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A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK, Krishnakumar S - Mol. Vis. (2012)

Bottom Line: The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values.The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion.The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

ABSTRACT

Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naïve and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells.

Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA-gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip.

Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome.

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Related in: MedlinePlus

This figure represents microarray heat map displaying deregulated genes and microRNAs in Y79 cells either or not co-cultured with microparticles. A: Heat map shows significantly upregulated and down-regulated genes in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). B: Heat map shows significantly upregulated and down-regulated microRNAs in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). Green color represents upregulated genes or microRNAs and red color represents down-regulated genes or microRNAs. Heat map shows gene IDs or microRNA names and their respective expression fold changes in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured with microparticles (2-D).
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f9: This figure represents microarray heat map displaying deregulated genes and microRNAs in Y79 cells either or not co-cultured with microparticles. A: Heat map shows significantly upregulated and down-regulated genes in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). B: Heat map shows significantly upregulated and down-regulated microRNAs in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). Green color represents upregulated genes or microRNAs and red color represents down-regulated genes or microRNAs. Heat map shows gene IDs or microRNA names and their respective expression fold changes in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured with microparticles (2-D).

Mentions: Results of the cDNA microarray revealed changes in the expression of 4,490 genes (overexpression of 2,490 genes; downregulation of 2,000 genes) in cells grown with PLGA–gelatin microparticles compared to those grown without microparticles (Figure 9A). Only genes with p≤0.05 and a log ratio of at least 2.0 for upregulation (≥1 fold) and a log ratio of 0.5 for downregulation (≤0.5 fold), keeping a median log ratio of 1 in both biologic replicates, were considered for expression analysis [11]. The important upregulated (Table 3 and Table 4) and downregulated (Table 5) genes are provided. We classified the altered transcripts based on their biologic roles. The data also identified several candidate genes that are found to be highly overexpressed in retinoblastoma tumors and that keep tumors in a proliferation state, such as Homo sapiens v-myc myelocytomatosis viral-related oncogene (MYCN) [12,13], H. sapiens jun oncogene (Jun) [11], H. sapiens v-erb-b2 (ERBB3) [14], and H. sapiens insulin-like growth factor binding protein 5 (IGFBP5) [15]. We also found enhanced expression of collagen, integrins, fibronectin, and laminin, which crosslink and stiffen the tissue stroma to promote transformation, tumor growth, motility, and invasion and enhance cancer cell survival [16].


A novel in vitro three-dimensional retinoblastoma model for evaluating chemotherapeutic drugs.

Mitra M, Mohanty C, Harilal A, Maheswari UK, Sahoo SK, Krishnakumar S - Mol. Vis. (2012)

This figure represents microarray heat map displaying deregulated genes and microRNAs in Y79 cells either or not co-cultured with microparticles. A: Heat map shows significantly upregulated and down-regulated genes in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). B: Heat map shows significantly upregulated and down-regulated microRNAs in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). Green color represents upregulated genes or microRNAs and red color represents down-regulated genes or microRNAs. Heat map shows gene IDs or microRNA names and their respective expression fold changes in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured with microparticles (2-D).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369889&req=5

f9: This figure represents microarray heat map displaying deregulated genes and microRNAs in Y79 cells either or not co-cultured with microparticles. A: Heat map shows significantly upregulated and down-regulated genes in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). B: Heat map shows significantly upregulated and down-regulated microRNAs in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). Green color represents upregulated genes or microRNAs and red color represents down-regulated genes or microRNAs. Heat map shows gene IDs or microRNA names and their respective expression fold changes in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured with microparticles (2-D).
Mentions: Results of the cDNA microarray revealed changes in the expression of 4,490 genes (overexpression of 2,490 genes; downregulation of 2,000 genes) in cells grown with PLGA–gelatin microparticles compared to those grown without microparticles (Figure 9A). Only genes with p≤0.05 and a log ratio of at least 2.0 for upregulation (≥1 fold) and a log ratio of 0.5 for downregulation (≤0.5 fold), keeping a median log ratio of 1 in both biologic replicates, were considered for expression analysis [11]. The important upregulated (Table 3 and Table 4) and downregulated (Table 5) genes are provided. We classified the altered transcripts based on their biologic roles. The data also identified several candidate genes that are found to be highly overexpressed in retinoblastoma tumors and that keep tumors in a proliferation state, such as Homo sapiens v-myc myelocytomatosis viral-related oncogene (MYCN) [12,13], H. sapiens jun oncogene (Jun) [11], H. sapiens v-erb-b2 (ERBB3) [14], and H. sapiens insulin-like growth factor binding protein 5 (IGFBP5) [15]. We also found enhanced expression of collagen, integrins, fibronectin, and laminin, which crosslink and stiffen the tissue stroma to promote transformation, tumor growth, motility, and invasion and enhance cancer cell survival [16].

Bottom Line: The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values.The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion.The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu, India.

ABSTRACT

Purpose: Novel strategies are being applied for creating better in vitro models that simulate in vivo conditions for testing the efficacy of anticancer drugs. In the present study we developed surface-engineered, large and porous, biodegradable, polymeric microparticles as a scaffold for three dimensional (3-D) growth of a Y79 retinoblastoma (RB) cell line. We evaluated the effect of three anticancer drugs in naïve and nanoparticle-loaded forms on a 3-D versus a two-dimensional (2-D) model. We also studied the influence of microparticles on extracellular matrix (ECM) synthesis and whole genome miRNA-gene expression profiling to identify 3D-responsive genes that are implicated in oncogenesis in RB cells.

Methods: Poly(D,L)-lactide-co-glycolide (PLGA) microparticles were prepared by the solvent evaporation method. RB cell line Y79 was grown alone or with PLGA-gelatin microparticles. Antiproliferative activity, drug diffusion, and cellular uptake were studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole (MTT) assay, fluorescent microscope, and flow cytometry. Extra cellular matrix (ECM) synthesis was observed by collagenase assay and whole genome miRNA-microarray profiling by using an Agilent chip.

Results: With optimized composition of microparticles and cell culture conditions, an eightfold increase from the seeding density was achieved in 5 days of culture. The antiproliferative effect of the drugs in the 3-D model was significantly lower than in the 2-D suspension, which was evident from the 4.5 to 21.8 fold differences in their IC(50) values. Using doxorubicin, the flow cytometry data demonstrated a 4.4 fold lower drug accumulation in the cells grown in the 3-D model at 4 h. The collagen content of the cells grown in the 3-D model was 2.3 fold greater than that of the cells grown in the 2-D model, suggesting greater synthesis of the extracellular matrix in the 3-D model as the extracellular matrix acted as a barrier to drug diffusion. The microarray and miRNA analysis showed changes in several genes and miRNA expression in cells grown in the 3-D model, which could also influence the environment and drug effects.

Conclusions: Our 3-D retinoblastoma model could be used in developing effective drugs based on a better understanding of the role of chemical, biologic, and physical parameters in the process of drug diffusion through the tumor mass, drug retention, and therapeutic outcome.

Show MeSH
Related in: MedlinePlus