Sulfide and polysulfide as pronociceptive mediators: Focus on Cav3.2 function enhancement and TRPA1 activation.

Reactive sulfur species including sulfides, polysulfides and cysteine hydropersulfide play extensive roles in health and disease, which involve modification of protein functions through the interaction with metals bound to the proteins, cleavage of cysteine disulfide (S-S) bonds and S-persulfidation of cysteine residues. Sulfides over a wide micromolar concentration range enhance the activity of Cav3.2 T-type Ca2+ channels by eliminating Zn2+ bound to the channels, thereby promoting somatic and visceral pain. Cav3.2 is under inhibition by Zn2+ in physiological conditions, so that sulfides function to reboot Cav3.2 from Zn2+ inhibition and increase the excitability of nociceptors. On the other hand, polysulfides generated from sulfides activate TRPA1 channels via cysteine S-persulfidation, thereby facilitating somatic, but not visceral, pain. Thus, Cav3.2 function enhancement by sulfides and TRPA1 activation by polysulfides, synergistically accelerate somatic pain signals. The increased activity of the sulfide/Cav3.2 system, in particular, appears to have a great impact on pathological pain, and may thus serve as a therapeutic target for treatment of neuropathic and inflammatory pain including visceral pain.

Opioid modulation of T-type Ca2+ channel-dependent neuritogenesis/neurite outgrowth through the prostaglandin E2/EP4 receptor/protein kinase A pathway in mouse dorsal root ganglion neurons.

Irregular regeneration or inappropriate remodeling of the axons of the primary afferent neurons after peripheral nerve trauma could be associated with the development of neuropathic pain. We analyzed the molecular mechanisms for the neuritogenesis and neurite outgrowth caused by prostaglandin E2 (PGE2) in mouse dorsal root ganglion (DRG) neurons, and evaluated their opioid modulation. PGE2 in combination with IBMX, a phosphodiesterase inhibitor, caused neuritogenesis/neurite outgrowth in DRG cells, an effect abolished by a prostanoid EP4, but not EP2, receptor antagonist, and inhibitors of adenylyl cyclase or protein kinase A (PKA). Blockers of T-type Ca2+ channels (T-channels), that are responsible for window currents involving the sustained low-level Ca2+ entry at voltages near the resting membrane potentials and can be functionally upregulated by PKA, inhibited the neuritogenesis/neurite outgrowth caused by PGE2/IBMX or dibutylyl cyclic AMP, a PKA activator, in DRG neurons, an inhibitory effect mimicked by ZnCl2 and ascorbic acid that block Cav3.2, but not Cav3.1 or Cav3.3, T-channels. Morphine and DAMGO, µ-opioid receptor (MOR) agonists, suppressed the neuritogenesis and/or neurite outgrowth induced by PGE2/IBMX in DRG neurons and also DRG neuron-like ND7/23 cells, an effect reversed by naloxone or ß-funaltrexamine, a selective MOR antagonist. Our data suggest that the EP4 receptor/PKA/Cav3.2 pathway is involved in the PGE2-induced neuritogenesis/neurite outgrowth in DRG neurons, which can be suppressed by MOR stimulation. We propose that MOR agonists including morphine in the early phase after peripheral nerve trauma might delay the axonal regeneration of the primary afferent neurons but prevent the development of neuropathic pain.

A hydrolysate of poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) suppresses Cav3.2-dependent pain by sequestering exogenous and endogenous sulfide.

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132), an organogermanium, is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP) in aqueous solutions, and reduces inflammation, pain and cancer, whereas the underlying mechanisms remain unknown. Sulfides including H2S, a gasotransmitter, generated from l-cysteine by some enzymes including cystathionine-γ-lyase (CSE), are pro-nociceptive, since they enhance Cav3.2 T-type Ca2+ channel activity expressed in the primary afferents, most probably by canceling the channel inhibition by Zn2+ linked via coordinate bonding to His191 of Cav3.2. Given that germanium is reactive to sulfur, we tested whether THGP would directly trap sulfide, and inhibit sulfide-induced enhancement of Cav3.2 activity and sulfide-dependent pain in mice. Using mass spectrometry and 1H NMR techniques, we demonstrated that THGP directly reacted with sulfides including Na2S and NaSH, and formed a sulfur-containing reaction product, which decreased in the presence of ZnCl2. In Cav3.2-transfected HEK293 cells, THGP inhibited the sulfide-induced enhancement of T-type Ca2+ channel-dependent membrane currents. In mice, THGP, administered systemically or locally, inhibited the mechanical allodynia caused by intraplantar Na2S. In the mice with cyclophosphamide-induced cystitis and cerulein-induced pancreatitis, which exhibited upregulation of CSE in the bladder and pancreas, respectively, systemic administration of THGP as well as a selective T-type Ca2+ channel inhibitor suppressed the cystitis-related and pancreatitis-related visceral pain. These data suggest that THGP traps sulfide and inhibits sulfide-induced enhancement of Cav3.2 activity, leading to suppression of Cav3.2-dependent pain caused by sulfide applied exogenously and generated endogenously.

Development of hepatic impairment aggravates chemotherapy-induced peripheral neuropathy following oxaliplatin treatment: Evidence from clinical and preclinical studies.

Oxaliplatin often induces peripheral neuropathy, a dose-limiting adverse reaction, and in rare cases leads to sinusoidal obstruction syndrome. We thus conducted a retrospective cohort study to examine the relationship between oxaliplatin-induced peripheral neuropathy (OIPN) and hepatic impairment, and then perform a fundamental study to analyze the underlying mechanisms. Analysis of medical records in cancer patients treated with oxaliplatin indicated that laboratory test parameters of hepatic impairment including AST, ALT and APRI (AST to platelet ratio index) moderately increased during oxaliplatin treatment, which was positively correlated with the severity of OIPN (grades 1-4), and associated with later incidence of survivors with OIPN grades ≥2. In mice, hepatic injury induced by CCl4 or ethanol accelerated OIPN in mice, an effect prevented by inactivation of high mobility group box 1 (HMGB1), known to participate in OIPN, by the neutralizing antibody or thrombomodulin alfa capable of promoting its thrombin-dependent degradation. Oxaliplatin also aggravated the hepatic injury in mice. CCl4 released HMGB1 from cultured hepatic parenchymal cells, and oxaliplatin at clinically achievable concentrations released HMGB1 from hepatic parenchymal and non-parenchymal cells. Our clinical and preclinical data suggest that the development of mild hepatic impairment during oxaliplatin treatment is associated with later aggravation of OIPN.

Role of neuron-derived ATP in paclitaxel-induced HMGB1 release from macrophages and peripheral neuropathy.

We examined the role of ATP and high mobility group box 1 (HMGB1) in paclitaxel-induced peripheral neuropathy (PIPN). PIPN in mice was prevented by HMGB1 neutralization, macrophage depletion, and P2X7 or P2X4 blockade. Paclitaxel and ATP synergistically released HMGB1 from macrophage-like RAW264.7 cells, but not neuron-like NG108-15 cells. The paclitaxel-induced HMGB1 release from RAW264.7 cells was accelerated by co-culture with NG108-15 cells in a manner dependent on P2X7 or P2X4. Paclitaxel released ATP from NG108-15 cells, but not RAW264.7 cells. Thus, PIPN is considered to involve acceleration of HMGB1 release from macrophages through P2X7 and P2X4 activation by neuron-derived ATP.

Caspase-Dependent HMGB1 Release from Macrophages Participates in Peripheral Neuropathy Caused by Bortezomib, a Proteasome-Inhibiting Chemotherapeutic Agent, in Mice.

Given the role of macrophage-derived high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel, we analyzed the role of HMGB1 and macrophages in the CIPN caused by bortezomib, a proteasome-inhibiting chemotherapeutic agent used for the treatment of multiple myeloma. Repeated administration of bortezomib caused CIPN accompanied by early-stage macrophage accumulation in the dorsal root ganglion. This CIPN was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin alfa capable of accelerating thrombin-dependent degradation of HMGB1, antagonists of the receptor for advanced glycation end-products (RAGE) and C-X-C motif chemokine receptor 4 (CXCR4), known as HMGB1-targeted membrane receptors, or macrophage depletion with liposomal clodronate, as reported in a CIPN model caused by paclitaxel. In macrophage-like RAW264.7 cells, bortezomib as well as MG132, a well-known proteasome inhibitor, caused HMGB1 release, an effect inhibited by caspase inhibitors but not inhibitors of NF-κB and p38 MAP kinase, known to mediate paclitaxel-induced HMGB1 release from macrophages. Bortezomib increased cleaved products of caspase-8 and caused nuclear fragmentation or condensation in macrophages. Repeated treatment with the caspase inhibitor prevented CIPN caused by bortezomib in mice. Our findings suggest that bortezomib causes caspase-dependent release of HMGB1 from macrophages, leading to the development of CIPN via activation of RAGE and CXCR4.

Macrophage as a Peripheral Pain Regulator.

A neuroimmune crosstalk is involved in somatic and visceral pathological pain including inflammatory and neuropathic components. Apart from microglia essential for spinal and supraspinal pain processing, the interaction of bone marrow-derived infiltrating macrophages and/or tissue-resident macrophages with the primary afferent neurons regulates pain signals in the peripheral tissue. Recent studies have uncovered previously unknown characteristics of tissue-resident macrophages, such as their origins and association with regulation of pain signals. Peripheral nerve macrophages and intestinal resident macrophages, in addition to adult monocyte-derived infiltrating macrophages, secrete a variety of mediators, such as tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, high mobility group box 1 and bone morphogenic protein 2 (BMP2), that regulate the excitability of the primary afferents. Neuron-derived mediators including neuropeptides, ATP and macrophage-colony stimulating factor regulate the activity or polarization of diverse macrophages. Thus, macrophages have multitasks in homeostatic conditions and participate in somatic and visceral pathological pain by interacting with neurons.

Estrogen decline is a risk factor for paclitaxel-induced peripheral neuropathy: Clinical evidence supported by a preclinical study.

We performed clinical retrospective study in female cancer patients and fundamental experiments in mice, in order to clarify risk factors for paclitaxel-induced peripheral neuropathy (PIPN). In the clinical study, 131 of 189 female outpatients with cancer undergoing paclitaxel-based chemotherapy met inclusion criteria. Breast cancer survivors (n = 40) showed significantly higher overall PIPN (grades 1-4) incidence than non-breast cancer survivors (n = 91). Multivariate sub-analyses of breast cancer survivors showed that 57 years of age or older and endocrine therapy before paclitaxel treatment were significantly associated with severe PIPN (grades 2-4). The age limit was also significantly correlated with overall development of severe PIPN. In the preclinical study, female mice subjected to ovariectomy received repeated administration of paclitaxel, and mechanical nociceptive threshold was assessed by von Frey test. Ovariectomy aggravated PIPN in the mice, an effect prevented by repeated treatment with 17ß-estradiol. Repeated administration of thrombomodulin alfa (TMα), known to prevent chemotherapy-induced peripheral neuropathy in rats and mice, also prevented the development of PIPN in the ovariectomized mice. Collectively, breast cancer survivors, particularly with postmenopausal estrogen decline and/or undergoing endocrine therapy, are considered a PIPN-prone subpopulation, and may require non-hormonal pharmacological intervention for PIPN in which TMα may serve as a major candidate.

Effects of Bepridil and Pimozide, Existing Medicines Capable of Blocking T-Type Ca2+ Channels, on Visceral Pain in Mice.

T-Type Ca2+ channels (T-channels), particularly Cav3.2, are now considered as therapeutic targets for treatment of intractable pain including visceral pain. Among existing medicines, bepridil, a multi-channel blocker, used for treatment of arrhythmia and angina, and pimozide, a dopamine D2 receptor antagonist, known as a typical antipsychotic, have potent T-channel blocking activity. We thus tested whether bepridil and pimozide could suppress visceral pain in mice. Colonic and bladder pain were induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) and systemic administration of cyclophosphamide (CPA), respectively. Referred hyperalgesia was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load with intracolonic water injection and spontaneous bladder pain were evaluated by observing nociceptive behaviors in conscious mice. The mice exhibited referred hyperalgesia and colonic hypersensitivity to distension on day 6 after TNBS treatment. Systemic administration of bepridil at 10-20 mg/kg or pimozide at 0.1-0.5 mg/kg strongly reduced the referred hyperalgesia on the TNBS-induced referred hyperalgesia and colonic hypersensitivity to distension. CPA treatment caused bladder pain-like nociceptive behavior and referred hyperalgesia, which were reversed by bepridil at 10-20 mg/kg or pimozide at 0.5-1 mg/kg. Our data thus suggest that bepridil and pimozide, existing medicines capable of blocking T-channels, are useful for treatment of colonic and bladder pain, and serve as seeds for the development of new medicines for visceral pain treatment.

Cystitis-Related Bladder Pain Involves ATP-Dependent HMGB1 Release from Macrophages and Its Downstream H2S/Cav3.2 Signaling in Mice.

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Cav3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X4/P2X7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X7/P2X4, and caused HMGB1 release via P2X7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Cav3.2-dependent nociceptor excitation, resulting in bladder pain.

HMGB1 and its membrane receptors as therapeutic targets in an intravesical substance P-induced bladder pain syndrome mouse model.

HMGB1, a nuclear protein, once released to the extracellular space, promotes somatic and visceral pain signals. We thus analyzed the role of HMGB1 in an intravesical substance P-induced bladder pain syndrome (BPS) mouse model. Intravesical administration of substance P caused referred hyperalgesia/allodynia in the lower abdomen and hindpaw without producing severe urothelial damage, which was prevented by an anti-HMGB1-neutralizing antibody, thrombomodulin α capable of inactivating HMGB1 and antagonists of RAGE or CXCR4. The HMGB1 inactivation or RAGE blockade also reversed the established bladder pain symptoms. HMGB1 and RAGE are thus considered to serve as therapeutic targets for BPS.

Role of HMGB1 in Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN), one of major dose-limiting side effects of first-line chemotherapeutic agents such as paclitaxel, oxaliplatin, vincristine, and bortezomib is resistant to most of existing medicines. The molecular mechanisms of CIPN have not been fully understood. High mobility group box 1 (HMGB1), a nuclear protein, is a damage-associated molecular pattern protein now considered to function as a pro-nociceptive mediator once released to the extracellular space. Most interestingly, HMGB1 plays a key role in the development of CIPN. Soluble thrombomodulin (TMα), known to degrade HMGB1 in a thrombin-dependent manner, prevents CIPN in rodents treated with paclitaxel, oxaliplatin, or vincristine and in patients with colorectal cancer undergoing oxaliplatin-based chemotherapy. In this review, we describe the role of HMGB1 and its upstream/downstream mechanisms in the development of CIPN and show drug candidates that inhibit the HMGB1 pathway, possibly useful for prevention of CIPN.

Role of non-macrophage cell-derived HMGB1 in oxaliplatin-induced peripheral neuropathy and its prevention by the thrombin/thrombomodulin system in rodents: negative impact of anticoagulants.

BACKGROUND: Macrophage-derived high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, plays a key role in the development of chemotherapy-induced peripheral neuropathy (CIPN) caused by paclitaxel in rodents. Endothelial thrombomodulin (TM) promotes thrombin-induced degradation of HMGB1, and TMα, a recombinant human soluble TM, abolishes peripheral HMGB1-induced allodynia in mice. We thus examined whether HMGB1, particularly derived from macrophages, contributes to oxaliplatin-induced neuropathy in mice and analyzed the anti-neuropathic activity of the TM/thrombin system. METHODS: CIPN models were created by the administration of oxaliplatin in mice and rats, and the nociceptive threshold was assessed by von Frey test or paw pressure test. Macrophage-like RAW264.7 cells were stimulated with oxaliplatin in vitro. Proteins were detected and/or quantified by Western blotting, immunostaining, or enzyme-linked immunosorbent assay. RESULTS: Intraperitoneal administration of an anti-HMGB1-neutralizing antibody (AB) at 1 mg/kg prevented the oxaliplatin-induced allodynia in mice and rats. Antagonists of Toll-like receptor (TLR) 4, receptor for advanced glycation end products (RAGE) and CXCR4 among the HMGB1-targeted pro-nociceptive receptors, also mimicked the anti-neuropathic activity of AB in mice. Macrophage accumulation in the sciatic nerve was observed in mice treated with paclitaxel, but not oxaliplatin, and neither macrophage depletion nor inhibitors of macrophage activation affected oxaliplatin-induced allodynia. Oxaliplatin was 10- to 100-fold less potent than paclitaxel in releasing HMGB1 from macrophage-like RAW264.7 cells. Like AB, TMα at 10 mg/kg prevented the oxaliplatin-induced allodynia in mice as well as rats, an effect abolished by argatroban at 10 mg/kg, a thrombin inhibitor. The anti-neuropathic activity of TMα in oxaliplatin-treated mice was suppressed by oral anticoagulants such as warfarin at 1 mg/kg, dabigatran at 75 mg/kg, and rivaroxaban at 10 mg/kg, but not antiplatelet agents such as aspirin at 50 mg/kg and clopidogrel at 10 mg/kg. Repeated administration of the anticoagulants gradually developed neuropathic allodynia and elevated plasma HMGB1 levels in mice treated with a subeffective dose of oxaliplatin. CONCLUSIONS: Our data thus suggests a causative role of HMGB1 derived from non-macrophage cells in oxaliplatin-induced peripheral neuropathy and a thrombin-dependent anti-neuropathic activity of exogenous TMα and, most probably, endogenous TM.

[HMGB1 as a target for prevention of chemotherapy-induced peripheral neuropathy].

Chemotherapy-induced peripheral neuropathy (CIPN) considerably impairs cancer patients' QOL, and may lead to discontinuation of drug treatment of cancer. Currently, there is no effective strategy against CIPN. Therefore, it is an urgent issue to develop clinically available drugs that prevent or treat CIPN. We have shown that high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, plays an essential role in the development of CIPN. Most interestingly, thrombomodulin α, approved as a medicine for treatment of disseminated intravascular coagulation (DIC) in Japan, causes thrombin-dependent degradation of extracellular HMGB1 that is released in response to chemotherapeutics, and prevents CIPN. Thus, we expect that targeting HMGB1 or its receptors would lead to prevention of CIPN in cancer patients in near future.

[Role of Cav3.2 T-type Ca2+ channels in prostate cancer cells].

Among voltage-gated Ca2+ channels, T-type Ca2+ channels, which are activated by low voltages, regulate neuronal excitability, spontaneous neurotransmitter release, hormone secretion, etc. and also participate in proliferation of distinct cancer cells. Among three isoforms of T-type Ca2+ channels, Cav3.2 is detectable in 100% of biopsy samples from prostate cancer patients. In general, prostate cancer cells are highly sensitive to androgen deprivation therapy, but often acquire hormone-therapy resistance. The androgen deprivation may trigger neuroendocrine (NE)-like differentiation of some prostate cancer cells. We have analyzed the expression and function of Cav3.2 in human prostate cancer LNCaP cells during NE-like differentiation. NE-like LNCaP cells overexpress Cav3.2 through the CREB/Egr-1 pathway and also cystathionine-γ-lyase (CSE), which generates H2S that enhances the channel activity of Cav3.2. H2S generated by upregulated CSE appears to enhance the activity of upregulated Cav3.2 after the differentiation. The enhanced Cav3.2 activity in NE-like cells may contribute to increased secretion of mitogenic factors essential for androgen-independent proliferation of surrounding prostate cancer cells. It is known that increased extracellular glucose levels enhance Cav3.2 activity through asparagine (N)-linked glycosylation of Cav3.2, which might contribute to diabetic neuropathy. We then found that high glucose accelerates the enhanced channel function and overexpression of Cav3.2 in NE-like LNCaP cells, which might be associated with clinical evidence for diabetes-related poor prognosis of prostate cancer and development of hormone therapy resistance. Thus, Cav3.2 is considered to play a role in the pathophysiology of prostate cancer, and may serve as a therapeutic target.

Critical role of Cav3.2 T-type calcium channels in the peripheral neuropathy induced by bortezomib, a proteasome-inhibiting chemotherapeutic agent, in mice.

Bortezomib, a first-line agent for treatment of multiple myeloma, exhibits anticancer activity through proteasome inhibition. However, bortezomib-induced peripheral neuropathy (BIPN) is one of the most serious side effects. Since decreased proteasomal degradation of Cav3.2 T-type calcium channels in the primary afferents is involved in persistent pain, we investigated whether BIPN involves increased protein levels of Cav3.2 in mice. Six repeated i.p. administrations of bortezomib for 12 days developed persistent mechanical allodynia. Systemic administration of novel T-type calcium channel blockers, (2R/S)-6-prenylnaringenin and KTt-45, and of TTA-A2, the well-known blocker, reversed the BIPN. Ascorbic acid, known to block Cav3.2, but not Cav3.1 or 3.3, and silencing of Cav3.2 gene also suppressed BIPN. Protein levels of Cav3.2 in the dorsal root ganglion (DRG) at L4-L6 levels increased throughout days 1-21 after the onset of bortezomib treatment. Protein levels of USP5, a deubiquitinating enzyme that specifically inhibits proteasomal degradation of Cav3.2, increased in DRG on days 3-21, but not day 1, in bortezomib-treated mice. In DRG-derived ND7/23 cells, bortezomib increased protein levels of Cav3.2 and T-channel-dependent currents, as assessed by a patch-clamp method, but did not upregulate expression of Cav3.2 mRNA or USP5 protein. MG-132, another proteasome inhibitor, also increased Cav3.2 protein levels in the cultured cells. Given the previous evidence for USP5 induction following nociceptor excitation, our data suggest that BIPN involves the increased protein levels of Cav3.2 in nociceptors through inhibition of proteasomal degradation of Cav3.2 by bortezomib itself and then by USP5 that is upregulated probably in an activity-dependent manner.

RagA, an mTORC1 activator, interacts with a hedgehog signaling protein, WDR35/IFT121.

Small Ras-like GTPases act as molecular switches for various signal transduction pathways. RagA, RagB/RagC and RagD are small Ras-like GTPases that play regulatory roles in mTORC1. Lack of proper activation of mTORC1 can lead to diseases, such as cancer and diabetes. In this study, we found an interaction between RagA and WDR35. Mutations of WDR35 may cause genetic diseases including Sensenbrenner syndrome. WDR35 seems to be a hedgehog signaling protein with a possible ciliary function and a possible upstream regulator of RagA. RagB is a homologue of RagA and is also associated with WDR35. WDR35 is present in the endoplasmic reticulum, but usually not in lysosomes, where Rag family proteins act as an mTORC1 switch. Over-expression of WDR35 results in decreased phosphorylation of ribosome S6 protein in a RagA-, RagB- and RagC-dependent manner. Thus, WDR35 is associated with RagA, RagB and RagC and might negatively influence mTORC1 activity.

Paclitaxel-induced HMGB1 release from macrophages and its implication for peripheral neuropathy in mice: Evidence for a neuroimmune crosstalk.

Given our recent evidence for the role of high mobility group box 1 (HMGB1) in chemotherapy-induced peripheral neuropathy (CIPN) in rats, we examined the origin of HMGB1 and the upstream and downstream mechanisms of HMGB1 release involved in paclitaxel-induced neuropathy in mice. Paclitaxel treatment developed mechanical allodynia in mice, as assessed by von Frey test, which was prevented by an anti-HMGB1-neutralizing antibody or thrombomodulin alfa capable of inactivating HMGB1. RAGE or CXCR4 antagonists, ethyl pyruvate or minocycline, known to inhibit HMGB1 release from macrophages, and liposomal clodronate, a macrophage depletor, prevented the paclitaxel-induced allodynia. Paclitaxel caused upregulation of RAGE and CXCR4 in the dorsal root ganglia and macrophage accumulation in the sciatic nerve. In macrophage-like RAW264.7 cells, paclitaxel evoked cytoplasmic translocation of nuclear HMGB1 followed by its extracellular release, and overexpression of CBP and PCAF, histone acetyltransferases (HATs), known to cause acetylation and cytoplasmic translocation of HMGB1, which were suppressed by ethyl pyruvate, N-acetyl-l-cysteine, an anti-oxidant, and SB203580 and PDTC, inhibitors of p38 MAP kinase (p38MAPK) and NF-κB, respectively. Paclitaxel increased accumulation of reactive oxygen species (ROS) and phosphorylation of p38MAPK, NF-κB p65 and I-κB in RAW264.7 cells. In mice, N-acetyl-l-cysteine or PDTC prevented the paclitaxel-induced allodynia. Co-culture of neuron-like NG108-15 cells or stimulation with their conditioned medium promoted paclitaxel-induced HMGB1 release from RAW264.7 cells. Our data indicate that HMGB1 released from macrophages through the ROS/p38MAPK/NF-κB/HAT pathway participates in the paclitaxel-induced peripheral neuropathy in mice, and unveils an emerging therapeutic avenue targeting a neuroimmune crosstalk in CIPN.

Involvement of Voltage-Gated Calcium Channels in Inflammation and Inflammatory Pain.

Voltage-gated calcium channels (VGCCs) are classified into high-voltage-activated (HVA) channels and low-voltage-activated channels consisting of Cav3.1-3.3, known as T ("transient")-type VGCC. There is evidence that certain types of HVA channels are involved in neurogenic inflammation and inflammatory pain, in agreement with reports indicating the therapeutic effectiveness of gabapentinoids, ligands for the α2δ subunit of HVA, in treating not only neuropathic, but also inflammatory, pain. Among the Cav3 family members, Cav3.2 is abundantly expressed in the primary afferents, regulating both neuronal excitability at the peripheral terminals and spontaneous neurotransmitter release at the spinal terminals. The function and expression of Cav3.2 are modulated by a variety of inflammatory mediators including prostanoids and hydrogen sulfide (H2S), a gasotransmitter. The increased activity of Cav3.2 by H2S participates in colonic, bladder and pancreatic pain, and regulates visceral inflammation. Together, VGCCs are involved in inflammation and inflammatory pain, and Cav3.2 T-type VGCC is especially a promising therapeutic target for the treatment of visceral inflammatory pain in patients with irritable bowel syndrome, interstitial cystitis/bladder pain syndrome, pancreatitis, etc., in addition to neuropathic pain.

Blockade of T-type calcium channels by 6-prenylnaringenin, a hop component, alleviates neuropathic and visceral pain in mice.

Since Cav3.2 T-type Ca2+ channels (T-channels) expressed in the primary afferents and CNS contribute to intractable pain, we explored T-channel-blocking components in distinct herbal extracts using a whole-cell patch-clamp technique in HEK293 cells stably expressing Cav3.2 or Cav3.1, and purified and identified sophoraflavanone G (SG) as an active compound from SOPHORAE RADIX (SR). Interestingly, hop-derived SG analogues, (2S)-6-prenylnaringenin (6-PNG) and (2S)-8-PNG, but not naringenin, also blocked T-channels; IC50 (µM) of SG, (2S)-6-PNG and (2S)-8-PNG was 0.68-0.75 for Cav3.2 and 0.99-1.41 for Cav3.1. (2S)-6-PNG and (2S)-8-PNG, but not SG, exhibited reversible inhibition. The racemic (2R/S)-6-PNG as well as (2S)-6-PNG potently blocked Cav3.2, but exhibited minor effect on high-voltage-activated Ca2+ channels and voltage-gated Na+ channels in differentiated NG108-15 cells. In mice, the mechanical allodynia following intraplantar (i.pl.) administration of an H2S donor was abolished by oral or i.p. SR extract and by i.pl. SG, (2S)-6-PNG or (2S)-8-PNG, but not naringenin. Intraperitoneal (2R/S)-6-PNG strongly suppressed visceral pain and spinal ERK phosphorylation following intracolonic administration of an H2S donor in mice. (2R/S)-6-PNG, administered i.pl. or i.p., suppressed the neuropathic allodynia induced by partial sciatic nerve ligation or oxaliplatin, an anti-cancer agent, in mice. (2R/S)-6-PNG had little or no effect on open-field behavior, motor performance or cardiovascular function in mice, and on the contractility of isolated rat aorta. (2R/S)-6-PNG, but not SG, was detectable in the brain after their i.p. administration in mice. Our data suggest that 6-PNG, a hop component, blocks T-channels, and alleviates neuropathic and visceral pain with little side effects.