EPZ015666, a selective protein arginine methyltransferase 5 (PRMT5) inhibitor with an antitumour effect in retinoblastoma.

Retinoblastoma (RB) is the most common intraocular malignant tumour in infants, and chemotherapy has been the primary therapy method in recent years. PRMT5 is an important member of the protein arginine methyltransferase family, which plays an important role in various tumours. Our study showed that PRMT5 was overexpressed in retinoblastoma and played an important role in retinoblastoma cell growth. EPZ015666 is a novel PRMT5 inhibitor, and we found that it inhibited retinoblastoma cell proliferation and led to cell cycle arrest at the G1 phase. At the same time, EPZ015666 regulated cell cycle related protein (P53, P21, P27, CDK2) expression. In brief, our study showed that PRMT5 promoted retinoblastoma growth, the PRMT5 inhibitor EPZ015666 inhibited retinoblastoma in vitro by regulating P53-P21/P27-CDK2 signaling pathways and slowed retinoblastoma growth in a xenograft model.

Anticancer Effects of Gingerol in Retinoblastoma Cancer Cells (RB355 Cell Line) Are Mediated via Apoptosis Induction, Cell Cycle Arrest and Upregulation of PI3K/Akt Signaling Pathway.

BACKGROUND The main aim of the current investigation was to study the antiproliferative activity of gingerol in RB355 human retinoblastoma cancer cells. The effects of gingerol on apoptosis induction, cell cycle arrest, and PI3K/Akt signaling pathway were also evaluated. MATERIAL AND METHODS MTT cell viability assay was used to assess the cytotoxic effects of gingerol in these cells while. To study apoptotic effects in these cells, we used inverted microscopy along with fluorescence microscopy using acridine orange/propidium iodide and Hoechst 33258 as staining dyes. Flow cytometry was used to study cell cycle phase distribution and Western blot assay indicated effects on PI3K/Akt protein expression levels. RESULTS Results showed that gingerol exerted dose-dependent and time-dependent growth inhibitory effects in these retinoblastoma cells. However, the growth inhibitory effects of gingerol were less pronounced against normal fr2 cells. As compared to the untreated control cells, gingerol-treated cells at concentrations of 25, 75, and 150 µM had drastic changes in cell morphology, including rounding and withering of cells, with disorganized cell layers. Gingerol-treated cells exhibited bright fluorescence, indicating rupture of the cell membrane. These results were further confirmed by acridine orange/propidium iodide staining, in which untreated cells showed normal green fluorescence and gingerol-treated cells showed yellow/red fluorescence. Gingerol also led to dose-dependent G2/M phase cell cycle arrest in RB355 retinoblastoma cells, as well as concentration-dependent activation of PI3K-related protein expressions. CONCLUSIONS Gingerol exhibits potent anticancer effects in RB355 human retinoblastoma cancer cells and these effects were mediated via apoptosis induction, cell cycle arrest, and modulation of the PI3K/Akt signaling pathway.

Prognostic significance of NADPH oxidase-4 as an indicator of reactive oxygen species stress in human retinoblastoma.

BACKGROUND: Reactive oxygen species (ROS) have been shown to enhance the proliferation of cancer cells. NADPH oxidases (NOX4) are a major intracellular source of ROS and are found to be associated with cancer and tumor cell invasion. Therefore, the purpose of this study is to evaluate the expression of NOX4 protein in human retinoblastoma. METHODS: Immunohistochemical expression of NOX4 protein was analyzed in 109 specimens from prospective cases of retinoblastoma and then correlated with clinicopathological parameters and patient survival. Western blotting confirmed and validated the immunoreactivity of NOX4 protein. RESULTS: In our study we found a male preponderance (55.9 %), and 25/109 (22.9 %) were bilateral. Massive choroidal invasion was the histopathological high-risk factor (HRF) most frequently observed, in 42.2 % of the cases. NOX4 protein was expressed in 67.88 % (74/109) of primary retinoblastoma cases and was confirmed by Western blotting. NOX4 was statistically significant with massive choroidal invasion and pathological TNM staging. There was a statistically significant difference in overall survival in patients with NOX4 expression (p = 0.0461). CONCLUSION: This is the first study to show the expression of NOX4 protein in retinoblastoma tumors. Hence, a retinoblastoma tumor may exhibit greater ROS stress. This protein may prove to be useful as a future therapeutic target for improving the management of retinoblastoma.

Prognostic significance of polo-like kinases in retinoblastoma: correlation with patient outcome, clinical and histopathological parameters.

BACKGROUND: Retinoblastoma is evolving, but it is still a therapeutic challenge for pediatric oncologists. Polo-like kinases (PLKs) plays an important role in cell cycle events. They play a crucial role in cell proliferation which may lead to tumour formation. The objective of this study is to investigate the role of PLK1 and PLK3 proteins in human retinoblastoma tissues. DESIGN: Non-randomized, prospective study was performed in the Dr R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India. PARTICIPANTS: This study included 74 primary enucleated retinoblastoma tissues. METHODS: Expression of PLK1 and PLK3 protein were assessed in primary enucleated retinoblastoma tissues by immunohistochemistry and western blotting. MAIN OUTCOME MEASURES: Expression of PLK1 and PLK3 protein were correlated with clinical and histopathological parameters, tumour staging and overall survival of patients. RESULTS: Immunohistochemical results revealed expression of PLK1 in 47/74 (63.51%) cases and PLK3 in 31/74 (41.89%) cases. Western blotting confirmed the immunoreactivity results. Expression of PLK1 showed correlation with poor differentiation and tumour invasion. In addition, PLK1 was statistically significant with massive choroidal invasion, whereas PLK3 did not correlate with any of the clinical or histopathological parameters. There was no statistical correlation in the overall survival of patients with PLK1 and PLK3 expression. CONCLUSIONS: PLK1 expression was associated with poor tumour differentiation and histopathological high-risk factors. These proteins may be involved in tumorigenesis and progression of disease. These results suggest that PLK1 may act as a potential therapeutic target and a promising marker for developing potent small molecule inhibitors of PLK isoforms in retinoblastoma.

Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors.

Retinoblastoma (RB) is a malignant intra-ocular neoplasm that affects children (usually below the age of 5years). In addition to conventional chemotherapy, novel therapeutic strategies that target metabolic pathways such as glycolysis and lipid metabolism are emerging. Fatty acid synthase (FASN), a lipogenic multi-enzyme complex, is over-expressed in retinoblastoma cancer. The present study evaluated the biochemical basis of FASN inhibition induced apoptosis in cultured Y79 RB cells. FASN inhibitors (cerulenin, triclosan and orlistat) significantly inhibited FASN enzyme activity (P<0.05) in Y79 RB cells. This was accompanied by a decrease in palmitate synthesis (end-product depletion), and increased malonyl CoA levels (substrate accumulation). Differential lipid profile was biochemically estimated in neoplastic (Y79 RB) and non-neoplastic (3T3) cells subjected to FASN inhibition. The relative proportion of phosphatidyl choline to neutral lipids (triglyceride+total cholesterol) in Y79 RB cancer cells was found to be higher than the non-neoplastic cells, indicative of altered lipid distribution and utilization in tumor cells. FASN inhibitor treated Y79 RB and fibroblast cells showed decrease in the cellular lipids (triglyceride, cholesterol and phosphatidyl choline) levels. Apoptotic DNA damage induced by FASN inhibitors was accompanied by enhanced lipid peroxidation.

The effect of ROCK-1 activity change on the adhesive and invasive ability of Y79 retinoblastoma cells.

BACKGROUND: Retinoblastoma (Rb) is the most common intraocular tumor in childhood worldwide. It is a deadly pediatric eye cancer. The main cause of death in Rb patients is intracranial and systemic metastasis. ROCK is the main downstream effector of Ras-homologous (Rho) family of GTPases which are involved in many cellular functions, such as cell proliferation, invasion and metastasis. Overexpression of ROCK promotes invasion and metastasis of many solid tumors. However, the effect of ROCK in Rb is largely unknown. METHODS: ROCK-1 and ROCK-2 mRNA expression in Y79 cell lines were examined by RT-PCR. Protein expression in the Y79 cell line were examined by western blot analyses. ROCK-1 and ROCK-2 siRNA were transfected into Y79 cells with Lipofectamine 2000. Cell proliferation was evaluated by CCK-8 assay after exposure to ROCK inhibitor (Y-27632). We examined the effect of ROCK inhibitors (Y-27632, ROCK-1 and ROCK-2 siRNA) on Y79 cell adhesive capacity by cell adhesion assay. Cell invasion assay through matrigel was used to study the effect of ROCK inhibitors on Y79 cell invasive capacity. RESULTS: The expression of mRNA of ROCK-1 was more than that of ROCK-2 in the Y79 cell line. The protein expression levels of ROCK-1 and ROCK-2 were downregulated in the cells transfected with siRNA. Y-27632 treatment didn't lead to any changes of Y79 cells proliferation. Adhesive ability of Y79 cells was enhanced following Y-27632 or ROCK-1 siRNA treatment. The invasive capacity of Y79 cells showed an inverse relationship with increasing Y-27632 concentration. Invasiveness of Y79 cells also decreased in Y79 cells transfected with ROCK-1 siRNA. However, there was no change in adhesive ability or invasive capacity in Y79 cells transfected with siRNA against ROCK-2. CONCLUSIONS: The findings of this study demonstrate that ROCK-1 protein plays a key role in regulating metastasis and invasion of Y79 cells, suggesting that the ROCK-1 dependent pathway may be a potential target for therapy of Rb.

High trefoil factor 1 (TFF1) expression in human retinoblastoma cells correlates with low growth kinetics, increased cyclin-dependent kinase (CDK) inhibitor levels and a selective down-regulation of CDK6.

Trefoil factor family (TFFs) peptides facilitate epithelial restitution, but also effect cell proliferation and apoptosis of normal and various cancer cell lines. In a recent study by our group, TFF2 expression was demonstrated in the murine retina, where it exhibits pro-proliferative and pro-apoptotic effects. In the present study, we investigated the expression and function of TFF peptides in eight human retinoblastoma cell lines. TFF1 was the only TFF peptide expressed at detectable levels in immunoblots of retinoblastoma cells. TFF1 expression levels were highly variable in different retinoblastoma cell lines and negatively correlated with cell growth curves. Recombinant human TFF1 had a negative effect on cell viability and caused a reduction in cell proliferation. Retinoblastoma cell lines with high TFF1 expression levels exhibited a selective down-regulation of cyclin-dependent kinase (CDK) 6, whereas CDK4 and CDK2 seem to be unaffected by TFF1 expression. In immunocytochemical studies, we observed a nuclear co-localization of TFF1 and CDK2 in Cajal bodies (CBs). In high TFF1 expressing human retinoblastoma cell lines CBs were smaller and higher in number compared to retinoblastoma lines with low TFF1 expression, indicating differences in cell cycle status between the different retinoblastoma cell lines. Our data further support the notion for a potential tumor suppressor function of TFF1. The nuclear localization of TFF1 in CBs--considered to play a role in cell cycle progression, potentially acting as a platform for CDK-cyclin function-offers a new impetus in the ongoing search for potential TFF1 interacting proteins.

2-Methoxyestradiol induced Bax phosphorylation and apoptosis in human retinoblastoma cells via p38 MAPK activation.

Retinoblastoma (Rb) is a common childhood intraocular cancer that affects approximately 300 children each year in the United States alone. 2-Methoxyestradiol (2ME), an endogenous metabolite of 17-ß-estradiol that dose not bind to nuclear estrogen receptor, exhibits potent apoptotic activity against rapidly growing tumor cells. Here, we report that 2ME induction of apoptosis was demonstrated by early fragmented DNA after 48 h of incubation with 10 µM 2ME in Rb cell lines. Subsequently, a decrease of proliferation was observed in a time- and dose-dependent manner. Further analysis of the mechanism indicates that p38 kinase plays a critical role in 2ME-induced apoptosis in Y79 cells, even though ERK was also activated by 2ME under the same conditions. Activation of p38 kinase also mediates 2ME induced Bax phosphorylated at Thr(167) after a 6 h treatment of 2ME, which in turn prevents formation of the Bcl-2-Bax heterodimer. Both p38 specific inhibitor, SB 203580, or p38 knockdown by specific siRNA, blocked 2ME induction of Bax phosphorylation. Furthermore, only transiently transfected mutant BaxT167A, but not Bax S163A, inhibited 2ME-induced apoptosis. In summary, our data suggest that 2ME induces apoptosis in human Rb cells by causing phosphorylation of p38 Mitogen-activated protein kinase (MAPK), which appears to be correlated with phosphorlation of Bax. This understanding of 2ME's ability may help develop it as a promising therapeutic candidate by inducing apoptosis in a Rb.

Clinico-pathological correlations of fatty acid synthase expression in retinoblastoma: an Indian cohort study.

Retinoblastoma (RB), the most common intra-ocular malignancy in children under 5 years of age, has an estimated incidence of about 2000 a year in India, where most cases are in advanced stage at the time of diagnosis. Newer therapeutic approaches would reduce the morbidity of chemotherapy in children with RB. Fatty Acid Synthase (FASN), a lipogenic multi-enzyme complex, is minimally expressed in normal human tissues and over expressed in many cancers, making it an attractive target for cancer therapy. We analyzed RB tissues for FASN protein expression by immunohistochemistry, western blot, and ELISA, and FASN mRNA expression by RT-PCR. FASN expression was correlated with the clinico-pathological characteristics of the tumors. FASN immunostaining was positive in all the 44 RB tissues analyzed (100%). However, FASN expression was heterogeneous within the tumor samples. Tumors with invasion of choroid, optic nerve, orbit and/or retinal pigment epithelium showed significantly higher FASN immunoreactivity than the tumors without invasion (P<0.05), supported by western analysis (P<0.05). FASN expression was significantly high in poorly differentiated retinoblastomas (P<0.05). FASN protein and FASN mRNA estimated by ELISA and RT-PCR respectively showed multi-fold expression over the non-neoplastic muller glial cells that varied quantitatively between tumor tissues. FASN mRNA over-expression was substantially lower than the corresponding FASN protein expression values. The present study reports (i) markedly high expression of FASN protein in poorly differentiated and in invasive retinoblastomas, and (ii) multi-fold over-expression of FASN mRNA and protein in RB tissues, although at varying levels, indicating FASN to be a potential therapeutic target in retinoblastoma management.

Aurora Kinase B Expression, Its Regulation and Therapeutic Targeting in Human Retinoblastoma.

Purpose: Aurora kinase B (AURKB) plays a pivotal role in the regulation of mitosis and is gaining prominence as a therapeutic target in cancers; however, the role of AURKB in retinoblastoma (RB) has not been studied. The purpose of this study was to determine if AURKB plays a role in RB, how its expression is regulated, and whether it could be specifically targeted. Methods: The protein expression of AURKB was determined using immunohistochemistry in human RB patient specimens and immunoblotting in cell lines. Pharmacological inhibition and shRNA-mediated knockdown were used to understand the role of AURKB in cell viability, apoptosis, and cell cycle distribution. Cell viability in response to AURKB inhibition was also assessed in enucleated RB specimens. Immunoblotting was employed to determine the protein levels of phospho-histone H3, p53, p21, and MYCN. Chromatin immunoprecipitation-qPCR was performed to verify the binding of MYCN on the promoter region of AURKB. Results: The expression of AURKB was found to be markedly elevated in human RB tissues, and the overexpression significantly correlated with optic nerve and anterior chamber invasion. Targeting AURKB with small-molecule inhibitors and shRNAs resulted in reduced cell survival and increased apoptosis and cell cycle arrest at the G2/M phase. More importantly, primary RB specimens showed decreased cell viability in response to pharmacological AURKB inhibition. Additional studies have demonstrated that the MYCN oncogene regulates the expression of AURKB in RB. Conclusions: AURKB is overexpressed in RB, and targeting it could serve as a novel therapeutic strategy to restrict tumor cell growth.

Xanthatin Selectively Targets Retinoblastoma by Inhibiting the PLK1-Mediated Cell Cycle.

Purpose: Retinoblastoma is the most common primary intraocular malignant tumor in children. Although intra-arterial chemotherapy and conventional chemotherapy have become promising therapeutic approaches for advanced intraocular retinoblastoma, the side effects threaten health and are unavoidable, making the development of targeted therapy an urgent need. Therefore, we intended to find a potential drug for human retinoblastoma by screening an in-house compound library that included 89 purified and well-characterized natural products. Methods: We screened a panel of 89 natural products in retinoblastoma cell lines to find the inhibitor. The inhibition of the identified inhibitor xanthatin on cell growth was detected through half-maximal inhibitory concentration (IC50), flow cytometry assay, and zebrafish model system. RNA-seq further selected the target gene PLK1. Results: We reported the discovery of xanthatin as an effective inhibitor of retinoblastoma. Mechanistically, xanthatin selectively inhibited the proliferation of retinoblastoma cells by inducing cell cycle arrest and promoting apoptosis. Interestingly, xanthatin targeted PLK1-mediated cell cycle progression. The efficacy of xanthatin was further confirmed in zebrafish models. Conclusions: Collectively, our data suggested that xanthatin significantly inhibited tumor growth in vitro and in vivo, and xanthatin could be a potential drug treatment for retinoblastoma.

Paclitaxel and beta-lapachone synergistically induce apoptosis in human retinoblastoma Y79 cells by downregulating the levels of phospho-Akt.

Paclitaxel (PTX) and beta-lapachone (LPC) are naturally occurring compounds that have shown a large spectrum of anticancer activity. In this article we show for the first time that PTX/LPC combination induces potent synergistic apoptotic effects in human retinoblastoma Y79 cells. Combination of suboptimal doses of PTX (0.3 nM) and LPC (1.5 microM) caused biochemical and morphological signs of apoptosis at 48 h of treatment. These effects were accompanied by potent lowering in inhibitor of apoptosis proteins and by activation of Bid and caspases 3 and 6 with lamin B and PARP breakdown. PTX/LPC combination acted by favoring p53 stabilization through a lowering in p-Akt levels and in ps166-MDM2, the phosphorylated-MDM2 form that enters the nucleus and induces p53 export and degradation. Treatment with wortmannin or transfection with a dominant negative form of Akt anticipated at 24 h the effects induced by PTX/LPC, suggesting a protective role against apoptosis played by Akt in Y79 cells. In line with these results, we demonstrated that Y79 cells contain constitutively active Akt, which forms a cytosolic complex with p53 and MDM2 driving p53 degradation. PTX/LPC treatment induced a weakness of Akt-MDM2-p53 complex and increased nuclear p53 levels. Our results suggest that phospho-Akt lowering is at the root of the apoptotic action exerted by PTX/LPC combination and provide strong validation for a treatment approach that targets survival signals represented by phospho-Akt and inhibitor of apoptosis proteins.

Discovery of a transcriptomic core of genes shared in 8 primary retinoblastoma with a novel detection score analysis.

PURPOSE: Expression microarrays are powerful technology that allows large-scale analysis of RNA profiles in a tissue; these platforms include underexploited detection scores outputs. We developed an algorithm using the detection score, to generate a detection profile of shared elements in retinoblastoma as well as to determine its transcriptomic size and structure. METHODS: We analyzed eight briefly cultured primary retinoblastomas with the Human transcriptome array 2.0 (HTA2.0). Transcripts and genes detection scores were determined using the Detection Above Background algorithm (DABG). We used unsupervised and supervised computational tools to analyze detected and undetected elements; WebGestalt was used to explore functions encoded by genes in relevant clusters and performed experimental validation. RESULTS: We found a core cluster with 7,513 genes detected and shared by all samples, 4,321 genes in a cluster that was commonly absent, and 7,681 genes variably detected across the samples accounting for tumor heterogeneity. Relevant pathways identified in the core cluster relate to cell cycle, RNA transport, and DNA replication. We performed a kinome analysis of the core cluster and found 4 potential therapeutic kinase targets. Through analysis of the variably detected genes, we discovered 123 differentially expressed transcripts between bilateral and unilateral cases. CONCLUSIONS: This novel analytical approach allowed determining the retinoblastoma transcriptomic size, a shared active transcriptomic core among the samples, potential therapeutic target kinases shared by all samples, transcripts related to inter tumor heterogeneity, and to determine transcriptomic profiles without the need of control tissues. This approach is useful to analyze other cancer or tissue types.

Regional assignment of genes for human esterase D and retinoblastoma to chromosome band 13q14.

The expression of human esterase D was evaluated quantitatively and qualitatively in five persons with partial deletions or duplications of chromosome 13. The results showed that the locus of this enzyme is at band 13q14. Deletion of this same band in other subjects has been found previously to indicate a predisposition to the development of retinoblastoma, which was present in the four individuals in this study who had partial deletions of chromosome 13. Because of this close synteny, esterase D evaluation should aid in the diagnosis and genetic counseling of retinoblastoma.

MicroRNA-129-5p suppresses proliferation, migration and invasion of retinoblastoma cells through PI3K/AKT signaling pathway by targeting PAX6.

BACKGROUND: Retinoblastoma (RB) is the most common primary intraocular malignancy in children. Accumulating evidences have clarified that microRNAs (miRNAs) modulated signaling molecules by acting as oncogenes or tumor-suppressor genes in RB. Thus, in our study, we aimed to investigate the function of miR-129-5p in RB cells through PI3K/AKT signaling pathway by targeting PAX6. Two RB cell lines, Y79 and WERI-Rb-1, were selected in our study, followed by transfection of miR-129-5p inhibitor or si-PAX6 to explore the regulatory role of miR-129-5p in RB cell proliferation, invasion and migration. MATERIAL AND METHODS: Dual-luciferase assay was used for the detection of targeting relationship between miR-129-5p and PAX6. Besides, western blot analysis was applied to detect expression of cell cycle-related factors (CDK2 and Cyclin E) and PI3K/AKT signaling pathway-related factors (p-AKT and AKT). Nude mice tumorigenesis experiment was used to evaluate the effect of miR-129a-5p on RB growth in vivo. RESULTS: miR-129-5p was down-regulated in RB cell lines. miR-129-5p directly targeted the 3'-untranslated region of PAX6. Artificial down-regulation of miR-129-5p promoted cell proliferation, migration and invasion in RB cell lines Y79 and WERI-Rb-1, and promoted RB growth in vivo via PI3K/AKT signaling pathway, which could be reversed by transfection with silencing PAX6. CONCLUSION: This study provides evidences that RB progression was suppressed by overexpressed miR-129-5p via direct targeting of PAX6 through PI3K/AKT signaling pathway, which may provide a molecular basis for better treatment for RB.

SRPK1: a cisplatin sensitive protein expressed in retinoblastoma.

Chemotherapy is an essential modality in the treatment of retinoblastoma (RB). Mammalian serine/arginine-rich protein-specific kinase 1 (SRPK1) is a cisplatin-sensitivity-related protein and its downregulation is known to be associated with decreased response to cisplatin and carboplatin. We investigated the expression of SRPK1 in 63 archival RB and correlated its expression with pathologic staging and exposure to chemotherapy. The majority of the RB (62/63) were advanced stage (Groups D and E) with intermediate to high risk of treatment failure according to the new international classification for intraocular RB and SRPK1 was reduced in 32/62 (51%) tumors. SRPK1 protein expression was reduced in (100%) 8/8 RB that had recurred in the orbit or had metastasized. SRPK1 protein expression is reduced in RB with advanced stage of presentation and this may add to drug resistance mechanisms in RB.

The down-regulation of microRNA-137 contributes to the up-regulation of retinoblastoma cell proliferation and invasion by regulating COX-2/PGE2 signaling.

MicroRNA-137 (miR-137) plays an important role in the development and progression of many types of human cancers; however, the role of miR-137 in retinoblastoma (RB) remains unclear. In this study, we aimed to investigate the functional significance and molecular mechanisms of miR-137 in RB. We reported that miR-137 was frequently down-regulated in RB tissues and cell lines. The overexpression of miR-137 inhibited RB cell proliferation and invasion, while the suppression of miR-137 promoted RB cell proliferation and invasion. Bioinformatic analysis predicted that cyclooxygenase-2 (COX-2) was a potential target gene of miR-137, which was validated by a dual-luciferase reporter assay. Moreover, our results showed that miR-137 negatively regulated the expression of COX-2 and the production of prostaglandin E2 (PGE2) in RB cells. The knockdown of COX-2 suppressed the proliferation and invasion of RB cells as well as the production of PGE2. The overexpression of COX-2 significantly reversed the inhibitory effect of miR-137 overexpression on RB cell proliferation and invasion. Taken together, these results suggest that miR-137 suppresses the proliferation and invasion of RB cells by targeting COX-2/PGE2. Our study reveals a tumor suppressive role of miR-137 in the progression of RB and suggests miR-137 as a potentially effective therapeutic target for the treatment of RB.

SYK-targeted dendritic cell-mediated cytotoxic T lymphocytes enhance the effect of immunotherapy on retinoblastoma.

PURPOSE: Retinoblastoma (RB) is the most common primary intraocular tumor in children. Chemotherapy is currently the main method of RB treatment. Unfortunately, RB often becomes chemoresistant and turns lethal. Here, we used in vitro cell immunotherapy to explore whether adoptive immunotherapy could be used as a potential treatment for RB. We focused on spleen tyrosine kinase (SYK), which is significantly upregulated in RB cells and serves as a marker for RB cells. METHODS: Using lentiviruses, we genetically modified dendritic cells (DCs) to express and present the SYK peptide antigen to cytotoxic T lymphocytes (CTLs) in vitro. We used SYK-negative cell lines (MDA-MB-231, MCF-10A, and hTERT-RPE1) and SYK-positive cell lines (MCF-7 and RB-Y79) to evaluate the specificity and cytotoxicity of DC presented CTLs using FACS, live-cell imaging, and RNA interference. RESULTS: The cytotoxicity of CTLs induced by SYK-overexpressing DCs (SYK-DC-CTLs) was enhanced more than three times in SYK-positive cell lines compared with SYK-negative cell lines. DCs primed with SYK could drive CTL cytotoxicity against SYK-positive cell lines but not against SYK-negative cell lines. Moreover, SYK-silenced RB-Y79 cells successfully evaded the cytotoxic attack from SYK-DC-CTLs. However, SYK-DC-CTLs could target SYK overexpressed hTERT-RPE1 cells, suggesting that SYK is a specific antigen for RB. Furthermore, SYK-DC-CTL exhibited specific cytotoxicity against carboplatin-resistant RB-Y79 cells in vitro. CONCLUSIONS: Our data showed that SYK could be a potential immunotherapy target mediated by DCs. We propose SYK as a candidate target for treatment of chemoresistant RB.

Anti-tumor activity of arginine deiminase via arginine deprivation in retinoblastoma.

In spite of recent advances in the treatment of retinoblastoma, chemotherapy is still challenging in high-stage intraocular retinoblastoma or metastatic retinoblastoma. Here, we investigated whether arginine deprivation via arginine deiminase (ADI) could be a new anti-tumor therapy in retinoblastoma cells. Expression of argininosuccinate synthetase (ASS) was detected in human retinoblastoma tissues. Even with a high expression of ASS, ADI effectively inhibited the proliferation of retinoblastoma cells and induced retinoblastoma cell death in a dose-dependent manner. These results indicate that arginine deprivation via ADI could be another treatment option for retinoblastoma due to low ASS activity in retinoblastoma cells.

Expression of genes involved in heme biosynthesis in the human retinoblastoma cell lines WERI-Rb-1 and Y79: implications for photodynamic therapy.

Photodynamic therapy (PDT), using protoporphyrin IX (PpIX) as a natural photosensitizer, may be a viable alternative therapy of retinoblastoma. In order to evaluate the potential value of PpIX, the expression profiles of genes involved in heme biosynthesis in human retinoblastoma WERI-Rb-1 and Y79 cells were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Expression levels were highest in protoporphyrinogen oxidase (PPOX), uroporphyrinogen synthase and aminolevulinic acid synthase. Ferrochelatase expression showed a reduction compared to PPOX. PpIX levels were 15- and 18-fold higher in WERI-Rb-1 and Y79 cells, respectively, following induction by delta-aminolevulinic acid. PDT may thus be a promising treatment in vitro, at least in these two retinoblastoma cell lines.