INTRODUCTION Prolactinomas are the most frequent pituitary tumor subtype. Despite most of them respond to medical treatment, a proportion are resistant and become a challenge in clinical management. Wnt/β-Catenin pathway has been implicated in several cancers including pituitary tumors and other sellar region malignancies. Interestingly, Wnt/β-Catenin inhibition augments the cytotoxicity of the chemotherapeutic agent Temozolomide (TMZ) in different cancers. TMZ is now being implemented as rescue therapy for aggressive pituitary adenoma treatment. However, the molecular mechanisms associated with TMZ action in pituitary tumors remain unclear. OBJECTIVES Our aims in the present study were to evaluate differential β-Catenin expression in human resistant prolactinomas and Wnt/β-Catenin signaling activation and involvement in Prolactin (PRL) secreting experimental models treated with TMZ. RESULTS We first evaluated by immunohistochemistry β-Catenin localization in human resistant prolactinomas in which we demonstrated reduced membrane β-Catenin in prolactinoma cells compared to normal pituitaries, independently of the Ki-67 proliferation indexes. In turn, in vivo 15 mg/kg of orally administered TMZ markedly reduced PRL production and increased prolactinoma cell apoptosis in mice bearing xenografted prolactinomas. Intratumoral β-Catenin strongly correlated with Prl and Cyclin D1, and importantly, TMZ downregulated both β-Catenin and Cyclin D1, supporting their significance in prolactinoma growth and as candidates of therapeutic targets. When tested in vitro, TMZ directly reduced MMQ cell viability, increased apoptosis and produced G2/M cell cycle arrest. Remarkably, β-Catenin activation and VEGF secretion were inhibited by TMZ in vitro. CONCLUSIONS We concluded that dopamine resistant prolactinomas undergo a β-Catenin relocalization in relation to normal pituitaries and that TMZ restrains experimental prolactinoma tumorigenicity by reducing PRL production and β-Catenin activation. Together, our findings contribute to the understanding of Wnt/β-Catenin implication in prolactinoma maintenance and TMZ therapy, opening the opportunity of new treatment strategies for aggressive and resistant pituitary tumors.

RESEARCH QUESTION:Does resveratrol exert a potent inhibitory effect on the development of endometriosis by interfering with some pivotal processes?DESIGN:In-vitro cultures of primary endometriotic stromal cells, immortalized endometrial stromal (St-T1b) and endometriotic epithelial (12Z) cells were used to assess the effects of resveratrol on endometrial cell mechanisms. The effects of resveratrol on 12Z and St-T1b cell viability were assessed by MTT assay, apoptosis by FITC Annexin V assay and cleaved caspase-3 levels and cell migration by wound healing assay. The effect of resveratrol on the expression of genes related to cell migration, angiogenesis and cell stemness was evaluated by qRT-PCR.RESULTS:Resveratrol significantly decreased cell viability (P= 0.0065 to P = 0.0180), cell migration (P < 0.001 to P = 0.0225) and increased the number of apoptotic cells (P = 0.0031 to P = 0.0432) in both cell lines. In cell lines and primary culture, the treatment reduced MMP-2/TIMP-1 (P < 0.001 to P = 0.0180), VEGF (P = 0.0052 to P = 0.0243) and Ang-1 mRNA (P < 0.001 to P = 0.0382) expression. Among the stem cell phenotype markers, resveratrol 100 µM increased mRNA expression levels of Notch-1 (P < 0.001 to P = 0.0018), KLF-4 (P = 0.0011 to P = 0.0137), SOX-2 (P < 0.001 to P = 0.0070) and TERT (P < 0.001 to P = 0.0193) in both cell lines and primary cultures. The mRNA expression level of Snail-1 increased in the cell lines (P < 0.001 to P = 0.0087), whereas OCT-4 mRNA expression increased in St-T1b (P = 0.0396) and primary cultures (P = 0.0148). Vimentin mRNA expression showed a significant upregulation in primary cultures (P < 0.001). The expression of Msi-1 (P = 0.0145) and NANOG (P = 0.0080) decreased only in St-T1b cells.CONCLUSION:Resveratrol showed inhibitory effects on cell behaviour related to the development of endometriosis by differentially affecting growth, apoptosis, migration and stem cell phenotype of endometrial and endometriotic cells in vitro.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

Anti-VEGF therapy is being implemented as second line drug for aggressive pituitary adenomas that do not respond to conventional treatments. Although it is approved for treatment of several cancers, many of them acquire resistance. In the present work, we evaluated Bevacizumab (BVZ) effect in an in vivo model of prolactinoma in different stages of tumor progression. Immunodeficient nu/nu mice were injected subcutaneously with MMQ tumoral lactotrophs and once xenografts developed, mice were divided in two groups: ‘Early Onset Treatment (EOT)’ or ‘Late Onset Treatment (LOT)’ depending on tumor size at the start of treatment (smaller than 30 mm3 or larger than 60 mm3, respectively). BVZ (25 mg/kg) or saline solution as control were administered twice a week for 2 weeks. When BVZ was administered in EOT group, treated mice showed a slower tumor growth rate than controls since the second week of treatment, although no significant differences in tumor volume were determined at the moment of sacrifice. On the contrary, this beneficial effect was not observed in LOT mice, which increased significantly tumor size in response to BVZ treatment (N=8 per condition in each group, p<0,05). In order to study the mechanisms of resistance involved in late stages of tumor progression, we analyzed VEGF serum levels, which augmented in LOT mice but were not modulated in EOT mice under BVZ treatment (ELISA, N=8, p<0,05). Furthermore, we detected a significant correlation between serum VEGF levels and tumor growth when all animals were considered (r=0,53, N=32, p<0,05). However, no differences were found when analyzing VEGF and bFGF intratumoral content between BVZ treated and controls animals from neither of both groups (ELISA, N=8, ns). A slight increase in SMA+ vessel size but not in their number was determined in BVZ treated animals from EOT group. On the other side, vessels of LOT treated mice tended to be smaller than controls (IHQ, N=8, ns). PCNA proliferation marker expression was increased in tumors which augmented their size in response to BVZ (WB, N=8, p<0,05) and it correlated with tumor growth when mice were evaluated altogether (r=0,4, N=32, p<0,05). Furthermore, we found that proliferation associated proteins as β-CATENIN and C-MYC were increased in BVZ treated-tumors from LOT but not from EOT group (WB, N=8, p<0,05). Tumor necrotic area showed a trend of reduction in BVZ-treated LOT mice but not in EOT mice regarding to controls (H&E, N=8, ns). Our results suggest that tumor progression stage impacts on BVZ response in prolactinomas and that the resistance mechanisms developed in later stages could be associated, not only with the activation of VEGF autocrine loop as observed, but also with the regulation of other cell processes as proliferation and cell death. Further insight into the mediators involved in these mechanisms could help to predict which patients could respond or not to anti-VEGF treatment.