Iron affects the sphere-forming ability of ovarian cancer cells in non-adherent culture conditions.

Introduction: Detachment from the extracellular matrix (ECM) is the first step of the metastatic cascade. It is a regulated process involving interaction between tumor cells and tumor microenvironment (TME). Iron is a key micronutrient within the TME. Here, we explored the role of iron in the ability of ovarian cancer cells to successfully detach from the ECM. Methods: HEY and PEO1 ovarian cancer cells were grown in 3D conditions. To mimic an iron rich TME, culture media were supplemented with 100 µM Fe3+. Cell mortality was evaluated by cytofluorimetric assay. The invasive potential of tumor spheroids was performed in Matrigel and documented with images and time-lapses. Iron metabolism was assessed by analyzing the expression of CD71 and FtH1, and by quantifying the intracellular labile iron pool (LIP) through Calcein-AM cytofluorimetric assay. Ferroptosis was assessed by quantifying mitochondrial reactive oxygen species (ROS) and lipid peroxidation through MitoSOX and BODIPY-C11 cytofluorimetric assays, respectively. Ferroptosis markers GPX4 and VDAC2 were measured by Western blot. FtH1 knockdown was performed by using siRNA. Results: To generate spheroids, HEY and PEO1 cells prevent LIP accumulation by upregulating FtH1. 3D HEY moderately increases FtH1, and LIP is only slightly reduced. 3D PEO1upregulate FtH1 and LIP results significantly diminished. HEY tumor spheroids prevent iron import downregulating CD71, while PEO1 cells strongly enhance it. Intracellular ROS drop down during the 2D to 3D transition in both cell lines, but more significantly in PEO1 cells. Upon iron supplementation, PEO1 cells continue to enhance CD71 and FtH1 without accumulating the LIP and ROS and do not undergo ferroptosis. HEY, instead, accumulate LIP, undergo ferroptosis and attenuate their sphere-forming ability and invasiveness. FtH1 knockdown significantly reduces the generation of PEO1 tumor spheroids, although without sensitizing them to ferroptosis. Discussion: Iron metabolism reprogramming is a key event in the tumor spheroid generation of ovarian cancer cells. An iron-rich environment impairs the sphere-forming ability and causes cell death only in ferroptosis sensitive cells. A better understanding of ferroptosis sensitivity could be useful to develop effective treatments to kill ECM-detached ovarian cancer cells.

Effects of external trigeminal nerve stimulation (eTNS) on laser evoked cortical potentials (LEP): A pilot study in migraine patients and controls.

Background Transcutaneous external supraorbital nerve stimulation has emerged as a treatment option for primary headache disorders, though its action mechanism is still unclear. Study aim In this randomized, sham-controlled pilot study we aimed to test the effects of a single external transcutaneous nerve stimulation session on pain perception and cortical responses induced by painful laser stimuli delivered to the right forehead and the right hand in a cohort of migraine without aura patients and healthy controls. Methods Seventeen migraine without aura patients and 21 age- and sex-matched controls were selected and randomly assigned to a real or sham external transcutaneous nerve stimulation single stimulation session. The external transcutaneous nerve stimulation was delivered with a self-adhesive electrode placed on the forehead and generating a 60 Hz pulse at 16 mA intensity for 20 minutes. For sham stimulation, we used 2 mA intensity. Laser evoked responses were recorded from 21 scalp electrodes in basal condition (T0), during external transcutaneous nerve stimulation and sham stimulation (T1), and immediately after these (T2). The laser evoked responses were analyzed by LORETA software. Results The real external transcutaneous nerve stimulation reduced the trigeminal N2P2 amplitude in migraine and control groups significantly in respect to placebo. The real stimulation was associated with lower activity in the anterior cingulate cortex under trigeminal laser stimuli. The pattern of LEP-reduced habituation was reverted by real and sham transcutaneous stimulation in migraine patients. Conclusions The present results could suggest that the external transcutaneous nerve stimulation may interfere with the threshold and the extent of trigeminal system activation, with a mechanism of potential utility in the resolution and prevention of migraine attacks.

A comparative study of cortical responses evoked by transcutaneous electrical vs CO(2) laser stimulation.

OBJECTIVES: A novel non-invasive method for nociceptive electrical stimulation of the skin has been recently introduced by using a planar concentric stimulating electrode (CE). We compared the cortical potentials induced by a CE vs laser stimulator in healthy subjects using a multichannel recording. METHODS: Cortical potentials were recorded in 11 healthy subjects by 54 scalp electrodes, stimulating the skin of the right hand and the right supra-orbital zone by the CE and laser stimulator settled two levels above the individual pain threshold. RESULTS: The latency difference between N1, N2, and P2 evoked by the CE vs laser stimulator was larger than the receptor activation time of 40 ms and larger following stimulation of the upper limbs than of the head. The amplitudes and topographic distribution of the cortical waves did not differ between the two stimulation types. CONCLUSIONS: A-beta fibre co-activation may be induced by CE electrodes, as suggested by latency gaps. Nevertheless, CE-evoked potentials showed similarity in amplitude, morphology and topographic representation with laser-induced ones. SIGNIFICANCE: At present, CE-evoked potentials cannot be considered a reliable measure of nociceptive pathway function.

Effects of high-frequency repetitive transcranial magnetic stimulation of primary motor cortex on laser-evoked potentials in migraine.

The aim of this study was to examine the effects of high-frequency (HF) repetitive transcranial magnetic stimulation (rTMS) of the left primary motor cortex (M1) on subjective pain and evoked responses induced by laser stimulation (LEPs) of the contralateral hand and supraorbital zone in a cohort of migraine patients without aura during the inter-critical phase, and to compare the effects with those of non-migraine healthy controls. Thirteen migraine patients and 12 sex- and age-matched controls were evaluated. Each rTMS session consisted of 1,800 stimuli at a frequency of 5 Hz and 90% motor threshold intensity. Sham (control) rTMS was performed at the same stimulation position. The vertex LEP amplitude was reduced at the trigeminal and hand levels in the sham-placebo condition and after rTMS to a greater extent in the migraine patients than in healthy controls, while the laser pain rating was unaffected. These results suggest that HF rTMS of motor cortex and the sham procedure can both modulate pain-related evoked responses in migraine patients.

Effects of topiramate and levetiracetam vs placebo on habituation of contingent negative variation in migraine patients.

Migraine is characterized by reduced habituation of multimodal evoked potentials, which in turn reflects an abnormal pattern of cortical excitability. We assessed the effects of a 2-month treatment with topiramate or levetiracetam vs placebo on contingent negative variation (CNV) habituation and amplitude in a cohort of migraine without aura (MO) patients. Forty-five MO patients were selected from a university-based outpatient clinic and randomly assigned to 100mg topiramate or 1000mg levetiracetam or placebo in a double-blind design. Twenty-four control subjects were also recruited. The initial CNV (iCNV) amplitude and habituation were assessed by Cz/A1-A2 derivation recordings in the basal condition (T0) and after 2 months of treatment (T1). Both topiramate and levetiracetam produced a significant reduction in migraine frequency compared to placebo, they also reversed the abnormal iCNV habituation pattern which characterized the MO patients in the basal condition and which was not present in controls. For migraine patients, the reduced migraine frequency and habituation index following treatment were significantly correlated. A lack of habituation of evoked responses is an interictal endophenotypic marker in migraine, the reversion of which may improve disease outcome. These results suggest a role for neurophysiological methods in the management of migraine.