Neurotoxic effects of antineoplastic drugs: the lesson of pre-clinical studies

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Dorsal root ganglia sensory neuron transporters and their role in drug-induced neurotoxicity

Drug transporters have been widely studied in cancer cells, mostly due to their importance in drug resistance. Given their clinical relevance, the role of drug transporters in chemotherapy-induced peripheral neurotoxicity (CIPN) is of particular interest, but knowledge is largely incomplete in dorsal root ganglia (DRG) sensory neurons. This critical aspect of CIPN has never been addressed until the observation in a single in vivo study that copper transporters are present and active in rat DRG neurons [1]. However, evidence is still missing regarding several crucial aspects of drug transporters, such as i) the possible co-localization of more than one transporter, ii) their relevance to each class of neurotoxic drugs, iii) their functionality and iv) the possibility to reduce toxicity through their pharmacological modulation in vivo. The precise mechanisms of cellular uptake of DNA-intercalating platinum drugs are partly unknown, although it is commonly supposed that both passive diffusion as well as facilitated or active transport mechanisms play substantial roles. Cisplatin transporters suggested to be involved in modulation of platinum accumulation are MRP2, the copper transporters CTR1, ATP7A and ATP7B. Moreover, a recent notion is that various anticancer drugs can enter mammalian cells by facilitated transport via solute carriers (SLCs). Among all the SLCs transporters, organic cationic transporter 2 (OCT2) is a critical determinant in the uptake and cytotoxicity of cisplatin in non-neuronal cells, and its activity modulation has been associated with reduced toxicity [2]. The antitubulin alkaloid paclitaxel is a substrate of the energy-dependent drug efflux pump P-glycoprotein (P-gp). P-gp is a member of the ATP-binding cassette (ABC) family of transporters widely expressed in many human cancers and the enhanced efflux of paclitaxel has been observed in drug-resistant tumor cells that overexpress P-gp. The role of P-gp in paclitaxel efflux has been confirmed by employing membrane modulating agents (such as verapamil and cyclosporin A) in combination with paclitaxel. The distribution of this transporter in the central nervous system is well known, but it has never been investigated in peripheral neurons [1]. Recently, paclitaxel has also been recognized as a substrate for the copper transporter ATP7A. The proteasome inhibitor bortezomib has only recently been described to be a substrate of ABCG2, another member of the ABC family, but only in human leukemia cells. Other cellular transportes might also be implicated in neuronal damage in CIPN. We recently performed a thorough investigation on glutamate transporters derived from the observation that glutamate production inhibition reduced in rat models the neurotoxicity of cisplatin, paclitaxel and bortezomib [3]. We demonstrated the presence in the DRG of the transporters, their functionality and the possibility to modulate this functionality using pharmacological inhibitors (DL-threo- -hydroxy-aspartate, dihydrokainate and L-serine-O-sulfate) in in vitro neuronal systems [4,5]. Preliminary results have already been obtained regarding OCT2 demonstrating the presence at the mRNA level and the possibility to modulate in vitro cisplatin cytotoxicity in DRG neurons using the transporter competitor cimetidine. Based on these observations, the possible role of the different families of drug transporters and their possible relevance to CIP onset and severity will be here revised

Erratum to: Distal pain and carpal tunnel syndrome diagnosis among cashiers: a longitudinal study

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Studies to Assess the Utility of Serum Neurofilament Light Chain as a Biomarker in Chemotherapy-Induced Peripheral Neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most common and disabling dose-limiting toxicities of chemotherapy. We report here the results of two separate non-interventional studies (49 patients), which evaluated blood neurofilament light chain (NfL) as a biomarker of CIPN in breast cancer patients treated with paclitaxel. All patients underwent a standard treatment protocol that was established independently of the present studies. NfL was measured in serum using an ultrasensitive single-molecule array and compared with the self-administered European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-CIPN twenty-item scale (CIPN20) and Total Neuropathy Score clinical version (TNSc), a clinician-reported measure of neuropathy progression. The TNSc increased with cumulative dose compared with baseline, and the NfL concentrations were also strongly associated with the cumulative dose of chemotherapy. The analysis showed a correlation between TNSc and NfL. Both TNSc and NfL showed weak to moderate associations with CIPN20 subscores, with a better association for the CIPN20 sensory compared with motor and autonomic subscores. Data from the two studies provide evidence that serum NfL has the potential to be used as a biomarker to monitor and mitigate CIPN. However, studies with additional patients planned in the ongoing clinical trial will determine the universal application of NfL as a biomarker in CIPN

Expression of CD38 in human neuroblastoma SH-SY5Y cells.

Human CD38 antigen is a 42–45 kDa type II transmembrane glycoprotein with a short N-terminal cytoplasmic domain and a long C-terminal extracellular region. It is widely expressed in different cell types including thymocytes, activated T cells, and terminally differentiated B cells (plasma cells) and it is involved in cellular proliferation and adhesion. CD38 acts as an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPR). It has been also demonstrated that increased extracellular levels of NAD+ and cADPR are involved in inflammatory diseases and in cellular damage, such as ischemia. In the present study, we have characterized the expression of CD38 in human neuroblastoma SH-SY5Y cell line. All-trans-retinoic acid (ATRA) treatment was used to induce cell differentiation. Our results indicate that: a) even if SH-SY5Y cells have a negative phenotype express CD38 at nuclear level, ATRA treatment does not influence this pattern; b) CD38 localizing to the nucleus may co-localize with p80-coilin positive nuclear-coiled bodies; c) purified nuclei, by Western blot determinations using anti-CD38 antibodies, display a band with a molecular mass of −42 kDa; d) SH-SY5Y cells show nuclear ADP-ribosyl cyclase due to CD38 activity; e) the basal level of CD38 mRNA shows a time-dependent increase after treatment with ATRA. These results suggest that the presence of constitutive fully functional CD38 in the SH-SY5Y nucleus has some important implications for intracellular generation of cADP-ribose and subsequent nucleoplasmic calcium release

The fundamental role of morphology in experimental neurotoxicology: the example of chemotherapy-induced peripheral neurotoxicity

The peripheral nervous system is a frequent target of toxic agents. The accurate identification of the sites of neurotoxic action through the morphological characterization of reliable in vivo models or in vitro systems can give fundamental clues when investigating the pathogenesis and interpreting the clinical features of drug-induced neuropathy. The morphological approach has been used to investigate almost all the anticancer drugs able to induce chemotherapy-induced peripheral neurotoxicity, i.e. platinum drugs, antitubulins and proteasome inhibitors. No models have ever been described for thalidomide. This review demonstrates that any pathogenetic study on chemotherapy-induced peripheral neurotoxicity must be based on solid morphological observations obtained in reliable animal and in vitro models. This is particularly true in this setting, since the availability of tissues of human origin is extremely limited. In fact, peripheral (generally sural) nerve biopsies are never required for diagnostic purposes in chemotherapy-treated cancer patients, and their use for a purely scientific aim, although potentially very informative, is not ethical. Moreover, several neurotoxic drugs target the dorsal root ganglia neurons, and it is very difficult to obtain high-quality specimens even from early autopsies. It is, therefore, our opinion that an extensive morphological assessment of the in vitro and in vivo effect of any potentially neurotoxic antineoplastic drugs, as well as of neuroprotectant agents, should be taken into consideration right from the earliest stages of their development

Expression, distribution and glutamate uptake activity of excitatory aminoacid transporters in vitro cultures of embryonic rat dorsal root ganglia cells

Glutamate is the major mediator of excitatory signalling in the mammalian central nervous system, but it has recently been shown to play also a role in the transduction of sensory input at the periphery and in peripheral neuropathies. New advances in research have demonstrated that rat peripheral sensory terminals and dorsal root ganglia (DRG) express molecules involved in glutamate signalling, including high-affinity membrane-bound glutamate transporters (Excitatory Aminoacid Transporters, EAATs) and that alterations in their expression and/or functionality can be implicated in several models of peripheral neuropathy, neuropathic pain and hyperalgesia. Since EAATS might represent an interesting target for pharmacological intervention, the knowledge of their distribution and functionality deserves to be improved. Here we describe, through immunofluorescence assays, immunoblotting and beta-counter analysis of (H3) L-glutamate uptake, the expression, distribution and activity of the EAATs in in vitro cultures of embryonic DRG sensory neurons, sensory neurons+satellite cells and satellite cells. In this study we demonstrated that EAATs are expressed in all cultures, but that their distribution recognizes a peculiar pattern for each of them, since EAATs immunolabelling was differentially expressed in the cytoplasm of neuronal or satellite cells. This result was further confirmed by immunoblotting. Moreover, both cell types showed a strong sodium-ATP-dependent (active) glutamate uptake activity. However, the net (i.e. active transport minus passive diffusion) glutamate transport was more marked in neuronal cultures when cells were grown and maintained without satellite cells. These results, that demonstrate that functionally active EAATs can be studied in DRG cell cultures, provide further evidence for a role of glutamatergic transport in the peripheral nervous system and will be useful for testing whether any change occurs in in vitro models of peripheral nervous system damage. This work was supported in part by an unrestricted research grant from the “Fondazione Banca del Monte di Lombardia”

Multimodal assessment of painful peripheral neuropathy induced by chronic oxaliplatin-based chemotherapy in mice

<p>Abstract</p> <p>Background</p> <p>A major clinical issue affecting 10-40% of cancer patients treated with oxaliplatin is severe peripheral neuropathy with symptoms including cold sensitivity and neuropathic pain. Rat models have been used to describe the pathological features of oxaliplatin-induced peripheral neuropathy; however, they are inadequate for parallel studies of oxaliplatin's antineoplastic activity and neurotoxicity because most cancer models are developed in mice. Thus, we characterized the effects of chronic, bi-weekly administration of oxaliplatin in BALB/c mice. We first studied oxaliplatin's effects on the peripheral nervous system by measuring caudal and digital nerve conduction velocities (NCV) followed by ultrastructural and morphometric analyses of dorsal root ganglia (DRG) and sciatic nerves. To further characterize the model, we examined nocifensive behavior and central nervous system excitability by <it>in vivo </it>electrophysiological recording of spinal dorsal horn (SDH) wide dynamic range neurons in oxaliplatin-treated mice</p> <p>Results</p> <p>We found significantly decreased NCV and action potential amplitude after oxaliplatin treatment along with neuronal atrophy and multinucleolated DRG neurons that have eccentric nucleoli. Oxaliplatin also induced significant mechanical allodynia and cold hyperalgesia, starting from the first week of treatment, and a significant increase in the activity of wide dynamic range neurons in the SDH.</p> <p>Conclusions</p> <p>Our findings demonstrate that chronic treatment with oxaliplatin produces neurotoxic changes in BALB/c mice, confirming that this model is a suitable tool to conduct further mechanistic studies of oxaliplatin-related antineoplastic activity, peripheral neurotoxicity and pain. Further, this model can be used for the preclinical discovery of new neuroprotective and analgesic compounds.</p

Influence of aging on peripheral nervous system: a morphological and morphometric study

It is well known that aging influences several functional and structural features of peripheral nerves (Verdù et al., 2000; Ceballos et al., 1999; Jeronimo et al., 2008). However, the role of these changes in the damage/repair mechanisms occurring in acquired peripheral neuropathies is still unclear. To this aim, a multimodal, long-term assessment in a mice model would represent an optimal tool to perform experimental neuropathy studies designed to evaluate the role of aging in relationship with a given nerve injury. In this study we used 40 females one-month-old C57B1/6 mice and we followed-up them for fifteen months. Digital and caudal nerve conduction velocity (NCV) studies were performed monthly to evaluate changes in electrophysiological features; moreover, four animals were sacrificed every two months in order to collect caudal nerve, sciatic nerve, dorsal root ganglia (DRG) and skin for morphological and morphometric analysis. The neurophysiological assessments showed a remarkable increase of caudal NCV until the age of 9 months and then it remained unchanged until the end of the observation period; in the same period, digital NCV increase was also present although less marked. At the pathological level, both caudal and sciatic nerves showed a decrease in fibres density related with age, whereas axon and fibres diameters tended to increase. These preliminary data can be considered a first step aiming at creating a background for future studies on the relationship between aging and peripheral nervous system induced damage

Functional Magnetic Resonance Imaging of Rats with Experimental Autoimmune Encephalomyelitis Reveals Brain Cortex Remodeling.

UNLABELLED: Cortical reorganization occurring in multiple sclerosis (MS) patients is thought to play a key role in limiting the effect of structural tissue damage. Conversely, its exhaustion may contribute to the irreversible disability that accumulates with disease progression. Several aspects of MS-related cortical reorganization, including the overall functional effect and likely modulation by therapies, still remain to be elucidated. The aim of this work was to assess the extent of functional cortical reorganization and its brain structural/pathological correlates in Dark Agouti rats with experimental autoimmune encephalomyelitis (EAE), a widely accepted preclinical model of chronic MS. Morphological and functional MRI (fMRI) were performed before disease induction and during the relapsing and chronic phases of EAE. During somatosensory stimulation of the right forepaw, fMRI demonstrated that cortical reorganization occurs in both relapsing and chronic phases of EAE with increased activated volume and decreased laterality index versus baseline values. Voxel-based morphometry demonstrated gray matter (GM) atrophy in the cerebral cortex, and both GM and white matter atrophy were assessed by ex vivo pathology of the sensorimotor cortex and corpus callosum. Neuroinflammation persisted in the relapsing and chronic phases, with dendritic spine density in the layer IV sensory neurons inversely correlating with the number of cluster of differentiation 45-positive inflammatory lesions. Our work provides an innovative experimental platform that may be pivotal for the comprehension of key mechanisms responsible for the accumulation of irreversible brain damage and for the development of innovative therapies to reduce disability in EAE/MS. SIGNIFICANCE STATEMENT: Since the early 2000s, functional MRI (fMRI) has demonstrated profound modifications in the recruitment of cortical areas during motor, cognitive, and sensory tasks in multiple sclerosis (MS) patients. Experimental autoimmune encephalomyelitis (EAE) represents a reliable model of the chronic-progressive variant of MS. fMRI studies in EAE have not been performed extensively up to now. This paper reports fMRI studies in a rat model of MS with somatosensory stimulation of the forepaw. We demonstrated modifications in the recruitment of cortical areas consistent with data from MS patients. To the best of our knowledge, this is the first report of cortical remodeling in a preclinical in vivo model of MS.This work was supported by grants from the National Multiple Sclerosis Society (NMSS; RG-4001-A1 to SP), the Italian Multiple Sclerosis Foundation (FISM; RG 2010/R/31 to SP and FISM Grant 10/12/F14/2011 to PM), the Italian Ministry of Health (GR08/7 to SP), the European Research Council (ERC) 2010-SIG (RG 260511-SEM_SEM to SP), the European Community (EC) 7th Framework Programme (FP7/2007-2013; RG 280772-iONE to SP), The Evelyn Trust (RG 69865 to SP), The Bascule Charitable Trust (RG 75149 to SP). LPJ is supported by a Wellcome Trust Research Training Fellowship (RRZA/057).This is the final version of the article. It first appeared from Society for Neuroscience via http://dx.doi.org/10.1523/JNEUROSCI.0540-15.201