The human breast cancer cell line MCF-7, the mouse breast cancer cell line 4T1 and the mouse colorectal cancer cell line CT26 used in this study were purchased from the China Center for Type Culture Collection (Shanghai, China). The human colorectal cancer cell line HCT116 was purchased from China Center for Type Culture Collection (Beijing, China). HCT116 and CT26 were cultured in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). 4T1 and MCF7 were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Carlsbad, CA, USA) containing 10% FBS. All cells were maintained in a humidified incubator containing 5% CO${}_2$ at 37 °C.

Cell viability was tested using the cell counting kit-8 (CCK8, Dojindo, Japan). Briefly, the cells were seeded at a density of 1 $\times$ 10${}^3$/well in 100 $\mu$L of medium into 96-well microwell culture plates and treated with indicated concentrations of juglone (0, 0.5, 1, 2, 4, 8, 16, 32, and 64 $\mu$M) for 24 hours. The medium was then carefully removed and 100 $\mu$L of serum-free medium with 10% CCK-8 reagent was added to each well. The cells were cultured with this reagent for 4 hours at 37 °C. Then the cell plates were the shaken mildly, and the absorbance was measured at 450 nm using a microtiter plate reader. The viability and IC50 were calculated using the equation calculated by [(As – Ab)/(Ac – Ab)] $\times$ 100% (As: absorbance of experimental group; Ac: absorbance of control group; Ab: absorbance of blank group, Supplementary Fig. 1).

Full-length human PIN1 (NM006221) cDNA was amplified using the primers Pin1-Forward (EcoRI): 5′-CTGAATTCGCCACCATGGCGGACGAGGAGAAG-3′ and Pin1-Reverse (BamHI): 5′-CGGATCCCTCAGTGCGGAGGATGATG-3′. Total RNA used in this amplification step was isolated from HCT116. RNA was isolated as previously describe [16]. Resultant cDNA was subsequently subcloned into pLVX-DsRed vector (donated by Dr. Jianfeng Jin) using the primer-encoded BamHI and EcoRI restriction sites. Lentivirus was subsequently packaged as previously described [16].

According to previously obtained IC50 values, various doses of juglone were selected to treat different cancer cells. The dose gradient for 4T1 was 0 $\mu$M, 1 $\mu$M and 2 $\mu$M; for MCF7 was 0 $\mu$M, 4.5 $\mu$M and 9 $\mu$M; for CT26 was 0 $\mu$M, 6 $\mu$M and 12 $\mu$M and for HCT116 was 0 $\mu$M, 1.5 $\mu$M and 3 $\mu$M.

500 cells/well of each line were seeded in a 24-well plate (Corning Life Science, MA, USA) and cultured in serum free DMEM supplemented with 20 ng/mL basic fibroblast growth factor (bFGF, Sigma, Darmstadt, Germany), 20 ng/mL epidermal growth factor (EGF, Sigma, Darmstadt, Germany) and 20 ng/mL B27 (Gibco, Carlsbad, CA, USA). 200 $\mu$L of this medium was replenished every other day and the plates gently shaken every 12 hours. The resultant tumor spheres were photographed using a light microscope after 7 days and the number of spheres with a diameter greater than 50 $\mu$m was counted.

Cultured cells were treated with differing doses of juglone for 24 hours as indicated above. Then the cells were subsequently seeded in 35 mm culture dishes (Corning Life Science, MA, USA) at a density of 500 cells/dish and incubated for 7 days. Following this, colonies were fixed and stained with 0.1% crystal violet (Sigma, Darmstadt, Germany) and counted.

A transwell chamber (8 $\mu$m pore size, Corning Costar, NY, USA) was used to examine cell migration potential. Cells were treated for 24 hours with various doses of juglone as indicated above and subsequently counted and seeded into the chambers (1 $\times$ 10${}^5$ cells for 4T1, 2 $\times$ 10${}^4$ cells for MCF7, 2 $\times$ 10${}^4$ cells for CT26 and 1 $\times$ 10${}^5$ cells for HCT116). Analysis of migration was conducted as previously described [16].

Twenty-one six-week-old BALB/c female mice were randomly divided into 3 groups. 4T1 cells were first treated with differing doses of juglone (0 $\mu$M, 1 $\mu$M and 2 $\mu$M) for 24 hours, then diluted to 1 $\times$ 10${}^7$/mL, 1 $\times$ 10${}^6$/mL, 1 $\times$ 10${}^5$/mL, 1 $\times$ 10${}^4$/mL concentration in 200 $\mu$L of serum free DMEM. Then the cells of four densities were subcutaneously injected with 100 $\mu$L of cell suspension in the bilateral axillary and groin of the same mouse. The mice were sacrificed 3 weeks after inoculation.

Each tumor injection was prepared using a 50 $\mu$L cell suspension containing 1 $\times$ 10${}^6$ cells. The CT26 cells were injected into the spleen of six-week-old BALB/c mice. Juglone was dissolved in DMSO to 20 mg/mL and diluted 100-fold with PBS. The mice were treated with juglone (2 mg/kg) every 2 days for 14 days or treated with an equivalent volume of DMSO diluted 100-fold in PBS as the control. Mice were subsequently sacrificed, and the liver was removed to evaluate metastasis. All animal experiments in this study were approved by the local ethics committee of Harbin Medical University.

Western blot analysis was conducted as previously described [16]. Briefly, 30–50 $\mu$g of total protein was loaded on polyacrylamide gels followed by electrophoresis and electrotransfer to nitrocellulose membranes. The primary antibodies used in this study are described in Supplementary Table 1, and the gray values of western blot see Supplementary Fig. 2.

All experiments were performed in, at least, triplicate, data are presented as the mean ($\pm$ SD) of these data points. Statistical significance was tested using Student’s t-test. A p-value 0.05 was considered statistically significant (*p 0.05, **p 0.01, ***p 0.001, the p-value see Supplementary Table 2).

Tumor sphere formation assays are a widely employed approach to assess cell capability of self-renewal [7]. Human breast and colorectal cancer cells were treated with differing concentrations of juglone for 24 hours, and a tumor sphere formation assay was subsequently performed. The appropriate concentrations of juglone used in different cell lines were selected according to an empirically obtained 50% inhibition concentration (IC50) (see Supplementary Fig. 1). The numbers and diameters of tumor spheres were counted after 7 days. Notably, juglone treatment significantly reduced the number and diameter of tumor spheres compared with the control (Fig. 1A,B). In a separate set of experiments, a colony formation assay also showed that juglone treatment reduced the number of colonies in a dose-dependent manner in both breast and colorectal cancer cells (Fig. 1C,D).

Fig. 1.

Juglone suppresses the self-renewal ability of different cancer cells. (A,B) MCF7 and HCT116 cells were treated with indicated doses of juglone and cultured in serum-free media under non-adherent conditions for 7 days. Scale bar, 100 $\mu$m. (C,D) A colony formation assay was performed, culture plates were stained with crystal violet. *p 0.05, **p 0.01, ***p 0.001.

To further determine the role of juglone in maintaining cancer cell stemness characteristics, we investigated the effect of juglone on the CSC signaling network. The Wnt/$\beta$-catenin pathway plays an important role in regulating both CSC and non-CSC dynamics [5]. It has been reported that activation of the Wnt/$\beta$-catenin axis could upregulate the expression of reprogramming factors such as Nanog, SOX2 and OCT4 [17, 18]. In the present study, western blot analysis indicated that juglone treatment significantly downregulated the expression of proteins related to maintaining stemness, including $\beta$-catenin, SOX2, OCT4, and Nanog (Fig. 2A).

Fig. 2.

Juglone suppresses stemness characteristics and EMT in cancer cells. (A) The expression of $\beta$-catenin, Nanog, OCT4 and SOX2 in breast and colorectal cancer cells was assessed by western blot analysis after juglone treatment. (B) The cells were treated with 0 $\mu$M, 1 $\mu$M and 2 $\mu$M of juglone for 24 hours, and serially diluted as 1 $\times$ 10${}^7$/mL, 1 $\times$ 10${}^6$/mL, 1 $\times$ 10${}^5$/mL, 1 $\times$ 10${}^4$/mL prior to injection into mice. Then the cells of four densities were subcutaneously injected with 100 $\mu$L of cell suspension in the bilateral axillary and groin of the same mouse. The mice were sacrificed 3 weeks after inoculation and the tumors were removed to evaluate the tumorigenicity. (C) The expression of E-cadherin, vimentin and N-cadherin was tested by western blot analysis after juglone treatment.

To examine the effect of juglone on cancer cell tumorigenicity in vivo, a limiting dilution cell transplantation assay was performed in BALB/c mice [19]. 4T1 breast cancer cells were treated with indicated concentrations of juglone (0 $\mu$M, 1 $\mu$M and 2 $\mu$M) for 24 hours and subcutaneously injected in the bilateral axillary and groin of the same mouse at four different cell concentrations (1 $\times$ 10${}^7$/mL, 1 $\times$ 10${}^6$/mL, 1 $\times$ 10${}^5$/mL, 1 $\times$ 10${}^4$/mL). The development of tumors, including the number and size of tumors, decreased significantly after juglone treatment compared with the control. Moreover, this decrease was found to occur in a juglone dose-dependent manner (Fig. 2B and Table 1).

EMT plays an important role in stemness maintenance and metastasis in cancer cells [17]. To further examine the effect of juglone on EMT, some typical EMT markers were assayed in cancer cells after juglone treatment. The results showed juglone treatment increased E-cadherin expression and decreased the vimentin and N-cadherin (Fig. 2C). These results are consistent with juglone downregulating EMT [9]. Taken together, these findings suggested that juglone could inhibit the self-renewal, tumorigenicity, and EMT of cancer cells in vitro and in vivo.

The transwell migration assay is a commonly used test to study the migratory response of cancer cells, a key characteristic for metastatic tumor spread [20]. To evaluate the role of juglone in downregulating tumor cell mobility, we performed a transwell assay with breast and colorectal cancer cells. The results showed juglone treatment could effectively reduce the mobility of both breast and colorectal cancer cells (Fig. 3A–D).

Fig. 3.

Juglone inhibits cancer cells metastasis both in vitro and in vivo. (A–D) Breast cancer cells (4T1, MCF7) and colorectal cancer cells (CT26, HCT116) were treated with indicated doses of juglone for 24 hours and subsequently counted and seeded into the chambers (1 $\times$ 10${}^5$ cells for 4T1, 2 $\times$ 10${}^4$ cells for MCF7, 2 $\times$ 10${}^4$ cells for CT26 and 1 $\times$ 10${}^5$ cells for HCT116). After 24 hours the cells passing through the chamber stained with 0.1% crystal violet and counted. Scale bar, 100 $\mu$m. (E) Liver metastasis model was established by injecting 1 $\times$ 10${}^6$ CT26 cells into spleen of every mouse. The mice were treated with either PBS or 2 mg/kg juglone by intraperitoneal injection every other day for 14 days. Mice were subsequently sacrificed, and the liver was removed to evaluate metastasis. Scale bar, 1 cm. (F) The ratio of liver weight vs. body weight were decreased in juglone treatment group compare with control group. *p 0.05, **p 0.01, ***p 0.001.

Next, we established a mouse model of colorectal cancer liver metastasis using a splenic injection model. 1 $\times$ 10${}^6$ CT26 cells were injected into the spleen of BALB/c mice. Subsequently, mice were treated with, or without, juglone (2 mg/kg) by intraperitoneal injection every 2 days for a total of 14 days. The results showed juglone treatment significantly reduced the severity of liver metastasis compared with control group (Fig. 3E). We found that the ratio of liver/body weight was decreased in the juglone treatment group (Fig. 3F). These results indicated juglone treatment could suppress the metastatic activity of colorectal cancer cells.

Juglone is a small molecule inhibitor of Pin1 [15] and it has been reported that Pin1 could play a role in the maintenance of stemness and induction of metastasis in breast cancer [21, 22]. To clarify whether Pin1 has the same function in colorectal cancer cells, we first verified the inhibitory effect of juglone on Pin1 expression in HCT116 cells (Fig. 4A). We subsequently tested the expression of $\beta$-catenin and Nanog in Pin1-overexpressing HCT116 cells. The results showed that Pin1 overexpression significantly upregulated the expression of stemness markers, including $\beta$-catenin and Nanog (Fig. 4B). These results demonstrate that Pin1 could upregulate CSC-related protein expression in colorectal cells. Moreover, a similar role for Pin1 has been documented in breast cancer [22]. A transwell migration assay was also performed, and demonstrated that overexpression of Pin1 significantly promoted the migratory ability of HCT116 cells (Fig. 4C,D).

Fig. 4.

Juglone suppresses the stemness characteristics of cancer cells through inhibiting Pin1. (A) The expression of Pin1 in four cell lines treated with different concentrations of juglone was assayed by western blot analysis. (B) HCT116 was transfected with either pLVX-DsRed vector (Mock group) or pLVX-Pin1 vector (Pin1 group). The expression of $\beta$-catenin, Nanog and Pin1 was assessed by western blot analysis. (C,D) A transwell migration assay was performed with transfected HCT116 cells. 1 $\times$ 10${}^5$ cells were seeded into the chambers. After 24 hours, the cells passing through the chamber stained with 0.1% crystal violet and counted. Scale bar, 100 $\mu$m. (E,F) Pin1-overexpressing HCT116 cells were treated with or without juglone (3 $\mu$M). 1 $\times$ 10${}^5$ cells were seeded into the chambers. After 24 hours, the cells passing through the chamber stained with 0.1% crystal violet and counted. Scale bar, 100 $\mu$m. (G) HCT116 was transfected with either pLVX-DsRed vector (Mock group) or pLVX-Pin1 vector (Pin1 group). And then these cells were treated with or without juglone. The expression of $\beta$-catenin, Nanog and Pin1 in the cells described above was assayed by western blot analysis. **p 0.01, ***p 0.001.

To clarify whether the effect of juglone on cancer cell stemness characteristics is related to Pin1 inhibition, we next performed a rescue test. Specifically, Pin1-overexpressing HCT116 cells were treated with or without juglone (3 $\mu$M). Transwell assays confirmed that Pin1 promoted the migratory ability of cancer cells compared with the suppressive activity displayed by juglone treatment (Fig. 4E,F). As shown in Fig. 4G, the results further demonstrated that Pin1 overexpression alleviated the inhibitory effect of juglone on the stemness-related proteins $\beta$-catenin and Nanog in HCT116 cells. These results suggest that the inhibitory effect of juglone on stemness characteristics may be achieved, at least in part, by inhibiting the expression of Pin1.