Research Paper Volume 13, Issue 2 pp 2912—2940

Systems pharmacology dissection of Epimedium targeting tumor microenvironment to enhance cytotoxic T lymphocyte responses in lung cancer

The effect of ICT on lung cancer cell lines. (A) Inhibition rates of ICT on H1975 and RAW264.7 cells. After incubated 24 h, H1975 and RAW264.7 cells viability was measured by CCK-8 assay after treated with control or ICT for 48h. (B) The expression of inflammatory factors (iNOS, TNF-α and COX-2) was measured by western blotting. (C) mRNA expression of inflammatory cytokines (IL-6, TNF-α and iNOS) was determined by RT-PCR. ICT reduced mRNA expression of IL-6, TNF-α and iNOS in LPS-induced RAW264.7 cells. *P P D) Western blotting showed the expression of classical apoptotic proteins (TNF-α, Bcl-2, Bax, CDK2) in H1975 cells. (E) Western blotting showed that ICT down-regulated the expression of migration proteins (eNOS, PKC, P38) in H1975 cells. (F) Apoptosis in H1975 cells was assessed after 24 h of treatment with ICT (0, 40, 60 and 80μM) by Annexin V-FITC/PI binding and measured by flow cytometry analysis. Numbers indicate the percentage of cells in each quadrant. The number of lower right quadrant and upper right quadrant represent the percentage of apoptosis and necrosis, respectively. (G) Bar plot represents the percentage of apoptosis and necrosis induced by ICT in flow cytometric image (F). *P H) Western blotting showed that the expression of extrinsic protein TNF-α increased and the expression of internal protein Bcl-2 decreased in ICT-treated tumor tissues. *P P P P

Figure 8. The effect of ICT on lung cancer cell lines. (A) Inhibition rates of ICT on H1975 and RAW264.7 cells. After incubated 24 h, H1975 and RAW264.7 cells viability was measured by CCK-8 assay after treated with control or ICT for 48h. (B) The expression of inflammatory factors (iNOS, TNF-α and COX-2) was measured by western blotting. (C) mRNA expression of inflammatory cytokines (IL-6, TNF-α and iNOS) was determined by RT-PCR. ICT reduced mRNA expression of IL-6, TNF-α and iNOS in LPS-induced RAW264.7 cells. *P < 0.05 vs LPS group, **P < 0.005 vs LPS group. (D) Western blotting showed the expression of classical apoptotic proteins (TNF-α, Bcl-2, Bax, CDK2) in H1975 cells. (E) Western blotting showed that ICT down-regulated the expression of migration proteins (eNOS, PKC, P38) in H1975 cells. (F) Apoptosis in H1975 cells was assessed after 24 h of treatment with ICT (0, 40, 60 and 80μM) by Annexin V-FITC/PI binding and measured by flow cytometry analysis. Numbers indicate the percentage of cells in each quadrant. The number of lower right quadrant and upper right quadrant represent the percentage of apoptosis and necrosis, respectively. (G) Bar plot represents the percentage of apoptosis and necrosis induced by ICT in flow cytometric image (F). *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 vs control group. (H) Western blotting showed that the expression of extrinsic protein TNF-α increased and the expression of internal protein Bcl-2 decreased in ICT-treated tumor tissues. *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001 vs control group. GAPDH was used as the loading control.