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. 2017 Nov 10;8(64):107374–107389. doi: 10.18632/oncotarget.22376

Figure 6. Folic acid conjugation is responsible for the enhanced chemosensitization potential of PPF-curcumin.

Figure 6

(A) Confocal microscopy image showing high expression of FOLR1 in HeLa cells. (B) Immunohistochemical staining of FOLR1 expression in HeLa xenografts. (C) Confocal microscopy image showing low expression of FOLR1 in HaCaT cells. (D) Uptake of PPF-curcumin nanoparticles in FOLR1 over-expressing HeLa cells. Cells were incubated with 5 μM PPF-curcumin nanoparticles for 2 h. Cells were fixed and nuclei were stained with PI and images were captured at a magnification of 40X. (E) Confocal microscopy image showing the reduced uptake of PPF-curcumin in HaCaT cells. Cells were incubated with 5 μM PPF-curcumin nanoparticles for 2 h. Cells were fixed and nuclei were stained with DAPI and images were captured at a magnification of 40X. (F) Uptake of PPF-curcumin in HeLa cells in the absence or presence of free folic acid. Reduced uptake of PPF-curcumin was observed in HeLa cells in the presence of free folic acid indicating FOLR1-targeted uptake of PPF-curcumin in FOLR1 over-expressing HeLa cells. Cells were incubated with free folic acid for 20 min under culture conditions as described in Materials and Methods. Later, the medium was removed and incubated with 5 μM PPF-curcumin nanoparticles for 2 h. For confocal microscopy, cells were fixed and nuclei were stained with DAPI and images were captured at a magnification of 40X. (G) Chemosensitization studies in HeLa cells using curcumin, PLGA-curcumin or PPF-curcumin in the presence or absence of folic acid. For cytotoxicity study, cells were cultured in the presence of 100 μg/ml and treated with curcumin or PLGA-curcumin or PPF-curcumin (5 μM) either alone in combination with paclitaxel (5 nM). Later, after 72 h of incubation, the cell viability was assessed using MTT. (H) Representative images of tumors from NOD-SCID mice bearing HeLa xenograft tumors with or without treatment after 4 weeks. (I) PPF-curcumin induced significant inhibition of NF-κB and AP-1 activation in vivo as assessed by EMSA.