Decursinol-mediated antinociception and anti-allodynia in acute and neuropathic pain models in male mice: Tolerance and receptor profiling.

Korean scientists have shown that oral administration of Angelica gigas Nakai (AGN) root alcoholic extract and the metabolite of its pyranocoumarins, decursinol, have antinociceptive properties across various thermal and acute inflammatory pain models. The objectives of this study were 1) to assess whether tolerance develops to the antinociceptive effects of once-daily intraperitoneally administered decursinol (50 mg/kg) in acute thermal pain models, 2) to establish its anti-allodynic efficacy and potential tolerance development in a model of chemotherapy-evoked neuropathic pain (CENP) and 3) to probe the involvement of select receptors in mediating the pain-relieving effects with antagonists. The results show that decursinol induced antinociception in both the hot plate and tail-flick assays and reversed mechanical allodynia in mice with cisplatin-evoked neuropathic pain. Tolerance was detected to the antinociceptive effects of decursinol in the hot plate and tail-flick assays and to the anti-allodynic effects of decursinol in neuropathic mice. Pretreatment with either the 5-HT2 antagonist methysergide, the 5-HT2A antagonist volinanserin, or the 5-HT2C antagonist SB-242084 failed to attenuate decursinol-induced antinociception in the tail-flick assay. While pretreatment with the cannabinoid inverse agonists rimonabant and SR144528 failed to modify decursinol-induced anti-allodynia, pretreatment with the opioid antagonist naloxone partially attenuated the anti-allodynic effects of decursinol. In conclusion, our data support decursinol as an active phytochemical of AGN having both antinociceptive and anti-allodynic properties. Future work warrants a more critical investigation of potential receptor mechanisms as they are likely more complicated than initially reported.

Optimization of Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids.

Three-dimensional (3D) cell culture models are widely used in tumor studies to more accurately reflect cell-cell interactions and tumor growth conditions in vivo. 3D anchorage-independent spheroids derived by culturing cells in ultra-low attachment (ULA) conditions is particularly relevant to ovarian cancer, as such cell clusters are often observed in malignant ascites of late-stage ovarian cancer patients. We and others have found that cells derived from anchorage-independent spheroids vary widely in gene expression profiles, proliferative state, and metabolism compared to cells maintained under attached culture conditions. This includes changes in mitochondrial function, which is most commonly assessed in cultured live cells by measuring oxygen consumption in extracellular flux assays. To measure mitochondrial function in anchorage-independent multicellular aggregates, we have adapted the Agilent Seahorse extracellular flux assay to optimize measurements of oxygen consumption and extracellular acidification of ovarian cancer cell spheroids generated by culture in ULA plates. This protocol includes: (i) Methods for culturing tumor cells as uniform anchorage-independent spheroids; (ii) Optimization for the transfer of spheroids to the Agilent Seahorse cell culture plates; (iii) Adaptations of the mitochondrial and glycolysis stress tests for spheroid extracellular flux analysis; and (iv) Suggestions for optimization of cell numbers, spheroid size, and normalization of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values. Using this method, we have found that ovarian cancer cells cultured as anchorage-independent spheroids display altered mitochondrial function compared to monolayer cultures attached to plastic dishes. This method allows for the assessment of mitochondrial function in a more relevant patho/physiological culture condition and can be adapted to evaluate mitochondrial function of various cell types that are able to aggregate into multicellular clusters in anchorage-independence. Graphic abstract: Workflow of the Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids.

Sex Differences in Tolerance to Delta-9-Tetrahydrocannabinol in Mice With Cisplatin-Evoked Chronic Neuropathic Pain.

Tolerance to the pain-relieving effects of cannabinoids limits the therapeutic potential of these drugs in patients with chronic pain. Recent preclinical research with rodents and clinical studies in humans has suggested important differences between males and females in the development of tolerance to cannabinoids. Our previous work found that male mice expressing a desensitization resistant form (S426A/S430A) of the type 1 cannabinoid receptor (CB1R) show delayed tolerance and increased sensitivity to the antinociceptive effects of delta-9-tetrahydrocannabinol (∆9-THC). Sex differences in tolerance have been reported in rodent models with females acquiring tolerance to ∆9-THC faster than males. However, it remains unknown whether the S426A/S430A mutation alters analgesic tolerance to ∆9-THC in mice with chemotherapy-evoked chronic neuropathic pain, and also whether this tolerance might be different between males and females. Male and female S426A/S430A mutant and wild-type littermates were made neuropathic using four once-weekly injections of 5 mg/kg cisplatin and subsequently assessed for tolerance to the anti-allodynic effects of 6 and/or 10 mg/kg ∆9-THC. Females acquired tolerance to the anti-allodynic effects of both 6 and 10 mg/kg ∆9-THC faster than males. In contrast, the S426A/S430A mutation did not alter tolerance to ∆9-THC in either male or female mice. The anti-allodynic effects of ∆9-THC were blocked following pretreatment with the CB1R antagonist, rimonabant, and partially blocked following pretreatment with the CB2R inverse agonist, SR144528. Our results show that disruption of the GRK/ß-arrestin-2 pathway of desensitization did not affect sensitivity and/or tolerance to ∆9-THC in a chronic pain model of neuropathy.

Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells.

Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase (SOD2). This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 upregulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.