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MHP-1 inhibits cancer metastasis and restores topotecan sensitivity via regulating epithelial-mesenchymal transition and TGF-[beta] signaling in human breast cancer cells.

ABSTRACT

Background: Cordyceps has long been used to treat cancer. However, its pharmacologically active components as well as the molecular mechanisms underlying its effects are still unclear.

Purpose: To investigate the effect of MHP-1, a newly isolated polysaccharide from Mortierella hepialid (the asexual structure of C. sinensis), on breast cancer metastasis.

Study design: The effect of MHP-1 on breast cancer cell migration, epithelial-mesenchymal transition (EMT) and TGF-[beta] signaling were investigated in vitro and in vivo. The effect of MHP-1 against topotecanresistant MCF-7 cells that developed an EMT-like phenotype was also examined.

Methods: The in vitro effect of MHP-1 on breast cancer cell proliferation and migration was evaluated by CCK8 and transwell assay. Morphological changes were observed and EMT markers were detected by western blot. The production of MMPs was measured by quantitative PCR and ELISA assay. To further investigate the mechanism that MHP-1 inhibited breast cancer EMT, western blot, ELISA, luciferase reporter gene assay, siRNA, quantitative PCR, immunohistochemistry, and xenograft tumor model were performed. Results: MHP-1 inhibited breast cancer cell migration but did not cause any cytotoxicity. MHP-1 significantly surpressed breast cancer EMT, and slightly decreased MMP-9 secretion. TGF-[beta] signaling was selectively inhibited after MHP-1 treatment, and other EMT-related pathways, like Wnt and Notch, were not affected. MHP-1 reduced the secretion of TGF-[beta]1, but rarely affected other EMT-induced cytokines. Dual luciferase assay and Smad2/3 phosphorylation analysis indicated that MHP-1 suppressed TGF-[beta] signaling. We further showed that MHP-1 restored sensitivity in topotecan (TPT)-resistant MCF-7 cells that developed an EMT-like phenotype. Similarly, the effect of TPT on resistant MCF-7 cells was also increased either by ALK5 (TGF[beta]RI) siRNA or by a small molecular inhibitor of ALK5, SB-431542. MHP-1 inhibited breast cancer metastasis in the MDA-MB-231 xenograft model, and the immunohistochemical staining showed dramatically decreased expression of ALK5 and vimentin, and increased expression of E-cadherin. Conclusion: MHP-1 significantly inhibited breast cancer metastasis and restored drug sensitivity in TPT-resistant cells via down-regulation of TGF-[beta] signaling and EMT program. The combination of non-toxic agents like MHP-1 and current anti-cancer drugs should be considered in the future treatment of cancer.

Keywords:

MHP-1

Cancer metastasis

EMT

TGF-[beta]

Topotecan resistance

Introduction

Epithelial-mesenchymal transition (EMT) is a biologic process that allows polarized epithelial cells to undergo multiple biological changes to acquire a mesenchymal cell phenotype (Kalluri and Weinberg, 2009). In the phenotypic change, cells undergo morphological changes, lose epithelial properties and exhibit mesenchymal characteristics. EMT generates functionally distinct cell types with increased capacity for cell migration (Tiwari et al., 2012). In addition, a large body of evidence suggests that EMT plays a critical role in different aspects of cancer progression, such as metastasis, stem cell traits, and chemoresistance (Wang et al., 2011). Hallmarks of cells undergoing EMT include disruption of cell-cell junctions, loss of epithelial markers, gain of mesenchymal markers, and alterations in ECM production. A number of cytokines, especially EGF, HGF, VEFG, PDGF and TGF-[beta], are found to initiate and activate EMT program (Thiery and Sleeman, 2006), therefore making them attractive targets for therapeutic purposes.

Cordyceps, one of a traditional Chinese medicine, is a potential harbor of natural drugs (Gu et al., 2007). Convincing scientific information has demonstrated significant pharmacological properties of Cordyceps, such as hepatoprotective, antioxidant, anti-cancer and immune boosting effects (Das et al., 2010; Yue et al., 2013). Cordyceps contains large amounts of polysaccharides. Although the pharmacologically active components of Cordyceps are still unclear, polysaccharides have been proposed to be important active constituents (Zhou et al., 2009). Cordyceps polysaccharides show hypoglycemic effects by increasing circulating insulin level in diabetic animals (Li et al., 2006). Cordyceps polysaccharides are also demonstrated to inhibit cancer cell growth (Rao et al., 2010; Zhu et al., 2013), and activate macrophages by converting M2 macrophage to Ml phenotype (Chen et al., 2012). However, the primary active ingredients of Cordyceps are not identified yet, and a detailed understanding of the underlying molecular mechanism remains elusive.

We previously reported a newly isolated neutral polysaccharide (MHP-1) from Mortierella hepiali, the asexual structure of C. sinensis (Wang et al., 2013). The molecular weight of MHP-1 was determined to be 8.8 x 103 Da, and monosaccharide analysis showed that MHP-1 was composed of mannose, galactose, and glucose at the ratio of 5:2:3. MHP-1 contained a repeating unit, as shown in Fig. 1. We found MHP-1 inhibited breast cancer cell migration, but did not affect cancer cell proliferation. Presently, it is considered that the anticancer effects of fungal polysaccharides arise from the activation of the body's immune system rather than direct cytocidal effects (Zhou et al., 2007), and the molecular mechanism of Cordyceps polysaccharides against cancer metastasis is unknown. In the current study we investigated the effect of MHP-1 on breast cancer cell migration and EMT, and identified MHP-1 as an effective inhibitor of TGF-[beta] signaling. We also showed that MHP-1 restored the sensitivity of resistant MCF-7 cells to topotecan by inhibition of EMT. We finally demonstrated that MHP-1 reduced breast cancer metastasis in immunodeficient nude mice, suggesting a potential therapeutic use of MHP-1 in the treatment of breast cancer.

Materials and methods

Chemicals and regents

The isolation and purification of MHP-1, and the NMR spectroscopy were previously reported (Wang et al., 2013). RPMI-1640, DMEM, fetal calf serum (FCS), and Lipofectamine 2000 were from Gibco/Invitrogen (Carlsbad, CA, USA). On-Target plus siRNAs and Dharmafect Duo transfection reagents were obtained from Dharmacon (Lafayette, CO, USA). TGF-[beta]1, bFGF, PDGF, HGF, VEGF ELISA kits and recombinant human TGF-[beta]1 were bought from R8)D Systems (Minneapolis, MN, USA). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SB-431542 and topotecan were purchased from Sigma (St. Louis, MO, USA).

Cell culture

Human breast cancer MDA-MB-231 cells and human breast epithelial cell line MCF10A were cultured as monolayers in RPMI1640 supplemented with 10% FCS. Human breast carcinoma MCF-7 and normal human fibroblasts MRC-5 were maintained in DMEM with 10% FCS. The topotecan-resistant MCF-7 cells were established by stepwise drug selection from the parental cells and maintained in 2 [micro]M Topotecan for at least 3 months.

Cell viability assays

A cell counting kit-8 assay (CCK8; Dojindo, Kumamoto, Japan) was used to evaluate relative cell viability. Cells (2 x [10.sup.3] cells per well) were seeded into 96-well microplates. After incubation for 24 h, culture medium was replaced by 10% FCS-medium containing the drug concentration indicated. After a further incubation for 24 h, 10 [micro]l of CCK-8 solution was added, cells were incubated for a further 4 h, and then absorbance at 450 nm was measured using an MRX 11 microplate reader (Dynex, Chantilly, VA, USA).

Migration assay

Cell migration assays were conducted in a double chamber transwell as previously described (Lin et al., 2012). The positive chemoattractant was 5% FCS; the negative chemoattractant was base medium alone.

Quantitative PCR

Total RNA was isolated from cultured cell lines using TRIzol reagent (Invitrogen). Quantitative polymerase chain reaction (PCR) was carried out on an iCycler Real-time PCR Detection System (BioRad, Hercules, CA, USA) as previously described (Lin et al., 2012). The primers used for quantitative PCR were as follows: MMP-2 forward

5'-CTTCTTCCCTCGCAAGCC-3'; reverse 5' ATGGATTCGAGAAAACCG-3,; MMP-9 forward 5'ACGCAGACATCGTCATCC-3'; reverse 5'-AACCGAGTTGAACCACG-3'; and 18S as internal control: forward 5'-GTAACCCGTTGAACCCCATT3'; reverse 5'-CCATCCAATGGGTAGTAGCG-3,.

Western blotting

Western blotting analysis was done as previously described (Lin et al., 2009a). Blots were incubated with appropriate antibodies, as indicated in the figure legends. Immunoreactive proteins on the membrane were visualized by enhanced chemiluminescence Western blotting detection reagents (Amersham, UK).

Enzyme-linked immunosorbent assay (ELISA)

Cells were treated with different concentrations of MHP-1 for 48 h, and the supernatants were harvested. Each supernatant was centrifuged at 2000 g and stored at -70 [degrees]C until analysis. Enzyme-linked immunosorbent assay was performed according to the manufacturer's instructions (R8jD Systems).

Luciferase reporter gene assay

The [(CAGA).sub.12] was cloned into the pGL-3 basic vector (Promega, Madison, WI, USA), between the Kpnl and Nhel site, immediately 5' upstream of the luciferase gene. Transfections were done using Lipofectamine 2000 as previously described (Lin et al., 2012). Briefly, breast cancer cells were cotransfected with reporter plasmids and renilla luciferase reporter vector pRL-TK. After 6h, the transfection mixture was replaced with fresh medium and cells were allowed to recover for 18 h. Cells were then treated with different concentrations of MHP-1, and luciferase activities were determined and data were normalized with respect to renilla luciferase activity.

RNA interference

On-Target plus siRNAs targeting human ALK5 (L-003929-000005) and snail (L-010847-01-0005) were purchased from Dharmacon (Lafayette, CO) and transfected using Dharmafect Duo transfection reagents, following the manufacturer's instructions. OnTarget plus GAPDH siRNA (D-001830-01) was the positive control and non-silencing On-Target (D-001810-10) was the negative control. ALK5 and snail knockdown were confirmed by quantitative PCR and western blot.

Plasmids construction and cell transfection

Full-length cDNA for snail was amplified and cloned into vector PCR4-TOPO. Cloned fragments were recovered and ligated into pcDNA3.1+ (Invitrogen). DNA transfectants were prepared using a commercially available kit (Endofree Maxi-Prep; QIAGEN). Transfections were done using Lipofectamine 2000 transfection reagents as previously described (Lin et al., 2012). Cells transfected with empty vectors (pcDNA3.1+) were used as control.

Animal study

Nude mice (body weight ~18g) were purchased from Shanghai Laboratory Animal Center, Chinese Academy of Science. Mice were maintained according to the guidelines for the welfare and use of animals in cancer research in a temperature-controlled room (22 [degrees]C). MDA-MB-231 cells (2 x [10.sup.6]) were transplanted into the second right mammary fat pads. After tumor volumes reached 50100 [mm.sup.3], tumor-bearing mice were treated with different doses (20, 40 and 60mg/kg) of MHP-1 (i.v.) every other day for 4 weeks. 10 mg/kg Taxol served as the positive control and saline (vehicle) was the negative control. To perform the cell extravasation assay, 2x[l0.sup.5] MDA-MB-231 cells were pretreated with 10 [micro]M MHP-1 for 24 h, and then injected into the tail vein of nude mice. After two weeks, animals were sacrificed and metastatic nodules on lung surfaces were counted.

Immunohistochemical analysis

The expression of ALK5, E-cadherin and vimentin within MDAMB-231 xenografts were detected by immunohistochemical techniques. Formalin-fixed and paraffin-embedded tumor samples were heated at 58 [degrees]C for 45 min. They were washed with xylene three times for 5 min each, followed by washes with 100%, 95%, and 75% ethanol; they were then rinsed with PBS. The detailed staining procedure was as previously described (Lin et al., 2009a). Stained tumor samples were analyzed independently by at least two pathologists.

Statistics

Results were mean [+ or -] SEM of at least three independent experiments. Unpaired Student's t-test and Spearman correlation coefficient analysis were used. P values were two-sided: 0.05 was considered statistically significant.

Results

Effect of MHP-1 on breast cancer cell proliferation and migration

The in vitro effect of MHP-1 on the viability of MDA-MB-231 and MCF-7 breast cancer cells was evaluated using the CCK8 assay. The human breast epithelial cell line MCF10A and normal human fibroblasts (MRC-5) were also investigated. The results revealed that MHP-1 had no significant inhibition on the growth of both breast cancer cells and normal human cells (Fig. 2A).

We next explored the effect of MHP-1 on breast cancer cell migration. To address this issue, the classic double-chamber transwell assay was employed. As shown in Fig. 2B-D, we observed significant decreases in migrated cell numbers in MDA-MB-231, MCF7 and MDA-MB-468 cells treated with different concentrations of MHP-1.

MHP-1 suppressed breast cancer cell EMT

The most comprehensive theory describing how cancer cells acquire metastatic potentials is EMT. The findings that MHP-1 inhibited breast cancer cell migration prompted us to assess the impact of MHP-1 on breast cancer cell EMT. As shown in Fig. 3A, when exposed to 10 [micro]M MHP-1 for 48 h, mesenchymal-like MDA-MB231 cells underwent cellular phenotypic changes, in which most cells lost their spindle-like shape. Along with the morphological changes, the expression of epithelial markers (E-cadherin and ZO1) were increased, whereas the expression of mesenchymal markers (vimentin and fibronectin) were greatly decreased (Fig. 3B). The expression of snail, a transcription factor that is essential for EMT, was significantly down-regulated. MHP-1 also moderately inhibited the protein levels of slug (Fig. 3B).

Consistent with previous reports, treatment with 5 ng/ml TGF-[beta]1 in luminal MCF-7 cells induced EMT. MHP-1 reversed TGF-[beta]1-induced EMT as shown by both morphological changes (Fig. 3C) and expression profiles of EMT markers and transcription factors (Fig. 3D). Furthermore, 5 ng/ml TGF-[beta]1 induced an increase of cell migration in both MDA-MB-231 and MCF-7 cells, which was also inhibited by MHP-1 (Fig. 3E and F). These results demonstrated that MHP-1 inhibited breast cancer cell EMT.

To link EMT with the ability of cancer cell migration, we inhibited the expression of snail in MDA-MB-231 cells through snail siRNA (Suppl. Figs. 1). The transcription factor snail is demonstrated to be an EMT driver in breast cancer (Mezencev et al., 2016; Smith et al., 2014). MHP-1 also significantly inhibited the expression of snail. Silencing of snail reduced the number of MDA-MB231 cells migrated to the lower chamber in the transwell assay (Fig. 3G). On the contrary, overexpression of snail in MCF-7 cells (Suppl. Figs. 2) stimulated cell migration (Fig. 3H). Taken together, these results showed that cancer cell migration was closely associated with EMT, and MHP-1 might decrease breast cancer cell migration by inhibition on EMT.

Effect of MHP-1 on MMP secretion

Except for EMT, extracellular matrix (ECM) also plays a critical role in cancer metastasis. Cancer cells secrete matrix metalloproteinases (MMPs), such as MMP-2 and MMP-9, which degrade the ECM and facilitate cancer cell invasion. We further examined the effect of MHP-1 on the production of MMP-2 and MMP-9 in breast cancer cells. MDA-MB-231 and MCF-7 cells were treated with different concentrations of MHP-1 for 24 h, and the relative gene expression of MMP-2 and MMP-9 was measured by quantitative PCR. As shown in Fig. 4A, MHP-1 moderately decreased the expression of MMPs. 10 [micro]M MHP-1 reduced the gene expression of MMP2 and MMP-9 by approximately 20% and 35% in MDA-MB-231 cells. In MCF-7 cells, 10 [micro]M MHP-1 obtained a 30% and 50% decrease of MMP-2 and MMP-9 gene expression, respectively. However, no significant changes of MMP-2 secretion were detected in both cell lines after MHP-1 treatment, as evidenced by ELISA assay (Fig. 4B). 10 [micro]M MHP-1 slightly decreased the secretion of MMP-9 (~15%) in MDA-MB-231 cells (Fig. 4B), reflecting a weak effect of MHP-1 to degrade the ECM. These data probably ruled out a critical role of MMPs in the anti-metastatic effect of MHP-1.

MHP-1 suppressed TGF-[beta] signaling

Cellular signaling pathways, like TGF-[beta], Wnt and Notch, are believed to be responsible for the underlying molecular mechanisms of EMT. To determine which signaling pathway was mediated by MHP-1, we first investigated the expressions of [beta]-catenin, non-phospho-[beta]-catenin, cleaved notchl, TGF[beta]RI and TGF[beta]RII in MDA-MB-231 cells. A dose-course analysis showed that the expression levels of [beta]-catenin, non-phospho-[beta]-catenin and cleaved notchl were not affected. MHP-1 significantly decreased TGF[beta]RI protein levels, and modestly reduced TGF[beta]RII expression in MDA-MB-231 cells (Fig. 5A). In luminal-like MCF-7 cells, treatment with MHP-1 also reduced the expression of TGF[beta]RI in a dose-dependent manner. TGF[beta]BII protein levels were much less efficiently down-regulated (Fig. 5B). We next measured the production of TGF-[beta]1 by enzyme-linked immunosorbent assay (ELISA). As shown in Fig. 5C, the levels of TGF-[beta]1 secreted by MDA-MB-231 cells were found down-regulated by 10 [micro]M MHP-1, while the expression of other EMT-induced cytokines, bFGF, PDGF, HGF and VEGF, were not affected. We finally showed MHP-1 reduced the production of TGF-[beta]1 in a dose-dependent manner (Fig. 5D).

We next determined the effect of MHP-1 on TGF-[beta] signaling. The luciferase reporter gene assay was performed using pGL3-[(CAGA).sub.12], a transcriptional reporter that can be driven by TGF-beta]/Smad3/Smad4 pathway (Chen et al., 2011). MDA-MB-231 and MCF-7 cells were transiently cotransfected with pGL3-[(CAGA).sub.12] and pRL-TK and exposed to 5ng/ml TGF-[beta]1 and different concentrations of MHP-1 for 24 h. We found that MHP-1 dose-dependently reduced the activity of TGF-[beta]-induced pGL3-[(CAGA).sub.12] transcriptional reporter activity (Fig. 6A). MCF-10A cells, an immortalized cell line that has been used extensively as an ideal model to assess TGF-[beta]-induced EMT, also showed lower TGF-[beta]-dependent gene transcriptional activity after MHP-1 treatment. To further investigate MHP1-mediated intracellular transduction mechanisms, MDA-MB-231 and MCF-7 cells were treated with MHP-1 under TGF-[beta]1 stimulation and phosphorylation of Smad2 and Smad3 were detected. As shown in Fig. 6B, MHP-1 impaired Smad2 and Smad3 phosphorylation in response to 5ng/ml TGF-[beta]1. Altogether, these data suggested that MHP-1 inhibited TGF-[beta] signaling in breast cancer cells.

MHP-1 restored sensitivity in topotecan-resistant MCF-7 cells by inhibition of EMT

The correlation between EMT and drug resistance has been implicated (Singh and Settleman, 2010). Inhibition of EMT is reported to decrease drug resistance in chemotherapy (Arumugam et al., 2009). As MHP-1 inhibited EMT in breast cancer cells, we were inspired to hypothesize that MHP-1 might restore sensitivity in resistant cancer cells that developed an EMT-like phenotype. MCF-7 cells consistently exposed to 2 [micro]M topotecan (TPT) displayed obvious EMT characters (Suppl. Figs. 3A and B), and acquisition of drug resistance was also confirmed (Suppl. Figs. 3C and D). As expected, treatment of resistant cells (TPT-R) with MHP-1 reversed the EMTlike phenotype as demonstrated by cellular morphological changes (Fig. 7A) and expression profiles of EMT markers (Fig. 7B). Cell viability assay was used to determine the synergistic effect of MHP-1 and TPT. As shown in Fig. 7C, the combination of these two compounds achieved significantly improved activities. Notably, addition of 10 [micro]M MHP-1 greatly decreased the [IC.sub.50] value of TPT against TPT-R cells, from 22.12 [+ or -] 2.84 [micro]M to 7.95 [+ or -] 1.33 [micro]M (Fig. 7D). Consistent with the above results, MHP-1 restored the ability of TPT to induce apoptosis in TPT-R cells, as demonstrated by increasing the cleavage of poly (ADPribose) polymerase (PARP) (Fig. 7E).

To further confirm the role of TGF-[beta] signaling in the acquisition of drug resistance, we inhibited TGF[beta]RI/ALK5 expression in TPT-R cells by more than 80% through ALK5 siRNA (Suppl. Figs. 4). Silencing of ALK5 significantly increased the inhibitory effect of TPT on TPT-R cells; the data presented in Fig. 8A demonstrated that the [IC.sub.50] value reduced from 22.12 [+ or -] 2.84 [micro]M to 6.75 [+ or -] 1.33 [micro]M. An inhibitor of ALK5, SB-431542, was also employed. The same as ALK5 siRNA, 10 [micro]M SB-431,542 also sensitized TPT-R cells to TPT (Fig. 8B). Western blot analysis showed that the cleavage of PARP caused by TPT was greatly enhanced either by siALK5 or by SB431542 (Fig. 8C). These data demonstrated a critical role of TGF-[beta] signaling in regulating breast cancer cell resistance to TPT, and indicated that the effect of MHP-1 to restore drug sensitivity was likely caused, at least in part, by changes of TGF-[beta] signaling.

MHP-1 inhibited breast cancer metastasis in vivo

We evaluated the effect of MHP-1 in vivo in a xenograft tumor model by subcutaneous inoculation of MDA-MB-231 cells into in the second right mammary fat pad area. As shown in Fig. 9A, 60mg/kg MHP-1 inhibited the growth of primary tumors, as evidenced by the tumor growth curve. No visible side effects or changes in body weight of the mice were observed (Fig. 9B). We then examined whether MHP-1 could inhibit breast cancer metastasis. MDA-MB-231 cells were pretreated with 10 [micro]M MHP1 for 24 h, and then injected into the tail vein of nude mice. MHP1-treated cells produced significantly fewer colonies in the lung compared to untreated control cells (Fig. 9C). The immuno-histochemical analysis revealed decreased expression of ALK5 and vimentin, and increased expression of E-cadherin in primary tumors from mice treated with 60 mg/kg MHP-1 (Fig. 9D).

Discussion

Cordyceps is a well-described remedy that has been used in Traditional Chinese Medicine for over 700 years. Although Cordyceps can inhibit cancer metastasis, little is known about the active ingredients as well as the mechanism underlying this effect. Most studies regarding the anti-tumor effect of Cordyceps emphasize on the modulation of innate immune responses. Here, we reported that MHP-1, a newly isolated neutral polysaccharide from C. sinensis, inhibited breast cancer metastasis and restored sensitivity in Topotecan-resistant MCF-7 cells. We also proposed a novel mechanism in which MHP-1 inactivated TGF-[beta] signaling and subsequently suppressed EMT in breast cancer cells.

Impressively, MHP-1 had no significant inhibition on the growth of breast cancer cells while at the same time blocked TGF-[beta] signaling, which could promote tumor cell proliferation by stimulating the production of autocrine mitogenic factors, e.g., PDGF (Bruna et al., 2007). A potential interpretation is that the effect of MHP1 on cancer cell proliferation depends on its effect on mitogenic factors; however, 10 [micro]M MHP-1 did not significantly affect the secretion of these factors, including PDGF, VEGF, bFGF and HGF, as evidenced in Fig. 5C, therefore these data might account for the fact that MHP-1 decreased breast cancer cell migration while did not dramatically affect cell viability. In fact, although Cordyceps has long been used for cancer treatment in China (Zhou et al., 2009) and the anti-cancer effect of Cordyceps has been confirmed in today's clinical researches (Niwa et al., 2013), the direct cytocidal effects of Cordyceps are not significant (Zhou et al., 2007). Cordyceps acts as an immune modulator, rather than a cytotoxic agent, in cancer therapy (Zhu et al., 1998). These reports suggest that Cordyceps may be non-cytotoxic. Thus, it made sense that MHP-1, as an active component of Cordyceps, inhibited breast cancer metastasis rather than proliferation. However, our in vivo data also showed that 60mg/kg MHP-1 moderately attenuated primary tumor growth (Fig. 9A), suggesting that high concentrations of MHP-1 might perturb pro-survival signals other than the above mentioned mitogenic factors.

Drug resistance is the main cause of the lack of chemotherapy effectiveness in most of the cancers. As with most chemotherapeutics, intrinsic and acquired drug resistance represents a hurdle that limits the success of Topotecan in cancer therapy. Licensed to be a second line anti-cancer agent, TPT suppresses DNA replication in cancer cells by inhibiting the nuclear enzyme topoisomerase I (topo 1), which plays a key role in DNA replication and transcription machinery. Presently, mechanisms of cancer cell resistance to TPT include mutations in Topo I, expression of DNA repair proteins and high rate of drug efflux (Tomicic and Kaina, 2013). To our knowledge, our study is the first demonstration that TPT resistance is associated with EMT. Interestingly, chronic TPT treatment induced EMT-like phenotype in MCF-7 cells, which differs entirely with our previous report that TPT inhibited cancer cell migration by down-regulation of CC chemokine receptor 7 and MMPs (Lin et al., 2009b). How could TPT exert either anti-metastatic or pro-metastatic effects in different cells? The discrepancy may indicate chronic TPT treatment activates pro-survival signals, e.g., TGF-[beta] signaling, which in turn triggers an EMT-like phenotype. Inhibition of TGF-[beta] signaling by either ALK5 siRNA or SB-431542 increased the inhibitory effect of TPT (Fig. 8), demonstrating a critical role of TGF-[beta] signaling in regulating breast cancer cell resistance to TPT. Our unpublished data also revealed that TPT-resistant MCF-7 cells showed increased levels of autophagy, which might protect cells from apoptosis and promote EMT through a TGF-[beta]/Smad3 signaling-dependent manner (Li et al., 2013).

Generally, basal release of TGF-[beta] by normal tissues may suffice for the maintenance of homeostasis. However, TGF-[beta] is abundantly secreted in cancer. TGF-[beta] levels have been demonstrated to highly correlate with tumor progression (Bierie and Moses, 2006). Recent studies have demonstrated that TGF-[beta] dictates neutrophil polarization in the tumor microenvironment, which may be detrimental for the host and beneficial for tumor growth, invasion and metastasis (Piccard et al., 2012). Importantly, tumor stroma cells, like leukocytes, macrophages, fibroblasts and bone marrow-derived endothelial, mesenchymal, and myeloid precursor cells, also produce TGF-[beta] and thus may be suspected sources of the accumulation of TGF-[beta] in tumor microenvironment (Yang et al., 2008). Whether MHP-1 abrogates TGF-[beta] signaling in these tumor-infiltrating cells, thereby repressing tumor progression by regulation of host-tumor interactions, remains to be investigated.

Conclusion

Our results demonstrated that MHP-1 significantly inhibited breast cancer metastasis and restored drug sensitivity in TPT-resistant cells via down-regulation of TGF-[beta] signaling and EMT program. As there is growing interests in TGF[beta]-based cancer therapy, the combination of non-toxic agents like MHP-1 and current anti-cancer drugs should be considered in the future treatment of cancer.

http://dx.doi.org/10.1016/j.phymed.2016.06.013

ARTICLE INFO

Article history:

Received 19 January 2016

Revised 14 June 2016

Accepted 18 June 2016

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was financially supported by the National Natural Science Foundation of China (81302794, 81573456), the National High Technoligy Research and Development Program of China (2014AA022208) and the Project of Science and Technology of Nanjing Science Committee (201303046).

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2016.06.013.

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Sensen Lin (a, 1), Xiaodan Lyu (a, 1), Jun Yu (b, 1), Li Sun (a), Danyu Du (a), Yanqi Lai (a), Hongyang Li (a), Ying Wang (c), Luyong Zhang (a), Hongping Yin (c), **, Shengtao Yuan (a), *

(a) Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China

(b) Jiangsu Cancer Hospital, Nanjing 210009, China

(c) School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China

Abbreviations: EMT, epithelial-mesenchymal transition; TGF-[beta], transforming growth factor-[beta]; MHP-1, an isolated polysaccharide from Mortierella hepialid; CCK8, cell counting kit-8 assay; TPT-R, topotecan-resistant MCF-7 cells; ALK5, activin receptor-like kinase 5.

* Corresponding author. Fax: +86 025 83271142.

** Corresponding author. Fax: +86 025 83271249.

E-mail addresses: yinhongping63@hotmail.com (H. Yin), 13914798635@163.com (S. Yuan).

(1) Sensen Lin, Xiaodan Lyu and Jun Yu contributed equally to this work.

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Title Annotation:Original article
Author:Lin, Sensen; Lyu, Xiaodan; Yu, Jun; Sun, Li; Du, Danyu; Lai, Yanqi; Li, Hongyang; Wang, Ying; Zhang,
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:Sep 15, 2016
Words:5355
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