Discovery of rafoxanide as a novel agent for the treatment of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), which accounts for approximately 85% of all lung cancer cases, is associated with a poor outcome. Rafoxanide is an anthelmintic drug that inhibits tumor growth in certain malignancies. However, its impact on NSCLC remains unknown. In this study, we examined the effect of rafoxanide on NSCLC and dissected the underlying mechanism. The results showed that rafoxanide significantly inhibited the growth, invasion, and migration of NSCLC cells. Besides, rafoxanide can induce NSCLC cell apoptosis and cell cycle arrest in a dose-dependent manner. RNA-seq analysis revealed that genes associated with endoplasmic reticulum stress (ER) stress responses were activated. Mechanistically, we found Rafoxanide can induce ER stress and activate the unfolded protein response (UPR). Apoptosis was activated by excessive ER stress, and autophagy was activated to partially alleviate ER stress. In vivo, we found that rafoxanide inhibited the growth of A549 and H1299 xenograft mouse models without severe side effects. Collectively, the present study indicates that rafoxanide may be a candidate drug for the treatment of NSCLC.

RHDV 3C protein antagonizes type I interferon signaling by cleaving interferon promoter stimulated 1 protein.

The host innate immune response to viral infection often involves the activation of type I interferons. Not surprisingly, many viruses have evolved various mechanisms to disable the interferon pathway and evade the antiviral response involving innate immunity. Rabbit hemorrhagic disease (RHD) is caused by RHD virus (RHDV), but whether it can antagonize the production of host interferon to establish infection has not been investigated. In this study, we found that during RHDV infection, the expressions of interferon and the interferon-stimulated gene were not activated. We constructed eukaryotic expression plasmids of all RHDV proteins, and found that RHDV 3C protein inhibited poly(I:C)-induced interferon expressions. Using siRNA to interfere with the expressions of TLR3 and MDA5, we found that the MDA5 signal pathway was used by the 3C protein to inhibit poly(I:C)-induced interferon expression. This effect was mediated by cleaving the interferon promoter stimulated 1 (IPS-1) protein. Finally, our study showed that interferon was effective against RHDV infection. In summary, our findings showed that the RHDV 3C protein was a new interferon antagonist. These results increase our understanding of the escape mechanism from innate immunity mediated by the RHDV 3C protein.

A positive feedback loop of heparanase/syndecan1/nerve growth factor regulates cancer pain progression.

Background: The purpose of this research was to assess the role of heparanase (HPSE)/syndecan1 (SDC1)/nerve growth factor (NGF) on cancer pain from melanoma. Methods: The influence of HPSE on the biological function of melanoma cells and cancer pain in a mouse model was evaluated. Immunohistochemical staining was used to analyze HPSE and SDC1. HPSE, NGF, and SDC1 were detected using western blot. Inflammatory factors were detected using ELISA assay. Results: HPSE promoted melanoma cell viability, proliferation, migration, invasion, and tumor growth, as well as cancer pain, while SST0001 treatment reversed the promoting effect of HPSE. HPSE up-regulated NGF, and NGF feedback promoted HPSE. High expression of NGF reversed the inhibitory effect of HPSE down-regulation on melanoma cell phenotype deterioration, including cell viability, proliferation, migration, and invasion. SST0001 down-regulated SDC1 expression. SDC1 reversed the inhibitory effect of SST0001 on cancer pain. Conclusions: The results showed that HPSE promoted melanoma development and cancer pain by interacting with NGF/SDC1. It provides new insights to better understand the role of HPSE in melanoma and also provides a new direction for cancer pain treatment.

Identification of the Acid-Sensitive Site Critical for Chloral Hydrate (CH) Activation of the Proton-Activated Chloride Channel.

The transmembrane protein TMEM206 was recently identified as the molecular basis of the extracellular proton-activated Cl- channel (PAC), which plays an essential role in neuronal death in ischemia-reperfusion. The PAC channel is activated by extracellular acid, but the proton-sensitive mechanism remains unclear, although different acid-sensitive pockets have been suggested based on the cryo-EM structure of the human PAC (hPAC) channel. In the present study, we firstly identified two acidic amino acid residues that removed the pH-dependent activation of the hPAC channel by neutralization all the conservative negative charged residues located in the extracellular domain of the hPAC channel and some positively charged residues at the hotspot combined with two-electrode voltage-clamp (TEVC) recording in the Xenopus oocytes system. Double-mutant cycle analysis and double cysteine mutant of these two residues proved that these two residues cooperatively form a proton-sensitive site. In addition, we found that chloral hydrate activates the hPAC channel depending on the normal pH sensitivity of the hPAC channel. Furthermore, the PAC channel knock-out (KO) male mice (C57BL/6J) resist chloral hydrate-induced sedation and hypnosis. Our study provides a molecular basis for understanding the proton-dependent activation mechanism of the hPAC channel and a novel drug target of chloral hydrate.SIGNIFICANCE STATEMENT Proton-activated Cl- channel (PAC) channels are widely distributed in the nervous system and play a vital pathophysiological role in ischemia and endosomal acidification. The main discovery of this paper is that we identified the proton activation mechanism of the human proton-activated chloride channel (hPAC). Intriguingly, we also found that anesthetic chloral hydrate can activate the hPAC channel in a pH-dependent manner. We found that the chloral hydrate activates the hPAC channel and needs the integrity of the pH-sensitive site. In addition, the PAC channel knock-out (KO) mice are resistant to chloral hydrate-induced anesthesia. The study on PAC channels' pH activation mechanism enables us to better understand PAC's biophysical mechanism and provides a novel target of chloral hydrate.

Ventral Morphology of the Non-Trilobite Artiopod Retifacies abnormalis Hou, Chen & Lu, 1989, from the Early Cambrian Chengjiang Biota, China.

The artiopodans represent a diverse group of euarthropods with a typically flattened dorsal exoskeleton that covers numerous pairs of biramous ventral appendages, and which are ubiquitous faunal components of the 518-million-year-old Chengjiang Lagerstätte in South China. Despite their abundance, several Chengjiang artiopodans remain poorly known, such as the large euarthropoda Retifacies abnormalis, Hou, Chen & Lu, 1989, which is distinguished by the presence of mesh-like ornamentation on its dorsal exoskeleton. Although only a few ventral details were described in a single study in 25 years, it has been frequently featured in phylogenetic analyses that explore the relationships between Cambrian euarthropods. Here, we employ micro-CT and fluorescent microphotography to investigate the exceptionally preserved ventral morphology of R. abnormalis and explore its phylogenetic implications through maximum parsimony and Bayesian inference. Detailed morphology revealed here better supports R. abnormalis as a sister group to the diminutive artiopod Pygmaclypeatus daziensis, also known from Chengjiang, and strengthens the close relationship of these taxa that have been suggested by previous studies as early-branching representatives of Trilobitomorpha. Cephalic appendages suggest this animal might be a scavenger, possibly feeding on soft-bodied organisms. Different pairs of pygidial appendages suggest an anamorphic post-embryonic ontogeny, which adds to the understanding of the developmental mode of Cambrian artiopods, and further supports the statement that post-hatching segment addition occurred in the ancestor of Euarthropoda.

Exites in Cambrian arthropods and homology of arthropod limb branches.

The last common ancestor of all living arthropods had biramous postantennal appendages, with an endopodite and exopodite branching off the limb base. Morphological evidence for homology of these rami between crustaceans and chelicerates has, however, been challenged by data from clonal composition and from knockout of leg patterning genes. Cambrian arthropod fossils have been cited as providing support for competing hypotheses about biramy but have shed little light on additional lateral outgrowths, known as exites. Here we draw on microtomographic imaging of the Cambrian great-appendage arthropod Leanchoilia to reveal a previously undetected exite at the base of most appendages, composed of overlapping lamellae. A morphologically similar, and we infer homologous, exite is documented in the same position in members of the trilobite-allied Artiopoda. This early Cambrian exite morphology supplements an emerging picture from gene expression that exites may have a deeper origin in arthropod phylogeny than has been appreciated.

IL-18 Contributes to Bone Cancer Pain by Regulating Glia Cells and Neuron Interaction.

Glial cell hyperactivity has been proposed to be responsible for chronic pain, however, the mechanisms remain unclear. Interleukin (IL)-18, released from glial cells, has been reported to be involved in neuropathic pain. In this study, we investigated the role of IL-18 in bone cancer pain. Bone cancer pain was mimicked by injecting Walker-256 mammary gland carcinoma cells into the intramedullary space of the tibia in rats. Expression and location of IL-18 and the IL-18 receptor were tested. To investigate the contribution of IL-18 signaling to bone cancer pain, IL-18 binding protein and recombinant IL-18 were used. To investigate the mechanisms of glial cells effects, MK801, N-methyl-D-aspartate (NMDA) receptor inhibitor, and Src kinase-specific inhibitor PP1 were used. Tumor cell implantation (TCI) treatment increased expression of IL-18 and IL-18 receptor in spinal cord. The time course of IL-18 upregulation was correlated with TCI-induced pain behaviors. Blocking the IL-18 signaling pathway prevented and reversed bone cancer-related pain behaviors. Meanwhile, blocking IL-18 signaling also suppressed TCI-induced glial cell hyperactivity, as well as activation of GluN2B and subsequent Ca2+-dependent signaling. Spinal administration of recombinant IL-18 in naive rat induced significant mechanical allodynia, as well as GluN2B activation. However, intrathecal injection of MK801 failed to suppress recombinant IL-18-induced GluN2B phosphorylation, whereas Src kinase inhibitor PP1 significantly inhibited IL-18-induced GluN2B activation. IL-18-mediated glial-glia and glial-neuron interaction may facilitate bone cancer pain. Blocking IL-18 signaling may effectively prevent and/or suppress bone cancer pain. PERSPECTIVE: IL-18 signaling may be a new target for cancer pain therapy.

Levo-Tetrahydropalmatine Attenuates Bone Cancer Pain by Inhibiting Microglial Cells Activation.

OBJECTIVE: The present study is to investigate the analgesic roles of L-THP in rats with bone cancer pain caused by tumor cell implantation (TCI). METHODS: Thermal hyperalgesia and mechanical allodynia were measured at different time points before and after operation. L-THP (20, 40, and 60 mg/kg) were administrated intragastrically at early phase of postoperation (before pain appearance) and later phase of postoperation (after pain appearance), respectively. The concentrations of TNF-α, IL-1ß, and IL-18 in spinal cord were measured by enzyme-linked immunosorbent assay. Western blot was used to test the activation of astrocytes and microglial cells in spinal cord after TCI treatment. RESULTS: TCI treatment induced significant thermal hyperalgesia and mechanical allodynia. Administration of L-THP at high doses significantly prevented and/or reversed bone cancer-related pain behaviors. Besides, TCI-induced activation of microglial cells and the increased levels of TNF-α and IL-18 were inhibited by L-THP administration. However, L-THP failed to affect TCI-induced astrocytes activation and IL-1ß increase. CONCLUSION: This study suggests the possible clinical utility of L-THP in the treatment of bone cancer pain. The analgesic effects of L-THP on bone cancer pain maybe underlying the inhibition of microglial cells activation and proinflammatory cytokines increase.

cGMP and cGMP-dependent protein kinase I pathway in dorsal root ganglia contributes to bone cancer pain in rats.

STUDY DESIGN: A prospective, randomized experimental research. OBJECTIVE: To demonstrate the role of cGMP (cyclic guanosine monophosphate)-cGKI (cGMP-dependent protein kinase I) pathway in dorsal root ganglia (DRG) in bone cancer pain. SUMMARY OF BACKGROUND DATA: Treating bone cancer pain continues to possess a major clinical challenge because the specific cellular and molecular mechanisms underlying bone cancer pain remain elusive. cGMP and cGMP-dependent protein kinases pathway in DRG plays important role in nerve injury-induced hyperexcitability of DRG neurons, as well as neuropathic pain, however, whether this pathway participates in bone cancer pain is unknown. METHODS: The rat model of bone cancer pain was produced by intramedullary injection of rat breast cancer cells (Walker 256) into right tibia. Thermal hyperalgesia and mechanical allodynia were measured before and after administration of inhibitor of cGMP-cGKs pathway (Rp-8-pCPT-cGMPS). Immunofluorescence and reverse transcription-polymerase chain reaction were used to reflect expression of cGKI in DRG neurons, whereas the concentration of cGMP in DRG was tested using enzyme-linked immunosorbent assay method. Whole-cell patch clamp was used to record the hyperexcitability of small neurons in DRG with or without cGKs inhibitor after tumor cell implantation (TCI). RESULTS: TCI treatment significantly increased the concentration of cGMP in DRG and activity of cGKs in DRG and the spinal cord. TCI treatment also induced upregulation of cGKI messenger ribonucleic acid and protein in DRG, as well as enhanced hyperexcitability in DRG neurons. Spinal administration of Rp-8-pCPT-cGMPS, cGMP-cGKs inhibitor, significantly suppressed TCI-induced activation of cGMP-cGKI signaling, and hyperexcitability of DRG neurons. Meanwhile, in vivo intrathecal delivery of the Rp-8-pCPT-cGMPS significantly prevented and suppressed TCI-induced hyperalgesia and allodynia. CONCLUSION: From these results, we confirm that TCI treatment activates cGMP-cGKI signaling pathway and continuing activation of this pathway in DRG is required for hyperalgesia and/or hyperalgesia and allodynia after TCI treatment. LEVEL OF EVIDENCE: N/A.