Jessian L. Munoz

Delivery of Functional Anti-miR-9 by Mesenchymal Stem Cell–derived Exosomes to Glioblastoma Multiforme Cells Conferred Chemosensitivity

Glioblastoma multiforme (GBM), the most common and lethal tumor of the adult brain, generally shows chemo- and radioresistance. MicroRNAs (miRs) regulate physiological processes, such as resistance of GBM cells to temozolomide (TMZ). Although miRs are attractive targets for cancer therapeutics, the effectiveness of this approach requires targeted delivery. Mesenchymal stem cells (MSCs) can migrate to the sites of cancers, including GBM. We report on an increase in miR-9 in TMZ-resistant GBM cells. miR-9 was involved in the expression of the drug efflux transporter, P-glycoprotein. To block miR-9, methods were developed with Cy5-tagged anti-miR-9. Dye-transfer studies indicated intracellular communication between GBM cells and MSCs. This occurred by gap junctional intercellular communication and the release of microvesicles. In both cases, anti-miR-9 was transferred from MSCs to GBM cells. However, the major form of transfer occurred with the microvesicles. The delivery of anti-miR-9 to the resistant GBM cells reversed the expression of the multidrug transporter and sensitized the GBM cells to TMZ, as shown by increased cell death and caspase activity. The data showed a potential role for MSCs in the functional delivery of synthetic anti-miR-9 to reverse the chemoresistance of GBM cells.

Immunological properties of mesenchymal stem cells and clinical implications

The rapid evolution of experimental data has acknowledged the critical relevance of immune biology in stem cell research. It appears that efficient transfer of stem cells to patients requires robust analyses of the immune properties as well as the responses of the stem cells to immune mediators. This review discusses the biology of adult human mesenchymal stem cells (MSCs) in the context of immunology. MSCs are pluripotent, self-renewing cells with the potential for tissue regeneration, for example the repair of bone, cartilage, tendon, ligament, skeletal muscle, and cardiac muscle. MSCs have also been shown to transdifferentiate into cells of ectodermal origin, such as neurons. MSCs are located in perfused areas of adult bone marrow, whereas hematopoietic stem cells are located in poorly perfused areas of the same organ. MSCs show bimodal, i.e. anti-inflammatory and immune-enhancing, immune responses. MSCs also regulate immune responses such as the regulation of antibody production by B cells, alterations in T cell subtypes, and immune tolerance of allogeneic transplants. MSCs also have the potential for gene delivery. This review explores the diverse clinical potential for MSCs and discusses the limitations and advantages of their immunomodulatory properties.

Temozolomide resistance in glioblastoma cells occurs partly through epidermal growth factor receptor- mediated induction of connexin 43

Glioblastoma Multiforme (GBM) is an aggressive adult primary brain tumor with poor prognosis. GBM patients develop resistance to the frontline chemotherapy, temozolomide (TMZ). As the connexins (Cx) have been shown to have a complex role in GBM, we investigated the role of Cx43 in TMZ resistance. Cx43 was increased in the TMZ-resistant low passage and cell lines. This correlated with the data in The Cancer Genome Atlas. Cx43 knockdown, reporter gene assays, chromatin immunoprecipitation assay, real-time PCR and western blots verified a role for Cx43 in TMZ resistance. This occurred by TMZ-resistant GBM cells being able to activate epidermal growth factor receptor (EGFR). In turn, EGFR activated the JNK-ERK1/2-AP-1 axis to induce Cx43. The increased Cx43 was functional as indicated by gap junctional intercellular communication among the resistant GBM cells. Cell therapy could be a potential method to deliver drugs, such as anti-EGF to tumor cells. Similar strategies could be used to reverse the expression of Cx43 to sensitize GBM cells to TMZ. The studies showed the potential for targeting EGF in immune therapy. These agents can be used in conjunction with stem cell therapy to treat GBM.

Loss of RE-1 silencing factor in mesenchymal stem cell-derived dopamine progenitors induces functional maturity

Stem cell-derived dopamine (DA) neurons hold great promise for Parkinsons disease (PD). Mesenchymal stem cells (MSCs) have great potential for clinical applications. The generation of DA cells from MSCs using sonic hedgehog (SHH) and fibroblast growth factors (FGF8 and bFGF) has been reported. However, the DA cells showed weak electrical properties, representing DA neuron progenitors. Since RE-1 Silencing Factor (REST), suppresses mature neuronal genes in neuronal progenitors, we studied its role in the maturation of MSC-derived DA cells. REST expression did not change during the induction process, thus we knocked down REST and subjected MSCs to the same neural induction cocktail. We observed increases in the protein level of the Na(+) voltage-gated channel and tyrosine hydroxylase (TH). Electrophysiological analyses showed spontaneous firings and spontaneous postsynaptic currents, similar to native DA neurons. Taken together, these results show REST as the limiting gene in the generation of functional mature neurons from MSCs.

Temozolomide Induces the Production of Epidermal Growth Factor to Regulate MDR1 Expression in Glioblastoma Cells

Glioblastoma multiforme (GBM) commonly resists the frontline chemotherapy treatment temozolomide. The multidrug resistance gene (MDR1) and its protein, P-glycoprotein (P-gp), are associated with chemoresistance. This study investigated the mechanisms underlying MDR1-mediated resistance by GBM to temozolomide. P-gp trafficking was studied by flow cytometry and Western blot analysis. MDR1 expression was analyzed by real-time PCR and reporter gene assays. AP-1 interaction with MDR1 was studied by chromatin immunoprecipitation assay. EGF production was analyzed by ELISA, EGFR signaling was determined by Western blot analysis, and in vivo response to erlotinib and/or temozolomide was studied in nude mice. During the early phase of temozolomide treatment, intracellular P-gp was trafficked to the cell membrane, followed by conformational change into active P-gp. At the later phase, gene transcription of MDR1 was induced by temozolomide-mediated production of EGF. EGF activated ERK1/2-JNK-AP-1 cofactors (c-jun and c-fos). An inhibitor of EGFR kinase (erlotinib) given to nude mice with GBM prevented temozolomide-induced resistance. The results identified an essential role for activated EGFR in the resistance of GBM to temozolomide. Temozolomide resistance occurred through a biphasic response; first, by a conformational change in P-gp into the active form and, second, by releasing EGF, which caused autocrine stimulation of GBM cells to induce MDR1. Pharmacologic inhibition of EGFR kinase blunted the ability of GBM cells to resist temozolomide. These findings may explain reports on the common occurrence of mutant EGFR (EGFRvIII) and EGFR expansion in the resistance of GBM cells. Mol Cancer Ther; 13(10); 2399–411. ©2014 AACR.

Temozolomide competes for P-glycoprotein and contributes to chemoresistance in glioblastoma cells

Chemotherapeutic resistance can occur by P-glycoprotein (P-gp), a 12-transmembrane ATP-dependent drug efflux pump. Glioblastoma (GBM) has poor survival rate and uniformly acquired chemoresistance to its frontline agent, Temozolomide (TMZ). Despite much effort, overcoming TMZ resistance remains a challenge. We reported on autonomous induction of TMZ resistance by increased transcription MDR1, the gene for P-gp. This study investigated how P-gp and TMZ interact to gain resistance. Using an experimental model of Adriamycin-resistant DC3F cells (DC3F/Adx), we showed that increased P-gp caused TMZ resistance. Increasing concentrations of TMZ competed with Calcein for P-gp, resulting in reduced efflux in the DC3F/Adx cells. Three different inhibitors of P-gp reversed the resistance to TMZ in two different GBM cell lines, by increasing active Caspase 3. Molecular modeling predicted the binding sites to be the intracellular region of P-gp and also identified specific amino acids and kinetics of energy for the efflux of TMZ. Taken together, we confirmed P-gp targeting of TMZ, a crucial regulator of TMZ resistance in GBM. This study provides insights on the effectiveness by which TMZ competes with other P-gp substrates, thereby opening the door for combined targeted therapies.

Feline bone marrow-derived mesenchymal stromal cells (MSCs) show similar phenotype and functions with regards to neuronal differentiation as human MSCs☆

Mesenchymal stromal cells (MSCs) show promise for treatment of a variety of neurological and other disorders. Cat has a high degree of linkage with the human genome and has been used as a model for analysis of neurological disorders such as stroke, Alzheimers disease and motor disorders. The present study was designed to characterize bone marrow-derived MSCs from cats and to investigate the capacity to generate functional peptidergic neurons. MSCs were expanded with cells from the femurs of cats and then characterized by phenotype and function. Phenotypically, feline and human MSCs shared surface markers, and lacked hematopoietic markers, with similar morphology. As compared to a subset of human MSCs, feline MSCs showed no evidence of the major histocompatibility class II. Since the literature suggested Stro-1 as an indicator of pluripotency, we compared early and late passages feline MSCs and found its expression in >90% of the cells. However, the early passage cells showed two distinct populations of Stro-1-expressing cells. At passage 5, the MSCs were more homogeneous with regards to Stro-1 expression. The passage 5 MSCs differentiated to osteogenic and adipogenic cells, and generated neurons with electrophysiological properties. This correlated with the expression of mature neuronal markers with concomitant decrease in stem cell-associated genes. At day 12 induction, the cells were positive for MAP2, Neuronal Nuclei, tubulin βIII, Tau and synaptophysin. This correlated with electrophysiological maturity as presented by excitatory postsynaptic potentials (EPSPs). The findings indicate that the cat may constitute a promising biomedical model for evaluation of novel therapies such as stem cell therapy in such neurological disorders as Alzheimers disease and stroke.

The bone marrow niche in support of breast cancer dormancy.

Despite the success in detecting breast cancer (BC) early and, with aggressive therapeutic intervention, BC remains a clinical problem. The bone marrow (BM) is a favorable metastatic site for breast cancer cells (BCCs). In BM, the survival of BCCs is partly achieved by the supporting microenvironment, including the presence of immune suppressive cells such as mesenchymal stem cells (MSCs). The heterogeneity of BCCs brings up the question of how each subset interacts with the BM microenvironment. The cancer stem cells (CSCs) survive in the BM as cycling quiescence cells and, forming gap junctional intercellular communication (GJIC) with the hematopoietic supporting stromal cells and MSCs. This type of communication has been identified close to the endosteum. Additionally, dormancy can occur by soluble mediators such as cytokines and also by the exchange of exosomes. These latter mechanisms are reviewed in the context of metastasis of BC to the BM for transition as dormant cells. The article also discusses how immune cells such as macrophages and regulatory T-cells facilitate BC dormancy. The challenges of studying BC dormancy in 2-dimensional (2-D) system are also incorporated by proposing 3-D system by engineering methods to recapitulate the BM microenvironment.

Mycoplasma genitalium: An Emerging Sexually Transmitted Infection

Mycoplasma genitalium has been recognized as a cause of male urethritis, and there is now evidence suggesting that it causes cervicitis and pelvic inflammatory disease in women. M. genitalium is a slow growing organism, and, with the advent of nucleic acid amplification test (NAAT), more studies are being performed, and knowledge about the pathogenicity of this organism elucidated. With NAAT detection, treatment modalities have been studied, and the next challenge is to determine the most effective antimicrobial therapy. Doxycycline, the first-line antibiotic for urethritis, is largely ineffective in the treatment of M. genitalium and furthermore, resistance to macrolide has also emerged. The most effective drug is Moxifloxacin although there are emerging reports of resistance to it in various parts of the world. This paper not only highlights the current research and knowledge, but also reviews the diversity of health implications on the health of men and women infected with M. genitalium. Alternate antibiotics and the impact of M. genitalium on infertility are areas that require more studies as we continue to research into this microorganism.

MicroRNA Cancer Therapeutics and the Challenge of Drug Delivery

MicroRNAs (miRNAs) have progressed to a separate field in cancer biology. As cancer cells undergo investigations to build a hierarchy, the dissection of miRNA in the developmental process occurs in parallel. There is no doubt that RNA interference (RNAi) will be in the clinic. However, the efficiency of the process remains a challenge. This chapter discusses the potential for RNAi in therapy and summarizes some of the different methods to package RNAi for delivery to the regions of cancer. The ability of mesenchymal stem cells to migrate to the site of tumors has been explored as a method to deliver RNAi. However, these stem cells can influence tumor growth and the immune response to tumors. This chapter briefly discusses the potential of using mesenchymal stem cells in RNAi delivery. This topic would require a separate chapter on stem cells and RNAi delivery. Confounds discussed in this chapter are applicable to any method developed with stem cells to target cancer cells with RNAi.