RESUMO
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.
Assuntos
Carcinoma/metabolismo , Carcinoma/patologia , Movimento Celular/fisiologia , Dipeptidil Peptidase 4/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Inibidores da Dipeptidil Peptidase IV/farmacologia , Feminino , Células HeLa , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosfato de Sitagliptina/farmacologiaRESUMO
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.
Assuntos
Monofosfato de Adenosina/biossíntese , Trifosfato de Adenosina/farmacologia , Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Neoplasias do Colo do Útero/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dipiridamol/farmacologia , Feminino , Células HeLa , Humanos , Proteínas de Transporte de Nucleosídeos/antagonistas & inibidores , Poli(ADP-Ribose) Polimerases/metabolismo , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Microambiente Tumoral , Proteína Supressora de Tumor p53/biossínteseRESUMO
Nucleotide excision repair (NER) is one of the most versatile DNA repair mechanisms, ensuring the proper functioning and trustworthy transmission of genetic information in all living cells. The phenotypic consequences caused by NER defects in humans are autosomal recessive diseases such as xeroderma pigmentosum (XP). This syndrome is the most sun-sensitive disorder leading to a high frequency of skin cancer. The majority of patients with XP carry mutations in the XPA or XPC genes that encode proteins involved in recognition of DNA damage induced by UV light at the beginning of the NER process. Cells cultured from XPA and XPC patients are hypersensitive to UV light, as a result of malfunctioning DNA repair. So far there is no effective long-term treatment for these patients. Skin cancer prevention can only be achieved by strict avoidance of sunlight exposure or by the use of sunscreen agents. We have constructed recombinant adenoviruses carrying the XPA and XPC genes that were used to infect XP-A and XP-C immortalized and primary fibroblast cell lines. UV survival curves and unscheduled DNA synthesis confirmed complete phenotypic reversion in XP DNA repair deficient cells with no trace of cytotoxicity. Moreover, transgene expression is stable for at least 60 days after infection. This efficient adenovirus gene delivery approach may be an important tool to better understand XP deficiency and the causes of DNA damage induced skin cancer.