An angiotensin system inhibitor (losartan) potentiates antitumor efficacy of cisplatin in a murine model of non–small cell lung cancer

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  Improved antitumor efficacy of chemotherapy with losartan.

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  Figure 1. A, Cell lines were characterized by expression levels of E-cadherin and vimentin. H441 and H358 expressed an epithelial phenotype–high E-cadherin and low vimentin. Lines H1299 and SW1573 expressed a mesenchymal phenotype–low E-cadherin and high vimentin. B, The lung cancer cell lines were separated into 4 subtypes: epithelial (E) (H441), intermediate epithelial (iE) (H358), intermediate mesenchymal (iM) (H1299), and mesenchymal (M) (SW1573). TC-1 was selected as a syngeneic cell line to balance differences of lung cancer cell phenotypes. Cell lines were exposed to losartan and significant decreases in vimentin (C) and E-cadherin (D) were observed (P < .05).

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  Figure 2. A, E-cadherin incrementally increased with cisplatin treatment, demonstrated dose-dependent increase with losartan, and significantly increased with combination losartan and cisplatin treatment over phosphate buffered saline (PBS) or single-drug treatment (P < .05). B, Vimentin decreased with cisplatin and showed a dose-dependent decrease with losartan. Combination losartan and cisplatin significantly reduced vimentin expression over PBS or single-drug treatment (P < .05). C, Cell viability was reduced with cisplatin and losartan individually, and combination treatment significantly reduced cell viability below single-drug treatment (P < .05). D through F, Epithelial-mesenchymal transition (EMT) mediators Twist 1, Snail 1, and Zeb 1 were significantly reduced with cisplatin and losartan monotreatment and demonstrated significant reduction with combination treatment below the single-agent treatment result (P < .05).

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  Figure 3. Representative image of cell migration assessed via scratch assay in the presence of losartan for each cell line (A), quantification of migratory cell number (B). At 24 hours, there was no significant difference in migratory cell number between losartan treated and control cells. At 48 hours, losartan treatment had significantly reduced the migratory cell number for each cell type (P < .05).

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  Figure 4. A through D, Allogeneic flank tumor model: Human SW1573 cells were injected into the flanks of nude mice on the day indicated by the red arrow. E through H, Syngeneic flank tumor model: Murine TC-1 cells were injected into the flank of C57BL/6 mice. In both flank tumor models, losartan did not influence tumor volume or weight. Cisplatin significantly reduced tumor weight and volume in both models.∗ Combination treatment with losartan and cisplatin significantly reduced tumor size and volume below that of phosphate buffered saline control, losartan alone, or cisplatin alone.∗I through K, Immunohistochemical quantification of epithelial-mesenchymal transition markers E-cadherin and vimentin in allogeneic and syngeneic flank tumor models. Losartan and cisplatin each increase E-cadherin separately, and the combination of losartan and cisplatin is associated with an increase in E-cadherin over individual treatment.∗ Vimentin is reduced by losartan and cisplatin individually, and combination treatment reduced vimentin below either individual treatment.∗L and N, E-cadherin and vimentin expression changes confirmed via polymerase chain reaction test. L through O, Zeb 1, programmed…

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  Figure 5. A, Representative images of immunofluorescence staining for E-cadherin and vimentin in each cell line. Cells were treated with losartan and angiotensin II (200×). B, Quantification of staining with image analysis. Losartan treatment significantly increased E-cadherin or reduced vimentin in each cell line (P < .05). Treatment with combination losartan and angiotensin II returned vimentin and E-cadherin levels to baseline.

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  Figure 6. A through D, Expression of proliferation markers AKT and Stat3 and their phosphorylated molecules in all 5 lung cancer cell lines treated with losartan, cisplatin, or combined losartan plus cisplatin. No treatment effect on AKT, AKT-p, Stat3, or Stat3-p levels were observed with single agent cisplatin or losartan. Combination losartan and cisplatin significantly reduced AKT, AKT-p, Stat3, and Stat3-p in all cell lines (∗P < .05). Programmed death-ligand (E) was not significantly influenced by either losartan or cisplatin, but combination treatment significantly reduced programmed death-ligand (PDL-1) in all cell lines (P < .05).

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  Figure E1. A, We assessed epithelial-mesenchymal transformation (EMT) makers (E-cadherin, vimentin, Zeb 1, and CYR-61) and identified primarily epithelial (H441, H358) and mesenchymal cell lines (H1299, SW1573). B, Angiotensin II type 1 receptor (AT1R) expression in all 5 lung cancer cell lines (magnification is 200 × ). C through E, AT1R expression in all 5 lung cancer cell lines treated with losartan alone or losartan combined with cisplatin. D, H441, ∗P = .001; H358, ∗P = .000; H1299, ∗P = .001; SW1573, ∗P = .003. E, H441, ∗P = .022, #P = .000; H358, ∗P = .000, #P = .013; H1299, ∗P = .043, #P = .029; SW1573, ∗P = .025, #P = .028. F, Zeb 1 expression with increasing losartan (H441, ∗P = .022; H358, ∗P = .014; H1299, ∗P = .001; SW1573, ∗P = .003; TC-1, ∗P = .001). G, Twist 1 expression with increasing losartan (H441, ∗P = .002; H358, ∗P = .024; H1299, ∗P = .020; SW1573, ∗P = .012; TC-1, ∗P = .001). H, Snail 1 expression with increasing losartan (H441, ∗P = .003; H358, ∗P = .001; H1299, ∗P = .001; SW1573, ∗P = .028; TC-1, ∗P = .024).

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  Figure E2. Dose ranging studies: The 5 lung cancer cell lines were treated with increasing doses of angiotensin II (A) and losartan (B) with cell viability measured. The first statistically significant point on the cell survival curve was shown by ∗. H441 ∗P = .014; H358 ∗P = .012; H1299 ∗P = .000; SW1573 ∗P = .019; TC-1 ∗P = .012. (H) H441 ∗P = .018; H358 ∗P = .010; H1299 ∗P = .000; SW1573 ∗P = .000; TC-1 ∗P = .005. C, Cell viability with increasing doses of cisplatin. The first statistically significant point on the cell survival curve was shown by ∗. H441, ∗P = .000; H358, ∗P = .002; H1299, ∗P = .000; SW1573, ∗P = .003; TC-1, ∗P = .008. Differences observed in the cell viability following cisplatin treatment between Figure E2, C, and Figure 2, C, were likely due to normal experimental variation because these experiments were conducted at different times.

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  Figure E3. A, P-stat3 expression with increasing losartan (H441, ∗P = .024; H358, ∗P = .000; H1299, ∗P = .000; SW1573, ∗P = .000; TC-1, ∗P = .032). B, Stat3 expression with increasing losartan (H441, ∗P = .030; H358, ∗P = .032; H1299, ∗P = .015; SW1573, ∗P = .040; TC-1, ∗P = .036). C, P-protein kinase B (AKT) expression with increasing losartan (H441, ∗P = .033; H358, ∗P = .047; H1299, ∗P = .002; SW1573, ∗P = .000; TC-1, ∗P = .024). D, AKT expression with increasing losartan (H441, ∗P = .003; H358, ∗P = .032; H1299, ∗P = .000; SW1573, ∗P = .005; TC-1, ∗P = .043). E, program death ligand 1 (PD-L1) expression with increasing losartan (H441, ∗P = .034; H358, ∗P = .005; H1299, ∗P = .038; SW1573, ∗P = .005; TC-1, ∗P = .005). F, Body weight of nude mice with SW1573. G, Body weight of C57BL/6 mice with TC-1. H, Vimentin, P-stat3, Stat3, P-AKT, AKT, and PD-L1 expression in mice with lung cancer.

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  Figure E4. A, Interleukin 6 (IL-6) expression with increasing losartan (H441, ∗P = .001; H358, ∗P = .000; H1299, ∗P = .000; SW1573, ∗P = .000; TC-1, ∗P = .000). B, Program death ligand 1 (PD-L1) expression with increasing losartan (H441, ∗P = .000; H358, ∗P = .000; H1299, ∗P = .043; SW1573, ∗P = .000; TC-1, ∗P = .020). C, Program death ligand 2 (PD-L2) expression with increasing losartan (H441, ∗P = .012; H358, ∗P = .000; H1299, ∗P = .001; SW1573, ∗P = .002; TC-1, ∗P = .000). D, IL-6 expression with increasing losartan and losartan combined cisplatin (H441, ∗P = .032, #P = .0.044; H358, ∗P = .021, #P = .025; H1299, ∗P = .042, #P = .042; SW1573, ∗P = .033, #P = .036; TC-1, ∗P = .001, #P = .013). E, PD-L1 expression with increasing losartan and combined cisplatin (H441, ∗P = .000, #P = .000; H358, ∗P = .000, #P = .023; H1299, ∗P = .000, #P = .000; SW1573, ∗P = .000, #P = .000; TC-1, ∗P = .005, #P = .007). F, PD-L2 expression with increasing losartan and combined with cisplatin (H441, ∗P = .000, #P = .000; H358, ∗P = .002, #P = .002; H1299, ∗P = .001, #P = .008; SW1573, ∗P = .046, #P = .033; TC-1, ∗P = .004, #P = .011).