RESUMEN
Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial.W In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G - cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.
Asunto(s)
Edición Génica/métodos , Antígenos HLA-G/genética , Antígenos HLA-G/metabolismo , Sistemas CRISPR-Cas , Línea Celular Tumoral , Antígenos HLA-G/inmunología , Humanos , Inmunoterapia/métodos , ARN Guía de Kinetoplastida , TransfecciónRESUMEN
OBJECTIVE: To test cell penetrating and interfering peptide Mut3DPT-PP2A/SET in interaction between serine threonine phosphatase PP2A and its physiological inhibitor, the oncoprotein SET. MATERIALS AND METHODS: Adult male C3H/S-strain mice, 60 days old, were given a graft of breast adenocarcinoma cells (TN60) into subcutaneous tissue. Mut3DPT-PP2A/SET peptide was used to block PP2A and SET oncoprotein interaction. The graft-bearing animals were divided into a control group (injected with saline buffer), and an intervention group injected intraperitoneally with Mut3DPT-PP2A/SET peptide (5 mg/kg) every day from day 5 to day 37. The variables we used to compare the outcome in both groups were tumor size in mm (length×width) and histological changes. In the statistical analysis we used ANOVA and Student-Keuls multiple comparisons test and Tuckey for the post-test analysis. RESULTS: 48 mice were grafted at day 0 with breast UNLP-C3H/S tumor cells, and after randomization, they were assigned to one of the two study groups. At day 5 all mice were injected either with placebo or with the peptide. The treated group showed significant tumor reduction (p<0.07). Histological changes showed presence of apoptosis and necrosis of tumor in treated group. CONCLUSION: The peptide Mut3DPT-PP2A/SET has demonstrated anti-tumor activity by reduction in vivo of tumor growth becoming a promising future in anticancer therapy.
Asunto(s)
Adenocarcinoma/patología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Péptidos de Penetración Celular/farmacología , Proteínas de Unión al ADN/efectos de los fármacos , Chaperonas de Histonas/efectos de los fármacos , Proteína Fosfatasa 2/efectos de los fármacos , Adenocarcinoma/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Chaperonas de Histonas/metabolismo , Ratones , Necrosis , Péptidos/farmacología , Proteína Fosfatasa 2/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Out of all the available methods for estimating age at death from immature human skeletal remains, those based on odontometric variables of deciduous dentition have proved to be one of the most accurate. The development of odontometric methods has been improved through the creation of documented human osteological collections, allowing their validation in different populations. The present study aims to test the regression equations for age estimation proposed by Liversidge et al. 1993, Irurita Olivares et al. 2014, and Cardoso et al. 2019, on the basis of the maximum length of deciduous teeth in an Argentinian sample of 35 infants of known age at death. The results showed that the absolute mean difference between estimated and chronological age was 5.76±6.33 weeks for Liversidge's method, 5.71±6.41 weeks for Irurita Olivares's method, and 6.79±5.80 for Cardoso's method. It was also found that, for Liversidge's method, the canines provided the most accurate and the least biased estimations. For Irurita Olivares's method, mandibular anterior teeth were the most accurate, while the first mandibular molars offered the least biased estimations. For Cardoso's method, the canines presented the most accurate estimations, while the lateral incisors the least biased ones. Finally, 95% confidence intervals of estimated ages were calculated for each method, finding that Irurita Olivares's method provided the most reliable age estimations when using mandibular central incisors and mandibular first molars.
Asunto(s)
Determinación de la Edad por los Dientes/métodos , Diente Primario , Argentina , Preescolar , Diente Canino/anatomía & histología , Femenino , Odontología Forense , Humanos , Incisivo/anatomía & histología , Lactante , Recién Nacido , Masculino , Mandíbula , Diente Molar/anatomía & histología , Análisis de RegresiónRESUMEN
Partial hepatectomy (PH) alters serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. Furthermore, angiogenesis is mainly stimulated by vascular endothelial growth factor (VEGF) and is a fundamental requirement either in liver regeneration or in tumours growth. This study looks at the expression of VEGF, DNA synthesis (DNAs) and mitotic activity (MA) in hepatectomised (H) and hepatectomised-tumour bearing (HTB) mice throughout a 24 h period. Adult male mice were sacrificed every 4 h from 26 to 50 h post-hepatectomy. H mice show a circadian rhythm in VEGF expression with a maximum value of 2.6 ± 0.1 at 08/46 h of day/hours posthepatectomy (HD/HPH); in DNAs, the maximum value was 3.4 ± 0.3 at 16/30 (HD/HPH) and in MA it was 2.3 ± 0.01 at 12/50 (HD/HPH). In HTB animals the peak of VEGF expression appears at 16/30 (HD/HPH) with a maximum value of 3.7 ± 0.1, the peak of DNAs was at 00/38 (HD/HPH) with a value of 4.6 ± 0.3 and the maximum value of MA of 08/46 (HD/HPH) with a value of 3.01 ± 0.3. We can conclude that the presence of the tumour induces modifications in the intensity and the temporal distribution of the circadian curves of VEGF expression, DNAs and MA of hepatectomised animals.
Asunto(s)
ADN/biosíntesis , Hepatectomía , Hepatocitos/metabolismo , Neoplasias Hepáticas/irrigación sanguínea , Neoplasias Hepáticas/cirugía , Mitosis , Neovascularización Patológica/metabolismo , Animales , Ritmo Circadiano , Hepatocitos/patología , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/fisiopatología , Masculino , Ratones , Neovascularización Patológica/patología , Neovascularización Patológica/fisiopatología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Transplanted tumours could modify the intensity and temporal distribution of the cellular proliferation in normal cell populations, and partial hepatectomy alters the serum concentrations of substances involved in cellular proliferation, leading to the compensatory liver hyperplasia. The following experiments were designed in order to study the SI (S-phase index) and VEGF (vascular endothelial growth factor) expression in regenerating liver (after partial hepatectomy) of adult male mice bearing a hepatocellular carcinoma, throughout one complete circadian cycle. We used adult male C3H/S-strain mice. After an appropriate period of synchronization, the C3H/S-histocompatible ES2a hepatocellular carcinoma was grafted into the subcutaneous tissue of each animal's flank. To determine the index of SI and VEGF expression of hepatocytes, we used immunohistochemistry. The animals were divided into two experimental groups: Group I, control, hepatectomized animals; Group II, hepatectomized tumour-bearing animals. The statistical analysis of SI and VEGF expression was performed using Anova and Tukey as a postcomparison test. The results show that in the second group, the curve of SI changes the time points for maximum and minimum activity, and the peak of VEGF expression appears before the first group. In conclusion, in the hepatectomized mice, the increases of hepatic proliferation, measured by the SI index, may produce a rise in VEGF expression with the object of generating a vascular network for hepatic regeneration. Lastly, as we have mentioned, in hepatectomized and tumour-bearing mice, the peak of VEGF expression appears before the one of DNA synthesis.