Overexpression of GPR35 confers drug resistance in NSCLC cells by β-arrestin/Akt signaling

Figures

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  Figure 1. GRP35 is overexpressed in NSCLC tissues and associated with poor prognosis. Notes: The expression of GRP35 according to public Gene Expression Omnibus database in Okayama’s cohort (A), Landi’s cohort (B), Selamat’s cohort (C), and Hou’s cohort (D). (E) The expression of GPR35 in NSCLC tissue microarray by IHC. Magnification, 200×. (F) The semiquantitative score of GPR35 staining in normal and tumor tissues. Kaplan–Meier analysis of the GPR35 expression in NSCLC overall survival (G) and progression-free survival (H). Abbreviation: NSCLC, non-small-cell lung cancer.

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  Figure 2. GPR35 is critical for NSCLC cells chemoresistance. Notes: (A) The effect of Dox in A549 and A549R cells. IC50 was measured by treating with gradient concentration of Dox for 48 hours. (B) The colony formation assay of A549 and A549R cells with Dox treatment. (C) Indicated proteins were measured in A549 and A549R cells. β-actin was internal control. A549R cells with knocking down of GRP35 and control cells were treated with gradient concentration of Dox and Cis for 48 hours. The cell viability (D) and the IC50 values (E) were showed. (F) Knocking down of GPR35 increased cell apoptosis in A549R cells both with and without Dox or Cis treatment. (G) A549R cells with knocking down of GRP35 and control cells were treated with Dox and the apoptosis markers cleaved PARP and cleaved caspase 3 proteins were measured. *P<0.05, compared with con group; #P<0.05, compared with con+siRNA group; ##P<0.01, compared with con+siRNA group. Abbreviations: con, control; NSCLC, non-small-cell lung cancer; Dox, doxorubicin; Cis, cisplatin; NC, negative control.

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  Figure 3. GPR35 depletion restores the sensitivity of A549R cells to chemodrug in vivo. Notes: (A) A549R cells and A549R with GPR35 knocking down cell-derived xenografts were treated with Cis (5 mg/kg/week) or DMSO. The tumor is shown. (B) The tumor volume of each group during the treatment. (C) The tumor weight of each group. (D) The Ki67 staining of tumor sections from each group. Magnification: 200×. (E) The statistics of Ki67-positive cells in each group. *P<0.05, **P<0.01, ***P<0.001 compared with con group. Abbreviations: Cis, cisplatin; con, control; DMSO, dimethyl sulfoxide.

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  Figure 4. Regulation of GPR35 in NSCLC chemoresistant cells. Notes: (A) Knocking down of GPR35 suppressed β-arrestin-2 (indicated as β-arr) expression and Akt signal activation. (B) Knocking down of β-arrestin-2 (si-arr) suppressed GPR35 expression and Akt signal activation. (C) Akt signal inhibitor suppressed GPR35 and β-arrestin-2 expression. (D) Overexpression of GPR35 in A549 cells enhances the expression of β-arrestin-2 and activates Akt signaling. Knocking down of β-arrestin-2 (E) or using Akt signal inhibitor (F) in A549R cells increased the apoptosis when treated with Dox or Cis. *P<0.05, **P<0.01, ***P<0.001 compared with control+drugs group. Abbreviations: Dox, doxorubicin; Cis, cisplatin; NC, negative control.