Caveolin-1 in the anterior cingulate cortex modulates chronic neuropathic pain via regulation of NMDA receptor 2B subunit.

Chronic pain is still a basic science and clinical challenge. Unraveling of the neurobiological mechanisms involved in chronic pain will offer novel targets for the development of therapeutic strategies. It is well known that central sensitization in the anterior cingulate cortex (ACC) plays a critical role in initiation, development, and maintenance of chronic pain. However, the underlying mechanisms still remain elusive. Here, we reported that caveolin-1 (Cav-1), a scaffolding protein in membrane rafts, was persistently upregulated and activated in the ACC neurons after chronic constriction injury (CCI) in mice. Knockdown or blocking of Cav-1 in the contralateral ACC to the injury side reversed CCI-induced pain behavioral and neuronal sensitization and overexpression of Cav-1 in the ipsilateral ACC-induced pain behavior in the unaffected hindpaw. Furthermore, we found that Cav-1 directly binding with NMDA receptor 2B subunit (NR2B) and promotion of NR2B surface levels in the ACC contributed to modulation of chronic neuropathic pain. Disrupting the interaction of Cav-1 and NR2B through microinjection of a short peptide derived from the C-terminal of NR2B into the ACC exhibited a significant anti-nociception effect associated with decrease of surface NR2B expression. Moreover, Cav-1 increased intracellular Ca(2+) concentration and activated the ERK/CREB signaling pathway in an NR2B-dependent manner in the ACC. Our findings implicate that Cav-1 in the ACC neurons modulates chronic neuropathic pain via regulation of NR2B and subsequent activation of ERK/CREB signaling, suggesting a possible caveolin-mediated process would participate in neuronal transmission pathways implicated in pain modulation.

Role of ERCC1 promoter hypermethylation in drug resistance to cisplatin in human gliomas.

Overexpression of ERCC1 mRNA is associated with drug resistance to cisplatin in human gliomas, but the role of the ERCC1 promoter in drug resistance has not been demonstrated. We have used sodium bisulfite sequencing to compare ERCC1 promoter methylation patterns in cisplatin-sensitive and cisplatin-resistant glioma cells. The levels of ERCC1 DNA methylation, mRNA and protein in 32 human glioma samples were examined by methylation specific PCR, real-time RT-PCR and immunohistochemistry, respectively. Meanwhile, cisplatin sensitivities to these human glioma samples were tested by histoculture drug response assay. Hypermethylation was observed in the upstream 5Kb region of the ERCC1 promoter of cisplatin-sensitive glioma cell lines. ERCC1 DNA methylation levels were highly variable in 32 human glioma samples ranging from 0.1 to 0.87, which have shown significant difference between cisplatin-sensitive samples and cisplatin-resistant samples (p < 0.05). The relative expression levels of ERCC1 mRNA in 32 glioma samples were also variable from 0.01 to 5.71. No detectable or low expression of ERCC1 protein was shown in 7 glioma samples. ERCC1 promoter methylation was inversely correlated to mRNA expression (r = -0.903 p = 0.001) as well as protein expression (r = -0.884 p = 0.001). Moreover, ERCC1 mRNA expression was significantly associated with protein levels (r = 0.840 p = 0.001). In summary, the aberrant CpG island methylation in ERCC1 promoter region exists in human glioma cell lines as well as clinical glioma samples. ERCC1 DNA methylation could regulate the expression of downstream mRNA and protein, and was associated with cisplatin chemosensitivity.

[Correlation of DNA-PK activity with anti-cancer drug-sensitivity in human gliomas].

OBJECTIVE: To investigate the relationship between DNA-dependent protein kinase (DNA-PK) activity and anti-cancer drug sensitivity in human glioma tissues. METHODS: Human glioma specimens were primarily cultured and its sensitivity to several anti-cancer drugs were evaluated by MTT assay. Nuclear protein was extracted from the glioma sample of the same patient and its DNA-PK activity was determined by a biotinylated DNA-PK assay with p53-derived peptide as a specific substrate. RESULTS: DNA-PK activity varied widely among these glioma samples. Of all 36 samples, 16 showed higher DNA-PK activity (relative activity > or = 0.40) and 20 samples with lower DNA-PK activity (relative activity < 0.40). The gliomas sensitive to DDP and VCR as evaluated by inhibition rate (IR > or = 50%) under plasma peak concentration (PPC) showed lower DNA-PK activity than the resistant ones (IR < 50%) (t = -3.445, P < 0.01). Furthermore, the gliomas with higher DNA-PK activity showed lower inhibition rate (IR < 50%) than those with lower DNA-PK activity ones (t = -2.145, P < 0.05). CONCLUSION: DNA-PK activity is significantly associated with anti-cancer drug sensitivity to DDP and VCR in human gliomas. DNA-PK activity could be used as a new biomarker for the chemotherapy sensitivity of human gliomas.

Co-application of lidocaine and QX-572 induces divergent pain behaviours in mice.

OBJECTIVES: We investigated the analgesic effects of lidocaine (LDC) and lidocane derivative, QX-572, co-application on the evoked pain behaviour (complete Freund's Adjuvant (CFA)-induced) and spontaneous pain behaviour (formalin-induced) in mice. METHODS: The experiments were performed using adult male Kunming mice. Formalin-induced acute pain model and CFA-induced chronic pain model was established by injecting formalin and CFA, respectively. Separate injections of LDC and QX-572, or co-injection of LDC and QX-572, were performed to observe the differences in neurobehavioural responses, paw withdrawal latency (PWL) and mechanical withdrawal threshold (MWT). KEY FINDINGS: QX-572 injection alone did not influence PWL and MWT, but injection of LDC alone led to a substantial, but short-lived, elevation in PWL and MWT (45 min). Co-injection of LDC and QX-572, however, resulted in a significant increase in PWL and MWT (120 min) compared with the LDC group. Injection of LDC and QX-572 combination in the adjacent sciatic nerve also produced a long-lasting sensory-specific nerve block. Additionally, intraplantar co-injection of LDC and QX-572 combination inhibited spontaneous pain in formalin-treated mice, but did not detectably attenuated hyperalgesia and allodynia in CFA-treated mice. CONCLUSIONS: Our results provide evidence that QX-572 induced sensory-selective blockade and co-injection of QX-572 and LDC enhance pain blockade, as evident from formalin-treated mice.

Role of the cerebrospinal fluid-contacting nucleus in the descending inhibition of spinal pain transmission.

The brainstem is well recognized as a critical site for integrating descending modulatory systems that both inhibit and facilitate pain at the level of the spinal cord. The cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) distributes and localizes in the ventral periaqueductal central gray of the brainstem. Although emerging lines of evidence suggest that the CSF-contacting nucleus may be closely linked to transduction and regulation of pain signals, the definitive role of the CSF-contacting nucleus in pain modulation remains poorly understood. In the present study, we determined the role of the CSF-contacting nucleus in rat nocifensive behaviors after persistent pain by targeted ablation of the CSF-contacting nucleus in the brainstem using the cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to cholera toxin subunit B. Compared with CB/SAP, CB-SAP induced complete ablation of the CSF-contacting nucleus, and the CB-SAP-treated rats showed hypersensitivity in responses to acute nociceptive stimulation, and exacerbated spontaneous nocifensive responses induced by formalin, thermal hyperalgesia and mechanical allodynia induced by plantar incision. Furthermore, immunohistochemical experiments showed that the CSF-contacting nucleus was a cluster of 5-HT-containing neurons in the brainstem, and the spinal projection of serotonergic axons originating from the CSF-contacting nucleus constituted the descending 5-HT pathway to the spinal cord. CB-SAP induced significant downregulation of 5-HT in the spinal dorsal horn, and intrathecal injection of 5-HT significantly reversed hypersensitivity in responses to acute nociceptive stimulation in the CB-SAP-treated rats. These results indicate that the CSF-contacting nucleus 5-HT pathway is an important component of the endogenous descending inhibitory system in the control of spinal nociceptive transmission.

Histone deacetylase inhibitor, 2-propylpentanoic acid, increases the chemosensitivity and radiosensitivity of human glioma cell lines in vitro.

BACKGROUND: Treatment for malignant glioma generally consists of cytoreductive surgery followed by radiotherapy and chemotherapy. In this study, we intended to investigate the effects of 2-propylpentanoic acid (VPA), a histone deacetylase inhibitor, on chemosensitivity and radiosensitivity in human glioma cell lines. METHODS: Human glioma cell lines, T98-G, and SF295, were treated with temozolomide (TMZ) or irradiation (IR), with or without VPA (1.0 mmol/L). Then, cytotoxicity and clonogenic survival assay was performed. Cell cycle stage, apoptosis, and autophagy were also detected using flow cytometry and dansyl monocadaverin (MDC) incorporation assay. One-way analysis of variance (ANOVA) and t-test were used to analyze the differences among variant groups. RESULTS: Mild cytotoxicity of VPA was revealed in both cell lines, T98-G and SF295, with the 50% inhibiting concentration (IC50) value of (3.85 ± 0.58) mmol/L and (2.15 ± 0.38) mmol/L, respectively; while the IC50 value of TMZ was (0.20 ± 0.09) mmol/L for T98-G and (0.08 ± 0.02) mmol/L for SF295. Moreover, if combined with VPA (1.0 mmol/L) for 96 hours, the sensitivity of glioma cells to TMZ was significant increased (P < 0.05). The surviving fractions at 2 Gy (SF2) of T98-G and SF295 cells exposed to IR alone were 0.52 and 0.58. However, when VPA was combined with IR, the SF2 of T98-G and SF295 dropped to 0.39 (P = 0.047) and 0.49 (P = 0.049), respectively. Treatment with VPA plus TMZ or IR also resulted in a significant decrease in the proportion of cells in the G2 phase and increased apoptotic rates as well as autophagy in T98-G and SF295 cell lines (P < 0.01). CONCLUSION: VPA may enhance the activities of TMZ and IR on glioma cells possibly through cell cycle block and promote autophagy, and thus could be a potential sensitizer of glioma treatment.

[A method of acutely isolating rat dorsal root ganglion neurons for patch-clamp study of single-channel].

OBJECTIVE: To establish a method of acutely isolating dorsal root ganglion (DRG) neurons for patch clamp study of single-channel. METHODS: DRG neurons of rats were acutely isolated by enzymatic digestion and mechanical blowing. RESULTS: The acutely isolated DRG cells were easy to form the higher sealing resistance (> 5G Omega), which lowered noise level, so that pA-level single channel currents could be recorded. CONCLUSION: The acutely isolated DRG neurons in this study are an ideal for patch-clamp study of single-channel.

Autophagy induced by valproic acid is associated with oxidative stress in glioma cell lines.

Autophagy represents an alternative tumor-suppressing mechanism that overcomes the dramatic resistance of malignant gliomas to radiotherapy and proapoptotic-related chemotherapy. This study reports that valproic acid (VPA), a widely used anti-epilepsy drug, induces autophagy in glioma cells. Autophagy, crucial for VPA-induced cell death, is independent of apoptosis, even though apoptotic machinery is proficient. Oxidative stress induced by VPA occurs upstream of autophagy. Oxidative stress also activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, whereas blocking this pathway inhibits autophagy and induces apoptosis. VPA-induced autophagy cannot be alleviated by inositol, suggesting a mechanism different from that for lithium. Moreover, VPA potentiates autophagic cell death, but not apoptosis, when combined with other autophagy inducers such as rapamycin, Ly294002, and temozolomide in glioma cells both in vitro and in vivo, which may warrant further investigation toward possible clinical application in patients with malignant gliomas.

Activities of DNA-PK and Ku86, but not Ku70, may predict sensitivity to cisplatin in human gliomas.

OBJECTIVE: This study was designed to investigate the relationship between activities of DNA-dependent protein kinase (DNA-PK), its subunits Ku86/Ku70, and sensitivities to cisplatin in human glioma samples. METHODS: Thirty-six glioma samples from patients without prior treatment before neurosurgery were included in this study. The sensitivities to cisplatin as indicated by IC(50) (the inhibitory concentration leading to 50% cell death) were assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenytetrazolium (MTT) assay; activities of DNA-PK and Ku70/Ku86 were analyzed by SigmaTECT DNA-Dependent Protein Kinase Assay System and Ku70/Ku86 DNA Repair Kit, respectively. RESULTS: Sensitivities to cisplatin correlated with the activities of DNA-PK/Ku86, but not with the Ku70 or other clinical parameters such as age, sex of the patients, pathological gradings of the tumors, or tumor size. The levels of DNA-PK activities also associated with pathological grading and Ku86, but not with other clinical parameters. The tumors of the patients who failed to respond to cisplatin-based chemotherapy tended to display higher activity levels of DNA-PK and Ku86. Furthermore, platinum-based chemotherapy did not result in significant changes of DNA-PK/Ku activities in four matched samples before and after chemotherapy. CONCLUSION: Pretreatment determination of DNA-PK/Ku86 activities might be helpful in identifying patients who will actually benefit from platinum-based treatment.

[Expression of costimulator 4-1BBL and B7-1 on glioma cell lines].

BACKGROUND & OBJECTIVE: Costimulatory molecules 4-1BBL and B7-1 have been proposed to be therapeutic targets to improve and sustain antitumor immune response. This study was to investigate the expression of 4-1BBL and B7-1 on several glioma cell lines. METHODS: The expression of 4-1BBL and B7-1 on glioma cell lines T98G, MGR2, MGR1, SF767, SKMG1, SKMG4, and UW28 was detected by flow cytometry. The cytotoxic effects of vincristine (VCR) on the glioma cell lines were assessed with MTT assay. The correlation of 4-1BBL expression to drug resistance was analyzed. RESULTS: The 7 glioma cell lines showed various levels of 4-1BBL expression, but none expressed B7-1. The positive rate of 4-1BBL was above 30% in T98G and MGR1 cells which were resistant to VCR; while it was below 10% in MGR2, SF767, SKMG1, SKMG4, and UW28 cells which were sensitive to VCR. CONCLUSIONS: All of the detected cell lines express 4-1BBL, but none expresses B7-1. The glioma cells with high 4-1BBL expression are resistant to VCR.