TRIzol^® buffer (catalog no. 87801), RevertAidTM first strand cDNA Synthesis Kit (catalog no. 00174486) and enhanced chemiluminescence (ECL) kit (catalog no. QE218149) were purchased from Thermo Fisher Scientific, Inc., (Waltham, MA, USA). Polyclonal anti-Wnt1 (catalog no. AA56134), anti-GSK3β (catalog no. CN33151), anti-phosphorylated (p)-GSK3β (catalog no. CN33151), anti-SFRP-2 (catalog no. CC22141) antibodies were purchased from Bioworld Technology, Inc., (St Louis Park, MN, USA). Monoclonal anti-Wnt2 (catalog no. GR203181-1), anti-Wnt5a (catalog no. CC36131), anti-β-catenin (catalog no. GR177612-31), anti-Survivin (catalog no. GR209492-1) and polyclonal anti-Dvl-1 antibodies (catalog no. 109145) were obtained from Abcam (Cambridge, UK). Anti-CD44V6 antibody (catalog no. bs-20756R) was purchased from BIOSS (Beijing, China). DAB kit (catalog no. K137726A) was obtained fromOrigene Technologies, Inc., (Beijing, China).

All crudes of QYSL decoction drugs and Cisplatin (DDP; catalog no. 411016CE) were purchased from dispensary of TCM and dispensary for western medicine in the First Affiliated Hospital of Anhui University of Chinese Medicine (Hefei, China). The decoction consisted of 30 g astragalus membranaceus (huangqi), 10 g polygonatum odoratum (yuzu), 6 g scolopendra subspinipes mutilans (tianlong), 6 g pberetima (dilong), 20 g solanum nigrum (longkui), 20 g herbahedyotis (baihushecao), 20 g semen coicis (yiyiren), 6 g euphorbia helioscopia (zeqi), 10 g curcuma longa (eshu) and 6 g tendril-leaved fritillary bulb (chuanbei). QYSL decoction was stored at 4°C at a concentration of 4.024 g/ml.

A total of 60 male SPF grade C57BL/6 mice (6–8 weeks old), weighing 20±2 g, were purchased from Cavens Laboratory Animal Co., Ltd. (Changzhou, China; catalog no. SCXK 2011-0003). Mice were housed in air-filtered laminar flow cabinets with a 12-h light cycle and food and water ad libitum. All the animal care and experimental procedures followed were approved by the Institutional Animal Care and Use Committee of Anhui University of Chinese Medicine. Lewis lung carcinoma LLCs were obtained from Institute of Shanghai Academy of Life Sciences, Chinese Academy of Sciences (Shanghai, China), and maintained in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 U/ml streptomycin (Origene Technologies, Inc.), and kept in an incubator at 37°C, in a humidified atmosphere containing 5% CO₂.

LLCs were suspended in serum-free medium and a total of 1×10⁵ (0.1 ml) LLCs per mouse were inoculated subcutaneously into the left dorsal flanks of nude mice. When tumors reached an average volume of approximately 3–5 mm³ (10 days following injection), the mice were considered as established with xenografts. The next day, mouse models were randomized into six groups (n=8 per group): i) Control group (C): Mice received dosage of 0.2 ml/10 g physiological saline via intragastric administration for 21 days, and 0.4 ml of physiological saline by intraperitoneal injection once a week. ii) Low dosage of QYSL decoction group (LQ): Mice underwent intragastric administration of 20.12 g/kg QYSL decoction for 21 days, and 0.4 ml physiological saline via intraperitoneal injection once a week. iii) Medium dosage of QYSL decoction group (MQ): Mice underwent intragastric administration of 40.24 g/kg QYSL for 21 days, and 0.4 ml physiological saline via intraperitoneal injection once a week. iv) High dosage of QYSL decoction group (HQ): Mice received intragastric administration with doses of 80.48 g/kg QYSL for 21 days, and 0.4 ml physiological saline via intraperitoneal injection once a week. v) DDP group (DDP): Mice received intragastric administration of 0.2 ml/10 g physiological saline for 30 days, and 0.4 ml DDP via intraperitoneal injection once a week. vi) HQ+DDP group: Mice received intragastric administration of 80.48 g/kg QYSL for 21 days, and 0.4 ml DDP via intraperitoneal injection once a week. Following a 22-day period, tumor xenografts were isolated, and measured with the precision electronic balance and the tumor inhibition ratio was calculated. The tumor inhibition ratio=(the average quality of tumor in C group-the average quality of tumor in experience group)/the average quality of tumor in C group × 100%.

Total RNA was extracted from xenograft tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The quality and quantity of RNA were determined by measuring the absorbance at wavelengths of 260 and 280 nm using Nano Drop-2000 ultra microspectrophotometer (Thermo Fisher Scientific, Inc.). A total of one µg of RNA was reverse transcribed into cDNA using RevertAidTM first strand cDNA Synthesis kit according to the manufacturer's protocol. C-myc, Cyclin D1 and Axin2 cDNAs were amplified using SYBR Premix Ex Taq kit (Takara Bio, Inc.) with LightCycler 480 PCR system (Roche Diagnostics, Indianopolis, IN, USA). The thermocycling conditions for the PCR assay cycles were as follows: 95°C for 5 min, 40 cycles of 95°C for 10 sec, 95°C for 10 sec and 60°C for 30 sec. The mRNA levels of the target gene were normalized to the level of β-actin using the 2^−ΔΔCq method (14) and were represented as fold induction. The primers used for RT-qPCR were as follows: Forward, 5′-TGAGGAAACGACGAGAACAG-3′ and reverse, 5′-ACGAGAGATTCCAGCTCCTC-3′ for c-myc; forward, 5′-ATCTCCCTTGATTCAAACGC-3′ and reverse, 5′-GCCCAATGAAAGACCAATCT-3′ for cyclin D1; forward, 5′-CAGTGAGCTGGTTGTCACCT-3′ and reverse, 5′-CTGAGCTGCTCCTTGAAGTG-3′ for Axin2; forward, 5′-CCTCTATGCCAACACAGTGC-3′ and reverse, 5′-GTACTCCTGCTTGCTGATCC-3′ for β-actin.

Proteins were extracted from xenograft tissues using radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The concentration of protein was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). A total of 50 µg protein was subjected to 10% SDS-polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). Subsequently, the membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in Tris-buffered saline with 0.05% Tween-20 (TBST) for 1 h at room temperature and then incubated overnight at 4°C with the primary antibodies against β-actin (1:1,000), Wnt1 (1:500), Wnt2 (1:500), Wnt5a (1:1,000), Dvl-1 (1:500), GSK3β (1:1,000), p-GSK3β (1:500) or SFRP-2 (1:1,000). Following washing with TBST three times, the membrane was probed with peroxidase conjugated affinity purified goat anti-mouse immunoglobulin G (Origene Technologies, Inc., Beijing, China, 1:1,000 dilution, cat. no. TA100015) at 37°C for 2 h. The level of β-actin was examined as an internal control. The membranes were developed with an ECL kit (Pierce; Thermo Fisher Scientific Inc.), and the gray value of the bands of interest was analyzed by FlourChem V2.0 (ProteinSimple, San Jose, CA, USA).

Tumor xenografts were fixed immediately following removal, in a 10% buffered formal in solution for a maximum of 48 h at room temperature, prior to being dehydrated and paraffin-embedded under vacuum conditions. Slides with paraffin sections (3–5 µm) were deparaffinized. Endogenous peroxidase activity was blocked by incubation in 3% H₂O₂ for 10 min at room temperature. The slides were incubated with the primary anti-CD44v6 antibody (1:200 dilution) or anti-Survivin antibody (1:500 dilution) overnight at 4°C. Sections were washed with PBS and incubated with biotinylated anti-rabbit secondary antibody for 2 h at 37°C. Diaminobenzidine (DAB)-H₂O₂ was used as the chromogen to reveal antibody binding sites. A total of five high-powered fields were randomly chosen using an inverted microscope. Positive cells were marked in brown or dark nankeen. The average optical density was quantified with JEDR801D software (Jetta Technology, Nanjing, Jiangsu, China). The staining results were evaluated and scored independently by two pathologists.

All experiments were performed at least three times using independent samples. All data are expressed as the mean ± standard deviation. Statistical analysis was conducted using SPSS software, version 12.0 software (SPSS, Inc., Chicago, IL, USA). The Student's t-test was used to compare the difference between two individual groups, and one-way analysis of variance followed by Dunnett's post hoc testwas used to compare the difference among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To explore the role of the QYSL decoction on lung cancer, the present studyestablished a C57BL/6 mouse xenograft model using LLC and administered different doses of QYSL decoction to treat mice. In Table I, there were no significant differences among C, LQ and MQ groups (P>0.05). However, the tumor size in HQ, DDP or HQ+DDP groups was decreased compared with the C group (P<0.05; P<0.01; P<0.01). No significant difference was observed between DDP and HQ+DDP groups, however the tumor masses in the DDP and HQ+DDP groups were lower compared with HQ group (P<0.01). Following this, the tumor inhibition ratio from different groups was analyzed. Compared with C group, the tumor inhibition ratios of LQ, MQ, HQ, DDP and HQ+DDP groups were increased (P<0.01). These data demonstrated that high doses of QYSL decoction and DDP significantly suppressed tumor growth, suggesting that QYSL decoction may function as a lung cancer therapeutic.

To examine the mechanism by which QYSL decoction exhibits its inhibitory effects in lung tumor development, the present study first detected the mRNA levels of c-myc, cyclin D1 and Axin 2 following different treatments. In Fig. 1, there was no significant difference between C and LQ groups in the mRNA levels of c-myc, cyclin D1 and Axin2 (P>0.05). Compared with C group, the levels of c-myc, cyclin D1 and Axin2 in MQ, HQ, DDP and HQ+DDP groups were significantly repressed (P<0.01). No significant differences in the expression of c-myc among HQ, DDP and HQ+DDP groups were observed. However, compared with HQ group, the level of cyclin D1 in DDP group were suppressed, however the expression of Axin2 was upregulated (P<0.01). These data demonstrated that the QYSL decoction and DDP inhibited the expression of c-myc, cyclin D1 and Axin2, however, the QYSL decoction and DDP demonstrated different degrees of inhibition, and the application of QYSL decoction affected the role of DDP to a certain degree.

To further determine the mechanism of QYSL decoction functioning in tumor inhibition, the protein levels of regulatory proteins in Wnt/β-catenin pathway were detected. In Fig. 2A, compared with C group, different dosages of QYSL decoction and DDP displayed different degrees of inhibition on the protein levels of Wnt1, Wnt2, Wnt5a, GSK3β, p-GSK3β, β-catenin and Dvl-1. Compared with C group, the protein levels of Wnt1, Wnt2, Wnt5a, GSK3β, p-GSK3β, β-catenin and Dvl-1 in LQ, MQ, HQ, DDP and HQ+DDP groups were significantly suppressed (Fig. 2B-H; P<0.01), and the HQ+DDP group demonstrated the most significant inhibitory effect on the expression of the aforementioned genes, except β-catenin. However, compared with the above genes, different doses of QYSL decoction and DDP resulted in different effect on the protein level of SFRP-2. Compared with C group, the levels of SFRP-2 in LQ, MQ, DDP and HQ+DDP groups were upregulated (P<0.01), and HQ markedly enhanced the expression of SFRP-2 (Fig. 2I; P<0.01). Overall, the results revealed that the QYSL decoction and DDP modulated the regulatory protein expression levels in Wnt/β-catenin pathway.

CD44 variant isoforms, particularly CD44v6, have been identified as protein markers for metastatic behavior innumerous malignant tumors (15). Elevated expression of Survivin correlates with poor prognosis, tumor recurrence and drug resistance in various human cancers, including lung cancer (16). The present study detected the signals of CD44v6 (Fig. 3) and Survivin (Fig. 4) via immunohistochemistry following different treatments, presented in Table II. In Fig. 3, the signals of CD44v6 in MQ, HQ, DDP and HQ+DDP groups decreased compared with C group (P<0.05), and there were no significant differences among MQ, HQ and DDP groups, however HQ+DDP group revealed the most significant inhibitory effect. Similarly, there was no significant differences observed in Survivin signals among C and LQ groups (P>0.05). However, the signals of Survivin in MQ, HQ, DDP and HQ+DDP groups were suppressed compared with the C group (Fig. 4; P<0.05), and there was no significant difference among the MQ, HQ, DDP and HQ+DDP groups (P>0.05). These data revealed that QYSL decoction and DDP inhibited the protein levels of CD44v6 and Survivin.