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. 2023 Apr 12;62:102700. doi: 10.1016/j.redox.2023.102700

Inhibition of spinal ferroptosis-like cell death alleviates hyperalgesia and spontaneous pain in a mouse model of bone cancer pain

Zhuofeng Ding a, Xiaoshen Liang a, Jian Wang a, Zongbin Song a, Qulian Guo a,b, Michael KE Schäfer c,d, Changsheng Huang a,b,∗
PMCID: PMC10141498  PMID: 37084690

Abstract

Bone cancer pain (BCP) impairs patients’ quality of life. However, the underlying mechanisms are still unclear. This study investigated the role of spinal interneuron death using a pharmacological inhibitor of ferroptosis in a mouse model of BCP. Lewis lung carcinoma cells were inoculated into the femur, resulting in hyperalgesia and spontaneous pain. Biochemical analysis revealed that spinal levels of reactive oxygen species and malondialdehyde were increased, while those of superoxide dismutase were decreased. Histological analysis showed the loss of spinal GAD65+ interneurons and provided ultrastructural evidence of mitochondrial shrinkage. Pharmacologic inhibition of ferroptosis using ferrostatin-1 (FER-1, 10 mg/kg, intraperitoneal for 20 consecutive days) attenuated ferroptosis-associated iron accumulation and lipid peroxidation and alleviated BCP. Furthermore, FER-1 inhibited the pain-associated activation of ERK1/2 and COX-2 expression and prevented the loss of GABAergic interneurons. Moreover, FER-1 improved analgesia by the COX-2 inhibitor Parecoxib. Taken together, this study shows that pharmacological inhibition of ferroptosis-like cell death of spinal interneurons alleviates BCP in mice. The results suggest that ferroptosis is a potential therapeutic target in patients suffering on BCP and possibly other types of pain.

Keywords: BCP, Ferroptosis, Spinal cord, GABA, Oxidative stress

Graphical abstract

Image 1

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1. Introduction

Cancer pain is one of the most frequent manifestations of cancer patients [1]. Most common cancers, such as prostate, breast, and lung cancer, are often associated with bone metastases, resulting in severe bone destruction and bone cancer pain (BCP) [2]. About 75–90% of cancer patients with bone metastases experience extreme pain and poor quality of life [3]. Increasingly potent analgesics, including opioids, and non-opioids such as non-steroidal anti-inflammatory drugs (NSAIDs), are used to relieve cancer pain [1]. Nevertheless, the pain therapy fails in about 33% of the patients [1,4]. Therefore, a better understanding of the underlying mechanisms and identification of new therapeutic targets is essential to develop effective BCP treatments.

The primary clinical manifestations of BCP are allodynia, hyperalgesia, and spontaneously persisting pain [5]. The loss of dorsal horn neurons in the spinal cord is a possible factor in the development of persistent hyperalgesia including BCP [6]. A likely cause is decreased production of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), which weakens restrictive control of nociceptive signaling in the superficial dorsal horn [7,8]. Several studies demonstrated that the loss of spinal dorsal horn neurons is caused by apoptosis and provided first insights into the underlying mechanisms, including NMDAR-mediated excitotoxicity and cytotoxic CD8+ T cells [9,10]. More recently, ferroptosis, an iron- and reactive oxygen species (ROS)-dependent form of cell death, has been shown to play a role in neuropathic and inflammatory pain [11,12]. Ferroptosis differs from other types of cell death such as necroptosis, apoptosis, and autophagy at the morphological, biochemical, and genetic levels [12]. Ferroptosis is controlled through the lipid repair enzyme glutathione peroxidase 4 (GPX4), transcriptional factors such as p53 and Nrf2 as well as Cyclooxygenase-2 (COX-2) and the phosphorylation of extracellular regulated protein kinases (ERK) [[13], [14], [15]]. Ultrastructural signs of ferroptosis include mitochondrial shrinkage and condensed mitochondrial membranes [16]. Ferroptosis has been implicated in the pathophysiology of several acute or chronic neurological disorders such as Alzheimer's disease, epilepsy, stroke and traumatic brain injury [17]. Moreover, ferroptosis has been also implicated in neuropathic pain, and the inhibition of spinal ferroptosis may alleviate neuropathic pain [18,19]. However, whether ferroptosis contributes to spinal cell death and neuropathic pain in the context of BCP remains unclear. Using the well characterized ferroptosis inhibitor ferrostatin-1 (FER-1) [20], we here tested the hypothesis that ferroptosis contributes to spinal cell death and BCP in a mouse model. We studied pain behavior as well as histological and biochemical features after administration of FER-1 and examined whether the inhibition of ferroptosis enhanced the analgesic action of NSAIDs. Our results suggest that ferroptosis is a potential therapeutic target in patients suffering on BCP.

2. Materials and methods

2.1. Animals

All trials employed male C57BL6/J mice weighing 20 g–25 g. Groups of 5 mice were kept in a cage with a 12 h light/dark cycle and unlimited accessibility to food and drink. All experimental techniques were authorized from the Ethics Committee of Xiangya Medical School, Central South University, and adhered to the guideline of International Association for the Study of Pain.

2.2. BCP model

To establish the model of BCP resulting from lung cancer metastasis [21], we used the murine luciferase expressing Lewis lung carcinoma (LLC-Luc) cell line obtained from the Hunan Fenghui Biotechnology Co., Ltd,. The cells were cultivated in high glucose (4.5 g/L) DMEM media with 10% (v/v) FBS (Gibco, Thermo Fisher Scientific) and 1% (v/v) penicillin and streptomycin (Gibco, Thermo Fisher Scientific) at 37 °C in an incubator with 5% CO2. After enzymatic digestion with 0.05% trypsin, cells were suspended in phosphate buffer saline (PBS) at 2 × 107/mL for subsequent inoculation. Mice were anesthetized using isoflurane (3%) and a cut of 0.5 cm was done on the knee. After exposure of the patellar ligament, a 30 G needle was used to make a hole at the intercondylar notch of the right femur. A bolus of tumor cells (5 μL, 1 ×105 cells) was slowly injected using a 10 μL microinjection syringe into the femoral cavity 5–7 mm distal to the knee joint. After injection, a 2 μL gelatin sponge solution was injected into the hole to avoid tumor cell leaking. The wound was closed in layers. The identical procedure was applied as a sham operation, except that boiling-denatured cells (5 μL, 1 ×105 cells) were administered. In vivo bioluminescence imaging utilizing the luciferase activity of tumor cells was performed 15 min after intraperitoneal administration of D-Luciferin (MCE, cat: HY-12591B) to assess the growing tumor burden. Bioluminescence images were captured under isoflurane anesthesia using a PerkinElmer VisEnFMT2500LX imaging system with an excitation bandpass filter at 670 nm and emission at 745 nm. The bioluminescent area intensity was determined using TrueQuant Imaging Software (PerkinElmer).

2.3. Drug treatment

FER-1 (cat: Hy-100579) and parecoxib (cat: SC 69124) were obtained from Med Chem Express (Shanghai, China). FER-1 (10 mg/kg) or Vehicle solution (10% DMSO in 90% corn oil) was injected intraperitoneally once daily for consecutive 20 days after cell inoculation [22,23].

For parecoxib treatment, 40 mg/kg parecoxib diluted in 0.5 ml saline or an equal volume of saline was injected intraperitoneally 30 min after FER-1 or Vehicle I.P. injection, respectively, on day 20 after LCC inoculation (n = 8, each group).

2.4. Behavioral testing

All behavioral tests were done between 9:30 a.m. and 3:30 p.m. in a separate room with temperature and lighting controls by the experimenter, who was blinded to the treatment. Prior to testing, mice were habituated to the test conditions for 20–30 min. The mechanical allodynia, thermal hyperalgesia, and spontaneous pain tests were conducted on the previous day of cell injection (initial) and on post operation day (POD) 1, 5, 10, 15, 20 after LLC-Luc inoculation. For parecoxib treatment, behavioral assays were performed 1 h after parecoxib or normal saline injection on the POD20.

2.5. Paw withdrawal mechanical threshold (PWT)

To evaluate mechanical allodynia, PWT and paw withdrawal frequency were examined using von Frey filaments (Stoelting, Wood Dale, IL) [21]. Mice were kept in 8 × 8 × 8 cm plastic compartments with a net steel flooring and permitted to habituate for 30 min. Von Frey filaments (0.02 g–2.0 g bending force) were vertically applied to the plantar surface of the right hind paw. A violent retraction or paw flinching was judged as a positive reaction. The initial bending force was 0.4 g and the stimulus intervals were at least 15 s. The 50% paw withdrawal threshold was calculated according the “up and down” method [24]. The paw withdrawal frequency was measured using a calibrated von Frey filament (0.4 g) which was perpendicularly put into the central plantar surface ten times, and was recorded as the number of withdrawal responses/10 × 100%.

2.6. Paw thermal withdrawal latency

Paw withdrawal thermal latency (PTL) assessed thermal hyperalgesia utilizing a plantar thermal pain tool (Ugo Basile, 07370, ITA). In brief, mice were separately habituated to the plexiglass compartments placed on the elevated glass plate for 30 min prior to the trial. In the right rear paw of mice, the radiant thermal stimulator was inserted below the glass palate underneath the plantar surface. Three heat stimuli were delivered 5 min apart. When the hind paw lifted, the stimulator was switched off, and the timer was terminated automatically. PTL was described as the average time to the endpoint. A time limit of 20 s was specified to prevent tissue harm.

2.7. Flinching behavior test

Spontaneous pain was assessed using the number of spontaneous flinches [21]. Mice were put in chambers on the elevated glass plate. Flinching behavior was described as the number of times the mouse kept the entire hind limb aloft, unrelated to walking or grooming in 10 min.

2.8. Histology

After the behavior test at POD20, the lumbar spinal cord (5 mm sections, n = 4 per group) was retrieved and preserved. Cancer cell growth in the femur was assessed by standard H&E staining. Spinal cord tissue was cut at 10 μm thick on a cryostat after post-fixation with 4% paraformaldehyde (PFA), 0.1 M PBS for 2 h, and cryoprotected in 30% sucrose in PBS at 4 °C overnight. The slices were stained with 1% thionin. Counts from Nissl-stained sections are expressed as the average count of positive cells per high magnification ( × 400) field of view. Stained slides were scanned by a high-resolution bright field slide scanner (Pannoramic P1000, 3DHistech Ltd., Budapest, Hungary). Immunohistochemistry was performed applying the primary antibodies rabbit anti-GAD65 (Abcam Cambridge, MA, ab239372, 1:200) at 4 °C overnight, and the secondary antibody goat anti-rabbit Alexa Fluor 488 (Abcam Cambridge, MA, ab150077, 1:200) for 2 h at room temperature. Images were acquired using a Leica DM5000B microscope (Leica Biosystems, Wetzlar, Germany).

2.9. Western blot

The spinal cord was quickly dissected and immediately frozen on dry ice. The frozen sample was retrieved and homogenized using RIPA lysis buffer (10 μL/mg tissue, supplemented with a protease inhibitor cocktail (Biotool Lab, Shanghai) at 4 °C. After homogenization, protein concentration was determined using BCA method, and 50 μg protein was loaded per lane, and separated using 10% polyacrylamide gels (Genscript, catalog M0012C) electrophoresis. Proteins were transferred to Immun-Blot PVDF membranes (Millipore, MA), and incubated with the primary antibodies rabbit anti-4-HNE (Abcam, ab46545, 1:1000), rabbit anti-pERK (CST, cat: 4370, 1:2000), rabbit anti-ERK (CST, cat: 4695, 1:2000) rabbit anti-GPX4 (Abcam, ab125066, 1:1000), rabbit anti-COX2 (Abcam, ab179800, 1:1000), rabbit anti-GAD65 (Abcam, ab239372, 1:500) or the reference rabbit anti-β-tubulin (Cell signaling technology, MA, cat: 2128, 1:3000) for 2h at room temperature. Secondary antibodies were anti-rabbit antibodies conjugated with HRP (1:5000, Jackson ImmunoResearch Laboratories West Grove, PA, cat: 615-035-214). Protein bands were revealed by enhanced chemiluminescence (Millipore) and quantified utilizing Image Lab software (Universal Hood III, Bio-Rad, Hercules). The optical protein band densities of all detected proteins were normalized to the corresponding optical protein band intensity of the reference protein β-tubulin.

2.10. Iron analysis

The iron level was assessed in the spinal tissue lysates of each group using a Total Iron Colorimetric Assay Kit according to the manufacturer's instructions (E-BC-K139-S, Elabscience, Wuhan, China).

2.11. ROS, SOD, malonaldehyde, and glutathione activity assays

ROS, superoxide dismutase (SOD), Glutathione (GSH), and malondialdehyde (MDA) concentrations were assessed using commercial kits according to the instructions of the manufacturer (E004-1-1 for ROS, A001-3 for SOD, A006-2-1 for GSH, and A003-1 for MDA, Jiancheng Biology, Nanjing, China).

2.12. Transmission Electron Microscopy

Transmission Electron Microscopy (TEM) was utilized to examine morphology of mitochondria in the spinal cord. Mice were perfused with 0.1 M PBS, 4% PFA, and 2% glutaraldehyde under deep anesthesia and the lumbar spinal cords were excised. Spinal tissue samples were cut to 100 nm-thick ultrathin slices and double-stained with uranyl acetate and lead citrate. Ultrathin slices were examined using a JEM1400 TEM (JEOL, Tokyo, Japan).

2.13. Statistical analysis

Values are expressed as mean ± SEM. Differences in behavioral parameters were calculated over the course of time using two-way variance analysis (ANOVA) with repeated measurements and post hoc Bonferroni testing. In other trials, comparisons between groups were conducted using one-way ANOVA and the post hoc Bonferroni test. Differences between two groups were tested using two-tailed unpaired Student's t-test. The percentage of normal appearing mitochondria was calculated in different treatment groups and analyzed using Chi-square test or Fisher's exact test. Graphpad Prism version 7 (San Diego, CA, USA) was used for statistical analysis, and P < 0.05 was considered significant.

3. Results

3.1. Tumor-bearing mice develop mechanical and therma l hyperalgesia and spontaneous pain

Luciferase-expressing LLC-Luc cells were inoculated into the femur of male C57BL/6J mice to establish the BCP model. First, to validate tumor development, the luciferase signal was detected by in vivo bioluminescence from POD5 to POD20 (Fig. 1A). Next, the femur of mice was examined after HE staining at POD20 showing heterotypic cells infiltrating the marrow cavity (Fig. 1B). To study consequences on sensation behavior, mice were subjected to mechanical or thermal plantar stimulation. In tumor-bearing mice, robust mechanical allodynia and thermal hyperalgesia were observed from POD5 to POD20 (Fig. 1C–E). Higher spontaneous rear limb elevation was also recorded in the tumor-bearing mice from POD5 to POD20, confirming that spontaneous pain was induced by cancer induction (Fig. 1F). Sham-operated mice did not display differences in pain sensitivity at any time point. Altogether, the results validated our mouse model of BCP.

Fig. 1.

Fig. 1

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Tumor-bearing mice develop mechanical and thermal hyperalgesia and spontaneous pain (A) In vivo bioluminescence imaging showing significant tumor cell growth in the femur 10 days after intrafemoral inoculation of LLC-Luc cells. (B) H&E staining showing structural bone destruction and infiltration of the marrow cavity by inoculated LLC-Luc cells in BCP mice as compared to sham mice which received heat-killed LLC-Luc cells. (C–F) Examination of mechanical and thermal pain behavior using von Frey test to determine PWT (C) and frequency (D) and Hargreaves tests to determine heat hyperalgesia (E) and spontaneous lifting of the right hind limb (F) in BCP mice and sham mice at the specified time points. BCP mice exhibited increased mechanical or thermal hypersensitivity and spontaneous pain onset on POD5 as compared to sham mice which did not show pain behavior. Data are expressed as the mean ± SEM and analyzed using two-way repeated-measures ANOVA with post hoc Bonferroni test; *P < 0.05, **P < 0.01, ***P < 0.001; n = 8 per group, scale: 200 μm (B).

3.2. Neuronal loss and ferroptosis in the spinal cord of BCP mice

We examined spinal cord sections after Nissl-staining at POD20 and found a significantly reduced number of Nissl-positive cells in BCP mice as compared to control mice (Fig. 2A−B). Next, anti-GAD65 immunostaining was performed to test whether the cell loss in BCP mice can be attributed to GABAergic interneurons as described before [9]. In agreement with these previous findings, the number of GAD65-positive neurons was significantly reduced in the dorsal horn of BCP mice (Fig. 2C–D).

Fig. 2.

Fig. 2

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Neuronal loss and ferroptosis in the spinal cord of BCP mice at POD20. (A) Nissl staining of spinal cord samples showing the dorsal horn. (B) Decreased number of Nissl positive cells in the dorsal horn of BCP mice as compared to sham mice (n = 4 per group). (C) Immunostaining of spinal cord at POD20 using an antibody specific for the GABAergic interneuron marker GAD65. (D) Decreased number of GAD65 positive cells in the dorsal horn of BCP mice as compared to sham mice (n = 4 per group). (E) Histograms showing ROS analyzed by DCFH-DA fluorescence, and activity levels of MDA and SOD. ROS and MDA levels were increased, whereas SOD levels were decreased in BCP mice as compared to sham mice (n = 8 per group). Data are shown as mean ± SEM and were analyzed by two-tailed unpaired Student's t-test. *P < 0.05, **P < 0.01. (F) Representative TEM images of mitochondria in spinal neurons of sham and BCP mice (red arrowheads, increased electron density of the bilayer membrane of classical shrunken mitochondria with decreased mitochondrial cristae and mitochondrial membrane rupture; blue arrowheads, reduced/vanishing cristae; black arrowheads, outer membrane rupture) (G) BCP mice show an increased percentage of shrunken mitochondria than sham mice (n = 4 per group). Data are shown as mean ± SEM and were analyzed by Fisher's exact test. *P < 0.05, **P < 0.01, ***P < 0.001. Scales: 200 μm (A, D), 1 μm (F). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)ences to colour in this figure legend, the reader is referred to the Web version of this article.)

We then examined ferroptosis-related oxidative stress, i.e. ROS and Fe2+-dependent lipid peroxidation. We found that BCP was accompanied by increased spinal activity levels of both, ROS, as determined by DCFH-DA fluorescence, and MDA, an end-product of lipid peroxides. Conversely, the activity of neuroprotective SOD was reduced in spinal cords from BCP mice compared to sham (P < 0.01, Fig. 2E).

Ferroptosis is characterized by morphological alterations of mitochondria. Indeed, ultrastructural analysis of mitochondria using TEM revealed more shrunken mitochondria with increased electron density of the bilayer membrane, decreased mitochondrial cristae or rupture of outer mitochondrial membrane of spinal cord neurons of BCP mice on POD20 (Fig. 2F). The quantitative data showed that the percentages of shrunken mitochondrial were increased in BCP mice (Fig. 2G). Distinguished from other forms of cell death, ferroptosis is associated with shrunken mitochondria [25]. These characteristic morphological changes of mitochondria suggested the occurrence of ferroptosis in spinal neurons in the BCP model.

3.3. FER-1 attenuates ferroptosis in the spinal cord of BCP mice

To study the druggability of spinal ferroptosis in BCP, we applied a pharmacological approach using the ferroptosis inhibitor FER-1 [23,25]. FER-1 was administered for 20 consecutive days (10 mg/kg) after LLC-Luc inoculation of LLC-Luc cells into the femur. In ferroptosis, the accumulation of lipid peroxidation products requires abundant iron [25]. Determination of spinal cord iron levels showed increased iron deposition in BCP mice and attenuated iron deposition after FER-1 therapy (Fig. 3A). 4-HNE has been implicated as a key mediator of oxidative stress-induced cell lipid peroxidation [26]. We determined 4-HNE modified protein expression in the spinal cord. BCP mice showed elevated 4-HNE levels with the typical pattern of 4-HNE protein adduction comprising various proteins with different molecular weights (Fig. 3B). Western blot data revealed that the expression of GPX4 was downregulated in the spinal cord of BCP mice at POD20 (Fig. 3C). BCP mice also showed reduced levels of the antioxidant GSH detected with a GSH assay Kit (Fig. 3D). Accordingly, FER-1 therapy prevented potentially pathogenic alterations in the levels of these biochemical markers of ferroptosis (Fig. 3B–D).

Fig. 3.

Fig. 3

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FER-1 attenuates ferroptosis in the spinal cord of BCP mice at POD20. (A) Quantitative analysis of iron content in the spinal cord of FER-1- or Vehicle-treated mice (n = 8 per group). (B) Representative western blot using anti-4-HNE and quantitative analysis of band intensities showing decreased levels of 4-HNE in the spinal cord of BCP mice after FER-1 treatment as compared to Vehicle treatment (n = 4 per group). (C) Representative western blot using anti-GPX4 and quantitative analysis of protein band intensities showing that FER-1 treatment prevented the increase of GPX4 in the spinal cord of BCP mice (n = 4 per group). (D) Histogram showing that FER-1 prevented the reduction of GSH in the spinal cord of BCP mice (n = 8 per group). (E) Representative western blot using anti-pERK and quantitative analysis of protein band intensities showing that FER-1 treatment prevented increased phosphorylation of ERK in BCP mice (n = 4 per group). (F) Representative western blot using anti-COX-2 and quantitative analysis of protein band intensities showing that FER-1 prevented increased COX-2 levels in BCP mice (n = 4 per group). Data are shown as mean ± SEM and were analyzed by One-way ANOVA and the post hoc Bonferroni test. *P < 0.05, **P < 0.01. (G) TEM images showing shrunken mitochondria in spinal neurons (arrows). (H) BCP mice treated with FER-1 displayed a lower percentage of shrunken mitochondria than BCP mice treated with vehicle and a similar percentage as compared to sham mice (n = 4 per group). Data are shown as mean ± SEM and were analyzed by Chi-square test. *P < 0.05, **P < 0.01, ***P < 0.001. Scale: 1 μm (G).

Phospho-ERK1/2 and COX-2 are considered markers for neural ferroptosis in neurodegenerative diseases and traumatic brain injury [14,27]. Therefore, we determined the levels of p-ERK1/2 and COX-2 by western blot. Both markers were significantly up-regulated in BCP mice as compared to control mice and FER-1 treatment prevented this effect (Fig. 3E–F). Moreover, the appearance of shrunken mitochondria in spinal neurons was clearly reduced after FER-1 treatment (Fig. 3G–H). These results demonstrated that FER-1 effectively attenuated spinal ferroptosis in BCP mice.

3.4. Inhibition of ferroptosis prevents the loss of GABAergic interneurons in the spinal cord of BCP mice

As the loss of GABAergic interneurons is a likely cause for BCP [9] and the reduction of GAD65 was associated with the loss of spinal interneurons and associated GABAergic inhibition [6], we examined the number of GAD65+ neurons in the spinal dorsal horn using immunohistochemistry. Treatment with FER-1 effectively inhibited the loss of GAD65+ neurons in BCP mice as compared to vehicle treatment (Fig. 4A–B). Furthermore, the changes of GAD65 expression in the BCP mice observed in immunofluorescence staining were confirmed by western blot analysis (Fig. 4C and D). These results indicated that inhibition of spinal ferroptosis prevented the loss of GABAergic interneurons in BCP mice.

Fig. 4.

Fig. 4

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Inhibition of ferroptosis prevents the loss of GABAergic interneurons in the spinal cord of BCP mice at POD20. (A) Images showing anti-GAD65 immunostaining in the spinal dorsal horn of sham mice, and BCP mice treated with Vehicle or FER-1. (B) Histogram showing the number of GAD65 positive spinal interneurons. FER-1 treatment of BCP mice prevented the loss of GAD65 positive spinal interneurons (n = 4 per group). (C–D) Examples and summary data of western blots showing that the decreased expression of GAD65 in the spinal cord of the BCP mice were prevented by intraperitoneal injection of FER-1 (n = 3 per group). Data are shown as mean ± SEM and were analyzed by one-way ANOVA and post hoc Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001. Scales: 200 μm (A).

3.5. FER-1 alleviates mechanical and thermal pain behavior independent of tumor burden in BCP mice

Compared to vehicle, the treatment of FER-1 reduced hind-paw mechanical allodynia and heat hyperalgesia from POD10 to POD20 (Fig. 5A–C). Furthermore, FER-1 also decreased the frequency of spontaneous hind limb lifting on days 10, 15, and 20 after LLC-Luc cell inoculation (Fig. 5D). We also assessed whether local tumor development was affected after FER-1 treatment. Bioluminescence imaging in vivo taken at POD 20 revealed that FER-1 treatment did not affect the total flux of tumor-bearing femurs compared to vehicle (Fig. 5E–F). Together, the results demonstrated that inhibition of ferroptosis attenuated the exaggerated pain behavior in our mouse model of BCP but did not affect the tumor burden.

Fig. 5.

Fig. 5

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FER-1 alleviates mechanical and thermal pain behavior independent of tumor burden in BCP mice at POD20. (A–D) Examination of mechanical and thermal pain behavior using von Frey test to determine PWT (A) and frequency (B) and and Hargreaves tests to determine heat hyperalgesia (C) and spontaneous lifting of the right hind limb (D) in sham mice, BCP mice treated with Vehicle or FER-1 (n = 8 per group). (E–F) In vivo bioluminescence imaging 15 min after intraperitoneal injection of D-luciferin sodium salt showing that FER-1 treatment did not affect tumor burden in BCP mice (n = 6 per group). Data are shown as mean ± SEM and were analyzed by two-way ANOVA and post hoc Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001.

3.6. FER-1 potentiates analgesic effects of parecoxib

NSAIDs are the first line analgesics for cancer pain. However, for terminal BCP，the analgesic effect of NSAIDs are diminished and often requires a combination of GABAergic agonists or opioids [1]. Since inhibition of spinal ferroptosis prevented the loss of the inhibitory tone of spinal cord in BCP, we investigated the analgesic effect of NSAIDs in BCP mice. To this end, Parecoxib (40 mg/kg), or an equal volume of normal saline, was intraperitoneally injected into the BCP + FER-1 and BCP + Vehicle mice on POD20. Behavioral assays were performed 1 h after parecoxib or normal saline injection. A dose of 40 mg parecoxib showed a negligible analgesic effect on Vehicle-treated BCP mice. In contrast, parecoxib significantly reduced PWMT, PWTL, and spontaneous lifting of BCP mice treated with FER-1 (Fig. 6A–D). Thus, FER-1 may potentiate analgesic effects of parecoxib.

Fig. 6.

Fig. 6

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FER-1 potentiates analgesic effects of parecoxib in BCP mice. Effects of parecoxib (40 mg/kg) on BCP mice were assessed at POD20. (A) compared with normal saline injection, Parecoxib 40 mg/kg has a non-significant effect on PWMT (A) in BCP + Vehicle mice, but it significantly increases PWMT in BCP + FER-1mice; parecoxib also decreased paw withdrawal frequency, (B) thermal hypersensitivity, (C) and spontaneous lifting times (D) in BCP mice treated with FER-1 (10 mg/kg) (n = 8 per group). Data are shown as mean ± SEM and were analyzed using Student's t-test. *P < 0.05, **P < 0.01.

4. Discussion

In this study, initial characterization of the BCP model demonstrated the development of increased pain behavior concomitantly with the loss of GABAergic neurons in the dorsal horn of spinal cord, and the occurrence of hallmarks of ferroptosis including iron accumulation, lipid peroxidation, COX-2 upregulation, increased levels of p-ERK1/2, and morphological alterations of mitochondria [13]. The major finding of this study is that FER-1 prevented pathogenic features associated with ferroptosis and alleviated BCP. Furthermore, FER-1 treatment facilitated analgesia with Parecoxib which was ineffective in vehicle-treated BCP mice. Overall, our results underscore the potential value of therapies targeting ferroptosis in BCP.

Spinal neuronal loss is a major process correlated with the onset of chronic pain [28,29]. As the major inhibitory interneurons in the dorsal horn, the death of GABAergic interneurons and the change in afferent excitability can lead to increased nerve excitability, and this “disinhibition” may improve signal transmission of neurons responsible for nociceptive projection, resulting in the occurrence of hyperalgesia and abnormal pain sensation [6,10]. Our research revealed that the number of GAD65 positive neurons in the dorsal horn of BCP mice was reduced, suggesting that GABAergic interneurons were lost. This is consistent with previous studies on BCP models [9,30] and suggests that the loss of GABAergic interneurons contributes to the enhancement of persistent bone cancer pain due to an impaired inhibitory tone. The specific mechanisms underlying the loss of GABAergic interneurons is not completely understood. However, it has become clear that GABAergic inhibitory interneurons have higher energy requirements and are particularly sensitive to oxidative stress, hypoxia and glutamate accumulation as compared with other types of neurons [31,32]. For example, cortical parvalbumin-positive interneurons, responsible for maintaining cortical inhibitory tone, are particularly vulnerable to oxidative stress and are thus disproportionately affected by brain injury [33]. In the spinal cord, interneurons degenerate prior to motoneurons in ALS-like model mice due to oxidative stress [34]. Along this line, repeated antioxidant treatment with a ROS scavenger, phenyl N-t-butylnitrone, in mice with the spinal nerve ligation model of neuropathic pain reduced pain behavior and attenuated the loss of GABAergic neurons in neuropathic pain condition [28]. These findings suggest that oxidative stress may account for the loss of GABAergic neurons in various neurological conditions. We found that the ROS homeostasis was unbalanced in the spinal cord of mice with BCP. Therefore, the loss of spinal cord GABAergic neurons in BCP might be related to oxidative stress.

Ferroptosis is a regulated cell death type driven by accumulation of ROS induced by excessive Fe2+ and subsequent lipid peroxidation which are closely related to oxidative stress. Various processes, but particularly iron overload associated with the loss of GPX4 activity and subsequent accumulation of iron, may contribute to ferroptosis. Indeed, iron overload plays a specific role in ferroptosis, generates ROS through the Fenton reaction and initiates liposome peroxidation [35]. FER-1 prevents spinal iron accumulation has previously been observed in several pathophysiological processes, including peripheral nerve injury induced by chronic constriction injury [15,19,36]. As expected, we detected a significant increase in spinal iron levels in BCP mice. Importantly, administration of FER-1, which forms a complex with iron in the cellular labile iron pool and reduce alkoxyl radicals produced by iron dependent lipid hydroperoxides [37], reduced spinal iron deposition, indicating that iron accumulation contributes to the process of BCP. As mentioned, GPX4 plays an important role in ferroptosis because it prevents detrimental phospholipid oxidation [35]. Excessive amounts of free iron in ferroptosis-induced cells compromises the activity of GPX4 activity which results in a reduction in GSH and increases membrane lipid peroxidation and oxidative stress [16]. Accordingly, the buildup of lipid ROS is mainly attributed to loss of GPX4 actions, which inhibits the synthesis of anti-oxidative glutathione and elevates levels of 4-HNE [38]. In the present study, lipid peroxidation induced oxidative stress was evidenced by increased levels of 4-HNE and downregulation of GPX4 and GSH. These results are consistent with previous findings showing that lipid peroxidation and oxidative stress contributed to the persistence of chronic pain [18,39]. Moreover, FER-1 administration not only prevented iron accumulation, but also the reduction of GPX4 levels and alleviated lipid peroxidation induced by BCP. Various compounds can directly or indirectly affect GPX4 activity via the MAPK signaling pathway, among which the ERK pathway is closely related to ferroptosis in neural cells [14]. p-ERK expression can be induced by noxious stimulation or tissue injury. We discovered a significant ERK stimulation in the spinal cord of animals with BCP. Therefore, ERK activation is considered an indicator of ferroptosis and neuron activation [40]. COX-2 is a major inducible enzyme that mediates pain and inflammation [41]. COX-2 is also a biomarker for ferroptosis and it is induced and produced in the presence of multiple inducing variables, including growth factors and cytokines [42]. In the present study, p-ERK and COX-2 levels were significantly downregulated after the treatment of FER-1. We therefore propose that spinal ferroptosis is related to COX-2 activation and ERK pathway in BCP.

Additionally, we observed that BCP altered mitochondrial morphology as characterized by condensed membrane densities and smaller volumes. The mitochondrial changes observed in our study are similar to those previously described [20,43]. Ferroptosis inhibitor FER-1 may protect mitochondria from BCP induced aberrant morphological changes. Therefore, the mitochondrial morphological changes provided further evidence that ferroptosis indeed occurred in the spinal cord of BCP mice.

Taken together, our results showed preventive effects of FER-1 on hallmark features of ferroptosis and BCP including the loss of GABAergic neurons. We propose that inhibition of GPX4 deficiency-induced lipid peroxidation and oxidative stress induced by iron overload in the process of spinal ferroptosis is of great significance in the pathogenesis of BCP.

Previous studies also showed that ferroptosis is a critical regulator of tumor growth [43]. However, the role of ferroptosis in lung cancer remains to be explored. Recently, it was reported that ferroptosis contributed to erianin-induced cell death in H460 or H1299 lung cancer cells, and pretreatment with FER-1 reduced erianin-induced cell death and suppressed lung cancer cell migration [44]. In our study, the bioluminescence imaging revealed that inhibition of ferroptosis by FER-1 had no effect on the tumor burden of metastatic lung cancer cells in male BCP mice. This is different from the study of Peng Chen et al. [44], and may be related to the different lung cancer cell line used in the present study.

Furthermore, it has been shown that estrogen may also affect the growth of lung cancer in a lung tumor model using LLCs [45]. Therefore, we only used male mice in this study, to avoid an effect of estrogen fluctuations on tumor growth in our model. This represents a limitation of this study, and the inclusion of both male and female animals would be an important objective for future studies to understand whether sex-related factors, such as estrogen, affect the pharmacological inhibition of ferroptosis in BCP.

Clinical management of BCP needs to be flexible depending on the stage of the disease due to its dynamic and complex mechanisms. At the early stage of BCP, as tumor's growth is accompanied by pronociceptive factors like Prostaglandin E2, the catalytic product of COX-2 and NSAIDs are commonly prescribed at this stage. When growing tumors cause pain by damaging the surrounding nerves and the related central sensitization forms, drugs used for neuropathic pain such as Gabapentin, a derivative of GABA, may provide improved pain relief. In later stages pain may stem from different mechanisms, requiring different drugs [46]. World Health Organization recommends the three-ladder analgesic drug treatment for cancer pain. NSAIDs run through the whole ladder, however, their analgesic effects lower as the tumor progresses in cancer pain [1,47]. We observed that injection of the NSAID COX-2 inhibitor parecoxib did not relieve late stage hyperalgesia in BCP mice, in accordance with other studies [47,48]. In contrast, combined with FER-1, parecoxib enhanced analgesia in BCP. This result suggests that FER-1 helped to maintain the inhibitory function of spinal cord and reduced its sensitization and inflammation in BCP mice, which improved the efficacy of NSAIDs in advanced cancer pain.

This study had several limitations. First, an aforementioned limitation is that only male mice were used in this study. Second, ferroptosis is probably not the sole mechanisms of cell death as other types of spinal neuron death have been associated with BCP [9,49]. Third, the systemic administration regime for FER-1 may have led to non-characterized peripheral effects or effects in CNS regions others than the spinal cord that are involved in pain behavior.

5. Conclusion

Utilizing a validated murine model, we identified a role of ferroptosis in BCP. Pharmacological inhibition of ferroptosis significantly reduced hyperalgesia and spontaneous pain, while enhancing the analgesic action of the NSAID parecoxib. Therefore, pharmacological treatment of ferroptosis in the spinal cord may represent a promising option to alleviate BCP.

Funding

This work was supported by National Natural Science Foundation of China (81901143 to Z.D.，82071249 and 81771207 to C.H., 82071248 to Z.S.，82001195 to J.W.) and Nature Science Foundation of Hunan Province (2022JJ30956 to Z.S.).

Author contributions

ZD and XS performed experiments, analyzed data, prepared figures and drafted the manuscript. MS participated in the study design and manuscript writing. JW and ZS performed experiments, analyzed data. QG and CH designed and supervised the experiments, and edited the manuscript. All authors read and approved the final manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2023.102700.

Appendix A. Supplementary data

The following is the Supplementary data to this article.

Fig.S1.

Fig.S1

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Data availability

Data will be made available on request.

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