Phosphatidylinositol 3-kinase/AKT pathway inhibition by doxazosin promotes glioblastoma cells death, upregulation of p53 and triggers low neurotoxicity

Glioblastoma is the most frequent and malignant brain tumor. Treatment includes chemotherapy with temozolomide concomitant with surgical resection and/or irradiation. However, a number of cases are resistant to temozolomide, as well as the human glioblastoma cell line U138-MG. We investigated doxazosin’s (an antihypertensive drug) activity against glioblastoma cells (C6 and U138-MG) and its neurotoxicity on primary astrocytes and organoptypic hippocampal cultures. For this study, the following methods were used: citotoxicity assays, flow cytometry, western-blotting and confocal microscopy. We showed that doxazosin induces cell death on C6 and U138-MG cells. We observed that doxazosin’s effects on the PI3K/Akt pathway were similar as LY294002 (PI3K specific inhibitor). In glioblastoma cells treated with doxasozin, Akt levels were greatly reduced. Upon examination of activities of proteins downstream of Akt we observed upregulation of GSK-3β and p53. This led to cell proliferation inhibition, cell death induction via caspase-3 activation and cell cycle arrest at G0/G1 phase in glioblastoma cells. We used in this study Lapatinib, a tyrosine kinase inhibitor, as a comparison with doxazosin because they present similar chemical structure. We also tested the neurocitotoxicity of doxazosin in primary astrocytes and organotypic cultures and observed that doxazosin induced cell death on a small percentage of non-tumor cells. Aggressiveness of glioblastoma tumors and dismal prognosis require development of new treatment agents. This includes less toxic drugs, more selective towards tumor cells, causing less damage to the patient. Therefore, our results confirm the potential of doxazosin as an attractive therapeutic antiglioma agent

Lithium Reversibly Inhibits Schwann Cell Proliferation and Differentiation Without Inducing Myelin Loss

This study was undertaken to examine the bioactivity, specificity, and reversibility of lithium?s action on the growth, survival, proliferation, and differentiation of cultured Schwann cells (SCs). In isolated SCs, lithium promoted a state of cell cycle arrest that featured extensive cell enlargement and c-Jun downregulation in the absence of increased expression of myelin-associated markers. In addition, lithium effectively prevented mitogen-induced S-phase entry without impairing cell viability. When lithium was administered together with differentiating concentrations of cyclic adenosine monophosphate (cAMP) analogs, a dramatic inhibition of the expression of the master regulator of myelination Krox-20 was observed. Likewise, lithium antagonized the cAMP-dependent expression of various myelin markers such as protein zero, periaxin, and galactocerebroside and allowed SCs to maintain high levels of expression of immature SC markers even in the presence of high levels of cAMP and low levels of c-Jun. Most importantly, the inhibitory action of lithium on SC proliferation and differentiation was shown to be dose dependent, specific, and reversible upon removal of lithium compounds. In SC-neuron cultures, lithium suppressed myelin sheath formation while preserving axonal integrity, SC-axon contact, and basal lamina formation. Lithium was unique in its ability to prevent the onset of myelination without promoting myelin degradation or SC dedifferentiation. To conclude, our results underscored an unexpected antagonistic action of lithium on SC mitogenesis and myelin gene expression. We suggest that lithium represents an attractive pharmacological agent to safely and reversibly suppress the onset of SC proliferation, differentiation, and myelination while maintaining the integrity of pre-existing myelinated fibers.Fil: Piñero, Gonzalo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Berg, Randall. The Miami Project To Cure Paralysis; Estados UnidosFil: Andersen, Natalia Denise. The Miami Project To Cure Paralysis; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Setton Avruj, Clara Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Monje, Paula. The Miami Project To Cure Paralysis; Estados Unido

Sciatic nerve regeneration after traumatic injury using magnetic targeted adipose-derived mesenchymal stem cells

Traumatic peripheral nerve injuries constitute a huge concern to public health. Nerve damage leads to a decrease or even loss of mobility of the innervated area. Adult stem cell therapies have shown some encouraging results and have been identified as promising treatment candidates for nerve regeneration. A major obstacle to that approach is securing a sufficient number of cells at the injured site to produce measurable therapeutic effects. The present work tackles this issue and demonstrates enhanced nerve regeneration ability promoted by magnetic targeted cell therapy in an in vivo Wallerian degeneration model. To this end, adipose-derived mesenchymal stem cells (AdMSC) were loaded with citric acid coated superparamagnetic iron oxide nanoparticles (SPIONs), systemically transplanted and magnetically recruited to the injured sciatic nerve. AdMSC arrival to the injured nerve was significantly increased using magnetic targeting and their beneficial effects surpassed the regenerative properties of the stand-alone cell therapy. AdMSC-SPIONs group showed a partially conserved nerve structure with many intact myelinated axons. Also, a very remarkable restoration in myelin basic protein organization, indicative of remyelination, was observed. This resulted in an improvement in nerve conduction, demonstrating functional recovery. In summary, our results demonstrate that magnetically assisted delivery of AdMSC, using a non-invasive and non-traumatic method, is a highly promising strategy to promote cell recruitment and sciatic nerve regeneration after traumatic injury. Last but not least, our results validate magnetic targeting in vivo exceeding previous reports in less complex models through cell magnetic targeting in vitro and ex vivo.This research was funded by CONICET (Grant numbers PIP11220170101059CO and 897), ANPCYT (Grant number PICT 3952), UNLP (Grant numbers X11/680 and X11/784), and UBACYT (Grant number 20020130100024).Peer reviewe

Nanoclusters of crystallographically aligned nanoparticles for magnetic thermotherapy: aqueous ferrofluid, agarose phantom and ex vivo melanoma tumour assessment

Magnetic hyperthermia is an oncologic therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings; 100 kHz and 9.3 kA m-1, was developed and the results were fully analysed in terms of nanoclusters structural and magnetic properties. A careful appraisal of the nanoclusters heating capacity in the three milieus clearly indicate that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict real tissue temperature rise or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, nanostructures distribution inside the tumour plays a key role in the effective heating. A suppression of magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be much increased, from the SAR values predicted from fluid or agarose to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards clinical translation of hyperthermia.This research was funded by CONICET (PIPs 897, 154, 524 and 567), UNLP X11/680 and X11/7884, and UBACYT 20020130100024 grants of Argentina and also partially funded by the Spanish Ministry of Economy (MAT2015-64442-R and SEV2015-0496 projects, co-funded with European Social Funds). We acknowledge, O. Moscoso-Londoño for SQUID data acquisition and M. Knobel for the use of Instituto de Física ‘Gleb Wataghin’, Universidade Estadual de Campinas (UNICAMP) magnetometry instrumentation, R. Peralta for her extreme care with cell biological sample preparation for TEM, the Brazilian Nanotechnology National Laboratory (LNNano) for the use of cryo-TEM (project: ME-22346) facilities, Y-TEC S.A. for the use of TEM TALOS F200X under the supervision of A. Floridia and A. Caneiro and F.H. Sánchez, G. Pasquevich and P. Mendoza Zélis for useful discussions during field inductor building. Monte Carlo simulation were performed in UnCaFiyQT-INIFTA-SNCAD. M.B.F.v.Raap, P. C. S. Avruj, L. Rogin, A. Veiga, E. Spinelli, V.Blank, G.P. Saracco and M.A Bab are members of CONICET, and P.A. Soto is a fellow of CONICET, Argentina.Peer reviewe