Say was performed. The gelatin zymography data indicated that MMP-2 enzyme activity was inhibited by 53 at the highest concentration of kaempferol (100 mM) (Figure 3A). Figure 3B shows a Western blotting analysis with the MMP-2 and TIMP-2 protein levels. The protein levels of both MMP-2 and TIMP-2 decreased substantially. The mRNA expression also demonstrated the same final results (Figure 3C). We also utilized a quantitative real-time PCR assay to confirm the MMP2 mRNA expression (Figure 3D). As a result, Kaempferol considerably inhibits MMP-2 mRNA expression within a concentration-dependent manner.Kaempferol Reduces MMP-2 in Oral CancerFigure five. Effects of kaempferol on the signaling pathways. SCC4 cells have been treated with a variety of doses of kaempferol (0, 20, 40, 60, 80, and one hundred mM) for 24 hours and whole cell lysates ready from these cells have been used for western blot analysis with (A) anti-ERK1/2, (B) anti-JNK1/2 and (C) anti-p38 (total and phosphorylated) antibodies as described inside the Components and Solutions section.Phosphatidylethano lamine The values represented the implies six SD of at least three independent experiments. *p,0.05 as compared together with the handle.Narasin doi:ten.1371/journal.pone.0080883.gKaempferol inhibits the transcriptional activity of MMP-To further investigate no matter whether the kaempferol regulated the promoter activity of MMP-2, we performed a luciferase reporter assay, along with a reporter gene that contained the MMP-2 promoter region was transfected in to the SCC4 cells. As shown in Figure 4A, the MMP-2 promoter activity was reduced inside a dose-dependentmanner, indicating that kaempferol inhibits MMP-2 expression in the transcriptional level.PMID:23916866 Kaempferol decreases c-Jun/AP-1 activation in SCC4 cellsBecause preceding data revealed that AP-1 was a key transcriptional regulator in MMP-2 promotion [33], EMSA and ChIP assays have been then performed to investigate the effect of kaempferolPLOS A single | www.plosone.orgKaempferol Reduces MMP-2 in Oral CancerFigure 6. Effects of MEK inhibitor (U0126) and kaempferol on MMP-2 activity and invasion of SCC4 cells. SCC4 cells were pre-treated with U0126 (10 or 20 mM) for 1 hour, then incubated within the presence or absence of kaempferol (60 mM) for 24 hours. (A) The culture media have been utilised as subjects for evaluation of MMP-2 activity, and cells had been made use of for invasion assay (B) as described within the Materials and Solutions section. The values represented the suggests six SD of at the least 3 independent experiments. *p,0.05 as compared using the control. doi:10.1371/journal.pone.0080883.gon AP-1 DNA-binding activity. As illustrated in Figure 4B, the binding activity of AP-1 towards the MMP-2 promoter decreased considerably in SCC4 cells just after getting treated with kaempferol within a concentration-dependent manner. Especially, the binding capacity of AP-1 on the promoter with the MMP-2 gene was repressed in SCC4 cells right after becoming treated with 60 mM kaempferol (Figure 4C).To determine the transcription aspects, we used a Western blot assay to detect the nuclear translocation of c-Jun and c-Fos. Treating SCC4 cells with kaempferol reduced the nuclear translocation of c-Jun, but made no effects on the level of c-Fos (Figure 4D).tion with the ERK1/2 signaling pathways may well result in a decreased expression of MMP-2, and reduced tumor cell migration.DiscussionThe consumption of fruits and vegetables containing flavonoids produces very important health benefits. Kaempferol, a flavonoid, demonstrates diverse biological and pharmacological effects, like antioxidant activity and anti-cancer-relat.