Selective anti-CXCR2 receptor blockade by AZD5069 inhibits CXCL8-mediated pro-tumorigenic activity in human thyroid cancer cells in vitro.

BACKGROUND: Thyroid cancer is the most common endocrine malignancy. Current therapies are successful, however some patients progress to therapeutically refractive disease. The immunotherapeutic potential of the CXCL8-chemokine/CXCR2-chemokine-receptor system is currently being explored in numerous human cancers. This study aimed to evaluate if the targeting of CXCR2 by its selective antagonist, AZD5069, could modulate CXCL8-mediated pro-tumorigenic effects in thyroid-cancer (TC) cells in vitro. METHODS: Normal human primary thyroid cells (NHT) and TC cell lines TPC-1 (RET/PTC), BCPAP, 8505C and 8305C (BRAFV600e) were treated with AZD5069 (100 pM-10 µM) over a time-course. Viability and proliferation were assessed by WST-1 and crystal violet assays. CXCL8 and CXCR2 mRNA were evaluated by RT-PCR. CXCL8-protein concentrations were measured in cell culture supernatants by ELISA. CXCR2 on cell surface was evaluated by flow-cytometry. Cell-migration was assessed by trans-well-migration chamber-system. RESULTS: AZD5069 exerted negligible effects on cell proliferation or viability. AZD5069 significantly reduced CXCR2, (but not CXCL8) mRNAs in all cell types. CXCR2 was reduced on the membrane of some TC cell lines. A significant reduction of the CXCL8 secretion was found in TPC-1 cells (basal-secretion) and NHT (TNFα-induced secretion). AZD5069 significantly reduced basal and CXCL8-induced migration in NHT and different TC cells. CONCLUSIONS: Our findings confirm the involvement of the CXCL8/CXCR2-axis in promoting pro-tumorigenic effects in TC cells, further demonstrating its immunotherapeutic significance in human cancer.

Serum cytokines and CXCR2: potential tumour markers in ovarian neoplasms.

PURPOSE: The aim was to investigate the systemic levels of cytokines and the expression of the chemokine receptor CXCR2 in circulating neutrophils in patients with non-neoplastic ovarian lesions, benign neoplasia or malignant neoplasia. MATERIALS AND METHODS: Controls and patients with ovarian tumours were pre-operatively compared for the production of cytokines (IL-2, IL-5, IL-6, IL-8, IL-10 and TNF-α) by ELISA, and for the expression of the chemokine receptor, CXCR2, in neutrophils, by flow cytometry. Randomly selected patients within the malignant group were re-evaluated for the inflammatory parameters at 30 days after surgery. RESULTS: The serum concentrations of IL-6, IL-8 and IL-10 were significantly higher in the benign and malignant neoplasia than in the control group, and their levels were significantly higher in ovarian cancer patients than in patients with non-neoplastic tumours or benign neoplasia. Treatment reduced IL-8 serum levels but did not affect CXCR2 expression in neutrophils. Cut-off values for IL-6, IL-8, and IL-10 comparing malignant vs. benign neoplasia were 11.3, 71.7, 14.8, and comparing malignant neoplasm vs. non-neoplastic lesions were 7.2, 43.5, 12.3, respectively. CONCLUSIONS: Serum IL-6, IL-8, and IL-10 levels, and expression of CXCR2 in circulating neutrophils seem promising for distinguishing ovarian cancer patients from patients with benign tumours.

Role of CINC-1 and CXCR2 receptors on LPS-induced fever in rats.

The classic model of fever induction is based on the administration of lipopolysaccharide (LPS) from Gram-negative bacteria in experimental animals. LPS-induced fever results in the synthesis/release of many mediators that assemble an LPS-fever cascade. We have previously demonstrated that cytokine-induced neutrophil chemoattractant (CINC)-1, a Glu-Leu-Arg (ELR) + chemokine, centrally administered to rats, induces fever and increases prostaglandin E2 in the cerebrospinal fluid. We now attempt to investigate the involvement of CINC-1 and its functional receptor CXCR2 on the fever induced by exogenous and endogenous pyrogens in rats. We also investigated the effect of reparixin, an allosteric inhibitor of CXCR1/CXCR2 receptors, on fever induced by either systemic administration of LPS or intracerebroventricular injection of CINC-1, as well as TNF-α, IL-1ß, IL-6, or ET-1, known mediators of febrile response. Our results show increased CINC-1 mRNA expression in the liver, hypothalamus, CSF, and plasma following LPS injection. Moreover, reparixin administered right before CINC-1 or LPS abolished the fever induced by CINC-1 and significantly reduced the response induced by LPS. In spite of these results, reparixin does not modify the fever induced by IL-1ß, TNF-α, and IL-6, but significantly reduces ET-1-induced fever. Therefore, it is plausible to suggest that CINC-1 might contribute to LPS-induced fever in rats by activating CXCR2 receptor on the CNS. Moreover, it can be hypothesized that CINC-1 is placed upstream TNF-α, IL-1ß, and IL-6 among the prostaglandin-dependent fever-mediator cascade and amidst the prostaglandin-independent synthesis pathway of fever.

Intracerebellar administration of the chemokine Cxcl3 reduces the volume of medulloblastoma lesions at an advanced stage by promoting the migration and differentiation of preneoplastic precursor cells.

The prognosis for many pediatric brain tumors, including cerebellar medulloblastoma (MB), remains dismal but there is promise in new therapies. We have previously generated a mouse model developing spontaneous MB at high frequency, Ptch1+/-/Tis21-/-. In this model, reproducing human tumorigenesis, we identified the decline of the Cxcl3 chemokine in cerebellar granule cell precursors (GCPs) as responsible for a migration defect, which causes GCPs to stay longer in the proliferative area rather than differentiate and migrate internally, making them targets of transforming insults. We demonstrated that 4-week Cxcl3 infusion in cerebella of 1-month-old mice, at the initial stage of MB formation, forces preneoplastic GCPs (pGCPs) to leave lesions and differentiate, with a complete suppression of MB development. In this study, we sought to verify the effect of 4-week Cxcl3 treatment in 3-month-old Ptch1+/-/Tis21-/- mice, when MB lesions are at an advanced, irreversible stage. We found that Cxcl3 treatment reduces tumor volumes by sevenfold and stimulates the migration and differentiation of pGCPs from the lesion to the internal cerebellar layers. We also tested whether the pro-migratory action of Cxcl3 favors metastases formation, by xenografting DAOY human MB cells in the cerebellum of immunosuppressed mice. We showed that DAOY cells express the Cxcl3 receptor, Cxcr2, and that Cxcl3 triggers their migration. However, Cxcl3 did not significantly affect the frequency of metastases or the growth of DAOY-generated MBs. Finally, we mapped the expression of the Cxcr2 receptor in human MBs, by evaluating a well-characterized series of 52 human MBs belonging to different MB molecular subgroups. We found that Cxcr2 was variably expressed in all MB subgroups, suggesting that Cxcl3 could be used for therapy of different MBs.

Cxcl1 monomer-dimer equilibrium controls neutrophil extravasation.

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.

Understanding the Virulence of Staphylococcus pseudintermedius: A Major Role of Pore-Forming Toxins.

Staphylococcus pseudintermedius is responsible for severe and necrotizing infections in humans and dogs. Contrary to S. aureus, the pathophysiological mechanisms involved in this virulence are incompletely understood. We previously showed the intracellular cytotoxicity induced after internalization of S. pseudintermedius. Herein, we aimed to identify the virulence factors responsible for this cytotoxic activity. After addition of filtered S. pseudintermedius supernatants in culture cell media, MG63 cells, used as representative of non-professional phagocytic cells (NPPc), released a high level of LDH, indicating that the cytotoxicity was mainly mediated by secreted factors. Accordingly, we focused our attention on S. pseudintermedius toxins. In silico analysis found the presence of two PSMs (δ-toxin and PSMε) as well as Luk-I leukotoxin, the presence of which was confirmed by PCR in all clinical strains tested (n = 17). Recombinant Luk-I leukotoxin had no cytotoxic activity on NPPc but the ectopic expression of the CXCR2 receptor in U937 cells conferred cytotoxity to Luk-I. This is in agreement with the lack of Luk-I effect on NPPc and the previous report of Luk-I cytoxic activity on immune cells. Contrary to Luk-I, synthetic δ-toxin and PSMε had a strong cytotoxic activity on NPPc. The secretion of δ-toxin and PSMε at cytotoxic concentrations by S. pseudintermedius in culture supernatant was confirmed by HPLC-MS. In addition, the supplementation of such supernatants with human serum, known to inhibit PSM, induced a complete abolition of cytotoxicity which indicates that PSMs are the key players in the cytotoxic phenotype of NPPc. The results suggest that the severity of S. pseudintermedius infections is, at least in part, explained by a combined action of Luk-I that specifically targets immune cells expressing the CXCR2 receptor, and PSMs that disrupt cell membranes whatever the cell types. The present study strengthens the key role of PSMs in virulence of the different species belonging to Staphylococcus genus.