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1.
Biomaterials ; 271: 120754, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33756215

RESUMO

High levels of reactive oxygen species (ROS) in tumors have been shown to exert anti-tumor activity, leading to the concept of ROS induction as therapeutic strategy. The organometallic compound ferrocene (Fc) generates ROS through a reversible one-electron oxidation. Incorporation of Fc into a tumor-targeting, bioactive molecule can enhance its therapeutic activity and enable tumor specific delivery. Therefore, we conjugated Fc to five synthetic, Arg-Gly-Asp (RGD)-based integrin binding ligands to enable targeting of the cell adhesion and signaling receptor integrin subtypes αvß3, α5ß1, or αvß6, which are overexpressed in various, distinct tumors. We designed and synthesized a library of integrin-ligand-ferrocene (ILF) derivatives and showed that ILF conjugates maintained the high integrin affinity and selectivity of their parent ligands. A thorough biological characterization allowed us to identify the two most promising ligands, an αvß3 (L2b) and an αvß6 (L3b) targeting ILF, which displayed selective integrin-dependent cell uptake and pronounced ferrocene-mediated anti-tumor effects in vitro, along with increased ROS production and DNA damage. Hence, ILFs are promising candidates for the selective, tumor-targeted delivery of ferrocene to maximize its anti-cancer efficacy and minimize systemic toxicity, thereby improving the therapeutic window of ferrocene compared to currently used non-selective anti-cancer drugs.


Assuntos
Integrinas , Neoplasias , Humanos , Integrina alfa5beta1 , Integrina alfaVbeta3 , Ligantes , Metalocenos , Neoplasias/tratamento farmacológico
2.
Mol Oncol ; 15(2): 503-522, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33155399

RESUMO

Epithelial ovarian cancer involves the shedding of single tumor cells or spheroids from the primary tumor into ascites, followed by their survival, and transit to the sites of metastatic colonization within the peritoneal cavity. During their flotation, anchorage-dependent epithelial-type tumor cells gain anoikis resistance, implicating integrins, including αvß3. In this study, we explored anoikis escape, cisplatin resistance, and prosurvival signaling as a function of the αvß3 transmembrane conformational activation state in cells suspended in ascites. A high-affinity and constitutively signaling-competent αvß3 variant, which harbored unclasped transmembrane domains, was found to confer delayed anoikis onset, enhanced cisplatin resistance, and reduced cell proliferation in ascites or 3D-hydrogels, involving p27kip upregulation. Moreover, it promoted EGF-R expression and activation, prosurvival signaling, implicating FAK, src, and PKB/Akt. This led to the induction of the anti-apoptotic factors Bcl-2 and survivin suppressing caspase activation, compared to a signaling-incapable αvß3 variant displaying firmly associated transmembrane domains. Dissecting the mechanistic players for αvß3-dependent survival and peritoneal metastasis of ascitic ovarian cancer spheroids is of paramount importance to target their anchorage independence by reversing anoikis resistance and blocking αvß3-triggered prosurvival signaling.


Assuntos
Anoikis , Integrina alfaVbeta3/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Feminino , Humanos , Integrina alfaVbeta3/genética , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/genética
3.
Oncotarget ; 9(5): 6369-6390, 2018 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-29464079

RESUMO

The tetraspanin and tumor suppressor KAI1 is downregulated or lost in many cancers which correlates with poor prognosis. KAI1 acts via physical/functional crosstalk with other membrane receptors. Also, a splice variant of KAI1 (KAI1-SP) has been identified indicative of poor prognosis. We here characterized differential effects of the two KAI1 variants on tumor biological events involving integrin (αvß3) and/or epidermal growth factor receptor (EGF-R). In MDA-MB-231 and -435 breast cancer cells, differential effects were documented on the expression levels of the tumor biologically relevant integrin αvß3 which colocalized with KAI1-WT but not with KAI1-SP. Cellular motility was assessed by video image processing, including motion detection and vector analysis for the quantification and visualization of cell motion parameters. In MDA-MB-231 cells, KAI1-SP provoked a quicker wound gap closure and higher closure rates than KAI1-WT, also reflected by different velocities and average motion amplitudes of singular cells. KAI1-SP induced highest cell motion adjacent to the wound gap borders, whereas in MDA-MB-435 cells a comparable induction of both KAI1 variants was noticed. Moreover, while KAI1-WT reduced cell growth, KAI1-SP significantly increased it going along with a pronounced EGF-R upregulation. KAI1-SP-induced cell migration and proliferation was accompanied by the activation of the focal adhesion and Src kinase. Our findings suggest that splicing of KAI1 does not only abrogate its tumor suppressive functions, but even more, promotes tumor biological effects in favor of cancer progression and metastasis.

4.
J Cell Physiol ; 220(2): 367-75, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19334037

RESUMO

We previously showed that integrin alphavbeta3 overexpression and engagement by its ligand vitronectin increased adhesion, motility, and proliferation of human ovarian cancer cells. In search of differentially regulated genes involved in these tumor biological events, we previously identified the integrin-linked kinase (ILK) to be under control of alphavbeta3. In the present investigation we demonstrated significantly upregulated ILK protein as a function of alphavbeta3 in two ovarian cancer cell lines, OV-MZ-6 and OVCAR-3, and proved co-localization at the surface of alphavbeta3-overexpressing cells adherent to vitronectin. Increase of ILK protein was reflected by enhanced ILK promoter activity, an effect, which we further characterized with regard to transcriptional response elements involved. Abrogation of NF-kappaB/c-rel or p53 binding augmented ILK promoter activity and preserved induction by alphavbeta3. The AP1-mutant exhibited decreased promoter activity but was also still inducible by alphavbeta3. Disruption of the two DNA consensus motifs for Ets proteins led to divergent observations: mutation of the Ets motif at promoter position -462 bp did not significantly alter promoter activity but still allowed response to alphavbeta3. In contrast, disruption of the second Ets motif at position -85 bp did not only lead to slightly diminished promoter activity but also, in that case, abrogated ILK promoter induction by alphavbeta3. Subsequent co-transfection studies with ets-1 in the presence of the second Ets motif led to additional induction of ILK promoter activity. Taken together, these data suggest that ets-1 binding to the second Ets DNA motif strongly contributes to alphavbeta3-mediated ILK upregulation. By increasing ILK as an important integrin-proximal kinase, alphavbeta3 may promote its intracellular signaling and tumor biological processes arising thereof in favor of ovarian cancer metastasis.


Assuntos
Regulação Neoplásica da Expressão Gênica , Integrina alfaVbeta3/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular Tumoral , Feminino , Humanos , Integrina alfaVbeta3/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética
5.
Int J Biochem Cell Biol ; 40(12): 2746-61, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18577466

RESUMO

Upon overexpression of integrin alphavbeta3 and its engagement by vitronectin, we previously showed enhanced adhesion, proliferation, and motility of human ovarian cancer cells. By studying differential expression of genes possibly related to these tumor biological events, we identified the epidermal growth-factor receptor (EGF-R) to be under control of alphavbeta3 expression levels. Thus in the present study we characterized alphavbeta3-dependent changes of EGF-R and found significant upregulation of its expression and activity which was reflected by prominent changes of EGF-R promoter activity. Upon disruption of DNA-binding motifs for the transcription factors p53, ETF, the repressor ETR, p50, and c-rel, respectively, we sought to identify DNA elements contributing to alphavbeta3-mediated EGF-R promoter induction. Both, the p53- and ETF-mutant, while exhibiting considerably lower EGF-R promoter activity than the wild type promoter, retained inducibility by alphavbeta3. Mutation of the repressor motif ETR, as expected, enhanced EGF-R promoter activity with a further moderate increase upon alphavbeta3 elevation. The p50-mutant displayed EGF-R promoter activity almost comparable to that of the wild type promoter with no impairment of induction by alphavbeta3. However, the activity of an EGF-R promoter mutant displaying a disrupted c-rel-binding motif did not only prominently decline, but, moreover, was not longer responsive to enhanced alphavbeta3, involving this DNA element in alphavbeta3-dependent EGF-R upregulation. Moreover, alphavbeta3 did not only increase the EGF-R but, moreover, also led to obvious co-clustering on the cancer cell surface. By studying alphavbeta3/EGF-R-effects on the focal adhesion kinase (FAK) and the mitogen activated protein kinases (MAPK) p44/42 (erk(-1)/erk(-2)), having important functions in synergistic crosstalk between integrins and growth-factor receptors, we found for both significant enhancement of expression and activity upon alphavbeta3/VN interaction and cell stimulation by EGF. Upregulation of the EGF-R by integrin alphavbeta3, both receptor molecules with a well-defined role as targets for cancer treatment, might represent an additional mechanism to adapt synergistic receptor signaling and crosstalk in response to an altered tumor cell microenvironment during ovarian cancer progression.


Assuntos
Receptores ErbB/metabolismo , Expressão Gênica , Integrina alfaVbeta3/metabolismo , Neoplasias Ovarianas/patologia , Regulação para Cima , Linhagem Celular Tumoral , Proliferação de Células , Receptores ErbB/genética , Feminino , Genes Reporter , Humanos , Imuno-Histoquímica , Integrina alfaVbeta3/genética , Luciferases de Renilla/metabolismo , Neoplasias Ovarianas/genética , Transfecção , Vitronectina/metabolismo
6.
Int J Biochem Cell Biol ; 37(3): 590-603, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15618016

RESUMO

We have recently described that integrin alphavbeta3 upon interaction with its major extracellular matrix ligand vitronectin induces adhesion, motility, and proliferation of human ovarian cancer cells. Due to the important function of alphavbeta3 in cancer cell biology, it has been the effort of many scientific approaches to specifically target alphavbeta3-mediated cell adhesion and tumorbiological effects arising thereof by synthetic integrin antagonists. More recently, proteins of the ADAM family have been recognized as naturally occurring integrin ligands. Among those, human ADAM15 which encompasses the integrin binding RGD motif was shown to interact with integrin alphavbeta3. Thus, we investigated in human ovarian OV-MZ-6 cancer cells, expressing both ADAM15 and alphavbeta3, whether ADAM15 might affect alphavbeta3-mediated tumorbiological effects. We stably (over)expressed ADAM15 or its extracellular domain in OV-MZ-6 cells as well as respective ADAM15 mutants containing the tripeptide SGA instead of RGD. Cells (over)expressing ADAM15-RGD exhibited a significantly reduced alphavbeta3-mediated adhesion to vitronectin. Also, a significant time-dependent decline in numbers of cells cultivated on vitronectin was noticed. This effect was found to be rather due to impaired alphavbeta3-mediated cell adhesion than decreased cell proliferation rates, since de novo DNA synthesis was not significantly altered by elevated ADAM15 expression. Moreover, a substantially decreased random cellular motility was noticed as a function of ADAM15 encompassing an intact RGD motif. In conclusion, our results point to a physiological role of ADAM15 as a natural binding partner of integrin alphavbeta3 thereby loosening tumor cell adhesion to the underlying matrix and regulating tumor cell migration and invasion.


Assuntos
Integrina alfaVbeta3/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Oligopeptídeos/metabolismo , Neoplasias Ovarianas/metabolismo , Vitronectina/metabolismo , Proteínas ADAM , Animais , Células CHO , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Cricetinae , Cricetulus , Feminino , Humanos , Imuno-Histoquímica , Integrina alfaVbeta3/antagonistas & inibidores , Cinética , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Microscopia Confocal , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia
7.
Biol Chem ; 384(7): 1073-83, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12956424

RESUMO

During tumor metastasis, a fine-tuned balance between the formation and loosening of adhesive cell contacts has to occur, a process based on the regulated expression of integrins. Human ovarian OV-MZ-6 cancer cells express the integrin alpha(v)beta3, which associates with vitronectin (VN) and correlates with ovarian cancer progression. Adhesion and spreading of OV-MZ-6 cells on VN was accompanied by the formation of focal adhesion contacts and the recruitment of activated tyrosine-phosphorylated focal adhesion kinase. Cultivation of OV-MZ-6 cells on VN resulted in a significantly induced cell proliferation. This VN effect could be mimicked by cultivating cells on the immobilized alpha(v)beta3 directed peptide cyclo-Arg-Gly-Asp-D-Phe-Val (cRGDfV). VN-dependent OV-MZ-6 cell adhesion and proliferation was significantly enhanced by overexpression of alpha(v)beta3 and was accompanied by rapid and transient tyrosine-phosphorylation of p44(erk-1)/p42(erk-2) mitogen-activated protein kinase. Moreover, overexpression of alpha(v)beta3 and OV-MZ-6 cell attachment to VN increased cell motility up to 5-fold accompanied by prominent changes in cytoskeletal organization and cell morphology. Upon alpha(v)beta3/VN interaction, by cDNA expression microarray analysis we identified altered mRNA levels of c-myc, epidermal growth factor receptor (EGF-R), transcription factor Fra-1, prothymosin-alpha (PTMA), integrin-linked kinase (ILK), and the cell adhesion molecule SQM-1, candidates which are possibly involved in changes of the adhesive, migratory, and proliferative phenotype of human ovarian cancer cells.


Assuntos
Integrina alfaVbeta3/metabolismo , Neoplasias Ovarianas/metabolismo , Vitronectina/metabolismo , Adesão Celular , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto/ultraestrutura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestrutura , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/química , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Transdução de Sinais , Vitronectina/química
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