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INTRODUCTION
Histone deacetylase (HDAC) is an enzyme that hydrolyzes the acetyl-L-lysine side chain in the N-terminal region of histones. Among the various HDAC subtypes, histone deacetylase 6 (HDAC6) is unique due to its dual catalytic domains and classification in the IIb group [1]. HDAC6 is instrumental in tumor progression and metastasis by modulating the stability of microtubules, which are vital for the cell cycle [2]. HDAC6 expression is upregulated in tumor cells, and HDAC6 inhibitors exert anticancer effects in vitro and in vivo by increasing intracellular α-tubulin acetylation [3].
Food and Drug Administration (FDA)–approved chemotherapy drugs for solid tumors include platinum-derived agents, proteasome inhibitors, taxanes, and vinca alkaloids. However, chemotherapy commonly induces peripheral neuropathy, characterized by nerve damage, and is known as chemotherapy-induced peripheral neuropathy (CIPN) [4–6].
Platinum-based chemotherapeutic agents are widely employed in cancer treatment due to their efficacy against various malignancies. However, their clinical use is often limited by the development of severe peripheral neuropathy, a debilitating side effect characterized by nerve damage and impaired sensory function. Cisplatin and oxaliplatin are two commonly used platinum-based drugs that are particularly notorious for inducing peripheral neuropathy. Cisplatin exhibits potent neurotoxicity, leading to alterations in sensory perception and bilateral degenerative changes in peripheral nerves. This neurotoxicity can significantly impair patients' quality of life and may even necessitate discontinuation of treatment [7–10]. Oxaliplatin also induces peripheral neuropathy through distinct mechanisms. It has been shown to increase mitochondrial biogenesis in peripheral nerves and activate astrocytes in the spinal cord. This astrocyte activation is associated with increased gap junction formation, leading to heightened pain sensitivity [11].
CIPN affects a significant portion of patients undergoing chemotherapy, occurring in 30%–80% of patients and severely negatively impacting quality of life of patients, often necessitating treatment discontinuation [6,12–15].
Bortezomib, a leading proteasome inhibitor, is a first-line therapy for multiple myeloma and targets the ubiquitin-proteasome pathway. In a phase 3 clinical trial involving patients with relapsed refractory multiple myeloma, 75% of the participants treated with bortezomib experienced grade 3/4 adverse events, with 8% developing peripheral neuropathy [16]. Bortezomib-induced peripheral neuropathy is not dose-dependent but age-dependent, with the risk increasing by 6% per year. HDAC6 inhibitors serves as an alternative multiple myeloma treatment to prevent bortezomib-induced peripheral neuropathy [17].
Currently, there are no FDA-approved treatments for CIPN, although anti-depressant drugs such as Duloxetine are used to manage symptoms. HDAC6 inhibitors could offer therapeutic benefits for CIPN [18,19].
Consequently, the aim of this study was to evaluate the therapeutic potential of novel HDAC6 inhibitors in ameliorating CIPN.
MATERIALS AND METHODS
1. Reagents
Chemotherapy drugs, including oxaliplatin, cisplatin, and paclitaxel were purchased from Tokyo Chemical Industry. The HDAC6 inhibitor ACY-1215 (Ricolinostat) was purchased from Selleckchem.
2. Animals
The animal study protocol was approved by the Institutional Animal Care and Use Committee at Inje University College of Medicine of Korea (2016-046).
Six- to 8-week-old male Sprague Dawley rats were obtained from Orient. The animals were maintained in a controlled laboratory environment with a consistent 12-hr light/dark cycle and provided unrestricted access to food and water. A CIPN rat model was established via chemotherapy drug administration. Subsequently, the degree of induction was confirmed using the von Frey filament test.
3. Drug administration
Bortezomib (TOKYO Chemical Industry), a proteasome inhibitor used as an anti-cancer drug, was used to establish the anticancer drug-induced CIPN model. Bortezomib was diluted with phosphate-buffered saline and administered intraperitoneally four times (Days 0, 2, 4, and 6) at 0.2 mg/kg. Oxaliplatin (5 mg/kg) was intraperitoneally administered on days 0, 3, and 6. Paclitaxel (8 mg/kg) was intraperitoneally administered on days 0, 2, 4, and 6. Cisplatin (3 mg/kg) was intraperitoneally administered on days 0, 6, 13, and 20. CKD-011 (developed by CKD pharm.), an HDAC6 inhibitor, was dissolved in saline and administered intraperitoneally once a day at doses of 5, 10, 20, and 40 mg/kg. Gabapentin (Tokyo Chemical Industry) was dissolved in saline and administered intraperitoneally at a dose of 60 mg/kg.
4. Measurement of mechanical hyperalgesia
This test was conducted with a dynamic plantar aesthesiometer (Ugo Basile). Following each drug administration, the rat was placed in a wire mesh-enclosed box and allowed to acclimate for a minimum of 15 minutes. As the rat adapted to the box, the strength of the plastic filament (0.5 mm in diameter) in the hind paw was gradually increased and a von Frey stimulus was applied. The intensity of the stimulus was increased from 1 g/sec by 2.5 g/sec, to a final 50 g/sec. The stimulus intensity was gradually increased until the animal exhibited a withdrawal response, which was recorded in grams (g) and seconds (sec), and the results were statistically analyzed [20].
5. Electron microscopy
Electron microscopy was performed for histopathological analysis. After drug administration, rat sciatic nerves were harvested and prepared into tissue sections for electron microscopy. Sciatic nerves were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and subsequently post-fixed in 1% osmium tetroxide. The samples were then dehydrated through a graded ethanol series and embedded in epoxy resin. Ultrathin sections (< 100 nm) were prepared using an ultramicrotome and mounted onto Formvar/carbon-coated grids to provide adequate support during imaging. Transmission electron microscopy was conducted using an H-7600 (Hitachi). Sciatic nerve damage was evaluated using a four-grade scoring system. Grade 0 (normal) was defined as intact axons with uniformly thick myelin sheaths. Grade 1 (mild damage) was characterized by axonal swelling or mild myelin thinning. Grade 2 (moderate damage) involved partial demyelination, irregular myelin thickness, and axonal shrinkage. Grade 3 (severe damage) was marked by complete axonal disintegration, total myelin loss, extensive extracellular debris, and disrupted Schwann cell morphology [20–23].
6. Histone extraction
The MM.1s cell line, a human multiple myeloma cell line obtained from ATCC, is widely used in research on multiple myeloma. For histone isolation, MM.1s cells were harvested 24 hours after drug reaction. Next, the cell pellet was resuspended in IP Lysis buffer (100 mM Tris-HCl at pH 7.5, 500 mM NaCl, 0.2% NP40, and 10 mM EDTA) and incubated on ice for 5 minutes. Following incubation, the cells were centrifuged at 10,000 × g and 10 minutes, and the supernatant was stored at –80°C. Histones were precipitated from the pallet, resuspended in TE buffer (10 mM Tris-HCl at pH 7.4 and 13 mM EDTA) and centrifuged at 600 × g for 5 minutes. After removing the supernatant, 0.4N H2SO4 was added, and the mixture was continuously rotated using a rotary mixer overnight at 4°C. Subsequently, the pallet was washed with acetone and air-dried. The histones were resuspended in 500 μL distilled water and quantified using bicinchoninic acid analysis.
7. Western blot analysis
Proteins and histones were separated by 4%–12% gradient SDS-PAGE using protease inhibitor (Roche)-containing RIPA buffer (Sigma, R0278) and transferred to a nitrocellulose membrane. The membranes were blocked with Tris-buffered saline containing 0.1% Tween 20 and 3% bovine serum albumin for 1 hour. Subsequently, the membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies at 37°C for 1 hour. The primary and secondary antibodies used in the study are as follow: acetylated tubulin (1:2,000, Sigma, T6793), alpha tubulin (1:2,000, Cell Signaling, 2144), ubiquitin (1:2,000, Enzo Life Sciences, BML-PW8810-0500), GAPDH (1:2,000, Thermo Fisher Scientific, PA19046), Acetyl-Histone H3 (1:2,000, Cell Signaling, 9649), and Histone H3 (1:2,000, Cell Signaling, 3638). For signal visualization, using the ChemiDocTM MP Imaging System (Bio-Rad) and the ECL validation kit (GE Healthcare).
RESULTS
1. HDAC6 inhibitor effectively reversed peripheral neuropathy induced by bortezomib
In this study, HDAC6 inhibitor activity was measured using α-tubulin acetylation. Acetylation levels of α-tubulin were measured following treatment of multiple myeloma cell, MM1.s, with various concentrations of the selective HDAC6 inhibitors CKD-011 and ACY-1215 (Ricolinostat). Acetylated tubulin levels increased at 5 nM (Fig. 1). Peripheral neuropathy significantly improved in the 40 mg/kg CKD-011 group (Fig. 2). In addition, CKD-011 administration (once daily for 15 days) significantly improved peripheral neuropathy at all doses (Fig. 2). These findings indicate that CKD-011 is a promising therapeutic option for managing bortezomib-induced peripheral neuropathy in the treatment of multiple myeloma.
2. HDAC6 inhibitor serves as a potential treatment for platinum-based drug-induced CIPN
The authors found that peripheral neuropathy was alleviated by CKD-011. In addition, by observing rat sciatic nerve tissue sections under an electron microscope, it was found that CKD-011 reversed axon and myelin damage. The efficacy of CKD-011 was confirmed to be similar to that of the control gabapentin (60 mg/kg) in a cisplatin-induced peripheral neuropathy rat model and oxaliplatin-induced peripheral neuropathy rat model (Figs. 3, 4) [24,25]. The authors found that CKD-011 alleviated peripheral neuropathy three hours after the last administration.
3. HDAC6 inhibitor, CKD-011, is a suitable treatment for paclitaxel-induced peripheral neuropathy
Paclitaxel induces changes in mitochondrial morphology and function, and disrupts axonal transport through inflammation. These pathological changes result in a loss of nerve fibers [26,27]. In the rat model, it was confirmed that paclitaxel-induced CIPN was alleviated by CKD-011. The effect of CKD-011 was comparable across different doses (5 mg/kg and 10 mg/kg; Fig. 5). In addition, the effect of CKD-011 was comparable to that of gabapentin (60 mg/kg), which was used as a positive control (Fig. 5). Rat weight was measured 24 hours after the last administration; a significant weight loss was observed in the CKD-011 10 mg/kg administration group (Fig. 5E).
4. Histopathological analysis
Analysis of rat sciatic nerve tissue sections confirmed that CKD-011 repaired axon and myelin damage. The authors found that CKD-011 administration significantly alleviated bortezomib-induced peripheral neurotoxicity. A decrease in axonal degeneration was observed starting from CKD-011 10 mg/kg, and statistical significance was observed in the 20 mg/kg and 40 mg/kg dose groups. The alleviation of myelin degeneration did not reach statistical significance; however, at doses of 20 and 40 mg/kg, the damage decreased to a level similar to that observed in the solvent group. Collectively, CKD-011 administration significantly reduced axonal and myelin damage induced by bortezomib (Fig. 6A, E, F) Furthermore, CKD-011 also reduced peripheral neurotoxicity induced by other platinum-based drugs like oxaliplatin and cisplatin, particularly showing excellent efficacy against cisplatin-induced neurotoxicity (Fig. 6B, C, G–J). However, in paclitaxel-induced peripheral neurotoxicity, CKD-011 did not significantly restore axonal and myelin damage (Fig. 6D, K, L).
DISCUSSION
HDAC6 inhibitors have been suggested as potential therapies for neurodegenerative diseases and are being considered as candidates for the treatment of CIPN. HDAC6 inhibitors act as therapeutic agents for neurodegenerative diseases by regulating key proteins involved in cell migration, alleviating cell damage, and recovering nerve damage and pain [28–30]. Acetylated tubulin formation was confirmed at the cellular level to confirm that CKD-011 effectively inhibits HDAC6, and it was found that CKD-011 exhibits excellent activity even at low doses.
CIPN is a side effect of anticancer medications that results in motor and sensory neuron abnormalities and degeneration of nerve fibers [31,32]. The main symptom of CIPN is numbness and cold pain, which in most cases is caused by cumulative administration of anti-cancer drugs. Many studies have confirmed that HDAC6 inhibitors can be used as a treatment for CIPN induced by various chemotherapy regimens [33]. Animal CIPN models were established using cisplatin, paclitaxel, oxaliplatin, and bortezomib, and the improvement of symptoms was confirmed by the developed HDAC6 inhibitor. Bortezomib induces peripheral neuropathy, and currently, there is no approved treatment for bortezomib-induced CIPN [34–36]. In a rat model, it was confirmed that CKD-011, at a dose of 40 mg/kg, significantly alleviated bortezomib-induced CIPN symptoms. These findings show that CKD-011, an HDAC6 inhibitor, can be used as a treatment for bortezomib-induced CIPN.
The incidence of peripheral neuropathy and pain resulting from the administration of platinum-based complexes is strongly correlated with the cumulative drug dosage and the frequency of anticancer treatments [37]. Oxaliplatin exhibits more pronounced neurotoxicity than other platinum-based drugs [38]. Taxane acute pain syndrome is also known to occur acutely after the administration of taxane drugs. When chemotherapy begins, muscle and joint pain occurs and lasts for several days. CIPN commonly occurs in patients undergoing treatment with paclitaxel, a widely utilized therapy for solid tumors. This is a significant factor contributing to dose reductions and treatment constraints [39–41]. Among platinum-based drugs, the CIPN recovery ability of CKD-011 was confirmed in a rat model treated with oxaliplatin and cisplatin. In this study, the recovery characteristics of CKD-011 were confirmed using a rat model treated with paclitaxel, a taxane drug-induced CIPN, and CKD-011, a new HDAC6 inhibitor, which could be used as a treatment for CIPN management.
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