RESUMEN
Dipeptidyl peptidase IV (DPPIV/CD26) is a transmembrane glycoprotein that inactivates or degrades some bioactive peptides and chemokines. For this reason, it regulates cell proliferation, migration and adhesion, showing its role in cancer processes. This enzyme is found mainly anchored onto the cell membrane, although it also has a soluble form, an enzymatically active isoform. In the present study, we investigated DPPIV/CD26 activity and expression in cervical cancer cell lines (SiHa, HeLa and C33A) and non-tumorigenic HaCaT cells. The effect of the DPPIV/CD26 inhibitor (sitagliptin phosphate) on cell migration and adhesion was also evaluated. Cervical cancer cells and keratinocytes exhibited DPPIV/CD26 enzymatic activity both membrane-bound and in soluble form. DPPIV/CD26 expression was observed in HaCaT, SiHa and C33A, while in HeLa cells it was almost undetectable. We observed higher migratory capacity of HeLa, when compared to SiHa. But in the presence of sitagliptin SiHa showed an increase in migration, indicating that, at least in part, cell migration is regulated by DPPIV/CD26 activity. Furthermore, in the presence of sitagliptin phosphate, SiHa and HeLa cells exhibited a significant reduction in adhesion. However this mechanism seems to be mediated independent of DPPIV/CD26. This study demonstrates, for the first time, the activity and expression of DPPIV/CD26 in cervical cancer cells and the effect of sitagliptin phosphate on cell migration and adhesion.
Asunto(s)
Carcinoma/metabolismo , Carcinoma/patología , Movimiento Celular/fisiología , Dipeptidil Peptidasa 4/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Femenino , Células HeLa , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosfato de Sitagliptina/farmacologíaRESUMEN
In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2 × 7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2 × 7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2 × 7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling-p53 increase, AMPK activation, and PARP cleavage-as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.
Asunto(s)
Adenosina Monofosfato/biosíntesis , Adenosina Trifosfato/farmacología , Adenosina/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Dipiridamol/farmacología , Femenino , Células HeLa , Humanos , Proteínas de Transporte de Nucleósidos/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasas/metabolismo , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Interferente Pequeño , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Microambiente Tumoral , Proteína p53 Supresora de Tumor/biosíntesisRESUMEN
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.