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Neuroactive steroids, molecules produced from cholesterol in steroidogenic cells (i.e., peripheral glands and nervous system) are physiological modulators and protective agents of nervous function. A possible role for neuroactive steroids in the sex-dimorphic clinical manifestation, onset and progression of Multiple Sclerosis (MS) has been recently suggested. To explore this possibility, we assessed the synthesis of the first steroidogenic product (pregnenolone; PREG) in the spinal cord of experimental autoimmune encephalomyelitis rats, a MS model. Data obtained indicate that the synthesis of PREG in the spinal cord is altered by the pathology in a sex-dimorphic way and depending on the pathological progression. Indeed, in male spinal cord the synthesis was already decreased at the acute phase of the disease (i.e., 14 days post induction - dpi) and maintained low during the chronic phase (i.e., 45 dpi), while in females this effect was observed only at the chronic phase. Substrate availability had also a role in the sex-dimorphic kinetics. Indeed, at the chronic phase, male animals showed a reduction in the levels of free cholesterol coupled to alteration of cholesterol metabolism into oxysterols; these effects were not observed in female animals. These findings suggest that the comprehension of the neurosteroidogenic processes could be relevant to better understand the sexual dimorphism of MS and to possibly design sex-oriented therapeutic strategies based on neuroactive steroids., We also acknowledge support from Agencia Estatal de Investigación, Spain (grant number BFU2017-82754-R), Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (CIBERFES), Instituto de Salud Carlos III, Madrid, Spain and Fondos Feder to LM.G-S.

Cholangiocarcinoma is the first most common cancer of the biliary tract. To date, surgical resection is the only potentially curative option, but it is possible only for a limited percentage of patients, and in any case survival rate is quite low. Moreover, cholangiocarcinoma is often chemotherapy-resistant, and the only drug with a significant benefit for patient’s survival is Gemcitabine. It is necessary to find new drugs or combination therapies to treat nonresectable cholangiocarcinoma and improve the overall survival rate of patients. In this work, we evaluate in vitro the antitumoral effects of Rigosertib, a multi-kinase inhibitor in clinical development, against cholangiocarcinoma EGI-1 cell lines. Rigosertib impairs EGI-1 cell viability in a dose- and time-dependent manner, reversibility is dose-dependent, and significant morphological and nuclear alterations occur. Moreover, Rigosertib induces the arrest of the cell cycle in the G2/M phase, increases autophagy, and inhibits proteasome, cell migration, and invasion. Lastly, Rigosertib shows to be a stronger radiosensitizer than Gemcitabine and 5-Fluorouracil. In conclusion, Rigosertib could be a potential therapeutic option, alone or in combination with radiations, for nonresectable patients with cholangiocarcinoma.

Oxaliplatin is a cornerstone chemotherapeutic used in the treatment of colorectal cancer, the third leading cause of death in Western countries. Most side effects of this platinum-containing drug are adequately managed in the clinic, although acute and long-term neurotoxicity still severely compromises the quality of life of patients treated with oxaliplatin. We have previously demonstrated that therapeutically relevant concentrations/doses of oxaliplatin lead to a reduction in intracellular pH in mouse dorsal root ganglion (DRG) neurons in vitro and in vivo and that this alteration sensitizes TRPA1 and TRPV1 channels, which most likely mediate the allodynia associated with treatment. In this study, we show that oxaliplatin leads to a reduction of intracellular pH by forming adducts with neuronal haemoglobin, which acts in this setting as a proton buffer. Furthermore, we show that FDA-approved drugs that inhibit carbonic anhydrase (an enzyme that is linked to haemoglobin in intracellular pH homeostasis), ie, topiramate and acetazolamide, revert (1) oxaliplatin-induced cytosolic acidification and TRPA1 and TRPV1 modulation in DRG neurons in culture, (2) oxaliplatin-induced cytosolic acidification of DRG of treated animals, and (3) oxaliplatin-induced acute cold allodynia in mice while not affecting OHP-induced cytotoxicity on cancer cells. Our data would therefore suggest that reversal of oxaliplatin-induced cytosolic acidification is a viable strategy to minimize acute oxaliplatin-induced symptoms.

Despite the advent of tyrosine kinase inhibitors, a proportion of chronic myeloid leukemia patients in chronic phase fails to respond to Imatinib or to second generation inhibitors and progress to blast crisis. Limited improvements in the understanding of the molecular mechanisms responsible for chronic myeloid leukemia transformation from chronic phase to the aggressive blast crisis were achieved until now. We present here a massive parallel sequencing analysis of 10 blast crisis samples and of the corresponding autologous chronic phase controls which reveals, for the first time, recurrent mutations affecting the ubiquitin-conjugating enzyme E2A gene (UBE2A, formerly RAD6A). Additional analyses on a cohort of 24 blast crisis, 41 chronic phase as well as 40 acute myeloid leukemia and 38 atypical chronic myeloid leukemia patients at onset confirmed that UBE2A mutations are specifically acquired during chronic myeloid leukemia progression with a frequency of 16.7% in advanced phases. In vitro studies show that the mutations here described cause a decrease in UBE2A activity, leading to an impairment of myeloid differentiation in chronic myeloid leukemia cells.

Despite advances in cancer therapies, nanomedicine approaches including the treatment of glioblastoma (GBM), the most common, aggressive brain tumor, remains inefficient. These failures are likely attributable to the complex and not yet completely known biology of this tumor, which is responsible for its strong invasiveness, high degree of metastasis, high proliferation potential, and resistance to radiation and chemotherapy. The intimate connection through which the cells communicate between them plays an important role in these biological processes. In this scenario, tunneling nanotubes (TnTs) are recently gaining importance as a key feature in tumor progression and in particular in the re-growth of GBM after surgery. In this context, we firstly identified structural differences of TnTs formed by U87-MG cells, as model of GBM cells, in comparison with those formed by normal human astrocytes (NHA), used as a model of healthy cells. Successively, we have studied the possibility to exploit U87-MG TnTs as drug-delivery channels in cancer therapy, using liposomes composed of cholesterol/sphingomyelin and surface functionalized with mApoE and chlorotoxin peptides (Mf-LIP) as nanovehicle model. The results showed that U87-MG cells formed almost exclusively thick and long protrusions, whereas NHA formed more thin and short TnTs. Considering that thick TnTs are more efficient in transport of vesicles and organelles, we showed that fluorescent-labeled Mf-LIP can be transported via TnTs between U87-MG cells and with less extent through the protrusions formed by NHA cells. Our results demonstrate that nanotubes are potentially useful as drug-delivery channels for cancer therapy, facilitating the intercellular redistribution of this drug in close and far away cells, thus reaching isolated tumor niches that are hardly targeted by simple drug diffusion in the brain parenchyma. Moreover, the differences identified in TnTs formed by GBM and NHA cells can be exploited to increase treatment precision and specificity.

ALK-positive Anaplastic Large Cell Lymphoma (ALCL) represents a subset of Non-Hodgkin Lymphoma whose treatment benefited from crizotinib development, a dual ALK/MET inhibitor. Crizotinib blocks ALK-triggered pathways such as PI3K/AKT/ mTOR, indispensable for survival of ALK-driven tumors. Despite the positive impact of targeted treatment in ALCL, resistant clones are often selected during therapy. Strategies to overcome resistance include the design of second generation drugs and the use of combined therapies that simultaneously target multiple nodes essential for cells survival. We investigated the effects of combined ALK/mTOR inhibition. We observed a specific synergistic effect of combining ALK inhibitors with an mTOR inhibitor (temsirolimus), in ALK+ lymphoma cells. The positive cooperation resulted in an increased inhibition of mTOR effectors, compared to single treatments, a block in G0/G1 phase and induction of apoptosis. The combination was able to prevent the selection of resistant clones, while longterm exposure to single agents led to the establishment of resistant cell lines, with either ALK inhibitor or temsirolimus. In vivo, mice injected with Karpas 299 cells and treated with low dose combination showed complete regression of tumors, while only partial inhibition was obtained in single agents-treated mice. Upon treatment stop the combination was able to significantly delay tumor relapses. Re-challenge of relapsed tumors at a higher dose led to full regression of xenografts in the combination group, but not in mice treated with lorlatinib alone. In conclusion, our data suggest that the combination of ALK and mTOR inhibitors could be a valuable therapeutic option for ALK+ ALCL patients.

Oxaliplatin induced peripheral neurotoxicity is characterized by an acute cold-induced syndrome characterized by cramps, paresthesias/dysesthesias in the distal limbs and perioral region, that develops rapidly and lasts up to one week affecting nearly all the patients as well as by long-lasting symptoms. It has been previously shown that pharmacological or genetic ablation of TRPA1 responses reduces oxaliplatin-induced peripheral neurotoxicity in mouse models. In the present report, we show that treatment with concentrations of oxaliplatin similar to those found in plasma of treated patients leads to an acidification of the cytosol of mouse dorsal root ganglia neurons in culture and this in turn is responsible for sensitization of TRPA1 channels, thereby providing a mechanistic explanation to toxicity of oxaliplatin. Reversal of the acidification indeed leads to a significantly reduced activity of TRPA1 channels. Last, acidification occurs also in vivo after a single injection of therapeutically-relevant doses of oxaliplatin.

Mesoporous silica materials (MSM) have been proposed as promising tools for cell specific drug delivery or fluorescent cell tracking. In cancer therapy there is an urgent need to develop a cancer cell specific drug carrier able to limit the non-specific uptake of the drug by normal cells thereby reducing serious side effects. Chemotherapy induced peripheral neurotoxicity (CIPN) is one of the most clinically relevant side effects linked to the use of several antineoplastic drugs. In this study we showed that the uptake of MSM (synthesized using a PEG surfactant-based interfacial synthesis procedure), functionalised with folic acid (MSM-FOL) after 1, 6 and 24 hours is very limited in neuronal-like cellular systems such as differentiated SH-SY5Y human neuroblastoma cells and rat embryonic dorsal root ganglia sensory neurons. By contrast, the nanoparticles are highly internalized in A549 and IGROV-1 cancer cells. The 6 hour-treatment of A549 and IGROV-1 cells with nanoparticles loaded with the antineoplastic drug cisplatin (CP) induced significant cytotoxicity with respect to CP alone. These results were observed treating IGROV-1 cells with 25 and 50 μg/ml nanoparticles doses (corresponding respectively to CP 6.25 and 12.5 μM) and treating A549 with 50 μg/ml.Our results demonstrated a selective uptake of functionalized MSM suggesting them as promising tools for targeted antineoplastic therapy. Further studies will be necessary in order to confirm if this approach may be useful in reducing neurotocity of anticancer drugs.

Most of the anaplastic large-cell lymphoma (ALCL) cases carry the t(2;5; p23;q35) that produces the fusion protein NPM-ALK (nucleophosmin-anaplastic lymphoma kinase). NPM-ALK-deregulated kinase activity drives several pathways that support malignant transformation of lymphoma cells. We found that in ALK-rearranged ALCL cell lines, NPM-ALK was distributed in equal amounts between the cytoplasm and the nucleus. Only the cytoplasmic portion was catalytically active in both cell lines and primary ALCL, whereas the nuclear portion was inactive because of heterodimerization with NPM1. Thus, about 50% of the NPM-ALK is not active and sequestered as NPM-ALK/NPM1 heterodimers in the nucleus. Overexpression or relocalization of NPM-ALK to the cytoplasm by NPM genetic knockout or knockdown caused ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) increased phosphorylation and cell death through the engagement of an ATM/Chk2- and γH2AX (phosphorylated H2A histone family member X)-mediated DNA-damage response. Remarkably, human NPM-ALK-amplified cell lines resistant to ALK tyrosine kinase inhibitors (TKIs) underwent apoptosis upon drug withdrawal as a consequence of ERK1/2 hyperactivation. Altogether, these findings indicate that an excess of NPM-ALK activation and signaling induces apoptosis via oncogenic stress responses. A 'drug holiday' where the ALK TKI treatment is suspended could represent a therapeutic option in cells that become resistant by NPM-ALK amplification.

Combined anticancer therapy using platinum compounds and antitubulins has increased the risk of neurotoxicity. However, the combination of low-dose cisplatin (CDDP) with toxic doses of paclitaxel significantly reduces cellular death in a human neuroblastoma SH-SY5Y cell line. To analyze the mechanisms of this protection, we evaluated various signaling molecules possibly involved in apoptosis and some relevant cell cycle regulatory proteins. CDDP does not interfere with the tubulin-stabilizing action of paclitaxel. The evaluation of molecular pathways involved in apoptosis indicates that the Bcl-2 but not the caspases may be involved in the CDDP protection of paclitaxel-induced apoptosis. The increase in p53 protein and its nuclear accumulation suggests a possible involvement of p53 in CDDP protection. The use of the chemical inhibitor of p53, pifithrin α, excluded this possibility. The study of cyclins and the flow cytometric analysis (fluorescence-activated cell sorting) suggest that CDDP exerts a protective action by blocking cells early in the cell cycle. The determination of the mitotic index indicates that CDDP prevents cells from reaching the mitosis. We concluded that low doses of CDDP are protective against toxic doses of paclitaxel and that the possible mechanism of this protection is that the CDDP prevents human neuroblastoma SH-SY5Y cells from achieving mitosis.

Several effective antineoplastic drugs are severely neurotoxic and induce the onset of disabling peripheral neuropathies. The clinical presentation of this side effect depends on the type of antineoplastic agent administered. The signs and symptoms shown are predominantly motor (e.g. in the case of vinca alkaloids), sensorimotor (e.g. with the taxanes) or sensory (e.g. using platinum-derived drugs). The pathogenesis of these antineoplastic drug-induced peripheral neuropathies is still poorly understood, although a relationship has been suggested between some neuronal growth factors (neurotrophins) and the toxic action of cisplatin (CDDP) and paclitaxel. In this study we correlated the changes in the circulating levels of Nerve Growth factor (NGF) in a series of 24 women affected by locally advanced squamous cervical carcinoma treated with cisplatin and paclitaxel-based chemotherapy with the severity of chemotherapy-induced peripheral neuropathy (CIPN) as assessed by the Total Neuropathy Score (TNS). After informed consent, blood samples were drawn before chemotherapy and after treatment and NGF circulating levels were determined by ELISA (EmaxTM ImmunoAssay System, Promega, USA; plasma working dilutions NGF 1:80) following the manufacturer protocols. The results obtained in this series of patients evidenced a significant correlation between the severity of CIPN and the reduction in the circulating levels of NGF (r = − 0.480, p = 0.017 by two-tailed Spearman correlation test, 95% CL −0.745 to −0.082), thus confirming similar results previously obtained in a rat model of CDDP peripheral neurotoxicity. In conclusion, our data suggest that a relationship exists between CIPN and NGF circulating levels in patients treated with cisplatin and paclitaxel. Therefore, this preliminary observation support the need for further studies in order to explore the possibility of reducing the severity of platinum-drug sensory neurotoxicity with treatments aimed at increasing the circulating levels of NGF.

Epothilones are a novel class of microtubule-targeting anticancer agents that are neurotoxic. In this study, we investigated the epothilone B toxic effect in vitro and we characterized in vivo the general and neurological side effects of epothilone B administration in Wistar and Fischer rats. The in vitro experiments made it possible to explore a wide concentration range (0.1 nM–1 μM) and evidenced a dose-dependent effect of epothilone B exposure on neuron neurite elongation. This dose-dependent neurotoxic effect was confirmed in both in vivo studies performed on two different rat strains at the neurophysiological, behavioral and pathological levels in the dose range 0.25–1.5 mg/kg iv weekly × 4 weeks and tubulin hyper-polymerization was demonstrated in sciatic nerve specimens. These are the first studies of the neurological effects of epothilone B and they can provide a basis for extending pre-clinical investigation to other members of the epothilone family.

Extracorporeal photochemotherapy (ECP) is a medical procedure effective in the treatment of several different T-cell mediated diseases such as cutaneous T-cell lymphoma and Graft-versus-Host Disease. During ECP treatment the patient's blood is processed by means of a cell separator to collect leukocytes (leukapheresis), mostly lymphocytes and monocytes, which are then incubated with the photoactive drug 8-methoxypsoralen (8-MOP), exposed to ultraviolet-A light (UV-A) and reinfused to the patient. It has been suggested that during ECP not only UV-A irradiation but also changes in the environmental condition may be relevant. Although ECP has been shown to have an in-vivo immunomodulatory effect, the mechanisms through which ECP exerts its effect remain elusive. One of the reasons for this incomplete knowledge is the absence of a reliable model for ECP. In order to investigate the effect of ECP on the peripheral immune system, we developed a new device which mimics the complete ECP cycle including blood transit through the cell separator. Peripheral blood samples (50ml) were obtained from volunteers and processed using a peristaltic pump. Peripheral blood mononuclear cells (PBMC) were then collected and treated with 8-MOP and UV-A under the same conditions used for the patients' therapy. Using this strategy we investigated 8-MOP, UV-A and their combined effect on the production of the pro-inflammatory cytokines interferon-gamma (IFN-gamma), interleukine-2 (IL-2) and tumor necrosis factor-alpha (TNF-alpha) in PBMC with and without polyclonal stimulation. We firstly demonstrated that our device does not affect total red and white blood cell counts. After 8-MOP and UV-A irradiation a significant decrease was observed in both activated CD4(+) and CD8(+) T lymphocytes producing IFN-gamma, IL-2 and TNF-alpha. Our findings are in line with those previously obtained in humans after complete ECP treatment, thus suggesting that our newly developed device is suitable for investigating the mechanism of action of ECP ex-vivo.

Pixantrone is less cardiotoxic and is similarly effective to mitoxantrone (MTX) as an antineoplastic drug. In our study, pixantrone reduced the severity of acute and decreased the relapse rate of chronic relapsing experimental allergic encephalomyelitis (EAE) in rats. A marked and long-lasting decrease in CD3+, CD4+, CD8+ and CD45RA+ blood cells and reduced anti-MBP titers were observed with both pixantrone and MTX. In vitro mitogen- and antigen-induced T-cell proliferation tests of human and rodents cells evidenced that pixantrone was effective at concentrations which can be effectively obtained after i.v. administration in humans. Cardiotoxicity was present only in MTX-treated rats. The effectiveness and the favorable safety profile makes pixantrone a most promising immunosuppressant agent for clinical use in multiple sclerosis (MS).

Background Bosutinib is a recently approved ABL inhibitor. In spite of the well-documented effectiveness of BCR-ABL inhibitors in treating chronic myeloid leukemia, development of resistance is a continuous clinical challenge. Transporters that facilitate drug uptake and efflux have been proposed as one potential source of resistance to tyrosine kinase inhibitor treatment. Our aim was to determine which carriers are responsible for bosutinib transport. Methods K562S cells overexpressing the drug transporters ABCB1, ABCG2, and SLC22A1 were generated, characterized and used in proliferation assay and intracellular uptake and retention assay (IUR). In vivo experiments were performed in nude mice injected with K562S, K562DOX cells (overexpressing ABCB1), and K562DOX silenced for ABCB1 (K562DOX/sh P-GP). Results The IUR assay using C-14 bosutinib showed that only ABCB1 was responsible for active bosutinib transport. K562DOX cells showed the lowest intracellular level of bosutinib, while K562DOX cells treated with the ABCB1 inhibitor verapamil showed intracellular bosutinib levels comparable with parental K562S. Proliferation assays demonstrated that K562DOX are resistant to bosutinib treatment while verapamil is able to restore the sensitivity to the drug. Nude mice injected with K562DOX and treated with bosutinib showed very limited response and quickly relapsed after stopping treatment while K562S as well as K562DOX/sh P-GP remained tumor-free. Conclusions Our data suggest that the analysis of ABCB1 expression levels might help determine treatment options for patients exhibiting resistance to bosutinib. Electronic supplementary material The online version of this article (doi:10.1186/s13045-015-0179-4) contains supplementary material, which is available to authorized users.