Gina Lungu

Simultaneous Molecular and Hypoxia Imaging of Brain Tumors In Vivo Using Spectroscopic Photoacoustic Tomography

Noninvasive molecular and functional imaging in vivo is promising for detecting and monitoring various physiological conditions in animals and ultimately humans. To this end, we present a novel noninvasive technology, spectroscopic photoacoustic tomography (SPAT), which offers both strong optical absorption contrast and high ultrasonic spatial resolution. Optical contrast allows spectroscopic separation of signal contributions from multiple optical absorbers (e.g., oxyhemoglobin, deoxyhemoglobin, and a molecular contrast agent), thus enabling simultaneous molecular and functional imaging. SPAT successfully imaged with high resolution the distribution of a molecular contrast agent targeting integrin overexpressed in human U87 glioblastomas in nude mouse brains. Simultaneously, SPAT also imaged the hemoglobin oxygen saturation and the total hemoglobin concentration of the vasculature, which revealed hypoxia in tumor neovasculature. Therefore, SPAT can potentially lead to better understanding of the interrelationships between hemodynamics and specific biomarkers associated with tumor progression.

Photoacoustic imaging of lacZ gene expression in vivo

In the postgenomic era, imaging techniques are playing an important role in visualizing gene expression in vivo. This work represents the first demonstration of photoacoustic tomography (PAT) for reporter gene imaging. Rats inoculated with 9L/lacZ gliosarcoma tumor cells are imaged with PAT before and after injection of X-gal, a colorimetric assay for the lacZ-encoded enzyme beta-galactosidase. Using far-red optical illumination, the genetically tagged tumors in rats are clearly visualized by PAT following the assay. The spatial resolution is quantified to be less than 400 microm, while 500-nM-level sensitivity is demonstrated. With the future development of new absorption-based reporter gene systems, it is anticipated that photoacoustic technology will provide a valuable tool for molecular imaging research.

MMP2 role in breast cancer brain metastasis development and its regulation by TIMP2 and ERK1/2

Matrix metalloproteinase 2 (MMP2) is important in breast cancer (BC) invasion and metastasis. We previously reported that BC brain metastases, in a rat syngeneic model developed in our laboratory, have high expression and activity of MMP2. The MMP2 mechanism of action in the brain is still under intense scrutiny. To study the role of MMP2 in the development of BC brain metastasis we transfected ENU1564 rat mammary adenocarcinoma cells with tissue inhibitor of MMP2 (TIMP2). Animals inoculated with ENU1564-TIMP2 cells had decreased orthotopic tumor growth, decreased orthotopic metastastic behavior and did not develop brain metastases. These results were associated with decreased MMP2 activity, demonstrated by gel zymography. Mitogen activated protein kinase (MAPK) pathway components, such as ERK1/2, have been correlated to MMP expression and/or astrocyte activity. We found that BC brain metastases have peripheral astrocyte reactivity and higher expression of glial fibrillary acidic protein (GFAP) and phosphorylated-ERK1/2 (p-ERK1/2). Additionally, rat astrocyte-conditioned media increased in vitro invasion of ENU1564 cancer cells and increased expression of MMP2 and p-ERK1/2. Blockage of ERK1/2 phosphorylation by treatment with MEK inhibitor (PD98059) decreased the expression of MMP2 in cancer cells grown in rat astrocyte-conditioned media. Our results are highly suggestive that MMP2 plays a role in the development of BC metastases, in particular to the brain. Furthermore, our results suggest that astrocyte factors and the ERK1/2 signaling pathway may be associated with BC brain metastasis development; and that ERK1/2 may regulate MMP2 in a way that is modifiable by astrocyte factors.

Identification of Cancer Stem Cells in Dog Glioblastoma

There is increasing evidence in some malignancies that the tumor clone is heterogeneous in regard to proliferation and differentiation. The cancer stem cell hypothesis implies that not all the cells in the tumor have the same capacity to proliferate and maintain the growth of the tumor. Only a relatively small fraction of cells in the tumor, termed cancer stem cells (CSCs), possess the ability to proliferate and self-renew extensively. In the past decade, several groups have reported the existence of a CSC population in different human brain tumors from both children and adults. We report here the identification of a CSC population from a Boxer dog with glioblastoma multiforme (GBM) that possesses a great capacity for proliferation, self-renewal, and differentiation. This cloned cell line is aneuploid, forms neurospheres in culture, possesses CSC markers, and reproduces the original dog GBM when inoculated into the nude mouse brain.

FGF‐1‐induced matrix metalloproteinase‐9 expression in breast cancer cells is mediated by increased activities of NF‐κB and activating protein‐1

Matrix metalloproteinase‐9 (MMP‐9) plays a critical role in tumor invasion and metastasis. Here, we investigate the effect of fibroblast growth factor‐1 (FGF‐1) on the expression of MMP‐9 in ENU1564, an ethyl‐N‐nitrosourea‐induced rat mammary adenocarcinoma cell line. We observed that FGF‐1 induces a dose‐dependent increase in MMP‐9 mRNA, protein, and activity in ENU1564 cells. To gain insight into the molecular mechanism of MMP‐9 regulation by FGF‐1, we investigated the role of components of PI3K‐Akt and MEK1/2‐ERK signaling pathways in our system since NF‐κB and AP‐1 transcription factor binding sites have been characterized in the upstream region of the MMP‐9 gene. We demonstrated that FGF‐1 increases Akt phosphorylation, triggers nuclear translocation of NF‐κBp65, and enhances degradation of cytoplasmic IκBα. Pretreatment of cells with LY294002, a PI3K inhibitor, significantly inhibited MMP‐9 protein expression in FGF‐1‐treated cells. Conversely, our data show that FGF‐1 increases ERK phosphorylation in ENU1564 cells, increases c‐jun and c‐fos mRNA expression in a time‐dependent manner, and triggers nuclear translocation of c‐jun. Pretreatment of cells with PD98059, a MEK1/2 inhibitor significantly inhibited MMP‐9 protein expression in FGF‐1 treated cells. Finally, we observed increased DNA binding of NF‐κB and AP‐1 in FGF‐1‐treated cells and that mutation of either NF‐κB or AP‐1 response elements prevented MMP‐9 promoter activation by FGF‐1. Taken together, these results demonstrated that FGF‐1‐induced MMP‐9 expression in ENU1564 cells is associated with increasing DNA binding activities of NF‐κB and AP‐1 and involve activation of a dual signaling pathway, PI3K‐Akt and MEK1/2‐ERK.

MicroRNA profiling and the role of microRNA-132 in neurodegeneration using a rat model

MicroRNAs (miRs) are endogenous small RNAs that regulate gene expression at the post-transcriptional level by mediating mRNA degradation or transcriptional inhibition. MiRs were implicated in the pathogenesis of numerous neurodegenerative diseases, including Parkinsons disease (PD). In this study we analyzed the possible role of miRs in the neurodegenerative process in a spontaneous autosomal recessive rat model for neurodegeneration developed in our laboratory. To investigate the role of miRs in the etiology of PD, we conducted miR expression profiling using microarrays. We found 20 miRs that are deregulated in affected rats and many of these are implicated in neurodegenerative disease, including PD. In this study we were particularly interested in the expression of miR-132, a miR that has been reported to be highly expressed in neurons, and to have a potential role in neurodegenerative diseases. We found a significant increase in miR-132 in affected rats by microarray and the result was confirmed by qPCR. Next we analyzed one of the known downstream targets of miR-132, nuclear receptor related 1 protein (Nurr1), which is essential in neurogenesis of midbrain dopaminergic neurons. Western blot analysis and immunohistochemistry revealed a significant decrease in Nurr1 protein expression in the mesencephalic neurons. Finally, we found a significant decrease in both serum and mesencephalon brain tissue of brain-derived neurotrophic factor (BDNF), which is known to be a direct target of Nurr1. Taken together, our findings suggest that miR-132 can regulate Nurr1 levels and might influence the development and function of midbrain dopaminergic neurons.

Up-regulation of pro-nerve growth factor, neurotrophin receptor p75, and sortilin is associated with retrovirus-induced spongiform encephalomyelopathy

The progressive spongiform encephalomyelopathy caused by ts1, a neuropathogenic temperature-sensitive mutant of Moloney murine leukemia virus (MoMuLV-ts1), results in motor neuronal loss without direct neuronal infection. We have previously reported that ts1-mediated neuronal degeneration in mice has a multifactorial pathogenesis. Here, we report that in the ts1-infected central nervous system (CNS) activated neural cells showed intense immunoreactivity for pro-nerve growth factor (proNGF), neurotrophin receptor p75 (p75(NTR)), and sortilin in the areas showing spongiform changes. Since recent studies suggested that proNGF is more active than mature NGF in inducing neuronal death after binding to co-receptors p75(NTR)/sortilin, we hypothesized that overexpression of proNGF, sortilin and p75(NTR) play a role in ts1-induced neurodegeneration. We found that proNGF and p75(NTR), but not sortilin, mRNA and protein were significantly elevated in ts1-infected brainstem compared to non-infected control tissue. There was extensive tyrosine phosphorylation of p75(NTR), a marker for its activation, in ts1-infected brainstem with abundance in degenerating neurons. We explored whether the increase in the in vivo proNGF expression also occurs in cultured immortalized C1 astrocytes infected by ts1 virus. The proNGF level was significantly increased in infected C1 cells compared to control cells only after addition of fibroblast growth factor (FGF-1). We also showed increased expression of FGF-1 in the CNS of ts1-infected mice. Our findings suggest that the FGF-1 signaling pathway may be responsible for the overexpression of proNGF in neural cells during pathogenesis of ts1-induced neurodegeneration. This study provides new in vivo insights into the possible role of proNGF and its receptors in ts1-induced neurodegeneration.

Curcumin induces apoptosis in a murine mammary gland adenocarcinoma cell line through the mitochondrial pathway

Curcumin, a phenol in turmeric (Curcuma longa), has been studied for the last decade as a potential anticancer drug. It has been shown to reduce viability of the highly malignant, metastatic rat mammary gland cell line ENU1564 in culture and reduce metastasis of these cells injected into nude mice. The purpose of this study was to identify the mechanisms by which curcumin induces apoptosis in these ENU1564 cells in vitro, and to examine its effects on mitochondrial membrane potential and mitochondrial Ca(2+) homeostasis. The results show that curcumin induced apoptosis in ENU1564 cells through the intrinsic pathway of apoptosis, as evident by an increase in mitochondrial Ca(2+) accumulation and a decrease in mitochondrial membrane potential. However, treatment of the ENU1564 cells with the mitochondrial uniporter inhibitor RU-360 prior to curcumin treatment partially inhibited the curcumin effects. SKF-96365, a store-operated Ca(2+) channel blocker, suppressed the curcumin effect on mitochondrial Ca(2+). In addition, curcumin down-regulated the expressions of Bcl-2 and procaspase-3 and increased the production of reactive oxygen species in ENU1564 cells. These data suggest that the mitochondrial Ca(2+) is the leading factor by which curcumin induced apoptosis in ENU1564 cells, followed by reactive oxygen species production and inhibition of Bcl-2 oncoprotein.

Metabolic Analysis of Striatal Tissues from Parkinson’s Disease-like Rats by Electrospray Ionization Ion Mobility Mass Spectrometry

Electrospray ionization ion mobility mass spectrometry (ESI-IMMS) was used to study the striatal metabolomes in a Parkinsons like disease (PD-like) rat model. Striatal tissue samples from Berlin Druckrey IV (BD-IV) with PD-like disease 20 dpn-affected and 15 dpn-affected rats (dpn: days postnatal) were investigated and compared with age-matched controls. An ion mobility mass spectrometer (IMMS) produced multidimensional spectra with mass to charge ratio (m/z), ion mobility drift time, and intensity information for each individual metabolite. Principle component analysis (PCA) was applied in this study for pattern recognition and significant metabolites selection (68% data was modeled in PCA). Both IMMS spectra and PCA results showed that there were clear global metabolic differences between PD-like samples and healthy controls. Nine metabolites were selected by PCA and identified as potential biomarkers using the Human Metabolome Database (HMDB). One targeted metabolite in this study was dopamine. Selected-mass mobility analysis indicated the absence of dopamine in PD-like striatal metabolomes. A major discovery of this work, however, was the existence of an isomer of dopamine. By using ion mobility spectrometry, the dopamine isomer, which has not previously been reported, was separated from dopamine.

Potential role of α-synuclein in neurodegeneration: studies in a rat animal model

J. Neurochem. (2012) 122, 812–822.