Enhancing Targeted Therapy in Breast Cancer by Ultrasound-Responsive Nanocarriers.

Currently, the response to cancer treatments is highly variable, and severe side effects and toxicity are experienced by patients receiving high doses of chemotherapy, such as those diagnosed with triple-negative breast cancer. The main goal of researchers and clinicians is to develop new effective treatments that will be able to specifically target and kill tumor cells by employing the minimum doses of drugs exerting a therapeutic effect. Despite the development of new formulations that overall can increase the drugs' pharmacokinetics, and that are specifically designed to bind overexpressed molecules on cancer cells and achieve active targeting of the tumor, the desired clinical outcome has not been reached yet. In this review, we will discuss the current classification and standard of care for breast cancer, the application of nanomedicine, and ultrasound-responsive biocompatible carriers (micro/nanobubbles, liposomes, micelles, polymeric nanoparticles, and nanodroplets/nanoemulsions) employed in preclinical studies to target and enhance the delivery of drugs and genes to breast cancer.

The Oligostilbene Gnetin H Is a Novel Glycolysis Inhibitor That Regulates Thioredoxin Interacting Protein Expression and Synergizes with OXPHOS Inhibitor in Cancer Cells.

Since aerobic glycolysis was first observed in tumors almost a century ago by Otto Warburg, the field of cancer cell metabolism has sparked the interest of scientists around the world as it might offer new avenues of treatment for malignant cells. Our current study claims the discovery of gnetin H (GH) as a novel glycolysis inhibitor that can decrease metabolic activity and lactic acid synthesis and displays a strong cytostatic effect in melanoma and glioblastoma cells. Compared to most of the other glycolysis inhibitors used in combination with the complex-1 mitochondrial inhibitor phenformin (Phen), GH more potently inhibited cell growth. RNA-Seq with the T98G glioblastoma cell line treated with GH showed more than an 80-fold reduction in thioredoxin interacting protein (TXNIP) expression, indicating that GH has a direct effect on regulating a key gene involved in the homeostasis of cellular glucose. GH in combination with phenformin also substantially enhances the levels of p-AMPK, a marker of metabolic catastrophe. These findings suggest that the concurrent use of the glycolytic inhibitor GH with a complex-1 mitochondrial inhibitor could be used as a powerful tool for inducing metabolic catastrophe in cancer cells and reducing their growth.

Microbubble-mediated delivery of human adenoviruses does not elicit innate and adaptive immunity response in an immunocompetent mouse model of prostate cancer.

BACKGROUND: Gene transfer to malignant sites using human adenoviruses (hAds) has been limited because of their immunogenic nature and host specificity. Murine cells often lack some of the receptors needed for hAds attachment, thus murine cells are generally non-permissive for human adenoviral infection and replication, which limits translational studies. METHODS: We have developed a gene transfer method that uses a combination of lipid-encapsulated perfluorocarbon microbubbles and ultrasound to protect and deliver hAds to a target tissue, bypassing the requirement of specific receptors. RESULTS: In an in vitro model, we showed that murine TRAMP-C2 and human DU145 prostate cancer cells display a comparable expression pattern of receptors involved in hAds adhesion and internalization. We also demonstrated that murine and human cells showed a dose-dependent increase in the percentage of cells transduced by hAd-GFP (green fluorescent protein) after 24 h and that GFP transgene was efficiently expressed at 48 and 72 h post-transduction. To assess if our image-guided delivery system could effectively protect the hAds from the immune system in vivo, we injected healthy immunocompetent mice (C57BL/6) or mice bearing a syngeneic prostate tumor (TRAMP-C2) with hAd-GFP/MB complexes. Notably, we did not observe activation of innate (TNF-α and IL-6 cytokines), or adaptive immune response (neutralizing antibodies, INF-γ+ CD8+ T cells). CONCLUSIONS: This study brings us a step closer to demonstrating the feasibility of murine cancer models to investigate the clinical translation of image guided site-specific adenoviral gene therapy mediated by ultrasound-targeted microbubble destruction.

Race-associated expression of MHC class I polypeptide-related sequence A (MICA) in prostate cancer.

Despite the lack of a complete understanding of the disparities involved, prostate cancer (PCa) has both higher incidence and death rates in African American Men (AAM) relative to those of Caucasian American Men (CAM). MHC class I polypeptide related sequence A (MICA) is an innate immunity protein involved in tumor immunoevasion. Due to a lack of reports of race-specific expression of MICA in PCa, we evaluated MICA expression in patients' tumors and in cell lines from a racially diverse origin. Immunohistochemistry was done on a tissue microarray (TMA) with antibodies against MICA. Tumor MICA mRNA was assessed by data mining using Oncomine and PROGeneV2. Surface MICA and release rate of soluble (s) MICA was evaluated in PCa cell lines originally derived from African American (MDA-PCa-2b) or Caucasian (LNCaP and DU-145) PCa patients. Prostate tumor tissue had a 1.7-fold higher MICA expression relative to normal tissue (p < .0001). MICA immunoreactivity in PCa tissue from AAM was 24% lower (p = .002) compared to CAM. Survival analysis revealed a marginal association of low MICA with poor overall survival (OS) (p = .058). By data mining analysis, a 2.9-fold higher level of MICA mRNA was evidenced in tumor compared to normal tissue (p < .0001). Tumors from AAM had 24% lower levels of MICA mRNA compared to tumors from CAM (p = .038), and poor prognosis was found for patients with lower MICA mRNA (p = .028). By flow cytometry analysis, cell fraction positive for surface MICA was of 3% in MDA-PCa-2b cells, 54% in DU-145 cells, and 67% in LNCaP cells (p < .0001). sMICA was detected in DU-145 and LNCaP cells, but was not detected in MDA-PCa-2b cells. Both LNCaP and DU-145 cells were sensitive to cytolysis mediated by Natural killer (NK) cells. MDA-PCa-2b cells, however were between 1.3-fold at 10:1 Effector:Target (E:T) ratio (p < .0001) and 2-fold at 50:1 E:T ratio (p < .0001) more resistant to NK-mediated cytolysis relative to cells from Caucasian origin. These results suggest that MICA expression may be related to the aggressive nature of PCa. Our findings also demonstrate for the first time that there are variations in MICA expression in the context of racial differences. This study establishes a rationale for further investigation of MICA as a potential race-specific prognostic marker in PCa.

Advances in molecular preclinical therapy mediated by imaging.

Several advances have been made toward understanding the biology of cancer and most of them are due to robust genetic studies that led to the scientific recognition that although many patients have the same type of cancer their tumors may have harbored different molecular alterations. Personalized therapy and the development of advanced techniques of preclinical imaging and new murine models of disease are emerging concepts that are allowing mapping of disease markers in vivo and in some cases also receptor targeted therapy. Aim of this review is to illustrate some emerging models of disease that allow patient tumor implantation in mice for subsequent drug testing and advanced approaches for therapy mediated by preclinical imaging. In particular we discuss targeted therapy mediated by high frequency ultrasound and magnetic resonance, two emerging techniques in molecular preclinical therapy.

Calcium ion cyclotron resonance (ICR), 7.0 Hz, 9.2 microT magnetic field exposure initiates differentiation of pituitary corticotrope-derived AtT20 D16V cells.

The aim of this work is the study of the effect of electromagnetic radiations (ELF-EMF) tuned to the calcium cyclotron resonance condition of 7.0 Hz, 9.2 microT on the differentiation process of pituitary corticotrope-derived AtT20 D16V cells. These cells respond to nerve growth factor by extending neurite-like processes. To establish whether exposure to the field could influence the molecular biology of the pituitary gland, a corticotrope-derived cells line (AtT20 D16V) was exposed to ELF-EMF at a frequency of 7.0 Hz, 9.2 microT electromagnetic field by a Vega Select 719 power supply. Significant evidence was obtained to conclude that as little as 36 h exposure to the Ca(2+) ICR condition results in enhanced neurite outgrowth, with early expression and aggregation of the neuronal differentiation protein NF-200 into neurite structures.

Cellular ELF signals as a possible tool in informative medicine.

According to Quantum Electro-Dynamical Theory by G. Preparata, liquid water can be viewed as an equilibrium between of two components: coherent and incoherent ones. The coherent component is contained within spherical so called "coherence domains" (CDs) where all molecules synchronously oscillate with the same phase. CDs are surrounded by the incoherent component where molecules oscillate with casual phases regarding each other. The existence of coherent domain in water has been demonstrated in a set of experiments on pure water exposed to high voltage, under this condition the electric field concentrates inside the water, arranging the water molecules to form high ordered structure. Recently has been studied the influence of combined static and alternating parallel magnetic fields on the current through the aqueous solution of glutamic acid; outlining the relevance of low frequency electro-magnetic field in interacting with biological target. Additional results demonstrate that at combined static and alternating parallel, magnetic fields matching the ion cyclotron energy resonance of a particular charged molecule into biological tissue an intrinsic weak magnetic field is generated by ion currents in the cell. These results should increase the reliability and the clinical feasibility of the use of electromagnetic field, tuned at ion cyclotron resonance of charged molecules, as a biophysical approach to interfere with biological mechanisms. We demonstrate that Exposure of human epithelial cell to ion cyclotron energy resonance generated by a commercial electromedical device (Vega select 719) tuned to calcium ion at 7 Hz act as a differentiation factor, thus opening up the possibility to use particular extremely low frequency electro magnetic field protocols, in informative medicine.

Calcium ion cyclotron resonance (ICR) transfers information to living systems: effects on human epithelial cell differentiation.

The specific aim of the present work concerns the effectiveness of low-frequency electromagnetic fields treatment to modify biochemical properties of human keratinocytes (HaCaT). Cells exposed to a 7 Hz electromagnetic field, tuned to calcium ion cyclotron resonance (ICR), showed modifications in the cytoskeleton. These modifications were related to different actin distributions as revealed by phalloidin fluorescence analysis. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression, indicating that exposure to electromagnetic field carries keratinocytes to an upper differentiation level. This study confirms our previous observation and supports the hypothesis that a 7 Hz calcium ICR electromagnetic field may modify cell biochemistry and interfere in the differentiation and cellular adhesion of normal keratinocytes, suggesting the possibility to use ICR electromagnetic therapy for the treatment of undifferentiated diseases.

Ion cyclotron resonance as a tool in regenerative medicine.

The identification of suitable stem cell cultures and differentiating conditions that are free of xenogenic growth supplements is an important step in finding the clinical applicability of cell therapy in two important fields of human medicine: heart failure and bone remodeling, growth and repair. We recently demonstrated the possibility of obtaining cardiac stem cells (CSCs) from human endomyocardial biopsy specimens. CSCs self-assemble into multi-cellular clusters known as cardiospheres (CSps) that engraft and partially regenerate infarcted myocardium. CSps and cardiosphere-derived-cells (CDCs) were exposed for five days in an incubator regulated for temperature, humidity, and CO(2) inside a solenoid system. This system was placed in a magnetically shielded room. The cells were exposed simultaneously to a static magnetic field (MF) and a parallel low-alternating frequency MF, close to the cyclotron frequency corresponding to the charge/mass ratio of the Ca(++) ion. In this exposure condition, CSps and CDCs modulate their differentiation turning on cardiogenesis and turning off vasculogenesis. Cardiac markers such as troponin I (TnI) and myosin heavy chain (MHC) were up-regulated. Conversely, angiogenic markers such as vascular endothelial growth factor (VEGF) and kinase domain receptor (KDR) were down-regulated as evidenced by immunocytochemistry. Exposure to the 7 Hz calcium ion cyclotron resonance (ICR) frequency can modulate the cardiogenic vs. angiogenic differentiation process of ex vivo expanded CSCs. This may pave the way for novel approaches in tissue engineering and cell therapy. With regard to bone remodeling, it has been suggested that bone marrow-derived mesenchymal stem cells (MSC) may be considered as a potential therapeutic tool. Using the Ca(++)-dependent specific differentiation potential of the ELF-MF 7 Hz ICR, we show here that exposure of human MSC to these same MF conditions enhanced the expression of osteoblast differentiation markers such as alkaline phosphatase, osteocalcin, and osteopontin, as analyzed by real-time quantitative PCR, without affecting cell proliferation. As expected, while the differentiation marker factors were up regulated, the ICR electromagnetic field down regulated osteoprotegerin gene expression, a critical regulator of postnatal skeletal development and homeostasis in humans as well as mice.

Omega-3 eicosapentaenoic acid decreases CD133 colon cancer stem-like cell marker expression while increasing sensitivity to chemotherapy.

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil.

Targeting a newly established spontaneous feline fibrosarcoma cell line by gene transfer.

Fibrosarcoma is a deadly disease in cats and is significantly more often located at classical vaccine injections sites. More rare forms of spontaneous non-vaccination site (NSV) fibrosarcomas have been described and have been found associated to genetic alterations. Purpose of this study was to compare the efficacy of adenoviral gene transfer in NVS fibrosarcoma. We isolated and characterized a NVS fibrosarcoma cell line (Cocca-6A) from a spontaneous fibrosarcoma that occurred in a domestic calico cat. The feline cells were karyotyped and their chromosome number was counted using a Giemsa staining. Adenoviral gene transfer was verified by western blot analysis. Flow cytometry assay and Annexin-V were used to study cell-cycle changes and cell death of transduced cells. Cocca-6A fibrosarcoma cells were morphologically and cytogenetically characterized. Giemsa block staining of metaphase spreads of the Cocca-6A cells showed deletion of one of the E1 chromosomes, where feline p53 maps. Semi-quantitative PCR demonstrated reduction of p53 genomic DNA in the Cocca-6A cells. Adenoviral gene transfer determined a remarkable effect on the viability and growth of the Cocca-6A cells following single transduction with adenoviruses carrying Mda-7/IL-24 or IFN-γ or various combination of RB/p105, Ras-DN, IFN-γ, and Mda-7 gene transfer. Therapy for feline fibrosarcomas is often insufficient for long lasting tumor eradication. More gene transfer studies should be conducted in order to understand if these viral vectors could be applicable regardless the origin (spontaneous vs. vaccine induced) of feline fibrosarcomas.

Nonionizing radiation as a noninvasive strategy in regenerative medicine: the effect of Ca(2+)-ICR on mouse skeletal muscle cell growth and differentiation.

Controlling cell differentiation and proliferation with minimal manipulation is one of the most important goals for cell therapy in clinical applications. In this work, we evaluated the hypothesis that the exposure of myoblast cells (C2C12) to nonionizing radiation (tuned at an extremely low-frequency electromagnetic field at calcium-ion cyclotron frequency of 13.75 Hz) may drive their differentiation toward a myogenic phenotype. C2C12 cells exposed to calcium-ion cyclotron resonance (Ca(2+)-ICR) showed a decrease in cellular growth and an increase in the G(0)/G(1) phase. Severe modifications in the shape and morphology and a change in the actin distribution were revealed by the phalloidin fluorescence analysis. A significant upregulation at transcriptional and translational levels of muscle differentiation markers such as myogenin (MYOG), muscle creatine kinase (MCK), and alpha skeletal muscle actin (ASMA) was observed in exposed C2C12 cells. Moreover, the pretreatment with nifedipine (an L-type voltage-gated Ca(2+) channel blocker) led to a reduction of the Ca(2+)-ICR effect. Consequently, it induced a downregulation of the MYOG, MCK, and ASMA mRNA expression affecting adversely the differentiation process. Therefore, our data suggest that Ca(2+)-ICR exposure can upregulate C2C12 differentiation. Although further studies are needed, these results may have important implications in myodegenerative pathology therapies.

Cord blood CD133 cells define an OV6-positive population that can be differentiated in vitro into engraftable bipotent hepatic progenitors.

Cell therapy represents the most promising alternative strategy for end-stage liver diseases and hepatic progenitors are the best candidates. We have identified a reservoir of immature hepatic precursors within human cord blood, which can derive engraftable bipotent progenitors. We isolated a stem cell subset CD133+/CD34+/OV6(low) expressing a surface-marker profile consistent with that of fetal liver cells. Upon induction of hepatic commitment by a medium containing cytokines and factors involved in vivo oval-cell activation, a heterogeneous cell population displaying characteristics of functional oval-cell-like bipotent hepatic progenitors was obtained. The cells expressed markers of hepatocytes and cholangiocytes and were highly enriched in OV6, c-Met, c-Kit, and Thy-1. They also displayed liver functional activity as glycogen storage, urea production, albumin secretion, and inducible CyP2B6 activity. When injected into liver-damaged severe-combined immunodeficient mice, induced bipotent hepatic progenitors appropriately engrafted livers of recipient animals, where they formed clusters of human-derived cells expressing human leucocyte antigen-class I, Hep-Par1, and OV6 antigens. Human-specific albumin, alpha-fetoprotein, and cytokeratin 19 were also expressed. In transplanted animals, AST serum levels showed a significative reduction with regard to controls. This human model for in vitro progenitor-cell activation may provide a powerful tool for elucidating the pathways and synergies that regulate this complex process and can represent a valuable source, exploitable for liver cell-based therapies and regenerative medicine.

Differentiation of human adult cardiac stem cells exposed to extremely low-frequency electromagnetic fields.

AIMS: Modulation of cardiac stem cell (CSC) differentiation with minimal manipulation is one of the main goals of clinical applicability of cell therapy for heart failure. CSCs, obtained from human myocardial bioptic specimens and grown as cardiospheres (CSps) and cardiosphere-derived cells (CDCs), can engraft and partially regenerate the infarcted myocardium, as previously described. In this paper we assessed the hypothesis that exposure of CSps and CDCs to extremely low-frequency electromagnetic fields (ELF-EMFs), tuned at Ca2+ ion cyclotron energy resonance (Ca2+-ICR), may drive their differentiation towards a cardiac-specific phenotype. METHODS AND RESULTS: A significant increase in the expression of cardiac markers was observed after 5 days of exposure to Ca2+-ICR in both human CSps and CDCs, as evidenced at transcriptional, translational, and phenotypical levels. Ca2+ mobilization among intracellular storages was observed and confirmed by compartmentalized analysis of Ca2+ fluorescent probes. CONCLUSIONS: These results suggest that ELF-EMFs tuned at Ca2+-ICR could be used to drive cardiac-specific differentiation in adult cardiac progenitor cells without any pharmacological or genetic manipulation of the cells that will be used for therapeutic purposes.