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

Abstract

Eupatorium fortunei has long been used to treat nausea and poor appetite, and has been prescribed as a diuretic and detoxifying drug in Chinese medicine. Recent studies have demonstrated that E. fortunei possesses anti-bacterial, anti-oxidant, and anti-diabetic activities, as well as cytotoxicity to human leukemia cells. However, at non-toxic concentrations, the effects of an aqueous extract of E. fortunei (WEF) on the metastatic and angiogenic potential of malignant tumor cells have not been reported. In this study, we found that WEF suppressed the metastatic properties, including anchorage-independent colony formation, migration, and invasion, by downregulating the proteolytic activity of MMP-9. NF-κB activation and the phosphorylation of p38 and JNK were reduced significantly by WEF. Additionally, WEF inhibited tumor-induced angiogenesis markedly, affecting HUVEC migration, tube formation by HUVECs, and microvessel sprouting from rat aortic rings via a reduction in VEGF in tumors. In a pulmonary metastasis model, daily administration of WEF at 50 mg/kg markedly decreased metastatic colonies of intravenously injected B16F10 cells on the lung surface in C57BL/6J mice. Further, none of the WEF-administered mice exhibited systemic toxicity. Taken together, our results indicate that WEF is a potential therapeutic herbal product that may be useful for controlling malignant metastatic cancer.

Figure 1. WEF inhibits anchorage-independent growth of B16F10 cells.

(A): Proliferation of cells (5 × 10³ cells/well) incubated with 10 to 500 μg/ml WEF for 48 h was measured by MTT assays. Data are the relative cell viability compared to untreated control and expressed as the mean ± SD. n.s, non-significant (B): Colony formation of B16F10 cells in soft agar in the presence of non-toxic concentrations of WEF (25, 50, and 100 μg/ml) was measured during 14-day-incubation. The diameters of 10 representative colonies were measured and expressed as the mean ± SD. Data are representative of two independent experiments. #, p<0.01 vs. untreated control.

Figure 2. WEF suppresses migration and invasion of B16F10 cells.

(A): Cells grown to 90% confluence were treated with mitomycin C for 30 min and then wounded by scraping. Cells were incubated in the presence of 25, 50, and 100 μg/ml WEF and migration was monitored using phase-contrast microscopy after 18 and 36 h. Based on the width of wound at 0 h, relative width at 18 and 36 h was calculated and expressed as the mean ± SD of four selected fields. (B): Cells pre-treated with 25, 50, and 100 μg/ml WEF for 12 h were subjected to migrate and invade through Transwell for 24 h and 36 h, respectively. Cells migrated and invaded to the lower surface of the Transwell membrane were stained and relative migration and invasion were quantified using ImageJ software. Data are representative of three independent experiments and expressed as the mean ± SD of five random fields of each well. #, p<0.01 vs. untreated control.

Figure 3. Oral administration of WEF inhibits pulmonary metastasis of B16F10 cells.

Five-week-old female C57BL/6J mice were intravenously injected via tail vein with B16F10 cells (3 × 10⁵). After daily oral administration with saline (control) or WEF (50 mg/kg) for 17 days, mice were sacrificed and black colonies settled on the lung surface counted macroscopically after fixation. Images show metastatic colonies on the front (F) and back (B) side of the lungs. Metastatic colonies were counted and represented as the mean ± SD of each group (each group, n = 4). #, p<0.05 vs. control.

Figure 4. WEF reduces PMA-induced MMP-9 expression and MMP-9 activity.

HT1080 cells pretreated with 25, 50, and 100 μg/ml WEF for 12 h in serum-free media were stimulated with 5 nM PMA for additional 24 h. (A): MMP-9 mRNA levels were measured by RT-PCR and relative expression was quantified after normalization to GAPDH. (B): CM were collected and analyzed for the MMP-9 level and MMP-9 activity by Western blotting and zymography, respectively. Gelatin and type I collagen were used as MMP-9 substrates. Data are expressed as the mean ± SD of two independent experiments. The full size blot was shown in the Supplementary Figure S3 and band of interest is indicated with an arrow. #, p<0.01 vs. untreated control. *, p<0.01 vs. PMA stimulation.

Figure 5. WEF suppresses PMA-induced p38 and JNK phosphorylation as well as NF-κB activation.

(A): After treatment with or without WEF (100 μg/ml) for 12 h, cells were stimulated with PMA (5 nM) for 15, 30, or 60 min. Total cell lysates were subjected to Western blotting to evaluate phosphorylation of p38, ERK, and JNK. Phosphorylation and degradation of IκBα were also measured. After normalization to tubulin, the relative ratios of phosphorylated protein/total protein were calculated. (B): Cells pre-treated with WEF (25, 50, or 100 μg/ml) for 12 h were stimulated with PMA for 30 min and then subjected to Western blotting. Total cell lysates were measured for IκBα phosphorylation and degradation. Cytosolic and nuclear fractions were prepared to evaluate nuclear translocation of the NF-κB p65 subunit after PMA stimulation. Tubulin and TBP were used as loading control for cytosolic and nuclear compartment, respectively. Data are expressed as the mean ± SD of two independent experiments. The full size blots were shown in the Supplementary Figure S4 and band of interest is indicated with an arrow. #, p<0.01 vs. untreated control. *, p<0.01 vs. PMA stimulation.

Figure 6. WEF suppresses tumor-induced angiogenesis.

(A): Capillary-like tube formation by HUVECs induced by control CM or WEF-treated CM of HT1080 or PC-3 cells was examined. The total tube area was measured using ImageJ software and expressed as the mean ± SD of three random fields. #, p<0.01 vs. untreated control. (B): PC-3 cells treated or untreated with WEF for 12 h were plated in the lower chamber of Transwell and incubated in 0.5% serum media. HUVECs suspended in 0.5% serum media were added to the upper chamber and allowed to migrate across Transwell. After 24 h, HUVECs migrated to the lower surface were stained and quantified using ImageJ software. #, p<0.01 vs. no PC-3 cells. *, p<0.01 vs. WEF-untreated PC-3 cells. (C): PC-3 cells (3 × 10⁶/100 μl) suspended in serum-free RPMI were mixed with Matrigel, heparin, and WEF (0, 50, and 100 μg/ml). Matrigel mixture was subcutaneously implanted into the ventral region of athymic nude mice. On day 14, Matrigel plugs were carefully removed and Hb contents in plugs were quantified using Drabkin's reagent kit. Data represent the mean ± SD (n = 3). #, p<0.01 vs. Matrigel alone. *, p<0.01 vs. WEF-untreated PC-3 cells. (D): Freshly prepared aortic rings were placed in a Matrigel-coated 48-well culture plate, incubated in EGM-2 for 3 days, and then replaced with control CM or WEF-treated CM of HT1080 or PC-3 cells. After 3 days, sprouts from rat aortic rings were photographed and sprout length was quantified. Data are expressed as the mean ± SD (n = 3) #, p<0.01 vs. untreated control.

Figure 7. WEF inhibits VEGF-α production and HIF-1α expression.

(A): HT1080 and PC-3 cells were treated with 25, 50, and 100 μg/ml WEF for 24 h and VEGF-α mRNA levels were measured by RT-PCR. Relative ratio was quantified after normalization to GAPDH. (B): HT1080 and PC-3 cells were treated with WEF (25, 50, and 100 μg/ml) for 48 h, and the level of VEGF-α in CM was determined by ELISA. #, p<0.01 vs. untreated control. (C): PC-3 cells were pretreated with WEF (25, 50, and 100 μg/ml) for 12 h, and then stimulated with 100 μM CoCl₂ 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. The full size blots were shown in the Supplementary Figure S10 and band of interest is indicated with an arrow. #, p<0.01 vs. untreated control.