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1.
OMICS ; 25(9): 545-566, 2021 09.
Article in English | MEDLINE | ID: mdl-34448651

ABSTRACT

MicroRNAs (miRNAs) that are mutually modulated by their interacting partners (interactome) are being increasingly noted for their significant role in pathogenesis and treatment of various human cancers. Recently, miRNA interactome dissected with multiomics approaches has been the subject of focus since individual tools or methods failed to provide the necessary comprehensive clues on the complete interactome. Even though single-omics technologies such as proteomics can uncover part of the interactome, the biological and clinical understanding still remain incomplete. In this study, we present an expert review of studies involving multiomics approaches to identification of miRNA interactome and its application in mechanistic characterization, classification, and therapeutic target identification in a variety of cancers, and with a focus on proteomics. We also discuss individual or multiple miRNA-based interactome identification in various pathological conditions of relevance to clinical medicine. Various new single-omics methods that can be integrated into multiomics cancer research and the computational approaches to analyze and predict miRNA interactome are also highlighted in this review. In all, we contextulize the power of multiomics approaches and the importance of the miRNA interactome to achieve the vision and practice of predictive, preventive, and personalized medicine in cancer research and clinical oncology.


Subject(s)
MicroRNAs , Neoplasms , Humans , MicroRNAs/genetics , Neoplasms/genetics , Precision Medicine , Proteomics
2.
Bioorg Med Chem Lett ; 30(22): 127594, 2020 11 15.
Article in English | MEDLINE | ID: mdl-33010449

ABSTRACT

New cisplatin analogs in which the diamminedichloro-Pt(II) unit is conjugated to dihydroquinoline- or tetrahydroquinoline frameworks were synthesized and subjected to biological evaluation in order to understand their effects on cellular redox homeostasis and cell viability. They exhibited better selectivity towards cancer cells (A549) compared to mice fibroblast NIH3T3 cells, with cytotoxicity in the same range as that of cisplatin. There was structure-dependent variation in the levels of ROS and were also able to induce cell death, as evidenced by accumulation of cells in sub-G1 phase.


Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Heterocyclic Compounds/pharmacology , Quinolines/pharmacology , A549 Cells , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cell Survival/drug effects , Cisplatin/chemical synthesis , Cisplatin/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Heterocyclic Compounds/chemical synthesis , Heterocyclic Compounds/chemistry , Humans , Mice , Molecular Structure , NIH 3T3 Cells , Oxidation-Reduction , Quinolines/chemistry , Structure-Activity Relationship
3.
Chem Biol Interact ; 252: 141-9, 2016 May 25.
Article in English | MEDLINE | ID: mdl-27062892

ABSTRACT

BACKGROUND: Retinoblastoma (RB) is a childhood retinal malignancy. Effective therapeutic strategies are still being investigated in RB disease management. Here, the anti-cancer effect of shepherdin, a peptido-mimetic inhibiting heat shock protein (HSP90)-Survivin interaction has been analyzed. METHODS: We analyzed HSP (HSP70/90) and Survivin protein expressions by immunohistochemistry (29 archival tumors), qRT-PCR, FACS and Western analysis (10 un-fixed RB tumors). We also analyzed cellular cytotoxicity and anti-proliferative effect in peptide treated RB cells (Y79, Weri Rb1) and MIO-M1 cells. RESULTS: Heterogeneous expressions of HSP70/90 and Survivin with a significant association between HSP70 and HSP90 (r(2) = 0.59, p = 0.001) was observed. In RB cells, anti-tumor effects were detected with 0.42 µg/ml of shepherdin at 4 h s of serum starvation. Decreased Survivin, Bcl2, MMP-2 activity with increased Bax, Bim, and Caspase-9 protein expressions were noticed. No significant changes were observed in shepherdin treated non-neoplastic MIO-M1, nor in scramble-peptide treated RB cells. CONCLUSION: The presence of HSPs (HSP70/90) and Survivin reveals multiple cellular mechanisms adopted by RB cells during cancer progression. Serum starvation induced HSP90 whose interactions with Survivin were specifically inhibited by shepherdin. The associated molecular shuffling has been reported. These findings strongly implicate the potential of targeting HSP90-Survivin interaction as an adjuvant therapy in RB management.


Subject(s)
Antineoplastic Agents/pharmacology , HSP90 Heat-Shock Proteins/metabolism , Peptide Fragments/pharmacology , Retina/drug effects , Retinal Neoplasms/drug therapy , Retinoblastoma/drug therapy , Apoptosis/drug effects , Cell Line, Tumor , Cell Survival/drug effects , HSP70 Heat-Shock Proteins/metabolism , Humans , Matrix Metalloproteinase 2/metabolism , Retina/metabolism , Retina/pathology , Retinal Neoplasms/metabolism , Retinal Neoplasms/pathology , Retinoblastoma/metabolism , Retinoblastoma/pathology
4.
PLoS One ; 11(1): e0146128, 2016.
Article in English | MEDLINE | ID: mdl-26812476

ABSTRACT

PURPOSE: To correlate the differentially expressed miRNAs with clinico-pathological features in uveal melanoma (UM) tumors harbouring chromosomal 3 aberrations among South Asian Indian cohort. METHODS: Based on chromosomal 3 aberration, UM (n = 86) were grouped into monosomy 3 (M3; n = 51) and disomy 3 (D3; n = 35) by chromogenic in-situ hybridisation (CISH). The clinico-pathological features were recorded. miRNA profiling was performed in formalin fixed paraffin embedded (FFPE) UM samples (n = 6) using Agilent, Human miRNA microarray, 8x15KV3 arrays. The association between miRNAs and clinico-pathological features were studied using univariate and multivariate analysis. miRNA-gene targets were predicted using Target-scan and MiRanda database. Significantly dys-regulated miRNAs were validated in FFPE UM (n = 86) and mRNAs were validated in frozen UM (n = 10) by qRT-PCR. Metastasis free-survival and miRNA expressions were analysed by Kaplen-Meier analysis in UM tissues (n = 52). RESULTS: Unsupervised analysis revealed 585 differentially expressed miRNAs while supervised analysis demonstrated 82 miRNAs (FDR; Q = 0.0). Differential expression of 8 miRNAs: miR-214, miR-149*, miR-143, miR-146b, miR-199a, let7b, miR-1238 and miR-134 were studied. Gene target prediction revealed SMAD4, WISP1, HIPK1, HDAC8 and C-KIT as the post-transcriptional regulators of miR-146b, miR-199a, miR-1238 and miR-134. Five miRNAs (miR-214, miR146b, miR-143, miR-199a and miR-134) were found to be differentially expressed in M3/ D3 UM tumors. In UM patients with liver metastasis, miR-149* and miR-134 expressions were strongly correlated. CONCLUSION: UM can be stratified using miRNAs from FFPE sections. miRNAs predicting liver metastasis and survival have been identified. Mechanistic linkage of de-regulated miRNA/mRNA expressions provide new insights on their role in UM progression and aggressiveness.


Subject(s)
Chromosome Disorders/genetics , Chromosomes, Human, Pair 3 , Melanoma/pathology , MicroRNAs/metabolism , Uveal Neoplasms/pathology , Adolescent , Adult , Aged , Child, Preschool , Chromosome Disorders/complications , Chromosome Disorders/mortality , Cohort Studies , Female , Follow-Up Studies , Gene Expression Profiling , Humans , In Situ Hybridization , India , Liver Neoplasms/pathology , Liver Neoplasms/secondary , Male , Melanoma/complications , Melanoma/mortality , Middle Aged , Monosomy , Oligonucleotide Array Sequence Analysis , Paraffin Embedding , RNA, Messenger/metabolism , Uveal Neoplasms/complications , Uveal Neoplasms/mortality , Young Adult
5.
Bioinform Biol Insights ; 9: 89-101, 2015.
Article in English | MEDLINE | ID: mdl-25983556

ABSTRACT

PURPOSE: Retinoblastoma (RB), a primary pediatric intraocular tumor, arises from primitive retinal layers. Several novel molecular strategies are being developed for the clinical management of RB. miRNAs are known to regulate cancer-relevant biological processes. Here, the role of selected miRNAs, namely, miR-532-5p and miR-486-3p, has been analyzed for potential therapeutic targeting in RB. METHODS: A comprehensive bioinformatic analysis was performed to predict the posttranscriptional regulators (miRNAs) of the select panel of genes [Group 1: oncogenes (HMGA2, MYCN, SYK, FASN); Group 2: cancer stem cell markers (TACSTD, ABCG2, CD133, CD44, CD24) and Group 3: cell cycle regulatory proteins (p53, MDM2)] using Microcosm, DIANALAB, miRBase v 18, and REFSEQ database, and RNA hybrid. The expressions of five miRNAs, namely, miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p, were analyzed by qRT-PCR on primary RB tumor samples (n = 30; including 17 invasive RB tumors and 13 noninvasive RB tumors). Detailed complementary alignment between 5' seed sequence of differentially expressed miRNAs and the sequence of target genes was determined. Based on minimum energy level and piCTAR scores, the gene targets were selected. Functional roles of these miRNA clusters were studied by using mimics in cultured RB (Y79, Weri Rb-1) cells in vitro. The gene targets (SYK and FASN) of the studied miRNAs were confirmed by qRT-PCR and western blot analysis. Cell proliferation and apoptotic studies were performed. RESULTS: Nearly 1948 miRNAs were identified in the in silico analysis, From this list, only 9 upregulated miRNAs (miR-146b-5p, miR-305, miR-663b, miR-299, miR-532-5p, miR-892b, miR-501, miR-142-5p, and miR-513b) and 10 downregulated miRNAs (miR-1254, miR-328, miR-133a, miR-1287, miR-1299, miR-375, miR-486-3p, miR-720, miR-98, and miR-122*) were found to be common with the RB serum miRNA profile. Downregulation of five miRNAs (miR-146b-5p, miR-532-5p, miR-142-5p, miR-328, and miR-486-3p) was confirmed experimentally. Predicted common oncogene targets (SYK and FASN) of miR-486-3p and miR-532-5p were evaluated for their mRNA and protein expression in these miRNA mimic-treated RB cells. Experimental overexpression of these miRNAs mediated apoptotic cell death without significantly altering the cell cycle in RB cells. CONCLUSION: Key miRNAs in RB pathogenesis were identified by an in silico approach. Downregulation of miR-486-3p and miR-532-5p in primary retinoblastoma tissues implicates their role in tumorigenesis. Prognostic and therapeutic potential of these miRNA was established by the miRNA mimic strategy.

6.
Bioinform Biol Insights ; 8: 177-91, 2014.
Article in English | MEDLINE | ID: mdl-25232279

ABSTRACT

Retinoblastoma (RB) is a primary childhood eye cancer. HMGA2 shows promise as a molecule for targeted therapy. The involvement of miRNAs in genome-level molecular dys-regulation in HMGA2-silenced RB cells is poorly understood. Through miRNA expression microarray profiling, and an integrated array analysis of the HMGA2-silenced RB cells, the dysregulated miRNAs and the miRNA-target relationships were modelled. Loop network analysis revealed a regulatory association between the transcription factor (SOX5) and the deregulated miRNAs (miR-29a, miR-9*, miR-9-3). Silencing of HMGA2 deregulated the vital oncomirs (miR-7, miR-331, miR-26a, miR-221, miR-17~92 and miR-106b∼25) in RB cells. From this list, the role of the miR-106b∼25 cluster was examined further for its expression in primary RB tumor tissues (n = 20). The regulatory targets of miR-106b∼25 cluster namely p21 (cyclin-dependent kinase inhibitor) and BIM (pro-apoptotic gene) were elevated, and apoptotic cell death was observed, in RB tumor cells treated with the specific antagomirs of the miR-106b∼25 cluster. Thus, suppression of miR-106b∼25 cluster controls RB tumor growth. Taken together, HMGA2 mediated anti-tumor effect present in RB is, in part, mediated through the miR-106b∼25 cluster.

7.
Pediatr Blood Cancer ; 60(10): 1598-601, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23804414

ABSTRACT

PURPOSE: Retinoblastoma (RB) is a malignant tumor of infancy and childhood. Unfavorable therapeutic response is still a quest in many tumors, including retinoblastoma. Hypoxic tumor microenvironment is one of the factors that determine the therapeutic response in many tumors. The purpose of this study was to determine the presence of hypoxia and its related proteins; Hypoxia inducible factor-1α (HIF-1α), Carbonic anhydrase IX (CA IX) and survivin in RB and their association with clinicopathological features. MATERIALS AND METHODS: We evaluated the expression of HIF-1α and survivin by immunohistochemistry in 42 archival retinoblastoma tumors and CA IX; a hypoxia marker in 33 tumors in the same cohort. The expression was correlated with tumor groups based on invasion, differentiation and IIRC. RESULTS: Expression of HIF-1α, survivin and CA IX was observed in 83% (35/42), 86% (36/42), and 93% (31/33) of tumors respectively. We observed no significance between HIF-1α and CA IX expression in tumors with invasion, differentiation and in IIRC tumor groups. An increased survivin expression was observed in group E tumors than in group D tumors (P = 0.044). A significant association was observed between HIF-1α and survivin in differentiated (r = -0.582; P = < 0.01) and undifferentiated tumors groups (r = 0.513; P = <0.012). A similar significant association was observed between HIF-1α and CA IX in tumors with high immunoreactivity for HIF-1α (r = 0.833; P = <0.01). CONCLUSION: Based on these observations, we propose that HIF-1α pathway is deregulated in RB. The role of drug resistance and the potential of targeting HIF-1α, CA IX, and survivin in RB should further examined.


Subject(s)
Antigens, Neoplasm/biosynthesis , Biomarkers, Tumor/biosynthesis , Carbonic Anhydrases/biosynthesis , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Inhibitor of Apoptosis Proteins/biosynthesis , Neoplasm Proteins/biosynthesis , Retinoblastoma/metabolism , Tumor Microenvironment , Antigens, Neoplasm/genetics , Carbonic Anhydrase IX , Carbonic Anhydrases/genetics , Cell Hypoxia , Child, Preschool , Female , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Inhibitor of Apoptosis Proteins/genetics , Male , Neoplasm Invasiveness , Neoplasm Proteins/genetics , Retinoblastoma/genetics , Retinoblastoma/pathology , Retinoblastoma/therapy , Survivin
8.
Bioinform Biol Insights ; 7: 21-34, 2013.
Article in English | MEDLINE | ID: mdl-23400111

ABSTRACT

Retinoblastoma (RB) is a malignant tumor of the retina seen in children, and potential non invasive biomarkers are in need for rapid diagnosis and for prognosticating the therapy. This study was undertaken to identify the differentially expressed miRNAs in the serum of children with RB in comparison with the normal age matched serum, to analyze its concurrence with the existing RB tumor miRNA profile, to identify its novel gene targets specific to RB, and to study the expression of a few of the identified oncogenic miRNAs in the advanced stage primary RB patient's serum sample. MiRNA profiling was performed on 14 pooled serum from children with advanced RB and 14 normal age matched serum samples, wherein 21 miRNAs were found to be upregulated (fold change ≤ -2.0, P ≤ 0.05) and 24 to be downregulated (fold change ≥ +2.0, P ≤ 0.05). Furthermore, intersection of 59 significantly deregulated miRNAs identified from RB tumor profiles with that of miRNAs detected in serum profile revealed that 33 miRNAs had followed a similar deregulation pattern in RB serum. Later we validated a few of the miRNAs (miRNA 17-92) identified by microarray in the RB patient serum samples (n = 20) by using qRT-PCR. Expression of the oncogenic miRNAs, miR-17, miR-18a, and miR-20a by qRT-PCR was significant in the serum samples exploring the potential of serum miRNAs identification as noninvasive diagnosis. Moreover, from miRNA gene target prediction, key regulatory genes of cell proliferation, apoptosis, and positive and negative regulatory networks involved in RB progression were identified in the gene expression profile of RB tumors. Therefore, these identified miRNAs and their corresponding target genes could give insights on potential biomarkers and key events involved in the RB pathway.

9.
Mol Vis ; 18: 2420-37, 2012.
Article in English | MEDLINE | ID: mdl-23077401

ABSTRACT

AIM: To explore the molecular mechanisms deregulated by high mobility group protein A2 (HMGA2) gene silencing in retinoblastoma (RB) cells. METHODS: Synthetic anti-HMGA2 short interfering RNA (siRNA) was used to silence the HMGA2 gene in cultured Y79 RB cells that were subjected to whole genome microarray analysis. The expression of differentially regulated key genes was confirmed with quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) in post-silenced RB cell lines (Y79 and WERI Rb1). These deregulated genes were compared for their constitutive expression in primary RB tumors (n=10). Zymographic determination of matrix metalloproteinase (MMP) activity was performed in RB cells. A cell cycle assay and a proliferation assay were performed in post-transfected RB cells. RESULTS: HMGA2 gene silencing in cultured RB cells results in reduced cell proliferation and transition in the G1/S phase. The whole genome microarray analysis of HMGA2 silenced Y79 cells revealed overall upregulation of 1,132 genes (≥ 1.0 fold) and downregulation of 1,562 genes (≤ -1.0 fold). Specific quantitative pathway analysis of the deregulated genes (using Biointerpreter) revealed 150 upregulated genes and 77 downregulated genes (≥ 1.0 fold) involved in vital pathways, namely, mitogen-activated protein kinase, Janus kinase/signal transducers and activators of transcription, Ras pathway, Ras-induced extracellular signal-regulated protein kinases 1 and 2, and tumor protein p53. The differential expression of genes obtained from microarray analysis (Homo sapiens ELK1, member of ETS oncogene family [ELK1], Homo sapiens cyclin-dependent kinase 6 [CDK6], Homo sapiens E2F transcription factor 4, p107/p130-binding [E2F4], Homo sapiens G-2 and S-phase expressed 1 [GTSE1], Damage-regulated autophagy modulator [DRAM], Homo sapiens cadherin 1, type 1,E-cadherin (epithelial) [CDH1], Homo sapiens snail homolog 1 (Drosophila) [SNAI1], Homo sapiens matrix metallopeptidase 2 [MMP2], and Homo sapiens matrix metallopeptidase 9 [MMP9]) was confirmed with quantitative reverse-transcriptase polymerase chain reaction in post-silenced RB cells. Zymographic analysis revealed that the increase in MMP mRNA expression in the post-silenced RB cells did not correlate with corresponding enzyme activity. CONCLUSIONS: Our study revealed molecular regulatory changes induced by HMGA2 silencing in RB cancer cells, offering mechanistic insights into the anticancer potential. HMGA2 may be considered a promising candidate for gene silencing therapy in RB.


Subject(s)
Eye Proteins/genetics , HMGA2 Protein/antagonists & inhibitors , HMGA2 Protein/genetics , Neoplasm Proteins/genetics , RNA, Small Interfering/genetics , Retinoblastoma/genetics , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Matrix Metalloproteinases/genetics , Molecular Targeted Therapy , Oligonucleotide Array Sequence Analysis , Retinoblastoma/metabolism , Retinoblastoma/pathology , Signal Transduction/genetics
10.
J Pediatr Hematol Oncol ; 31(3): 209-14, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19262251

ABSTRACT

Retinoblastoma (RB) is the commonest primary intraocular tumor in children. Overexpression of the high mobility group (HMG) A2 protein has been observed in a variety of malignant tumors and often correlates with poor prognosis. We studied the expression of HMGA2 in primary tumor samples and correlated with clinicopathologic features such as invasion, differentiation, and laterality of the tumors. Among 64 tumors, there were 29 tumors with invasion of the optic nerve, choroid, and/or orbit and 35 tumors without invasion. HMGA2 immunoreactivity was evaluated on archival paraffin sections and the results confirmed by Western blotting on 12 fresh tumor samples. Among 29 tumors with invasion, HMGA2 was strongly positive (++) in 10 tumors, moderately positive (+) in 11 tumors. Among 35 tumors without invasion, HMGA2 was strongly positive (++) in 6 tumors, moderately positive (+) in 6 tumors. Tumors with invasion showed significantly higher expression of HMGA2 compared with tumors without invasion (P<0.01). Non-neoplastic retina was negative for HMGA2. There was no correlation between HMGA2 expression and differentiation/laterality. Western blotting revealed that 7 tumors were strongly positive, 2 were moderately positive, and 1 was faintly positive for HMGA2. Our study has demonstrated the HMGA2 expression in a large cohort of primary retinoblastoma tumors and its correlation with invasiveness.


Subject(s)
HMGA2 Protein/biosynthesis , Retinal Neoplasms/metabolism , Retinal Neoplasms/pathology , Retinoblastoma/metabolism , Retinoblastoma/pathology , Biomarkers, Tumor/analysis , Blotting, Western , Child, Preschool , Female , Humans , Immunohistochemistry , Infant , Male , Neoplasm Invasiveness , Prognosis
11.
J Pediatr Hematol Oncol ; 31(1): 8-13, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19125079

ABSTRACT

Recent studies have shown the presence of human papillomavirus (HPV) genome in retinoblastoma (RB) tumor samples. There is no information on the HPV status in the RB tumors of Indian patients. We studied the presence of HPV genome in RB tumor samples from patients with unilateral tumor. Forty-four fresh RB tumor samples and 30 non-neoplastic donor retinas were analyzed for the presence of HPV 16 and 18 genome by nested and seminested polymerase chain reaction. Tumor tissue sections were also used to assess the expression of the retinoblastoma (Rb) protein. All 30 control tissues were negative for HPV genome. Among the 44 tumor samples, there were 23 tumors with invasion of optic nerve/choroid and 21 tumors with no invasion. HPV DNA was present in 21/44 (47%) RB tumors. Among 21 unilateral RB tumors that were positive for HPV DNA, HPV 16 was detected in 12/21 (57%) tumors. However, HPV 18 was negative in all the tumors. Rb protein was absent in 16 (71%) of 21 tumors that had HPV DNA. However, Rb was also absent in 20 (86%) of 23 tumors that were HPV negative. Children younger than 18 months old were significantly associated with the presence of HPV DNA compared with children above 24 months old (P<0.014). Our study shows the presence of HPV and HPV 16 in a subset of RB tumor samples. However, further studies are in progress to know the role played by HPV in RB.


Subject(s)
DNA, Viral/analysis , Eye Infections, Viral/virology , Human papillomavirus 16/genetics , Human papillomavirus 18/genetics , Papillomavirus Infections/virology , Retinal Neoplasms/virology , Retinoblastoma/virology , Child , Child, Preschool , Eye Infections, Viral/diagnosis , Female , Humans , Immunoenzyme Techniques , Infant , Male , Papillomavirus Infections/diagnosis , Polymerase Chain Reaction , Retinal Neoplasms/diagnosis , Retinoblastoma/diagnosis , Retinoblastoma Protein/metabolism , Sensitivity and Specificity
12.
Pediatr Blood Cancer ; 50(2): 402-6, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17091485

ABSTRACT

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.


Subject(s)
Antineoplastic Agents/pharmacology , Cisplatin/pharmacology , Protein Serine-Threonine Kinases/biosynthesis , Retinal Neoplasms/enzymology , Retinoblastoma/enzymology , Adolescent , Child , Child, Preschool , Female , Humans , Immunohistochemistry , Infant , Male , Neoplasm Staging , Retinal Neoplasms/drug therapy , Retinal Neoplasms/pathology , Retinoblastoma/drug therapy , Retinoblastoma/pathology
13.
Exp Eye Res ; 83(6): 1446-52, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17027002

ABSTRACT

The Rho GTPases are the molecular regulators of the cell motility processes and are involved in cell cycle progression and gene transcription. We studied the expression of Rho-like GTPases molecules, particularly Rac, Tiam1 and cdc42, in retinoblastoma and correlated these with clinicopathological parameters of the tumors. Sixty-seven tumors were included which were divided in to two groups; group A: tumors with optic nerve/choroidal/orbital invasion (n=35) and group B: tumors with no invasion (n=32). Immunohistochemistry was done on paraffin sections for all the proteins and were confirmed by Western blot on fresh tumor samples. In group A tumors, Rac was positive in 10/35 (28%), cdc42 was positive in 12/35 (34%) and Tiam1 was positive in 30/35 (85%) tumors. In group 2 tumors, Rac was positive in 5/32 (15%), cdc42 was positive in 4/32 (12%) and Tiam1 was positive in 30/32 (93%) tumors. Two groups (both invasive and non-invasive tumors) showed decreased expression of Rac1 and cdc42 whereas Tiam1 was significantly expressed in invasive tumors compared to non-invasive tumors (P<0.0001). We observed a 70K cleavage product of Tiam1 along with an 110K product by blotting in RB samples. Caspase-3 was also demonstrated in RB samples, which showed Tiam1 cleavage products. This is the first study that showed the expression patterns of Rac, cdc42 and Tiam1 in retinoblastoma tumors. Thus, further studies are required to prove the involvement of caspase-3 in the cleavage of Tiam1 in vitro in RB cells and to trace out alternative pathways involved in tumor progression.


Subject(s)
Eye Proteins/metabolism , Neoplasm Proteins/metabolism , Retinal Neoplasms/metabolism , Retinoblastoma/metabolism , Adolescent , Blotting, Western/methods , Caspase 3/metabolism , Cell Differentiation , Child , Child, Preschool , Female , Guanine Nucleotide Exchange Factors/metabolism , Humans , Infant , Male , Neoplasm Invasiveness , Retinal Neoplasms/pathology , Retinoblastoma/pathology , T-Lymphoma Invasion and Metastasis-inducing Protein 1 , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism
14.
Ocul Immunol Inflamm ; 14(4): 207-13, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16911982

ABSTRACT

PURPOSE: Human leukocyte antigen (HLA) mediates interactions of tumor cells with cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Retinoblastoma (RB) is the most common intraocular malignant tumor in childhood and is characterized by direct spread to the optic nerve and orbit as well as hematogenous and lymphatic spread. Earlier, we observed that invasive RB showed reduced HLA, which could contribute to its escape from the immune system. Little is known about the role of the nonclassical HLA molecule, HLA-G, in RB and its role in tumor escape mechanisms in RB. METHODS: Forty archival paraffin-embedded RB tumors were analyzed for the expression of HLA-G by immunohistochemistry using a monoclonal antibody; fresh tumor samples were also subjected to Western blot analysis. There were 22 tumors with no invasion and 18 with invasion of the choroid/optic nerve. Immunoanalysis was performed based on the International Histocompatibility Working Group Project Description. RESULTS: HLA-G was negative in the non-neoplastic retina, reduced in 22/22 tumors with no invasion, and positive in 15/18 with invasion. The immunohistochemistry results were confirmed by Western blot analysis. The difference in expression between the two groups was significant ( p < 0.001). There was no correlation of HLA-G expression with differentiation of the tumors. CONCLUSION: Increased expression of HLA-G was observed in invasive RB. This preliminary observation deserves further investigation and may shed more light on the immune escape mechanisms of this tumor and thus enable novel therapeutic strategies.


Subject(s)
HLA Antigens/metabolism , Histocompatibility Antigens Class I/metabolism , Retinal Neoplasms/metabolism , Retinoblastoma/metabolism , Antibodies, Monoclonal , Blotting, Western , Child , Child, Preschool , Female , HLA-G Antigens , Humans , Immunoenzyme Techniques , Infant , Male , Neoplasm Invasiveness , Retinal Neoplasms/pathology , Retinoblastoma/pathology
15.
Exp Eye Res ; 83(4): 736-40, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16716300

ABSTRACT

We examine the immunoreactivity of the non-receptor tyrosine kinase, c-Src kinase and its downstream molecule, signal transducer and activator of transcription 3 (pStat3) in retinoblastoma (RB), and correlation with invasiveness and differentiation. Tumor samples from 40 patients with RB were available for the study. There were 18 tumors in group 1 (non-invasive) and 22 tumors in group 2 (invasive). The immunoreactivity of c-Src kinase and pStat3 was compared in the two groups of tumors. Group 1 (non-invasive) RB showed intermediate c-Src kinase immunoreactivity (Allred score 4-5) in 14/18 tumors and low immunoreactivity (Allred score 2-3) in 4/18 tumors. pStat3 was intermediate (Allred score 4-5) in 6/18 tumors and negative (Allred score 0) in 12/18 tumors. Group 2 (invasive) RB showed high c-Src kinase immunoreactivity (Allred score 6-8) in 22/22 tumors and high pStat3 (Allred score 6-8) in 19/22 tumors. The expression of c-Src kinase (P<0.001) and pStat3 (P<0.001) was significantly higher in group 2 RB. Src kinase expression (P<0.05) and pStat3 expression (P<0.05) was higher in the poorly differentiated tumors compared to moderately- and well-differentiated tumors. The increased expression of c-Src kinase and pStat3 expression could play a role in the invasiveness of group 2 tumors. Further characterization of the pathways involved in the pathogenesis of RB will shed light on fundamental mechanisms of tumorigenesis.


Subject(s)
Biomarkers, Tumor/metabolism , Retinal Neoplasms/metabolism , Retinoblastoma/metabolism , STAT3 Transcription Factor/metabolism , src-Family Kinases/metabolism , Cell Differentiation , Child , Child, Preschool , Female , Humans , Infant , Male , Neoplasm Invasiveness , Neoplasm Proteins/metabolism , Retinal Neoplasms/pathology , Retinoblastoma/pathology
16.
Invest Ophthalmol Vis Sci ; 45(12): 4247-50, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15557427

ABSTRACT

PURPOSE: This study was conducted to investigate the potential of targeting epithelial cell adhesion molecules (EpCAMs) in the treatment of retinoblastoma. It was first determined whether EpCAM is expressed in retinoblastoma and then whether EpCAM reactivity correlates with tumor aggressiveness. METHODS: EpCAM reactivity was evaluated by immunohistochemistry in 43 retinoblastoma specimens from 43 patients, by using the monoclonal antibody GA733.2. The tumors were divided into two groups. There were 20 tumors with no invasion of the choroid and optic nerve (group A) and 23 tumors with invasion of the choroid, optic nerve, and orbit (group B). EpCAM reactivity was correlated with invasion and differentiation of the tumors. RESULTS: Among the 43 tumors, EpCAM reactivity was observed in 100% (43/43) tumors. EpCAM reactivity was significantly higher in the invasive than the noninvasive tumors (P < 0.05) and in poorly differentiated than in well-differentiated tumors (P < 0.005). Non-neoplastic retina also expressed EpCAM. CONCLUSIONS: The results confirm that EpCAM is vastly expressed in retinoblastoma and point to its use as a target for therapy in the future.


Subject(s)
Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/metabolism , Retinal Neoplasms/metabolism , Retinoblastoma/metabolism , Adult , Cell Differentiation , Child , Child, Preschool , Epithelial Cell Adhesion Molecule , Female , Humans , Immunohistochemistry , Infant , Male , Neoplasm Invasiveness , Retina/metabolism , Retinal Neoplasms/pathology , Retinal Neoplasms/therapy , Retinoblastoma/pathology , Retinoblastoma/therapy
17.
Cancer ; 101(7): 1672-6, 2004 Oct 01.
Article in English | MEDLINE | ID: mdl-15378505

ABSTRACT

BACKGROUND: The importance of the Fas-Fas ligand (FasL) mechanism for the immune evasion by tumors provided a strong rationale for the examination of FasL expression in retinoblastoma. In an earlier publication, the authors reported that invasive retinoblastomas decreased Fas expression. Because to the authors' knowledge there is not much information regarding the effect of FasL expression on retinoblastoma, the authors studied the expression of FasL in retinoblastoma and correlated it with invasiveness. METHODS: Thirty-six archival retinoblastoma specimens were divided into 2 groups. Group A (n = 17) was comprised of specimens from tumors with no invasion and Group B (n = 19) was comprised of specimens from tumors with invasion of the choroid (focal, diffuse), optic nerve (laminar, postlaminar, surgical end), and orbit. Sections were immunostained with a monoclonal antibody to FasL and the immunoreactivity was assessed. RESULTS: In Group A, FasL was negative in 100% (17 of 17) of the tumor specimens. In Group B, FasL was expressed in 79% (15 of 19) of the tumor specimens (positive in 9 tumors and heterogeneous in 6 tumors). The difference in FasL expression between the two groups was significant (P < 0.001) CONCLUSIONS: Increased expression of FasL was observed in specimens taken from patients with aggressive tumors. Thus, Loss of Fas and gain of aberrant FasL expression were common features of malignant transformation. The data suggested that the Fas/FasL pathway is potentially immunosuppressive and may be involved in the escape of retinoblastoma cells from immune destruction.


Subject(s)
Membrane Glycoproteins/analysis , Retinal Neoplasms/chemistry , Retinoblastoma/chemistry , Adolescent , Cell Transformation, Neoplastic , Child , Child, Preschool , Fas Ligand Protein , Female , Humans , Immunohistochemistry , Infant , Male , Neoplasm Invasiveness , Retinal Neoplasms/immunology , Retinal Neoplasms/pathology , Retinoblastoma/immunology , Retinoblastoma/pathology
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