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1.
J Robot Surg ; 18(1): 82, 2024 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-38367193

RESUMO

Robotic surgery may decrease surgeon stress compared to laparoscopic. To evaluate intraoperative surgeon stress, we measured salivary alpha-amylase and cortisol. We hypothesized robotic elicited lower increases in surgeon salivary amylase and cortisol than laparoscopic. Surgical faculty (n = 7) performing laparoscopic and robotic operations participated. Demographics: age, years in practice, time using laparoscopic vs robotic, comfort level and enthusiasm for each. Operative data included operative time, WRVU (surgical "effort"), resident year. Saliva was collected using passive drool collection system at beginning, middle and end of each case; amylase and cortisol measured using ELISA. Standard values were created using 7-minute exercise (HIIT), collecting saliva pre- and post-workout. Linear regression and Student's t test used for statistical analysis; p values < 0.05 were significant. Ninety-four cases (56 robotic, 38 laparoscopic) were collected (April-October 2022). Standardized change in amylase was 8.4 ± 4.5 (p < 0.001). Among operations, raw maximum amylase change in laparoscopic and robotic was 23.4 ± 11.5 and 22.2 ± 13.4; raw maximum cortisol change was 44.21 ± 46.57 and 53.21 ± 50.36, respectively. Values normalized to individual surgeon HIIT response, WRVU, and operative time, showing 40% decrease in amylase in robotic: 0.095 ± 0.12, vs laparoscopic: 0.164 ± 0.16 (p < 0.02). Normalized change in cortisol was: laparoscopic 0.30 ± 0.44, robotic 0.22 ± 0.4 (p = NS). On linear regression (p < 0.001), surgeons comfortable with complex laparoscopic cases had lower change in normalized amylase (p < 0.01); comfort with complex robotic was not significant. Robotic may be less physiologically stressful, eliciting less increase in salivary amylase than laparoscopic. Comfort with complex laparoscopic decreased stress in robotic, suggesting laparoscopic experience is valuable prior to robotic.


Assuntos
Laparoscopia , Procedimentos Cirúrgicos Robóticos , Cirurgiões , Humanos , Procedimentos Cirúrgicos Robóticos/métodos , Hidrocortisona/análise , Amilases
2.
Cancers (Basel) ; 15(12)2023 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-37370863

RESUMO

Cancer progression depends on an accumulation of metastasis-supporting physiological changes, which are regulated by cell-signaling molecules. In this regard, a disintegrin and metalloproteinase 8 (Adam8) is a transmembrane glycoprotein that is selectively expressed and induced by a variety of inflammatory stimuli. In this study, we identified Adam8 as a sox2-dependent protein expressed in MDA-MB-231 breast cancer cells when cocultured with mesenchymal-stem-cell-derived myofibroblast-like cancer-associated fibroblasts (myCAF). We have previously found that myCAF-induced cancer stemness is required for the maintenance of the myCAF phenotype, suggesting that the initiation and maintenance of the myCAF phenotype require distinct cell-signaling crosstalk pathways between cancer cells and myCAF. Adam8 was identified as a candidate secreted protein induced by myCAF-mediated cancer stemness. Adam8 has a known sheddase function against which we developed an RNA aptamer, namely, Adam8-Apt1-26nt. The Adam8-Apt1-26nt-mediated blockade of the extracellular soluble Adam8 metalloproteinase domain abolishes the previously initiated myCAF phenotype, or, termed differently, blocks the maintenance of the myCAF phenotype. Consequently, cancer stemness is significantly decreased. Xenograft models show that Adam8-Apt-1-26nt administration is associated with decreased tumor growth and metastasis, while flow cytometric analyses demonstrate a significantly decreased fraction of myCAF after Adam8-Apt-1-26nt treatment. The role of soluble Adam8 in the maintenance of the myCAF phenotype has not been previously characterized. Our study suggests that the signal pathways for the induction or initiation of the myCAF phenotype may be distinct from those involved with the maintenance of the myCAF phenotype.

3.
Cureus ; 14(9): e29354, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36284815

RESUMO

BACKGROUND: Myofibroblast-like cancer-associated fibroblasts (myCAF) in the tumor microenvironment (TME) promote cancer stemness, growth, and metastasis. Cancer cell-derived osteopontin (OPN) has been reported as a biomarker related to malignant cancer growth. In this study, we confirm that cancer cell stemness is required for the maintenance of an OPN-induced myCAF phenotype. METHODS: MDA-MB-231 or HepG2 cells and Sox2 knockout variants were co-cultured with human mesenchymal stem cells (MSC). In selected instances, the OPN bioactivity inhibitor OPN-R3 aptamer (APT), OPN-R3 mutant aptamer (MuAPT), or cancer cell stemness inhibitor BBI-608 were added separately. MDA-MB-231 cancer stemness and myCAF markers were quantified by real-time PCR. Stemness-lacking cancer cell mice models were created to confirm that stemness is required for the maintenance of the OPN-induced myCAF phenotype in vivo. RESULTS: In an MDA-MB-231 co-culture system, myCAF and stemness markers increased. Osteopontin and stemness blockade in this co-culture system decreased both myCAF and stemness marker expression, but OPN blockade after 72 hours had no effect. In contrast, when BBI608 was added at 72 hours, myCAF markers were abated after 36-hour treatment. Replacing wildtype with MDA-MB-231(-/-sox2) in co-cultures at 72 hours decreased myCAF marker expression to baseline despite the Western blot confirming the presence of OPN. Conversely, replacing MDA-MB-231(-/-sox2) cells with wildtype increased myCAF marker expression to a level equivalent to the MDA-MB-231+MSC co-culture system. In vivo osteopontin blockade diminished stemness and myCAF marker expression and stemness lacking cancer cell models, indicated by decreasing myCAF presence. Experiments were repeated in a HepG2 cell line with identical results. CONCLUSIONS: Cancer and myCAF crosstalk increases myCAF maintenance and cancer cell stemness. In this study using human breast and liver cancer cell lines, maintenance of the OPN-induced myCAF phenotype also requires cancer stemness. This indicates that the myCAF phenotype requires two distinct signaling pathways: initiation and maintenance.

4.
Exp Suppl ; 113: 59-87, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35165860

RESUMO

The understanding of how normal cells transform into tumor cells and progress to invasive cancer and metastases continues to evolve. The tumor mass is comprised of a heterogeneous population of cells that include recruited host immune cells, stromal cells, matrix components, and endothelial cells. This tumor microenvironment plays a fundamental role in the acquisition of hallmark traits, and has been the intense focus of current research. A key regulatory mechanism triggered by these tumor-stroma interactions includes processes that resemble epithelial-mesenchymal transition, a physiologic program that allows a polarized epithelial cell to undergo biochemical and cellular changes and adopt mesenchymal cell characteristics. These cellular adaptations facilitate enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of ECM components. Indeed, it has been postulated that cancer cells undergo epithelial-mesenchymal transition to invade and metastasize.In the following discussion, the physiology of chronic inflammation, wound healing, fibrosis, and tumor invasion will be explored. The key regulatory cytokines, transforming growth factor ß and osteopontin, and their roles in cancer metastasis will be highlighted.


Assuntos
Células Endoteliais , Neoplasias , Transição Epitelial-Mesenquimal/genética , Humanos , Neoplasias/genética , Células Estromais , Microambiente Tumoral/genética
5.
Surgery ; 166(4): 515-523, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31301870

RESUMO

BACKGROUND: Osteopontin acts thru myeloid zinc finger-1 and transforming growth factor-ß to drive the adoption of a cancer-associated fibroblast phenotype by local mesenchymal stem cells. Cancer-associated fibroblasts increase cancer cell stemness. METHODS: Mesenchymal stem cells were exposed to osteopontin or were cocultured with MB231 human breast cancer cells (high osteopontin producer) in the presence or absence of aptamer (inactivates extracellular osteopontin). Myeloid zinc finger-1 phosphorylation sites were identified, and phosphomutants of T134 (SCAN domain) and S453 (zinc finger DNA binding domain) were constructed. Transforming growth factor-ß F and cancer-associated fibroblast markers (smooth muscle actin, vimentin, and tenascin-c) were measured in mesenchymal stem cells. In MB231, stemness markers Sox2, Nanog, and Oct4 were measured. RESULTS: Mesenchymal stem cells plus osteopontin increased transforming growth factor-ß and cancer-associated fibroblast markers (P < .05 vs mesenchymal stem cells alone); this was abolished by aptamer inactivation of osteopontin. In mesenchymal stem cells transfected with phosphoresistant myeloid zinc finger-1, osteopontin did not increase cancer-associated fibroblast markers or transforming growth factor-ß. In contrast, phosphomimetic myeloid zinc finger-1 increased cancer-associated fibroblast markers and transforming growth factor-ß (P < .05 vs mesenchymal stem cells alone). In mesenchymal stem cells plus MB231, MB231 stemness markers were increased (P < .05 vs MB231 alone). In MB231 plus mesenchymal stem cells expressing phosphoresistant myeloid zinc finger-1, MB231 stemness markers were not increased in comparison with MB231 plus mesenchymal stem cells. CONCLUSION: Myeloid zinc finger-1 phosphorylation in mesenchymal stem cells drives the osteopontin-mediated cancer-associated fibroblast phenotype, which then increases the cancer cell stemness profile.


Assuntos
Neoplasias da Mama/genética , Fibroblastos Associados a Câncer/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/genética , Osteopontina/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/citologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Feminino , Humanos , Células-Tronco Mesenquimais , Sensibilidade e Especificidade , Fator de Crescimento Transformador beta1/metabolismo , Microambiente Tumoral
6.
Surgery ; 163(2): 330-335, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28988933

RESUMO

BACKGROUND: Cancer cells metastasize to the bone marrow to create the premetastatic niche. Cancer stemness (expression of stem cell characteristics) is regulated by the tumor microenvironment and associated with self-renewal and poor clinical outcomes. Osteopontin induces mesenchymal stem cells in the tumor microenvironment to adopt a cancer-associated fibroblast phenotype to potentiate cancer growth and metastasis. The mechanisms by which cancer cells and tumor microenvironment regulate stemness in the bone marrow premetastatic niche is unknown. METHODS: Human breast cancer cell lines, MDA-MB-231 and MCF-7 were used in an orthotopic murine xenograft model. NOD-scid mice were implanted with 2 × 106 tumor cells in the presence and absence of human mesenchymal stem cells-green fluorescent protein cells and/or osteopontin aptamer, which blocks and inactivates extracellular osteopontin, or mutant aptamer (osteopontin mutant aptamer). In select instances, MCF-7 cells transfected to express osteopontin were coimplanted instead of MCF-7. Stemness markers (Nanog, Oct4, Sox2) in the tumor cells and cancer-associated fibroblast (α-smooth muscle actin, Vimentin) markers in the mesenchymal stem cells were measured in femoral bone marrow via real-time polymerase chain reaction. Cell number was determined by titrating cell number to Ct value in vitro. RESULTS: Tumor cells and mesenchymal stem cells migrate from the primary tumor site to the bone marrow. Migration of mesenchymal stem cells is osteopontin dependent. In both MDA-MB-231 and MCF-7 cell lines, levels of both cancer-associated fibroblast and stemness markers were 3 to 4 times greater under conditions wherein mesenchymal stem cells were present with osteopontin. Inactivation of extracellular osteopontin with an aptamer decreased migration of mesenchymal stem cells and expression of both cancer-associated fibroblast and stemness markers. Cancer cells exhibited a significantly increased stem cell profile in the presence of cancer-associated fibroblast in the bone marrow. In the presence and absence of osteopontin, Sox2 knockdown abolished expression of both Nanog and Oct4. CONCLUSION: We conclude that osteopontin-dependent migration of cancer-associated fibroblast is required for increased cancer cell stemness in the bone marrow premetastatic niche.


Assuntos
Medula Óssea/patologia , Neoplasias da Mama/patologia , Fibroblastos Associados a Câncer/fisiologia , Células-Tronco Mesenquimais/fisiologia , Micrometástase de Neoplasia , Osteopontina/fisiologia , Animais , Movimento Celular , Feminino , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Células-Tronco Neoplásicas
11.
J Clin Med ; 5(4)2016 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-27023622

RESUMO

Osteopontin (OPN) plays an important functional role in both physiologic and pathologic states. OPN is implicated in the progression of fibrosis, cancer, and metastatic disease in several organ systems. The epithelial-mesenchymal transition (EMT), first described in embryology, is increasingly being recognized as a significant contributor to fibrotic phenotypes and tumor progression. Several well-established transcription factors regulate EMT and are conserved across tissue types and organ systems, including TWIST, zinc finger E-box-binding homeobox (ZEB), and SNAIL-family members. Recent literature points to an important relationship between OPN and EMT, implicating OPN as a key regulatory component of EMT programs. In this review, OPN's interplay with traditional EMT activators, both directly and indirectly, will be discussed. Also, OPN's ability to restructure the tissue and tumor microenvironment to indirectly modify EMT will be reviewed. Together, these diverse pathways demonstrate that OPN is able to modulate EMT and provide new targets for directing therapeutics.

12.
Biochim Biophys Acta ; 1862(1): 135-44, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-26529285

RESUMO

INTRODUCTION: Liver fibrosis develops when hepatic stellate cells (HSC) are activated into collagen-producing myofibroblasts. In non-alcoholic steatohepatitis (NASH), the adipokine leptin is upregulated, and promotes liver fibrosis by directly activating HSC via the hedgehog pathway. We reported that hedgehog-regulated osteopontin (OPN) plays a key role in promoting liver fibrosis. Herein, we evaluated if OPN mediates leptin-profibrogenic effects in NASH. METHODS: Leptin-deficient (ob/ob) and wild-type (WT) mice were fed control or methionine-choline deficient (MCD) diet. Liver tissues were assessed by Sirius-red, OPN and αSMA IHC, and qRT-PCR for fibrogenic genes. In vitro, HSC with stable OPN (or control) knockdown were treated with recombinant (r)leptin and OPN-neutralizing or sham-aptamers. HSC response to OPN loss was assessed by wound healing assay. OPN-aptamers were also added to precision-cut liver slices (PCLS), and administered to MCD-fed WT (leptin-intact) mice to determine if OPN neutralization abrogated fibrogenesis. RESULTS: MCD-fed WT mice developed NASH-fibrosis, upregulated OPN, and accumulated αSMA+ cells. Conversely, MCD-fed ob/ob mice developed less fibrosis and accumulated fewer αSMA+ and OPN+ cells. In vitro, leptin-treated HSC upregulated OPN, αSMA, collagen 1α1 and TGFß mRNA by nearly 3-fold, but this effect was blunted by OPN loss. Inhibition of PI3K and transduction of dominant negative-Akt abrogated leptin-mediated OPN induction, while constitutive active-Akt upregulated OPN. Finally, OPN neutralization reduced leptin-mediated fibrogenesis in both PCLS and MCD-fed mice. CONCLUSION: OPN overexpression in NASH enhances leptin-mediated fibrogenesis via PI3K/Akt. OPN neutralization significantly reduces NASH fibrosis, reinforcing the potential utility of targeting OPN in the treatment of patients with advanced NASH.


Assuntos
Leptina/metabolismo , Cirrose Hepática/metabolismo , Fígado/patologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Osteopontina/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Deleção de Genes , Hepatócitos/metabolismo , Hepatócitos/patologia , Leptina/genética , Fígado/metabolismo , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Osteopontina/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais , Regulação para Cima
13.
Ann Surg ; 262(4): 683-91, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26366549

RESUMO

OBJECTIVE: We hypothesized that perioperative hospital resources could overcome the "weekend effect" (WE) in patients undergoing emergent/urgent surgeries. SUMMARY BACKGROUND DATA: The WE is the observation that surgeon-independent patient outcomes are worse on the weekend compared with weekdays. The WE is often explained by differences in staffing and resources resulting in variation in care between the week and weekend. METHODS: Emergent/urgent surgeries were identified using the Healthcare Cost and Utilization Project State Inpatient Database (Florida) from 2007 to 2011 and linked to the American Hospital Association (AHA) Annual Survey Database to determine hospital level characteristics. Extended median length of stay (LOS) on the weekend compared with the weekdays (after controlling for hospital, year, and procedure type) was selected as a surrogate for WE. RESULTS: Included were 126,666 patients at 166 hospitals. A total of 17 hospitals overcame the WE during the study period. Logistic regression, controlling for patient characteristics, identified full adoption of electronic medical records (OR 4.74), home health program (OR 2.37), pain management program [odds ratio (OR) 1.48)], increased registered nurse-to-bed ratio (OR 1.44), and inpatient physical rehabilitation (OR 1.03) as resources that were predictors for overcoming the WE. The prevalence of these factors in hospitals exhibiting the WE for all 5 years of the study period were compared with those hospitals that overcame the WE (P < 0.001). CONCLUSIONS: Specific hospital resources can overcome the WE seen in urgent general surgery procedures. Improved hospital perioperative infrastructure represents an important target for overcoming disparities in surgical care.


Assuntos
Disparidades em Assistência à Saúde/organização & administração , Tempo de Internação/estatística & dados numéricos , Centro Cirúrgico Hospitalar/organização & administração , Procedimentos Cirúrgicos Operatórios , Adulto , Idoso , Emergências , Feminino , Florida , Cirurgia Geral , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Avaliação de Processos e Resultados em Cuidados de Saúde , Admissão e Escalonamento de Pessoal , Fatores de Tempo
14.
Surgery ; 158(4): 1039-47; discussion 1047-8, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26189955

RESUMO

INTRODUCTION: Osteopontin (OPN) mediates metastasis and invasion of hepatocellular carcinoma (HCC). Epigallocatechin-3-gallate (EGCG), found in green tea, suppresses HCC tumor growth in vitro. We sought to investigate the role of EGCG in modulating OPN in cell lines of metastatic HCC. METHODS: Experimental HCC cell lines included HepG2 and MHCC-97H HCC cells, which express high levels of OPN, and the Hep3B cells, which express lesser levels of OPN. Cells were treated with EGCG (0.02-20 µg/mL) before measurement of OPN with enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction. Scratch assay measured cell migration. Binding of the OPN promoter to RNA pol II was evaluated by the use of Chromatin-IP assay after EGCG treatment. Transcriptional regulation of OPN was investigated with luciferase reporter plasmids containing various deletion fragments of the human OPN promoter. Measurement of the half-life of OPN mRNA was conducted using actinomycin D. RESULTS: Treatment of MHCC-97H and HepG2 cells with 2 µg/mL and 20 µg/mL EGCG caused a ∼6-fold and ∼90-fold decrease in secreted protein levels of OPN (All P < .001). OPN mRNA was decreased with EGCG concentrations of 0.2-20 µg/ml (All P < .001). The 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (ie, MTT) assay revealed that differences in OPN expression were not due to viability of the HCC cell lines. Promoter assay and chromatin immunoprecipitation analysis revealed no effect of EGCG on the transcriptional regulation of OPN. Posttranscriptionally, EGCG decreased the half-life of OPN mRNA from 16.8 hours (95% confidence interval 9.0-125.1) to 2.5 hours (95% confidence interval 2.1-3.2) (P < .001). Migration was decreased in EGCG treated cells at 24 hours (8.0 ± 2.4% vs 21.2 ± 10.8%, P < .01) and at 48 hours (13.2 ± 3.6% vs 53.5 ± 19.8%, P < .001). CONCLUSION: We provide evidence that EGCG decreases OPN mRNA and secreted OPN protein levels by decreasing the half-life of OPN mRNA in MHCC-97H cells. The translatability of EGCG for patients with HCC is promising, because EGCG is an inexpensive, easily accessible chemical with an extensive history of safety.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Catequina/análogos & derivados , Neoplasias Hepáticas/tratamento farmacológico , Osteopontina/metabolismo , RNA Mensageiro/metabolismo , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Catequina/farmacologia , Catequina/uso terapêutico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Meia-Vida , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metástase Neoplásica , Osteopontina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
J Biol Chem ; 290(16): 9959-73, 2015 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-25713073

RESUMO

Alcohol (EtOH) intoxication is a risk factor for increased morbidity and mortality with traumatic injuries, in part through inhibition of bone fracture healing. Animal models have shown that EtOH decreases fracture callus volume, diameter, and biomechanical strength. Transforming growth factor ß1 (TGF-ß1) and osteopontin (OPN) play important roles in bone remodeling and fracture healing. Mesenchymal stem cells (MSC) reside in bone and are recruited to fracture sites for the healing process. Resident MSC are critical for fracture healing and function as a source of TGF-ß1 induced by local OPN, which acts through the transcription factor myeloid zinc finger 1 (MZF1). The molecular mechanisms responsible for the effect of EtOH on fracture healing are still incompletely understood, and this study investigated the role of EtOH in affecting OPN-dependent TGF-ß1 expression in MSC. We have demonstrated that EtOH inhibits OPN-induced TGF-ß1 protein expression, decreases MZF1-dependent TGF-ß1 transcription and MZF1 transcription, and blocks OPN-induced MZF1 phosphorylation. We also found that PKA signaling enhances OPN-induced TGF-ß1 expression. Last, we showed that EtOH exposure reduces the TGF-ß1 protein levels in mouse fracture callus. We conclude that EtOH acts in a novel mechanism by interfering directly with the OPN-MZF1-TGF-ß1 signaling pathway in MSC.


Assuntos
Etanol/efeitos adversos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteopontina/farmacologia , Tíbia/efeitos dos fármacos , Fraturas da Tíbia/metabolismo , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Animais , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Diferenciação Celular , Consolidação da Fratura/efeitos dos fármacos , Regulação da Expressão Gênica , Humanos , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Luciferases/genética , Luciferases/metabolismo , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Osteopontina/metabolismo , Fosforilação , Transdução de Sinais , Tíbia/lesões , Tíbia/metabolismo , Fraturas da Tíbia/genética , Fraturas da Tíbia/patologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
16.
Clin Transl Med ; 3: 35, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25343018

RESUMO

The epithelial to mesenchymal transition (EMT) is implicated in many processes, ranging from tissue and organogenesis to cancer and metastatic spread. Understanding the key regulatory mechanisms and mediators within this process offers the opportunity to develop novel therapeutics with broad clinical applicability. To date, several components of EMT already are targeted using pharmacologic agents in fibrosis and cancer. As our knowledge of EMT continues to grow, the potential for novel therapeutics will also increase. This review focuses on the role of EMT both as a necessary part of development and a key player in disease progression, specifically the similarity in pathways used during both processes as targets for drug development. Also, the key role of the tumor microenvironment with EMT is outlined, focusing on both co-factors and cell types with the ability to modulate the progression of EMT in cancer and metastatic disease. Lastly, we discuss the current status of clinical therapies both in development and those progressed to clinical trial specifically targeting pathologic EMTs including small molecule inhibitors, non-coding RNAs, exogenous co-factors, and adjunctive therapies to current chemotherapeutics.

17.
Clin Sci (Lond) ; 126(12): 845-55, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24438228

RESUMO

OPN (osteopontin)) is a Hh (Hedgehog)-regulated cytokine that is up-regulated during chronic liver injury and directly promotes fibrosis. We have reported that Hh signalling enhances viral permissiveness and replication in HCV (hepatitis C virus)-infected cells. Hence we hypothesized that OPN directly promotes HCV replication, and that targeting OPN could be beneficial in HCV. In the present study, we compared the expression of OPN mRNA and protein in HCV (JFH1)-infected Huh7 and Huh7.5 cells, and evaluated whether modulating OPN levels using exogenous OPN ligands (up-regulate OPN) or OPN-specific RNA-aptamers (neutralize OPN) leads to changes in HCV expression. Sera and livers from patients with chronic HCV were analysed to determine whether OPN levels were associated with disease severity or response to therapy. Compared with Huh7 cells, Huh7.5 cells support higher levels of HCV replication (15-fold) and expressed significantly more OPN mRNA (30-fold) and protein. Treating Huh7 cells with OPN ligands led to a dose-related increase in HCV (15-fold) and OPN (8-fold) mRNA. Conversely, treating Huh7.5 cells with OPN-specific RNA aptamers inhibited HCV RNA and protein by >50% and repressed OPN mRNA to basal levels. Liver OPN expression was significantly higher (3-fold) in patients with advanced fibrosis. Serum OPN positively correlated with fibrosis-stage (P=0.009), but negatively correlated with ETBCR (end-of-treatment biochemical response), ETVR (end-of-treatment virological response), SBCR (sustained biochemical response) and SVR (sustained virological response) (P=0.007). The OPN fibrosis score (serum OPN and presence of fibrosis ≥F2) may be a predictor of SVR. In conclusion, OPN is up-regulated in the liver and serum of patients with chronic hepatitis C, and supports increased viral replication. OPN neutralization may be a novel therapeutic strategy in chronic hepatitis C.


Assuntos
Hepacivirus/fisiologia , Hepatite C Crônica/fisiopatologia , Osteopontina/fisiologia , Regulação para Cima , Replicação Viral , Adulto , Sequência de Bases , Primers do DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteopontina/sangue , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Surgery ; 154(2): 404-10, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23889968

RESUMO

BACKGROUND: Twist is an epithelial-mesenchymal transition (EMT) transcription factor that instigates cell invasion. Our research has shown that osteopontin (OPN) regulates the EMT factor Twist. The underlying signaling pathway is unknown. We hypothesized that OPN activates Twist to induce EMT in human breast cancer. METHODS: Potential kinases for Twist were identified using NetPhosK. Inhibitors of MEK1/2, JNK, p38 MAPK, and PI3K were applied to human breast cancer cells MDA-MB231 (OPN high). After 24 h, Twist was immunoprecipitated and incubated with phosphoserine. Expression of the Twist target protein, Bmi-1, was determined following 24-h osteopontin aptamer (APT) treatment; mutant aptamer (MuAPT) was used as the control. Scratch-wound assay was imaged 12, 24, and 48 h after APT and MuAPT treatment. RESULTS: MEK1/2 inhibition caused ≈ twofold decrease in Twist serine phosphorylation (P < .05). APT blockade of OPN in MB231 decreased Bmi1 protein twofold (P < .05). Aptamer-treated cells were significantly decreased in cell migration and wound closure in the scratch wound-assay (P < .001). CONCLUSION: We demonstrate that OPN extracellular binding to MB231 activates an autocrine MAPK intracellular signaling pathway resulting in Twist activation and promoting Bmi1 expression to further EMT initiation and cellular migration. Our results elucidate a previously undescribed role for OPN as a prime regulator of EMT in human breast cancer cells.


Assuntos
Neoplasias da Mama/patologia , Transição Epitelial-Mesenquimal , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína 1 Relacionada a Twist/fisiologia , Linhagem Celular Tumoral , Movimento Celular , Feminino , Humanos , Proteína Quinase 7 Ativada por Mitógeno/fisiologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Osteopontina/antagonistas & inibidores , Osteopontina/fisiologia
19.
J Am Coll Surg ; 217(1): 17-26; discussion 26, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23619316

RESUMO

BACKGROUND: Tumor cells undergoing epithelial-mesenchymal transition (EMT) develop cellular properties leading to stroma invasion and intravasation. We have previously shown in a xenograft breast cancer model that blocking osteopontin (OPN), a secreted phosphoprotein, decreases EMT. This study examines OPN's role in EMT initiation through its regulation of EMT transcription factors (TFs) Snail, Slug, and Twist. OPN's role in Twist activation is examined through immunoprecipitation and Western blot. STUDY DESIGN: MDA-MB-231 breast cancer cells secreting high levels of OPN were treated with OPN aptamer (APT) or mutant APT. Osteopontin APT binds to and inhibits extracellular OPN. Low-OPN-secreting breast cancer cells, MCF-7, were treated with OPN, OPN+APT, or OPN+mutant APT. Twist was isolated in MDA-MB-231 with immunoprecipitation. Phospho-serine antibody detected activated Twist in Western blot. Activation of Twist was confirmed by chromatin immunoprecipitation. RESULTS: Analysis through quantitative polymerase chain reaction demonstrated APT inhibition of OPN in MDA-MB-231 cells caused a decrease in EMT-TF expression (MDA-MB-231 vs MDA-MB-231+APT: *Twist ΔΔCT: 1.0 vs 0.07; *Snail ΔΔCT: 1.0 vs 0.11; *Slug ΔΔCT: 1.0 vs 0.11; *p < 0.001). Mutant APT did not change EMT-TF expression (NS). Treatment of MCF-7 cells with OPN caused an increase in EMT-TF expression (MCF-7 vs MCF-7+OPN: Twist ΔΔCT: 1.0 vs 9.1; *Snail ΔΔCT: 1.0 vs 11.2; *Slug ΔΔCT: 1.0 vs 10.9; *p < 0.001). The EMT-TF expression in MCF-7 treated with OPN+APT did not differ significantly from MCF-7 alone. Phosphorylated Twist protein was reduced 2-fold with APT in MDA-MB-231 compared with MDA-MB-231 and MDA-MB-231+mutant APT. Twist phorphorylation induced binding to the promoter regions of Twist-regulated gene, B lymphoma Mo-MLV insertion region 1 homolog, a critical protein for EMT progression. CONCLUSIONS: This study shows that OPN is critical in EMT initiation through activation of Twist via serine phosphorylation. These unique observations indicate that OPN APT can serve a clinical role as a novel therapeutic agent by diminishing breast cancer oncogenesis.


Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Transição Epitelial-Mesenquimal , Osteopontina/metabolismo , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Adenocarcinoma/patologia , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imunoprecipitação , Células MCF-7 , Invasividade Neoplásica , Osteopontina/antagonistas & inibidores , Fenótipo , Complexo Repressor Polycomb 1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Regulação para Cima
20.
Surgery ; 152(3): 449-54, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22938903

RESUMO

BACKGROUND: The microenvironments of neoplasms influence both mesenchymal stem cell differentiation into cancer-associated fibroblasts (CAF) and tumor cell line differentiation to mesenchymal phenotypes via epithelial-to-mesenchymal transition (EMT). Using direct cell-cell contact approximating the microenvironment of a neoplasm, we investigated the role of this interaction in human mesenchymal stem cells (hMSCs) and epithelial hepatic carcinoma SK-Hep1 cells by evaluating CAF differentiation and EMT. METHODS: hMSCs and SK-Hep1 cells were homogenously cultured for 12 hours with media only, OPN-R3 aptamer blockade of OPN, or RGD peptide blockade of integrin receptor, negative control mutant OPN-R3 aptamer, and RGE peptide blockade. mRNA was isolated from each subpopulation, and real-time-polymerase chain reaction was performed for CAF markers and EMT transcription factors and structural proteins. RESULTS: SK-Hep1 cells in admixture with hMSCs showed increased EMT marker vimentin expression that was ablated with OPN-R3 aptamer or RGD blockade. SK-Hep1 cells when cultured with hMSC admixture increased Snail and Slug expression that was hindered with OPN-R3 aptamer. hMSCs acquired CAF markers tenascin-c and SDF-1 in admixture that was ablated with either OPN-R3 aptamer or RGD blockade. All SK-Hep1 and hMSC negative control subpopulations were statistically equivalent to media-only groups. Fluorescence photography exhibited the critical cell-cell interfaces and acquired EMT traits of SK-Hep1. CONCLUSION: We conclude that direct interaction of cell lines closely replicates the native neoplasm microenvironment. Our experiments reveal soluble OPN or integrin receptor blockade independently prevents progression to metastatic phenotype by acquisition of CAF and EMT markers.


Assuntos
Carcinoma Hepatocelular/patologia , Transição Epitelial-Mesenquimal , Fibroblastos/patologia , Neoplasias Hepáticas/patologia , Células-Tronco Mesenquimais/patologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Epiteliais/patologia , Humanos , Microscopia de Fluorescência , Microambiente Tumoral
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