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Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

ABSTRACT: RNA interference (RNAi) is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh) RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL) alone (Control) and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i) and EGFP (EGFPi, also served as a control) shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing) and 0.2-3 µm (Control). While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (~2.5% cumulative release) of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ~51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ~40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

SUBMITTER: Achille C

PROVIDER: S-EPMC3527537 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

Achille Clément C Sundaresh Sowmya S Chu Benjamin B Hadjiargyrou Michael M

PloS one 20121220 12

RNA interference (RNAi) is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh) RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolacton ...[more]

PMID: 23285007

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Project description:The determination of potential transplantable substrates and substitution cells for corneal endothelium transplantation may compensate for the shortage of cornea donors. Appropriate biodegradable and biocompatible tissue-engineered substratum with seed cells for endothelial keratoplasty has been increasingly studied. In the present study, electrospun gelatin/polycaprolactone (PCL) and collagen/PCL scaffolds were successfully established. Bone marrow endothelial progenitor cells (BEPCs) were cultured on these scaffolds to determine whether the scaffolds may promote the proliferation of BEPCs as well as maintain stem cell characteristics. Two variations of hybrid scaffolds, collagen/PCL (70% collagen and 30% PCL) and gelatin/PCL (70% gelatin and 30% PCL), were established via electrospinning. Microscopic structure, hydrophilicity and wettability of the two scaffolds were subsequently investigated. BEPCs were separately cultured on the scaffolds and were also seeded on glass slides to establish the control group. Furthermore, cell morphology; adherence, as determined by investigation of F-actin expression levels; proliferation, as determined via Cell Counting Kit-8 assays, Ki-67 staining and bromodeoxyuridine (BrdU) staining; and stem cell markers, as determined by cluster of differentiation (CD)-34 and CD-133 protein expression levels; were investigated. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine gene expression. The two nanofiber scaffolds were established using electrospun techniques with expected hydrophilicity, wettability and biocompatibility. BEPCs were revealed to spread well on and strongly adhere to the collagen/PCL (70:30) and gelatin/PCL (70:30) scaffolds. Furthermore, Ki-67 and BrdU staining results revealed greater levels of positive dots on the two hybrid scaffolds compared with the control group. CD-34 and CD-133 protein staining demonstrated increased levels of fluorescence intensity on scaffolds compared with the control group. Furthermore, increased expression levels of differentiation markers, such as ATP binding cassette subfamily G member 2, leucine rich repeat containing G protein-coupled receptor 5 and CD166, were detected on both scaffolds. RT-qPCR results demonstrated that the expression of caspase-3, which is associated with apoptosis, was decreased on the two scaffolds compared with in the control group. The expression of inflammatory factors, including interleukin (IL)-1, exhibited a significant decrease on the gelatin/PCL scaffold compared with in the control group; whereas the difference between the expression level of IL-1 exhibited by the collagen/PCL group and the control group were not markedly different. Electrospun collagen/PCL and gelatin/PCL scaffolds exhibited the potential to enhance the adherence and proliferation of BEPCs. BEPCs cultured on the two scaffolds demonstrated increased stem cell characteristics and differentiation potential. Electrospun gelatin/PCL and collagen/PCL scaffolds may represent a promising substratum in tissue-engineered corneal endothelium.

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Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

Publications

Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

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