Multi-targeting chemoprevention of Chinese herb formula Yanghe Huayan decoction on experimentally induced mammary tumorigenesis

The YHHY Decoction is an aqueous preparation from 8 Chinese medicinal herbs: CCP 15 g, RB 9 g, SB 6 g, CC 3 g, BG 3 g, EV 9 g, JRL 6 g, and GU 6 g. The raw herb materials were purchased from Bozhou Huqiao Pharmaceutical Co Ltd. (Anhui, China) in September 2015, and were authenticated by Dr. Feng Li at the Department of Pharmacognosy, Shandong University of TCM. A voucher specimen was deposited at TCM Pharmacy, Affiliated Hospital of Shandong University of TCM, with the voucher numbers as CCP 1508160116, RB 1507180227, SB 1507270896, CC 1508030119, BG 1506080417, EV 1507210325, JRL 1508200021, and GU 1507290632. Each raw herb was homogenized to fine powder and the eight herbs were mixed thoroughly. A 8.4 g sample of the mixed fine-ground powder was accurately weighted and extracted with 84 mL of boiling H₂O for 6 h and then centrifuged at 12,000 rpm for 15 min, both the volatile oil and the supernatant were collected. The pellet was resolved in 60% ethanol (1:1.5 volume) and statically stewed for 24 h. The mixture was then centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The two supernatants were combined and dried in the rotary evaporator at 160 rpm at 30 °C. Then the dried powder was accurately weighed and dissolved in the volatile oil Tween-80 solution (1:1 volume) at 1 mg/ml.

DMBA was purchased from Sigma-Aldrich (St. Louis, MO). Primary antibodies, c-myc (NCL-cMYC, RRID:AB_563665, from Leica Microsystems), phosphor-c-myc (PA5–35814, RRID:AB_2553124, from Thermo Fisher Scientific), CD-105/Endoglin (NCL-CD105, RRID:AB_563482, from Leica Microsystems), and Ki-67 (sc-23,900, RRID:AB_627859), Bax (sc-70,407, RRID:AB_1119412), Bcl-2 (sc-7382, RRID:AB_626736), ERK1/2 (sc-135,900, RRID:AB_2141283), pERK1/2 (sc-136,521, RRID:AB_10856869), PI3K (sc-365,290, RRID:AB_10846944), AKT (sc-5298, RRID:AB_626658), KDR (sc-101,559, RRID:AB_1123212) and β-actin (sc-47,778, RRID:AB_2714189) from Santa Cruz Biotechnology (Santa Cruz, CA) were used. Antioxidant assay kit (cs0790) and In Situ Cell Death Detection Kit (11,684,817,910 ROCHE) were purchased from Sigma-Aldrich (St. Louis, MO). Ventana Basic DAB (3,3-diaminobenzidine) Detection kit was from Boster Biotechnology, Wuhan, China.

The animal study was conducted upon the approval by the Institutional Animal Care and Use Committee of Shandong University of TCM (SDUTCM2012042001). All experiments were performed in accordance with relevant guidelines and regulations. Pathogen-free virgin female Sprague-Dawley rats (MGI Cat# 5651135, RRID:MGI:5651135), approximately 42 days of age, weight 150 ± 10 g, were provided by Experimental Animal Center of Shandong University of TCM and housed in an animal facility accredited by the Chinese Association for the Accreditation of Laboratory Animal Care. The rats were acclimatized to standard housing conditions, including ambient temperature of 22 ± 2 °C, relative humidity at 30–50%, and a 12-h light-dark cycle, in plastic cages (maximum 4 animals/cage) for 1 wk. before initiation of the experiment. The animals had free access to the nutrition formula rodent diet (NTP-2000 standard) and drinking water.

The potential chemopreventive role of YHHY Decoction was investigated using a well-established DMBA-induced rat mammary tumorigenesis model [27, 29]. Following 1-wk acclimatization period, the rats were randomly divided into 4 groups with n = 6 per each group: group A-normal control with vehicle treatment; group B-DMBA induction with vehicle treatment, group C-DMBA induction with YHHY Decoction treatment and group D-normal control with YHHY Decoction treatment. The experiments were independently repeated three times, so the final sample size for each group is n = 18 rats. YHHY Decoction was fed through oral gavage (p.o.) once daily for 5 wks (1 wk. before and 4 wk. following DMBA treatment). A human equivalent dose of YHHY Decoction at 1 mg/ml, given by 0.02 ml/g body weight, was used based on the human to animal dose conversion formula (https://​www.​fda.​gov/​downloads/​drugs/​guidances/​ucm078932.​pdf, page 7). Following 1 wk. feeding with the YHHY Decoction, rats in the Groups B and C were administered a single dose of DMBA at 50 mg/kg body weight (dissolved in corn oil) by oral gavage to induce mammary carcinogenesis. This dose of DMBA was chosen so that substantial tumor incidence could be produced but not so high as to overwhelm chemopreventive action of YHHY Decoction. The specific time for DMBA exposure is based on previous carcinogenic bioassays indicating that rats at this age possess high frequency of terminal end buds that are sensitive to the established mammary carcinogen DMBA [30]. Rats in Group C were continually fed with YHHY Decoction for another 4 weeks after the DMBA administration (totally for 5 wks). Food, water intake and behavioral patterns were monitored daily, body weights were recorded twice a week. Palpation of mammary glands started 4 wks following DMBA treatment with a frequency of twice per week. The length of time before palpable tumor is examined is defined as tumor-free survival time. The experiments were terminated at 16 wks post-DMBA administration.

Animals were euthanized by cervical dislocation after overnight fast. The mammary tumors were carefully excised from mammary gland parenchyma and rinsed with phosphate-buffered saline (PBS) (pH = 7.4). Each tumor was measured in 2 perpendicular directions by a caliper to obtain an average diameter, then was cut into 2 halves. One half was immediately snap frozen in liquid nitrogen and used for molecular analysis. The other half was fixed in 4% paraformaldehyde and used for histopathological and immunohistochemical analysis. Serial tumor sections at 10 μm thickness were prepared for hematoxylin and eosin (H&E) and relevant immunohistochemistry staining. The mammary tumors were classified according the established criteria [31].

Tumor sections were stained with primary Ki-67 antibody, and CD-105 antibody with the Ventana Basic DAB Detection kit for cell proliferation and microvessel detection. Apoptotic cells were detected by TUNEL reaction with In Situ Cell Death Detection Kit. The slides were evaluated by an independent pathologist who was blinded to group assignment and outcome assessment using a light microscope (BX41, Olympus). Five random fields under 20× objective for each slide, and at least 5 slides for each tumor sample were analyzed.

Similar as previous described [32], two sets of measurements were performed on the excised mammary tissues: (i) antioxidant enzyme activities – superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT); and (ii) biochemicals – malondialdehyde (MDA) and total nitrate (NOx). Each frozen mammary tissue was divided into two portions (each approximately 0.5 g). One portion was homogenized in 1.5% ice-cold KCl solution to give a 10% suspension and used for the MDA assay. The other portion was cut into several small pieces, homogenized in PBS (pH 7.4), with a w/v ratio of 1:5, and spun at 13,000×g for 15 min, at 4 °C. The supernatant was separated for the measurements of SOD, CAT, GPx activities and NOx levels. The protein level in the supernatant was determined spectrophotometrically by the method of Lowry et al. [33] using BSA as a standard. SOD activity was expressed as the amount causing 50% inhibition of the reduction of cytochrome c per milligram of protein (U/mg of protein), with bovine copper–zinc SOD (Cu/Zn SOD) as standard [34]. CAT activity was measured by the method of Luck [35]. The decomposition of the substrate H₂O₂ was monitored spectrophotometrically at 240 nm. Specific activity was defined as micromole substrate decomposed per minute per milligram of protein (expressed as U/mg protein). GPx activity was measured as before [36]. Specific GPx activity (U/mg protein) was calculated as micromole NADPH consumed per minute per milligram of protein using an appropriate molar absorption coefficient (6220/M per cm). The level of MDA, determined using the method of Mihara and Uchiyama [37], was expressed as nmol/mg protein. The level of NOx was measured by the method developed by Sastry et al. [38]. A calibration standard involving potassium nitrate was used to calculate the total concentrations of nitrate, which was expressed as μmol/mg protein.

Equal quantities of protein from the tumor tissue lysate were processed for Western blotting. Each sample was denatured, electrophoresed, and transferred onto a PVDF membrane. After blocking the membrane, blots were incubated in specific primary antibodies and then secondary antibodies following the manufacturer’s instructions. Densitometric analysis was conducted using ImageJ software. The primary antibodies used include: anti-c-myc (1:5000), anti-phospho-c-myc (0.5 μg/ml), anti-Bax (1:1000), anti-Bcl-2 (1:2000), anti-ERK1/2 (1:500), anti-pERK1/2 (1:500), anti-PI3K (1:1000), anti-AKT (1:800), anti-KDR (1:400) and anti-β-actin (1:2000). No grouping of gels/blots cropped from different parts of the same gel or from different gels, fields, or exposures was performed.

The reference standards of eight chemical constituents, including adenosine, uracil, tubeimoside A, allyl isothiocyanate, trans-cinnamic acid, cinnamic aldehyde, 6-shogaol and polysaccharides (purity≥98%) were purchased from Chengdu Biopurify Phytochemicals Ltd., (Chengdu, China). Methanol, acetonitrile (Fisher Scientific, USA), and acetic acid (Tianjin Chemical Regent Co., Ltd., Tianjin, China) were of HPLC grade. The distilled water was obtained from Wahaha Co., Ltd. (Hangzhou, China).

An Agilent 1100 liquid chromatography system was used for the analysis, equipped with a diode array detector working in the range of 190-400 nm, a quaternary solvent delivery system, a column temperature controller, and an autosampler. The chromatographic data was recorded and processed with Agilent Chromatographic Work Station software.

For HPLC analysis and quantification of each constituents, 1 mg/ml decoction was filtered through a membrane filter (0.45 μm pore size) prior to injection and analyzed three times. Adenosine and uracil, cinnamic acid and cinnamaldehyde were examined simultaneously in one aliquot of the decoction sample, and other constituents were examined individually. Previously validated chromatographic conditions were applied to detect the concentrations of each constituent [39‐45]:

For simultaneously detection of adenosine and uracil analysis, C18 analytical column (TIANHE Kromasil C18, 4.6 mm × 250 mm, 5 μm) was used. The mobile phase consisted of water (A)-acetonitrile (B); A:B was as follows: o min, 99:1; 5 min, 98:2; 10 min, 98:2; 20 min, 96:4; 42 min, 45:55; 55 min, 40:60; 60 min, 0:100. The flow rate and column temperature were set constantly at 0.3 ml/min and 30 °C. The detection wavelength was at 260 nm.

For detection of tuberimoside A, C18 analytical column (RP C18, 4.6 mm × 250 mm, 5 μm) was used. The mobile phase consisted of methanol-water (V:V = 68:32). The flow rate and column temperature were set constantly at 0.1 ml/min and 25 °C. The detection wavelength was at 214 nm.

For detection of allyl isothiocyanate, sphereclone ODS-2 column (4.6 mm × 15cmm, 5 μm) was used. The mobile phase was acetonitrile. The flow rate and column temperature were set constantly at 0.1 ml/min and 25 °C. The detection wavelength was at 242 nm.

For detection of 6-shogaol, C18 analytical column (Alltima HP C18, 4.6 mm × 250 mm, 5 μm) was used. The mobile phase consisted of water (A) and acetonitrile (B). The A:B flow was as follows; 0 min, 55:45; 8 min, 50:50; 17 min, 35:65; 32 min, 0:100 B, 38 min, 0:100, 43 min, 55:45, 48 min, 55:45. The flow rate and column temperature were set constantly at 1 ml/min and 30 °C. The detection wavelength was at 230 nm.

The reference standards were accurately weighed and dissolved in 60% ethanol, and then diluted to appropriate concentration ranges for the establishment of calibration curves. All stock and working standard solutions were stored in brown bottles at 4 °C until used for analysis.

The well-established DMBA-induced rat mammary tumorigenesis model was used in the current study [27, 29]. There were 4 animal groups: group A-normal control with vehicle treatment; group B-DMBA induction with vehicle treatment, group C-DMBA induction with YHHY Decoction treatment and group D-normal control with YHHY Decoction treatment. The rats received YHHY Decoction gained their weight at similar rates as the group A rats (Fig. 1a). Food and water intake was monitored periodically throughout the study and did not differ among the treatment groups. There were no observed behavioral changes among animal groups (data not shown).

The development of palpable tumors was first observed in the DMBA control rats 7 wks after DMBA induction (Fig. 1b). In contrast, rats fed with YHHY Decoction did not develop mammary tumors until wk. 10 after DMBA treatment (P = 0.015, log-rank test). At the end of the study which was 16 wk. post DMBA induction, while 94% (17/18) rats in the vehicle group were detected with palpable tumors, there was only 61% (11/18) rats receiving YHHY Decoction were found to bear tumors (P = 0.041, fisher exact test). Power analysis showed that having 18 animals in each group provided 100% power to detect the increase of the mean tumor-free survival time from 10.788 ± 0.646 wks in the DMBA control group to 12.944 ± 0.646 wks in the YHHY+DMBA group with alpha = 5%. These results indicated that YHHY Decoction treatment not only delayed the tumor latency, but also prevented the occurrence of mammary tumors in some rats. Tumors in the YHHY treatment group also showed 74% less of the average weight than those in the DMBA controls (5.1 ± 1.7 g vs. 19.5 ± 4.5 g, P < 0.05).

Histopathological examination on the tumor sections indicated that tumors in the group B (DMBA control) rats showed extensive epithelial proliferation and enlargement of the alveolus as a fused glandular pattern (Fig. 1c), as well as a presence of nuclear pleomorphism (Fig. 1c). Epithelial cells demonstrated various nuclear sizes and irregular chromatin (Fig. 1c). The YHHY Decoction treatment improved the cellular architecture remarkably. Most of the tissue appeared normal ductal and alveolar structure without hyperplastic changes on epithelial cells (Fig. 1c).

The terminal end buds in mammary gland comprise epithelial stem cells that are sensitive to carcinogen DMBA for malignant transformation [46]. Since myc proto-oncogene is a hallmark of cancer initiation [47], we first examined the expression of phosphorylated c-myc protein in the mammary glands upon DMBA induction and YHHY prevention. The results showed that two weeks after DMBA induction the mammary epithelial cells already exhibited elevated expression of phospho-c-myc, but its expression was absent in all the examined animals in the YHHY group (n = 3) (Fig. 1d). Besides tumor initiation, sustained myc activation also contributes to autonomous tumor proliferation and growth [47]. When examining the c-myc expression in all the endpoint tumors in the two groups (Fig. 1e), the results showed a significant decrease in the levels of phosphor-c-myc in tumors of YHHY-treated rats vs. tumors of the control rats (526 ± 83 vs. 831 ± 126, in relative densitometric units, P < 0.05). The concentrations of total c-myc protein were also decreased dramatically in the tumors of YHHY-treated rats (834 ± 118 vs. 1215 ± 185 in relative densitometric units, p < 0.05).

HPLC analyses identified eight major chemical compositions of the decoction (6-shogaol, cinnamic acid, cinnamic aldehyde, polysaccharide, adenosine, uracil, tubeimoside A and allyl isothiocyanate) (Table. 1).

The multifarious chemical constitutes of Chinese herbal cocktail may indicate complex mechanisms of action that warrant a systemic exploration. After searching in the IPA database, 7 out of the 8 chemicals were found and their direct-interacting molecules, totally 293 molecules, were used to analyze the disease or function networks related to the chemicals. Overall, the 293 molecules were significantly (P < 0.001) enriched in the top function networks of cell death and survival (apoptosis), cellular function and maintenance (cellular homeostasis), inflammatory response, organism injuries and abnormalities (inflammation of organ), free radical scavenging (synthesis of reactive oxygen species), cardiovascular system development and function (development of vasculature, angiogenesis), cell cycle (cell cycle progression), cellular movement (invasion of cells), and so on. Table. 2 listed the detailed category, disease and function annotation, p value and number of molecules for each network. Although needs further experimental validation on specific DMBA tumors, these results gave us an overall picture about the functions or mechanisms of the formula cocktail as a whole. In the following studies, we examined the activities of four major functions of the YHHY formula in the DMBA tumors.

TUNEL assay was performed to evaluate the extent of cell apoptosis in the mammary tumor specimens. In the tumors from DMBA control rats, the presence of apoptotic cells was extremely rare. However, a significant increase of the apoptotic cell population was examined in the YHHY plus DMBA group (Fig. 2a, b).

To investigate the molecular signalings involved in the apoptosis-inducing effect of YHHY Decoction, Bax and Bcl2, two apoptosis-related proteins, both were in the 293 molecule list, were evaluated in the mammary tumors by Western blotting analysis. The Bax immuno-reactive band was detected extremely low in the DMBA control tumors (Fig. 2c, d). A significant increase of Bax expression was found in the tumors obtained from YHHY treatment group (Fig. 2c, d). The mammary tumors from DMBA control group showed extensive Bcl2 protein expression (Fig. 2c, d), and YHHY treatment exhibited considerable inhibition of Bcl2 expression (Fig. 2c, d). Oral administration of YHHY Decoction elevated the Bax/Bcl2 ratio under DMBA exposure (Fig. 2e). These data clearly indicated a pro-apoptotic mechanism of YHHY-mediated prevention of rat mammary tumorigenesis.

To determine whether YHHY Decoction impacted cell proliferation in the DMBA-induced mammary tumors, immunohistochemical staining of Ki-67 was assessed in the tumor sections. An abundant Ki-67-positive cells were found in the tumors from DMBA control rats, indicating active cell proliferation. YHHY Decoction suppressed cell proliferation to 30% of the DMBA tumors (Fig. 3a, b).

The activation of MAPK/ERK and PI3K/AKT signalings has been reported to contribute to both tumor initiation and oncogenic phenotype of mammary tumors through the effects on cell proliferation [1, 48]. ERK and AKT were both identified as the interacting molecules among the 293 molecules of the chemicals. Expressions of total ERK1/2, PI3K and AKT were examined to be 2–3 folds up-regulated in the DMBA control tumors compared to the normal mammary glands, and YHHY Decoction significantly suppressed their expressions (Fig. 3c, d).

The onset of angiogenesis is believed to be an early event in tumorigenesis. Like human breast pathology, the progression of DMBA-initiated tumor in the rat is associated with an angiogenic phenotype [27] (Fig. 4a). CD-105/Endoglin antibody was used here to label only newly formed blood vessels [49]. A remarkable reduction (80%) of CD-105/Endoglin positive microvessel density was observed in the YHHY Decoction-treated DBMA tumors in comparing with the control DMBA tumors (Fig. 4a, b).

Vascular endothelial growth factor (VEGF) and its receptor VEGFR2 or KDR play major roles in promoting the formation of new blood vessels [50, 51]. VEGFA and the receptor KDR were both examined to be expressed much lower in the YHHY treated tumors than those in the DMBA controls (Fig. 4c-e). While the less expressions of VEGF and its receptor may due to the less blood vessels in the YHHY treated DMBA tumors, we found that YHHY treatment blocked the activation of KDR at its major autophosphorylation site, i.e., Tyr951/996 [52] (Fig. 4c-e). These results suggested that the continuous use of YHHY Decoction in rats blocked the initiation and development of neoangiogenesis.

The IPA analysis also revealed that regulation of free radical scavenging is one of the possible mechanisms of action of the YHHY Decoction. Numerous studies have shown that free radicals reactive oxygen species are generated by exposure to DMBA [53, 54]. A balance between free radicals and antioxidants is necessary for proper physiological function. If free radicals overwhelm the body’s ability to regulate them, a condition known as oxidative stress ensues. Free radicals thus adversely alter lipids, proteins, and DNA and trigger a number of human diseases including cancer [55]. Thus, the antioxidant capacity of YHHY Decoction was measured by two sets of analyses: (1) anti-oxidant enzyme activities of SOD, GPx and CAT; and (2) biochemicals of MDA and total NOx. As shown in Table. 3, DMBA induction significantly stimulated the enzyme activity of SOD and GPx in the mammary tumors by 54 and 61%, respectively (P < 0.01). Treatment with YHHY Decoction completely blocked the enzymatic activation, reduced the mean value of the SOD enzyme activity similar to those of the control group (P = 0.41) and an even lower of the GPx activity (P = 0.02). DMBA induction decreased the CAT activity by 17% in comparing to the control group (P < 0.01), and the application of YHHY Decoction had no further effect (P = 0.44). In addition, DMBA significantly increased MDA and NOx levels by 36 and 54%, respectively (P < 0.01), but these increases were completely blocked by YHHY Decoction for MDA (P = 0.47) and NOx (P = 0.38). These results indicated a strong capacity of YHHY Decoction on anti-oxidative stress.