Dissecting the novel abilities of aripiprazole: The generation of anti-colorectal cancer effects by targeting G α q via HTR2B

Abstract

Colorectal cancer (CRC) is a type of malignant tumor that seriously threatens human health and life, and its treatment has always been a difficulty and hotspot in research. Herein, this study for the first time reports that antipsychotic aripiprazole (Ari) against the proliferation of CRC cells both in vitro and in vivo, but with less damage in normal colon cells. Mechanistically, the results showed that 5-hydroxytryptamine 2B receptor (HTR2B) and its coupling protein G protein subunit alpha q (Gαq) were highly distributed in CRC cells. Ari had a strong affinity with HTR2B and inhibited HTR2B downstream signaling. Blockade of HTR2B signaling suppressed the growth of CRC cells, but HTR2B was not found to have independent anticancer activity. Interestingly, the binding of Gαq to HTR2B was decreased after Ari treatment. Knockdown of Gαq not only restricted CRC cell growth, but also directly affected the anti-CRC efficacy of Ari. Moreover, an interaction between Ari and Gαq was found in that the mutation at amino acid 190 of Gαq reduced the efficacy of Ari. Thus, these results confirm that Gαq coupled to HTR2B was a potential target of Ari in mediating CRC proliferation. Collectively, this study provides a novel effective strategy for CRC therapy and favorable evidence for promoting Ari as an anticancer agent.

Keywords: 5-Hydroxytryptamine 2B receptor; 5-Hydroxytryptamine receptor; Aripiprazole; Colorectal cancer; Extracellular regulated protein kinases; G protein subunit alpha q; Phosphoinositide 3-kinase/the serine threonine kinase AKT.

Graphical abstract

Figure 1

Ari inhibited CRC cell proliferation in vitro. (A)–(E) Inhibitory ability of Ari on HT29, LoVo, SW480, HCT116 FHC cells. These results are expressed as mean ± SD, n = 6; ∗P < 0.05, ∗∗P < 0.01 (^ for 24 h, ^# for 48 h, ∗ for 72 h) vs. control group. (F) Ari reduced the number of HT29 and HCT116 cells' monoclonal formation at a concentration of 12 μmol/L for 14 days. (G, H) The protein expression of PI3K/AKT/mTOR pathway after Ari treatment in HT29 and HCT116 cells. Data are expressed as mean ± SEM, n = 3; ∗P < 0.05, ∗∗P < 0.01 vs. control group.

Figure 2

Ari inhibited subcutaneous xenograft tumor growth. (A) Animal experiment protocol. (B) The body weight of mice during Ari administration. (C, D) Tumor-bearing mass weight was significantly reduced by Ari. (E, F) H&E staining results of kidney and liver sections. (G) Ki67 was reduced in xenograft tumors. (H) The relative expression of p-mTOR and p-85 in xenograft tumors was changed after Ari treatment. Data are expressed as mean ± SEM; n = 3 for Western blot; n = 6 for animal experiment. ∗∗P < 0.01 vs. control group.

Figure 3

Ari inhibited CRC cell growth not perturbed by BRAF mutation. (A) Cex was ineffective on BRAF-mutated HT29 cells. (B) The effect of Cex on HCT116. (C) The effects of Cex and/or Ari on HT29 cells. (D) Animal experimental protocol. (E) The body weight of mice during Ari administration. (F) Cex was ineffective against xenograft tumors in vivo. (G) The changes of PI3K/AKT and ERK pathway-related factors after CEX and/or Ari treatment. Data are expressed as mean ± SEM; n = 3 for Western blot; n = 6 for animal experiment. ∗P < 0.05, ∗∗P < 0.01 vs. control group; ^##P < 0.01 vs. the Cex group; ⁺P < 0.05, ⁺⁺P < 0.01 vs. the Ari group.

Figure 4

Ari inhibited CRC cell proliferation through HTR2B signaling. (A, B) 5-HT promoted HT29 growth. (C) The expression of HTR2B in FHC, HT29, LoVo, SW480, and HCT116. (D) The relative expression of PKC, CaMKII, and p-ERK1/2 after a brief Ari treatment. (E) SB 204741 inhibited HT29 cell activity in a dose-dependent manner. (F) SB decreased the relative expression of p-ERK1/2 in HT29 cells. (G) The relative expression of HTR2B and Gαq in HT29 cells after Ari treatment. (H) HTR2B KD did not arouse the signaling pathway changes. Data are expressed as mean ± SEM; n = 6 for MTT, n = 3 for Western blot; ∗P < 0.05, ∗∗P < 0.01 vs. the control group.

Figure 5

Ari targeted Gαq to inhibit CRC cell proliferation. (A) Distribution of Gαq in CRC cells. (B) The relative expression of HTR2B and Gαq after Ari intervention for 30 min. (C) Gαq bound to HTR2B was decreased after Ari treatment. (D) The inhibitory effect of Ari on HCT116 was enhanced after Gαq overexpression. (E) Gαq KD inhibited HCT116 cell growth. (F, G) The relative expression of p-85, p-AKT, p-ERK1/2 in Gαq KD and Gαq overexpression cells. (H) CETSA experiment examined the thermal stability of Ari-treated Gαq. Data are expressed as mean ± SEM, n = 3. ∗P < 0.05, ∗∗P < 0.01 vs. the control group; ^##P < 0.01 vs. the Gαq group; ⁺⁺P < 0.01 vs. the Ari group; ^&&P < 0.01 vs. the Gαq group.

Figure 6

The structure of the Gαqβγ heterotrimer in complex with Ari. (A) Gαq consists of the GTPase (yellow) and the helical domains (green) connected by two linker regions, Linker 1 and Linker 2 (red), Gβ and Gγ are blue and orange, GDP (purple) and Ari (cyan) are shown as cartoon models. (B) Surface representation of the heterotrimer as shown in 6A. (C) On the left, it is a surface representation of the front view of Gαq, and on the right without Ari in the pocket. (D) The chemical bond formed by Ari with Gαq amino acid is shown. (E) Amino acid sequence of Gαq, Gαq-60, Gαq-183 and Gαq-190. (F) The effect of Ari on HCT116 cells transfected with Gαq, Gαq-60, Gαq-183 and Gαq-190. Data are expressed as mean ± SEM, n = 3; ∗∗P < 0.01 vs. control group; ^#P < 0.05 vs. the Gαq group.

Figure 7

A working model summarizes how Ari suppresses CRC cell proliferation.