Normal Retinal Cells Research Articles

Functionalization of polymeric nanomicelles and mixed nanomicelles for targeted retinal delivery in the management of retinoblastoma.

The current research discusses polymer conjugation, formulation development, and evaluation of sorafenib-loaded polymeric nanomicelles of conjugated soluplus (solu-tin) and polymeric mixed nanomicelles of conjugated soluplus (solu-tin) with conjugated poloxamer 188 (polo-tin) for site-specific posterior segment delivery to the retina in managing retinoblastoma. Firstly, the soluplus and poloxamer 188 were conjugated with biotin by Fischer esterification reaction and evaluated by FTIR and 1H NMR for confirmation of covalent bond formation involving the carboxyl group of biotin and hydroxyl group of polymers. Secondly, the sorafenib-loaded solu-tin nanomicelles and mixed nanomicelles of solu-tin with polo-tin were formulated by the thin film hydration method. Thereafter, these nanomicelles were evaluated and displayed suitable outcomes for particle size (78.53nm and 73.17nm), PDI (0.089 and 0.074), zeta potential (-3.65mV and -4.17 mv), entrapment efficiency (99.23% and 99.83%), in vitro drug release (4h and 8h), solid-state analysis, osmolality (290mOsm/kg and 293mOsm/kg), pH (7.4 and 7.4), TEM (spherical) and residual solvent analysis (287.90ppm and 363.49ppm). The ex vivo transcleral permeation at 8h was found to be 548.45ng/cm2 and 281.61ng/cm2, respectively. Both the drug-loaded nanomicelles displayed a dose-dependent anticancer effect on Y-79 cells at all time points i.e. 6, 12, 18, and 24h, and were non-toxic to normal retinal pigmented epithelial cell line (ARPE-19) when incubated for 24h. Furthermore, the formulations were non-irritant (HET-CAM) and stable for 6months. Hence, the developed technology is safe and efficacious for targeting the retina in managing retinoblastoma.

Molecular mechanism of RB progression and Circ_0082415 inhibits MKLN1 translation to suppress retinoblastoma progression: Changes in mRNA and protein levels.

The molecular mechanism of retinoblastoma (RB) is complex, involving the abnormal regulation of many genes and signaling pathways. The objective of this research was to explore the molecular mechanisms underlying the role of Circ_0082415 in the progression of retinoblastoma (RB), with particular emphasis on its suppressive impact on the translation of MKLN1 and its consequent influence on the advancement of RB. The study quantified the expression levels of Circ_0082415 in both RB cells and normal retinal cells using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and subsequently analyzed the trend of Circ_0082415 expression throughout the progression of RB. The interaction mechanism between Circ_0082415 and MKLN1 mRNA was investigated by bioinformatics analysis and double luciferase reporter gene experiment. The expression of Circ_0082415 was markedly reduced in retinoblastoma (RB) cells, and its expression level exhibited a negative correlation with RB progression. Notably, the upregulation of Circ_0082415 significantly suppressed the proliferation, migration, and invasion of RB cells. A dual luciferase reporter gene assay substantiated the interaction between Circ_0082415 and MKLN1 mRNA, elucidating the mechanism through which Circ_0082415 exerts its anti-tumor effects by impeding the translation of MKLN1.

EXTH-07. UNRAVELING THE ROLE OF SHH SIGNALING PATHWAY IN RETINOBLASTOMA

Abstract Retinoblastoma (RB) is a pediatric cancer affecting the retina, often requiring surgical removal of one or both eyes due to the limited effectiveness of current treatments. There is an urgent need for advanced therapeutic strategies capable of effectively eliminating tumors, thereby obviating the need for surgical removal of eyes in affected children. Achieving this goal requires a deeper understanding of the cellular and molecular mechanisms governing RB initiation and progression. Using a transgenic mouse model, we identified the crucial role of the Shh signaling pathway in Rb1 mutation-induced tumorigenesis, highlighting its potential as a novel therapeutic target for RB treatment. We have observed frequent overexpression of Shh signaling proteins (SHH, GLI1, and GLI2) in human RB, correlating with aggressive clinicopathological features. GLI2 overexpression is necessary for Rb1 deletion-induced RB tumor initiation in mouse retinal progenitor cells (RPCs). These mouse intraocular tumors accurately replicate human RB at both cellular and molecular levels. Additionally, Shh pathway activation extends to normal retinal cells adjacent to human RB tumors, suggesting the involvement of Shh signaling in RB-microenvironment interactions. Importantly, our studies have identified a novel small molecular inhibitor targeting the Shh pathway, effectively inhibiting RB growth in vitro and in vivo by targeting both tumor cells and the tumor microenvironment (TME). The proposed research aims to provide unprecedented insights into the role of SHH signaling pathway in RB initiation and progression and the supporting communication network between RB tumors and TME. These outcomes have the potential to treat RB effectively while improving the well-being of young individuals with RB and those at risk of RB.

Auxiliary effect of trolox on coenzyme Q10 restricts angiogenesis and proliferation of retinoblastoma cells via the ERK/Akt pathway

Reactive oxygen species (ROS) are essential for cancer signalling pathways and tumour maintenance, making ROS targeting a promising anti-cancer strategy. Coenzyme Q10 (CoQ10) has been shown to be effective against various cancers, but its impact on retinoblastoma, alone or with trolox, remains unreported. Cytotoxicity of CoQ10 alone and with trolox was evaluated in normal human retinal pigment epithelium cells (ARPE-19) and Y79 retinoblastoma cells using CCK-8. Flow cytometry was used to assess apoptosis, cell cycle, ROS, and mitochondrial membrane potential (MMP). Anti-angiogenic potential was tested using human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM) assays. Mechanistic studies were conducted via RT-PCR and western blotting. CoQ10, alone and with trolox, reduced Y79 cell viability, induced apoptosis through excess ROS generation, and decreased MMP significantly. Both treatments caused G2/M phase cell arrest. The CAM assay showed a significant reduction in endothelial cell proliferation, evidenced by fewer number of co-cultured HUVECs when exposed to CoQ10 or CoQ10 with trolox. The combination of CoQ10 and trolox significantly reduced VEGF-A, ERK, and Akt receptor levels, while CoQ10 alone significantly inhibited ERK and Akt phosphorylation. Together, CoQ10 and trolox reduced protein expression of VEGFA. CoQ10 alone and with trolox, induces apoptosis in Y79 retinoblastoma cells by inhibiting the ERK/Akt pathway and downregulating VEGFA. This study is the first to report the in vitro and in-ovo anti-cancer potential of CoQ10 alone or when combined with trolox, on human retinoblastoma Y79 cells.

In Vitro Methylene Blue and Carboplatin Combination Triggers Ovarian Cancer Cells Death.

Ovarian cancer presents a dire prognosis and high mortality rates, necessitating the exploration of alternative therapeutic avenues, particularly in the face of platinum-based chemotherapy resistance. Conventional treatments often overlook the metabolic implications of cancer, but recent research has highlighted the pivotal role of mitochondria in cancer pathogenesis and drug resistance. This study delves into the metabolic landscape of ovarian cancer treatment, focusing on modulating mitochondrial activity using methylene blue (MB). Investigating two epithelial ovarian cancer (EOC) cell lines, OV1369-R2 and OV1946, exhibiting disparate responses to carboplatin, we sought to identify metabolic nodes, especially those linked to mitochondrial dysfunction, contributing to chemo-resistance. Utilizing ARPE-19, a normal retinal epithelial cell line, as a control model, our study reveals MB's distinct cellular uptake, with ARPE-19 absorbing 5 to 7 times more MB than OV1946 and OV1369-R2. Treatment with 50 µM MB (MB-50) effectively curtailed the proliferation of both ovarian cancer cell lines. Furthermore, MB-50 exhibited the ability to quell glutaminolysis and the Warburg effect in cancer cell cultures. Regarding mitochondrial energetics, MB-50 spurred oxygen consumption, disrupted glycolytic pathways, and induced ATP depletion in the chemo-sensitive OV1946 cell line. These findings highlight the potential of long-term MB exposure as a strategy to improve the chemotherapeutic response in ovarian cancer cells. The ability of MB to stimulate oxygen consumption and enhance mitochondrial activity positions it as a promising candidate for ovarian cancer therapy, shedding light on the metabolic pressures exerted on mitochondria and their modulation by MB, thus contributing to a deeper understanding of mitochondrial dysregulation and the metabolic underpinnings of cancer cell proliferation.

Exploring the Potential of Psephellus huber-Marathi (Wagenitz) Wagenitz: A Comprehensive UHPLC-MS/MS Analysis of Phytochemical Composition and Evaluation of Antioxidant, Antimicrobial, and Antiproliferative Activities

Psephellus huber-morathii (PH) is an endemic species in the Eastern Anatolia Region. In this study, besides the biological activities of the 70% methanol extract of PH, its comprehensive phytochemical composition was investigated. The phenolic composition of the PH extract was analyzed using the UHPLC-MS/MS method. To evaluate its antimicrobial characteristics, the microdilution technique was employed. The antioxidant capabilities and total phenolic and flavonoid contents of the extract were determined using the spectrophotometer. Additionally, the effects of the extracts on cell proliferation and NCI-60 survival parameter values were assessed using the MTT assay. Quinic acid and chlorogenic acid were major compounds. The extract showed high antioxidant activity in DPPH (IC50:13.9±0.4 μg mL-1) and FRAP (61.3±2.3 mg TE g extract-1) methods. The total phenolic and flavonoid contents of the extract were found as 52.2±1.9 mg GAE g extract-1 and 28.6±0.9 mg QE g extract-1, respectively. Discussion: The extract showed low antiproliferative activity against normal lung and retinal cell lines, promising anticancer effects on gynecological and colon cancer cells, and moderate antimicrobial activity against Bacillus cereus and Enterococcus faecium. The study demonstrated the medicinal potential and value of PH extract against infectious diseases and cancer.

Evaluation of acrylonitrile rubber containing zinc oxide as safety products

AbstractThe goal of this research is to find the ideal formulation with the best antimicrobial performance in terms of rheological, physicomechanical, and lowest toxic properties to produce safety rubber for the children toy's and other industrial products. In order to accomplish this, a number of formulations containing ZnO as activator for vulcanization process as well as an antimicrobial agent at concentrations ranging from 1.5 to 40 phr in the presence of 40 phr of different fillers were prepared. The rheological characteristics of prepared compounded rubber samples were assessed and the cure rate was calculated. The overall rheological test results indicate a raise in minimum and maximum torque along with a fall in scorch and optimum cure time. The compounded NBR was vulcanized as estimated curing time. The results illustrated that the NBR vulcanizates containing various amounts of ZnO and 40 phr talc have the best physicomechanical properties. Also, the scanning electron micrographs indicated surface homogeneity and good dispersion of the NBR components. The cytotoxicity test of the investigated vulcanizates on normal retina cell line gave no cytotoxic effect which assessed their safety. These findings offer useful information for antimicrobial NBR vulcanizates for kid's toys and other safety products.Highlights Synthesis of antimicrobial NBR rubber vulcanizates for safety applications. The antimicrobial agent used was ZnO. ZnO nanoparticles and other ingredients were incorporated with NBR on the ordinary rubber mixer. Rheological, physicomechanical properties and antimicrobial activity of NBR rubber were tested. The products have better mechanical and antimicrobial safety properties.

ETMR-25. UNRAVELING THE ROLE OF SHH SIGNALING PATHWAY IN RETINOBLASTOMA

Abstract BACKGROUND Retinoblastoma (RB) is a pediatric cancer affecting the retina, often requiring surgical removal of one or both eyes due to the limited effectiveness of current treatments. There is an urgent need for advanced therapeutic strategies capable of effectively eliminating tumors, thereby obviating the need for surgical removal of eyes in affected children. Achieving this goal requires a deeper understanding of the cellular and molecular mechanisms governing RB initiation and progression. METHODS Using a transgenic mouse model, we identified the crucial role of the Shh signaling pathway in Rb1 mutation-induced tumorigenesis, highlighting its potential as a novel therapeutic target for RB treatment. RESULTS We have observed frequent overexpression of Shh signaling proteins (SHH, GLI1, and GLI2) in human RB, correlating with aggressive clinicopathological features. GLI2 overexpression is necessary for Rb1 deletion-induced RB tumor initiation in mouse retinal progenitor cells (RPCs). These mouse intraocular tumors accurately replicate human RB at both cellular and molecular levels. Additionally, Shh pathway activation extends to normal retinal cells adjacent to human RB tumors, suggesting the involvement of Shh signaling in RB-microenvironment interactions. Importantly, our studies have identified a novel small molecular inhibitor targeting the Shh pathway, effectively inhibiting RB growth in vitro and in vivo by targeting both tumor cells and the tumor microenvironment (TME). CONCLUSIONS The proposed research aims to provide unprecedented insights into the role of SHH signaling pathway in RB initiation and progression and the supporting communication network between RB tumors and TME. These outcomes have the potential to treat RB effectively while improving the well-being of young individuals with RB and those at risk of RB.

ALYREF/THOC4 expression and cell growth modulation in retinoblastoma

In this study, we tested the hypothesis that ALYREF/THOC4, a poor prognostic factor in different cancer types, has potential as a drug target and prognostic biomarker for retinoblastoma (RB). Immunostaining (IHC), Western blot, and RT-qPCR analyses detected overexpression of ALYREF in the RB cell lines Y79, RB143, WERI-RB1, and RB116. IHC analysis on RB tumor array showed that 11/14 of RB tumors were ALYREF+ to varying degrees, with eight tumors at maximum 3+ intensity. The IHC analysis also detected ALYREF+ cells in normal retina, mainly in the inner nuclear and ganglion cell layer, while some tumor-bearing human eyes were ALYREF+ in the optic nerve suggesting a role in optic invasion/tumor invasion. The expression of ALYREF within the tumor itself, in the optic nerve, as well as in adjacent "normal" retina, suggest that this pattern of expression may lead to ALYREF being a potentially useful prognostic indicator for RB, as it is for other tumors. siRNA knockdown of ALYREF resulted in a 40 % decrease in cell growth in both WERI-RB1 and Y79 cells (p<0.05) and this was associated with decreased expression of mRNAs for the cell proliferation markers Ki67 and PCNA (p<0.005). These results suggest a role for ALYREF in RB cell growth regulation and its potential as both a target and a biomarker for tumor growth inhibition by anti-cancer therapies.

Efficient malachite green biodegradation by Pseudomonas plecoglossicide MG2: process optimization, application in bioreactors, and degradation pathway

Microbial degradation of synthetic dyes is considered a promising green dye detoxification, cost-effective and eco-friendly approach. A detailed study on the decolorization and degradation of malachite green dye (MG) using a newly isolated Pseudomonas plecoglossicide MG2 was carried out. Optimization of MG biodegradation by the tested organism was investigated by using a UV–Vis spectrophotometer and the resultant degraded products were analyzed by liquid chromatography–mass spectrometry and FTIR. Also, the cytotoxicity of MG degraded products was studied on a human normal retina cell line. The optimum conditions for the significant maximum decolorization of MG dye (90–93%) by the tested organism were pH 6–7, inoculum size 4–6%, and incubation temperature 30–35 °C, under static and aerobic conditions. The performance of Pseudomonas plecoglossicide MG2 grown culture in the bioreactors using simulated wastewater was assessed. MG degradation (99% at 100 and 150 mg MG/l at an optimal pH) and COD removal (95.95%) by using Pseudomonas plecoglossicide MG2 culture were the best in the tested culture bioreactor in comparison with that in activated sludge or tested culture-activated sludge bioreactors.The FTIR spectrum of the biodegraded MG displayed significant spectral changes, especially in the fingerprint region 1500–500 as well as disappearance of some peaks and appearance of new peaks. Twelve degradation intermediates were identified by LC–MS. They were desmalachite green, didesmalachite green, tetradesmalachite green, 4-(diphenylmethyl)aniline, malachite green carbinol, bis[4-(dimethylamino)phenyl]methanone, [4-(dimethylamino)phenyl][4-(methyl-amino)phenyl]methanone, bis[4-(methylamino)phenyl]methanone, (4-amino- phenyl)[4-(methylamino)phenyl]methanone, bis(4-amino phenyl)methanone, (4-amino phenyl)methanone, and 4-(dimathylamino)benzaldehyde. According to LC–MS and FTIR data, two pathways for MG degradation by using Pseudomonas plecoglossicide MG2 were proposed. MG showed cytotoxicity to human normal retina cell line with LC50 of 28.9 µg/ml and LC90 at 79.7 µg/ml. On the other hand, MG bio-degraded products showed no toxicity to the tested cell line. Finally, this study proved that Pseudomonas plecoglossicide MG2 could be used as an efficient, renewable, eco-friendly, sustainable and cost-effective biotechnology tool for the treatment of dye wastewater effluent.

Cytotoxicity of Ruthenium(II) Arene Complexes Containing Functionalized Ferrocenyl β-Diketonate Ligands.

The synthesis and characterization of 24 ruthenium(II) arene complexes of the type [(p-cym)RuCl(Fc-acac)] (where p-cym = p-cymene and Fc-acac = functionalized ferrocenyl β-diketonate ligands) are reported, including single-crystal X-ray diffraction for 21 new complexes. Chemosensitivity studies have been conducted against human pancreatic carcinoma (MIA PaCa-2), human colorectal adenocarcinoma p53-wildtype (HCT116 p53+/+) and normal human retinal epithelial cell lines (APRE-19). The most active complex, which contains a 2-furan-substituted ligand (4), is 5x more cytotoxic than the analogs 3-furan complex (5) against MIA PaCa-2. Several complexes were screened under hypoxic conditions and at shorter-time incubations, and their ability to damage DNA was determined by the comet assay. Compounds were also screened for their potential to inhibit the growth of both bacterial and fungal strains.

Memantine mitigates ROS/TXNIP/NLRP3 signaling and protects against mouse diabetic retinopathy: Histopathologic, ultrastructural and bioinformatic studies

Diabetic retinopathy (DRET) triggers vision loss in adults, however, little therapeutic options are existing. Memantine is an anti-Alzheimer drug that antagonizes the activity of glutamate at N-methyl-D-aspartate (NMDA) receptors. Glutamate and thioredoxin-interacting protein (TXNIP) are known to be overexpressed in diabetic retinas and can produce activation of NOD-like receptor protein 3 (NLRP3) with subsequent secretion of interlukin-1β. This study repurposed memantine for its neuroprotective effect in experimental DRET and tested its impact on ROS/TXNIP/NLRP3. In addition, KEGG pathway database and STRING database identified the protein-protein interaction between glutamate receptors and TXNIP/NLRP3. Male Swiss albino mice received alloxan (180mg/kg) to induce DRET. After 9 weeks, we divided the mice into groups: (a) saline, (ii) DRET, (iii and iv) DRET +oral memantine (5 or 10mg per kg) for 28 days. Then, mice were euthanized, and eyeballs were removed. Retinal samples were utilized for biochemical, histopathological, and electron microscopy studies. Retinal levels of glutamate, TXNIP, NLRP3 and interlukin-1β were estimated using ELISA technique as well as retinal malondialdehyde. Histopathological and ultrastructural examination demonstrated that oral memantine attenuated vacuolization and restored normal retinal cell layers. Moreover, memantine reduced TXNIP, NLRP3, interleukin-1β and MDA concentrations. These results provide evidence demonstrating memantine' efficacy in alleviating DRET via suppressing reactive oxygen species/TXNIP/NLRP3 signaling cascade. Therefore, memantine might serve as a potential therapy for retinopathy after adequate clinical research.

Trogocytosis of neurons and glial cells by microglia in a healthy adult macaque retina

Microglial cells are the primary resident immune cells in the retina. In healthy adults, they are ramified; that is, they have extensive processes that move continually. In adult retinas, microglia maintain the normal structure and function of neurons and other glial cells, but the mechanism underlying this process is not well-understood. In the mouse hippocampus, microglia engulf small pieces of axons and presynaptic terminals via a process called trogocytosis. Here we report that microglia in the adult macaque retina also engulf pieces of neurons and glial cells, but not at sites of synapses. We analyzed microglia in a volume of serial, ultrathin sections of central macaque retina in which many neurons that ramify in the inner plexiform layer (IPL) had been reconstructed previously. We surveyed the IPL and identified the somas of microglia by their small size and scant cytoplasm. We then reconstructed the microglia and studied their interactions with other cells. We found that ramified microglia frequently ingested small pieces of each major type of inner retinal neuron and Müller glial cells via trogocytosis. There were a few instances where the interactions took place near synapses, but the synapses, themselves, were never engulfed. If trogocytosis by retinal microglia plays a role in synaptic remodeling, it was not apparent from the ultrastructure. Instead, we propose that trogocytosis enables these microglia to present antigens derived from normal inner retinal cells and, when activated, they would promote antigen-specific tolerance.

Effects of antimicrobial agents on the processing and performance properties of rubber material formulations with no cytotoxicity

AbstractThe present work aims to develop antimicrobial rubber for safe industrial toys. For this purpose, natural rubber (NR) and synthetic rubber as styrene butadiene rubber (SBR) and ethylene propylene diene monomer (EPDM) were examined. Rubber and their ingredients as well as antimicrobial agents (doxycycline and cephalexin) were mixed in a rubber mixer. The rheological properties of compounded rubber were studied, and the curing time was determined. Mechanical properties and cytotoxicity were evaluated at optimally cured rubber compounds. Scanning electron micrographs of vulcanizates showed good dispersion of ingredients throughout the investigated matrices. Rheology study for the investigated vulcanizates in presence of tested antimicrobial species exhibited no significant change in their flow behaviors. It is significant to remember that the desired physical characteristics of rubber products, including their chemical and mechanical characteristics (elongation at break and tensile strength) enhanced when doxycycline and cephalexin are present, depending on their nature and concentration. Similar results were obtained for both the SBR and EPDM rubber vulcanizates. The cytotoxicity of the prepared vulcanizates towards human normal retina cell line (RPI‐1) indicated good safety of these rubber products. Furthermore, developed rubber vulcanizates showed good antimicrobial efficacy towards the test bacteria and fungi strains.”

PRELP Regulates Cell-Cell Adhesion and EMT and Inhibits Retinoblastoma Progression.

Simple SummaryMutation of the RB1 tumor suppressor gene is fundamental in retinoblastoma initiation and progression although its downstream mechanism has not been well elucidated. Here, we found that expression of proline/arginine-rich end leucine-rich repeat protein (PRELP) is strongly downregulated in human retinoblastoma and highly expressed in Müller glial cells in normal retina. Deletion of PRELP in mice resulted in retinal dysplasia associated with enhanced proliferation. mRNA expression profiling revealed that cancer pathways were strongly activated in PRELP−/− retina. Additionally, cell–cell adhesion was inhibited while epithelial mesenchymal transition (EMT) and inflammation were activated. On the other hand, application of PRELP protein to retinoblastoma cell lines enhances cell–cell and cell–substrate adhesion and inhibits anchorage independent growth by reversing EMT. These observations indicate that PRELP downregulation in human retinoblastoma can contribute cancer progression through regulation of cell adhesion and EMT suggested that PRELP application might be a novel strategy for retinoblastoma treatment.Retinoblastoma (RB) is the most common intraocular pediatric cancer. Nearly all cases of RB are associated with mutations compromising the function of the RB1 tumor suppressor gene. We previously demonstrated that PRELP is widely downregulated in various cancers and our in vivo and in vitro analysis revealed PRELP as a novel tumor suppressor and regulator of EMT. In addition, PRELP is located at chromosome 1q31.1, around a region hypothesized to be associated with the initiation of malignancy in RB. Therefore, in this study, we investigated the role of PRELP in RB through in vitro analysis and next-generation sequencing. Immunostaining revealed that PRELP is expressed in Müller glial cells in the retina. mRNA expression profiling of PRELP−/− mouse retina and PRELP-treated RB cells found that PRELP contributes to RB progression via regulation of the cancer microenvironment, in which loss of PRELP reduces cell–cell adhesion and facilitates EMT. Our observations suggest that PRELP may have potential as a new strategy for RB treatment.

OTHR-34. Identifying mechanisms of microglia-tumor cell interactions in retinoblastoma

Abstract BACKGROUND: Treatment-related long-term sequelae and chemotherapy resistance diminish the success of retinoblastoma (RB) treatment. To unravel the mechanisms leading to tumor progression and resistance we examined the intratumoral cellular heterogeneity of RB and its interactions with cells of the tumor microenvironment (TME). METHODS: We used single-cell RNA (scRNA-seq) and ATAC sequencing (scATAC-seq) as well as spatial transcriptomics to analyze and compare RB samples from patients with or without previous chemotherapy (chemo-treated vs. naïve). In addition, we developed a 3D model by injection of RB and TME cells into retinal organoids, which mimics the heterogeneous surrounding of the tumor in a spatially and functionally organized manner. RESULTS: ScRNA-seq revealed a high intratumoral heterogeneity of tumor cells representing distinct developmental stages from progenitors to more differentiated photoreceptor cells. The predominant cell type in the TME was M2-activated microglia (MG). M2-MG exerted multiple receptor-ligand interactions with RB tumor cells which were not found in non-diseased retinas. These tumor-specific cellular interactions regulate multiple signaling pathways (e. g. VEGF-, WNT-, BMP-, PGF- signaling) known to be involved in RB progression. By comparing chemo-treated and chemo-naïve RB samples we were able to identify treatment-resistant and -sensitive subpopulations of tumor cells. Finally, data from our RB-retina in vitro model highlighted the impact of RB cells on gene expression programs of normal retinal cells. CONCLUSION: In summary, we deciphered the intratumoral heterogeneity of RB, uncovered an intricate network of microglia-tumor cell interactions that connects numerous important signaling pathways, and further identified chemotherapy-resistant/sensitive tumor cell populations. The latter observation could prove to be very helpful in the development of novel therapeutic approaches in the treatment of RB.

LncRNA HOTAIR facilitates proliferation and represses apoptosis of retinoblastoma cells through the miR-20b-5p/RRM2/PI3K/AKT axis

PurposeRetinoblastoma (RB) represents an adolescent eye malignancy. Long non-coding RNA (LncRNA) HOTAIR shows aberrant expression in many malignancies. This research investigated the mechanism of HOTAIR in RB.MethodsNormal retinal cell lines (ARPE-19 and RPE-1) and RB cell lines (ORB50, Y79, HXO-RB44, and WERI-RB) were selected for detection of HOTAIR expression by qRT-PCR. sh-HOTAIR was delivered into Y79 and HXO-RB44 cells. Cell-cycle distribution, proliferation, and apoptosis were detected by CCK-8 assay and flow cytometry. Binding relationships among HOTAIR, miR-20b-5p, and RRM2 were confirmed using dual-luciferase assay. Roles of miR-20b-5p and RRM2 in RB cell-cycle distribution, proliferation, and apoptosis were ascertained by functional rescue experiments. Murine model of xenograft tumor was established, followed by detection of tumor growth and counting of Ki67-positive cells. Expressions of proliferation- and apoptosis-associated proteins and PI3K/AKT pathway-related proteins were determined by Western blot.ResultsHOTAIR was elevated in RB cells relative to that in normal retinal cells and showed relatively high expression in Y79 and HXO-RB44 cells. sh-HOTAIR induced RB cell-cycle arrest, restrained proliferation, and strengthened apoptosis. HOTAIR functioned as the ceRNA of miR-20b-5p and targeted RRM2. RB cells had poorly-expressed miR-20b-5p and highly-expressed RRM2. miR-20b-5p downregulation or RRM2 overexpression facilitated RB cell-cycle and proliferation, suppressed apoptosis, and reversed the protective effect of sh-HOTAIR on RB. sh-HOTAIR reduced tumor growth and Ki67-positive cells in vivo and inactivated PI3K/AKT pathway.ConclusionLncRNA HOTAIR upregulated RRM2 by competitively binding to miR-20b-5p and activated PI3K/AKT pathway, thereby facilitating proliferation and repressing apoptosis of RB cells.

MTOR inhibition by TAK-228 is effective against growth, survival and angiogenesis in preclinical retinoblastoma models.

We and others have shown that aberrant activation of the mammalian target of rapamycin (mTOR) signalling is essential for retinoblastoma progression and has potential therapeutic value. TAK‐228 is a potent inhibitor of mTOR1 and 2 with preclinical activity in a variety of cancers. In this study, we report that TAK‐228 is a dual inhibitor of retinoblastoma and angiogenesis. TAK‐228 inhibits growth and induces apoptosis in a panel of retinoblastoma cell lines, with IC50 at ~0.2 μM. Under the same experimental conditions, TAK‐228 was less effective in inhibiting growth and survival in normal retinal and fibroblast cells than retinoblastoma cells. In addition, TAK‐228 inhibited retinal endothelial cell capillary network formation, migration, growth and survival. We further demonstrate that TAK‐228 inhibits retinoblastoma and retinal angiogenesis through inhibiting mTOR signalling. Rescue studies confirm that mTOR is the target of TAK‐228 in both retinoblastoma and retinal endothelial cells. Finally, we confirm the inhibitory effects of TAK‐228 on tumor and angiogenesis in retinoblastoma xenograft mouse model. Our findings provide a preclinical rationale to explore TAK‐228 as a strategy to treat retinoblastoma and highlight the therapeutic value of targeting mTOR in retinoblastoma.

Ribonucleotide reductase subunit M2 promotes proliferation and epithelial–mesenchymal transition via the JAK2/STAT3 signaling pathway in retinoblastoma

ABSTRACT Retinoblastoma (RB) is an intraocular malignant tumor that often occurs in children. Along with the improvement of treatment strategies, the cure rate of RB has increased significantly. However, the treatment of advanced and recurrent RB remains as a critical challenge. Therefore, studying the molecular mechanisms underlying the progression of RB is essential for the development of novel and effective therapeutic strategies. Through the analysis of a previously published microarray study, we found that ribonucleotide reductase subunit M2 (RRM2) was highly expressed in RB tissues as compared to normal tissues. The purpose of this study is to clarify the role and mechanism of RRM2 in regulating the progression of RB. We first demonstrated that RRM2 expression level in RB tissues and cell lines was significantly higher when compared to that in normal retinal tissue and cell lines, and high RRM2 expression level was associated with a poorer overall survival of patients. In RB cells, RRM2 overexpression promoted cell proliferation, migration, invasion and epithelial–mesenchymal transformation (EMT), while RRM2 silencing suppressed these biological features. Silencing RRM2 reduced the activation of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, and the presence of JAK2/STAT3 signaling pathway inhibitor INCB attenuated the effect of RRM2 overexpression. Collectively, our data indicate that RRM2 promotes the progression of RB by activating JAK2/STAT3 signaling pathway. Targeting RRM2/JAK2/STAT3 axis lays a theoretical foundation for the formulation of novel RB therapy.

Ubiquitin specific peptidase 33 promotes cell proliferation and reduces apoptosis through regulation of the SP1/PI3K/AKT pathway in retinoblastoma

ABSTRACT Ubiquitin-specific protease 33 (USP33), a deubiquitinating enzyme (DUB), has been identified to serve as a tumor suppressor or an oncogene in different cancers. However, its role in retinoblastoma (RB) remains unknown. Here, we aimed to uncover USP33 expression profile and function in RB, and disclose the underlying mechanism. USP33 levels in RB tissues and cells were determined using RT-qPCR and western blotting assays. USP33 effects on cell growth, cycle, apoptosis and tumorigenesis were studied using MTT, Edu, cycle and western blotting and in vivo assays. The results showed that USP33 expression levels were elevated in RB tissues and cells as compared with normal retinal tissues and cells. Downregulation of USP33 in RB Y79 and WERI-RB1 cells leaded to significant increases in cell apoptosis, G1 phase arrest and tumorigenesis, and reductions in cell growth and G2 and S phase arrest, as well as inhibited the activation of the PI3K/AKT signaling. SP1 overexpression abolished the roles of USP33 downregulation in modulating the activation of PI3K/AKT signaling, cell growth, apoptosis, and cell cycle. This study uncovered that USP33 promoted the progression of RB through regulation of the SP1/PI3K/AKT pathway.