Cervical cancer cells respond to high extracellular ATP. There is cooperation between ATP and its metabolites with regard to cytotoxicity, with adenosine necessary, but not sufficient, to induce cell death in the whole population of cells, which is significant in the context of cancer therapeutics.
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.
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.
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.
Cervical cancer is the third most commonly diagnosed malignancy and the fourth leading cause of cancer death in females worldwide. The high mortality rate is largely due to the lack of effective therapies for eliminating disease in women with high-grade cervical cancer and the lack of response to chemotherapy of inoperable disease. Thus, to understand the mechanisms involved on cervical cancer development and find a molecular target to prevent it is strongly desirable. Until now, researchers show that HPV infection and suppression of cell death mechanisms like apoptosis are the most important factors involving of cervical carcinogenesis. Anyway, the mechanism that leads cells infected by HPV escape to apoptosis remains unknown. A role for purinergic signaling on control of cell growth and death, mainly through P2X7 receptor activation by extracellular ATP, has been described. We hypothesized that a disruption in this signaling could be involved with cervical cancer resistance to apoptosis and development. Previous work, using human carcinoma cell line SiHa, showed that ATP 5mM induces cell death through apoptosis by P2X7 activation. However, the mechanism involved in this cytotoxic effect remained unclear. To answers this question, we investigate the role of P2X7 on ATP induce cell death. We started analyzing P2X7 protein expression in cells resistant to ATP treatment. In this case, SiHa cells were treated with ATP 5mM for 24, 48, and 72h, and the adherent survival cells were removed and analyzed by Western Blot. After, we knockdown P2X7 receptor in SiHa cell line and evaluate this effect on cell viability after ATP treatment. In addition, we studied the mechanistic way involved in this cell death pathway using EGTA and caspase-3 inhibitor previous to ATP treatment. Unexpectedly, SiHa wild type (WT) cells that remained adherent after treatment with ATP showed less expression of P2X7, suggesting a defense way to apoptosis. Corroborating with this, SiHa cells knockdown (KD) for P2X7 showed increased cell viability when compared to SiHa WT and SiHa KD control, after exposure to ATP 5mM for 24, 48, and 72h. The mechanism of ATP induces apoptosis through P2X7 don't seems to be by increase intracellular calcium and caspase-3 activation, but in an independent caspase way. These findings suggest that P2X7 receptor could be involved with death and resistance cell mechanisms, indicating a possible new target on therapeutical research in cervical cancer and on tumor aggressiveness. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B208. Citation Format: Paola de Andrade Mello, Eduardo Cremonese Filippi-Chiela, Jessica Nascimento, Franciele Cristina Kipper, Aline Beckenkamp, Danielle Bertodo Santana, Alessandra Nejar Bruno, Guido Lenz, Andréia Buffon. Reduction in P2X7 receptor expression is a marker of resistance to ATP treatment in human cervical carcinoma cell line. [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 B208.
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