Pien Tze Huang Overcomes Multidrug Resistance and Epithelial-Mesenchymal Transition in Human Colorectal Carcinoma Cells via Suppression of TGF-β Pathway

Abstract

The traditional Chinese medicine formula Pien Tze Huang (PZH) has long been used as a folk remedy for cancer. To elucidate the mode of action of PZH against cancer, in the present study we used a 5-FU resistant human colorectal carcinoma cell line (HCT-8/5-FU) to evaluate the effects of PZH on multidrug resistance (MDR) and epithelial-mesenchymal transition (EMT) as well as the activation of TGF-β pathway. We found that PZH dose-dependently inhibited the viability of HCT-8/5-FU cells which were insensitive to treatment of 5-FU and ADM, demonstrating the ability of PZH to overcome chemoresistance. Furthermore, PZH increased the intercellular accumulation of Rhodamine-123 and downregulated the expression of ABCG2 in HCT-8/5-FU cells. In addition, drug resistance induced the process of EMT in HCT-8 cells as evidenced by EMT-related morphological changes and alteration in the expression of EMT-regulatory factors, which however was neutralized by PZH treatment. Moreover, PZH inhibited MDR/EMT-enhanced migration and invasion capabilities of HCT-8 cells in a dose-dependent manner and suppressed MDR-induced activation of TGF-β signaling in HCT-8/5-FU cells. Taken together, our study suggests that PZH can effectively overcome MDR and inhibit EMT in human colorectal carcinoma cells via suppression of the TGF-β pathway.

Figure 1

Effect of PZH on the viability of 5-FU resistant colorectal cancer cells. Cell viability was determined by MTT assay after HCT-8/5-FU and parental HCT-8 cells were treated with the indicated concentrations of 5-FU (a), ADM (b), and PZH (c) for 48 h. The data were normalized to the viability of control cells. Data are averages with S.D. (error bars) from at least three independent experiments. ^# P < 0.05, versus controls; ^* P < 0.05, versus HCT-8/5-FU cells.

Figure 2

Effect of PZH on accumulation of Rhodamine-123 in HCT-8/5-FU cells. Intercellular accumulation of Rhodamine-123 was determined by FACS after treated with the indicated concentrations of PZH for 24 h (a). The accumulation of Rhodamine-123 present by relative values was averaged with S.D. (error bars) from at least three independent experiments and is summarized as histograms in (b). ^* P < 0.05, versus untreated HCT-8/5-FU cells.

Figure 3

Effect of MDR on the morphological changes, the expression of ABCG2, EMT-related factors, and the activation of TGF-β pathway. (a) Morphology of HCT-8/5-FU and parental HCT-8 cells was observed using phase-contrast microscopy. The photographs were taken at a magnification of 400x. Images are representative of three independent experiments. (b-c) The mRNA and protein expression levels of ABCG2, E-cadherin, N-cadherin, ZEB1, ZEB2, TGF-β, and SMAD4 in parental HCT-8 and HCT-8/5-FU cells were determined by RT-PCR and Western Blot analyses. GAPDH or β-actin was used as the internal control for RT-PCR or Western Blot, respectively. Images are representatives of three independent experiments.

Figure 4

Effect of PZH on the migration of HCT-8/5-FU cells. HCT-8/5-FU cells were treated with indicated concentrations of PZH for 24 h. (a) The migration of HCT-8/5-FU or parental HCT-8 cells was determined using transwell cell culture chambers. Cells were stained with crystal violet; the photographs were taken at a magnification of 200x. (b) The average number of migrated cells was counted in 3 randomly selective fields. The data were normalized to the migration of HCT-8/5-FU cells (100%). Data are averages with S.D. (error bars) from three independent experiments. ^# P < 0.05, versus parental HCT-8 cells; ^* P < 0.05, versus HCT-8/5-FU cells without PZH treatment.

Figure 5

Effect of PZH on the invasion of HCT-8/5-FU cells. HCT-8/5-FU cells were treated with indicated concentrations of PZH for 24 h. (a) The invasion of HCT-8/5-FU or parental HCT-8 cells was determined using transwell cell culture chambers with membranes (8 μM) coated with Matrigel matrix. Cells were stained with crystal violet; the photographs were taken at a magnification of 200x. (b) The average number of invaded cells was counted in 3 randomly selective fields. The data were normalized to the invasion of HCT-8/5-FU cells (100%). Data are averages with S.D. (error bars) from three independent experiments. ^# P < 0.05, versus parental HCT-8 cells; ^* P < 0.05, versus HCT-8/5-FU cells without PZH treatment.

Figure 6

Effect of PZH on the expression of ABCG2, EMT-related factors, and activation of TGF-β pathway in HCT-8/5-FU cells. HCT-8/5-FU cells were treated with indicated concentrations of PZH for 24 h. The mRNA (a) and protein (b) expression levels of ABCG2, E-cadherin, N-cadherin, ZEB1, ZEB2, TGF-β, and SMAD4 in HCT-8/5-FU cells were determined by RT-PCR and Western Blot analyses. GAPDH or β-actin was used as the internal control for RT-PCR or Western Blot, respectively. Images are representatives of three independent experiments.