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. 2020 Aug 15;11(1):161–179. doi: 10.1016/j.jcmgh.2020.08.004

Figure 8.

Figure 8

ProAgio decreases Cda in cancer cells by decreasing IGF1 signaling. (A) Representative images of IHC staining of pIGF1R (left panel; 4× and 10× enlarged images) and Cda (right panel, 4× and 10× enlarged images) in tumor sections of PDAC patients (n = 40). (B) Correlation of pIGF1R protein expression with Cda protein expression. Regression analysis for expression of the pIGF1R (IHC) vs Cda (IHC) in tumor sections of PDAC patients (n = 12). (C) The levels of Cda (IB:Cda) in KPC 961 cells that were treated with IGF-1 (0 ng/mL and 10 ng/mL) were analyzed by immunoblot. (D) Levels of IGF1 secreted by inactivated PaSC (without transforming growth factor [TGF]-β, grey bar) or activated PaSC (with 5 ng/mL TGF-β, black bar). (E) Levels of IGF1 secreted by the indicated cells. (F) Levels of IGF1 in the serum of a subcutaneous xenograft mouse model of Panc1 or Panc1 co-implanted with activated human PaSC treated with vehicle or ProAgio (n = 5). IGF1 levels were analyzed by enzyme-linked immunosorbent assay. (G and H) Representative images of IHC staining of IGF1 (G) and quantitation of IHC staining of IGF1 (H) (IGF1+ area [X], fold change by comparison with the mean value of the ProAgio group as 1) in tumor sections of GEM-KPC mice treated with the indicated agents. (I and J) Representative images of IHC staining of pIGF1R (I) and quantitation of pIGF1R-positive staining (J, pIGFR+ cells/HPF, pIGFR-positive cells per view field) in tumor sections of vehicle or ProAgio-treated OrKPC mice. (K) Apoptosis of KPC 961 cells after treatment with Gem (1 μmol/L, grey bar, or 1 mmol/L, black bar) in the presence (+IGF1) or absence (-IGF1) of 10 ng/mL IGF1 was measured by an apoptosis kit. Cell apoptosis is presented as the percentage of apoptosis by defining the apoptosis of untreated cells as 0%. (L) The levels of Cda (IB:Cda) in KPC 961 cells after Cda knockdown (Cda small interfering RNA [siRNA]) compared with control (ctrl siRNA) were analyzed by immunoblot. (M) Cell viability of KPC 961 cells with (Cda siRNA) or without (ctrl siRNA) knockdown of Cda treated with the indicated concentrations of Gem was measured by MTT assay. (C and L) Immunoblot of β-actin (IB:β-actin) is a loading control. (D, E, F, K, and M) Error bars represent means ± SEM. MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; pIGF1R, Phospho-IGF-I Receptor; X, times.