An extraction from Trametes robiniophila Murr. (Huaier) inhibits non-small cell lung cancer proliferation via targeting to epidermal growth factor receptor

ABSTRACT An extraction from Trametes robiniophila Murr. (Huaier) is a kind of natural fungus growing from the sophora japonica tree. Huaier is widely applied to cure the hepatocellular cancer (HCC). However, the medicinal fungus’ curative result on non-small-cell lung cancer (NSCLC) is not well elaborated. In this study, we applied in vitro experiments to study Huaier’s curative result on NSCLC. The potential Huaier targets were predicted using bioinformatics and validated by western blotting. We further elucidated the mechanism of Huaier targeting by molecular docking, kinase activity assay, CEllular Thermal Shift Assays (CETSAs). At last, in vivo curative result was verified further. Huaier weakened proliferation and promoted apoptosis of the NSCLC cell lines. According to bioinformatics, Epidermal Growth Factor Receptor (EGFR) may be the target of Huaier. Western blotting showed that Huaier can attenuate the activation of EGFR and we found that Huaier can dock to EGFR. Huaier significantly inhibited the tumor growth by weakening the expression of p-EGFR in vivo. This study offers a new idea for further understanding of Huaier and shows its potential as a therapeutic agent.


Introduction
Eighty percent of lung cancer-related deaths are due to non-small-cell lung cancer (NSCLC) [1]. Chemotherapy is the preferred therapeutic method for advanced NSCLC, although its median overall survival (OS) does not exceed the upper limit of approximately one year [2]. The Epidermal Growth Factor Receptor -Tyrosine Kinase Inhibitor (EGFR-TKI), such as gefitinib, significantly extend survival and improve quality of life in NSCLC patients [3]. However, drug resistance limits the use of these drugs [4]. Therefore, the search for more effective and safe therapeutic drugs has become a hot topic in NSCLC research.
Traditional Chinese medicine cancer treatments have the advantages of few adverse effects, high safety, and improved quality of life [5,6]. Huaier is an extraction from Trametes robiniophila Murr., the main ingredient of which is a polysaccharide protein (PS-T), containing about 40% of polysaccharides, 10% of amino acids, six types of monosaccharides, and 18 amino acids [7]. In a prospective study of more than 1,000 patients, Chen [8] confirmed the curative result of Huaier granules as adjuvant therapeutic method for hepatocellular cancer (HCC) after hepatectomy. Patients who received Huaier granule adjuvant therapy were more prone to relapse-free survival (RFS) and overall survival (OS) than those who received placebo. Another study showed that liver cancer patients treated with Huaier experienced significantly prolonged duration recurrence time compared with those with transcatheter arterial chemoembolization (TACE) treatment alone [9]. Using microarray analysis, Wang [10] found that lncRNA H19 expression was down-regulated after breast cancer cell lines were administered with Huaier. Other studies have shown MAPK [11], Twist [12], and Wnt [13] are also Huaier targets. Besides, it also showed that Huaier inhibits triplenegative breast cancer metastasis [14]. However, the precise mechanism remains unknown.
Bioinformatics is a research method used in the identification of new drug targets based on a network perspective [15]. In pharmacological research of traditional Chinese medicine (TCM), bioinformatics can integrate the complex components, targets, and diseases. Here, PharmMapper and other databases were used to study the Huaier targets for the first time, revealing EGFR as the main target. A good spatial and energy match was found between Huaier and EGFR using molecular docking.
In this study, we explored the anti-cancer roles of Huaier in vitro and in vivo and the Huaier targets were predicted and verified. Huaier inhibited EGFR activity by binding to them directly. This study sharpened the understanding of Huaier's molecular biological function and indicated that Huaier may be a promising adjunct therapy for NSCLC.

Cell lines and reagents
Human NSCLC cell lines A549 and H1299 were purchased from Cell Bank of Representative Culture Preservation Committee of Chinese Academy of Sciences (Shanghai, China). The NSCLC cell lines were cultured in standard cell culture medium and kept in an incubator at 37°C with 5% CO 2 .

Colony formation
Colony formation was conducted as described before [17]. The formed colony under Huaier (and/or Gefitinib) administration was stained and photographed with a digital camera and analyzed using ImageJ (National Institutes of Health (NIH), Bethesda, USA).

Flow cytometry
We measured changes in apoptosis and cell cycle after Huaier (and Gefitinib) administration by flow cytometry and the data were analyzed using FlowJo (Version X; TreeStar, Ashland, OR) and ModFit LT (Verity Software House; Topsham, ME).

EdU staining
The effect of Huaier on DNA replication was measured through EdU/Apollo488 staining (Ribobio, C10301-1), which was conducted as manufacturer's protocal. Images were acquired by fluorescence microscope and analyzed by ImageJ.

Western blotting
Western blotting was done according to the protocal described before [17]. Proteins treated with different concentrations of Huaier were extracted, separated by electrophoresis, transferred to membranes, blocked and incubated with antibodies. Finally, membranes were visualized using the chemiluminescence reagent and densitimetric analysis was done by ImageJ.

Molecular docking
Molecular docking was conducted using Schrödinger software. EGFR crystal structure (PDB ID: 2ITY) [24] were prepared using RCSB database [25] and optimized with the Protein Preparation Wizard module. Different conformational states for Huaier ligand molecules was generated by Ligprep module. Glide module [26,27] was applied to calculate the docking between Huaier and EGFR in standard precision (SP).

EGFR enzymatic assay
We performed EGFR enzymatic assay using the Kinase Activity Assay Kit (Abace, Beijing, China), following the manufacturer's protocol. We treated EGFR protein with different concentrations of Huaier (or adenosine, daucosterol) and measure the fluorescence intensity by an automatic microplate reader.

CElluar thermal shift assays
We conducted CETSAs to reveal the binding between Huaier and EGFR in celluar. Briefly, cells, exposed to Huaier beforehand, were transferred into 1.5 ml PCR tubes and heated. Next, cells were lysed, separated then detected by western blot assays.

In vivo experiments
4-6 weeks old BALB/c nude mice were purchased from the National Laboratory Animal Center (Shanghai, China). After 6 days of acclimatization, we injected 5*10 6 A549 cells in the flank of each nude mouse subcutaneously. We assigned randomly the mice to vehicle control (saline) or Huaier alone (five animals in each group, 50 mg Huaier/100 µL solution) groups when the tumor volume reached approximately 60 mm 3 . We administered the drug gavagely once every three days and calculated tumor volume from the width and length of tumor body and sacrificed the mice after 21 days to remove their xenografts for immunohistochemical staining. The liver and kidneys in each group were dissected and embedded in paraffin to prepare sections for the hematoxylin and eosin (H&E) staining. In another experiment panel, mouse survival was evaluated. The study was performed in accordance with the Guidelines for Care and Use of Laboratory Animals of 'National Institutes of Health' and approved by the Ethics Committee of Shengjing Hospital of China Medical University (Supplementary Material 1).

Statistical methods
We repeated all experiments independently at least three times and analyzed results by SPSS software (SPSS, Chicago, IL, USA). The data were presented as the mean ± Standard Deviatio (SD) and standardized. We determined statistical significance between Huaier and control groups using student's two-tailed t-test and one-way ANOVA. In vivo experiments, survival was evaluated using the Kaplan-Meier method. Differences were considered significant when p-values < 0.05.

Results
In this study, we examined Huaier's anti-NSCLC effects in vitro and in vivo, then explored it's specific mechanism by molecular docking, kinase activity assay and CETSAs. We found that Huaier killed NSCLC cells by binding to EGFR directly. These findings provide new insights into the treatment of NSCLC.

Huaier inhibits NSCLC cell proliferation and promotes apoptosis
Different concentrations of Huaier were added to lung cancer cell lines to test inhibitory activity of Huaier on proliferation. MTT assay was applied to determine the cell viability of Huaier at different time points and at different concentrations, to detect Huaier's inhibitory effect on the proliferation of lung cancer cells. As displayed in Figure 1(a), Huaier inhibited proliferation of lung cancer cells concentration-and time-dependently. We further conducted colony formation to determine Huaier's anti-proliferation effect. As displayed in Figure 1(b), Huaier effectively inhibited colony formation. Then, we performed flow cytometry to test whether Huaier caused changes in the apoptotic cell number. Addition of the Huaier increased apoptotic cells and promoted late apoptosis (late apoptosis rate: A549: 0 mg/ml: 4.51% ± 0.8%, 4 mg/ml: 9.36% ± 1.1%, 8 mg/ml: 16.4% ± 1.9%. H1299: 0 mg/ml: 4.21% ± 0.9%, 4 mg/ml: 6.64% ± 1.4%, 8 mg/ml: 9.22% ± 2.0%. Figure 1(c)).
To investigate effects of Huaier on EGFR mutant NSCLC cells, we employed PC9 cell line, an EGFR 19 exon deletion mutant lung adenocarcinoma cell line that are highly dependent on EGFR activation, for further study. In MTT assay, Huaier inhibited proliferation of PC9 cells in a concentration-manner (IC 50 : 24 h 7.98 mg/ ml, Supplementary Material 2A). To further evaluate the inhibitory effect of Huaier on the growth of PC9 cells, we selected Gefitinib, a commonly used EGFR tyrosine kinase inhibitor (TKI) [28], as the standard reference drug. The results of the MTT assay showed that 8 mg/ml Huaier and 50 nM Gefitinib (a commonly used concentration of Gefitinib [29,30]) had similar inhibitory effects (Supplementary Material 2B), while the combination of Huaier and Gefitinib sharply decreased the viability of PC9 cells, and the extent of reduced cell viability by the combination therapy was significantly greater than either of the monotherapies. As shown in Supplementary Material 2C, similar results were observed in colony formation assay. PI-annexin V staining showed that apoptotic cells increased after the addition of Lyc and/or Gefitinib, with significant differences between the different treatments (Supplementary Material 2D).
In conclusion, these in vitro experiments showed that Huaier inhibited the growth of NSCLC cells.

Identification of Huaier targets using bioinformatics
Five structural formulas for Huaier components were uploaded to PharmMapper, where duplicate results were removed to obtain a total of 575 possible targets. The official symbol for the drug targets was obtained from UniProt. For a more in-depth understanding of the target protein, the GO function and KEGG pathway analyses were applied in the WebGestalt. The metabolic process was the most significant biological process (BP) term, membrane was the cellular component (CC) term with highest significance, and the most significant (MF) term was protein binding (Figure 3(a-c)). The KEGG pathway showed that the targets were involved in many metabolic pathways (Figure 3(d)). A total of 376 NSCLC-related targets were obtained from CooLGen. The Huaier targets were intersected with the NSCLC-related targets. Thirty-two possible targets for Huaier inhibition of lung cancer were obtained (Figure 3(e)). Among them, EGFR were the classical kinases driving lung cancer. Therefore, it was speculated that Huaier inhibits lung cancer through EGFR (Figure 3(f)).

Huaier targets EGFR
As mentioned above, EGFR was verified as Huaier targets verified using bioinformatics prediction. We applied western blotting to discover the protein expression changes to clarify the Huaier's regulatory effect on the EGFR pathways. The total-EGFR expression did not change significantly after the Huaier treatment. The tyrosine 1068 phosphorylation of EGFR (p-EGFR) expression level was downregulated ( Figure 4(a,b)). When lung cancer cells were treated with Huaier (6 mg/mL) and pretreated with EGF (25 ng/mL), it was evident that Huaier can inhibit the EGF-induced EGFR activation (Figure 4  (c,d)). These results suggest that Huaier can inhibit the activation of EGFR.

Huaier docks to EGFR
Molecular docking is to judge the characteristics of receptors and the interaction between receptors and drug molecules. We used molecular docking to explore the specific mechanism of Huaier on the targets. To investigate geometrical complementarity and energy matching of Huaier and EGFR crystal structure, we chose gefitinib as a positive control. Previous reports on the five components of Huaier and molecular docking showed that adenosine and EGFR have the best binding capacity (Table 1), with a G-score of −8.805 kcal/mol, which is similar to that of gefitinib (G-score: −8.626 kcal/mol). Both adenosine and gefitinib can form hydrogen bonds with the EGFR Aspartate (Asp 555), suggesting that adenosine and gefitinib may have similar effects on EGFR (Figure 5(a)). Daucosterol and adenosine have similar binding with EGFR. Daucosterol also form hydrogen bonds with the EGFR Aspartate (Asp 555), with a G-score of −6.826 kcal/mol. Almost no steric hindrance exists between adenosine and EGFR. Similar to the molecular docking results, the kinase activity assay showed that Huaier inhibits EGFR with IC 50 value of approximately 1.72 mg/mL ( Figure 5(b)). Subsequently, we performed CETSAs to determine the docking EGFR protein and Huaier in cellulo. As shown in Figure 5(c),

Huaier inhibits NSCLC in vivo
To validate the results of cell experiments, a xenotransplantation model of human NSCLC in mice was established in mice using A549 cells. Huaier was administered for 3 weeks before the mice were sacrificed. The volume of the xenograft tumors decreased after the Huaier administration compared with the control group ( Figure 6(a)). The xenograft tumors was also significantly lighter than that of the control group. The xenograft tumors average weight was 1.5 ± 0.41 g in the Huaier treatment group and was 2.4 ± 0.54 g in the placebo treatment group (Figure 6(b)). The transplanted tumors growth rate in the Huaier treatment group was slower than that in the placebo treatment group. After approximately 18 days, transplanted tumors size between two groups differed significantly (Figure 6(c)). The expression levels of p/t-EGFR and cell proliferation marker Ki-67 were detected using immunohistochemistry to explore Huaier's mechanism on tumor growth inhibition. Huaier reduced the expression of p-EGFR and proliferation, while it had no effect on total-EGFR expression ( Figure 6(d)). During the experiment, the weight changes in mice were regularly monitored. The average weights of the two groups were similar (Supplementary Material 5B). The mice treated with Huaier had no clinical symptoms of disease and discomfort, and no obvious drug-induced liver and kidney damage (Supplementary Material 5C) was found by H&E staining, which indicated good Huaier tolerance. These results demonstrate that Huaier is effective and safe in vivo.

Discussion
One of the leading cause of cancer-related death worldwide is lung cancer and its effective treatment is still scarce. Approximately 80% of traditional Chinese medicines come from plants, among which fungal Chinese medicines play an important role. The Huaier fungus extract was discovered during a treatment of hepatocellular ascites. But, how Huaier works on lung cancer remains unclear. Chen [31] found that Huaier inhibited lung cancer proliferation and metastasis through MTDH, JAK2/STAT3, and MAPK. In addition, other studies [32] have shown that Huaier regulates lung cancer sensitivity to cisplatin via the mTOR pathway. In addition, several microRNAs have also been identified as targets of Huaier (e.g. let-7d-5p [33], miRNAs-203 [34] and miRNAs-26b-5p [35]), suggesting that Huaier has multitarget and multipathway pharmacological effects in anti-cancer thearapy. The specific mechanism involved in Huaier's inhibition of lung cancer is still not clear. In this study, we found for the first time that Huaier inhibited EGFR's activiation by binding to the ATP-pocket directly. Investigation of pharmacodynamic substances and mechanisms by bioinformatics can reveal new research directions and ideas for traditional experimental research methods [36]. Molecular docking is a process of recognition by space and energy matching between proteins and drugs, which can also simulate drug-target binding [37]. In this study, nearly 600 Huaier targets were obtained through the PharmMapper database. The GO and KEGG pathway enrichment analyses showed these targets were involved in many tumor-associated biological processes. Using screening, EGFR was predicted to be Huaier's targets for NSCLC inhibition. Through virtual verification of molecular docking, it was shown that EGFR can bind to Huaier. Additionally, using molecular docking, we found that Huaier active ingredients not only bind to wild-type EGFR, but also bind to exon 21 mutant EGFR (L858R) (data not shown). Therefore, we conclude that Huaier can also inhibit the activity of mutant EGFR, but this observation needs to be confirmed by further experiments. We speculated that daucosterol may be the effective components of Huaier in inhibition of EGFR. Adenosine and EGFR had a higher G-score, but failed to inhibit EGFR activity, while daucosterol had a lower G-score but inhibited EGFR activity. This result may be attributed to the higher molecular weight (MW) of daucosterol (adenosine: 267.24, daucosterol: 576.85), which may be able to better enter the ATP pocket of EGFR. On the other hand, it is limited to evaluate the binding affinity of ligand and receptor by Glide score or docking score.
The imbalance of proliferation and apoptosis is one of the characteristics of cancer, which indicates that inhibiting proliferation and promoting apoptosis is an important anti-cancer therapy strategy. In this study, it was found Huaier can inhibit the growth of A549 and H1299 cell lines. EGFR is a tumor proliferation-related molecule. Huaier can inhibit the activation of EGFR. The molecular docking results showed that the binding sites of Huaier's active components for EGFR were located near the tyrosine 1068 phosphorylation sites, which may be one of the mechanisms of Huaier's inhibition of Growth Factor Receptor-Bound protein 2 (GRB2), an adaptor protein that forms stable complexes with tyrosinephosphorylated EGFR, activating intracellular signaling pathways [38]. In the next study, we will explore if Huaier influence expression or location of GRB2.

Conclusion
TCM has unique advantages in cancer treatment, but its effective substances and mechanisms are not yet clear. In this study, using bioinformatics prediction, molecular docking, and in vitro and in vivo experiments, it was revealed that Huaier attenuated the EGFR activation. We preliminarily showed that daucosterol is the component that inhibit EGFR, but this needs further demonstration. Possible Huaier impact on other targets, especially other tyrosine kinase, needs to be further investigated. We look forward to a large sample of clinical trials to verify the efficacy of Huaier on NSCLC patients for improved lung cancer patients' survival.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.