Reduction of metastatic and angiogenic potency of malignant cancer by Eupatorium. fortunei via suppression of MMP-9 activity and VEGF production

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1 Supplementary Information Reduction of metastatic and angiogenic potency of malignant cancer by Eupatorium fortunei via suppression of MMP-9 activity and VEGF production Aeyung Kim, Minju Im, Nam-Hui Yim and Jin Yeul Ma* - Supplementary Table S1 - Supplementary Table S2 - Supplementary Table S3 - Supplementary Table S4 - Supplementary Figure S1 - Supplementary Figure S2 - Supplementary Figure S3 - Supplementary Figure S4 - Supplementary Figure S5 - Supplementary Figure S6 - Supplementary Figure S7 - Supplementary Figure S8 - Supplementary Figure S9 - Supplementary Figure S10 -

2 Table S1. Body weights of mice administrated with WEF or saline Treatment Body weight (g) Day 0 Day 7 Day 14 Control ± ± ± mg/kg ± ± ± 0.19 Data are presented as mean S.D. Each group of female C57BL/6J mice (n=3) were orally administrated with 50 mg/kg of WEF or same volume of saline daily and weighed body weight at 0, 7, and 14 days.

3 Table S2. Organ weights of mice administrated with WEF or saline Treatment Weight of organs (g) Liver Heart Lung spleen Kidney (L) Kidney (R) Control mg/kg Data are presented as mean S.D. Each group of female C57BL/6J mice (n=3) were orally administrated with 50 mg/kg of WEF or same volume of saline daily, sacrificed at 15 days, and weighed organs.

4 Table S3. Chemical analysis of serums obtained from mice administrated with WEF or saline. Treatment GOT (IU/L) GPT (IU/L) BUN (mg/dl) CRE (mg/dl) Control mg/kg Data are presented as mean S.D. Each group of female C57BL/6J mice (n=3) were orally administrated with 50 mg/kg of WEF or same volume of saline daily, sacrificed at 15 days, and analyzed the levels of GOT, GPT, BUN, and CRE. GOT, glutamic oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; BUN, blood urea nitrogen; CRE, creatinine.

5 Table S4. Hematological analysis of bloods obtained from mice administrated with WEF or saline. Parameter control 50 mg/kg WBCP ( 10 3 cells/ l) WBCB ( 10 3 cells/ l) RBC ( 10 6 cells/ l) Means HGB (g/dl) HCT (%) MCV (fl) MCH (pg) MCHC (g/dl) PLT ( 10 4 cells/ l) % NEUT % LYM % MONO Data are presented as mean S.D. Each group of mice (n=3) were orally administrated with 50 mg/kg of WEF or saline daily, sacrificed at 15 days, and analyzed hematologic parameters. CBC, complete blood cell count; WBCP, white blood cell count peroxidase method; WBCB, white blood cell count basophile method; RBC, red blood cell count; HGB, hemoglobin, HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet; NEUT, neutrophil; LYM, lymphocyte; MONO, monocyte.

6 Figure S1. WEF inhibits the migration and invasion of HT1080 cells. (A): After making wounds on the confluent cell monolayers, cells were incubated in the presence of 25, 50, and 100 g/ml WEF for 36 h. Cell migration was calculated based on the relative width of wound at 0 h and expressed as the mean SD of four selected fields. (B): Cells were pre-treated with 25, 50, and 100 g/ml WEF for 12 h. After harvest, cells ( /100 l) suspended in serum-free RPMI was added into upper chamber of Transwell and lower chamber was filled with 600 l 10% FBS/RPMI. For invasion assay, Matrigel-coated Transwell was used. Cells migrated and invaded to the lower surface of the Transwell membrane were measured at 12 h and 24 h incubation, respectively. Data are representative of two independent experiments and expressed as the mean SD of five random fields of each well. #, p 0.01 vs. untreated control.

7 Figure S2. WEF reduces transcriptional expression of MMPs under PMA stimulation. HT1080 cells pretreated with 25, 50, and 100 g/ml WEF for 12 h in serum-free media were stimulated with 5 nm PMA for additional 24 h. MMPs mrna levels were measured by RT-PCR.

8 Figure S3. WEF suppresses MMP-9 gelatinolytic activity under PMA stimulation. This is a full length image of the cropped blot presented in the Figure 4B.

9 Figure S4. WEF suppresses PMA-induced p38 and JNK phosphorylation as well as NF-κB activation. These are full length images of the cropped blot presented in the Figure 5A and 5B.

10 Figure S5. WEF reduces TNF-α-induced MMP-9 expression and activity via suppression of NFκB activation in B16F10 cells. (A): B16F10 cells pretreated with 25, 50, and 100 g/ml WEF for 12 h in serum-free media were stimulated with TNF-α (10 ng/ml) for additional 24 h. MMP-9 mrna level and MMP-9 activity were measured by RT-PCR and gelatin zymography, respectively. (B): B16F10 cells were pretreated with WEF (100 g/ml) for 12 h and then stimulated with TNF-α (10 ng/ml) for 30, 60, and 180 min. Total cell lysates were subjected to Western blotting and relative ratios were calculated after normalization to tubulin. Data are representative of two independent experiments.

11 Figure S6. Inhibitory effect of WEF on vessel sprout formation is reversible. Aortic rings were first incubated with WEF-treated CMs of HT1080 or PC-3 and continued to culture in EGM-2 for another 72 h after CMs were removed.

12 Figure S7. WEF inhibits HIF-1α expression. HT1080 cells were pretreated with WEF (25, 50, and 100 g/ml) for 12 h, and then stimulated with 100 M CoCl 2 for 6 h. Total cell lysates were subjected to Western blotting to measure the levels of HIF-1α, phosphorylated Akt and mtor. After normalization to tubulin, the relative ratios were calculated. Data are expressed as the mean SD of two independent experiments. *, p 0.01 vs. untreated control.

13 Figure S8. WEF inhibits VEGF-α and HIF-1α expression. (A): B16F10 cells were pretreated with WEF (25, 50, and 100 g/ml) for 24 h and VEGF-α mrna levels were measured by RT- PCR. Relative ratio was quantified after normalization to GAPDH. (B): B16F10 cells were pretreated with WEF (25, 50, and 100 g/ml) for 12 h, and then stimulated with 100 M CoCl 2 for 3 h. Total cell lysates were subjected to Western blotting to measure the levels of HIF-1α, phosphorylated Akt and mtor. After normalization to tubulin, the relative ratios were calculated. Data are representative of two independent experiments.

14 Figure S9. No inhibitory effects of WEF on HUVEC migration and tube formation. (A): Matrigels mixed with bfgf (1 g/ml) and VEGF-α (100 ng/ml) in the presence of WEF were implanted and angiogenesis was examined after 14 days. (B): HUVEC capillary-like tube formation induced by bfgf (1 g/ml) and VEGF-α (100 ng/ml) in the presence of WEF was examined. (C): EGM-2 was added to the lower chamber, and HUVECs in EBM-2 were allowed to migrate in the presence of WEF (0, 25, 50, and g/ml) for 24 h.

15 Figure S10. WEF inhibits HIF-1α expression and phosphorylation of Akt/mTOR. These are full length images of the cropped blot presented in the Figure 7C.