken all together these results suggest that sigma receptors mediates opposite biological effects but they also share common pharmacological effect on apoptosis and autophagy in uveal melanoma. In conclusion, these data provide the first evidence that sigma receptors may represent a “druggable” target to develop new chemotherapic agent for uveal melanoma.

Verteporfin-induced formation of protein cross-linked oligomers and high molecular weight complexes is mediated by light and leads to cell toxicity

Verteporfin (VP) was first used in Photodynamic therapy, where a non-thermal laser light (689 nm) in the presence of oxygen activates the drug to produce highly reactive oxygen radicals, resulting in local cell and tissue damage. However, it has also been shown that Verteporfin can have non-photoactivated effects such as interference with the YAP-TEAD complex of the HIPPO pathway, resulting in growth inhibition of several neoplasias. More recently, it was proposed that, another non-light mediated effect of VP is the formation of cross-linked oligomers and high molecular weight protein complexes (HMWC) that are hypothesized to interfere with autophagy and cell growth. Here, in a series of experiments, using human uveal melanoma cells (MEL 270), human embryonic kidney cells (HEK) and breast cancer cells (MCF7) we showed that Verteporfin-induced HMWC require the presence of light. Furthermore, we showed that the mechanism of this cross-linking, which involves both singlet oxygen and radical generation, can occur very efficiently even after lysis of the cells, if the lysate is not protected from ambient light. This work offers a better understanding regarding VP’s mechanisms of action and suggests caution when one studies the non-light mediated actions of this drug.

Podophyllotoxin–pterostilbene fused conjugates as potential multifunctional antineoplastic agents against human uveal melanoma cells

ConjugateB1showed potential multifunctional antineoplastic activity against human uveal melanoma cells.