Intest Res.  2019 Oct;17(4):527-536. 10.5217/ir.2019.00031.

Parthenolide inhibits transforming growth factor β1-induced epithelial-mesenchymal transition in colorectal cancer cells

• Zhu SM
• Park YR
• Seo SY
• Kim IH
• Lee ST
• Kim SW

• KMID: 2465818
• DOI: http://doi.org/10.5217/ir.2019.00031

Abstract

BACKGROUND/AIMS
Transforming growth factor-β1 (TGF-β1) induction of epithelial-mesenchymal transition (EMT) is one of the mechanisms by which colorectal cancer (CRC) cells acquire migratory and invasive capacities, and subsequently metastasize. Parthenolide (PT) expresses multiple anti-cancer and anti-inflammatory activities that inhibit nuclear factor κB by targeting the IκB kinase complex. In the present study, we aimed to investigate whether PT can inhibit TGF-β1-induced EMT in CRC cell lines.
METHODS
HT-29 and SW480 cell lines were used in the experiment. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and sub-G1 analysis was measured by flow cytometry. The induction of EMT by TGF-β1 and inhibition of the process by PT was analyzed by phase contrast microscopy, wounding healing, cellular migration and invasion assays, and Western blotting.
RESULTS
TGF-β1 inhibits HT-29 cell proliferation, but has no effect on SW480 cell proliferation; different concentrations of TGF-β1 did not induce apoptosis in HT-29 and SW480 cells. PT attenuates TGF-β1-induced elongated, fibroblast-like shape changing in cells. PT inhibits TGF-β1-induced cell migration and cell invasion. In addition, other EMT markers such as β-catenin, Vimentin, Snail, and Slug were suppressed by PT, while E-cadherin was increased by PT.
CONCLUSIONS
Our findings show that PT inhibits TGF-β1-induced EMT by suppressing the expression of the mesenchymal protein and increasing expression of the epithelial protein. These findings suggest a novel approach for CRC treatment by suppression of TGF-β1-induced EMT.

Keyword

Transforming growth factor beta 1; Epithelial-mesenchymal transition; Parthenolide; Colorectal neoplasms

MeSH Terms

Apoptosis
Blotting, Western
Cadherins
Cell Line
Cell Movement
Cell Proliferation
Cell Survival
Colorectal Neoplasms*
Epithelial-Mesenchymal Transition*
Flow Cytometry
Gastropoda
HT29 Cells
Humans
Microscopy, Phase-Contrast
Phosphotransferases
Snails
Transforming Growth Factors*
Vimentin
Wounds and Injuries
Cadherins
Phosphotransferases
Transforming Growth Factors
Vimentin

Figure

• 

  Fig. 1. Effect of transforming growth factor β1 (TGF-β1) on cell proliferation and apoptosis in colorectal cancer cell lines. HT-29 and SW480 cells were treated with different concentrations of TGF-β1 or with 5 ng/mL TGF-β1 combined with different concentrations of parthenolide (PT) for 48 hours. Subsequently, the cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (A, B) and cell apoptosis (sub-G1 fraction) was detected by flow cytometry (C, D). Data are shown as the mean±SD and based on 3 independent experiments. aP<0.01.

• 

  Fig. 2. Parthenolide (PT) attenuates transforming growth factor β1 (TGF-β1)-induced elongated, fibroblast-like shape changing in colon cancer cell lines. Phase-contrast photomicrographs of control cells, cells treated with 5 ng/mL TGF-β1, cells treated with 5 ng/mL TGF-β1+5 μM PT for 48 hours.

• 

  Fig. 3. Parthenolide (PT) represses transforming growth factor β1 (TGF-β1)-induced cell migration and invasion in colon cancer cell lines. HT-29 and SW480 cells were treated with 5 ng/mL TGF-β1 or 5 ng/mL TGF-β1+5 μM PT for 48 hours, and subsequently, the changes in migratory capacity were measured by the (A) wound healing assay and (B) Transwell migration assay. Scratch closure was monitored for 48 hours; microscopic images were taken at 0- and 48-hours post-scratching is shown. The percentage of wound area is shown in the histogram (A). The result is presented as the mean±SD, and the graph bar represent the mean±SD of 3 independent experiments. aP<0.05.

• 

  Fig. 4. Parthenolide (PT) inhibits transforming growth factor β1 (TGF-β1) induces epithelial-mesenchymal transition pathway in colon cancer cell lines. Western blotting analysis of all protein expression in total lysates of untreated cells and cells treated with 5 ng/mL TGF-β1 or 5 ng/mL TGF-β1+5 μM PT for 48 hours by the indicated primary antibodies. Actin was used as a loading control. The results represent the mean of their independent experiments. Values represent mean±SD. Significant difference versus control group. aP<0.05, bP<0.01, and cP<0.001.

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