Huaier was provided by Gaitianli Pharmaceutical (Qidong, Jiangsu, China); the Huaier grain was dissolved in RPMI-1640 medium (Procell Company, Wuhan, China) and sterilised with a 0.22 µm filter (Merck Millipore, Darmstadt, Germany).

The Wnt agonist 1 was purchased from the MedChemExpress Company (Monmouth Junction, NJ, USA), and chloroquine (CQ) was obtained from the Sigma Company (St. Louis, MO, USA).

The HCT8 cells were purchased from the Procell Company, and the resistant cells of HCT8/L were purchased from the Aolu Biological Company (Shanghai, China); all cell lines were cultured in Dulbecco’s-Modified-Eagle-Medium-High Glucose supplemented with 10% fetal bovine serum (FBS, Procell Company) and 100 mg/mL of penicillin/streptomycin/glutamine (Procell Company) at 37 °C with 5% CO${}_2$. All cell lines were tested using a mycoplasma kit (Sigma) and were free of mycoplasma contamination. Furthermore, all cell lines were authenticated using short tandem repeat technology performed by the Shanghai Xuanyi Technical Service Centre shortly before use.

The CCK-8 assay was employed to detect cell viability using a Cell Counting Kit-8 (MCE, NJ, USA). Specifically, cells at the logarithmic stage were digested with trypsin and diluted to 1 $\times$ 10${}^3$–5 $\times$ 10${}^3$cells/mL; next, the cells were seeded into 96-well plates. This was followed by treatment with Huaier at different doses (0, 6, 9, 12, 15 and 18 mg/mL) for 24 or 48 h; the mixed solution of the Cell Counting Kit-8 per well was then added and placed in an incubator for 2 h. Finally, the plates were measured at an absorbance of 450 nm. This experiment was performed in triplicate.

The cells were seeded in 24-well plates. After 24 h, the culture medium was discarded, and a sensitive strain control group, a drug-resistant strain control group and a drug-resistant strain plus different concentrations of Huaier were cultured for 24 h. Next, the cells were repeatedly washed with phosphate-buffered saline (PBS), added with a 500 µL acridine orange (AO) mixed solution in each well, stained for 15 min, washed twice with PBS and immediately analysed using a fluorescence microscope (Olympus, Tokyo, Japan). The proportion of positive AO staining was determined by counting the number of fluorescent cells.

The cells were seeded on coverslips. After treatment with Huaier or 10% complete medium, the cells were fixed with 4% paraformaldehyde; 5% bovine serum albumin (BSA, Solarbio, Beijing, China) was used to block for 1 h, and LC3-B antibody (Abclonal, Woburn, MA, USA) was applied for incubation overnight at 4 °C. Next, the cells were incubated with Alexa Fluor 488 (Bioss, Beijing, China) for 1 h and stained with 4${}^{\prime }$,6-diamidino-2-phenylindole for 5 min. Finally, an anti-fluorescence quenching agent was added. The cells were immediately observed under a fluorescence microscope. The proportion of positive LC3 staining was determined by counting the number of fluorescent cells.

The cells were digested with trypsin and diluted in 6-well plates containing 10% complete medium. Next, the cells were cultured in a 5% CO${}_2$ 37 °C incubator overnight. The cells were treated with Huaier at different concentrations (0, 6, 9 and 12 mg/mL) for 24 h. The TRIzol (Biosharp, Shanghai, China) reagent was used to isolate total RNA, and cDNA was synthesised using a polymerase chain reaction (PCR) kit (Sparkjade, Shandong, China). The mRNA expression levels were detected using the SYBR Green qPCR Mix (Sparkjade); the data were analysed using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method. The specific primers are listed as follows: (1) p-gp forward, 5${}^{\prime }$-TCTATGGTTGGCAACTAACACT-3${}^{\prime }$ and reverse, 5${}^{\prime }$-CTCCTGAGTCAAAGAAACAACG-3${}^{\prime }$; and (2) GAPDH forward, 5${}^{\prime }$-GCACCGTCAAGGCTGAGAAC-3${}^{\prime }$ and reverse, 5${}^{\prime }$-TGGTGAAGACGCCAGTGGA-3${}^{\prime }$.

Prior to use, phenylmethylsulfonyl fluoride was added to the cytoplasmic protein extraction reagent A and the nuclear protein extraction reagent at a working concentration of 1 mM. The PBS-washed cells were used 2–3 times, then scraped, blown with a pipette gun and transferred to a centrifuge tube for centrifugation; the supernatant was discarded every 20 µL. For precipitation, cytoplasmic protein extraction reagent A containing PMSF was added, shaken vigorously for 5 s, and soaked in ice for 15 min. Then, 10 µL cell protein extraction reagent B was added into the precipitate, shaken well for 5 s and soaked in ice for 1 min before vigorous shaking again for 5 s at 4 °C. Subsequently, the mixture was centrifuged for 5 min at 15,000 g. A pipette gun was used to transfer the extracted cytoplasmic protein supernatant to a new Eppendorf (EP) tube for analysis. A nuclear protein extraction reagent was added to the remaining precipitate and shaken vigorously for 5 s to completely disperse the cell precipitate. Under the condition of an ice bath, it was shaken forcefully for 30 s/2 min for a total of 30 min and then centrifuged for 10 min at 4 °C and 15,000 g. The extracted supernatant of the nuclear protein was transferred to a new precooled EP tube with a pipette gun for standby.

Cells were lysed using radio-immunoprecipitation assay buffer (Solarbio) to collect proteins. The BCA protein assay kit (Solarbio) was used determined the protein concentration, and the proteins were subsequently separated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene fluoride membranes, 5% BSA was used to block the membranes for 2 h, and the mixture was incubated with primary antibody to p-gp (1:1000, CST, MA, USA), LC3B (1:2000, Abclonal), P62 (1:2000, Abclonal), Wnt 3a (1:1000, Bioss), $\beta$-catenin (1:10,000, CST), proliferating cell nuclear antigen (1:3000, Abclonal), GAPDH (1:10,000, Abclonal) and $\beta$-actin (1:100,000, Abclonal) overnight at 4 °C. Then, the secondary antibody (1:10,000, Abclonal) was incubated for 1 h. Finally, an enhanced chemiluminescence reagent (Biosharp) was used and developed using a gel imaging machine. The ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to analyse the protein grey values.

All data were shown as mean $\pm$ standard error of the mean (SEM). A Student’s t-test, one-way analysis of variance (ANOVA) and two-way ANOVA were performed. The SPSS (version 20, Cary, NC, USA) and GraphPad 8 (San Diego, CA, USA) software were used to analyse the data; a p value of 0.05 was considered statistically significant.

A comparative plot showed the cell inhibition rates of HCT-8 and HCT-8/L strains in response to different concentrations of OXA. As shown in the Supplementary Fig. 1, the IC${}_{50}$ value of HCT-8 was 0.5 µg/mL, the IC${}_{50}$ value of HCT-8/L was 7.58 µg/mL, and the resistive index was 15.16, indicating that HCT-8/L was a highly OXA-resistant cell line. The CCK-8 assay results showed that (1) Huaier decreased the viability of drug-resistant cells (Fig. 1A,B) and (2) the IC${}_{50}$ values for HCT-8 and HCT-8/L cells treated with Huaier for 24 h were 13.92 mg/mL and 15.76 mg/mL, respectively, whereas those treated with Huaier for 48 h were 9.64 mg/mL and 10.97 mg/mL, respectively. Moreover, the inhibitory effect of Huaier on cell viability was significantly enhanced in HCT-8 cells, whereas Huaier weakened the viability of HCT-8/L cells (Fig. 1C,D). Huaier inhibited cell viability; the inhibition of sensitive strains was greater than that of drug-resistant strains.

Fig. 1.

Huaier reduced the viability of colorectal cancer (CRC) cells. (A) Effect of Huaier on the activity of HCT-8 cells. (B) Effect of Huaier on the activity of HCT-8/L cells. (C) Huaier treatment for 24 h. (D) Huaier treatment for 48 h. Data means $\pm$ standard error of measurement (SEM), each group was triplicated, two-way ANOVA analysis.

Subsequently, qRT-PCR and western blotting (WB) were used to detect the effect of Huaier on the expression of resistance-associated protein p-gp in HCT-8/L cells (Fig. 2A,B). At the protein level, the expression level of p-gp in HCT-8/L cells decreased with increasing Huaier concentrations; the expression level of p-gp mRNA was significantly lower in HCT8 cells than in HCT8/L cells, whereas the expression of p-gp mRNA decreased with the increase in Huaier concentrations (Fig. 2C). The above results showed that Huaier could reverse the drug resistance of HCT-8/L cells by decreasing the p-gp expression.

Fig. 2.

Effect of Huaier on the expression of p-gp. (A) Western blotting results of p-gp in HCT-8 and HCT-8/L cells under the effect of different concentrations of Huaier. (B) Effect of Huaier on the relative quantification protein expressions of p-gp. (C) On the gene level, the change of p-gp and the change of the expression level of p-gp in HCT-8/L cells under the effect of different concentrations of Huaier. Data means $\pm$ SEM, two-tailed Student’s t-test, n $\ge$3. *p 0.05, ***p 0.001, ****p 0.0001.

To investigate the effects of Huaier on autophagy, AO staining assay was performed. Acridine orange is a specific dye that can penetrate acidic organelles, such as autophagic lysosomes. Owing to its sensitivity to pH, AO emits red fluorescence when the pH is low. The AO staining results are presented in Fig. 3A,C. Compared with HCT-8/L cells, HCT-8 cells had more acidic vesicular organelles, which were stained red by AO. After treatment of HCT-8/L cells with different Huaier concentrations, the number of acid vesicular organelles stained red with AO increased significantly.

Fig. 3.

Effect of Huaier on cell autophagy. (A,C) Acridine orange staining was used to observe the changes of acidic vesicular organelles induced by Huaier (white arrow: acidic vesicular organelles). (B,D) Detection of Huaier-induced autophagy via immunofluorescence staining. (E,F) Western blotting for P62 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) treated with Huaier. (G,H) Western blotting for LC3-II and GAPDH in HCT-8 and HCT-8/L cells treated with Huaier. Data means $\pm$ SEM, two-tailed Student’s t-test, n $\mathrm{\ge }$3. *p 0.05, **p 0.01, ***p 0.001.

The findings of the immunofluorescence staining assay further support the AO staining assay results. Although the HCT-8 cytoplasm exhibited LC3 green fluorescence, the staining intensity was weak. Meanwhile, in the Huaier group, the LC3 staining intensity increased with increasing Huaier concentrations (Fig. 3B,D).

Based on the above assays, the effect of Huaier on autophagy was further researched. After 24 h of treatment of HCT-8/L cells with Huaier, the P62 protein expression level significantly decreased, whereas the LC3-II protein expression significantly increased in each group. The expression of LC3-II was higher in the HCT-8 group than in the HCT-8/L group (Fig. 3G,H). However, the P62 protein expression was lower in the HCT-8 group than in the HCT-8/L group (Fig. 3E,F). The above results showed that Huaier can affect the viability of HCT-8/L cells through autophagy.

To determine whether Huaier affected the Wnt/$\beta$-catenin protein expression of HCT-8/L cells, the Wnt/$\beta$-catenin protein was detected using WB assay following treatment with Huaier for 24 h. Cytosolic $\beta$-catenin and Wnt3a were less expressed in HCT-8 cells (Fig. 4A–D). Huaier significantly reduced the expression of $\beta$-catenin and Wnt3a proteins in the HCT-8/L group.

Fig. 4.

Effect of Huaier on the Wnt/$\beta$-catenin signalling pathway. (A,B) Western blotting for $\beta$-catenin and $\beta$-actin treated with Huaier. (C,D) Western blotting for Wnt3a and $\beta$-actin treated with Huaier. Data means $\pm$ SEM, two-tailed Student’s t-test, n $\mathrm{\ge }$3. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

In this study, the autophagy inhibitor CQ was used to further clarify whether Huaier leads to the transformation of cellular drug resistance by enhancing the degree of autophagy. Acridine orange and immunofluorescence staining assays were performed to determine the effects of Huaier and CQ on autophagy. Chloroquine treatment decreased autophagy in HCT-8/L cells, which was significantly increased in HCT-8/L cells after Huaier treatment; furthermore, when used alongside Huaier, it did not reduce its effect on autophagy in HCT-8/L cells (Fig. 5A–D). The effects of Huaier and CQ on autophagy and drug resistance were detected using WB (Fig. 5E–J). The results showed that Huaier treatment significantly enhanced autophagy. After addition of the autophagy inhibitor CQ, the amount of LC3-II increased. Some studies have indicated that an increase in LC3-II is related to the fusion of autophagosomes and lysosomes, and/or lysosome degradation [15]. As demonstrated in Fig. 5B,D, CQ significantly inhibited autophagy, and the combination of Huaier and CQ promoted autophagy. Huaier reduced the expression of p-gp, CQ enhanced the expression of p-gp, and the combination of Huaier and CQ reduced the expression of p-gp (Fig. 5K).

Fig. 5.

Effect of Huaier and CQ on HCT-8/L. (A,C) Acridine orange staining was used to observe the changes of acidic vesicular organelles in HCT-8/L cells induced by Huaier and CQ. Control group. (B,D) Detection of Huaier and CQ induced autophagy by immunofluorescence staining. (E,F) Western blotting for P62 and $\beta$-actin in HCT-8/L cells treated with Huaier and CQ. (G,H) Western blotting for LC3-Ⅱ and $\beta$-actin in HCT-8/L cells treated with Huaier and CQ. (I,J) Western blotting for p-gp in HCT-8/L cells treated with Huaier and CQ. (K) A qRT-PCR for p-gp mRNA in HCT-8/L cells treated with Huaier and CQ. CQ, chloroquine. Data means $\pm$ SEM, two-tailed Student’s t-test, n $\mathrm{\ge }$3. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.

The Wnt agonist 1 was used in the present study to further elucidate that Huaier affected autophagy through inhibiting the Wnt/$\beta$-catenin signalling pathway. Acridine orange and immunofluorescence staining were also used to detect the effects of Huaier and Wnt agonist 1 on autophagy. The effects of Huaier and Wnt agonist 1 on autophagy and drug resistance were determined using WB. As presented in Fig. 6A–L, the degree of autophagy was significantly enhanced by Huaier, significantly inhibited by Wnt agonist 1, and promoted by the combination of Huaier and Wnt agonist 1. As demonstrated in Fig. 6M–O, the p-gp expression was reduced by Huaier, enhanced by Wnt agonist 1, and reduced by the combination of Huaier and Wnt agonist 1. The above results indicated that Huaier can regulate autophagy and drug resistance in HCT-8/L cells by regulating the Wnt/$\beta$-catenin signalling pathway.

Fig. 6.

Effect of Huaier and Wnt agonist 1 on HCT-8/L. (A,C) AO staining was used to observe the changes of acidic vesicular organelles in HCT-8/L cells induced by Huaier and Wnt agonist 1. (B,D) Detection of autophagy induced by Huaier and Wnt agonist 1 via immunofluorescence staining. (E,F) Western blotting for P62 and $\beta$-actin in HCT-8/L cells treated with Huaier and Wnt agonist 1. (G,H) Western blotting for LC3-Ⅱ and $\beta$-actin in HCT-8/L cells treated with Huaier and Wnt agonist 1. (I,J) Western blotting for $\beta$-catenin and $\beta$-actin in HCT-8/L cells treated with Huaier and Wnt agonist 1. (K,L) Western blotting for Wnt3a and $\beta$-actin in HCT-8/L cells treated with Huaier and Wnt agonist 1. (M,N) Western blotting for p-gp in HCT-8/L cells treated with Huaier and Wnt agonist 1. (O) A qRT-PCR for p-gp mRNA in HCT-8/L cells treated with Huaier and Wnt agonist 1. CQ, chloroquine. Data means $\pm$ SEM, two-tailed Student’s t-test, n $\mathrm{\ge }$3. *p 0.015, **p 0.01, ***p 0.001, ****p 0.0001.

The $\beta$-catenin expression in the nuclei was detected using WB. The results showed that the $\beta$-catenin nuclear expression was significantly higher in HCT-8/L cells than in HCT-8 cells; the $\beta$-catenin expression decreased after the addition of Huaier (Fig. 7A,B). Subsequently, HCT-8/L resistance to Oxaliplatin was determined following treatment with Huaier. As shown in Fig. 7C, the IC${}_{50}$ of HCT-8/L treated with Huaier against oxaliplatin was 3.11 µg/mL, which was less than that of 7.58 µg/mL untreated with Huaier. The results indicated that Huaier inhibited $\beta$-catenin entry into the nucleus by affecting the Wnt/$\beta$-catenin signalling pathway to reverse drug resistance of HCT-8/L cells.

Fig. 7.

Effect of Huaier on the entry of $\beta$-catenin into the nucleus. (A,B) Western blotting for $\beta$-catenin in the nucleus of HCT-8/L cells treated with Huaier. (C) Effect of Oxaliplatin on the activity of HCT-8/L cells after Huaier treatment. Data means $\pm$ SEM, two-tailed Student’s t-test, n $\ge$3. *p 0.05, **p 0.01.