Brexpiprazole, a novel Dopamine and Serotonin modulator in antipsychotic medication was purchased from Bidepharmtech Ltd. (Shanghai, China). Cetuximab was acquired from Merck Serono China (Beijing, China). Oxaliplatin was kindly provided by Heng Rui Pharmaceuticals (Jiangsu, China). For in vitro experiments, Brexpiprazole was dissolved in dimethylsulfoxide to 40 mM. For in vivo applications, Brexpiprazole was solubilized in 5% Arabic gum in sterile Phosphate Buffered Saline (PBS).

The human normal colonic epithelial cells (2 $\times$ 10${}^6$ NCM460 cells) and human CRC cells (1 $\times$ 10${}^6$ HCT116 cells and 1 $\times$ 10${}^6$ SW620 cells) were obtained from ATCC. NCM460 was maintained in RPMI-1640, HCT116 was cultured in McCoy’s 5A medium (Solarbio, Pleasanton, CA, USA), and SW620 was grown in DMEM with 10% FBS and 1% pen/strep (Boster, Wuhan, China). All cells were grown at 37 °C and 5% CO${}_2$. All cells were validated through STR and tested negative for mycoplasma.

Cells were placed in a 96-well plate (6 $\times$ 10${}^3$ cells/well), and were treated with 5, 10, 20, and 40 µM Brexpiprazole for 24 or 48 h, or treated with 1.5 mg/mL Cetuximab for 72 h, or received both Cetuximab (1.5 mg/mL) and Brexpiprazole (10 µM) for 72 h, respectively. After treatment, 10 µL CCK-8 (Beyotime, Suzhou, China) was provided and kept at 37 °C for 1.5 h, and OD450 was recorded.

HCT116 and SW620 cells were cultured in 6-well plates (8 $\times$ 10${}^5$ cells/well) for 12 h. After making a scratch with a 200 µL pipette tip, cells were PBS-washed twice, and treated with 20 µM Brexpiprazole in a serum-free medium for 48 h. Wound closure was represented by the formula (the scratched area before incubation – the scratched area after incubation)/(the scratched area before incubation) $\times$ 100%.

Migration assay was carried out with transwell inserts of an 8-µm filter. HCT116 and SW620 cells were resuspended in a serum-free medium after starvation. Cell suspension (5 $\times$ 10${}^4$ cells) was inoculated in the upper transwell chambers and incubated in the lower chamber with 600 µL complete medium containing Brexpiprazole (20 µM) for 48 h. Cells on transwell inserts were fixed with methanol and stained with a 1% crystal violet solution. Cells on the lower surface of the chamber membrane were photographed with a Leica DMI3000 microscope at 200$\times$ magnification. The average counting number of 5 random high-power fields was recorded.

Cells were incubated with 0, 10, 20, and 40 µM Brexpiprazole for 24 h, collected, and washed. After re-suspension in 500 µL binding buffer, the cells were stained with Annexin V-FITC and PI (Beyotime) in the dark for 15 min and apoptosis was monitored with a BD Accuri C6 cytometer. The raw data were analyzed with FlowJo v10.3 (Tree Star Inc., Ashland, OR, USA).

Four-week-old male nude mice and weight of 20 $\pm$ 2 g from Beijing Laboratory Animal Center were routinely maintained at the Animal Experimental Center of North Sichuan Medical College. All procedures followed the guidelines of Sichuan and were approved by the Experimental Animal Ethics Committee of our university. After one week of adaption, 1 $\times$ 10${}^7$ HCT116 cells were inoculated subcutaneously into each mouse. When the tumor reached 100 mm${}^3$, the mice were divided into four groups, 6 nude mice per group: Group 1, 5% gum arabic was gavaged and PBS was intraperitoneally injected. Group 2, Brexpiprazole in 5% gum Arabic was gavaged (5 mg/kg/day). Group 3, cetuximab was injected intraperitoneally with a dose of 1 mg twice a week. In group 4, both Brexpiprazole and cetuximab were administered. Tumor volume was calculated by measuring the length (a) and width (b) with a caliper every 2 days. After 3-week treatment, nude mice were anesthetized with 1% pentobarbital and then killed by cervical dislocation, and tumors were collected for immunochemical staining or Western blotting.

RIPA buffer, containing 1% PMSF (Dingguo, Guangzhou, China) and 1% phosphatase inhibitor (Solarbio), was used for total protein extraction from tumor tissues or treated cells. The supernatant was taken after the centrifugation. After quantification via BCA assay, proteins (30 µg) were resolved by 10% SDS-PAGE, and electro-blotted into PVDF membranes. After blocking, membranes were probed with first antibodies at 4 °C, followed by second antibodies such as goat anti-rabbit IgG HRP (Huabio, HA1031, Hangzhou, China, 1:5000), goat anti-mouse IgG HRP (Huabio, HA1006, Hangzhou, China, 1:5000). ECL reagent (Bio-Rad, Hercules, CA, USA) was used for development, and images were quantified with ImageJ 1.52a Software (National Institutes of Health, Washington, DC, USA).

Primary antibodies that were used in this study were ERK1/2 (Abcam, ab28481, Cambridge, UK) and Ser473 (Proteintech, 28731-1, Wuhan, China).

Tissue was fixed with 4% formalin, paraffin-embedded, and cut to 4 µm sections, followed by deparaffin and rehydration, and antigen retrieval. After blocking with 3% H${}_2$O${}_2$, sections were probed with antibodies against survivin (Abcam, ab76424, Cambridge, UK, 1:500). After PBS-washing, sections were probed with a second antibody goat anti-rabbit IgG HRP (Huabio, HA1031, Hangzhou, China, 1:200). DAB kit (ZSGB-BIO) was applied to stain the slides. After hematoxylin counter-staining and mounting, sections were observed under the Olympus microscope. Five random fields ($\times$400) were selected for intensity analysis. Image-Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD, USA) was used for quantitation.

Data were presented as mean $\pm$ standard deviation (SD). SPSS 24.0 software (SPSS Inc., Chicago, IL, USA) was used for one-way ANOVA ($>$2 groups) and Student t-test (2 groups) analysis. p-value 0.05 was defined as significant.

The effect of brexpiprazole on human CRC cells’ proliferation was first measured. Brexpiprazole treatment dose- and time-dependently inhibited HCT116 and SW620 cells’ viability (Fig. 1A,B), while HCT116 cells were more sensitive to this treatment. The toxicity of brexpiprazole in human normal colonic epithelial cells (NCM460) was also measured. Only a high concentration of Brexpiprazole (40 µM) significantly reduced the cell viability of NCM460 cells at 24 h, other concentrations (5, 10, and 20 µM) did not affect the cell viability of NCM460 cells at 24 h (Fig. 1C). Morphological observation showed that the HCT116 and SW620 cells in the control group were adherent to the wall, appearing colorless and transparent, connected in sheets and densely packed with cells; after Brexpiprazole treatment, the HCT116 and SW620 cells showed a small amount of cell shrinkage and rounding, and the number of cells was relatively reduced, especially after the treatment with 40 µM Brexpiprazole (Fig. 1D,E). The data suggest that brexpiprazole can selectively inhibit CRC cells’ proliferation.

Fig. 1.

Brexpiprazole remarkably inhibited the proliferation of colorectal cancer (CRC) cells. Cells were treated with Brexpiprazole at various concentrations for different time courses. (A) Viability of HCT116, (B) viability of SW620, (C) morphology of HCT116, (D) morphology of SW620 cells, and (E) NCM460 (scale: 50μm). *p 0.05, **p 0.01, ***p 0.001. (n = 3)

Knowing that brexpiprazole inhibits CRC cells’ proliferation triggers our interest in studying how brexpiprazole affects CRC cells’ migration. HCT116 and SW620 cells were treated with brexpiprazole (20 µM) for 48 h, and cell migration was monitored using wound healing and transwell assay. Wound healing experiments showed that brexpiprazole markedly inhibited the migration of HCT116 and SW620 cells, and HCT116 cells were more sensitive to brexpiprazole than SW620 cells (Fig. 2A,B). Meanwhile, results from transwell assays demonstrated that brexpiprazole dramatically suppressed HCT116 cells’ migration (Fig. 2C), indicating that brexpiprazole can suppress the migration of CRC cells.

Fig. 2.

Brexpiprazole suppressed CRC cells’ migration. Cells were treated by brexpiprazole (20 µM) for 48 h. (A) Wound healing assay of HCT116 cells, (B) wound healing assay of SW620 cells (scale: 50μm), and (C) transwell migration assay of HCT116 cells (scale: 500μm). **p 0.01. (n = 3)

Annexin FITC/V and PI staining were performed to measure how brexpiprazole affects the apoptosis of CRC cells. As depicted in Fig. 3A, brexpiprazole treatment increased the percentage of apoptotic cells in a dose-dependent manner, indicating that brexpiprazole could induce apoptosis of CRC cells. Next, we checked the effect of brexpiprazole on apoptosis-related proteins in these cells through Western blot. Brexpiprazole dose-dependently enhanced survivin but decreased cleaved caspase-9, indicating the activation of caspase-9 by brexpiprazole (Fig. 3B). These findings demonstrate that brexpiprazole can induce the apoptosis of CRC cells by regulating survivin and caspase-9.

Fig. 3.

Brexpiprazole induced apoptosis in colon cancer cells. Cells were treated with Brexpiprazole at various concentrations. (A) Flow cytometry analysis of apoptosis, where Q4 is viable cells, Q1 is necrotic cells, Q2 represents late apoptotic cells, and Q3 represents early apoptotic cells. Apoptotic cells include late apoptotic cells and early apoptotic cells. (B) Western blotting analysis of apoptosis-related proteins. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. (n = 3)

The activation status of the PI3K pathway in HCT116 and NCM460 was determined through Western blot. As shown in Fig. 4A, phosphor-PI3K subunit P85 in HCT116 was significantly higher than that of NCM460, indicating that the pathway is activated. To determine how brexpiprazole affects downstream pathways of EGFR, proteins related to PI3K and MAPK pathways were measured. Brexpiprazole significantly down-regulated the expression of phosphorylated AKT, P85, P38, ERK1/2, and RAF in HCT116 and SW620 cells without affecting their total expression levels. However, brexpiprazole only down-regulated the expression level of RAS protein in HCT116 cells but had no significant effect on RAS protein in SW620 cells (Fig. 4B,C). Taking the data together, brexpiprazole might regulate CRC cells’ survival and proliferation by affecting the signal transduction of PI3K and MAPK pathways.

Fig. 4.

Brexpiprazole inhibited the signal transition of the EGFR pathway in colon cancer cells. Immunoblotting analysis of (A) the activation of the PI3K pathway, (B) the effect of brexpiprazole on PI3K and MAPK pathway-related proteins in (B) HCT116 cells, and (C) SW620 cells. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. (n = 3)

The anti-colon cancer effect of brexpiprazole was also analyzed in vivo. HCT116 tumor-bearing mice received brexpiprazole (5 mg/kg) daily through intragastrical administration (i.g.) for 17 days. Positive control mice received oxaliplatin (5 mg/kg) twice a week. Tumors developed rapidly in control mice. Both brexpiprazole and oxaliplatin significantly attenuated tumor growth (Fig. 5A). Next, we investigated the change of survivin expression in tumor tissues via immunohistochemical staining. The proportion of survivin-positive cells in brexpiprazole group was less than that of vehicle group, indicating that brexpiprazole increased apoptosis in tumor sections (Fig. 5B). Furthermore, data showed that the expression of phosphorylated AKT, P85, P38, ERK1/2, RAF in tumor tissues was remarkably decreased by brexpiprazole treatment, but the expression of RAS did not change significantly (Fig. 5C). In summary, these data suggest that brexpiprazole inhibits colon cancer xenograft tumor proliferation, possibly through affecting the signal transduction of PI3K and MAPK pathways.

Fig. 5.

Brexpiprazole inhibited tumor growth in HCT116 xenograft models. HCT116 tumor-bearing mice received brexpiprazole (5 mg/kg) daily through intragastrical administration (i.g.) for 17 days. (A) Tumor growth curve, (B) immunohistochemical staining of surviving (scale: 50μm), (C) immunoblotting of PI3K and MAPK pathway-related proteins. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. NC represents the untreated group, injected intraperitoneally with an equal dose of PBS and gavaged with gum arabic solvent; Brexpiprazole group was represented by intraperitoneal injection of an equal doses of PBS and gavage of brexpiprazole 5 mg/kg prepared with gum arabic; Oxaliplatin group was represented by intraperitoneal injection of an equal dose of oxaliplatin 5 mg/kg prepared with PBS and gavage of gum arabic solvent. (n = 6)

Previous data showed that HCT116 cells had different sensitivity to brexpiprazole treatment. Therefore, in subsequent studies, we chose HCT116 cells, which are more sensitive to brexpiprazole, as the research object. We first verified the effect of brexpiprazole combined with cetuximab on HCT116 cells’ proliferation. As shown in Fig. 6A, compared with the vehicle group, brexpiprazole group, or cetuximab group, the combination of brexpiprazole and cetuximab significantly reduced the proliferation activity of HCT116 cells. Next, we detected the effect of the combination of the two drugs on the PI3K and MAPK pathway in HCT116. Compared with the vehicle group, brexpiprazole group, or cetuximab group, the combination of brexpiprazole and cetuximab dramatically reduced the expression of RAS, phosphorylated AKT, P85, P38, ERK1/2, and RAF proteins (Fig. 6B). These data indicate that brexpiprazole increased the sensitivity of HCT116 cells to cetuximab.

Fig. 6.

Brexpiprazole synergized with cetuximab to reduce cell viability and downregulate RAS, phosphorylated AKT, P85, P38, ERK1/2, and RAF in HCT116 cells. HCT116 cells were treated with brexpiprazole, cetuximab, or a combination of brexpiprazole and cetuximab. (A) Cell viability, (B) immunoblotting of the effect of brexpiprazole, cetuximab, or their combination on PI3K and MAPK pathway-related proteins in HCT116 cells. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. There were four groups in total: Group 1, Colorectal cancer cells were added to equal volumes of DMSO and PBS (brexpiprazole – & cetuximab –); Group 2, Equal volumes of 10 μM of brexpiprazole prepared in DMSO and PBS(brexpiprazole + & cetuximab –); Group 3, Equal volumes of DMSO and 0.5 mg/kg of cetuximab prepared in PBS (brexpiprazole – & cetuximab +); Group 4, Equal volumes of 10 μM of brexpiprazole prepared in DMSO and 0.5 mg/kg of cetuximab prepared in PBS. (n = 3)

Next, we validated the effect of brexpiprazole and cetuximab on tumor cell proliferation in the HCT116 xenografts. Mice in the single medication group were given 5 mg/kg of brexpiprazole daily via gavage or 25 mg/kg of cetuximab intraperitoneally twice a week, and the combination medication group received both treatments. Compared with single medication groups, the combined medication observably suppressed HCT116 xenograft growth (Fig. 7A). To verify its underlying mechanism, we also performed immunohistochemical analysis and Western blot. Data indicated that compared to single medication groups, combination medication drastically down-regulated the expression of survivin in tumor tissues (Fig. 7B). Combination medication also remarkably down-regulated the phosphorylation expression levels of PI3K- and MAPK-related proteins in tumor tissues but did not affect the expression of RAS protein (Fig. 7C). Taking the data together, brexpiprazole combined with cetuximab can produce a synergistic anti-tumor effect.

Fig. 7.

Brexpiprazole synergized with cetuximab to inhibit xenograft tumor growth in CRC. HCT116 tumor-bearing mice received brexpiprazole, cetuximab, or the combination of brexpiprazole and cetuximab. (A) Tumor growth curve, (B) immunohistochemical staining of surviving (scale: 50μm), and (C) immunoblotting of PI3K and MAPK pathway-related proteins. *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. NC represents the untreated group, injected intraperitoneally with an equal dose of PBS and gavaged with gum arabic solvent; Brexpiprazole group was represented by intraperitoneal injection of an equal doses of PBS and gavage of brexpiprazole 5 mg/kg prepared with gum arabic; Cetuximab group was represented by intraperitoneal injection of an equal dose of ceximab 25 mg/kg prepared with PBS and gavage of gum arabic solvent; Brex+Cetu group was represented by intraperitoneal injection of an equal dose of ceximab 25 mg/kg prepared with PBS and gavage of brexpiprazole 5 mg/kg prepared with gum arabic. (n = 6)