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Braz. j. biol ; 84: e250151, 2024. tab, graf
Article in English | LILACS, VETINDEX | ID: biblio-1350306


Abstract Mammals have a limited capacity to regenerate their tissues and organs. One of the mechanisms associated with natural regeneration is dedifferentiation. Several small molecules such as vitamin C and growth factors could improve reprogramming efficiency. In this study, the NTERA2-D1 (NT2) cells were induced towards differentiation (NT2-RA) with 10-5 M retinoic acid (RA) for three days and then subjected to various amounts of vitreous humor (VH). Results show that the growth rate of these cells was reduced, while this rate was partly restored upon treatment with VH (NT2-RA-VH). Cell cycle analysis with PI method also showed that the numbers of cells at the S phase of the cell cycle in these cells were increased. The levels of SSEA3 and TRA-1-81 antigens in NT2-RA were dropped but they increased in NT2- RA-VH to a level similar to the NT2 cells. The level of SSEA1 had an opposite pattern. Expression of OCT4 gene dropped after RA treatment, but it was recovered in NT2-RA-VH cells. In conclusion, we suggest VH as a potent mixture for improving the cellular reprogramming leading to dedifferentiation.

Resumo Os mamíferos têm uma capacidade limitada de regenerar seus tecidos e órgãos. Um dos mecanismos associados à regeneração natural é a desdiferenciação. Várias moléculas pequenas, como vitamina C e fatores de crescimento, podem melhorar a eficiência da reprogramação. Neste estudo, as células NTERA2-D1 (NT2) foram induzidas à diferenciação (NT2-RA) com ácido retinóico (RA) 10-5 M por três dias e depois submetidas a várias quantidades de humor vítreo (VH). Os resultados mostram que a taxa de crescimento dessas células foi reduzida, enquanto essa taxa foi parcialmente restaurada após o tratamento com VH (NT2-RA-VH). A análise do ciclo celular com o método PI também mostrou que o número de células na fase S do ciclo celular nessas células estava aumentado. Os níveis de antígenos SSEA3 e TRA-1-81 em NT2-RA diminuíram, mas aumentaram em NT2-RA-VH a um nível semelhante ao das células NT2. O nível de SSEA1 teve um padrão oposto. A expressão do gene OCT4 diminuiu após o tratamento com AR, mas foi recuperado em células NT2-RA-VH. Em conclusão, sugerimos o VH como uma mistura potente para melhorar a reprogramação celular levando à desdiferenciação.

Humans , Vitreous Body , Cell Proliferation , Cell Dedifferentiation , Tretinoin , Tumor Cells, Cultured , Cell Differentiation , Cell Division , Cell Line
Rev. invest. clín ; 73(1): 39-51, Jan.-Feb. 2021. graf
Article in English | LILACS | ID: biblio-1289743


ABSTRACT Background: Cancer gene therapy using a nonviral vector is expected to be repeatable, safe, and inexpensive, and to have long-term effectiveness. Gene therapy using the E3 and C1 (E3C1) domain of developmental endothelial locus-1 (Del1) has been shown to improve prognosis in a mouse transplanted tumor model. Objective: In this study, we examined how this treatment affects angiogenesis in mouse transplanted tumors. Materials and methods: Mouse transplanted tumors (SCCKN human squamous carcinoma cell line) were injected locally with a nonviral plasmid vector encoding E3C1 weekly. Histochemical analysis of the transplanted tumors was then performed to assess the effects of E3C1 on prognosis. Results: All mice in the control group had died or reached an endpoint within 39 days. In contrast, one of ten mice in the E3C1 group had died by day 39, and eight of ten had died or reached an endpoint by day 120 (p < 0.01). Enhanced apoptosis in tumor stroma was seen on histochemical analyses, as was inhibited tumor angiogenesis in E3C1-treated mice. In addition, western blot analysis showed decreases in active Notch and HEY1 proteins. Conclusion: These findings indicate that cancer gene therapy using a nonviral vector encoding E3C1 significantly improved life-span by inhibiting tumor angiogenesis. (REV INVEST CLIN. 2021;73(1):39-51)

Animals , Rabbits , Calcium-Binding Proteins/therapeutic use , Carcinoma, Squamous Cell/blood supply , Carcinoma, Squamous Cell/therapy , Cell Adhesion Molecules/therapeutic use , Epidermal Growth Factor/therapeutic use , Discoidin Domain/genetics , Calcium-Binding Proteins/genetics , Tumor Cells, Cultured , Genetic Therapy , Cell Adhesion Molecules/genetics , Amino Acid Motifs , Epidermal Growth Factor/genetics , Mice, Nude , Neoplasm Transplantation , Neovascularization, Pathologic/therapy
Biol. Res ; 53: 13, 2020. tab, graf
Article in English | LILACS | ID: biblio-1100919


BACKGROUND: Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry. RESULTS: After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones (PUC-GBC1, PUC-GBC2 and PUC-GBC3) by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice. CONCLUSIONS: The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent.

Humans , Animals , Male , Middle Aged , Antigens, Tumor-Associated, Carbohydrate/genetics , Indians, South American/genetics , Gallbladder Neoplasms/genetics , Ascitic Fluid/metabolism , Tumor Cells, Cultured , Carcinogenicity Tests , Chile , DNA Fingerprinting , Tumor Suppressor Protein p53/genetics , Cisplatin/pharmacology , Mice, Inbred NOD , Clone Cells/drug effects , Clone Cells/metabolism , Sequence Analysis, RNA , Receptor, ErbB-2/genetics , Genes, erbB-2/genetics , Gene Expression Profiling , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Epithelial Cells/metabolism , Keratin-19/genetics , Keratin-7/genetics , Carcinogenesis/genetics , Gallbladder Neoplasms/metabolism , Antineoplastic Agents/pharmacology
Rev. invest. clín ; 71(6): 369-380, Nov.-Dec. 2019. tab, graf
Article in English | LILACS | ID: biblio-1289708


ABSTRACT Cancer is the second-leading cause of death in the world, accounting for one out of six deaths. Consequently, there is an urgent need for new and more effective therapeutic options as well as drug screening methods. Immortal, “stable” cancer cell lines have been employed since the past century to assess drug response but face several disadvantages. They often accumulate new genetic aberrations due to long-term culture and lack the indisputable heterogeneity of solid tumors, therefore, compromising the recapitulation of molecular features from parental tumors. Primary cancer cells have emerged as an attractive alternative to commercial cell lines since they can preserve such properties more closely. Here, we provide an overview of the basic concepts underlying generation and characterization of primary cell cultures from tumor samples. We emphasize the advantages and disadvantages of using these types of cancer cell cultures, and we make a comparison with other types of cultures used for personalized therapy. Finally, we consider the use of primary cancer cell cultures in personalized therapy as a means to improve drug response prediction and therapeutic outcomes.

Humans , Cell Culture Techniques , Precision Medicine/methods , Neoplasms/therapy , Tumor Cells, Cultured , Neoplasms/pathology
Bol. latinoam. Caribe plantas med. aromát ; 18(5): 480-491, sept. 2019. ilus, tab
Article in English | LILACS | ID: biblio-1008273


In the present study, we investigated the antiproliferative activity of essential oil from leaves of Melissa officinalis L. grown in Southern Bosnia and Herzegovina. In vitro evaluation of antiproliferative activity of the M. officinalis essential oil was carried out on three human tumor cell lines: MCF-7, NCI-H460 and MOLT-4 by MTT assay. M. officinalis essential oil was characterized by high percentage of monoterpenes (77,5%), followed by the sesquiterpene fraction (14,5%) and aliphatic compounds (2,2%). The main constituents of the essential oil of M. officinalis are citral (47,2%), caryophyllene oxide (10,2%), citronellal (5,4%), geraniol (6,6%), geranyl acetate (4,1%) and ß- caryophyllene (3,8%). The essential oil showed significant antiproliferative activity against three cancer cell lines, MOLT-4, MCF-7, and NCI-H460 cells, with GI50 values of <5, 6±2 and 31±17 µg/mL, respectively. The results revealed that M. officinalis L. essential oil has a potential as anticancer therapeutic agent.

En el presente estudio, investigamos la actividad antiproliferativa del aceite esencial de las hojas de Melissa officinalis L. cultivadas en el sur de Bosnia y Herzegovina. La evaluación in vitro de la actividad antiproliferativa del aceite esencial de M. officinalis se llevó a cabo en tres líneas celulares de tumores humanos: MCF-7, NCI-H460 y MOLT-4 utilizando el ensayo de MTT. El aceite esencial de M. officinalis se caracterizó por un alto porcentaje de monoterpenos (77,5%), seguido de la fracción sesquiterpénica (14,5%) y compuestos alifáticos (2,2%). Los principales constituyentes del aceite esencial de M. officinalis fueron citral (47,2%), óxido de cariofileno (10,2%), citronelal (5,4%), geraniol (6,6%), acetato de geranilo (4, 1%), y ß-cariofileno (3,8%). El aceite esencial mostró una actividad antiproliferativa significativa contra las líneas celulares de cáncer MOLT-4, MCF-7 y NCI-H460, con valores GI50 de <5, 6±2 y 31±17 µg/mL, respectivamente. Los resultados revelaron que el aceite esencial de M. officinalis L. tiene potencial como agente terapéutico contra el cáncer.

Oils, Volatile/pharmacology , Melissa , Antineoplastic Agents/pharmacology , Sesquiterpenes/analysis , In Vitro Techniques , Oils, Volatile/chemistry , Tumor Cells, Cultured , Plant Leaves , Monoterpenes/analysis , Cell Line, Tumor/drug effects , Cell Proliferation/drug effects , Gas Chromatography-Mass Spectrometry , Antineoplastic Agents/chemistry
Electron. j. biotechnol ; 40: 58-64, July. 2019. graf, tab, ilus
Article in English | LILACS | ID: biblio-1053475


Background: Prodigiosin has been demonstrated to be an important candidate in investigating anticancer drugs and in many other applications in recent years. However, industrial production of prodigiosin has not been achieved. In this study, we found a prodigiosin-producing strain, Serratia marcescens FZSF02, and its fermentation strategies were studied to achieve the maximum yield of prodigiosin. Results: When the culture medium consisted of 16.97 g/L of peanut powder, 16.02 g/L of beef extract, and 11.29 mL/L of olive oil, prodigiosin reached a yield of 13.622 ± 236 mg/L after culturing at 26 °C for 72 h. Furthermore, when 10 mL/L olive oil was added to the fermentation broth at the 24th hour of fermentation, the maximum prodigiosin production of 15,420.9 mg/L was obtained, which was 9.3-fold higher than the initial level before medium optimization. More than 60% of the prodigiosin produced with this optimized fermentation strategy was in the form of pigment pellets. To the best of our knowledge, this is the first report on this phenomenon of pigment pellet formation, which made it much easier to extract prodigiosin at low cost. Prodigiosin was then purified and identified by absorption spectroscopy, HPLC, and LCMS. Purified prodigiosin obtained in this study showed anticancer activity in separate experiments on several human cell cultures: A549, K562, HL60, HepG2, and HCT116. Conclusions: This is a promising strain for producing prodigiosin. The prodigiosin has potential in anticancer medicine studies.

Prodigiosin/biosynthesis , Prodigiosin/pharmacology , Serratia marcescens/metabolism , Antineoplastic Agents/pharmacology , Arachis/chemistry , Powders , Prodigiosin/isolation & purification , Mass Spectrometry , Tumor Cells, Cultured/drug effects , Chromatography, High Pressure Liquid , Chromatography, Liquid , Cell Culture Techniques , Fermentation , Olive Oil/chemistry , Acetates , Nitrogen
Article in Chinese | WPRIM | ID: wpr-773088


The aim of this paper was to investigate the effect of SIRT1/TSC_2 signal axis on leukemia stem cell senescence induced by ginsenoside Rg_1. CD34~+CD38~- leukemia stem cells(CD34~+CD38~-LSCs) was isolated by magnetic cell sorting(MACS) and divided into two groups. The control group cells were routinely cultured, 40 μmol·L~(-1) ginsenoside Rg_1 was added to the control group for co-culture in Rg_1 group. The effect of Rg_l to induce CD34~+CD38~-LSCs senescence were evaluated by senescence-associated β-Galactosidase(SA-β-Gal) staining, cell cycle assay, CCK-8 and Colony-Assay. The expression of senescence associated SIRT1, TSC_2 mRNA and protein was examined by Real-time fluorescence quantitative PCR(FQ-PCR) and Western blot. The results showed that the CD34~+CD38~-LSCs could effectively be isolated by MACS, and the purity of CD34~+CD38~-LSCs is up to(95.86±3.04)%. Compared with the control group, the percentage of positive cells expressed SA-β-Gal in the Rg_1 group is increased, the senescence morphological changes were observed in the CD34~+CD38~-LSCs in the Rg_1 group. The proliferation inhibition rate and the number of cells entered G_0/G_1 phase in the Rg_1 group were increased, but the colony-formed ability was decreased, Rg_1 could significantly inhibit the proliferation and self-renewal ability of CD34~+CD38~-LSCs. The expression of SIRT1 and TSC_2 mRNA and protein were down regulated in the Rg_1 group compared with the control group. Our research implied that Rg_1 may induce the senescence of CD34~+CD38~-LSCs and SIRT1/TSC_2 signal axis plays a significant role in this process.

Cellular Senescence , Ginsenosides , Pharmacology , Humans , Leukemia, Myeloid, Acute , Neoplastic Stem Cells , Signal Transduction , Sirtuin 1 , Metabolism , Tuberous Sclerosis Complex 2 Protein , Metabolism , Tumor Cells, Cultured
Article in English | WPRIM | ID: wpr-776893


Resistance to cisplatin (DDP)-based chemotherapy is a major cause of treatment failure in human gastric cancer (GC). It is necessary to identify the drugs to re-sensitize GC cells to DDP. In our previous research, Zuo Jin Wan Formula (ZJW) has been proved could increase the mitochondrial apoptosis via cofilin-1 in a immortalized cell line, SGC-7901/DDP. Due to the immortalized cells may still difficult highly recapitulate the important molecular events in vivo, primary GC cells model derived from clinical patient was constructed in the present study to further evaluate the effect of ZJW and the underlying molecular mechanism. Immunofluorescent staining was used to indentify primary cultured human GC cells. Western blotting was carried out to detect the protein expression. Cell Counting Kit-8 (CCK-8) was used to evaluate cell proliferation. Flow cytometry analysis was performed to assess cell apoptosis. ZJW inhibited proliferation and induced apoptosis in primary DDP-resistant GC cells. Notably, the apoptosis in GC cells was mediated by inducing cofilin-1 mitochondrial translocation, down-regulating Bcl-2 and up-regulating Bax expression. Surprisingly, the level of p-AKT protein was higher in DDP-resistant GC cells than that of the DDP-sensitive GC cells, and the activation of AKT could attenuate ZJW-induced sensitivity to DDP. These data revealed that ZJW can increase the chemosensitivity in DDP-resistant primary GC cells by inducing mitochondrial apoptosis and AKT inactivation. The combining chemotherapy with ZJW may be an effective therapeutic strategy for GC chemoresistance patients.

Adult , Aged , Aged, 80 and over , Apoptosis , Cell Proliferation , Cisplatin , Pharmacology , Therapeutic Uses , Cofilin 1 , Metabolism , Drug Resistance, Neoplasm , Drugs, Chinese Herbal , Pharmacology , Female , Humans , Male , Middle Aged , Mitochondria , Metabolism , Pathology , Proto-Oncogene Proteins c-akt , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Stomach Neoplasms , Drug Therapy , Metabolism , Pathology , Tumor Cells, Cultured
Article in Chinese | WPRIM | ID: wpr-776050


Objective To determine the effect of denticleless E3 ubiquitin protein ligase(DTL)on the proliferation and clone formation of multiple myeloma(MM)cells and investigate the related mechanism. Methods Mononuclear cells were extracted from 34 MM patients.Mononuclear cells harvested from 14 healthy volunteers were used as controls.Quantitative polymerase chain reaction was used to detect the change of DTL at mRNA level.Furthermore,12 MM patients and 2 controls were selected,in whom the change of DTL at protein level was detected by Western blot.Human MM cell line RPMI8226 was divided into control(CON)group and DTL-short hairpin RNA(DTL-shRNA)group,which was infected with the CON and DTL-shRNA virus,respectively,for 48 hours.The infection efficiency was detected by using flow cytometry,the knock-down efficiencies at mRNA and protein levels were detected by quantitative polymerase chain reaction and Western blot,the change of cell counts in the next 0,24,48,72,96 hours were measured with CCK8 assay.The CON and DTL-shRNA cells were cultured in semisolid medium.Ten days later,inverted phase microscopy was used to measure the number of colones that contain more than 50 cells,annexin V/propidium iodide double staining to detect apoptosis,and propidium iodide staning to detect cell cycle.Finally,Western blot was empoyed to detect the phosphorylation of P65 and inhibitory subunit-κBα(IκBα)in nuclear factor-κB(NF-κB)pathway and electrophoretic mobility shift assay(EMSA)to detect the NF-κB transcriptional ability. Results The DTL expression was(1.00±0.12)and(9.36±3.71),respectively in the bone marrow mononuclear cells of healthy volunteers and in the CD138+cells of MM patients(t=3.65,P=0.0024).DTL was also highly expressed in MM CD138+positive cells at protein level.After RPMI8226 was infected by CON and DTL-shRNA virus for 48 hours,green fluorescent protein-positive cells accounted for more than 90%.The relative expression of DTL was(1.00±0.01)and(0.21±0.04)(t=33.19,P<0.0001)at mRNA level and(0.52±0.13)and(0.11±0.02)at protein level(t=5.399,P=0.0057).CCK8 revealed that CON and DTL-shRNA cells proliferated by(1.00±0.03)vs.(1.00±0.02),(2.19±0.28)vs.(1.47±0.13),(3.50±0.14)vs.(2.24±0.19),(5.43±0.41)vs.(3.08±0.14),(7.42±0.17)vs.(4.29±013)after 0,24,48,72,and 96 hours(F=24.58,P=0.001).The number of colone containing more than 50 cells was in 76±4 in CON group and 0 in DTL-shRNA group(P<0.01).The proportion of G1 stage cells was(28.61±8.64)% in CON group and(57.25±10.37)% in DTL-shRNA group(t=3.675,P=0.0213).The proportion of annexin V+in CON and DTL-shRNA groups was(3.21±0.89)% vs.(34.71±18.68)%(t=2.895,P=0.0443).After RPMI8226 was infected with CON or DTL-shRNA virus for 48 hours,the relative expression of phosphorylation P65 was(1.52±0.14)vs.(0.82±0.11)(t=6.81,P=0.0024),the P65 relative expression was(0.25±0.04)vs.(0.24±0.08)(t=0.19,P=0.85),the CON and DTL-shRNA phosphorylation-IκBα relative expression was(0.19±0.03)vs.(0.13±0.02)(t=2.882,P=0.0449),and the IκBα was(0.22±0.05)vs.(1.01±0.06)(t=17.52,P<0.0001).Detection of the transcriptional ability of DTL-shRNA NF-κB by EMSA further confirmed the down-regulation of DTL suppressed the NF-κB transcriptional ability. Conclusions DTL is highly expressed in MM cells,and down-regulation of DTL suppresses the cell proliferation,inhibit the colony formation,and induce cell apoptosis and cell cycle arrest.The effect of DTL on the biological functions of MM cells is related to the change of NF-κB pathway.

Apoptosis , Case-Control Studies , Cell Line, Tumor , Cell Proliferation , Humans , Multiple Myeloma , Pathology , NF-kappa B , Metabolism , Signal Transduction , Tumor Cells, Cultured , Ubiquitin-Protein Ligases , Metabolism
Rev. bras. cancerol ; 64(1): 93-98, Jan/Fev/Mar 2018.
Article in Portuguese | LILACS | ID: biblio-969213


Introdução: Bisfosfonatos são fármacos utilizados para o tratamento de enfermidades ósseas, como a osteoporose e metástases ósseas, em razão do seu mecanismo de ação, que consiste na diminuição do processo de reabsorção do osso. Outros estudos verificaram que bisfosfonatos de alta potência, como o zoledronato, poderiam auxiliar no tratamento de outras enfermidades malignas por causa da promoção de um efeito antiproliferativo. Objetivo: Este estudo in vitro objetivou avaliar a atividade antiproliferativa de zoledronato em diferentes linhagens de células tumorais. Método: Nove linhagens humanas (U251; MCF7; NCI/ADR-RES; 786-0; NCI-H460; PC-3; OVCAR-3; HT29; K-562 e HaCaT) foram submetidas ao tratamento com as concentrações de 0,12; 1,2; 12 e 120 µM de zoledronato e tiveram sua atividade proliferativa avaliada após 48 horas, utilizando-se o corante sulforrodamina B. Resultados: Verificou-se que as concentrações de 12 µM e 120 µM de zoledronato foram eficazes para a redução em 50% e 100%, respectivamente, da proliferação das células 786-0 (carcinoma renal). A maior concentração de zoledronato (120 µM) promoveu um efeito citostático (redução da proliferação celular em 50%) para as células HaCaT (queratinócito humano não tumoral), HT-29 (carcinoma de cólon), NCI-ADR/ RES (adenocarcinoma de ovário com fenótipo de multirresistência) e NCI-H460 (carcinoma pulmonar). Conclusão: Esses resultados sugerem um promissor efeito auxiliar do zoledronato para o tratamento de alguns tipos de tumores; estudos complementares in vitro e in vivo são necessários para a validação dessa hipótese.

Introduction: Bisphosphonates are used in the treatment of bone diseases such as osteoporosis and bone metastases, because of their ability to inhibit bone resorption. There is evidence that high-potency bisphosphonates, such as zoledronate, are useful in the treatment of other malignancies because they have an antiproliferative effect. Objective:To evaluate the antiproliferative activity of zoledronate in different tumor cell lines. Method: This was an in vitro study in which nine human cell lines (U251, MCF7, NCI/ ADR-RES, 786-0, NCI-H460, PC-3, OVCAR-3, HT29, K-562, and HaCaT) were treated with of 0.12, 1.2, 12, and 120 µM of zoledronate, their proliferative activity being evaluated 48 h later with sulforhodamine B assay. Results: At the 12 µM and 120 µM doses, zoledronate effectively reduced the proliferation of 786-0 (renal carcinoma) cells by 50% and 100%, respectively. At the highest concentration (120 µM), zoledronate had a cytostatic effect (50% reduction in cell proliferation) on HaCaT (non-tumor human keratinocyte), HT-29 (colon carcinoma), NCI-ADR/ RES (multidrug-resistant ovarian adenocarcinoma), and NCI-H460 (lung carcinoma) cells. Conclusion: These results suggest a promising auxiliary effect of zoledronate for the treatment of some tumors. Further in vitro and in vivo studies are needed in order to test that hypothesis.

Introducción: Los bisfosfonatos son fármacos utilizados para el tratamiento de enfermedades óseas, como la osteoporosis y metástasis óseas debido a su mecanismo de acción, que consiste en la disminución del proceso de reabsorción del hueso. Otros estudios observaron que los bisfosfonatos de alta potencia, como el zoledronato, podrían ayudar en el tratamiento de otras enfermedades malignas debido a la promoción de un efecto antiproliferativo. Objetivo: Este estudio in vitro objetivó evaluar la actividad antiproliferativa de zoledronato en diferentes linajes de células tumorales. Método: Los nueve humano linajes (U251, MCF7, NCI / ADR-RES, 786-0, NCI-H460, PC-3, OVCAR-3, HT29, K-562 and HaCaT) se sometieron al tratamiento con las concentraciones de 0,12; 1,2; 12 y 120 µM de zoledronato y tuvieron su actividad proliferativa evaluada después de 48 horas utilizando el colorante sulforrodamina B. Resultados: Se comprobó que las concentraciones de 12 µM y 120 µM de zoledronato fueron efectivas para reducir en un 50% y un 100%, respectivamente, de la proliferación de las células 786-0 (carcinoma renal). La mayor concentración de zoledronato (120 µM) promovió un efecto citostático (reducción de la proliferación celular en un 50%) para las células HaCaT (queratinocito humano no tumoral), HT-29 (carcinoma de colon), NCI-ADR/RES (adenocarcinoma de ovário con fenótipo de multirresistencia) y NCI-H460 (carcinoma pulmonar). Conclusión: Estos resultados sugieren un prometedor efecto auxiliar del zoledronato para el tratamiento de algunos tumores; se requieren más estudios in vitro e in vivo para validar esta hipótesis

Humans , Cell Proliferation/drug effects , Diphosphonates , In Vitro Techniques , Tumor Cells, Cultured/drug effects , Cell Line, Tumor/drug effects
Protein & Cell ; (12): 867-878, 2018.
Article in English | WPRIM | ID: wpr-757003


Human epidermal growth factor receptor 2 (HER2) proteins are overexpressed in a high proportion of gastric cancer (GC) cases and affect the maintenance of cancer stem cell (CSC) subpopulations, which are used as targets for the clinical treatment of patients with HER2-positive GC. Despite improvements in survival, numerous HER2-positive patients fail treatment with trastuzumab, highlighting the need for more effective therapies. In this study, we generated a novel type of genetically modified human T cells, expressing a chimeric antigen receptor (CAR), and targeting the GC cell antigen HER2, which harbors the CD137 and CD3ζ moieties. Our findings show that the expanded CAR-T cells, expressing an increased central memory phenotype, were activated by the specific recognition of HER2 antigens in an MHC-independent manner, and effectively killed patient-derived HER2-positive GC cells. In HER2-positive xenograft tumors, CAR-T cells exhibited considerably enhanced tumor inhibition ability, long-term survival, and homing to targets, compared with those of non-transduced T cells. The sphere-forming ability and in vivo tumorigenicity of patient-derived gastric cancer stem-like cells, expressing HER2 and the CD44 protein, were also inhibited. Our results support the future development and clinical application of this adoptive immunotherapy in patients with HER2-positive advanced GC.

Animals , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasms, Experimental , Allergy and Immunology , Pathology , Therapeutics , Receptor, ErbB-2 , Allergy and Immunology , Receptors, Antigen, T-Cell , Allergy and Immunology , Stomach Neoplasms , Allergy and Immunology , Pathology , Therapeutics , Tumor Cells, Cultured
Protein & Cell ; (12): 693-716, 2018.
Article in English | WPRIM | ID: wpr-756928


Hypertonia is a neurological dysfunction associated with a number of central nervous system disorders, including cerebral palsy, Parkinson's disease, dystonia, and epilepsy. Genetic studies have identified a homozygous truncation mutation in Trak1 that causes hypertonia in mice. Moreover, elevated Trak1 protein expression is associated with several types of cancers and variants in Trak1 are linked to childhood absence epilepsy in humans. Despite the importance of Trak1 in health and disease, the mechanisms of Trak1 action remain unclear and the pathogenic effects of Trak1 mutation are unknown. Here we report that Trak1 has a crucial function in regulation of mitochondrial fusion. Depletion of Trak1 inhibits mitochondrial fusion, resulting in mitochondrial fragmentation, whereas overexpression of Trak1 elongates and enlarges mitochondria. Our analyses revealed that Trak1 interacts and colocalizes with mitofusins on the outer mitochondrial membrane and functions with mitofusins to promote mitochondrial tethering and fusion. Furthermore, Trak1 is required for stress-induced mitochondrial hyperfusion and pro-survival response. We found that hypertonia-associated mutation impairs Trak1 mitochondrial localization and its ability to facilitate mitochondrial tethering and fusion. Our findings uncover a novel function of Trak1 as a regulator of mitochondrial fusion and provide evidence linking dysregulated mitochondrial dynamics to hypertonia pathogenesis.

Adaptor Proteins, Vesicular Transport , Metabolism , Animals , HeLa Cells , Humans , Membrane Fusion , Mice , Mitochondria , Metabolism , Mitochondrial Proteins , Metabolism , Muscle Proteins , Metabolism , Tumor Cells, Cultured
Biol. Res ; 51: 13, 2018. graf
Article in English | LILACS | ID: biblio-950899


BACKGROUND: Neuroblastoma (NB) represents the most common extracranial solid tumor in children. Accumulating evidence shows that microRNAs (miRs) play an important role in the carcinogenesis of NB. Here, we investigated the biological function of miR-1247 in NB in vitro. METHODS/RESULTS: We found miR-1247 was downregulated in NB tissues and cells using quantitative PCR analysis. Gain- and loss-of-function studies demonstrated that miR-1247 significantly suppressed cell proliferation and induced cell cycle G0/G1 phase arrest and cell apoptosis of NB cells in vitro by using MTT, colony formation assay and Flow cytometry analysis. Luciferase assay suggested ZNF346 was the target of miR-1247 and its expression could be down-regulated by miR-1247 overexpression using Western blotting. Furthermore, downregulation of ZNF346 by siRNA performed similar effects with overexpression of miR-1247 in NB cells. CONCLUSIONS: Our findings suggested miR-1247 directly targeted to repress ZNF346 expression, thus suppressing the progression of NB, which might be a novel therapeutic target against NB.

Humans , Male , Female , RNA-Binding Proteins/metabolism , MicroRNAs/metabolism , DNA-Binding Proteins/metabolism , Neuroblastoma/metabolism , Phenotype , Time Factors , Tumor Cells, Cultured , Down-Regulation , Gene Expression Regulation, Neoplastic , Child, Preschool , RNA-Binding Proteins/genetics , Colony-Forming Units Assay , MicroRNAs/genetics , Cell Proliferation/genetics , DNA-Binding Proteins/genetics , Real-Time Polymerase Chain Reaction , Flow Cytometry , Neuroblastoma/genetics , Neuroblastoma/pathology
Bauru; s.n; 2018. 98 p. ilus, graf, tab.
Thesis in Portuguese | LILACS, BBO | ID: biblio-885097


O osteossarcoma (OS) é o tumor maligno primário mais comum do tecido ósseo, caracterizado pela formação de osteócitos anormais. Apesar do avanço nas terapias convencionais (quimioterapia e retirada do tumor), essas não conseguem eliminar totalmente as células tumorais e impedir a progressão da doença. Recentemente, agentes derivados de fontes naturais ganharam considerável atenção por causa de sua segurança, eficácia e disponibilidade imediata. Nesse sentido, a apocinina, inibidor do complexo NADPH-oxidase, vem sendo estudada como agente antitumoral em alguns tipos de câncer como: pâncreas, próstata, pulmão e mama. Apocinina é um pró-fármaco e sua ação parece estar relacionada à sua conversão produzindo a diapocinina, a qual se mostrou mais efetiva do que a apocinina. Portanto, o objetivo desse estudo é avaliar, in vitro, o potencial antitumoral da apocinina e diapocinina em células de osteossarcoma humano. Para isso, foram utilizados osteoblastos humanos normais (HOb) e osteossarcoma humano imortalizadas (SaOS-2) tratados ou não com apocinina e diapocinina em diversas concentrações. Foram realizados os ensaios de viabilidade celular, alterações morfológicas, apoptose celular, produção de espécies reativas de oxigênio (EROs), formação de colônias, migração, invasão e expressão do fator indutor de hipóxia-1alfa (HIF-1). Também foram conduzidos ensaios para verificar a atividade de metaloproteinase de matriz (MMP) 2 e 9. Os resultados em SaOS-2 mostraram que o tratamento com apocinina nas concentrações de 1,5 e 3 mM; e diapocinina nas concentrações de 0,75 e 1,5 mM reduziram a viabilidade; aumentaram o número de células em apoptose e diminuíram a produção de EROs; sem causar danos às células HOb. Além disso, essas mesmas concentrações inibiram a migração e invasão celular; diminuíram a expressão de HIF-1; e reduziram a atividade de MMP-2 em SaOS-2. Considerando os resultados obtidos, concluímos que a apocinina e diapocinina podem atuar como possíveis moduladores de células tumorais, sendo que a diapocinina mostrou ser mais efetiva nos parâmetros testados.(AU)

Osteosarcoma (OS) is the most common primary malignant tumor of bone tissue, characterized by the formation of abnormal osteocytes. Despite advances in conventional therapies (chemotherapy and surgery) they cannot completely eliminate tumor cells and prevent the progression of the disease. Recently, agents derived from natural sources have achieved considerable attention because of their safety, efficacy and immediate availability of therapies. In this way, apocynin, an inhibitor of the NADPH-oxidase complex, has been studied as an antitumor agent in some types of cancer, such as pancreas, prostate, lung and breast. Apocynin is a prodrug and its action indicate to be related to its conversion to diapocynin, which has been shown to be more efficient than apocynin itself. Thus, the aim of this study is to evaluate, in vitro, the antitumor potential of apocynin and diapocynin in human osteosarcoma cells. For this, normal human osteoblasts (HOb) and immortalized human osteosarcoma cells (SaOS-2) were treated or no-treated with apocynin and diapocynin in various concentrations. Cell viability assay, morphological alterations, cellular apoptosis, reactive oxygen species (ROS) production, colony formation, migration, invasion and expression of hypoxia-inducible factor-1 alpha (HIF-1) were performed. We also performed assays to verify the activity of matrix metalloproteinase (MMP) 2 and 9. The results in SaOS-2 showed that treatment with apocynin at concentrations of 1,5 e 3 mM; and diapocynin at concentrations of 0,75 e 1,5 mM reduced cell viability; increased the number of cells in apoptosis and decreased the production of ROS; without damaging HOb cells. Moreover, these same concentrations inhibited cell migration and invasion; decreased HIF-1 expression; and reduced MMP 2 activity in SaOS-2. Considering the results, we suggest that apocynin and diapocynin may act as possible modulators of tumor cells, and diapocynin has been shown to be more effective.(AU)

Humans , Acetophenones/pharmacology , Antineoplastic Agents/pharmacology , Biphenyl Compounds/pharmacology , Osteosarcoma/drug therapy , Apoptosis/drug effects , Cell Movement/drug effects , Cell Survival/drug effects , Matrix Metalloproteinase 2/drug effects , Matrix Metalloproteinase 9/drug effects , Osteoblasts/drug effects , Reactive Oxygen Species/analysis , Reproducibility of Results , Tumor Cells, Cultured
Braz. j. med. biol. res ; 51(5): e6889, 2018. graf
Article in English | LILACS | ID: biblio-889078


2-Methyl-2-butanol (MBT) is a chemical compound from the group of alcohols more specifically pentanols, which has shown an excellent anti-cancer activity in our previous study. However, its mechanism of action remains unclear. The present study was designed to investigate the anti-cancer effect of MBT on human retinoblastoma cells. The results showed that the use of MBT leads to HXO-RB44 cell death but is cytotoxic to normal cells at higher concentrations. It showed a dose- as well as a time-dependent inhibition of HXO-RB44 cells. P27 is a cell cycle inhibitory protein, which plays an important role in cell cycle regulation whereas cyclin-B1 is a regulatory protein involved in mitosis. MBT increased the cell cycle arrest in a dose-dependent manner by augmenting p27 and reducing cyclin B1 expression. Moreover, it also accelerated apoptosis, increased light chain-3 (LC-3) conversion in a dose-dependent manner, and helped to debulk cancerous cells. LC3 is a soluble protein, which helps to engulf cytoplasmic components, including cytosolic proteins and organelles during autophagy from autophagosomes. In order to verify the effect of MBT, bafilomycin A1, an autophagy inhibitor, was used to block the MTB-induced apoptosis and necrosis. Additionally, a specific Akt agonist, SC-79, reversed the MBT-induced cell cycle arrest and autophagy. Thus, from the present study, it was concluded that MBT induced cell cycle arrest, apoptosis and autophagy through the PI3K/Akt pathway in HXO-RB44 cells.

Humans , Antineoplastic Agents/pharmacology , Autophagy/drug effects , Cell Cycle Checkpoints/drug effects , Pentanols/pharmacology , Retinoblastoma/pathology , Blotting, Western , Phosphatidylinositol 3-Kinases/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Tumor Cells, Cultured
Article in English | WPRIM | ID: wpr-772285


To screen for additional treatment targets against tongue cancer, we evaluated the contributions of extracellular signal-related kinase (ERK), AKT and ezrin in cancer development. Immunohistochemical staining showed that ERK and ezrin expressions were significantly higher in invasive squamous cell carcinoma than in carcinoma in situ. To investigate the roles of ERK and ezrin in cancer development, we used the non-woven silica fibre sheet Cellbed with a structure resembling the loose connective tissue morphology in a novel 3D culture system. We confirmed that the 3D system using Cellbed accurately mimicked cancer cell morphology in vivo. Furthermore, cell projections were much more apparent in 3D-cultured tongue cancer cell lines than in 2D cultures. Typically, under conventional 2D culture conditions, F-actin and cortactin are colocalized in the form of puncta within cells. However, in the 3D-cultured cells, colocalization was mainly observed at the cell margins, including the projections. Projections containing F-actin and cortactin colocalization were predicted to be invadopodia. Although suppressing ezrin expression with small interfering RNA transfection caused no marked changes in morphology, cell projection formation was decreased, and the tumour thickness in vertical sections after 3D culture was markedly decreased after suppressing ERK activity because both the invasion ability and proliferation were inhibited. An association between cortactin activation as well as ERK activity and invadopodia formation was detected. Our novel 3D culture systems using Cellbed™ are simple and useful for in vitro studies before conducting animal experiments. ERK contributes to tongue cancer development by increasing both cancer cell proliferation and migration via cortactin activation.

Carcinoma in Situ , Metabolism , Pathology , Carcinoma, Squamous Cell , Metabolism , Pathology , Cell Culture Techniques , Methods , Cell Movement , Cell Proliferation , Cytoskeletal Proteins , Metabolism , Extracellular Signal-Regulated MAP Kinases , Metabolism , Humans , Neoplasm Invasiveness , Pathology , Phosphorylation , Podosomes , Pathology , Proto-Oncogene Proteins c-akt , Metabolism , Silicon Dioxide , Tongue Neoplasms , Metabolism , Pathology , Tumor Cells, Cultured
Frontiers of Medicine ; (4): 726-734, 2018.
Article in English | WPRIM | ID: wpr-771271


Bromodomain-containing 4 (BRD4) has been considered as an important requirement for disease maintenance and an attractive therapeutic target for cancer therapy. This protein can be targeted by JQ1, a selective small-molecule inhibitor. However, few studies have investigated whether BRD4 influenced acute promyelocytic leukemia (APL), and whether BRD4 had interaction with promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein to some extent. Results from cell viability assay, cell cycle analysis, and Annexin-V/PI analysis indicated that JQ1 inhibited the growth of NB4 cells, an APL-derived cell line, and induced NB4 cell cycle arrest at G1 and apoptosis. Then, we used co-immunoprecipitation (co-IP) assay and immunoblot to demonstrate the endogenous interaction of BRD4 and PML/RARα in NB4 cells. Moreover, downregulation of PML/RARα at the mRNA and protein levels was observed upon JQ1 treatment. Furthermore, results from the RT-qPCR, ChIP-qPCR, and re-ChIP-qPCR assays showed that BRD4 and PML/RARα co-existed on the same regulatory regions of their target genes. Hence, we showed a new discovery of the interaction of BRD4 and PML/RARα, as well as the decline of PML/RARα expression, under JQ1 treatment.

Apoptosis , Azepines , Pharmacology , Cell Differentiation , Down-Regulation , Gene Expression Regulation, Neoplastic , Humans , Leukemia, Promyelocytic, Acute , Drug Therapy , Genetics , Nuclear Proteins , Genetics , Promyelocytic Leukemia Protein , Genetics , RNA, Messenger , Genetics , Retinoic Acid Receptor alpha , Genetics , Transcription Factors , Genetics , Triazoles , Pharmacology , Tumor Cells, Cultured
Article in Chinese | WPRIM | ID: wpr-813189


To determine the effect of andrographolide (Andro) on angiogenesis of human umbilical vein endothelial cells (HUVECs).
 Methods: HUVECs were treated with different concentrations of Andro and the cell viability was detected with Cell Counting Kit-8 (CCK-8). HUVECs were treated with half lethal dose (IC50) of Andro. Matrigel was used to make capillary formation of HUVECs and the effect of Andro on capillary formation was evaluated by calculating the percentage of capillary formation. Moreover, the effects of Andro and the supernatant from cultured A549 tumor cells on capillary formation were evaluated by calculating the percentage of capillary formation. The effect of Andro on the expression of matrix metalloproteinase-9 (MMP-9) was determined with Western blot.
 Results: The cell viability of HUVECs decreased with the increase of Andro concentrations. IC50 was 20 μmol/L. The capillary formation of HUVECs was inhibited when treated with 20 μmol/L Andro for 24 hours. Moreover, Andro was able to antagonize the promotion of the capillary formation induced by the supernatant from cultured tumor cells. Andro could suppress the expression of MMP-9 and antagonize the capillary formation.
 Conclusion: Andro inhibits the capillary formation of HUVECs and can antagonize the promotion of angiogenesis induced by the supernatant from cultured tumor cells.

Capillaries , Cell Survival , Collagen , Culture Media , Diterpenes , Pharmacology , Drug Combinations , Human Umbilical Vein Endothelial Cells , Humans , Laminin , Matrix Metalloproteinase 9 , Metabolism , Neovascularization, Pathologic , Proteoglycans , Tumor Cells, Cultured
São Paulo; s.n; s.n; 2018. 79 p. graf, ilus, tab.
Thesis in Portuguese | LILACS | ID: biblio-883230


O câncer primário de fígado (CPF) apresenta mau prognóstico, o que torna importante sua quimioprevenção. Nesse sentido, a tributirina (TB), um pró-fármaco do ácido butírico (AB), presente em laticínios e no mel, mostrou-se um agente quimiopreventivo promissor da hepatocarcinogênese experimental. Os efeitos inibitórios da TB têm sido relacionados à inibição do desenvolvimento de lesões pré-neoplásicas, bem como indução de apoptose e hiperacetilação de histonas. A quimioterapia é uma das abordagens mais comuns para o tratamento de diversos tipos de câncer, inclusive o CPF. Neste caso, o tratamento com sorafenibe (SO) é capaz de prolongar a sobrevida média dos pacientes com a doença em fases avançadas em aproximadamente apenas três meses. Em vista disso, são necessários estudos da associação do sorafenibe com outros compostos que possam aumentar a eficácia do tratamento quimioterápico. Desta forma, a associação de fármacos anti-neoplásicos com compostos bioativos dos alimentos pode consistir em uma estratégia potencial para aumentar a eficácia contra o câncer. No presente estudo, foi avaliada a atividade anticarcinogênica da TB e do SO, isoladamente ou em associação, na etapa de progressão da hepatocarcinogênese. Para tanto, foram realizados implantes singênicos no flanco de ratos Fischer-344 a partir de células da linhagem tumoral GP7TB. Quando as neoplasias atingiram 1 cm3, os animais foram aleatorizados em grupos experimentais: Grupo controle (CO), constituído por 10 ratos Fischer 344 que receberam Maltodextrina (300mg/ 100 g. p. c.), controle isocolarico e solução de etanol à 12,5% e Cremofor à 12,5% em agua estéril; Grupo Tributirina (TB), constituído por 9 ratos Fischer 344 que receberam TB (200mg/ 100 g. p. c.) e solução de etanol à 12,5% e Cremofor à 12,5% em água estéril; Grupo sorafenibe (SO) constituído por 9 ratos Fischer 344 que receberam Maltodextrina (300 mg/ 100 g. p. c.), controle isocalorico e tosilato de sorafenibe (3mg / 100 g. p. c. ) em água estéril; Grupo associação da tributirina com o sorafenibe (AS) constituído por 9 ratos Fischer 344 que receberam TB (20 mg/ 100 g. p. c.) e tosiliato de sorafenibe (3mg/ 100 g. p. c.); tratados por administração intragástrica (i.g) diariamente por 5 semanas consecutivas. As concentrações de AB e SO foram analisadas por cromatografia gasosa associada à espectrometria de massa e as neoplasias foram caracterizadas por imunoistoquímica. Em relação à evolução do tamanho das neoplasias o grupo AS apresentou menor (p=0,009) tamanho das mesmas em relação ao grupo CO. No entanto, estas diferenças não atingiram diferenças significativas (p>0,05) entre os grupos TB e CO, bem como entre os grupos SO e CO. Contudo, quando ajustados os valores do tamanho da neoplasia pela latência, observou-se alterações significativas (p<0,05) nos diversos grupos quando comparados ao grupo CO. O grupo SO aumentou a área necrótica das neoplasias, embora esta diferença não tenha atingido diferença significativa (p>0,05), enquanto que o grupo TB reduziu essa área necrótica em relação ao grupo CO (p=0,005). O grupo TB e AS apresentaram significativamente maiores (p<0,05) concentrações hepáticas e neoplásicas de AB em relação ao grupo CO. O grupo SO e AS apresentaram significativamente maiores (p<0,05) concentrações neoplásicas de SO em relação ao grupo CO. Os grupos SO e AS reduziram a expressão de PTEN, quando comparados ao grupo CO, embora esta diferença não tenha atingido diferença significativa (p>0,05). O grupo TB por sua vez expressou maiores niveis de PTEN, embora esta diferença não tenha atigindo significância estatística (p>0,05). Todos os grupos expressaram maiores niveis de caspase 3 clivada quando comparada ao grupo CO (p>0,05). OS grupos TB e SO reduziram a expressão de pERK ½ quando comparados ao grupo CO. embora estas diferenças não tenham atingidos diferença estatística (p>0,05). O grupo AS apresentou maior expressão de pERK ½ quando comparada ao grupo CO, embora esta diferença não tenha atingido diferença significativa (p>0,05). A caracterização das neoplasias do grupo CO foi padronizada por imunoistoquímica, apresentando-se positivas para CK 7, CK8, CK19 e Arginase e negativas para HepPar1 e CK18. Assim, os resultados sugerem que as neoplasias obtidas por implantes com células da linhagem GP7TB apresentam características de CPF oriundo de células tronco neoplásicas. Além disso, os grupos experimentais TB e AS apresentaram atividade anticarcinogênica promissora no modelo de implantes singênicos com células GP7TB, que eventualmente envolvem mecanismos de ação distintos da atividade quimioterápica apresentada pelo SO

Primary liver cancer (PLC) presents poor prognosis, which makes its chemoprevention important. In this sense, tributyrin (TB), a prodrug of butyric acid (AB), present in dairy products and honey, has been shown to be a promising chemopreventive agent for experimental hepatocarcinogenesis. The inhibitory effects of TB have been related to inhibition of the development of pre-neoplastic lesions, as well as induction of apoptosis and hyperacetylation of histones. Chemotherapy is one of the most common approaches for treating various types of cancer, including PLC. In this case, treatment with sorafenib (SO) is able to prolong the average survival of patients with the disease in advanced stages in approximately three months. In view of this, studies of the association of sorafenib with other compounds that may increase the efficacy of chemotherapeutic treatment are necessary. Thus, the association of anti-neoplastic drugs with bioactive compounds in food may be a potential strategy to increase efficacy against cancer. In the present study, the anticarcinogenic activity of TB and SO was evaluated, alone or in combination, in the progression stage of hepatocarcinogenesis. For this purpose, syngenic implants were performed on the flank of Fischer-344 mice from GP7TB tumor cells. When the neoplasms reached 1 cm3, the animals were randomized into experimental groups: Control group (CO), consisting of 10 Fischer 344 rats receiving Maltodextrin (300mg / 100 g.p.c), isocaloric control and 12.5% ethanol solution, and Cremofor to 12.5% in sterile water; Tributyrin group (TB), consisting of 9 Fischer 344 rats that received TB (200mg / 100 g.p.c.) and 12.5% ethanol solution and Cremofor 12.5% in sterile water; Sorafenib group (SO) consisting of 9 Fischer 344 rats receiving maltodextrin (300 mg / 100 g, w / w), isocaloric control and sorafenib tosylate (3 mg / 100 g, w / w) in sterile water; The association group of tributyrin and sorafenib (AS) consisted of 9 Fischer 344 rats receiving TB (20 mg / 100 g p.o.) and sorafenib tosylate (3 mg / 100 g p.o.); treated intragastric (i.g) daily for 5 consecutive weeks. The concentrations of AB and SO were analyzed by gas chromatography associated with mass spectrometry and the neoplasms were characterized by immunohistochemistry. In relation to the evolution of the size of the neoplasias, the AS group presented smaller (p = 0.009) size of the same ones in relation to the CO group. However, these differences did not reach significant differences (p> 0.05) between the TB and CO groups, as well as between the SO and CO groups. However, when adjusted for size of the neoplasm by latency, significant changes (p <0.05) were observed in the different groups when compared to the CO group. The SO group increased the necrotic area of the neoplasias, although this difference did not reach a significant difference (p> 0.05), while the TB group reduced this necrotic area in relation to the CO group (p = 0.005). The TB and AS groups presented significantly higher (p <0.05) hepatic and neoplastic AB concentrations than the CO group. The SO and AS groups presented significantly higher (p <0.05) neoplastic concentrations of SO in relation to the CO group. The SO and AS groups reduced the PTEN expression when compared to the CO group, although this difference did not reach a significant difference (p> 0.05). The TB group in turn expressed higher levels of PTEN, although this difference did not increase statistical significance (p> 0.05). All groups expressed higher levels of caspase 3 cleaved when compared to the CO group (p> 0.05). The TB and SO groups reduced the expression of pERK ½ when compared to the CO group. although these differences did not reach statistical difference (p> 0.05). The AS group presented higher pERK ½ expression when compared to the CO group, although this difference did not reach a significant difference (p> 0.05). Characterization of the neoplasias of the CO group was standardized by immunohistochemistry, presenting positive for CK 7, CK8, CK19 and Arginase and negative for HepPar1 and CK18. Thus, the results suggest that the neoplasias obtained by implants with GP7TB cells present CPF characteristics originating from neoplastic stem cells. In addition, the experimental groups TB and AS presented promising anticarcinogenic activity in the model of syngeneic implants with GP7TB cells, which eventually involve mechanisms of action distinct from the chemotherapy activity presented by SO

Animals , Male , Rats , Pharmaceutical Preparations/analysis , Anticarcinogenic Agents/analysis , Liver Neoplasms/prevention & control , Immunohistochemistry/methods , Tumor Cells, Cultured/classification , Blotting, Western/instrumentation , Data Interpretation, Statistical , Carcinoma, Hepatocellular , Butyric Acid/agonists , Gas Chromatography-Mass Spectrometry/methods
Mem. Inst. Oswaldo Cruz ; 113(11): e180267, 2018. graf
Article in English | LILACS | ID: biblio-1040585


The Bacille Calmette-Guérin (BCG) vaccine comprises a family of genetically different strains derived by the loss of genomic regions (RDs) and other mutations. In BCG Moreau, loss of RD16 inactivates rv3405c * , encoding a transcriptional repressor that negatively regulates the expression of Rv3406, an alkyl sulfatase. To evaluate the impact of this loss on the BCG and host cell viability and the cytokine profile, THP-1 cells were infected with BCG Moreau (harbouring the empty vector) and a complemented strain carrying a functional copy of rv3405c. Viability of the host cells and bacteria as well as the pattern of cytokine secretion were evaluated. Our results show that the viability of BCG Moreau is higher than that of the complemented strain in an axenic medium, suggesting a possible functional gain associated with the constitutive expression of Rv3406. Viability of the host cells did not vary significantly between recombinant strains, but differences in the profiles of the cytokine secretion (IL-1β and IL-6) were observed. Our results suggest an example of a functional gain due to gene loss contributing to the elucidation of the impact of RD16 on the physiology of BCG Moreau.

Humans , Transcription, Genetic/genetics , BCG Vaccine/pharmacology , Cell Survival/genetics , Cytokines/drug effects , Gain of Function Mutation/genetics , Macrophages/microbiology , Mycobacterium bovis/genetics , Time Factors , Transcription, Genetic/drug effects , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/microbiology , BCG Vaccine/genetics , Cell Survival/drug effects , Cytokines/genetics , Gain of Function Mutation/drug effects , Mycobacterium bovis/physiology