Inhibition of C5aR1 as a promising approach to treat taxane-induced neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common side effect of several antitumor agents resulting in progressive and often irreversible damage of peripheral nerves. In addition to their known anticancer effects, taxanes, including paclitaxel, can also induce peripheral neuropathy by activating microglia and astrocytes, which release pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin 1-beta (IL-1ß), and chemokine (C-C motif) ligand 2 (CCL-2). All these events contribute to the maintenance of neuropathic or inflammatory response. Complement component 5a (C5a)/C5a receptor 1 (C5aR1) signaling was very recently shown to play a crucial role in paclitaxel-induced peripheral neuropathy. Our recent findings highlighted that taxanes have the previously unreported property of binding and activating C5aR1, and that C5aR1 inhibition by DF3966A is effective in preventing paclitaxel-induced peripheral neuropathy (PIPN) in animal models. Here, we investigated if C5aR1 inhibition maintains efficacy in reducing PIPN in a therapeutic setting. Furthermore, we characterized the role of C5aR1 activation by paclitaxel and the CIPN-associated activation of nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. Our results clearly show that administration of the C5aR1 inhibitor strongly reduced cold and mechanical allodynia in mice when given both during the onset of PIPN and when neuropathy is well established. C5aR1 activation by paclitaxel was found to be a key event in the induction of inflammatory factors in spinal cord, such as TNF-α, ionized calcium-binding adapter molecule 1 (Iba-1), and glial fibrillary acidic protein (GFAP). In addition, C5aR1 inhibition significantly mitigated paclitaxel-induced inflammation and inflammasome activation by reducing IL-1ß and NLRP3 expression at both sciatic and dorsal root ganglia level, confirming the involvement of inflammasome in PIPN. Moreover, paclitaxel-induced upregulation of C5aR1 was significantly reduced by DF3966A treatment in central nervous system. Lastly, the antinociceptive effect of C5aR1 inhibition was confirmed in an in vitro model of sensory neurons in which we focused on receptor channels usually activated upon neuropathy. In conclusion, C5aR1 inhibition is proposed as a therapeutic option with the potential to exert long-term protective effect on PIPN-associated neuropathic pain and inflammation.

Oral sodium butyrate supplementation ameliorates paclitaxel-induced behavioral and intestinal dysfunction.

Paclitaxel (PTX) is one of the most broadly used chemotherapeutic agents for the treatment of several tumor types including ovarian, breast, and non-small cell lung cancer. However, its use is limited by debilitating side effects, involving both gastrointestinal and behavioral dysfunctions. Due to growing evidence showing a link between impaired gut function and chemotherapy-associated behavioral changes, the aim of this study was to identify a novel therapeutic approach to manage PTX-induced gut and brain comorbidities. Mice were pre-treated with sodium butyrate (BuNa) for 30 days before receiving PTX. After 14 days, mice underwent to behavioral analysis and biochemical investigations of gut barrier integrity and microbiota composition. Paired evaluations of gut functions revealed that the treatment with BuNa restored PTX-induced altered gut barrier integrity, microbiota composition and food intake suggesting a gut-to-brain communication. The treatment with BuNa also ameliorated depressive- and anxiety-like behaviors induced by PTX in mice, and these effects were associated with neuroprotective and anti-inflammatory outcomes. These results propose that diet supplementation with this safe postbiotic might be considered when managing PTX-induced central side effects during cancer therapy.

Binding site requirements for pea nuclear protein factor GT-1 correlate with sequences required for light-dependent transcriptional activation of the rbcS-3A gene.

Nuclear protein factor GT-1 binds to sequence boxes II, III, II* and III* upstream of the light-responsive pea rbcS-3A gene. We have shown previously that box II and box III are required for expression of rbcS-3A when redundant elements upstream of -170 (relative to the transcription start site) are removed. Here we present evidence that deletion and substitution mutations downstream of -170 which eliminate expression also decrease binding. Using a series of 2 bp substitution mutations we have defined a core of six residues (GGTTAA) within box II (GTGTGGTTAATATG) that are critical for binding. The most detrimental mutation for binding, which changes the double Gs to Cs, is sufficient to eliminate detectable expression in vivo when only 170 bp of 5' flanking sequences are present. The simplest interpretation of these data is that GT-1 is an activator of rbcS-3A transcription. Footprinting experiments show that GT-1 from both light-grown and dark-adapted plants binds to the same sequences in vitro. Therefore, the lack of expression of rbcS-3A in the dark is not due to the absence of GT-1. In our analysis of the sequence elements upstream of -170, we have mapped two additional GT-1 sites (boxes II** and III**) between -330 and -410. The similarities and differences among the GT-1 sites located upstream and downstream of -170 are discussed in terms of the different sequence requirements for rbcS-3A expression during development.

Localization and conditional redundancy of regulatory elements in rbcS-3A, a pea gene encoding the small subunit of ribulose-bisphosphate carboxylase.

Expression of the pea rbcS-3A gene, one of a family of genes encoding the small subunit of ribulose-bisphosphate carboxylase [EC 4.1.1.39], is regulated by light and is restricted to chloroplast-containing cells. We analyzed the effects of light and development on rbcS-3A expression in transgenic plants. Two highly conserved sequences ("boxes" II and III) around nucleotide position -150 (relative to the transcription initiation site, +1) are required for rbcS-3A expression. The so-defined positive elements overlap with previously identified negative light-regulatory elements. In the case of box II, which has sequence similarity to the core enhancer motif of simian virus 40, a GG----CC transversion is sufficient to abolish expression. The effect of mutations in boxes II and III can only be measured when sequences upstream of -170 are removed, and because sequences both 5' and 3' of -170 can direct light-regulated and organ-specific expression. This implies that there is a redundancy of cis-acting elements in the 5' noncoding region of rbcS-3A. However, we show that the sequences upstream of -170 are dispensable only in the mature leaves of a green plant. In contrast, in the young, expanding leaves at the top of a green plant, as well as in seedlings, the distal elements are required for high-level expression. Therefore, redundancy is not absolute, and the requirements for rbcS-3A expression change during plant development.

Characterization of the F plasmid TraJ protein synthesized in F' and Hfr strains of Escherichia coli K-12.

Using purified F plasmid TraJ protein (Cuozzo, M., Silverman, P., and Minkley, E. (1984) J. Biol. Chem. 259, 6659-6666), we prepared rabbit anti-TraJ protein antibodies to analyze for the first time the TraJ protein as it is synthesized in normal F' and Hfr conjugal donor strains. Using affinity-purified antibody, we identified the protein on immuno-overlay blots of whole cell proteins separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. In contrast to the TraJ protein synthesized in large quantity by heat-induced lambda (traJ) lysogens, the TraJ protein synthesized in normal donor cells was soluble, even after sedimentation at 100,000 X g. The soluble protein was found with the cytoplasmic fraction after separation of cytoplasmic and periplasmic proteins. Velocity sedimentation analysis indicated an S20,w of 3.5 for the single molecular species composed of or including all the TraJ polypeptide in crude extracts. Quantitative analyses showed that conjugal donor strains normally contain 2000-4000 TraJ monomers/cell. However, that level depended on other plasmid and chromosomal genes.

Overproduction in Escherichia coli K-12 and purification of the TraJ protein encoded by the conjugative plasmid F.

The TraJ protein encoded by the conjugative plasmid F has been designated an Escherichia coli K-12 envelope protein that participates in a mechanism of gene regulation. We have purified the TraJ protein, using an Flac::lambda traJ lysogen that overproduces the protein after heat induction of the prophage. Sufficient TraJ protein was synthesized within 40 min after induction to follow the purification by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Our purification exploited the observation that the TraJ protein remains insoluble after repeated Triton X-100/EDTA extractions of crude envelope fractions. The protein was then solubilized in sodium dodecyl sulfate at 60 degrees C and fractionated further by gel filtration and hydroxylapatite chromatography, both in the presence of sodium dodecyl sulfate. After hydroxylapatite chromatography, the protein was greater than 95% pure. The identity of the purified protein was confirmed by analysis of its NH2-terminal amino acid sequence, which was the same as that predicted from the partial nucleotide sequence of the traJ gene (Thompson, R., and Taylor, L. (1982) Mol. Gen. Genet. 188, 513-518). This analysis also indicated that the TraJ protein is localized in the cell without proteolytic modification of its NH2-terminus. We discuss the possible significance of these observations with respect to the cellular functions of the TraJ protein.