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Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines.

ABSTRACT: Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans Brevican, Neurocan, and Versican in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins ?1-Laminin, Fibronectin, Tenascin-C, and Tenascin-R as well as Collagen IV, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases MMP2, MMP7, MMP9, the tissue-inhibitor of metalloproteinase TIMP2, the Integrin receptor subunits ITGA4, ITGA5 and ITGB1, and all receptor protein tyrosine phosphatase ?/? isoforms. Downregulation of Brevican, Collagen IV, Tenascin-R, MMP2, TIMP2, and ITGA5 was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB.

SUBMITTER: Reinhard J

PROVIDER: S-EPMC7352646 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines.

Reinhard Jacqueline J Wagner Natalie N Krämer Miriam M MM Jarocki Marvin M Joachim Stephanie C SC Dick H Burkhard HB Faissner Andreas A Kakkassery Vinodh V

International journal of molecular sciences 20200617 12

Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 an ...[more]

PMID: 32560557

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Project description:BackgroundRetinoblastoma (RB) is the most frequent pediatric retinal tumor. In the present study, to elucidate chemoresistance mechanisms and identify potential biomarkers in RB, we utilized RNA sequencing (RNAseq) technological platforms to reveal transcriptome profiles and identify any differentially expressed genes (DEGs) between an etoposide drug-resistant subline (Y79/EDR) and parental Y79 cells.MethodsTo test whether Y79/EDR cells showed resistance to antineoplastic agents for RB, we treated the cells with etoposide, carboplatin and vincristine and analyzed them with a Cell Counting Kit-8 (CCK-8). Y79/EDR and parental Y79 cells were used for RNAseq and bioinformatics analysis to enable a genome-wide review of DEGs between the two lines using the DESeq R package (1.10.1). Then, DEG enrichment in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was analyzed with KOBAS software. Next, real-time quantitative reverse transcription polymerase chain reaction (real time QRT-PCR) and cytotoxicity assays were performed to experimentally and functionally validate the identified candidate biomarkers.ResultsY79/EDR cells showed resistance to etoposide, carboplatin and vincristine at different concentrations. In total, 524 transcripts were differentially expressed in Y79/EDR cells based on analysis of fragments per kilobase of transcript per million fragments mapped (FPKM); among these, 57 genes were downregulated and 467 genes were upregulated in Y79/EDR cells compared to parental Y79 cells. We selected candidate DEGs, including ARHGAP9, HIST1H4H, RELN, DDIT4, HK2, STC1 and PFKFB4, for mRNA expression validation with real time QRT-PCR assays and found that the expression levels determined by real time QRT-PCR were consistent with the RNAseq data. Further studies involving downregulation of ARHGAP9 with a specific siRNA showed that ARHGAP9 altered the cellular sensitivity of Y79 cells to etoposide and carboplatin.ConclusionOur initial findings provided a genomic view of the transcription profiles of etoposide-induced acquired resistance in RB. Follow-up studies indicated that ARHGAP9 might be a chemoresistance biomarker in RB, providing insight into potential therapeutic targets for overcoming acquired chemoresistance in RB. These findings can aid in understanding and overcoming chemoresistance during treatment of RB in the clinic.

Project description:There are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca2+ levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca2+ homeostasis. Accordingly, we determined if Asc induced intracellular Ca2+ influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca2+ imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca2+, 1 mM Asc induced larger intracellular Ca2+ transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La3+, 10 mM NAC, or 100 µM 2-APB, these Ca2+ transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca2+ influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca2+ influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.

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Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines.

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Expression Changes and Impact of the Extracellular Matrix on Etoposide Resistant Human Retinoblastoma Cell Lines.

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