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1.  Peptide Carrier-Mediated Non-Covalent Delivery of Unmodified Cisplatin, Methotrexate and Other Agents via Intravenous Route to the Brain 
PLoS ONE  2014;9(5):e97655.
Background
Rapid pre-clinical evaluation of chemotherapeutic agents against brain cancers and other neurological disorders remains largely unattained due to the presence of the blood-brain barrier (BBB), which limits transport of most therapeutic compounds to the brain. A synthetic peptide carrier, K16ApoE, was previously developed that enabled transport of target proteins to the brain by mimicking a ligand-receptor system. The peptide carrier was found to generate transient BBB permeability, which was utilized for non-covalent delivery of cisplatin, methotrexate and other compounds to the brain.
Approach
Brain delivery of the chemotherapeutics and other agents was achieved either by injecting the carrier peptide and the drugs separately or as a mixture, to the femoral vein. A modification of the method comprised injection of K16ApoE pre-mixed with cetuximab, followed by injection of a ‘small-molecule’ drug.
Principal findings
Seven-of-seven different small molecules were successfully delivered to the brain via K16ApoE. Depending on the method, brain uptake with K16ApoE was 0.72–1.1% for cisplatin and 0.58–0.92% for methotrexate (34-50-fold and 54–92 fold greater for cisplatin and methotrexate, respectively, with K16ApoE than without). Visually intense brain-uptake of Evans Blue, Light Green SF and Crocein scarlet was also achieved. Direct intracranial injection of EB show locally restricted distribution of the dye in the brain, whereas K16ApoE-mediated intravenous injection of EB resulted in the distribution of the dye throughout the brain. Experiments with insulin suggest that ligand-receptor signaling intrinsic to the BBB provides a natural means for passive transport of some molecules across the BBB.
Significance
The results suggest that the carrier peptide can non-covalently transport various chemotherapeutic agents to the brain. Thus, the method offers an avenue for pre-clinical evaluation of various small and large therapeutic molecules against brain tumors and other neurological disorders.
doi:10.1371/journal.pone.0097655
PMCID: PMC4029735  PMID: 24847943
2.  A Carrier for Non-Covalent Delivery of Functional Beta-Galactosidase and Antibodies against Amyloid Plaques and IgM to the Brain 
PLoS ONE  2011;6(12):e28881.
Background
Therapeutic intervention of numerous brain-associated disorders currently remains unrealized due to serious limitations imposed by the blood-brain-barrier (BBB). The BBB generally allows transport of small molecules, typically <600 daltons with high octanol/water partition coefficients, but denies passage to most larger molecules. However, some receptors present on the BBB allow passage of cognate proteins to the brain. Utilizing such receptor-ligand systems, several investigators have developed methods for delivering proteins to the brain, a critical requirement of which involves covalent linking of the target protein to a carrier entity. Such covalent modifications involve extensive preparative and post-preparative chemistry that poses daunting limitations in the context of delivery to any organ. Here, we report creation of a 36-amino acid peptide transporter, which can transport a protein to the brain after routine intravenous injection of the transporter-protein mixture. No covalent linkage of the protein with the transporter is necessary.
Approach
A peptide transporter comprising sixteen lysine residues and 20 amino acids corresponding to the LDLR-binding domain of apolipoprotein E (ApoE) was synthesized. Transport of beta-galactosidase, IgG, IgM, and antibodies against amyloid plques to the brain upon iv injection of the protein-transporter mixture was evaluated through staining for enzyme activity or micro single photon emission tomography (micro-SPECT) or immunostaining. Effect of the transporter on the integrity of the BBB was also investigated.
Principal Findings
The transporter enabled delivery to the mouse brain of functional beta-galactosidase, human IgG and IgM, and two antibodies that labeled brain-associated amyloid beta plaques in a mouse model of Alzheimer's disease.
Significance
The results suggest the transporter is able to transport most or all proteins to the brain without the need for chemically linking the transporter to a protein. Thus, the approach offers an avenue for rapid clinical evaluation of numerous candidate drugs against neurological diseases including cancer. (299 words).
doi:10.1371/journal.pone.0028881
PMCID: PMC3244419  PMID: 22216132
3.  Sleeping Beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high grade astrocytomas 
Cancer research  2010;70(9):3557-3565.
The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using RT-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes, and they imply an important role for CSF1 in the development of these tumors.
doi:10.1158/0008-5472.CAN-09-4674
PMCID: PMC2862088  PMID: 20388773
Astrocytoma; Sleeping Beauty; Mutagenesis; Colony stimulating factor-1
4.  Preferential Expression of the Secreted and Membrane forms of Tumor Endothelial Marker 7 transcripts in Osteosarcoma 
Anticancer research  2009;29(11):4317-4322.
Background
High expression of tumor endothelial marker 7 (TEM7) is correlated with osteogenic sarcoma (OS) metastasis and poor survival of patients. The TEM7 gene produces four alternatively spliced transcripts with distinct functional domains; the expression pattern of these transcripts in OS is unknown.
Materials and Methods
mRNA expression was assessed in 5 OS cell lines, 7 normal bone, and 9 OS tumor specimens by reverse transcriptase polymerase chain reaction.
Results
All OS cell lines, 6/9 tumors but none of the bone specimens expressed mRNA of TEM7 secreted forms 1 and 2. A total of 3/5 OS cell lines, 8/9 of tumors and 4/7 of bone specimens expressed mRNA of the TEM7 intracellular form. One out of 5 cell lines, 2/7 tumors and none of the bone specimens expressed mRNA of the TEM7 membrane form. The secreted forms had 20-fold higher expression in metastatic (LM7) compared to non-metatstatic (SAOS-2) cells.
Conclusion
The mRNA of secreted and the membrane forms of TEM7 are preferentially expressed in OS.
PMCID: PMC2800050  PMID: 20032373
TEM7; alternative splicing; osteosarcoma; PCR; metastasis
5.  Osteoblastic and Osteolytic Human Osteosarcomas can be Studied with a new Xenograft Mouse Model Producing Spontaneous Metastases 
Cancer investigation  2009;27(4):435-442.
There is no animal model that reflects the histological and radiographical heterogeneity of osteosarcoma. We assessed seven osteosarcoma cell lines for their potential to develop orthotopic tumors and lung metastasis in SCID mice. Whereas radiologically, 143B developed osteolytic tumors, SaOS-LM7 developed osteoblastic primary tumors. The mineralization status was confirmed by assessing the alkaline phosphatase activity and the microarray expression profile. We herein report a xenograft orthotopic osteosarcoma mouse model to assess osteoblastic and osteolytic lesions, which may contribute in the search for new diagnostic and therapeutic approaches.
doi:10.1080/07357900802491477
PMCID: PMC2723944  PMID: 19212826
Osteosarcoma; Animal Model; Xenograft; Orthotopic; Lung metastasis
6.  Cellular Uptake of Gold Nanoparticles Directly Cross-linked with Carrier Peptides by Osteosarcoma Cells 
Nanoparticles have been extensively used for a variety of biomedical applications and there is a growing need for highly specific and efficient delivery of the nanoparticles into target cells and subcellular location. We attempted to accomplish this goal by modifying gold particles with peptide motif’s that are known to deliver a ‘cargo’ into chosen cellular location specifically, we intended to deliver nanogold particles into cells through chemical cross-linking with different peptides known to carry protein into cells. Our results suggest that specific sequence of such ‘carrier peptides’ can efficiently deliver gold nanoparticles into cells when chemically cross-linked with the metal particles.
doi:10.1007/s10856-008-3588-x
PMCID: PMC2824438  PMID: 18807262
7.  2-Methoxyestradiol-induced Cell Death in Osteosarcoma Cells is Preceded by Cell Cycle Arrest 
2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17β-Estradiol (E2), induces cell death in osteosarcoma cells. To further understand the molecular mechanisms of action, we have investigated cell cycle progression in 2-ME-treated human osteosarcoma (MG63, SaOS-2 and LM8) cells. At 5 μM, 2-ME induced growth arrest by inducing a block in cell cycle; 2-ME-treatment resulted in 2-fold increases in G1 phase cells and a decrease in S phase cells in MG63 and SaOS-2 osteosarcoma cell lines, compared to the appropriate vehicle controls. 2-ME-treatment induced a 3-fold increase in the G2 phase in LM8 osteosarcoma cells. The results demonstrated steroid specificity, as the tumorigenic metabolite, 16α-hydroxyestradiol (16-OHE), did not have any effect on cell cycle progression in osteosarcoma cells. The cell cycle arrest coincided with an increase in expression of the cell cycle markers p21, p27 and p53 proteins in 2-ME-treated osteosarcoma cells. Also, MG63 cells, transiently transfected with cDNA for a ‘loss of function mutant” RNA-dependent protein kinase (PKR) protein, were resistant to 2-ME-induced cell cycle arrest. These results suggest that 2-ME works in concert with factors regulating cell cycle progression, and cell cycle arrest precedes cell death in 2-ME-treated osteosarcoma cells.
doi:10.1002/jcb.21758
PMCID: PMC2821714  PMID: 18384113
estrogen metabolite; MG63 cells; cell cycle arrest; PKR
8.  High expression of Tumor Endothelial Marker 7 is associated with metastasis and poor survival of patients with osteogenic sarcoma 
Gene  2007;399(2):137-143.
Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metastasis is the primary cause of mortality among patients with OGS. To identify such genes, we first created a database of differentially expressed genes between six low-grade and six high-grade OGS tumors, and between a normal immortalized osteoblast cell line (FOB) and four commercially available OGS-derived cell lines. We specifically searched for surface-proteins over-expressed in high-grade OGS, since we hypothesize that tumor-cell specific surface markers are key to metastasis. A gene encoding Tumor Endothelial Marker7 (TEM7) was selected as a candidate for further study. TEM7 expression pattern was assessed by RT-PCR, Western blotting and immunostaining. TEM7 mRNA was abundantly expressed in SAOS cells (derived from high-grade OGS), but not in FOB or MG63 cells (derived from low-grade OGS). Virtually no expression of TEM7 protein was observed in FOB cells but abundant expression was noted in SAOS and TE85 cells. Employing immunostaining of 92 human OGS specimens (50 high grade and 42 low-grade) collected before chemotherapy show 97% (37 of 38) of high-grade OGS specimens with metastasis have high TEM7 staining. Further, we found that elevated expression of TEM7 correlated with poor survival (p<0.04) of affected patients. Inhibiting TEM7 function by siRNA inhibited invasion and migration of OGS cells with metastatic potential. Our results suggest TEM7 expression level closely parallels histology-based prognostication of OGS metastasis and, therefore, it is a therapeutic target. This is the first demonstration of a link between TEM7 and cancer metastasis.
doi:10.1016/j.gene.2007.05.003
PMCID: PMC2066185  PMID: 17560052
TEM7; Osteogenic sarcoma; Metastasis; siRNA; Tumor marker
9.  TASR-1 Regulates Alternative Splicing of Collagen Genes in Chondrogenic Cells 
During the differentiation of chondroprogenitors into mature chondrocytes, the alternative splicing of collagen genes switches from longer isoforms to shorter ones. To investigate the underlying mechanisms, we infected mouse ATDC5 chondroprogenitor cells with retrovirus for stable expression of two closely related SR splicing factors. RT-PCR analysis revealed that TASR-1, but not TASR-2, influenced alternative splicing of type II and type XI collagens in ATDC5 cells. The effect of TASR-1 on splicing could be reversed with the addition of insulin. Results from our microarray analysis of ATDC5 cells showed that TASR-1 and TASR-2 differentially affect genes involved in the differentiation of chondrocytes. Of special interest is the finding that TASR-1 could down-regulate expression of type X collagen, a hallmark of hypertrophic chondrocytes. Immunohistostaining demonstrated that TASR-1 protein is more abundantly expressed than TASR-2 in mouse articular chondrocytes, raising the possibility that TASR-1 might be involved in phenotype maintenance of articular chondrocytes.
doi:10.1016/j.bbrc.2007.02.159
PMCID: PMC1887518  PMID: 17367759
SR protein; splicing factor; collagen genes; chondrocyte differentiation; chondrogenesis; articular chondrocyte

Results 1-9 (9)