Emodin Suppresses Migration and Invasion through the Modulation of CXCR4 Expression in an Orthotopic Model of Human Hepatocellular Carcinoma

10.1371/journal.pone.0057015PLoS ONE83

Emodin inhibits TNF-α inducible NF-κB activation in HepG2 cells.

<p>A, HepG2 cells were incubated with 50 µM emodin for the indicated time points and then stimulated with TNF-α (1 nM) for 24 h. The nuclear extracts were assayed for NF-κB activation by TransAM p65 transcription factor assay kit. Bars indicate standard error. * indicates p value <0.05. B, HepG2 cells were transiently transfected with an NF-κB luciferase plasmid and co-transfected with β-galactosidase and then treated with 50 µM emodin for the indicated time points and then stimulated with TNF-α (1 nM) for 24 h. Cell supernatants were thereafter collected and assayed for luciferase activity as described in Materials and Methods. Representative results of two independent experiments are shown. Results are expressed as fold activity over the activity of the vector control. Bars indicate standard error. * indicates p value <0.05; ** indicates p value <0.001. C, Emodin inhibits binding of NF-κB to the CXCR4 promoter. Hep3B cells were treated with 50 µM emodin for indicated time intervals and the proteins were crosslinked with DNA by formaldehyde and then subjected to ChIP assay using an anti-p65 antibody with the CXCR4 primer. Reaction products were resolved by electrophoresis.</p

Emodin suppresses CXCR4 mRNA level in HCC cells.

<p>A and B, Emodin suppresses CXCR4, neither by proteasomal nor by lysosomal degradation. HepG2 cells (1×10⁶) were treated with indicated concentrations of ALLN or chloroquine for 1 h at 37°C, followed by treatment of 50 µM emodin for 6 h. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against CXCR4. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. Representative results of two independent experiments are shown. C, Emodin suppresses the expression of CXCR4 mRNA in HepG2 cells. HepG2 cells were treated with 50µM emodin for the indicated time intervals, after which cells were harvested after treatment and total RNA samples were extracted. 1µg portions of the respective RNA extracts then proceed for Reverse Transcription to generate corresponding cDNA. Real time PCR was performed to measure the relative quantities of CXCR4 mRNA using targeted TaqMan probes, with GAPDH as endogenous control for measurement of equal loading of RNA samples. Results were analyzed using Sequence Detection Software version 1.3 provided by Applied Biosystems. Relative gene expression was obtained after normalization with endogenous GAPDH and determination of the difference in threshold cycle (Ct) between treated and untreated cells using 2-ΔΔCt method.</p

Emodin suppresses migration and invasion of HepG2 cells.

<p>A, Wound-healing assay was performed for evaluating the inhibitory effect of emodin on HepG2 cell migration. Confluent monolayers of HepG2 cells were scarred, and repair was monitored microscopically after 12 h of pre-treatment with emodin (50 µM) before being exposed to 100ng/mL CXCL12 for 24 h. Width of wound was measured at time zero and 24 h of incubation with and without emodin in the absence or presence of CXCL12. The representative photographs showed the same area at time zero and after 24 h of incubation. B, HepG2 (2×10⁵ cells) were seeded in the top-chamber of the Matrigel. After pre-incubation with or without emodin (50 µM) for 12 h, transwell chambers were then placed into the wells of a 24-well plate, in which we had added either the basal medium only or basal medium containing 100 ng/mL CXCL12 for 24 h. After incubation, they were assessed for cell invasion as described in Materials and Methods. Columns represent percentage of invaded cells; bars, S.E. *indicates p value <0.05. Representative results of two independent experiments are shown.</p

Emodin suppresses lung metastasis and CXCR4 expression in orthotopic mice model.

<p>Spontaneous metastatic model of human liver cancer cells orthotopically implanted in nude mice. Cubes of HCCLM3_Luc tumor were implanted orthotopically into the liver of female Balb/c nude mice. Mice were euthanized when the detectable liver tumor signal was >1×10¹¹ p/s. A, At the time of necropsy, bioluminescent imaging was employed to monitor signals from lung metastasis. The lung biopsy was imaged to detect metastasis in the lungs. The color bar depicts the photon flux (p/s) emitted. # = mouse ID; D = day at necropsy. Lung and liver signals are in p/s unit. B, Comparison of the lung metastasis signals detected at time of necropsy for the untreated and emodin-treated groups. C, Tumor tissue extracts were prepared as described in Methods. 30 µg of protein was taken and analyzed by Western blot analysis with antibodies against CXCR4. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading. D, Immunohistochemical analysis of representative section of tumor tissues stained with anti-CXCR4 antibody demonstrate strong cytoplasmic staining for CXCR4 in the control section and weak cytoplasmic staining in the emodin treated section E, Emodin decreases CXCR4 levels in tumor tissues. Tumor tissue homogenate was prepared as described in Methods. 150 µg of the tissue supernatant was taken for ELISA assay. Assay was performed according to manufacturer’s instructions using ELISA kit to determine the levels of CXCR4 in the control and treated group. Data is represented as Mean ± SE. * indicates p value <0.05.</p

Emodin suppresses migration and invasion of Hep3B cells.

<p>A, Wound-healing assay for evaluating the inhibitory effect of emodin on cell migration. Confluent monolayers of Hep3B cells were scarred, and repair was monitored microscopically after 12 h of pre-treatment with emodin (50 µM) before being exposed to 100 ng/mL CXCL12 for 24 h. Width of wound was measured at time zero and 24 h of incubation with and without emodin in the absence or presence of CXCL12. The representative photographs of two independent experiments showed the same area at time zero and after 24 h of incubation. B, Hep3B (2×10⁵ cells) were seeded in the top-chamber of the Matrigel. After pre-incubation with or without emodin (50 µM) for 12 h, transwell chambers were then placed into the wells of a 24-well plate, in which we had added either the basal medium only or basal medium containing 100 ng/mL CXCL12 for 24 h. After incubation, the chambers were assessed for cell invasion as described in Materials and Methods. Columns represent percentage of invaded cells; bars, SE. * indicates p value <0.05. Representative results of two independent experiments are shown.</p

Emodin suppresses CXCR4 in HCC cells.

<p>A, The chemical structure of emodin. B, Western blot analysis of CXCR4 and HER2 expression in HCC cells. Whole-cell extracts of HepG2, Hep3B, HUH7, and PLC/PRF5 (30 µg) were resolved on SDS-PAGE gel and probed with anti-CXCR4 and HER2 antibodies. As a loading control, stripped membrane was probed with β-actin antibody. C, Emodin suppresses CXCR4 levels in a dose-dependent manner. HepG2 cells (1×10⁶) were treated with the indicated concentrations of emodin for 6 h. Whole cell extracts were then prepared, and 30 µg of protein was resolved on SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed for CXCR4. The same membrane was stripped and reprobed with β-actin antibody to show equal protein loading. D, Emodin suppresses CXCR4 levels in a time-dependent manner. HepG2 cells (1×10⁶) were treated with 50 µM emodin for the indicated times, after which Western blotting was done as described above. E, Effect of emodin on HER2 expression in HepG2 cells. HepG2 cells (1×10⁶) were treated with 50 µM emodin for the indicated times, after which Western blotting for HER2 was done as described above. The same membrane was stripped and reprobed with β-actin antibody to show equal protein loading. The representative results of two independent experiments are shown. F, Emodin suppresses expression of CXCR4 protein expression in Hep3B cells. Cells were incubated with 50 µM emodin for indicated times. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against CXCR4. The same membrane was stripped and reprobed with β-actin antibody to show equal protein loading. Representative results of two independent experiments are shown. G, Emodin suppresses expression of CXCR4 mRNA expression in Hep3B cells. Hep3B cells were treated with 50 µM emodin for indicated times. Total RNA was isolated and analyzed by quantitative real time PCR assay as described in Materials and Methods. GAPDH was shown to equal loading of total RNA. Representative results of two independent experiments are shown.</p

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