Emodin suppresses pulmonary metastasis of breast cancer cells accompanied with decreased macrophage recruitment and M2 polarization in the lungs
Xuemei Jia1,2, FangYu2,3, Junfeng Wang2,4, Stephen Iwanowycz2, Fatma Saaoud2, Yuzhen Wang2, Jun Hu5, Qian Wang5, and Daping Fan2,*
1Department of Gynecology, the First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China
2Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, Columbia, SC 29209
3Department of Nutrition and Food Hygiene, Fourth Military Medical University, Xi'an, 710032, China
4Centre for Stem Cell Research and Application, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
5Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208
*Correspondence: Daping Fan, Department of Cell Biology and Anatomy, University of South Carolina School of Medicine, 6439 Garners Ferry Road, Columbia, SC 29208. Phone: 803-216-3806; Fax: 803-216-3846; E-mail:
Journal: Breast Cancer Research and Treatment
Materials and methods
LDH cytotoxicity assay
1x104 EO771 or 4T1 cells or 1x105 mouse peritoneal macrophages were seeded onto 96-well plates and maintained in DMEM containing 10% FBS overnight. After washing with PBS, the media was replaced with DMEM containing 1% DMSO (Control group) or varying concentrations of Emodin (0-100 µM). After 24 or 48 h of incubation, the cell viability was determined using a LDH Cytotoxicity Detection Kit (Clontech, Mountain View, CA) according the manufacturer’s instructions. Briefly, 50 µl of supernatant was removed from each well and placed in a new well on the same plate. 50 µl of cell lysis buffer (2% triton in DMEM) was then added to each well containing the cancer cells or macrophages, and the cells were incubated at room temperature for 15 min. The reaction mixture was then added to each well (cell lysate+supernatant) and incubated for 5 min before the stop solution was added. The absorbance was measured at 490nm on a Spectra Max M5 Microplate Reader (Molecular Devices, Sunnyvale, CA). The percent viability was then calculated as the ratio of LDH in the cell lysate to the total amount of LDH in the lysate plus the supernatant. There were three wells per group.
Suppl. Fig. S1. A. Effects of Emodin on breast cancer cell viability in vitro. 1x104 EO771 or 4T1 cells were seeded onto 96-well plates in DMEM+10% FBS. The cells were incubated overnight at 37°C. The media was then removed and the cells were washed with PBS. DMEM containing varying concentrations of Emodin (0-100µM) was then added to the cells. There were three wells per group. The Control group contained an equal volume of DMSO. Plates of cells were then incubated for 24 or 48 h; then the viability of the cells was determined using an LDH Cytotoxicity Detection Kit.B. Effects of Emodin on breast cancer cell migration in vitro. Tumor cells (2x105 cells) were seeded onto the top chamber of transwell inserts with 8 μm pores (Corning). The inserts were then placed into 24-well plates that contained 600 µl of serum-free RPMI1640 with Emodin at indicated concentrations for 6 h. Cells that migrated across the inserts were stained with DAPI and counted under a fluorescence microscope at 40 x magnification (twenty fields per well, triplicate for each group). n=6; *p<0.05 vs Control.
Suppl. Fig. S2. Immunofluorescence staining was performed to detectF4/80+ (A) and Ym1+ (B) macrophages in primary tumors of BALB/c mice inoculated with 4T1 cells. FITC-labeled rabbit anti-F4/80 and anti-Ym1+antibodies were applied. Sections were counterstained with DAPI nuclear dye (Blue). Magnification, 40x. The quantification data was shown in the right panels.C. Lung slides of 4T1 tumor-bearing mice were co-stained for Mac2 (Green) and YM1 (Red). The ratio of YM1 positivity in Mac2 positive cells was calculated and shown on the right. n=5 in each group, *p<0.05 vs Control.
Suppl. Fig. S3. Effects of Emodin on macrophage viability in vitro. 1x105 mouse peritoneal macrophages were seeded onto 96-well plates in DMEM+10% FBS. The cells were incubated overnight at 37°C. The media was then removed and the cells were washed with PBS. DMEM containing varying concentrations of Emodin (0-100µM) was then added to the cells. There were three wells per group. The Control group contained an equal volume of DMSO. Plates of cells were then incubated for 24 or 48 h; then the viability of the cells was determined using an LDH Cytotoxicity Detection Kit.
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