Reagents, experimental animals, cell and cell culture
Fetal bovine serum (FBS, cat. no. SV30087.02) and L15culture medium (cat. no. SH30525.01) were obtained from Hyclone (USA). Minimum essential medium (MEM, cat. no. 41500-034) were purchased from Gibco company (USA). Cycle TESTTM PLUS DNA Resgent Kit and Annexin V-FITC/PI apoptosis kit (cat. no. 556547) were provided by BD Biosciences (USA). TUNEL kit (cat. no. c10618) and Antiquenching glue (cat. no. S36963) were obtained from Thermo Fisher Scientific (USA). RNAiso Plus (cat. no. 9108), SYBR Premix Ex Taq (cat. no. RR420A) and Prime Script RT reagent Kit (cat. no. RR037A) were purchased from TaKaRa Bio (Japan). Hoechst 33342/PI (cat. no. G023, Jiancheng Bioengineering Institute, China). The BCA kit (cat. no. CW0014) were obtained from ComWin Biotechnology (China). H&E kit (cat. no. C0105S) was provided by Beyotime Biotechnology (China).
BALB/c naked mice were acquired by Beijing Vital River Laboratory Animal Technology Co., Ltd., SCXK(Jing)2016-0011 and were kept in the experimental animal center of Qiqihaer Medical University (SYXK(hei)2016-001).
Human MCF-7 cells and MDA-MB-453 cells were purchase by the cell resource center of Shanghai institute of life sciences, Chinese academy of sciences. MCF-7 cells were fostered in MEM culture medium with 10% FBS, and maintained in an incubator for 37℃ and a moist environment of 5% CO2. MDA-MB-453 cells were fostered in L15 culture medium with 10% FBS, and maintained in a moist incubator for 37℃. After about 1 to 2 days MCF-7 cells or MDA-MB-453 cells were digested and passed on when they filled the bottom of the cell culture dish. The cells at logarithmic growth stage were used for the test.
Experimental drug
The ingredients of ESZM extract formula include E. fischeriana 100 g, Z. jujuba 100 g, and distilled water 1000 mL. E. fischeriana was identified by professor Lina Guo of the natural pharmaceutical chemistry center of Qiqihar medical university, and was picked from Taikang county, Daqing city, Heilongjiang province, China. Z. jujuba were purchased from the wholesale market of medicinal materials in Qiqihar city. The formula were boiled for 120 min under sustaining stirring status. The procedures were repeated two times following that the polyjuices were collected centrifugation (Eppendorf, 5804R, Germany) at 8000×g. The supernatants were enriched and kept by low pressure for 70℃ to evaporation until 200 mL by rotary evaporator (Buchi, R-220, Switzerland). The same dosage of E. fischeriana extract and Z. jujuba extract were prepared by the above method.
HPLC analysis
HPLC analysis was executed via Waters 2489 system (Waters Technologies), which facility with a waters 2545 quaternary pump and a waters 2545 autosampler. ESZM extract samples were separated by Diamonil-C18 column (250×4.6 mm, 5 µm) at 35 ℃. The mobile phase system was comprised of (A) water with 0.1% formic acid and (B) methanol. 0–20 min, 98%A, 2%B; 20–32 min, 95%A, 5%B; 32–38 min, 90%A, 10%B; 38–39 min, 60%A, 40%B; 39–40 min, 100%B. The mobile phase flow rate was 1.0 mL per minute and the UV spectrum was 210 nm. 20 microlitre of volume was injected. Data acquisition was executed by Waters software system.
Establishment of xenograft nude mice
Five week-old BALB/c female naked mice were given 0.2 mL MCF-7 cells suspension (4×107 cell/mL) or 0.2 mL (containing 0.1 mL matrix adhesive) MDA-MB-453 cells suspension (6×107 cell/mL) to establish the nude mouse xenograft models of ER(-) or ER(+) breast cancer. The neoplastic mice were stochastically divided into treatment group that were given with intra-gastric administration of ESZM extract or E. fischeriana extract, and model group that were given with intra-gastric administration of saline or Z. jujuba extract once a day for 4 weeks or 8 weeks.
Inhibition of tumor growth
Tumor diameter was monitored weekly. The tumor volumes were computed by a formula, V = (L × S2) /2 (mm3) (L: tumor long diameter, S: tumor short diameter). After 4 weeks of medication (model group, 2.5, 5.0, 10.0 g/kg groups of ESZM extract), nude mice were sacrificed using tumor inhibition rate detection. Tumor inhibition rate was computed by a formula: 1-mean tumor weight of drug treatment group / mean tumor weight of model group.
Hoechst 33342/PI staining analysis
Tumor tissues was dehydrated, deparaffinized and prepared into paraffin sections. Hoechst 33342 dye and PI dye were diluted with a buffer solution and severally dropped on the sections in 37℃ for 15 min, then the sections were rinsed with a buffer solution once. After slides with antiquenching glue were uesd to seal the sections. Last, the sections were observed and photographed with fluorescence microscope (Zeiss, Observer A1, Germany).
TUNEL assay analysis
Paraffin sections preparation were the same. TUNEL assay protocol as previously described [18]. The sections dropped antiquenching glue was encapsulated with slides, observed and photographed with an laser confocal microscopy (Zeiss, LSM710, Germany) to assess apoptosis index of TUNEL dyed tumour tissues.
TEM analysis
TEM protocol: A small piece of tumor tissue from each group were collected in containing 2.5% glutaraldehyde of EP tubes at 4°C for passthenight. Then, the ultrathin slices was prepared and fixed with 2% osmium tetroxide, soaked with Epon812, embedded, dyed with uranyl acetate and lead citrate, observed and photographed with a TEM (Hitachi Limited, HT7700, Japan) .
Detection of cell cycle and apoptosis in tumor tissues
Cell cycle and Apoptosis of tumor tissue was detected through flow cytometry (BD, FACSCalibur, USA) combined with PI or Annexin V-FITC/PI staining. Tumor tissue were made into cell suspensions. The suspensions were washed for 3 times with cooled PBS and collected in 12×75-mm capped polypropylene tubes. According to the manufacturer’s instruction, add 250 µL of Solution A (trypsin buffer) to each tube and gently mix by tapping the tube, to react for 10 min at room temperature, and 200 µL of Solution B (trypsin inhibitor and RNase buffer) to react for 10 min at room temperature, and then add 200 µL of cold Solution C (PI stain solution) to react for 10 min in the dark on ice. The cell cycle distribution were measured by using a flow cytometer.
The cell suspensions washed with cooled PBS for 3 times were resuspended with 300 µl buffer solution. Ten microlitre Annexin V-FITC and five microlitre PI were added to the suspensions. The samples was gentle mixed and fostered for 10 min at lightless room temperature. The apoptosis rate was evaluated by using a flow cytometer.
Immunohistochemical analysis
Paraffin sections preparation were the same. Immunohistochemical analysis on tumor tissue from mice treated with or without ESZM extract. The antigen was retrieved by a sodium citrate buffer. This was followed by endogenous peroxidase blocking and incubation in normal goat serum for 25 mins according to UltraSensitiveTM SP(Mouse/Rabbit) manufacturer’s protocol (cat. no. KIT-9710, MaiXin Bio, China). These sections were incubated with Bcl-2 (1:400, Cell Signaling, USA), Bax (1:400,Cell Signaling, USA), caspase 3 (1:1000, Proteintech, USA), caspase 9 (1:400, Cell Signaling, USA) overnight at 4°C and then with the peroxidase-conjugated secondary antibody. Color development was performed with DAB, and slides were counterstained with hematoxylin. Neutral resin sealing was performed. The results of immunohistochemical staining were observed under a microscope (OLYMPUS, IX51, Japan).
Immunofluorescence analysis
The p-PI3k (1:250, Abcam, USA), p-Akt (1:50, Cell Signaling, USA) and p-FoxO3a (1:500, Abcam, USA) proteins expression levels were monitored with immunofluorescence method as previously described (Ma et al., 2021).
Western blot analysis
The tumor tissues were fetched from liquid nitrogen, and ground in lysis buffer with a pre-cooling EP tube until invisible precipitate. The suspension with buffer were enriched into 4 mL EP tubes for 30 min in ice bath, and then centrifuged at 12000 rcf for 15 min at 4℃ to acquire protein. The BCA kit was used to evaluate protein concentrations. The 20 µg of protein were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with BSA buffer solution at room temperature for 2 h, and fostered with antibodies of p-Her2 (1:500, Abcam, USA), p-PI3k (1:250, Abcam, USA), p-Akt (1:2000, Cell Signaling, USA), p-FoxO3a (1:500, Abcam, USA), p-FoxO1a (1:500, Abcam, USA) and GAPDH (1:1000, Cell Signaling, USA) passthenight at 4℃, and washed three times with trisbuffered buffer solution, and incubated with a horseradish peroxidase conjugated secondary antibody for 2.0 h. At last, the imaging system (Bio-Rad, ChemiDoc MP, USA) was used to detect and analyse the intensity of protein bands.
Quantitative real-time polymerase chain reaction (qRT-PCR) test
The tumor tissues were fetched from liquid nitrogen, and ground into RNAiso Plus with a pre-cooling EP tube until invisible precipitate to obtain total RNA. Ultraviolet spectrophotometry (Shimadzu, Biospecnano, Japan) was used to detect purity and concentration of RNA at A260 nm/A280 nm. All RNA were reverse-transcribed into cDNA by Prime Script RT reagent Kit, and the levels of Her2, Bcl-2, Bax, caspse 3, caspase 9, FoxO3a, FoxO1a and Bim gene expression were evaluated via qRT-PCR Detection System (ABI, Q5, USA) with Premix Ex Taq Kit. The sequences of primers for Bcl-2 (Forward: 5'-GTTGTCGACGACGAGCG-3'; Reverse: 5'-CTGAG TACCTGAACCGGCA-3', 106 bp), Bax (Forward: 5'-AGCTTCTTGGTGGACGCAT − 3'; Reverse: 5'-CAGAGGCGGGGTTTCATC-3', 101 bp), Bim (Forward: 5’-GATAGTGGTTGAAGGCCTGG − 3’; Reverse: 5’-CCTCCCTACAGACAGAGCCA-3’, 102 bp), caspase 3 (Forward: 5’-TCGCTT CCATGTATGATCTTTG-3’; Reverse: 5’-CTGCCTCTTCCCCCATTCT-3’, 110 bp), caspase 9 (Forward: 5’-CATGCTCAGGATGTAAGCCA-3’; Reverse: 5’-AGGTTCTCAGACCGGAAACA − 3’, 116 bp), FoxO3a (Forward: 5’-AGTTCCCTCATTCTGGACCC-3’; Reverse: 5’-CTTCAAGG ATAAGGGCGACA-3’, 102 bp), FoxO1a (Forward: 5’-GCACACGAATGAACTTGCTG-3’; Reverse: 5’-AAGAGCGTGCCCTACTTCAA-3’, 106 bp), Her2 (Forward: 5’-AGCATGTCCAG GTGGGTCT-3’; Reverse: 5’-CTCCTCCTCGCCCTCTTG-3’, 109 bp) and β-actin (Forward: 5'-G TTGTCGACGACGAGCG-3'; Reverse: 5'-GCACAGAGCCTCGCCTT-3', 93 bp). The cycling conditions for all genes were as follows: 95℃ for 30 s followed by 40 cycles of denaturation at 95℃for 5 s, annealing and extension at 65℃ for 34 s. The each candidate gene expression were normalized to β-actin. The fold change in gene expression was calculated according to the 2−△△Ct method.
Drug toxicity analysis
After 8 weeks of medication (model group, 2.5, 5.0, 10.0g/kg groups of ESZM extract, 5.0 g/kg groups of E. fischeriana extract and Z. jujuba extract), the toxicity of the drug was examined in ER(-) or ER(+) breast tumor-bearing mice. The body weight, serum biochemical indexes, liver and kidney pathological indexes of the tumor-bearing mice were detected by an automatic biochemical analyzer or H&E staining analysis. H&E (cat. no. C0105, Beyotime Biotechnology, China) staining process follows. Paraffin sections of liver or kidney tissues preparation were the same. Hematoxylin staining, 1% hydrochloric acid alcohol differentiation, Eosin staining, gradient alcohol for dehydration, and seal slide. The sections pathological changes were assessed under a phase-contrast microscopy (OLYMPUS, IX51, Japan).
Statistical analysis
All data were presented as means ± standard deviation. SPSS19.0 statistical software was applied to evaluate statistical significance. Distinction between two groups was analyzed via two-tailed Student's T-Test, and distinction among three or more groups was analyzed with one-way ANOVA multiple comparisons. The distinction was conducted statistically significant when P < 0.05, P < 0.01 or P < 0.001 .