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. 2020 Jan 1;11(2):421-431.
doi: 10.7150/jca.31245. eCollection 2020.

Blockade of Adenosine A2b Receptor Reduces Tumor Growth and Migration in Renal Cell Carcinoma

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Free PMC article

Blockade of Adenosine A2b Receptor Reduces Tumor Growth and Migration in Renal Cell Carcinoma

Ye Yi et al. J Cancer. .
Free PMC article

Abstract

Adenosine A2b receptor (A2bR) is a member of the G protein-coupled receptor superfamily members, which has been considered involved in the pathogenesis of various cancers. However, little is known about the role of A2bR renal cell carcinoma (RCC). The A2bR expression levels in RCC 769-P and Caki-1 cell lines compared with HK-2 were analyzed by qRT-PCR. 769-P and Caki-1 cells were transfected with shRNA-A2bR to knock down the expression of A2bR. Cell proliferation was detected by MTT assays and colony formation assays. Wounding healing assays and transwell assays were used to evaluate the effects of A2bR on cell capacity of invasion and migration. Finally, potential mechanisms involved in A2bR blockade's effects on altered tumor behaviors were evaluated by western blotting. We showed that A2bR were significantly up-regulated in RCC cells compared to HK-2 cell. Functionally, MRS1754, a selective A2bR antagonist, and knocking-down the expression of A2bR by shRNA reduced proliferation and migration in vitro and tumor growth in vivo. Furthermore, we demonstrated that A2bR blockade inhibited tumor progression in part via the MAPK/JNK pathway. Conclusion: Our findings suggest the A2bR potentially plays an important role in RCC progression and A2bR blockade may be a promising candidate for therapeutic intervention for renal cell carcinoma.

Keywords: Adenosine A2b Receptor; MAPK/JNK signal pathway; MRS1754; Renal cell carcinoma..

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
A2bR was relatively high expressed in renal cell carcinoma cell lines. (A) qRT-PCR showed that A2bR in 769-P and Caki-1 cell lines is over-expressed than HK-2 cells. (B) The representative images of shRNA-A2bR transfected cells. (C and E) qRT-PCR results showed that MRS1754 had no significant effects on expression of A2bR of cell lines. qRT-PCR (D and E) results showed that transfection of shRNA-A2bR knocked down the mRNA and protein expression of adenosine A2b receptor. ***p<0.001, **p<0.01, *p<0.05.
Figure 2
Figure 2
A2bR blockade inhibited proliferation of RCC 769-P and Caki-1 cell lines. MRS1754 inhibited proliferation of 769-P and Caki-1 cell lines evaluated by MTT assays (A) and colony formation assays (C), which could be rescued by Bay60-6583 significantly. Down-regulation of A2bR expression inhibited cell proliferation of RCC cells as detected by MTT (B) and colony formation assays (C). ***p<0.001, **p<0.01, *p<0.05.
Figure 3
Figure 3
A2bR blockade inhibited cell migration and invasion of RCC cells. (A) MRS1754 inhibited RCC cells migrating into the scratching area in a dose-dependent manner measured by wound healing assays. (B) Suppression of A2bR inhibited 769-P and Caki-1 cells migrating into the scratching area. (C) Transwell assays showed that A2bR blockade reduced 769-P and Caki-1 cells invasion ability. ***p<0.001, **p<0.01, *p<0.05.
Figure 4
Figure 4
Bay60-6583 rescued the inhibitory effects of MRS1754 on cell growth and migration. (A)Colony formation assay showed Bay60-6583 rescued the MRS1754-induced reduction in cell growth. (B)Wound healing assay showed Bay60-6583 (10 nM) reversed the inhibitory effects of MRS1754 on cell migration capability. ***p<0.001, **p<0.01, *p<0.05.
Figure 5
Figure 5
A2bR blockade inhibited cell growth of 769-P and Caki-1 cell lines in vivo. Nude mice were sacrificed at the 40th days after subcutaneous injection (n=5 for each group) and tumors were removed. The volume of tumors were measured as width2×length/2.
Figure 6
Figure 6
A2bR blockade decreased expression of MAPK/JNK pathway proteins in 769-P and Caki-1 cell lines. Down-regulation of adenosine A2b receptor inhibited the phosphorylation level of MAPK/JNK protein. While MRS1754 inhibited the phosphorylation level of MAPK/JNK protein in a dose-dependent manner.
Figure 7
Figure 7
Bay60-6583 could rescue the decreased expression of MAPK/JNK pathway protein in 769-P and Caki-1 cell lines. MRS1754 inhibited the phosphorylation level of MAPK/JNK protein, which could be rescued by Bay60-6583(10nM), an A2bR selective agonist.

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