br Introduction br Cervical cancer CeCa represents a

1. Introduction
Cervical cancer (CeCa) represents a major public health problem worldwide, as more than 500,000 new cases and more than 250,000 deaths are detected each year, of which over 80% occur in developing countries [1]. Infection with high-risk human papillomavirus (HR-HPV) is considered the main etiological agent of CeCa [2]. Although the immune response against HPV Dihexa eliminates most infections and precursor lesions [3], only a small number of women exposed to HR-HPV develop cancer, suggesting that other risk factors may be involved [4]. A growing number of studies have suggested that immunoregula-tion may play an important role in the carcinogenesis of CeCa. In this context, it has been reported that transforming growth factor-β (TGF-β) plays a crucial role in the development of CeCa. TGF-β generates a local
immunosuppressive microenvironment in the cervix infected by HR-HPV to inhibit the proliferation and activation of T cells with antitumor activity [5]. However, TGF-β also favors the persistence of HR-HPV infection and promotes tumor formation, epithelial–mesenchymal transition (EMT), CeCa tumor cell invasion and CeCa tumor cell me-tastasis [6]. TGF-β has been detected in serum and tissues of patients with HPV-AR infection, in low-grade squamous intraepithelial lesions (SILs), in high-grade SILs and mainly in patients with advanced CeCa. In addition, TGF-β levels correlate directly with the degree of lesion progression [7,8]. The presence of TGF-β in the tumor microenviron-ment (TME) stabilizes hypoxia-inducible factor 1α (HIF-1α) [9], which in turn induces the expression of CD39 ectonucleotidases (ectonucleo-side triphosphate diphosphohydrolase-1, ENTPD1; EC 3.6.1.5) and 5′-ectonucleotidase (CD73, EC 3.1.3.5), which hydrolyze ATP/ADP, AMP
Abbreviations: Ado, adenosine; Anti-TGF-β, anti-human neutralizing antibody against TGF-β1, -β2, and -β3; ARs, adenosine receptors; ATCC, American Type Culture Collection; CeCa, cervical cancer; CTLs, cytotoxicT lymphocytes; EMT, epithelial–mesenchymal transition; HIF-1α, hypoxia-inducible factor 1α; HR-HPV, high-risk human papillomavirus; Ino, inosine; MDSCs, myeloid-derived suppressor cells; MFI, mean fluorescence intensity; NK, natural killer cells; rh-TGF-β1, recombinant human TGF-β1; SILs, low-grade squamous intraepithelial lesions; T regs, regulatory T cells; TGF-β, transforming growth factor-β
Corresponding author.
E-mail address: lulumora@yahoo.com (M. de Lourdes Mora-García).
R. García-Rocha et al.
Fig. 1. CeCa cells cultured in the presence of AMP and Ado are induced to secrete TGF-β1. CaSki and HeLa cells (1 × 105) were cultured for 96 h in the presence of 1 μM, 10 μM, 100 μM and 1 mM AMP (A and C) or Ado (B and D). Samples of the supernatants were collected every 24 h to analyze TGF-β1 content by ELISA. Data are representative of three independent experiments, and the averages ± SEM are shown. * Indicates significant difference (p < 0.05) in the TGF-β1 content of the supernatants of the treated cells with respect to the supernatant of the untreated cells for each culture time.
and AMP in adenosine (Ado), respectively, and generate high con-centrations of Ado in the TME [10–12]. Moreover, TGF-β is a factor that induces the expression of CD39/CD73 in T lymphocytes and dendritic cells [13], and it contributes to the high expression of ectonucleotidases in myeloid-derived suppressor cells (MDSCs) and regulatory T cells (T regs) within the TME [14]. Most of the extracellular signaling activities performed by Ado are mediated by adenosine receptors (ARs; namely, A1, A2A, A2B and A3) coupled to G proteins and found in the mem-branes of target cells [15]. CD73 expression in tumor cells has been reported in the following different types of cancer: bladder cancer, leukemia, glioma, glioblastoma, melanoma, ovarian cancer, esophagus, thyroid, prostate, breast, colorectal and CeCa [16–29]. It has been es-timated that the concentration of Ado in the TME is 10–100 μM. Ado exerts the following effects in the host: suppresses CD8+ cytotoxic T lymphocytes (CTLs), natural killer cells (NK) cells, macrophages, B cells, neutrophils and dendritic cells through interaction with high af-finity A2A receptors [15,30]; and promotes tumor growth by inducing the proliferation, invasion and metastasis of tumor cells, mainly through interaction with A1R, A2AR and A2BR [31–33]. When inter-acting with A2AR and A2BR, Ado induces the production of TGF-β in several cell types to cause immunoregulatory and anti-inflammatory effects [34–36]. Taking into account that we recently reported that CeCa tumor cells are capable of generating large amounts of Ado from the hydrolysis of adenine nucleotides through the hydrolytic activity of CD73 [29], the ability of Ado, generated through the functional activity of the adenosinergic pathway in CeCa cells, to induce the expression and secretion of TGF-β1 as well as maintain CD73 expression in CeCa cells was analyzed in the present study.