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Base editing introduces precise single-nucleotide edits in genomic DNA and has the potential to treat genetic diseases such as the blistering skin disease recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by mutations in the COL7A1 gene and type VII collagen (C7) deficiency. Adenine base editors (ABEs) convert A-T base pairs to G-C base pairs without requiring double-stranded DNA breaks or donor DNA templates. Here, we use ABE8e, a recently evolved ABE, to correct primary RDEB patient fibroblasts harboring the recurrent RDEB nonsense mutation c.5047 C > T (p.Arg1683Ter) in exon 54 of COL7A1 and use a next generation sequencing workflow to interrogate post-treatment outcomes. Electroporation of ABE8e mRNA into a bulk population of RDEB patient fibroblasts resulted in remarkably efficient (94.6%) correction of the pathogenic allele, restoring COL7A1 mRNA and expression of C7 protein in western blots and in 3D skin constructs. Off-target DNA analysis did not detect off-target editing in treated patient-derived fibroblasts and there was no detectable increase in A-to-I changes in the RNA. Taken together, we have established a highly efficient pipeline for gene correction in primary fibroblasts with a favorable safety profile. This work lays a foundation for developing therapies for RDEB patients using ex vivo or in vivo base editing strategies.
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Codón sin Sentido , Epidermólisis Ampollosa Distrófica , Humanos , Codón sin Sentido/genética , Adenina , Colágeno Tipo VII/genética , Colágeno Tipo VII/metabolismo , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/terapia , Epidermólisis Ampollosa Distrófica/patología , MutaciónRESUMEN
Uterine Fibroids, or leiomyomata, affect millions of women world-wide, with a high incidence of 75% within women of reproductive age. In ~30% of patients, uterine fibroids cause menorrhagia, or heavy menstrual bleeding, and more than half of the patients experience symptoms such as heavy menstrual bleeding, pelvic pain, or infertility. Treatment is symptomatic with limited options including hysterectomy as the most radical solution. The genetic foundations of uterine fibroid growth have been traced to somatic driver mutations (MED12, HMGA2, FH -/-, and COL4A5-A6). These also lead to downstream expression of angiogenic factors including IGF-1 and IGF-2, as opposed to the VEGF-driven mechanism found in the angiogenesis of hypoxic tumors. The resulting vasculature supplying the fibroid with nutrients and oxygen is highly irregular. Of particular interest is the formation of a pseudocapsule around intramural fibroids, a unique structure within tumor angiogenesis. These aberrations in vascular architecture and network could explain the heavy menstrual bleeding observed. However, other theories have been proposed such as venous trunks, or venous lakes caused by the blocking of normal blood flow by uterine fibroids, or the increased local action of vasoactive growth factors. Here, we review and discuss the evidence for the various hypotheses proposed.
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INTRODUCTION: Millions of women suffer from the consequences of endometriosis and uterine fibroids, with fibroids the cause for over 50% of hysterectomies in the USA, and direct costs for their treatment estimated at between US$4 and US$9 billion. Endometriosis commonly affects millions of women worldwide predominantly during reproductive age, with severe menstrual and non-menstrual pain and subfertility the main symptoms. Due to the 'unhappy triad' of endometriosis-lack of awareness, lack of clinically relevant biomarkers and the unspecific nature of symptoms-women wait on average for 8-12 years before the definitive endometriosis diagnosis is made. Treatment options for both conditions are not satisfactory at the moment, especially with a view to preserving fertility for the women and families affected. In the Fibroids and Endometriosis Oxford (FENOX) study, we combine the investigation of fibroids and endometriosis, and plan to collect high-quality tissue samples and medical data of participants over a time frame of 5 years after surgical intervention. METHODS AND ANALYSIS: Biological samples such as blood, saliva, urine, fat, peritoneal fluid and-if found-endometrial tissue or fibroids as well as detailed clinical and intraoperative data will be collected from women undergoing surgery and participating in the study after informed consent. We plan to recruit up to 1200 participants per disease arm (ie, endometriosis and uterine fibroids) over 5 years. Participants will fill in detailed and validated questionnaires on their medical history and quality of life, with follow-ups for 5 years. Enrolment started on 2 April 2018, and FENOX will close on 31 March 2028. We will analyse the biological samples using state-of-the-art molecular biology methods and correlate the findings with the medical records and questionnaire data. ETHICS AND DISSEMINATION: The findings will be published in high-ranking journals in the field and presented at national and international conferences. TRIAL REGISTRATION NUMBER: ISRCTN13560263.
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Endometriosis/fisiopatología , Leiomioma/fisiopatología , Calidad de Vida , Adulto , Femenino , Humanos , Estudios Longitudinales , Estudios Prospectivos , Proyectos de InvestigaciónRESUMEN
BACKGROUND: Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Annonacin, a natural pure compound extracted from the seeds of Annona muricata, is a potential alternative therapeutic agent to treat EC. OBJECTIVE: To study the antitumor activity of annonacin and its mechanism of action in EC cells (ECCs). MATERIALS AND METHODS: Viability of ECCs treated with annonacin for 72 h was determined using methyl thiazolyl tetrazolium assay. The induction of cell cycle arrest and apoptotic cell death was evaluated using propidium iodide and annexin V-PE/7-AAD assay, respectively. DNA strand breaks were visualized using transferase dUTP nick end labeling assay, and the effects of annonacin on survival signaling were determined using western blotting. RESULTS: Annonacin exhibited antiproliferative effects on EC cell lines (ECC-1 and HEC-1A) and primary cells (EC6-ept and EC14-ept) with EC50values ranging from 4.62 to 4.92 µg/ml. EC cells were shown arrested at G2/M phase after treated with 4 µg/ml of annonacin for 72 h. This led to a significant increase in apoptotic cell death (65.7%) in these cells when compared to vehicle-treated cells (P < 0.005). We further showed that annonacin-mediated apoptotic cell death was associated with an increase in caspase-3 cleavage and DNA fragmentation. Cell apoptosis was accompanied with downregulation of extracellular signal-regulated kinase survival protein expression and induction of G2/M cell cycle arrest. CONCLUSION: Annonacin may be a potential novel therapeutic agent for EC patients. SUMMARY: We aimed to study the antitumor activity of annonacin and its mechanism of action in endometrial cancer cells. Annonacin exerted antiproliferation effects on both endometrial cancer cell lines and primary cells via induction of apoptosis and inhibition of extracellular signal-regulated kinase. Our data represented that annonacin could be an alternative therapeutic treatment to combat endometrial cancer. Abbreviations Used: 7-AAD: 7-Amino-Actinomycin, ATP: Adenosine diphosphate, BSA: Bovine serum albumin, DNA: Deoxyribonucleic acid, EC: Endometrial cancer, ECC-1: Endometrial cancer cell-1, EC50: Half maximal effective concentration, Ept: Epithelial, FBS: Fetal bovine serum, HEC-1A: Human endometrial carcinoma-1A, MTT: Methyl thiazolyl tetrazolium, NaCl: Sodium chloride, NADH: Nicotinamide adenine dinucleotide, RPMI 1640: Roswell Park Memorial Institute Medium, SDS: Sodium dodecyl sulfate.
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Cancer-associated fibroblasts (CAFs) secrete various pro-tumorigenic cytokines, yet the role of these cytokines in the progression of endometrial cancer remains unclear. We found that CAFs isolated from human endometrial cancer (EC) tissues secreted high levels of interleukin-6 (IL-6), which promotes EC cell proliferation in vitro. Neutralizing IL-6 in CAF-conditioned media reduced (47% inhibition) while IL-6 recombinant protein increased cell proliferation (~2.4 fold) of both EC cell lines and primary cultures. IL-6 receptors (IL-6R and gp130) were expressed only in EC epithelial cells but not in CAF, indicating a one-way paracrine signaling. In the presence of CAF-conditioned media, Janus kinase/signal transducers and activators of transcription (JAK/STAT3) pathway was activated in EC cells. Treatment with JAK and STAT3 specific inhibitors, AD412 and STATTIC, respectively, significantly abrogated CAF-mediated cell proliferation, indicating the role of IL-6 activation in EC cell proliferation. We further showed that one of STAT-3 target genes, c-Myc, was highly induced in EC cells after exposure to CAF-conditioned medium at both mRNA (>105-fold vs. control) and protein level (>2-fold vs. control). EC cell proliferation was dependent on c-Myc expression, as RNAi-mediated c-Myc down-regulation led to a significant 46% reduction in cell viability when compared with scrambled control. Interestingly, CAF-conditioned media failed to promote proliferation in EC cells with reduced c-Myc expression, suggesting that CAF-mediated cell proliferation was also dependent on c-Myc expression. Subcutaneous tumor xenograft model showed that EC cells grew at least 1.4 times larger when co-injected with CAF, when compared to those injected with EC cells alone. Mice injected with EC cells with down-regulated c-Myc expression, however, showed at least 2.5 times smaller tumor compared to those in control group. Notably, there was no increase of tumor size when co-injected with CAFs. Further immunohistochemical staining on human tissues showed positive expression of IL-6 receptors, phosphorylated-STAT3 and c-Myc in human EC tissues with less signals in benign endometrium. Taken together, our data suggests that IL-6 secreted by CAF induces c-Myc expression to promote EC proliferation in vitro and in vivo. IL-6 pathway can be a potential target to disrupt tumor-stroma interaction in endometrial cancer progression.
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Pancreatic stellate cells (PSC), a prominent stromal cell, contribute to the progression of pancreatic ductal adenocarcinoma (PDAC). We aim to investigate the mechanisms by which PSC promote cell proliferation in PDAC cell lines, BxPC-3 and AsPC-1. PSC-conditioned media (PSC-CM) induced proliferation of these cells in a dose- and time-dependent manner. Nrf2 protein was upregulated and subsequently, its transcriptional activity was increased with greater DNA binding activity and transcription of target genes. Downregulation of Nrf2 led to suppression of PSC-CM activity in BxPC-3, but not in AsPC-1 cells. However, overexpression of Nrf2 alone resulted in increased cell proliferation in both cell lines, and treatment with PSC-CM further enhanced this effect. Activation of Nrf2 pathway resulted in upregulation of metabolic genes involved in pentose phosphate pathway, glutaminolysis and glutathione biosynthesis. Downregulation and inhibition of glucose-6-phosphate-dehydrogenase with siRNA and chemical approaches reduced PSC-mediated cell proliferation. Among the cytokines present in PSC-CM, stromal-derived factor-1 alpha (SDF-1α) and interleukin-6 (IL-6) activated Nrf2 pathway to induce cell proliferation in both cells, as shown with neutralization antibodies, recombinant proteins and signaling inhibitors. Taken together, SDF-1α and IL-6 secreted from PSC induced PDAC cell proliferation via Nrf2-activated metabolic reprogramming and ROS detoxification.
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Proliferación Celular/genética , Factor 2 Relacionado con NF-E2/genética , Células Estrelladas Pancreáticas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Western Blotting , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL12/metabolismo , Medios de Cultivo Condicionados/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/metabolismo , Redes y Vías Metabólicas/efectos de los fármacos , Redes y Vías Metabólicas/genética , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.