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

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.


Introduction
Colorectal cancer (CRC) is a serious public health problem, with more than one million new cases and over a half million deaths worldwide each year [1,2]. Although surgical resection to completely remove the cancer offers the best prognosis for long-term survival, a substantial portion of CRC patients is not suitable for surgery because of the presenting of metastasis at the time of diagnosis; and surgery cannot always extirpate cancer recurrence [3,4]. Therefore, chemotherapy, especially 5-fluorouracil-(5-FU-) based regimens, remains an important therapeutic option for advanced CRC. However, due to multidrug resistance and an unacceptable level of toxicity against normal cells, systemic chemotherapy using 5-FU-based regimens produces objective response rates of only 10-20% [5][6][7][8]. These problems highlight the urgent need for the development of novel therapeutic strategies and agents.
Multidrug resistance (MDR), the cellular resistance to numerous drugs differing in mechanisms of action and/or chemical structures, is a major cause of failure of cancer chemotherapy. MDR is mediated by multiple mechanisms, including overexpression of energy-dependent transporters that eject anticancer drugs from cells and acquisition of epithelial-mesenchymal transition (EMT) [9][10][11][12]. ATPbinding cassette (ABC) family of transporter proteins can 2 Evidence-Based Complementary and Alternative Medicine pump various xenobiotics out of the cell, reducing the intracellular accumulation of chemotherapeutic drugs [13,14]. As a half-transporter of the G subfamily of ABC transporter, breast cancer resistance protein (BCRP/ABCG2) is known to play a crucial role in multidrug resistance. The overexpression of ABCG2 protects cells from xenobiotic-and toxin-induced damages by increasing efflux of these compounds [15]. Thus, inhibition of ABC transporter activity is a potential approach to overcome the chemoresistance [16].
EMT is a biological process in which epithelial cells lose their polarity and cell-cell adhesion, and acquire migratory and invasive properties of mesenchymal cells. The process of EMT is observed during embryonic development, wound healing, organ fibrosis, and cancer progression and metastasis [17][18][19][20][21]. Epithelial and mesenchymal cells are different in both phenotype and function. Epithelial cells have an apical-basal polarity, express high levels of epithelial markers such as Ecadherin, and are closely connected to each other forming epithelial adherent junctions. In contrast, mesenchymal cells lack the cell polarity, highly express mesenchymal markers such as N-cadherin and vimentin, display a spindleshaped morphology, and interact with each other through focal points [17]. After acquiring a mesenchymal phenotype through the process of EMT, carcinoma cells obtain the capacities to invade adjacent tissues, break through the basement membrane, and eventually enter the bloodstream [22][23][24]. Furthermore, accumulating evidence has shown that the process of EMT is also strongly associated with MDR in various types of human malignancies including CRC [25][26][27][28][29][30][31][32][33][34]. Thus, EMT not only confers cancer cells, the unique advantage of migration and invasion, leading to cancer progression and metastasis, but also plays an important role in drug resistance, resulting in the failure of clinical chemotherapies.
EMT in cancer progression and metastasis is highly regulated by a diverse array of cytokines and growth factors. Prominent among these regulatory factors is the transforming growth factor (TGF-) superfamily [12,34,35] that consists of TGF-proteins, bone morphogenetic proteins (BMPs), growth differentiation factors (GDFs), and various other polypeptide morphogens [36]. The prototypic member of this superfamily is TGF-1. The activation of TGFsignaling pathway is initiated by the binding of ligands to a type II receptor, which recruits, phosphorylates, and activates a type I receptor. The activated type I receptor then phosphorylates receptor-regulated SMADs (R-SMADs, e.g., SMAD2/3) that in turn bind the coSMAD (e.g., SMAD4). The R-SMAD/coSMAD complex translocates to the nucleus to regulate the expression of target genes, including three families of transcription factors, Snail, ZEB, and bHLH families [37,38]. Upon activation, these transcription factors suppressed epithelial marker gene expression and upregulated mesenchymal gene expression leading to EMT [35]. Thus, inhibiting EMT via suppression of TGF-may represent a novel therapeutic strategy for reversing MDR.
Natural products, including traditional Chinese medicines (TCM), have recently received great interest since they have relatively few side-effects as compared to modern chemotherapeutics and have long been used to treat various diseases including cancer [39,40]. TCM formula is a complex combination of many natural products, each of which contains numerous chemical compounds. TCM formulas therefore are considered to be multicomponent and multitarget agents that may exert their therapeutic activities in a more holistic way. Pien Tze Huang (PZH) is a well-known TCM formula prescribed by a royal physician more than 450 years ago during the Chinese Ming Dynasty [41]. PZH has also been used in China and Southeast Asia for centuries as a folk remedy for various cancers. Previously, we reported that PZH inhibits colorectal cancer growth both in vivo and in vitro via promotion of cancer cell apoptosis, inhibition of cell proliferation, and tumor angiogenesis [42][43][44][45][46][47]. To further elucidate the mechanism of its antitumor activities, in the present study we evaluated the effects of PZH on MDR and EMT in a 5-FU resistant human colorectal carcinoma cell line and investigated the underlying mechanisms of its action.

Preparations of PZH. PZH was obtained from and authenticated by the sole manufacturer Zhangzhou Pien Tze
Huang Pharmaceutical Company Limited, China (Chinese FDA approval number: Z35020242). Stock solutions of PZH were prepared just before use by dissolving the PZH powder in PBS (phosphate buffered saline) to a concentration of 20 mg/mL. The working concentrations of PZH were made by diluting the stock solution in the culture medium.

Evaluation of Cell Viability by MTT Assay.
Viability of HCT-8 cells and HCT-8/5-FU cells was examined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay. Cells were seeded into 96-well plates at a density of 6 × 10 3 cells/well in 0.1 mL medium and then treated with various concentrations of 5-FU, Adriamycin (ADM), or PZH for indicated periods of time. 100 L MTT (0.5 mg/mL in PBS) was added to each well, and the samples were incubated for an additional 4 h at 37 ∘ C. The purple-blue MTT formazan precipitate was dissolved in 100 L DMSO. The absorbance was measured at 570 nm using an ELISA reader (BioTek, Model ELX800, USA).

Observation of Morphological Changes. HCT-8 cells and
HCT-8/5-FU cells were seeded into 6-well plates at a density of 5 × 10 5 cells/well in 2 mL medium. The cell morphology was observed by using a phase-contrast microscope (Leica, German). The photographs were taken at a magnification of ×400.
2.6. Measurement of Rhodamine-123 Accumulation. HCT-8/5-FU cells were seeded in 6-well plates at a density of 2.5 × 10 5 cells/mL in 2 mL medium, and then the cells were treated with indicated concentration of PZH (0-0.75 mg/mL) for 24 h. 1 × 10 6 cells were resuspended in 1 mL medium with 5 g/mL Rhodamine-123 and incubated for an additional 10 min at 37 ∘ C with 5% CO 2 . The accumulation of Rhodamine-123 was stopped by cooling on ice and cells were washed in ice-cold PBS before fluorescence-activated cell sorting (FACS) analysis. The accumulation of Rhodamine-123 was present as relative values.

Measurement of Cell Migration and Invasion by
Transwell Assay. The migration assay and invasion assay were performed using transwell cell culture chambers, coated without (Corning Life Sciences) or with Matrigel Matrix (BD Biosciences). The inserts were placed within a 24-well chamber containing 0.7 mL RPMI-1640 with 10% fetal bovine serum as a chemoattractant. As before, HCT-8 and HCT-8/5-FU cells were seeded into 6-well plates and HCT-8/5-FU cells  Table 1.  with 5% skimmed milk and probed with primary antibodies ABCG2, N-cadherin, E-cadherin, TGF-, SMAD4, ZEB1, ZEB2, and -actin (1 : 1,000) overnight at 4 ∘ C and subsequently with the appropriate HRP-conjugated secondary antibody followed by enhanced chemiluminescence detection. the data was performed with Student's -test and ANOVA. Differences with < 0.05 were considered statistically significant.

PZH Inhibits the Proliferation of 5-FU Resistant Human
Colorectal Carcinoma Cell Line HCT-8/5-FU. To evaluate MDR activity in cancer cells and therapeutic effects of PZH, HCT-8/5-FU and parental cells were exposed to different concentrations of 5-FU, ADM, or PZH for 48 h and the MTT assay was performed to determine the cell viability. As shown in Figures 1(a) and 1(b), the viability of parental HCT-8 cells was significantly decreased by treatment with 5-FU or ADM, whereas HCT-8/5-FU cell viability did not remarkably change  (Figure 1(c)), suggesting that PZH may possess the ability to overcome colorectal cancer cell chemoresistance.

PZH Inhibits Drug Efflux and ABCG2 Expression in HCT-8/5-FU Cells.
To evaluate the effect of PZH on drug efflux, Rhodamine-123 accumulation was determined as a measure of intracellular drug accumulation by FACS analysis. As shown in Figures 2(a) and 2(b), comparing with untreated control, PZH treatment increased the fluorescence intensity of Rhodamine-123, indicating that PZH is able to enhance the accumulation of Rhodamine-123 and suggesting that inhibition of drug efflux to increase the intercellular accumulation of chemotherapeutics might be one of the mechanisms for PZH to overcome MDR. To explore the underlying mechanism, we determined the expression of ABCG2 in HCT-8 cells and HCT-8/5-FU cells treated with or without PZH using RT-PCR and Western Blot analyses. As shown in Figures 3 and 6, drug resistance induced expression of ABCG2 was remarkably inhibited by PZH treatment.

PZH Inhibits MDR-Induced EMT in HCT-8/5-FU Cells.
MDR-induced EMT was evaluated by observing the morphological changes in HCT-8/5-FU cells. As shown in Figure 3(a), compared with parental cells, 5-FU resistant HCT-8 cells (HCT-8/5-FU) displayed typical EMT morphological characteristics, including loss of cell polarity, spindleshaped fibroblastoid-like morphology, and formation of pseudopodia. To further verify these results, we determined the expression of EMT-related factors in HCT-8/5-FU and parental cells using RT-PCR and Western Blot analyses. As shown in Figure 3, acquisition of drug resistance significantly decreased the mRNA and protein expression levels of epithelial marker E-cadherin, whereas those of mesenchymal markers such as N-cadherin, ZEB1, and ZEB2 were remarkably increased. However, PZH treatment profoundly neutralized the MDR-induced alteration in the expression of EMTregulatory factors in both transcriptional and translational levels ( Figure 6).

PZH Suppresses MDR-Induced Activation of TGF-Pathway in HCT-8/5-FU Cells.
To explore the underlying mechanisms of inhibitory activities of PZH on cancer MDR and EMT, we determined its effect on the activation of the TGF-pathway. As shown in Figure 3(b), acquisition of drug resistance significantly increased the mRNA expression of TGF-and SMAD4 in HCT-8 cells, which however was suppressed by PZH treatment in a dose-dependent manner (Figure 6(a)). The protein expression patterns of TGFand SMAD4 in different cell groups were similar to the patterns observed for the respective mRNA (Figures 3(c) and 6(b)), suggesting that PZH inhibits colorectal cancer cell MDR/EMT probably through suppression of TGFsignaling pathway.

Discussion
Multidrug resistance (MDR) and toxicity profoundly limit the effectiveness of currently used chemotherapeutic regimens for many human malignancies including colorectal cancer (CRC) [5][6][7][8]. Thus, the development of safer therapeutic agents with the ability to overcome the MDR could be a promising strategy to improve the effectiveness of anticancer therapies. Traditional Chinese medicines (TCM) have received recent interest since they have relatively few side-effects as compared to modern chemotherapeutics and have been used for thousands of years to clinically treat various diseases including cancer. Pien Tze Huang (PZH), a well-known TCM formula first prescribed in the Ming Dynasty, has long been used as a folk remedy for cancers in China and Southeast Asia. We recently demonstrated that PZH suppresses colorectal cancer growth in vivo and in vitro via modulation of multiple CRC-related signaling pathways, promotion of cancer cell apoptosis, and inhibition of cell proliferation and tumor angiogenesis [42][43][44][45][46][47]. These data suggest that PZH possesses a broad range of anticancer activities due to its ability to affect multiple intracellular targets. To further elucidate the antitumor mechanisms of PZH, in the present study we used a 5-FU resistant human colorectal carcinoma cell line (HCT-8/5-FU) to evaluate PZH's effects on drug resistance. We found that PZH significantly and dosedependently inhibits the viability of 5-FU and ADM resistant HCT-8/5-FU cells. Moreover, PZH increased the accumulation of Rhodamine-123 and downregulated the drug resistance induced expression of ABCG2 in HCT-8/5-FU cells, thus demonstrating the inhibitory effect of PZH on chemoresistance of colorectal cancer cells.
Since the process of epithelial-mesenchymal transition (EMT) plays important roles in drug resistance [25][26][27][28][29][30][31][32][33], we examined the effect of PZH on EMT in HCT-8/5-FU cells. Consistent with previous studies that MDR induces EMT-related properties in cancer cells [48], we observed that acquisition of drug resistance in HCT-8 cells resulted in EMT-related morphological changes (e.g., loss of cell polarity, spindle-shaped fibroblastoid-like morphology, and formation of pseudopodia) and alteration in the expression of EMT-regulatory factors, including the decrease of epithelial marker E-cadherin expression and the upregulation of expression of mesenchymal markers such as N-cadherin, ZEB1, and ZEB2. However, these EMT-related changes in HCT-8/5-FU cells were significantly neutralized by PZH treatment. Moreover, we found that drug resistance significantly enhanced the capacities of migration and invasion in colorectal cancer cells. PZH treatment significantly inhibited the migration and invasion of HCT-8/5-FU cells in a dosedependent manner. Cancer EMT is mediated by multiple intracellular pathways including TGF-signaling. To further investigate the mechanisms of inhibitory activities of PZH on cancer MDR and EMT, we determined its effect on the activation of TGF-signaling pathway. As expected, acquisition of drug resistance significantly increased mRNA and protein expression levels of TGF-and SMAD4. However, MDRinduced upregulation of TGF-and SMAD4 expression was significantly suppressed by PZH treatment, suggesting that PZH suppresses TGF-pathway in drug-resistant colorectal cancer cells.
In conclusion, we demonstrate for the first time that PZH can effectively overcome MDR and inhibit EMT in CRC cells via suppression of the TGF-pathway. Together with our previous studies that PZH suppresses the activation of several CRC-related signaling pathways including STAT3, Akt, and MAPKs, we speculate that PZH may exert its inhibitory activities on MDR and EMT in a holistic way by modulating multiple cellular signal transduction pathways. This intriguing observation should be addressed in future studies to fully elucidate the molecular mechanism of the tumoricidal activity of PZH, which may help to develop better multitarget drugs for cancer therapy.