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1.
Tumour Biol ; 41(7): 1010428319860728, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31264534

RESUMO

Colon cancer represents one of the most common cancers in the world. Despite improved treatment, mortality remains high. In order to improve the assessment of prognosis for colon cancer patients, identifying new prognostic markers remains necessary. We analyzed preoperative serum samples from 148 colon cancer patients surgically treated at Helsinki University Hospital from 1998 through 2002 using a multiplex proximity extension assay (Oncology II panel, Olink Bioscience, Uppsala, Sweden), a panel constituting 92 immunological and oncological markers. We performed univariate and multivariate analyses on these patients and calculated the disease-specific survival among patients using the log-rank test for Kaplan-Meier estimates. In the univariate survival analysis of 92 biomarkers, 26 resulted in p < 0.1. Among these, eight biomarkers emerged as statistically significant (p < 0.05). Patients with low levels of kallikrein 13 had a poor prognosis. Moreover, patients with high levels of amphiregulin, carcinoembryonic antigen-related adhesion molecule 5, interleukin 6, mucin 16, syndecan 1, transforming growth factor alpha, and vimentin also had a poor prognosis. In the multivariate analysis, kallikrein 13 and mucin 16 emerged as independent prognostic markers. The role of kallikrein 13, a member of the serine protease kallikrein biomarker family, in tumorigenesis remains unclear. Mucin 16 is also known as carbohydrate antigen 125, a well-known ovarian cancer biomarker. Patients with low levels of kallikrein 13 (hazard ratio: 0.36; 95% confidence interval: 0.14-0.92; p = 0.033) and high levels of mucin 16 (hazard ratio: 3.15; 95% confidence interval: 1.68-5.93; p < 0.005) had a poor prognosis. Mucin 16 and kallikrein 13 represent independent prognostic markers for colon cancer. Furthermore, the clinical utility of mucin 16 and kallikrein 13 serum tests warrants additional investigation.


Assuntos
Antígeno Ca-125/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Calicreínas/metabolismo , Proteínas de Membrana/metabolismo , Idoso , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Imunoensaio/métodos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Análise de Sobrevida
2.
Acta Crystallogr F Struct Biol Commun ; 75(Pt 5): 385-391, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31045568

RESUMO

The inhibition of kallikrein 5 (KLK5) has been identified as a potential strategy for treatment of the genetic skin disorder Netherton syndrome, in which loss-of-function mutations in the SPINK5 gene lead to down-regulation of the endogenous inhibitor LEKTI-1 and profound skin-barrier defects with severe allergic manifestations. To aid in the development of a medicine for this target, an X-ray crystallographic system was developed to facilitate fragment-guided chemistry and knowledge-based drug-discovery approaches. Here, the development of a surrogate crystallographic system in place of KLK5, which proved to be challenging to crystallize, is described. The biochemical robustness of the crystallographic surrogate and the suitability of the system for the study of small nonpeptidic fragments and lead-like molecules are demonstrated.


Assuntos
Benzamidinas/química , Calicreínas/química , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Benzamidinas/farmacologia , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Descoberta de Drogas , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Calicreínas/antagonistas & inibidores , Calicreínas/genética , Calicreínas/metabolismo , Cinética , Modelos Moleculares , Mutação , Síndrome de Netherton/tratamento farmacológico , Síndrome de Netherton/enzimologia , Inibidores de Proteases/farmacologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Eletricidade Estática , Especificidade por Substrato
3.
Artif Cells Nanomed Biotechnol ; 47(1): 1224-1233, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30945557

RESUMO

In this study, we identified hsa_circ_0051240 was significantly increased in ovarian cancer (OC) tissues. Our results indicated that silencing of hsa_circ_0051240 inhibited OC cell proliferation, migration and invasion in vitro, and also prevented OC tumour formation in vivo. In addition, the inhibitory effects by blockage of hsa_circ_0051240 could be attenuated by the miR-637 inhibitor. Furthermore, we also identified that hsa_circ_0051240 act as a sponge of miR-637, and miR-637 directly targeted KLK4 mRNA in OC cells. Altogether, hsa_circ_0051240 promotes OC cell proliferation, migration and invasion through inhibiting the miR-637/KLK4 axis. Therefore, these results demonstrated that the hsa_circ_0051240/miR-637/KLK4 axis might serve as a therapeutic target for OC treatment.


Assuntos
Movimento Celular/genética , Calicreínas/metabolismo , MicroRNAs/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , RNA/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica , Humanos , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA/metabolismo , Transdução de Sinais/genética
4.
Acta Med Okayama ; 73(1): 1-6, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30820048

RESUMO

Excessive protease activity is a characteristic abnormality that affects the epidermal barrier in patients with atopic dermatitis (AD). Kallikrein-related peptidases (KLKs) are excessively expressed in AD lesions, and it is suggested that the abnormal action of KLKs is involved in the skin barrier dysfunction in AD. In other words, overexpressed KLKs disrupt the normal barrier function, and due to that breakdown, external substances that can become antigens of AD easily invade the epidermis, resulting in dermatitis, coupled with the induction of Th2 cytokines. Further investigations are required to elucidate the role of KLKs in AD; this knowledge could contribute to the design of new therapeutic and prophylactic drugs for AD.


Assuntos
Dermatite Atópica/enzimologia , Dermatite Atópica/patologia , Calicreínas/metabolismo , Humanos , Pele/enzimologia
5.
Int J Mol Sci ; 20(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925705

RESUMO

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.


Assuntos
Ensaios Enzimáticos/métodos , Calicreínas/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Cinética , Neoplasias/enzimologia , Biblioteca de Peptídeos , Peptídeos/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
6.
J Dairy Res ; 86(1): 114-119, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30729897

RESUMO

The experiments reported in this research paper examine the potential of digestion using acidic enzymes Protease A and Protease M to selectively hydrolyse α-lactalbumin (α-La) whilst leaving ß-lactoglobulin (ß-Lg) relatively intact. Both enzymes were compared with pepsin hydrolysis since its selectivity to different whey proteins is known. Analysis of the hydrolysis environment showed that the pH and temperature play a significant role in determining the best conditions for achievement of hydrolysis, irrespective of which enzyme was used. Whey protein isolate (WPI) was hydrolysed using pepsin, Acid Protease A and Protease M by randomized hydrolysis conditions. Reversed-phase high performance liquid chromatography was used to analyse residual proteins. Regarding enzyme selectivity under various milieu conditions, all three enzymes showed similarities in the reaction progress and their potential for ß-Lg isolation.


Assuntos
Calicreínas/metabolismo , Lactalbumina/metabolismo , Lactoglobulinas/isolamento & purificação , Pepsina A/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas do Soro do Leite/química , Hidrólise , Cinética , Especificidade por Substrato
7.
Methods Mol Biol ; 1929: 111-125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710270

RESUMO

Early diagnosis, noninvasive detection, and staging of various diseases, remain one of the major clinical barriers to effective medical treatment and prevention of disease progression toward major clinical consequences. Molecular imaging technologies play an indispensable role in the clinical field in overcoming these major barriers. The increasing application of imaging techniques and agents in early detection of different diseases such as cancer has resulted in improved treatment response and clinical patient management. In this chapter we will first introduce criteria for the design and engineering of calcium-binding protein (CaBP) parvalbumin as a protein Gd-MRI contrast agent (ProCA) with unprecedented metal selectivity for Gd3+ over physiological metal ions. We will then discuss the further development of targeted MRI contrast agent for molecular imaging of PSMA biomarker for early detection of prostate cancer.


Assuntos
Meios de Contraste/síntese química , Gadolínio/química , Calicreínas/metabolismo , Parvalbuminas/química , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/diagnóstico por imagem , Engenharia de Proteínas/métodos , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Meios de Contraste/química , Meios de Contraste/farmacologia , Detecção Precoce de Câncer , Humanos , Imagem por Ressonância Magnética/métodos , Masculino , Camundongos , Modelos Moleculares , Conformação Molecular , Imagem Molecular/métodos , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo
8.
Mater Sci Eng C Mater Biol Appl ; 96: 436-445, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30606553

RESUMO

In this study, prostate-specific membrane antigen (PSMA)-targeted and core-crosslinked micelles were developed based on prostate cancer-binding peptide (PCP) modified glycol chitosan-lipoic acid (PGC-LA) conjugate. The degree of substitution was 5.2 PCP groups and 10.7 lipoic acid groups per 100 sugar residues of glycol chitosan in PGC-LA copolymer. Docetaxel (DTX) was chosen as a model anti-tumor drug. The DTX-loaded micelles were prepared by an o/w method, and core-crosslinked micelles were further constructed by using a catalytic amount of dithiothreitol. The mean diameter of DTX-loaded core-crosslinked PGC-LA (DTX-PGC-LA/cc) micelles was 397 nm determined by dynamic light scattering (DLS). In vitro DTX released from core-crosslinked micelles was slower than that from non-crosslinked counterpart. Blank micelles exhibited good biocompatibility. Additionally, cellular uptake and cytotoxcity of PCP-modified micelles were higher than those of micelles without PCP in PSMA-positive LNCaP cells. Importantly, DTX-PGC-LA/cc demonstrated the stronger anti-tumor efficacy against LNCaP tumor xenograft models than DTX injection and other DTX-loaded micelles. Taken together, this study provides a potential way in developing actively targeted and core-crosslinked micelles for hydrophobic drug delivery in cancer therapy.


Assuntos
Quitosana , Docetaxel , Sistemas de Liberação de Medicamentos/métodos , Calicreínas/metabolismo , Micelas , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Quitosana/química , Quitosana/farmacocinética , Quitosana/farmacologia , Docetaxel/química , Docetaxel/farmacocinética , Docetaxel/farmacologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Oncogene ; 38(18): 3387-3401, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30643192

RESUMO

Blood circulating cell-free DNA (ccfDNA) is a suggested biosource of valuable clinical information for cancer, meeting the need for a minimally-invasive advancement in the route of precision medicine. In this paper, we evaluated the prognostic and predictive potential of ccfDNA parameters in early and advanced breast cancer. Groups consisted of 150 and 16 breast cancer patients under adjuvant and neoadjuvant therapy respectively, 34 patients with metastatic disease and 35 healthy volunteers. Direct quantification of ccfDNA in plasma revealed elevated concentrations correlated to the incidence of death, shorter PFS, and non-response to pharmacotherapy in the metastatic but not in the other groups. The methylation status of a panel of cancer-related genes chosen based on previous expression and epigenetic data (KLK10, SOX17, WNT5A, MSH2, GATA3) was assessed by quantitative methylation-specific PCR. All but the GATA3 gene was more frequently methylated in all the patient groups than in healthy individuals (all p < 0.05). The methylation of WNT5A was statistically significantly correlated to greater tumor size and poor prognosis characteristics and in advanced stage disease with shorter OS. In the metastatic group, also SOX17 methylation was significantly correlated to the incidence of death, shorter PFS, and OS. KLK10 methylation was significantly correlated to unfavorable clinicopathological characteristics and relapse, whereas in the adjuvant group to shorter DFI. Methylation of at least 3 or 4 genes was significantly correlated to shorter OS and no pharmacotherapy response, respectively. Classification analysis by a fully automated, machine learning software produced a single-parametric linear model using ccfDNA plasma concentration values, with great discriminating power to predict response to chemotherapy (AUC 0.803, 95% CI [0.606, 1.000]) in the metastatic group. Two more multi-parametric signatures were produced for the metastatic group, predicting survival and disease outcome. Finally, a multiple logistic regression model was constructed, discriminating between patient groups and healthy individuals. Overall, ccfDNA emerged as a highly potent predictive classifier in metastatic breast cancer. Upon prospective clinical evaluation, all the signatures produced could aid accurate prognosis.


Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Ácidos Nucleicos Livres/sangue , Metilação de DNA/fisiologia , DNA de Neoplasias/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Epigênese Genética/fisiologia , Feminino , Humanos , Calicreínas/metabolismo , Pessoa de Meia-Idade , Prognóstico
10.
Proc Natl Acad Sci U S A ; 116(1): 271-276, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30559188

RESUMO

Blood-brain barrier (BBB) disruption and transendothelial trafficking of immune cells into the central nervous system (CNS) are pathophysiological hallmarks of neuroinflammatory disorders like multiple sclerosis (MS). Recent evidence suggests that the kallikrein-kinin and coagulation system might participate in this process. Here, we identify plasma kallikrein (KK) as a specific direct modulator of BBB integrity. Levels of plasma prekallikrein (PK), the precursor of KK, were markedly enhanced in active CNS lesions of MS patients. Deficiency or pharmacologic blockade of PK renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by a remarkable reduction of BBB disruption and CNS inflammation. In vitro analysis revealed that KK modulates endothelial cell function in a protease-activated receptor-2-dependent manner, leading to an up-regulation of the cellular adhesion molecules Intercellular Adhesion Molecule 1 and Vascular Cell Adhesion Molecule 1, thereby amplifying leukocyte trafficking. Our study demonstrates that PK is an important direct regulator of BBB integrity as a result of its protease function. Therefore, KK inhibition can decrease BBB damage and cell invasion during neuroinflammation and may offer a strategy for the treatment of MS.


Assuntos
Bradicinina/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Calicreínas/metabolismo , Receptor PAR-2/metabolismo , Animais , Barreira Hematoencefálica , Western Blotting , Bradicinina/fisiologia , Encefalomielite Autoimune Experimental/fisiopatologia , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Humanos , Calicreínas/antagonistas & inibidores , Calicreínas/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Esclerose Múltipla/metabolismo , Receptor PAR-2/fisiologia
11.
J Mol Histol ; 49(5): 499-507, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30066216

RESUMO

Enamel is the hardest tissue with the highest degree of mineralization protecting the dental pulp from injury in vertebrates. The ameloblasts differentiated from ectoderm-derived epithelial cells are a single cell layer and are important for the enamel formation and mineralization. Wnt/ß-catenin signaling has been proven to exert an important role in the mineralization of bone, dentin and cementum. Little was known about the regulatory mechanism of Wnt/ß-catenin signaling pathway in ameloblasts during amelogenesis, especially in the mineralization of enamel. To investigate the role of ß-catenin in ameloblasts, we established Amelx-Cre; ß-catenin∆ex3fl/fl (CA-ß-catenin) mice, which could constitutive activate ß-catenin in ameloblasts. It showed the delayed mineralization and eventual hypomineralization in the incisor enamel of CA-ß-catenin mice. Meanwhile, the amelogenesis-related proteinases Mmp20 and Klk4 were decreased in the incisors of CA-ß-catenin mice. These data indicated that ß-catenin plays an essential role in differentiation and function of ameloblasts during amelogenesis.


Assuntos
Ameloblastos/metabolismo , Hipoplasia do Esmalte Dentário/etiologia , Esmalte Dentário/química , beta Catenina/metabolismo , Amelogênese , Animais , Calicreínas/metabolismo , Metaloproteinase 20 da Matriz/metabolismo , Camundongos , Via de Sinalização Wnt
12.
Clin Biochem ; 59: 78-85, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29958881

RESUMO

BACKGROUND: Human tissue kallikrein 15 (KLK15) is the latest member of the kallikrein-related peptidase family. Little is known about the pathophysiological roles of KLK15. Previous studies implied a role of KLK15 in prostate cancer. METHODS: In the present study, we examined KLK15 protein expression using a new immunoassay (ELISA) and immunohistochemistry (IHC). RESULTS: Highest KLK15 levels were detected in the testis and seminal fluid, whereas lower levels were observed in prostate and other tissues. Immunohistochemical analysis of testis suggests that KLK15 is strongly expressed in mature spermatids, but not in immature germ cells. KLK15 displayed predominantly nuclear localization in the basal cell layer of the prostatic epithelium. We also measured KLK15 in supernatants of various cell lines. Highest KLK15 levels were primarily detected in prostate cancer cell lines and KLK15 expression was hormone-independent, in contrast to KLK3. CONCLUSIONS: Collectively, our data provide insights into the localization and possible role of KLK15 in human physiology.


Assuntos
Calicreínas/biossíntese , Calicreínas/genética , Próstata/enzimologia , Testículo/enzimologia , Adulto , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Células Jurkat , Calicreínas/metabolismo , Masculino , Neoplasias da Próstata/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sêmen/enzimologia , Transcriptoma
13.
Oncol Rep ; 40(3): 1459-1466, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30015976

RESUMO

Colorectal cancer is one of the most common types of malignancies worldwide, and as it is often diagnosed at an advanced stage, it is a serious threat to human health. MicroRNAs are important regulators of the growth and metastasis of colon cancer (CC). In the present study, the results demonstrated that kallikrein­related peptidase 6 (KLK6) plays a critical role in suppressing colon carcinoma progression. To further investigate whether microRNAs affect the impact of KLK6, a bioinformatics approach was employed, which indicated that let­7i­5p may directly target KLK6. Furthermore, the expression level of let­7i­5p was significantly negatively correlated with the expression of KLK6 at the mRNA and protein levels in CC. Functionally, overexpression of let­7i­5p inhibited the proliferation and invasion of CC cells, and suppressed the growth of CC in vitro. The luciferase reporter assays revealed that let­7i­5p targeted the KLK6 3'­untranslated region. Collectively, these results indicated that let­7i­5p inhibited the proliferation and metastasis of CC cells by targeting KLK6, thereby blocking the cell cycle and promoting apoptosis in colon cells. Therefore, the present study revealed that the let­7i­5p/KLK6 axis may be a potential target for new therapeutic strategies to treat colon tumors.


Assuntos
Biomarcadores Tumorais/metabolismo , Movimento Celular , Proliferação de Células , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Calicreínas/metabolismo , MicroRNAs/genética , Adulto , Idoso , Animais , Apoptose , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Feminino , Seguimentos , Humanos , Calicreínas/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Molecules ; 23(6)2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29914143

RESUMO

Since cells in solid tumors divide less rapidly than cells in the bone marrow or cells of the immune system, mitotic inhibitors often cause severe side effects when used for treatment of diseases like prostate cancer and breast cancer. One approach to overcome this problem involves attempts at developing drugs based on general cytotoxins, like calicheamicin and thapsigargin, which kill cells at all phases of the cell cycle. However, such toxins can only be used when efficient targeting to the malignant tissue is possible. In the case of thapsigargin, selectivity for tumor-associated cells is achieved by conjugating the drug to a peptide that is only cleaved in the vicinity of tumors to release the cytotoxic drug or an analog with retained activity. Solid-phase synthesis protocols were developed for preparation of three already validated prodrugs of thapsigargin: one prodrug cleavable by human kallikrein 2, one prodrug cleavable by prostate-specific antigen, and one prodrug cleavable by prostate-specific membrane antigen.


Assuntos
Antineoplásicos/síntese química , Pró-Fármacos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Tapsigargina/síntese química , Antígenos de Superfície/metabolismo , Antineoplásicos/química , Sistemas de Liberação de Medicamentos , Glutamato Carboxipeptidase II/metabolismo , Humanos , Calicreínas/metabolismo , Estrutura Molecular , Peptídeos/química , Pró-Fármacos/química , Antígeno Prostático Específico/metabolismo , Tapsigargina/química
15.
Clin Biochem ; 58: 108-115, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29928903

RESUMO

OBJECTIVE: Human tissue kallikrein 15 (KLK15) is the last cloned member of the KLK-related gene family. Despite being implicated in multiple cancers, its pathophysiological role remains unknown. We aimed to biochemically characterize KLK15 and preliminarily study its role in cancer. DESIGN & METHODS: Recombinant KLK15 protein was produced, purified to homogeneity and quantified by mass spectrometry (parallel reaction monitoring analysis). We profiled the enzymatic activity of KLK15 using fluorogenic peptide substrates, and performed kinetic analysis to discover the cleavage sites. As KLK15 has mainly been associated with prostate cancer, we used a degradomic approach and subsequent KEGG pathway analysis to identify a number of putative protein substrates in the KLK15-treated prostate cancer cell line PC3. RESULTS: We discovered trypsin-like activity in KLK15, finding that it cleaves preferentially after arginine (R). The enzymatic activity of KLK15 was regulated by different factors such as pH, cations and serine protease inhibitors. Notably, we revealed that KLK15 most likely interacts with the extracellular matrix (ECM) receptor group. CONCLUSION: To our knowledge, this is the first study that experimentally verifies the trypsin-like activity of KLK15. We show here for the first time that KLK15 may be able to cleave many ECM components, similar to several members of the KLK family. Thus the protease could potentially be linked to tumorigenesis by promoting metastasis via this mechanism.


Assuntos
Matriz Extracelular/enzimologia , Calicreínas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/enzimologia , Linhagem Celular , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Calicreínas/química , Calicreínas/genética , Masculino , Metástase Neoplásica , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
Bioorg Med Chem ; 26(12): 3639-3653, 2018 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-29884582

RESUMO

A series of 1,3,6-trisubstituted 1,4-diazepan-7-ones were prepared as kallikrein 7 (KLK7, stratum corneum chymotryptic enzyme) inhibitors. Previously reported compounds 1-3 were potent human KLK7 inhibitors; however, they did not exhibit inhibitory activity against mouse KLK7. Comparison of the human and mouse KLK7 structures reveals the cause of this species differences; therefore, compounds that could inhibit both KLK7s were designed, synthesized, and evaluated. Through this structure-based drug design, compound 22g was identified as an inhibitor against human and mouse KLK7, and only one of the enantiomers, (-)-22g, exhibited potent inhibitory activity. Furthermore, the crystal structure of mouse KLK7 complexed with 22g enabled the elucidation of structure-activity relationships and justified 22g as a valuable compound to overcome the species differences.


Assuntos
Azepinas/química , Calicreínas/metabolismo , Inibidores de Proteases/síntese química , Sequência de Aminoácidos , Animais , Azepinas/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Desenho de Drogas , Humanos , Calicreínas/antagonistas & inibidores , Camundongos , Inibidores de Proteases/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade da Espécie , Estereoisomerismo , Relação Estrutura-Atividade
17.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 34(3): 230-236, 2018 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-29773104

RESUMO

Objective To study the effect of muscle segment homeodomain homeobox 2 (MSX2) on the expression of enamel matrix protein and the formation of enamel. Methods Immunohistochemical staining was used to detect the expression of MSX2 in mouse tooth embryos and its localization in ameloblasts. The short hairpin RNA (shRNA) of the MSX2 gene was designed and synthesized, and then the annealed double stranded DNA was constructed into the pGMLV-SC5 RNAi lentivirus vector, and finally it was packaged with lentivirus. The lentivirus was used to infect ameloblasts. Real-time fluorescent quantitative PCR was performed to screen the best interference fragment, and detect the mRNAs of amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam), amelotin (Amtn) and kallikrein 4 (Klk4). The embryos were isolated for 18.5 days and then infected with RNAi recombinant lentivirus targeting MSX2. The tooth germ was implanted under the renal capsule of the mouse. Ten weeks later, the tissue was harvested to separate and observe the tooth form and contour. Results MSX2 was expressed in the secretory phase and maturation phase of mouse ameloblasts, but the expression signal was weaker in the secretory phase and was stronger in the mature stage. The lentivirus of MSX2-shRNA targeting MSX2 gene we constructed inhibited the expression of Amelx and Klk4 mRNAs. The RNAi lentivirus targeting MSX2 gene infected the tooth enamel and led to a decrease in the degree of enamel mineralization. Conclusion The MSX2 gene is expressed in ameloblasts. The knockdown of MSX2 can inhibit the expression of enamel matrix proteins and the enamel mineralization.


Assuntos
Amelogênese , Amelogenina/genética , Proteínas do Esmalte Dentário/genética , Esmalte Dentário/metabolismo , Proteínas de Homeodomínio/genética , Calicreínas/genética , Ameloblastos/metabolismo , Amelogenina/metabolismo , Animais , Esmalte Dentário/embriologia , Proteínas do Esmalte Dentário/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Calicreínas/metabolismo , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Dente/embriologia , Dente/metabolismo
18.
Biochem Biophys Res Commun ; 499(3): 584-593, 2018 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-29621546

RESUMO

Pancreatic Ductal Adenocarcinoma (PADC) metastasis is the leading cause of morality of this severe malignant tumor. Proteases are key players in the degradation of extracellular matrix which promotes the cascade of tumor metastasis. As a kind of serine proteases, the kallikrein family performs vital function on the cancer proteolysis scene, which have been proved in diverse malignant tumors. However, the specific member of kallikrein family and its function in PDAC remain unexplored. In this study, by data mining of GEO datasets, we have identified KLK10 is upregulated gene in PDAC. We found that KLK10 was significantly overexpressed in tissues of pancreatic intraepithelial neoplasia (PanIN) and PDAC from Pdx1-Cre; LSL-KrasG12D/+ mice (KC) and Pdx1-Cre; LSL-KrasG12D/+; LSL-Trp53R172H/+ mice (KPC) by immunohistochemical analysis. Moreover, KLK10 is extremely elevated in the PDAC tissues, especially that from the PDAC patients with lymphatic and distant metastasis. Aberrant KLK10 expression is significantly correlated with poor prognosis and shorter survival by univariable and multivariable analysis. Functionally, knockdown of KLK10 observably inhibits invasion and metastatic phenotype of PDAC cells in vitro and metastasis in vivo. In addition, blockade of KLK10 attenuates epithelial-mesenchymal transition and activation of FAK-SRC-ERK signaling, which explains the mechanism of KLK10 in promoting metastasis. Collectively, KLK10 should be considered as a promising biomarker for diagnosis and potential target for therapy in PDAC.


Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Transição Epitelial-Mesenquimal/genética , Calicreínas/genética , Neoplasias Pancreáticas/genética , Regulação para Cima/genética , Adenocarcinoma/patologia , Animais , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Progressão da Doença , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Calicreínas/metabolismo , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias Pancreáticas/patologia , Fenótipo , Prognóstico , Transdução de Sinais , Quinases da Família src/metabolismo
19.
Biochim Biophys Acta Proteins Proteom ; 1866(5-6): 602-607, 2018 May - Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29563071

RESUMO

PURPOSE: Cancer cells consume more glucose than normal human cells and convert most glucose into lactate. It has been proposed that deregulated glycolysis is triggered by the posttranslational modification of 85 kDa muscle-type 6-phosphofructo-1-kinase (PFK-M) which is cleaved by a specific protease to form shorter, highly active, feedback-inhibition-resistant PFK-M fragments. PRINCIPAL RESULTS: To find the protease involved in PFK-M modification, analyses of the protease target sites on the human PFK-M enzyme yielding 45-47 kDa fragments were performed in silico. The results suggested that an enzyme in the kallikrein (KLK) family may be involved. Kallikreins can be self-activated in the cytosol and are often overexpressed in cancer cells. After incubating the internally quenched FRET peptide with a sequence characteristic of the target site, along with the active KLK6, the cleavage of the peptide was observed. The ability of KLK6 to cleave native PFK-M and form highly active citrate-resistant 45 kDa fragments was further confirmed by enzymatic tests and SDS-PAGE. A role of KLK6 in the posttranslational modification of native PFK-M was ultimately confirmed in vivo. A yeast strain that encoded native human PFK-M as the only PFK1 enzyme was additionally transformed with proKLK6 or KLK6 genes under the control of an inducible promoter. The transformants growth rate was found to increase after the induction of proKLK6 gene expression as compared to the strain with the native PFK-M enzyme. CONCLUSION: KLK6 may be the key protease involved in the modification of PFK-M and trigger deregulated glycolytic flux in cancer cells.


Assuntos
Calicreínas/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfofrutoquinase-1 Muscular/metabolismo , Processamento de Proteína Pós-Traducional , Eletroforese em Gel de Poliacrilamida , Transferência Ressonante de Energia de Fluorescência , Humanos , Calicreínas/genética , Cinética , Fragmentos de Peptídeos/genética , Fosfofrutoquinase-1 Muscular/genética , Proteólise , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Especificidade por Substrato
20.
Br J Radiol ; 91(1091): 20170893, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29565650

RESUMO

Theranostic nuclear oncology is on the cusp of adoption into routine clinical management of neuroendocrine tumours (NETs) following publication of the Phase 3 randomised controlled trial, NETTER-1. For the first time, level 1b evidence of efficacy and safety of 68-gallium/177-lutetium-DOTA-octreotate peptide receptor radionuclide therapy, of mid-gut neuroendocrine tumours was established. Multicentre Phase 2 studies of 68-gallium/177-lutetium-prostate specific membrane antigen theranostic approaches to management of end-stage metastatic castrate-resistant prostate cancer, are also very encouraging. However, the retrospective uncontrolled data currently available are inadmissible for formal regulatory agency evaluation. The challenge is to engage with oncologists and urologists, and to collaborate with the pharmaceutical industry, to design and perform the controlled clinical trials required for regulatory approval, and eventual reimbursement for theranostic nuclear oncology procedures. Strategies to facilitate timely establishment of an evidence base are considered in this review of theranostic advances over the past year. The prime objective is the provision of novel, effective, safe, personalised, tumour-targeted molecular theranostic management of metastatic castrate-resistant prostate cancer, and other cancers, such as non-Hodgkin lymphoma, which express the appropriate molecular receptor tumour targets. It would also be desirable to offer theranostic treatments at an earlier stage of malignant disease when the benefit is likely to be greater. The ultimate goal of theranostic nuclear oncology is to prolong survival and to improve quality of life for cancer patients worldwide. This may only be achieved through close collaboration between oncologists, nuclear physicians, radiologists, dosimetric physicists, Pharma, and, above all, with the patients themselves, in ways which are explored in this review.


Assuntos
Terapia de Alvo Molecular/tendências , Nanomedicina Teranóstica/tendências , Ensaios Clínicos como Assunto , Dipeptídeos/uso terapêutico , Previsões , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Humanos , Calicreínas/metabolismo , Masculino , Tumores Neuroendócrinos/terapia , Peptídeos Cíclicos/uso terapêutico , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/terapia , Traçadores Radioativos , Radiometria , Compostos Radiofarmacêuticos/uso terapêutico , Dosagem Radioterapêutica
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