Ruyong Formula (RYF) Attenuates the Abnormal Phenotypic Changes of Thymic Epithelial Cells in 4T1 Breast Cancer Mice

Abstract


Background
Worldwide, breast cancer is the most common cancer affecting women and remains a major health problem.It is an aggressive disease with approximately 17 million new cases each year [1].However, there is still no gold standard treatment for metastatic breast cancer, such as triple-negative breast cancer, so far in spite of great improvements have been made in detection and diagnosis [2].Especially the patients of advanced breast cancer, the tumor microenvironment and chemoradiotherapy for patients with malignant diseases often lead to serious immune and hematopoietic system damage.Immunologic dysfunction, which is di cult for patients to recover from trauma, cachexia and other problems, seriously affect the survival and survival rate of patients.However, for patients with advanced breast cancer, there is no recognized treatment methods at present.
Medicinal herbs, such as Traditional Chinese Medicine (TCM), have been used as an adjunct treatment for improving the quality of life in cancer patients and exhibit potential anti-cancer activity against various type cancer including breast cancer [3].And there are other research under way on the treatment of cancer by TCM [4][5].Ruyong Formula (RYF) has been used for thousands of years, with the earliest description being recorded in 1481 in "dan xi xin fa", (recorded the clinical experiences and prescriptions of Zhu Danxi, a famous doctor of Yuan Dynasty in China).It is composed of Radix Codonopsis, Astragalus membranaceus, Angelica Sinensis, Radix Bupleuri, Rhizoma Chuanxiong, raw Radix Paeoniae Alba, Fructus Forsythiae, Radix Glycyrrhizae, Trichosanthes kirilowii Maxim and Pericarpium Citri Reticulatae Viride.Pharmacological studies have showed that the Radix Codonopsis, Astragalus membranaceus and Angelica Sinensis in RYF have effective therapeutic effect in anti-in ammatory, antitumor, immunity enhancement and so on [6][7][8][9][10].Meanwhile, the extract of Radix Codonopsis [11], Astragalus membranaceus [12,13], Angelica Sinensis [14], raw Radix Paeoniae Alba [15], Fructus Forsythiae [16] and Trichosanthes kirilowii Maxim [17] could inhibit EMT (epithelial-mesenchymal transition), which is an important development process of cell adhesion and migration.Our primary study shown that Ruyong Formula in uence the phenotypic changes of thymic epithelial cells in 4T1 breast cancer mice, but the mechanisms of action and its effect on mouse breast cancer metastasis are still unknown [18].
In this study, we explored the effect and mechanism of action of RYF on phenotypic changes of thymic epithelial cells in breast cancer mice.We studied the changes of anti-tumor rate, pulmonary metastasis, thymus histopathology features and related proteins expression (E-acdherin, Vimentin) in 4T1 breast cancer mice after RYF treated in vivo.We evaluated whether RYF could in uence the proliferation and reverse the phenotypic changes caused by TGF-β1 in iTECs in vitro.We also examined the change of TGFβ/Smad pathway related to the EMT process.
The extract was ltered, and the extraction was repeated once.Subsequently, the ltrates were combined and evaporated under vacuum (RV8V, IKA, Werke GmbH & CO.KG, Staufen, Germany) and then lyophilized with a freeze dryer (VirTis, AD 2.0 EL, SP Scienti c, USA) into powder.The percentage yield of RYF aqueous extract was 9.61% (w/w).

Animals and cells
BALB/c mice (4-6 w, Female) were purchased from BK Corporation (China).All animal experiment was performed under semi-sterile conditions.The animals were stored in special-pathogen-free (SPF) environment during entire experiment.Water and food were provided normally.Animals were maintained under the standard 12 light/dark cycle at 22-23℃ with relative humidity of 50-60%.All animals' protocols were approved by the Institutional Animal Care and Use Committee at Zhejiang Chinese Medical University Laboratory Animal Research Center.
The mice breast cancer cell line (4T1), Plasmid SSR#69 (designed by Professor Westerman) and PAMPHO plasmid, were favored from Professor Tong-chuan He from the University of Chicago.

Mice mammary tumor model establishment
After two days of acclimatization, mice were with 100 μl of 4T1 cell suspension subcutaneously into the 2rd pair mammary fat pad on the right at a cell density of 1.0×10 7 cells/ml (the normal group were injected with a vehicle of PBS).Two days after tumor inoculation, animals (n=4) were randomly divided into ve groups (four mice per group): (i) cyclophosphamide (CTX, Baxter, Shanghai, China) group (30 mg/kg, i.p. once every two days); (ii) model group (0.2 ml, intragastrically, normal saline, once a day); (iii) RYF-L (low concentration) group (0.145g crude herbs/kg, intragastrically, once a day); (iv) RYF-M (medium concentration) group (0.29g crude herbs/kg, intragastrically, once a day); (v) RYF-H (high concentration) group (0.58g crude herbs/kg, intragastrically, once a day).Four health mice were prepared in normal group (0.2 ml, intragastrically, normal saline, once a day).All treatments lasted for 20 days.Throughout the study, body weights were monitored and the longest (L, mm) and shortest (W, mm) diameter of tumor were measured with a vernier caliper weekly.Tumor volumes (V, mm 3 ) were calculated with Eq. 1. Mice were sacri ced 2 hours after the last administration.The visceral weight and index of thymus, lung, spleen and lymph node were measured with Eq. 2. The anti-tumor rate were calculated with Eq. 3 after collected and weighed the tumor.Calculated the pulmonary metastasis rate according to the number and volume of nodes on the lung.

Immunohistochemical analysis
Thymus tissue sections were in ated with 4% paraformaldehyde, embedded in para n and cut at a thickness of 5μm.Tissue sections of each specimen were stained using hematoxylin and eosin (H&E, Sangon Biotech, Shanghai) according to a standard protocol for histopathological examination using uorescence microscope (Axio Scope.A1, Zeiss, Germany).The pathological changes in the structure of the tissue sections in each group were evaluated.The numbers of cells in thymus cortex and medulla regions were measured using Image J. 3% hydrogen peroxide was used to block endogenous peroxidase activity at room temperature for 15 min.Subsequently, the unspeci c binding sites were blocked with 5% BSA/PBA for 40 min.The sections were then incubated for overnight with primary antibody at 4℃ in a humid chamber for E-cadherin (Cell Signaling Technology) and Vimentin (Cell Signaling Technology).PBS was used as negative control.The dilutions used for the primary antibodies were 1:200.The sections were subsequently incubated with rabbit anti-mouse enhanced polymer antibodies (Zsbio Co., Ltd.) at 37℃ for 2 h.Then the reaction products were developed with 3, 30-diaminobenzidine (DAB), counterstained with haematoxylin, differentiated with 1% hydrochloric acid ethanol, washed with distilled water, dehydrated with graded ethanol, vitri ed by xylene and sealed with neutral gum.The stained sections were observed with uorescence microscope after the sections dried.Immunoreactivity was analyzed with Image J to evaluate the expression of proteins.

Cell proliferation analysis
The establishment and evaluation of iTECs were described in our previous literature [19].Logarithmically grown iTECs were seeded in 96-well plates (1×10 4 /well).Cells were divided into different concentration treatment groups of RYF extraction (100 μl of 25, 50, 100, 200, 400 μg/ml).There was no cells and only 100 μl cell cultural medium in control group.Negative control group used 100 μl of cell cultural medium with 0.1% DMSO (Sangon Biotech) replaced RYF extraction.There were three wells of cells used as repeated in each group.The cells were incubated at 37 ℃ with 5% CO 2 for 48 h.20 μl of MTT solution (5 mg/ml, Biofrox) was then added into each well.After 4 h incubation, cell culture medium was discarded, and 150 μl of DMSO was added into each well.Plates were shaken at room temperature for 10 min.Absorbance value was measured at 570 nm using Synergy H1 Multi-Mode Microplate Reader (Bio-Tek, USA).Then calculated the inhibition rate of cell proliferation (%) with MTT assay.
Logarithmically grown iTECs cells were seeded in 24-well plates (3×10 4 /well) for 24 h.There was 100 μl of cell cultural medium with 0.1% DMSO (Sangon Biotech) added in to iTECs negative control group and different concentration treatment groups of RYF extraction (300 μl of 50, 100, 200 μg/ml).The cells were incubated at 37 ℃ with 5% CO 2 for 48 h, and then cell culture medium was discarded.300 μl of crystal violet staining solution was added into each well and stained at room temperature for 30 min.Subsequently repeated washed with water and dried naturally and then observed the proliferation.
Results were expressed as percentage of cell proliferation with respect to negative control group cells (as 100%).
TGF-β1 induced iTECs EMT TGF-β1 was used to induce EMT of iTECs.Brie y, iTECs in logarithmic growth phase were seeded in 24well plates (3×10 4 /well) for 24 h, and then starved with serum-free medium for 24 h.Cells were divided into ve groups: (i) normal group (DMED high sugar medium); (ii) TGF-β1 group (10 ng/ml TGF-β 1 ); (iii) RYF-L group (50 μg/ml RYF extraction and 10 ng/ml TGF-β 1 ); (iv) RYF-M group (100 μg/ml RYF extraction and 10 ng/ml TGF-β1); (v) RYF-H group (200 μg/ml RYF extraction and 10 ng/ml TGF-β1).There were three wells of cells used as repeated in each group.The TGF-β1 was administered rst, then after 48 h the RYF extraction were added and cultured for 24 h.The morphology of iTECs cells induced by TGF-β1 was observed with microscope.The phenotype marker of E-cadherin, Vimentin and a-Tubulin was observed by immuno uorescence staining method described in 2.7.Quantitative real-time PCR analysis detected the expression level of Zeb-1 and Snail 1 and other mRNA for phenotypic markers and phenotype-related transcription factors.

Quantitative Real-time Pcr Analysis
The cells were seeded on 6-well plates (1×10 5 /well) for 24 h respectively.The steps of total RNA extraction, reverse transcription, and uorescence quantitative detection of SV40 mRNA was as described [19].Trizol Reagent (Thermo); First Strand cDNA Synthesis Kit (Sangon Biotech).PCR ampli cation was performed with GAPDH as a reference to detect the expression of target mRNA by the 2 −ΔΔCt method in different group cells.There were three wells of cells used as repeated in each group.The primer sequences were listed in Table 1.Primer synthesis was performed by Shanghai Sangon.Logarithmically grown iTECs cells were seeded in 24-well plates (3×10 4 /well) until the cell density reached 80%-90%.Then discarded the culture medium, washed by serum-free DMEM, added 1.2-2.0ml serum-free DMEM and put in the culture incubator for 20 min.The pBGLuc-Smad plasmid was infected and transfected by LipofectAMINE 2000.Subsequently, the successful infection and transfection iTECs were divided and treated by the method described in 2.8.The luciferase level of all groups cell supernatant liquid (50 µl) were analyz ed with single-channel bioluminescent detector according to the Luciferase gene kit's instruction and calculated the relative activation degree of Smad pathway (%).The expression of Smad mRNA of each group was detected by PCR.

Statistical Analysis
The statistical analysis was performed with SPSS 20.0 software and the results were expressed as mean ± SD.One way ANOVA were used to compare means of multiple groups and least signi cant difference test was used for comparison between two groups.Differences were considered to be statistically signi cant when P < 0.05.

HPLC analysis of RYF extract
Four components were analyzed and determined by HPLC by comparing with the referenced solution.In Fig.  3A).Meanwhile, the cortex regions had darker staining, indicate it has more thymocytes than medulla regions.On the contrary, the boundaries between thymus cortex and medulla were indistinct and the cells arranged disordered in model group with cortex regions cells reducing notably and medulla regions cells increasing remarkably compared to normal group.In addition, CTX and RYF-treated groups all had clear boundaries between thymus cortex and medulla.Moreover, the cells in cortex increased signi cantly and in medulla cells decreased notably compared with model group as seen in Fig. 3B.

RYF in uenced the expression of E-cadherin and Vimentin in thymus tissue
E-cadherin is the characteristic markers of epithelial cells and Vimentin is the interstitial cells.The Ecadherin positive cells in model group reduced signi cantly and Vimentin positive cells increased notably compared with normal group (Fig. 4A).Conversely, compared to the model group, the cells with Ecadherin positive expression increased and with Vimentin positive expression reduced in RYF-treated groups and CTX group.(Fig. 4B).
Ryf Altered Morphology Of Itecs After Tgf-β1 Treatment The morphology observation (Fig. 5C) results showed iTECs were short fusiform and lined up tightly in normal group.Besides, the number of iTECs induce by TGF-β1 reduced and intercellular spaces got fewer with the morphology changed to long fusiform.Simultaneously, RYF treated could reverse the change of intercellular space and morphology.
RYF in uenced the mRNA levels and protein expressions of E-cadherin, Vimentin, Snai1 and Zeb1 in TGF-β1 induced iTECs Compared with normal group, the E-cadherin's expression and mRNA's level decreased and Vimentin's expression and mRNA's level were up-regulation in TGF-β1 group.Moreover, the level of Snail 1 and Zeb 1 increased signi cantly.Furthermore, compared with TGF-β1 group, RYF-treated groups reversed the change in relative proteins expression and mRNA levels.The results showed in Fig. 6A, B, C and D.
As seen in Fig. 6E, F, G, immuno uorescence staining results showed the target proteins presented in green uorescence and the cell nucleus presented in blue.TGF-β1 group and RYF-M group' α-Tubulin uorescent expression results suggested that RYF could resist the change of the TGF-β1-induced iTECs' morphology and could maintain normal cell morphology.
Ryf Smad Pathway showed in Fig. 7, PCR results suggested that the level of Smad mRNA was up-regulated signi cantly compared to the normal group in iTECs after TGF-β1 induced.Interestingly, the level of Smad2 mRNA was down-regulated after RYF treated compared to the TGF-β1 group and differences were considered to be statistically signi cant (P<0.01).
Luciferase assay results showed the expression of luciferase increased signi cantly in TGF-β1 group compared to the normal group, and it indicated that Smad pathway got activated.Furthermore, the luciferase expression in RYF-treated groups were down-regulated.The difference between RYF-L and TGF-β1 group were considered to be statistically signi cant (P<0.05).
It was reported that the activation of Smad pathway could induce the phenomenon of cell type II EMT [20].Moreover, it is suggested that RYF can be confronted the phenotypic transition of iTECs induced by TGF-β1.This change may be caused by TGF-β1 activation of the Smad pathway and induced thymic epithelial cell type II EMT.

Discussion
Our research found that the tumor index and lung metastases of RYF was signi cantly lower while compared with the model group (P < 0.05).Moreover, the boundary between medulla and cortex in mouse thymus in RYF was clearer, and the cell number was signi cantly increased in cortical area and decreased in medulla area than that in the model group.RYF could increase the expression of E-cadherin and decrease Vimentin in vivo.The above results were con rmed by cell experiments, after induced with TGF-β1, the mRNA levels of Smad was up-regulated, after treatment with RYF, the mRNA levels of Smad were down-regulated, and the activation of signaling pathways was inhibited.It is suggested that RYF could confront the phenotypic transition of iTECs induced by TGF-β1, and its mechanism is related to the inhibition of Smad pathway.
As an important part of prescription, the Radix Codonopsis, Astragalus membranaceus and Angelica Sinensis in RYF have better therapeutic effects in improving immune and anti-tumor.As one of the most important immune organs, the abnormal changes of thymus usually use to evaluate body's immune function.The ndings and results of this study indicated that the thymus index of tumor-bearing mice increased slightly but the pathological changes of thymus were reduced noticeably after RYF treated, what reminded RYF could repair the thymus function of tumor-bearing body and played an important role in adjuvant or combination therapy of advanced breast cancer.
Consequently, TGF-β has been widely used to induce phenotypic changes of epithelial cells, and mediates the transformation of epithelial cells into mesenchymal cells, thus inhibiting the migration of tumor cells.
As an important development process of cell adhesion and migration during tumor invasion, the hallmarks of EMT is the functional loss of E-cadhetrin [23,24].E-cadherin is one of cadherin members, which is a calcium-dependent cell adhesion molecule of the epithelial cells, maintains the epithelial tissues but also plays a pivotal role in organogenesis and morphogenesis [25].Vimentin is an intermediate lament characteristically present only in certain epithelial of mesenchymal cells [26].In general, E-cadherin is the epithelial marker and Vimentin is regarded as the mesenchymal marker.Gain of Vimentin and loss of E-cadherin is a hallmark of EMT and E-cadherin is a good prognostic marker whereas Vimentin expression indicates a poor prognosis [27].This study showed RYF therapy could effectively reverse the cell morphology changes and the abnormal expression of E-cadherin and Vimentin in TGF-β1-induced iTECs, what suggested RYF could confront the TGF-β1-induced EMT in iTECs.
EMT is regulated by several transcription factors, including Snail and Zeb, which downregulate E-cadherin expression [28].As two key transcription factors for cell motility, Snail and Zeb trigger EMT by repression of epithelial markers and activation of mesenchymal properties.This study showed the expression of Snai1 and Zeb1 in TGF-β1-induced iTECs increased while they reduced after RYF treated and then inhibited EMT.It also adds to the proof that RYF could confront the TGF-β1-induced EMT in iTECs.
Smad signaling pathways are necessary for EMT [29], and TGF-β could induce EMT in NMuMG cells through activation of Smad, while in renal tubular epithelial cells, non-Smad pathways can not be activated of EMT-related transcription factors [30,31].Our study results showed Smad pathway got activated in the phenotypic transition process of TGF-β1-induced iTECs.Meanwhile, the expression of Smad luciferase and level of mRNA reduced after RYF treated, which suggested TGFβ1-induced thymic epithelial cell EMT through activation of the TGFβ/Smad pathway of RYF.

Figure 1 The
Figure 1

Figure 2 The
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Figure 3 Changes
Figure 3

Table 1
Sequences of primer pairs for quantitative real-time PCR.
Anti-tumor Effects Of Ryf In Mammary Tumor Model MiceFigure2showed the thymus, lung, spleen, lymph node and tumor index in mammary tumor model mice increased signi cantly compared with normal group.Compared with model group, the spleen, thymus and tumor index in CTX group decreased signi cantly, and the anti-tumor rate was 51.33%.In addition, a notable decrease of spleen index in RYF-L group and tumor index in RYF-M and RYF-H group compared with model group, and the anti-tumor rate in RYF-L, RYF-M and RYF-H were 6.49%, 31.43% and 33.45%, respectively.As showed in Table2and Fig.2G, compared with model group, both of the number of pulmonary metastasis (diameter > 2 mm and < 2 mm) in CTX and RYF treated groups reduced signi cantly.The pulmonary metastasis rate in model group was 100%, and in CTX group, low, medium and high concentration RYF was 50%, 75%, 75% and 100%, respectively.RYF altered histomorphology of thymus in mammary tumor model miceIt was clear-out of the thymus cortex and medulla boundaries and cells had a regular arrangement in normal group (Fig.