Uveal Melanoma Cells Research Articles

SGSM2 in Uveal Melanoma: Implications for Survival, Immune Infiltration, and Drug Sensitivity.

The abnormal expression of small G protein signaling modulator 2 (SGSM2) is related to the occurrence of thyroid cancer and breast cancer. However, the role of SGSM2 in uveal melanoma (UVM) is unclear. To elucidate this ambiguity, our study utilized bioinformatics analysis and experimental validation. The expression of SGSM2 was detected in UVM cell lines through quantitative real-- time PCR (qRT-PCR). We utilized the Cancer Genome Atlas (TCGA) database to assess the relationship between SGSM2 expression and clinical characteristics, as well as its prognostic significance in UVM. Furthermore, the study examined potential regulatory networks involving SGSM2 in relation to immune infiltration, immune checkpoint genes, microsatellite instability (MSI), and drug sensitivity in UVM. The study also examined SGSM2 expression in UVM single-cell sequencing data. SGSM2 was highly expressed in UVM cell lines. Moreover, elevated levels of SGSM2 in UVM patients were significantly linked to poorer overall survival (OS) (p < 0.001), progress- free survival (PFS) (p < 0.001), and disease-specific survival (DSS) (p < 0.001). Additionally, SGSM2 expression was identified as an independent prognostic factor in UVM patients (p < 0.001). SGSM2 was associated with several pathways, including the calcium signaling pathway, natural killer cell-mediated cytotoxicity, cell adhesion molecules (CAMs), and others. The study revealed that SGSM2 expression in UVM is linked to immune infiltration, immune checkpoint genes, and MSI. Additionally, a significant inverse correlation was observed between SGSM2 expression and the compounds GSK690693, TL-2-105, PHA-793887, Tubastatin A, and SB52334 in UVM patients. SGSM2 may not only serve as an important indicator for prognostic assessment. Still, it may also be a key target for the development of new therapeutic approaches, providing new perspectives on the treatment of UVM patients.

Injectable nanocomposite hydrogel with cascade drug release for treatment of uveal melanoma

Uveal melanoma (UM) is the most common malignant intraocular tumor with the trait of distant metastases. Currently, the standard clinical therapy results in suboptimal outcomes due to ineffective inhibition of tumor metastasis. Thus, developing novel therapeutic modalities for UM remains a critical priority. Herein, we have developed an injectable nanocomposite hydrogel (HA-DOX/LAP gel) through integrating hyaluronic acid-based drug-loaded nanoparticles into an alginate-dopamine gel, delivering the chemotherapeutic drugs, lapatinib and doxorubicin for combinational treatment of UM. HA-DOX/LAP gel is fabricated in situ by a simple injection of the mixed precursor solution into tumor sites and maintains in vivo for more than 21 days. The entrapped drug-loaded nanoparticles can gradually release from HA-DOX/LAP gel, enhancing tumor targeting and penetration, and synchronously releasing lapatinib and doxorubicin into the acidic intracellular environment to synergistically destroy UM cells. In vivo anti-tumor studies conducted in MuM-2B tumor models demonstrated that HA-DOX/LAP gel significantly impedes tumor growth, diminishes postoperative recurrence, and prolongs overall survivals of UM tumor-bearing mice through only single injection. Remarkably, the escaped drug-loaded nanoparticles effectively reduce the risk of tumor metastases. Our findings provide new insights for the development of multifunctional nanocomposite-incorporating combination therapy against UM by targeting tumor recurrence and metastases.

Expression of Markers Associated with Epithelial-Mesenchymal Transition and Extracellular Matrix Degradation in Human Uveal Melanoma.

The expression of markers associated with epithelial-mesenchymal transition (EMT) and extracellular matrix degradation in human uveal melanoma tissue samples and postequatorial zone of the choroid was assessed by immunohistochemical staining. Increased expression of EMT markers E-cadherin and vimentin was observed in the tumor. The ratio of MMP-9 to TIMP-1 proteins related to the extracellular matrix degradation was higher in the tumor. These results may indicate activation of EMT-like process in the uveal melanoma cells and degradation of the extracellular matrix, which can contribute to the development of collective invasion in uveal melanoma.

Azacytidine treatment affects the methylation pattern of genomic and cell-free DNA in uveal melanoma cell lines

BackgroundUveal melanoma (UM) is the most common primary intraocular tumour in adults, and approximately 50% of patients will develop metastasis. Epigenetic changes are a major factor in cancer progression. We aimed to determine whether methylation profiles could be altered using a DNA methyltransferase (DNMT) inhibitor in UM cell lines.MethodsFour primary and metastatic UM cell lines were treated with azacytidine and analysed for cell proliferation, colony formation, and BAP1 protein expression. Genomic and cell-free (cf)DNA methylation were compared.ResultsIn all cell lines, azacytidine treatment resulted in dose-dependent effects on proliferation, colony formation, and radiosensitivity. Methylation profiling revealed differences in methylation between cell lines according to BAP1 expression. Matched primary and metastatic cell lines showed very similar patterns. Alterations were seen in pathways known to be important in UM progression, such as PI3K/Akt and MAPK signaling, and in pathways involved in cancer progression, such as regulation of stemlike potential, cell motility, and invasion. These changes were maintained in genomic and cell-free DNA.ConclusionsThis data suggests that DNMT inhibitors cause changes in UM cells that are maintained in cfDNA. The results suggest that targeting methylation in UM treatment and monitoring response to treatment using cfDNA methylation could be a valuable tool.

Neuropeptide Precursor VGF Promotes Liver Metastatic Colonization of Gαq Mutant Uveal Melanoma by Facilitating Tumor Microenvironment via Paracrine Loops.

Uveal melanoma (UM), the predominant primary ocular malignancy, often progresses to liver metastasis with limited therapeutic options. The interplay of the tumor microenvironment, encompassing secreted soluble factors, plays a crucial role in facilitating liver metastasis. In this study, the role is elucidated of the neural growth factor-inducible gene (VGF), a secreted neuropeptide precursor, in Gαq mutant UM. Employing a multiomics approach, encompassing transcriptomic and secretomic analyses, the intricate involvement of VGF in UM progression is unveiled. VGF is upregulated in Gαq mutant UM cells and associated with poor prognosis of UM patients. Targeting VGF significantly suppressed the growth of UM in vitro and in vivo. Further evidence shows that VGF is regulated by Gαq through MAPK/CREB pathway. Mechanistically, CREB modulates VGF expression by directly binding to consensus DNA response elements in the promoters of the VGF gene. Combined inhibition of Gαq and MEK remarkably reduces tumor burden in the UM xenograft model. Notably, VGF triggers liver metastatic colonization of UM and activates the fibrosis of hepatic stellate cells (HSCs), creating a favorable microenvironment, through an autocrine and paracrine loop. Furthermore, VGF directly binds to TGFBR2 and regulates TGF-β-SMAD signaling pathway, thereby regulating genes associated with endothelial-mesenchymal transition (EMT) to promote metastasis. Taken together, these findings identify VGF as a pivotal driver in the progression and metastasis of Gαq mutant UM and confers a promising therapeutic target and strategy for UM patients.

Identification of targetable epigenetic vulnerabilities in uveal melanoma.

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults, which preferentially metastasizes to the liver in approximately half of all cases. Metastatic UM is notoriously resistant to therapy and is almost uniformly fatal. UM metastasis is most strongly associated with mutational inactivation of the BAP1 tumor suppressor gene. Given the role of BAP1 in epigenetic regulation as the ubiquitin hydrolase subunit of the polycomb repressive deubiquitinase (PR-DUB) complex, we conducted high-throughput drug screening using a well-characterized epigenetic compound library to identify new therapeutic vulnerabilities. We identified several promising new lead compounds, in particular the extra-terminal domain protein (BET) inhibitor mivebresib (ABBV-075). Mivebresib significantly improved survival rates in a metastatic uveal melanoma xenograft mouse model and entirely prevented detectable metastases to the bones, spinal cord, and brain. RNA sequencing revealed a notable overlap between the genes and pathways affected by HDAC and BET inhibition, including the reversal of gene signatures linked to high metastatic risk and upregulation of genes associated with a neuronal phenotype. Together, we found that UM cells are particularly vulnerable to class I HDAC and BET inhibition, and highlight the BET inhibitor mivebresib as a promising candidate for further clinical evaluation.

Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma.

Uveal melanoma (UM) is a highly aggressive disease with very few treatment options. We previously demonstrated that mUM is characterized by high oxidative phosphorylation (OXPHOS). Here we tested the anti-tumor, signaling and metabolic effects of imipridones, which are CLPP activators, which inhibit OXPHOS indirectly and have demonstrated safety in patients. We assessed CLPP expression in UM patient samples. We tested the effects of imipridones (ONC201 and ONC212) on the growth, survival, signaling and metabolism of UM cell lines in vitro, and for therapeutic efficacy in vivo in UM liver metastasis models. CLPP expression was detected in primary and mUM patient samples. ONC201 and 212 decreased OXPHOS effectors, inhibited cell growth and migration, and induced apoptosis in human UM cell lines in vitro. ONC212 inhibited OXPHOS, increased metabolic stress and apoptotic pathways, inhibited amino acid metabolism, and induced cell death-related lipids. ONC212 also decreased tumor burden and increased survival in vivo in two UM liver metastasis models. Imipridones are a promising strategy for further testing and development in mUM.

CYSLTR1 antagonism displays potent anti-tumor effects in uveal melanoma

Uveal Melanoma (UM) is the most common primary intraocular malignancy in adults. Although rare, it is a deadly tumor, with a long-term prognosis of death occurring in more than 50% of the cases. It is characterized by frequent (∼80%) driver mutations in GNAQ and GNA11 genes, both of which are activated by cysteinyl leukotriene receptors (CYSLTRs). CYSLTR1 is upregulated and participated in the progression of several cancers. In the present study, we sought to determine the expression levels of CYSLTR1 in 31 human UM specimens and cell lines (3 primary and 1 metastatic), and its role in the proliferation and viability of these cells by analyzing cell metabolic activity, cell confluence and apoptosis levels. We show that all analyzed UM specimens and cells expressed CYSLTR1 at high levels. Notably, the pharmacological blockage of this receptor, using the inverse agonist MK571, reduced the growth and metabolic activity, and increased the apoptotic cell death of all analyzed UM cell lines. We provide evidence that CYSLTR1 is expressed in human UM and plays a significant role in UM progression behavior. Our data highlight the potential beneficial effects of targeting CYSLTR1 in the control of UM progression.

Hypoxia-related lncRNA correlates with prognosis and immune microenvironment in uveal melanoma

BackgroundHypoxia-related genes are linked to the prognosis of various solid malignant tumors. However, the role of hypoxia-related long non-coding RNAs (HRLs) in uveal melanoma (UVM) remains unclear. This study aimed to identify HRLs associated with UVM prognosis and develop a novel risk signature to predict patient outcomes.MethodsData from 80 UVM samples were obtained from The Cancer Genome Atlas. Prognostic HRLs were screened using Cox univariate and Pearson correlation analyses. HRL signature were constructed using Lasso analysis, and gene enrichment analysis was performed to explore the association between HRLs and immune features. Cell Counting Kit-8 assay was used to measure the propagation of human uveal melanoma (MuM2B) cells, while tumor invasion and migration were evaluated using Transwell and wound-healing experiments. Inflammatory factors and macrophage polarization were evaluated using quantitative PCR.ResultsIn total, 621 prognostic HRLs were screened and constructed in 12 HRLs. The risk score showed a significant correlation with the survival time of patients with UVM. Additionally, HRL correlated with diverse key immune checkpoints, revealing possible targets for immunotherapy. Immune-related pathways were highly enriched in the high-risk group. LINC02367, a protective HRL, was associated with the tumor microenvironment and survival time of patients with UVM. In vitro, LINC02367 significantly influenced MuM2B proliferation and migration. It also modulated macrophage polarization by regulating inflammatory factor levels, thereby affecting the immune microenvironment.ConclusionsWe developed a novel HRL signature to predict prognosis in patients with UVM. HRLs are potential biomarkers and therapeutic targets for the treatment of UVM.

The Impact of Spliceosome Inhibition in SF3B1-Mutated Uveal Melanoma.

Unfortunately, treatment of patients with uveal melanoma (UM) with metastatic disease is limited. Twenty percent of patients with UM harbor a mutation in the splicing factor gene SF3B1, suggesting that aberrant spliceosome function plays a vital role in tumorigenesis. Splicing inhibitors exploit the preferential sensitivity of spliceosome-compromised leukemic cells to these compounds. We studied the effect of the splicing inhibitor E7107 using two UM cell lines and ex vivo cultured SF3B1- and BAP1-mutated primary UM tumor slices. These UM cell lines and ex vivo tumor slices were exposed for 24 hours to different concentrations of E7107. Tumor slices were stained with hematoxylin and eosin (H&E) and incubated with BAP1, MelanA, MIB-1, and caspase-3 antisera. The E7107-exposed UM cell lines exhibited decreased cell viability and increased apoptosis, with the greatest effect on SF3B1-mutated UM cells. A similar effect on UM tumor slices was observed upon exposure to E7107. Additionally, RNA was isolated for differential isoform expression analysis. No significant difference in isoform usage was found genome-wide. However, specific genes were differentially expressed after E7107 treatment in the SF3B1-mutated samples. Moreover, E7107 had the greatest effect on intron retention. This study indicates/suggests that mutated SF3B1 UM cells are more sensitive to the splicing inhibitor E7107 than wild-type SF3B1 UM cells.

YAP promotes global mRNA translation to fuel oncogenic growth despite starvation.

Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) play fundamental roles in stem/progenitor cell expansion during homeostasis, and their dysregulation often leads to tissue overgrowth. Here, we show that YAP activation is sufficient to overcome the restriction of global protein synthesis induced by serum starvation, enabling cells to sustain proliferation and survival despite an unfavorable environment. Mechanistically, YAP/TAZ selectively promoted the mTORC1-dependent translation of mRNAs containing 5' terminal oligopyrimidine (5'TOP) motifs, ultimately increasing the cellular polysome content. Interestingly, DNA damage-inducible transcript 4 (DDIT4), a negative regulator of mTORC1, was upregulated by serum starvation but repressed by YAP/TAZ. DDIT4 was sufficient to suppress the translation and transformative potential of uveal melanoma cells, which are often serum unresponsive due to G protein mutations. Our findings reveal a vital role for protein synthesis as a key modality of YAP/TAZ-induced oncogenic transformation and indicate the potential for targeting mTORC1 or translation to treat YAP/TAZ-driven malignancies.

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.

Pathogenic Germline Variants in Uveal Melanoma Driver and BAP1-Associated Genes in Finnish Patients with Uveal Melanoma.

Uveal melanoma (UM) is a rare yet aggressive eye cancer causing over 50% mortality from metastasis. Familial UM, amounting to 1%-6% of patients in Finland and the United States, mostly lack identified genetic cause, while 8% show associations with other cancer syndromes. We searched novel genetic associations for predisposition to UM, additional to already studied BAP1 and MBD4, by using targeted amplicon sequencing of 19 genes associated with UM, BAP1, or renal cell carcinoma in 270 consecutively enrolled Finnish patients with UM. Key UM drivers GNAQ, GNA11, CYSLTR2, PLCB4, EIF1AX, and SF3B1 lacked pathogenic germline variants. One patient carried the pathogenic BRCA1 variant c.3626del p.(Leu1209*), and one harbored a novel truncating MET variant c.252C > G p.(Tyr84*), classified as likely pathogenic. FLCN and BRCA2, previously identified with pathogenic variants in patients with UM, did not have such variants in our cohort. Two patients were heterozygous for a pathogenic recessive BLM variant c.2824-2A > T. None of the carriers of identified variants had familial UM. We identified BRCA1 and MET as genes with pathogenic germline variants in Finnish UM patients, each with a frequency of 0.4% (95% confidence interval, 0-2).

Local anesthetic tetracaine hydrochloride induces pyroptosis via caspase-3/gasdermin E in uveal melanoma

BackgroundEvasion of pyroptosis is an effective survival strategy employed by cancer cells to evade immune cell attacks and drug-induced cytotoxicity. Exploring potent molecules capable of inducing pyroptosis in cancer cells has significant clinical implications for the control of cancer progression. Unexpectedly, we found that the local anesthetic tetracaine hydrochloride (TTC) induced pyroptosis, specifically in uveal melanoma but not in acral or cutaneous melanoma. MethodsWe investigated the effects of TTC on various melanoma cell lines and performed transcriptome sequencing of TTC-treated uveal melanoma cells. The role of gasdermin E (GSDME), an executive protein responsible for pyroptosis, was explored using CRISPR-Cas13d knockdown, caspase-3 inhibitor treatment, and western blot analysis. Differential gene expression and pathway enrichment analyses were performed. Furthermore, we used tissue microarrays to assess GSDME expression levels in melanoma tissues from different anatomical sites. ResultsTTC significantly induced pyroptosis specifically in uveal melanoma cells with high GSDME expression levels. TTC treatment could lead to GSDME cleavage by the caspase-3 in uveal melanoma C918 cells. GSDME knockdown or caspase-3 inhibition suppressed TTC-induced pyroptosis. Transcriptome analysis revealed differentially expressed genes enriched in signaling pathways related to pyroptosis, immunity, and cytokines. ConclusionsThis study showed that the local anesthetic TTC effectively induces pyroptosis in uveal melanoma through the caspase-3/GSDME pathway, highlighting its potential application in immunotherapy. Notably, the use of TTC has potential as an agent for inducing pyroptosis and as an adjuvant anticancer therapy in uveal melanoma.

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

Uveal melanoma (UM) is a rare melanoma originating in the eye’s uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases.

Uveal melanoma (UM) is a rare melanoma originating in the eye's uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients with uveal melanoma metastases succumb to disease within 2 years. This study aimed to provide a path toward enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA-seq of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR-seq, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs, and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

ZNF197-AS1/miR-425/GABARAPL1 Axis: A Novel Regulatory Mechanism in Uveal Melanoma

This study investigates the role of the long non-coding RNA (lncRNA) ZNF197-AS1 in uveal melanoma (UM), focusing on its function within a competing endogenous RNA (ceRNA) network. Utilizing the UM-related TCGA dataset, we analyzed the expression levels of ZNF197-AS1 and its correlation with miR-425 and GABARAPL1, an essential autophagy-related gene. Our analysis revealed that ZNF197-AS1 acts as a ceRNA by competitively binding to miR-425, resulting in the upregulation of GABARAPL1. This interaction plays a crucial role in the growth and metastasis of UM. The expression of GABARAPL1 showed a strong correlation with the clinical outcomes of UM patients. Furthermore, in vitro assays confirmed that ZNF197-AS1 impedes UM cell proliferation, migration, and invasion by modulating the miR-425/GABARAPL1 axis. These findings suggest that ZNF197-AS1 can effectively inhibit UM progression through this ceRNA regulatory network. This study provides valuable insights into the molecular mechanisms underlying UM and highlights the potential of targeting the ZNF197-AS1/miR-425/GABARAPL1 axis as a therapeutic strategy for UM.

The Phenotypical Characterization of Dual-Nature Hybrid Cells in Uveal Melanoma.

Metastasis, occurring years after primary diagnosis, represents a poor prognosis in uveal melanoma (UM)-affected individuals. The nature of cells involved in this process is under debate. Circulating hybrid cells that have combined tumor and immune cell features found in blood were predictive of metastasis and may correspond to dual-nature cells (DNC) in the primary tumor. Herein, we sought to determine the presence of DNCs in primary UM tumors, the cell types involved in their genesis, and their ability to be formed in vitro. UM lesions (n = 38) were immunolabeled with HMB45 in combination with immune-cell-specific antibodies. In parallel, we co-cultured UM cells and peripheral blood mononuclear cells (PBMCs) to analyze DNC formation. HMB45+/CD45+ DNCs were present in 90% (26/29) of the tumors, HMB45+/CD8+ DNCs were present in 93% (26/28), and HMB45+/CD68+ DNCs were present in 71% (17/24). DNCs formed with CD8+ and CD68+ cells were positively correlated to the infiltration of their respective immune cells. Notably, UM cells were prone to hybridize with PBMCs in vitro. This phenotypical characterization of DNCs in UM demonstrates that CD8+ T-cells and macrophages are capable of DNC formation, and they are important for better understanding metastatic dissemination, thus paving the path towards novel therapeutic avenues.

Three-dimensional cell spheroid culture and cell viability study of uveal melanoma cell line C918 with luteolin treatment

Three-dimensional cell spheroid culture and cell viability study of uveal melanoma cell line C918 with luteolin treatment

Evaluation of the effects of vitamin D analogs, bevacizumab, and radiotherapy in uveal melanoma cells

Due to the lack of a definitive effective treatment method that provides a complete cure and increases survival rates in uveal melanoma, the search for alternative treatments at the molecular level continues. In this context, we aimed to comparatively analyze the therapeutic effects of 25-hydroxyvitamin D3 (D2), 1a, 25-dihydroxyvitamin D3 (D3), bevacizumab and radiotherapy (RT) in a uveal melanoma cell line (MP41). Cytotoxicity was evaluated using XTT cell proliferation kit and Xcelligence cell analyzer system. RT dose was determined after a clonogenic assay. Annexin V/PI staining and Western blot analyses for caspase-3, -8, and -9 were performed to analyze apoptosis. Additionally, cell cycle analyses were also conducted. As a result, we found that D2 and D3 did not show cytotoxic effects, while bevacizumab and RT showed time and dose-dependent cytotoxicity. IC50 concentration of bevacizumab was 6.945 mg/mL. Radiotherapy and bevacizumab significantly reduced cell survival and induced apoptosis when administered both as monotherapy and in combination. A significant increase in caspase proteins was detected at high bevacizumab concentrations. However, the combination of bevacizumab and radiotherapy caused a substantial decrease in caspase-3, -8 and -9 expressions. No significant difference in cell cycle distribution was detected in any treatment. Our results showed that bevacizumab inhibited MP41 cell proliferation and had an additive effect when administered with RT. In conclusion, our study offers a different perspective on the treatment of uveal melanoma, and these results, when supported by animal experiments and clinical studies in the future, might be a new step in the treatment of this challenging ocular tumor.