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Ectonucleotidase expression profile and activity in human cervical cancer cell lines

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Cervical cancer is the third most frequent cancer in women worldwide. Adenine nucleotide signaling is modulated by the ectonucleotidases that act in sequence, forming an enzymatic cascade. Considering the relationship between the purinergic signaling and cancer, we studied the E-NTPDases, ecto-5′-nucleotidase, and E-NPPs in human cervical cancer cell lines and keratinocytes. We evaluated the expression profiles of these enzymes using RT-PCR and quantitative real-time PCR analysis. The activities of these enzymes were examined using ATP, ADP, AMP, and p-nitrophenyl-5′-thymidine monophosphate (p-Nph-5′-TMP) as substrate, in a colorimetric assay. The extracellular adenine nucleotide hydrolysis was estimated by HPLC analysis. The hydrolysis of all substrates exhibited a linear pattern and these activities were cation-dependent. An interesting difference in the degradation rate was observed between cervical cancer cell lines SiHa, HeLa, and C33A and normal imortalized keratinocytes, HaCaT cells. The mRNA of ecto-5′-nucleotidase, E-NTPDases 5 and 6 were detectable in all cell lines, and the dominant gene expressed was the Entpd 5 enzyme, in SiHa cell line (HPV16 positive). In accordance with this result, a higher hydrolysis activity for UDP and GDP nucleotides was observed in the supernatant of the SiHa cells. Both normal and cancer cells presented activity and mRNAs of members of the NPP family. Considering that these enzymes exert an important catalytic activity, controlling purinergic nucleotide concentrations in tumors, the presence of ectonucleotidases in cervical cancer cells can be important to regulate the levels of extracellular adenine nucleotides, limiting their effects.
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Keywords: NTPDase5; biomarkers; biomarqueurs; cancer du col utérin; cervical cancer; ectonucleotidases; ectonucléotidases; purinergic signaling; signalisation purinergique

Document Type: Research Article

Affiliations: 1: LABC – Laboratory of Biochemical and Cytological Analysis, Analysis Department, Faculty of Pharmacy, Federal University of Rio Grande do Sul, Av. Ipiranga 2752, bairro Santana, CEP 90610-000, Porto Alegre, RS, Brazil. 2: Federal Institute of Education, Science and Technology of Rio Grande do Sul, Porto Alegre, RS, Brazil. 3: Department of Biochemistry, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. 4: International Centre for Genetic Engineering and Biotechnology (ICGEB), Cancer Genomics Group, Cape Town, South Africa. 5: Laboratório de Sinalização Celular, Departamento de Biofísica, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. 6: Laboratório de Biologia Celular, Department of Basic Health Sciences, Federal University of Health Sciences of Porto Alegre, RS, Brazil.

Publication date: January 1, 2014

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