LUIZ FELIPE TEIXEIRA DA SILVA

LUIZ FELIPE TEIXEIRA DA SILVA

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Evaluation of the antiangiogenic activity of zinc in renal adenocarcinoma in a hypoxic microenvironment
2024 - CARLOS, G.O.; NETO, B.M.; FERREIRA, L.S.; TEIXEIRA, L.F.S.; BELLINI, N.H.
Renal cancer is a common urologic malignancy, and therapeutic options for metastatic disease are limited. Most clear cell renal cell carcinomas (ccRCC) are associated with loss of Von Hippel-Lindau tumor suppressor (VHL) function and deregulation of hypoxia pathways. This deregulation induces angiogenesis, promoting tumor growth and metastasis. Vascular endothelial growth factor (VEGF), a pro-angiogenic factor, is overexpressed in ccRCC, contributing to its aggressive nature. Zinc (Zn), an essential micronutrient, has garnered attention for its potential in cancer therapy. While Zn deficiency increases cancer risk, Zn supplementation exhibits anticancer effects, inhibiting proliferation and migration of some types of cancer. However, conflicting evidence regarding Zn's association with RCC necessitates further research to elucidate its role in renal carcinogenesis. This study aimed to evaluate the effect of Zinc Chloride on angiogenesis regulator genes in ccRCC under normoxic and hypoxic conditions. The MTS (3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium-bromide) assay was used to define cell viability of different Zn concentrations on ccRCC cells. The expression of HIF-1a and VEGF-a genes from the 786-0, 786-0 + Zn, 786-0 hypoxia and 786-0 hypoxia + Zn samples was evaluated by RT-qPCR and Western blot. Cell viability assays demonstrated that 100mM of Zn on ccRCC cells showed a significant inhibitory effect. Gene expression analysis by RT-qPCR comparing 786-0 with and without Zn supplementation, under normoxia and hypoxia conditions, showed decreased levels of HIF-1a - 73.75 ± 20.32% (P<0.001 vs. 786-0); 67.99 ± 4.26% (P<0.001 vs. 786-0 hypoxia) and VEGF-a - 60.72 ± 17.58% (P<0.05 vs. 786-0); 56.62 ± 8.07% (P<0.05 vs. 786-0 hypoxia). Western Blot data corroborated the RT-qPCR results. Zinc Chloride demonstrates an antiangiogenic potential in ccRCC cells. Additional experiments are going to be done to comprehensively understand the pathophysiological mechanisms of Zinc Chloride as a novel treatment for ccRCC. Keywords: Angiogensis, Cancer, Zinc
Development of a ready-to-use PSMA-11 kit formulation and biological evaluation of binding affinity
2024 - FREIRE, MARIA R.V.B.; BEXIGA, NATALIA M.; STEPHANO, MARCO A.; OLIVEIRA, JOAO E.; SOARES, CARLOS R.J.; MIRANDA, ANA C.C.; BARBOSA, ANNA C. de A.; BARBOZA, MARYCEL F. de; GARCIA, LAURA F.; TEIXEIRA, LUIZ F.S.; BELLINI, MARIA H.M.; SILVA, NATANAEL G. da; BALIEIRO, LUIZA M.; MENDES, JOEL; ARAUJO, ELAINE B. de
Introduction: 68Ga-PSMA-11 is considered the gold standard in detection of micro and oligometastases in advanced prostate cancer, being used for therapeutic planning, as well as, potentially, for evaluating response to treatment. The development of ready-to-use lyophilized kit of PSMA-11 adds quality and safety to the routine use of this radiopharmaceutical and represents a pharmacotechnical challenge as it must preserve the integrity and specificity of the ligand. Methods: PSMA-11 kit formulation was proposed, considering radiolabeling parameters and the preservation of the peptide during the lyophilization process, using mannitol as an excipient. Critical temperature characterization studies were carried out using DSC equipment and the freeze-drying process was developed. The direct radiolabeling conditions were evaluated and standardized using 68Ge/68Ga generator eluate from two different manufacturers (ITG and Eckert & Ziegler). The radiochemical purity was evaluated by TLC and HPLC. Biological evaluation was carried out with lyophilized PSMA-11 to demonstrate the integrity of the peptide and preservation of biological activity after the lyophilization process. Results: Based on critical temperature characterization studies, the freeze-drying cycle was designed to reach a freezing temperature of around −40˚C and primary drying at 2˚C. Using 20 mg of mannitol, an intact and elegant lyophilized cake was obtained. PSMA-11 lyophilized kit was directly labeled with 68Ga eluate from 68Ge/68Ga GMP generators (ITG and Eckert & Ziegler) resulting in % RP > 95% at pH 4.0 to 4.5. The results obtained from in vitro and in vivo biological competition studies confirmed the preservation of PSMA-11 affinity for the receptor after lyophilization. Conclusion: A lyophilized formulation (Kit) of PSMA-11 was successfully obtained, which preserved the integrity and biological activity of the peptide and guaranteed radiolabeling efficiency.
ZIP11 and ZnT1 are differentially expressed in human renal cell carcinoma
2024 - FERREIRA, LUANA da S.; TEIXEIRA, LUIZ F.S.; CARLOS, GUILHERME O.; BELLINI, MARIA H.
Renal cell carcinoma (RCC) is a solid, malignant, and heterogeneous tumor originating in the renal tubular epithelium. There are several histopathological classifications, with clear-cell carcinoma (ccRCC) being the most common. Its occurrence is associated with mutations in the von Hippel-Lindau gene, which regulates cellular responses to stress, division, death, and differentiation. The loss of functionality affects the degradation of hypoxia-inducible factor (HIF). In turn, the HIF1α subunit becomes highly expressed. Zinc (Zn) is the second most abundant trace element in the human body, and small variations in its concentration can lead to disturbances in the cellular environment. Zn transporters play a fundamental role in Zn homeostasis. In this study, the expression and localization of the Zn channels ZIP11 and ZnT1 were verified in normal tubular renal cells (HK-2) and a clear cell renal adenocarcinoma line (786-0). RT-PCR and western blotting data showed that the ZIP-11 channel was highly expressed in tumor cells. In contrast, the ZnT1 channel was expressed at lower levels in tumor cells. Immunofluorescence data revealed different localization patterns between the two lines. The expression of ZIP-11 in HK-2 cells was predominantly cytoplasmic, while in 786-0 tumor cells, in addition to strong cytoplasmic staining, nuclear staining was also observed. ZnT1 expression in HK-2 cells was cytoplasmic and accentuated on the cell membrane, while that in 786-0 tumor cells was nuclear. These results demonstrated different expression patterns of the key Zn transporters, ZIP11 and ZnT1, in RCC cells compared to those in the normal kidney epithelium. Further elucidation of the molecular mechanisms underlying Zn dysregulation in RCC may reveal potential therapeutic targets.
Micro-Raman spectroscopy identification of hydroxyapatite in dental pulp stem cells
2023 - SILVA, FLAVIA R.O.; PASCOAL, DIEGO R.C.; BERECZKI, ALLAN; SIPERT, CARLA R.; BRAGA, ROBERTO R.; BELLINI, MARIA H.; SILVA, LUIS F.T.; FREITAS, ANDERSON Z.; WETTER, NIKLAUS U.
Cell differentiation using calcium phosphate nanoparticles was studied. The hydroxyapatite was internalized in human dental pulp stem cells and characterized by Raman spectroscopy. Raman spectra showed the hydroxyapatite distribution in nanoparticles nodules in the cells.
NF-ĸΒ1 knockout reduces IL6 expression under hypoxia in renal cell carcinoma
2023 - TEIXEIRA, LUIZ F.S.; BELLINI, MARIA H.
Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer, accounting for more than 90% of all renal neoplasms. Clear cell RCC (ccRCC) is the most common subtype of RCC. Most patients with ccRCC have a mutation in the von Hippel-Lindau (VHL) tumor suppressor gene, which encodes a protein that downregulates various intracellular proteins, including hypoxia-inducible factor (HIF). Many molecules have been identified to be responsible for the aggressive phenotype of ccRCC, including the transcription factor nuclear factor kappa B (NF-кB). The increase in NF-кB activity observed in RCC is correlated with an increase in angiogenesis markers, such as interleukin 6 (IL-6). In recent years, several groups have demonstrated the functional role of NF-кB1 in RCC tumorigenicity. Herein, we used the CRISPR/Cas-9 technique to obtain an NF-кB1 knockout-human renal adenocarcinoma cell line. Expression of IL-6 at the mRNA and protein levels was analyzed under normoxia and hypoxia by real time-polymerase chain reaction and multiplex assay, respectively. The CRISPR/Cas9 technique was effective in producing 786-0 knockout cells for NF-κB1 (p105/p50), as confirmed by western blot analysis. Suppression of p50 expression in 786-0 single guide RNA (sg)1, 786-0 sg2 and 786-0 sg3 cells downregulated IL-6 mRNA and protein expression under normoxia and hypoxia. The observed decrease in the differential expression of IL-6 in hypoxia/normoxia is suggestive of a change in cellular responsiveness to hypoxia with respect to IL-6.
Evaluation of angiogenic capacity of human adenocarcinoma cell line knockout for NF-ĸΒ1 protein
2022 - TEIXEIRA, LUIZ F.S.; BELLINI, MARIA H.
INTRODUCTION: Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer. The most frequent subtype of RCC is clear cell (ccRCC). Most of ccRCC patients have a mutation in the Von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene encodes a protein, the VHL, which can up-regulate a series of intracellular proteins, including the hypoxia inducible factor (HIF). The transcription factor NF-кB is increased in the ccRCC. OBJECTIVES: To evaluate the impact of the NF-кB1 gene knockout on the VEGF and IL6 expression in the human RCC cells under normoxia and hypoxia. MATERIALS AND METHODS: The CRISPR/Cas-9 technique was used to obtain 786-0 cells knockout for the NF-кB1 protein. Western Blot assay was used to selected the clones. A hypoxia-inducing humid chamber was used and its effectiveness was validated its effectiveness was certified by the analysis of HIF-2α expression levels. The quantification of VEGF and IL-6 levels was measured using Real Time-PCR and MILLIPLEX assay. DISCUSSION AND RESULTS: The VEGF gene expression in the clones was significantly lower than that presented by the control both in normoxia (786-0-sg1 99.68±0.09%, 786-0-sg2 78.55±0.85%, 786-0-sg3 91.70±0.87%) and in hypoxia (786-0-sg1 98.30±1.49%, 786-0-sg2 75.21±4.14%, 786-0-sg3 98.44±0.18%). The expression of IL-6 gene was also significant lower in normoxia (786-0-sg1 49.03±0.80%, 786-0-sg2 76.59±12.43%, 786-0-sg3 66.98±10.89%) and in hypoxia (786-0-sg1 95.85±0.36%, 786-0-sg2 96.45±0.49%, 786-0-sg3 91.08±1.42%). The MILLIPLEX results show that there was a significant reduction of both VEGF and IL-6 in the culture medium of cells knocked out in normoxia and hypoxia compared to control group. CONCLUSION: Suppression of p50 expression in the clones resulted in the reduction of VEGF and IL6 in both conditions. The reduction in the IL-6 relative expression hypoxia/normoxia demonstrates a change in cellular responsiveness to decreased levels of oxygen.
Micro-Raman spectroscopy characterization of dental pulp stem cells differentiation induced by calcium phosphate nanoparticles
2022 - SILVA, FLAVIA R.O.; PASCOAL, DIEGO R.C.; BERECZKI, ALLAN; SIPERT, CARLA R.; BRAGA, ROBERTO R.; BELLINI, MARIA H.; SILVA, LUIS F.T. da; FREITAS, ANDERSON Z.; WETTER, NIKLAUS U.
Calcium phosphates are chemical compounds used in medicine for tissue engineering. This work analyzes the process of cell differentiation by nanoparticles in dental pulp stem cells for tissues regeneration and the development of new therapeutic methods. The most widely used synthetic calcium phosphate based bioceramic is hydroxyapatite [HA, Ca10(PO4)6(OH)2]. Micro-Raman spectroscopy assays are a powerful tool for measuring and characterizing calcium phosphates nanoparticles internalized by cells because of its capability to detect the chemical bonds of nanoparticles and collagen simultaneously and evidencing their interaction within the cell-nanoparticle system. Microscope images (Fig.1a) and Raman spectra (Fig.1b) were obtained for HA-incorporated stem cells where it was possible to observe the formed nodules of calcium phosphate and the matrix in the incorporated samples. HA and collagen peaks were detected in the samples, showing that the nanoparticles induced osteogenic differentiation of the stem cells. The spectra of the nodules showed HA characteristic peaks, while matrix spectra displayed characteristic collagen peaks. Studies have been carried out for the development of new and modified calcium phosphate nanoparticles that should further improve biostimulation.
Identification of appropriate housekeeping genes for gene expression studies in human renal cell carcinoma under hypoxic conditions
2022 - TEIXEIRA, LUIZ F.S.; GIGLIOTTI, RODRIGO; FERREIRA, LUANA da S.; BELLINI, MARIA H.
Background: Hypoxia pathways are deregulated in clear renal cell carcinoma (ccRCC) because of the loss of the von Hippel-Lindau tumor suppressor function. Quantitative PCR is a powerful tool for quantifying differential expression between normal and cancer cells. Reliable gene expression analysis requires the use of genes encoding housekeeping genes. Therefore, in this study, eight reference candidate genes were evaluated to determine their stability in 786-0 cells under normoxic and hypoxic conditions. Methods and Results: Four different tools were used to rank the most stable genes—geNorm, NormFinder, BestKeeper, and Comparative Ct (ΔCt), and a general ranking was performed using RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable. There was no agreement among the results from the algorithms for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes for use in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. Conclusions: To the best of our knowledge, this is the first study to identify reliable genes that can be used for gene expression analysis in ccRCC in a hypoxic environment.
Lanthanide-based β-tricalcium phosphate upconversion nanoparticles as an effective theranostic nonviral vectors for image-guided gene therapy
2022 - SILVA, FLAVIA R.O.; LIMA, NELSON B.; BELLINI, MARIA H.; TEIXEIRA, LUIZ F.S.; DU, ERIC Y.; JAMSHIDI, NILOUFAR; GOODING, JUSTIN; MARTIN, ADAM D.; MACMILLAN, ALEXANDER; MARQUIS, CHRISTOPHER P.; THORDARSON, PALL
Lanthanide-based beta-tricalcium phosphate (β-TCP) upconversion nanoparticles are exploited as a non-viral vector for imaging guided-gene therapy by virtue of their unique optical properties and multi-modality imaging ability, high transfection efficiency, high biocompatibility, dispersibility, simplicity of synthesis and surface modification. Ytterbium and thulium-doped β-TCP nanoparticles (βTCPYbTm) are synthesized via co-precipitation method, coated with polyethylenimine (PEI) and functionalized with a nuclear-targeting peptide (TAT). Further, in vitro studies revealed that the nanotheranostic carriers are able to transfect cells with the plasmid eGFP at a high efficiency, with approximately 60% of total cells producing the fluorescent green protein. The optimized protocol developed comprises the most efficient βTCPYbTm/PEI configuration, the amount and the order of assembly of βTCPYbTm:PEI, TAT, plasmid DNA and the culturing conditions. With having excellent dispersibility and high chemical affinity toward nucleic acid, calcium ions released from βTCPYbTm:PEI nanoparticles can participate in delivering nucleic acids and other therapeutic molecules, overcoming the nuclear barriers and improving the transfection efficacy. Equally important, the feasibility of the upconversion multifunctional nanovector to serve as an effective contrast agent for imaging modality, capable of converting low-energy light to higher-energy photons via a multi-photons mechanism, endowing greater unique luminescent properties, was successfully demonstrated.
In vitro and in vivo response of PSMA-617 radiolabeled with CA and NCA lutetium-177
2022 - BOAS, CRISTIAN A.W.V.; SILVA, JEFFERSON de J.; DIAS, LUIS A.P.; FREIRE, MARIA R.B.; BALIEIRO, LUIZA M.; SANTOS, CAROLINA S.F. dos; VIVALDINI, BIANCA F.; BENEDETTO, RAQUEL; VIEIRA, DANIEL P.; PASSOS, PRISCILA de Q.S.; MARUMO, MARIA H.; TEIXEIRA, LUIS F.S.; ARAUJO, ELAINE B. de
The PSMA-targeted radionuclide therapy has been explored since 2015 with radioisotope lutetium-177, whose β− emission range is adequate for micrometastases treatment. This radioisotope is obtained by two different production routes that directly affect the specific activity of lutetium-177 (non-carrier added and carrier added) and, consequently, the specific activity of radiopharmaceuticals, like 177Lu-PSMA-617. The influence of the specific activity of lutetium-177 on the properties of the radiopharmaceutical PSMA-617 was evaluated through pre-clinical studies. The in vitro study pointed to a lower constant of dissociation with non-carrier added lutetium-177 due to the difference in the specific activity. However, competition and internalization assays resulted in similar results for both lutetium-177. Based on these pre-clinical experiments, the total in vitro tumor cell binding and tumor uptake in vivo were similar, with no influence of the specific activity of the 177Lu-PSMA-617. Regardless the specific activity did not directly affect tumor uptake, the tumor/non-target organs ratios were higher for the radiopharmaceutical labeled with carrier added lutetium-177, which had the lowest specific activity.