DataSheet1_Involvement of P-gp on Reversing Multidrug Resistance Effects of 23-Hydroxybetulinic Acid on Chemotherapeutic Agents.DOCX

Betulinic acid (BA) and 23-Hydroxybetulinic acid (23-HBA) are natural products with similar structures, which show a range of biological effects including cytotoxicity activity. The aim of current research was to investigate and evaluate the combinational cytotoxicity of BA and 23-HBA with chemotherapeutic agents in vitro, and to clarify the potential interaction and related mechanism with P-gp. Instead of BA, 23-HBA could increase cytotoxicity of MCF-7/ADR cells to adriamaycin (ADR) and vincristine (VCR). The intracellular accumulation of ADR/VCR in MCF-7/ADR cells was obviously increased in the presence of 23-HBA. Furthermore, 23-HBA could show dose-dependent increase on the transport of VCR and digoxin, which are typical P-gp substrates, in both MDCK-MDR1 and Caco-2 cells. However, the transport of BA and 23-HBA was not influenced by P-gp inhibition in MDCK-MDR1 cells. MDR1 shift assay and molecular docking model suggested that both compounds showed interaction with P-gp, yet the binding affinity and sites are different. In conclusion, 23-HBA could strongly improve the efficacy of anti-tumor agents in multidrug resistance (MDR) cells, which was related to P-gp inhibition. The MDR1 shift assay and molecular docking study further revealed that 23-HBA and BA showed different interaction modes with P-gp.</p

DataSheet1_Integrative Metabolomics, Proteomics and Transcriptomics Analysis Reveals Liver Toxicity of Mesoporous Silica Nanoparticles.docx

As pharmaceutical excipients, mesoporous silica nanoparticles (MSNs) have attracted considerable concern based on potential risks to the public. The impact of MSNs on biochemical metabolism is poorly understood, and few studies have compared the effects of MSNs administered via different routes. To evaluate the hepatotoxicity of MSNs, metabolomics, proteomics and transcriptomic analyses were performed in mice after intravenous (20 mg/kg/d) or oral ad-ministration (200 mg/kg/d) of MSNs for 10 days. Intravenous injection induced significant hepatic injury based on pathological inspection and increased the levels of AST/ALT and the inflammatory factors IL-6, IL-1β and TNF-a. Omics data suggested intravenous administration of MSNs perturbed the following metabolites: succinate, hypoxanthine, GSSG, NADP+, NADPH and 6-phosphogluconic acid. In addition, increases in GPX, SOD3, G6PD, HK, and PFK at proteomic and transcriptomic levels suggested elevation of glycolysis and pentose phosphate pathway, synthesis of glutathione and nucleotides, and antioxidative pathway activity, whereas oxidative phosphorylation, TCA and mitochondrial energy metabolism were reduced. On the other hand, oral administration of MSNs disturbed inflammatory factors and metabolites of ribose-5-phosphate, 6-phosphogluconate, GSSG, and NADP+ associated with the pentose phosphate pathway, glutathione synthesis and oxidative stress albeit to a lesser extent than intravenous injection despite the administration of a ten-fold greater dose. Overall, systematic biological data suggested that intravenous injection of nanoparticles of pharmaceutical excipients substantially affected hepatic metabolism function and induced oxidative stress and inflammation, whereas oral administration exhibited milder effects compared with intravenous injection.</p

Effect of apigenin on LPS-induced proinflammatory cytokines mRNA expression in mouse J774A.1 macrophages.

<p>Cells were pretreated with different concentrations of apigenin (A, 6.25, 12.5,25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 hours. Total cellular RNA was isolated and reverse transcribed. The relative mRNA levels of IL-1β, IL-6, and TNF-α were detected by real-time RT-PCR as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ##p<0.01; ###p<0.001; Statistical significance relative to LPS group, **p<0.01, ***p<0.001. <b>A</b>. IL-1β; <b>B</b>. IL-6; <b>C</b>. TNF-α.</p

Effects of apigenin on LPS-mediated mRNA stabilization in mouse J774A.1 macrophages.

<p>Cells were pretreated with apigenin (A, 25 µM) for 2 h and then treated with LPS (100 ng/mL) for 2 h, followed by treatment with actinomycin D (5 µg/ml). Total cellular RNA was isolated at 0, 0.5,1, 2, 4 and 6 h after actinomycin D treatment. The mRNA levels of IL-1β and IL-6 were determined by real-time RT-PCR as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">METHODS</a>”. Values are the means ± S.E. from three independent experiments. <b>A</b>. IL-1β; <b>B</b>. IL-6.</p

Effect of apigenin on caspase-1 activation in human THP-1-derived macrophages.

<p>Cells were pretreated with apigenin (A, 6.25, 12.5, 25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 h. Total cell lysates were prepared for Western blot analysis for caspase-1 protein level as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. β-Actin was used as a loading control. The relative protein levels of caspase-1 were normalized to β-Actin and analyzed using Odyssey V3.0 software. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ###p<0.001; Statistical significance relative to LPS group, *p<0.05, **p<0.01, ***p<0.001. <b>A</b>. Representative immunoblots of Pro-caspase-1 and active caspase-1 p20. <b>B</b>. The relative protein levels of total caspase-1. <b>C</b>. The relative protein levels of active caspase-1.</p

Effect of apigenin on LPS-induced IL-1β protein expression and maturation in human THP-1-derived macrophages.

<p>Cells were pretreated with apigenin (A, 6.25, 12.5, 25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 h. The Pro-IL-1β protein level was detected by Western blot analysis. β-Actin was used as a loading control. The mature IL-1β protein level was detected by ELISA as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ###p<0.001; Statistical significance relative to LPS group, ***p<0.001. <b>A</b>. Representative immunoblots of Pro-IL-1β and β-Actin; <b>B</b>. The relative protein levels of pro-IL-1β were analyzed using Odyssey V3.0 software. <b>C</b>. Mature IL-1β level in the media.</p

Effect of apigenin on pro-inflammatory cytokine-induced activation of NF-κB.

<p>Human 293 Cells were stably transfected with pGL4.32 [luc2P/NF-κB-RE/Hygro] luciferase reporter. Cells were pretreated with apigenin (6.25, 12.5,25 µM) for 2 h and then treated with human IL-1β (10 ng/mL) or TNF-α (10 ng/mL) for 4 h. The luciferase activity was detected as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">METHODS</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ###p<0.001; Statistical significance relative to LPS group, **p<0.01, ***p<0.001. <b>A</b>. IL-1β; <b>B</b>. TNF-α.</p

Real-time PCR primers.

<p>Real-time PCR primers.</p

Effect of apigenin on LPS-induced caspase-1 mRNA expression in mouse J774A.1 macrophages.

<p>Cells were pretreated with different concentrations of apigenin (A, 6.25, 12.5,25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 hours. Total cellular RNA was isolated and reverse transcribed. The relative mRNA level of caspase-1 was detected by real-time RT-PCR as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ##p<0.01; ###p<0.001; Statistical significance relative to LPS group, **p<0.01, ***p<0.001.</p

Effect of apigenin on LPS-induced IL-6 and TNF-α protein expression in in mouse J774A.1 macrophages.

<p>Cells were pretreated with different concentrations of apigenin (A, 6.25, 12.5,25 µM) for 2 h and then treated with LPS (100 ng/mL) for 24 h. At the end of treatment, each cell culture medium was collected. The protein levels of IL-6 and TNF-α were determined by ELISA as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107072#s2" target="_blank">Methods</a>”. Values are mean ± S.E. of three independent experiments. Statistical significance relative to vehicle control, ###p<0.001; Statistical significance relative to LPS group, **p<0.01, ***p<0.001. <b>A</b>. IL-6; <b>B</b>.TNF-α.</p