Abstract A43: Investigation of dipeptidyl peptidase IV/CD26 role in cervical cancer cell lines.
Andréia BuffonAline BeckenkampDanielle Bertodo SantanaJéssica NascimentoJuliano D. PaccezLuiz F. ZerbiniMichaël WinkAlessandra Nejar Bruno
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Abstract Cervical cancer is a common type of cancer in women and an important public health problem, mainly in developing countries. The glycoprotein DPPIV/CD26 is expressed at the membrane of many cells. It is able to inactivate some bioactive peptides and chemokines, thereby regulating cell proliferation and survival, being involved in processes related to cancer. This enzyme is also present in a soluble form (DPPIV/sCD26), in biological fluids. In this study, we evaluated, the expression and enzymatic properties of the DPPIV/CD26 and its relation with tumoral mechanisms in cervical cancer cell lines. The cervical cancer cells exhibit DPPIV/CD26 enzymatic activity in both membrane bound and, its soluble form, which was confirmed by the inhibition of the enzymatic DPPIV/CD26 activity for sitagliptin phosphate. The CD26 mRNA expression was observed in the SiHa and C33A cells, while the HeLa cells showed no transcripts. The DPPIV/CD26 expression was correlated with the enzymatic activity in these cell lines. We also observed a higher migratory capacity of HeLa (cervical cancer cell line that less express CD26) compared to SiHa (cervical cancer cell line that more express CD26), moreover in the presence of the specific inhibitor we observed that SiHa cell line shows an increase in migration, thus confirming a relationship of this enzyme with the migratory ability. Our results show, that cervical cancer cells present DPPIV/CD26 enzymatic activity in both membrane bound form, as in its soluble form, revealing a differential expression as well as its relationship to cell migration and adhesion. Considering that DPPIV/CD26 plays an important role in tumor biology, this protein may become an important therapeutic target although additional work is required to elucidate the molecular mechanisms associated with DPPIV/CD26 in cervical cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A43. Citation Format: Andréia Buffon, Aline Beckenkamp, Danielle Bertodo Santana, Jéssica Nascimento, Juliano Paccez, Luiz Fernando Zerbini, Márcia Rosangela Wink, Alessandra Nejar Bruno. Investigation of dipeptidyl peptidase IV/CD26 role in cervical cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A43.Keywords:
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Dipeptidyl peptidase
Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) has been purified 18,000-fold in a yield of 2.2% from human serum. Serum DPPIV, a serine enzyme with an apparent mass of 250 kDa, consists of two identical subunits with an apparent mass of 100 kDa and is inhibited by DPPIV-specific inhibitor Diprotin A and also by p-chloromercuribenzoate (p-CMB), 2-mercaptoethanol, HgCl2, CdCl2, SrCl2, and ZnCl2. One of the remarkable properties of DPPIV is that its activity is greatly enhanced by Gly-X (X: especially, Gly, Gln, Glu and Ser) dipeptides. Gly-X dipeptides increase not only an apparent Km of serum DPPIV for glycyl-L-proline 3,5-dibromo-4-hydroxyanilide nearly 10-fold, but also an apparent kcat nearly 4-fold. This mechanism is unclear, but one possibility is that Gly-Pro from substrate might bind amino acids or dipeptides instead of water molecules as DPPIV transpeptidyl activity reported previously. Another remarkable property of DPPIV is the ability to bind adenosine deaminase-I and -II, as is the case with recombinant soluble CD26 (rsCD26). This probably indicates that DPPIV purified from human serum by our method originates from T-lymphocytes. © 1996 Wiley-Liss, Inc.
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目的 : 甲状腺腫瘍に対する診断的マーカーの検索を研究目的とする. その候補の一つとして CD26/DPPIV を多施設, 多年度のデータから評価する.方法 : 1991∼2011 年の 21 年間, 研究関連病院において組織診断が確定した延べ 1901 例を対象とした. 方法は, 酵素活性染色, 酵素活性定量, 免疫組織化学染色, ノーザンブロットおよび RT-PCR による mRNA 解析である.成績 : CD26/DPPIV は, 正常および良性病変の甲状腺細胞においては発現量が低いが, 乳頭癌や濾胞癌などの高分化癌ではほとんどの症例で高発現していた. 濾胞腺腫の中に高発現する症例が存在したが, その出現頻度は低率であった.結論 : 多施設における共同研究から, CD26/DPPIV は甲状腺腫瘍マーカーとして有用であることが確証できた. CD26/DPPIV 活性染色は, 迅速性・簡便性などの点において, 他の診断的マーカーにはない特質を有している.
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Colorimetrical rate assay for urinary dipeptidyl peptidase iv (dppiv) activity using a new substrate
We synthesized a new substrate glycyl-L-proline 3,5-dibromo-4-hydroxyanilide (Gly-Pro-DBAP), for dipeptidyl peptidase IV (DPPIV). Its hydrolysis by DPPIV resulted in the formation of a chromophore, 2,6-dibromophenol-indo-p-xylenol, and its maximal absorption wavelength (600 nm) was longer than that of p-nitroaniline (415 nm) released from conventional substrate, glycyl-L-proline p-nitroanilide (Gly-Pro-pNA). We also established the rate assay for urinary DPPIV activity using Gly-Pro-DBAP. The optimum pH was between 8.5 and 9.0. The apparent Km was 1.1 mmol/1. The detectable range was 2.5-350 U/l. No changes in blank values occurred throughout the enzyme reaction in the optimum pH. Its value was also much lower than Gly-Pro-pNA. CVs for within-run and between-run were 1.1% (n = 10) and 3.0% (n = 10), respectively. Among tested peptidases, only DPPIV could hydrolyze Gly-Pro-DBAP. Among the protease inhibitors, only two, diprotin-A and phenylmethylsulfonyl fluoride (PMSA), could inhibit DPPIV activity. The present method did not interfere with urinary ingredients such as hemoglobin. The correlation between the present (y) and conventional (x) methods is presented by the equation y = 1.121x + 0.096 (r = 0.993). Thus the present method provides practical advantages over the conventional method for routine laboratory use.
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Cervical cancer is a serious threat to women’s health. In recent years, the incidence rate has increased year by year, and the incidence of cervical cancer tends to be younger. Because of its characteristics of easy recurrence and easy metastasis, the drug therapy of cervical cancer has attracted increasing attention. Coixen ester has anti-cancer and immunomodulatory effects. After years of clinical research, it has been shown to be effective against a variety of cancers and has a growth inhibitory effect on tumor cells. This article aims to study the mechanism of Coixen ester-induced apoptosis of human cervical cancer HeLa cells. This article puts forward what are the causes of cervical cancer, and analyzes the positive effects of traditional Chinese medicine in the prevention and treatment of cervical cancer. During the experiment, the MTT method was used to study the effect of the complex on the proliferation of HeLa cells. The experimental results in this article show that a certain concentration of Coixen ester solution can inhibit cell proliferation and can also reduce cell viability to about 0.35.
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Abstract Dipeptidyl peptidase IV (DPPIV)/CD26 is by far the most extensively studied member of the prolyl oligopeptidase family of serine proteases. The discovery of the related enzymes DPP8 and DPP9 necessitates a (re-)evaluation of the DPPIV-like enzymatic activity in cells and organs. In this study, we aimed (1) to investigate the expression of the individual dipeptidyl peptidases in different types of endothelial cells (ECs) and (2) to reconsider published data in relation to our findings. Examination of DPP expression in rat primary ECs of aortic, endocardial and cardiac microvascular origin revealed the presence of DPPIV-like activity in all cell lysates. More than half of this activity could be attributed to DPP8/9. Western blot analysis revealed an abundance of the DPP8 protein as compared to DPP9. The expression of DPPIV and DPP8 was significantly higher in the cardiac microvascular endothelium than in the other ECs, suggesting a more pronounced role of these DPPs in the microvasculature. In situ , DPP activity in ventricular microvasculature was completely inhibited by sitagliptin, indicating that DPPIV is the predominant DPPIV-like enzyme in this organ. By contrast, immunohistochemical studies indicated DPP9 as the predominant DPP in human carotid artery ECs. In conclusion, our results support a highly regulated expression of individual DPPs in ECs, with a spatial heterogeneity in the cardiovascular tree.
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Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a serine type protease with an important modulatory activity on a number of chemokines, neuropeptides and peptide hormones. It is also known as CD26 or adenosine deaminase (ADA; EC 3.5.4.4) binding protein. DPPIV has been demonstrated on the plasmamembranes of T cells and activated natural killer or B cells as well as on a number of endothelial and differentiated epithelial cells. A soluble form of CD26/DPPIV has been described in serum. Over the past few years, several related enzymes with similar dipeptidyl peptidase activity have been discovered, raising questions on the molecular origin(s) of serum dipeptidyl peptidase activity. Among them attractin, the human orthologue of the mouse mahogany protein, was postulated to be responsible for the majority of the DPPIV‐like activity in serum. Using ADA‐affinity chromatography, it is shown here that 95% of the serum dipeptidyl peptidase activity is associated with a protein with ADA‐binding properties. The natural protein was purified in milligram quantities, allowing molecular characterization (N‐terminal sequence, glycosylation type, CD‐spectrum, pH and thermal stability) and comparison with CD26/DPPIV from other sources. The purified serum enzyme was confirmed as CD26.
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[Objective] The aim was to discuss the effect of composite PSP on the proliferation of HeLa cells of human cervical cancer.[Method] PSP and GBE were compounded according to different doses and 3 proportions of 1∶1,1∶2 and 2∶1,and the inhibitory effect of the composite PSP on HeLa cells of human cervical cancer was studied by MTT method.[Result] The inhibitory effects of using PSP and GBE compoundly according to 3 proportions on HeLa cells of human cervical cancer were all obviously better than that of using single component at corresponding doses and the tumor inhibition rate of group at the same proportion showed positive correlation with dose.The combined use of PSP and GBE could produce synergy in inhibiting HeLa cells of human cervical cancer.Among them,the effect of PSP and GBE with the proportion at 2∶1 was most prominent and the tumor inhibition rates at high,middle and low doses were 67.92%,58.93% and 34.86%(P0.01),resp.[Conclusion] The study laid a foundation for curing human cervical cancer.
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