Hydrogen sulfide (H(2)S), an endogenous gas molecule synthesized by cystathionine-β-synthetase (CBS), is involved in inflammation and nociceptive signaling. However, the molecular and epigenetic mechanisms of CBS-H(2)S signaling in peripheral nociceptive processing remain unknown. We demonstrated that peripheral inflammation induced by intraplantar injection of complete Freund adjuvant significantly up-regulated expression of CBS at both protein and mRNA levels in rat dorsal root ganglia (DRG). The CBS inhibitors hydroxylamine and aminooxyacetic acid attenuated mechanical hyperalgesia in a dose-dependent manner and reversed hyperexcitability of DRG neurons in inflamed rats. Intraplantar administration of NaHS (its addition mimics CBS production of H(2)S) or l-cysteine in healthy rats elicited mechanical hyperalgesia. Application of NaHS in vitro enhanced excitability and tetrodotoxin (TTX)-resistant sodium current of DRG neurons from healthy rats, which was attenuated by pretreatment of protein kinase A inhibitor H89. Methylation-specific PCR and bisulfite sequencing demonstrated that promoter region of cbs gene was less methylated in DRG samples from inflamed rats than that from controls. Peripheral inflammation did not alter expression of DNA methyltransferase 3a and 3b, the 2 major enzymes for DNA methylation, but led to a significant up-regulation of methyl-binding domain protein 4 and growth arrest and DNA damage inducible protein 45α, the enzymes involved in active DNA demethylation. Our findings suggest that epigenetic regulation of CBS expression may contribute to inflammatory hyperalgesia. H(2)S seems to increase TTX-resistant sodium channel current, which may be mediated by protein kinase A pathway, thus identifying a potential therapeutic target for the treatment of chronic pain.

The cancer stem cell theory hypothesizes that cancer stem cells (CSCs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor formation, recurrence and metastasis. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. In this study, we enriched gastric cancer stem cells through spheroid body formation by cultivating the human gastric cancer cell line MKN-45 in defined serum-free medium. The stemness characteristics of spheroid body-forming cells, including self-renewal, proliferation, chemoresistance, tumorigenicity of the MKN-45 spheroid body-forming cells were evaluated, and the expression levels of stemness genes and related proteins in the MKN-45 spheroid body-forming cells were assessed. Furthermore, immunofluorescence staining for the stem cell markers on spheroid body-forming cells was examined to evaluate the association between stemness factors (Oct4, Sox2, Nanog) and the proposed CSC marker CD44. Our data demonstrated that non-adherent spheroid body-forming cells from the gastric cancer cell line MKN-45 cultured in stem cell-conditioned medium possessed gastric CSC properties, such as persistent self-renewal, extensive proliferation, drug resistance, high tumorigenic capacity and overexpression of CSC-related genes and proteins (Oct4, Sox2, Nanog and CD44), compared with the parental cells. More importantly, CD44-positive cells co-expressing the pluripotency genes Oct4, Sox2 and Nanog may represent gastric CSCs. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify CSCs.

No abstract

BackgroundHydrogen sulfide (H2S), an endogenous gaseotransmitter/modulator, is becoming appreciated that it may be involved in a wide variety of processes including inflammation and nociception. However, the role and mechanism for H2S in nociceptive processing in trigeminal ganglion (TG) neuron remains unknown. The aim of this study is to investigate distribution of endogenous H2S synthesizing enzyme cystathionine-β-synthetase (CBS) expression and role of H2S on excitability and voltage-gated potassium channels of TG neurons.MethodsImmunofluorescence studies were carried out to determine whether CBS was co-expressed in Kv1.1 or Kv1.4-positive TG neurons. Whole cell patch clamp recordings were employed on acutely isolated TG neurons from adult male Sprague Dawley rats (6–8 week old). von Frey filaments were used to examine the pain behavioral responses in rats following injection of sodium hydrosulfide.ResultsIn rat TG, 77.3±6.6% neurons were immunoreactive for CBS, 85.1±3.8% for Kv1.1 and 97.8±1.1% for Kv1.4. Double staining showed that all CBS labeled cells were Kv1.1 and Kv1.4 positive, but only 92.2±6.1% of Kv1.1 and 78.2±9.9% of Kv1.4 positive cells contained CBS. Application of H2S donor NaHS (250 μM) led to a significant depolarization of resting membrane potential recorded from TG neurons. NaHS application also resulted in a dramatic reduction in rheobase, hyperpolarization of action potential threshold, and a significant increase in the number of action potentials evoked at 2X and 3X rheobase stimulation. Under voltage-clamp conditions, TG neurons exhibited transient A-type (IA) and sustained outward rectifier K+ currents (IK). Application of NaHS did suppress IK density while did not change IA density of TG neurons (n=6). Furthermore, NaHS, a donor of hydrogen sulfide, produced a significant reduction in escape threshold in a dose dependent manner.ConclusionThese data suggest that endogenous H2S generating enzyme CBS was co-localized well with Kv1.1 and Kv1.4 in TG neurons and that H2S produces the mechanic pain and increases neuronal excitability, which might be largely mediated by suppressing IK density, thus identifying for the first time a specific molecular mechanism underlying pain and sensitization in TG.