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  • br CaSki pS siRNA CD

    2020-08-30

    
    CaSki-pS/siRNA-CD73 and HeLa-pS/siRNA-CD73 ZD-1839 showed sig-nificantly decreased levels of CD73 protein (Fig. 2A and C) and mRNA (Fig. 2B and D). The ability of these cells to generate Ado by the hy-drolysis of AMP (Fig. 2E) was strongly reduced in relation to the wild-type (wt) cells.
    When culturing CaSki-pS/siRNA-CD73 and HeLa-pS/siRNA-CD73 cells in the presence of 1 mM AMP or Ado, only Ado induced TGF-β1
    ng g
    g
    ng
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    n
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    Fig. 5. Functional activity of TGF-β pro-
    CaSki (A) or HeLa (B) conditioned media that
    had been cultured with 1 mM Ado and in the
    presence or absence of neutralizing anti-TGF-β.
    Mv1Lu cells cultured in the presence of rh-
    used as positive and negative controls, respec-
    tively. The percentage of proliferation was de-
    termined. Data are representative of three in-
    dependent experiments, and the
    averages ± SEM are shown. Significant dif-
    M
    M M
    M
    M
    M
    M
    M
    µ
    µ
    µ
    µ
    µ
    µ
    m
    m
    production (Fig. 3), indicating that the induction of TGF-β1 in CeCa cells is dependent on Ado generated through the hydrolysis of AMP.
    The expression of A2AR and A2BR receptors was examined by RT-PCR (Supplementary Fig. 1) to analyze their participation in the in-duction of TGF-β1 production. CaSki and HeLa cells were cultured for 30 min in the presence of 10 μM ZM241385 or MRS1754, selective antagonists of A2AR and A2BR, respectively, and subsequently in the presence of 1 mM AMP or Ado. Interestingly, the ZM241385 and MRS1754 antagonists strongly reversed the production of TGF-β1 in-duced by the presence of AMP or Ado in the CaSki (Fig. 4A and B) and HeLa (Fig. 4D and E) cells. Similarly, the increase in TGF-β1 mRNA expression induced by Ado was reversed in CaSki (Fig. 4C) and HeLa (Fig. 4F) cells cultured in the presence of ZM241385 or MRS1754. These results suggested that extracellular Ado participates in the in-duction of TGF-β1 production in CeCa tumor cells through interaction with A2AR and A2BR.
    3.2. TGF-β1 produced by CeCa cells is required to maintain CD73 expression
    Several studies have shown that TGF-β regulates CD73 expression in different cells of the immune system [13,14]. Because Ado induced the secretion and expression of TGF-β1 in CeCa cells, the ability of this factor produced by CaSki and HeLa cells to regulate CD73 expression was investigated. Initially, the functional activity of TGF-β1 produced by CeCa cells cultured in the presence of Ado was determined using the TGF-β-sensitive Mv1Lu cell line [38]. Supernatants from CaSki (Fig. 5A) and HeLa (Fig. 5B) cells cultured for 96 h in the presence of 1 mM Ado inhibited the proliferation of Mv1Lu cells by more than 30%, 
    which was comparable to the addition of 12.5 ng/ml rh-TGF-β1 (Fig. 5C). In contrast, the addition of anti-TGF-β1-neutralizing anti-bodies significantly reversed the inhibitory effect of these supernatants. It is important to mention that the presence of synthetic Ado at con-centrations < 1 mM inhibited the proliferation of Mv1Lu cells by < 10% (Fig. 5D). To analyze the effect of TGF-β content in the su-pernatants from CeCa cells cultured in the presence of Ado on CD73 expression in the tumor cells, anti-TGF-β-neutralizing antibodies were added. The increase in CD73 expression (< 30% with respect to basal) observed in CaSki cells cultured in the presence of Ado was reversed by the addition of the anti-TGF-β-neutralizing antibody (Fig. 6A). In con-trast, HeLa cells treated with either rh-TGF-β1 or Ado showed no in-crease in CD73 expression (Fig. 6B). However, the addition of SB505124, selective inhibitor of TGF-β type I receptors, to the cultures of both CaSki (Fig. 7A) and HeLa (Fig. 7B) treated with Ado decreased the expression of CD73 by more than 40% relative to the basal ex-pression of CD73 in both cell lines. In addition, a significant increase in the TGF-β1 content was detected in the supernatants from cells cultured under these conditions (Fig. 7C) and (Fig. 7D). These results suggested that TGF-β autocrinously produced by CeCa cells through the CD73-adenosine pathway may be an indispensable factor in maintaining or inducing CD73 expression in these tumor cells.
    4. Discussion
    TGF-β is a pleiotropic cytokine that controls several biological functions, such as cell proliferation, apoptosis, stem cell maintenance, EMT and immune response suppression [39,40]. In addition, TGF-β plays an important role in the progression and tumor metastasis of
    Fig. 6. Anti-TGF-β1 reverses the increase in CD73 expression induced by Ado in CeCa cells. CaSki (A) and HeLa (B) cells (1 × 105) were cultured with 1 mM Ado in the presence or absence of anti-TGF-β. CaSki and HeLa cells cultured in the presence of 20 ng/ml TGF-β-hr were used as the positive control. After 96 h, CD73 expression on the membrane of cells was analyzed by flow cytometry. Data are representative of three independent experiments, and the averages ± SEM are shown. Significant differences in CD73 expression with respect to the basal expression are shown as *(p < 0.05) and **(p < 0.005).