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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 shows the real time quantitative PCR validation of selected genes from microarray results. A: Graph shows real time quantitative PCR analysis showing upregulated genes (dark bars) in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). The fold change of respective genes in microarray was also displayed (gray bars) for comparison. The genes that were upregulated in microarray analysis were also found to be upregulated in quantitative real time PCR analysis. B: Graph shows real time quantitative PCR analysis showing down-regulated genes (dark bars) in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). The fold change of respective genes in microarray was also displayed (gray bars) for comparison. The genes that were down-regulated in microarray analysis were also found to be down-regulated in quantitative real time PCR analysis. The error bars represent the data in triplicates.
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f10: This figure shows the real time quantitative PCR validation of selected genes from microarray results. A: Graph shows real time quantitative PCR analysis showing upregulated genes (dark bars) in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). The fold change of respective genes in microarray was also displayed (gray bars) for comparison. The genes that were upregulated in microarray analysis were also found to be upregulated in quantitative real time PCR analysis. B: Graph shows real time quantitative PCR analysis showing down-regulated genes (dark bars) in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). The fold change of respective genes in microarray was also displayed (gray bars) for comparison. The genes that were down-regulated in microarray analysis were also found to be down-regulated in quantitative real time PCR analysis. The error bars represent the data in triplicates.

Mentions: Seven upregulated genes-erythroblastic leukemia viral oncogene homolog 3(ERBB-3), B-cell leukemia/lymphoma 2 (BCL-2), v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), ATP-binding cassette, sub-family C-member 6 (ABCC6), High mobility group AT-hook 2 (HMGA-2), Matrix metallopeptidase 9 (MMP-9), ATP-binding cassette, sub-family A-member 3 (ABCA-3) and four down-regulated genes- Cyclin-dependent kinase inhibitor 2A (CDKN-2), Cyclin-dependent kinase inhibitor 1A (CDKN-1A), Programmed cell death 2 (PDCD-2) and apoptotic peptidase activating factor 1 (APAF-1)-from the microarray data have been confirmed by real-time quantitative RT–PCR. The results are consistent with the microarray data. The fold changes of all the genes studied was only slightly higher when measured using quantitative RT–PCR compared to the microarray fold changes, reflecting the better dynamic range of quantitative RT–PCR (Figure 10).


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 shows the real time quantitative PCR validation of selected genes from microarray results. A: Graph shows real time quantitative PCR analysis showing upregulated genes (dark bars) in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). The fold change of respective genes in microarray was also displayed (gray bars) for comparison. The genes that were upregulated in microarray analysis were also found to be upregulated in quantitative real time PCR analysis. B: Graph shows real time quantitative PCR analysis showing down-regulated genes (dark bars) in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). The fold change of respective genes in microarray was also displayed (gray bars) for comparison. The genes that were down-regulated in microarray analysis were also found to be down-regulated in quantitative real time PCR analysis. The error bars represent the data in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f10: This figure shows the real time quantitative PCR validation of selected genes from microarray results. A: Graph shows real time quantitative PCR analysis showing upregulated genes (dark bars) in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). The fold change of respective genes in microarray was also displayed (gray bars) for comparison. The genes that were upregulated in microarray analysis were also found to be upregulated in quantitative real time PCR analysis. B: Graph shows real time quantitative PCR analysis showing down-regulated genes (dark bars) in Y79 cells co-cultured with microparticles (3-D) when compared with Y79 cells cultured without microparticles (2-D). The fold change of respective genes in microarray was also displayed (gray bars) for comparison. The genes that were down-regulated in microarray analysis were also found to be down-regulated in quantitative real time PCR analysis. The error bars represent the data in triplicates.
Mentions: Seven upregulated genes-erythroblastic leukemia viral oncogene homolog 3(ERBB-3), B-cell leukemia/lymphoma 2 (BCL-2), v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN), ATP-binding cassette, sub-family C-member 6 (ABCC6), High mobility group AT-hook 2 (HMGA-2), Matrix metallopeptidase 9 (MMP-9), ATP-binding cassette, sub-family A-member 3 (ABCA-3) and four down-regulated genes- Cyclin-dependent kinase inhibitor 2A (CDKN-2), Cyclin-dependent kinase inhibitor 1A (CDKN-1A), Programmed cell death 2 (PDCD-2) and apoptotic peptidase activating factor 1 (APAF-1)-from the microarray data have been confirmed by real-time quantitative RT–PCR. The results are consistent with the microarray data. The fold changes of all the genes studied was only slightly higher when measured using quantitative RT–PCR compared to the microarray fold changes, reflecting the better dynamic range of quantitative RT–PCR (Figure 10).

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