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. 2017 Mar 14;8(11):17593-17609.
doi: 10.18632/oncotarget.4307.

The neuroleptic drug pimozide inhibits stem-like cell maintenance and tumorigenicity in hepatocellular carcinoma

Jia-Jie Chen  1   2   3 , Nan Cai  1   2 , Guan-Zhong Chen  1   4 , Chang-Chang Jia  1   2 , Dong-Bo Qiu  4   2 , Cong Du  4   2 , Wei Liu  1   4 , Yang Yang  1 , Zi-Jie Long  3 , Qi Zhang  1   4   2
Affiliations

The neuroleptic drug pimozide inhibits stem-like cell maintenance and tumorigenicity in hepatocellular carcinoma

Jia-Jie Chen et al. Oncotarget. .

Abstract

Drug repurposing is currently an important approach for accelerating drug discovery and development for clinical use. Hepatocellular carcinoma (HCC) presents drug resistance to chemotherapy, and the prognosis is poor due to the existence of liver cancer stem-like cells. In this study, we investigated the effect of the neuroleptic agent pimozide to inhibit stem-like cell maintenance and tumorigenicity in HCC. Our results showed that pimozide functioned as an anti-cancer drug in HCC cells or stem-like cells. Pimozide inhibited cell proliferation and sphere formation capacities in HCC cells by inducing G0/G1 phase cell cycle arrest, as well as inhibited HCC cell migration. Surprisingly, pimozide inhibited the maintenance and tumorigenicity of HCC stem-like cells, particularly the side population (SP) or CD133-positive cells, as evaluated by colony formation, sphere formation and transwell migration assays. Furthermore, pimozide was found to suppress STAT3 activity in HCC cells by attenuating STAT3-dependent luciferase activity and down-regulating the transcription levels of downstream genes of STAT3 signaling. Moreover, pimozide reversed the stem-like cell tumorigenic phenotypes induced by IL-6 treatment in HCC cells. Further, the antitumor effect of pimozide was also proved in the nude mice HCC xenograft model. In short, the anti-psychotic agent pimozide may act as a novel potential anti-tumor agent in treating advanced HCC.

Keywords: STAT3 inhibitor; STAT3 signaling; hepatic cancer stem-like cells; pimozide; self-renewal.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The neuroleptic drug pimozide inhibits HCC cell proliferation in dose- and time-dependent manners by inducing G0/G1 phase cell cycle arrest
A. MHCC-97L (a), Hep 3B (b), Hep G2 (c) and Huh7 (d) cells were treated with various concentrations of pimozide for various times, and cell viability was determined by MTT assay. B. Then, the cells were subjected to flow cytometric analysis to determine the level of CFSE staining. C. The cells stained with PI were subjected to flow cytometric analysis to determine the cell distributions at each phase of the cell cycle. The results are shown as the mean values ± SD of 3 independent experiments. *p < 0.05, **p < 0.01, compared with the control. D. Western blot analysis of the expression of cell cycle-related genes. Cell extracts were probed with antibodies against p21, p27, Cyclin D1 and GAPDH (loading control) as indicated.
Figure 2
Figure 2. Pimozide inhibits the self-renewal capacity of HCC cells
MHCC-97L and Hep 3B cells were treated with pimozide at the indicated concentrations, incubated for extra 10-14 day and then subjected to colony formation assay. Images were taken at a magnification of 100× A. The numbers of colonies were counted after staining with crystal violet and the histogram indicated the number of colonies. The results are from 3 independent transfection experiments (B). (C & D). Sphere formation assay of HCC cells treated with pimozide. The spheres were imaged under a light microscope (magnification, 100× ), and the statistical results are shown. E. Western blot analysis of the expression of self-renewal genes. Cell extracts were probed with antibodies against c-Myc, Bmi1, Nanog, Oct3/4 and GAPDH. F. MHCC-97L and Hep 3B cells were incubated with the indicated doses of pimozide for 48h before subjected to RT-PCR to detect the expression of the self-renewal genes Bmi1, Nanog and Oct4. *p < 0.05, **p < 0.01, compared with the control.
Figure 3
Figure 3. Pimozide suppresses HCC cell migration
Transwell migration assays of HCC cells treated with pimozide for 48 h. A. Images shown at a magnification of 100×. B. The data are summarized from 3 independent experiments, *p < 0.05, **p < 0.01, compared to the control. C. Western blotting analysis of the expression of EMT-related markers. Cell extracts were probed with antibodies directed against E-cadherin, N-cadherin, vimentin and β-actin (loading control) as indicated.
Figure 4
Figure 4. Pimozide inhibits the self-renewal and migration capacities of SP HCC cells
A. Fluorescence-activated cell sorting isolation of SP MHCC-97L cells. B. Colony formation assay of SP MHCC-97L cells treated with 5μM or 10μM pimozide. The images (a) and statistical results (b) are shown. C. Sphere formation assay of SP MHCC-97L cells treated with pimozide. D. Transwell migration assay of SP MHCC-97L cells treated with 10 μM pimozide for 48h. Images are shown at a magnification of 100×, and the statistical results are shown. The data are summarized from 3 independent experiments, *p < 0.05, **p < 0.01, compared to the control.
Figure 5
Figure 5. Pimozide inhibits the self-renewal and migration capacities of CD133-positive HCC cells
A. Fluorescence-activated cell sorting isolation of CD133-positive cells and negative cells from MHCC-97L cells (a). The purity of fluorescence-activated cell sorting isolation was confirmed (b). B. Colony formation assay of CD133-positive and negative MHCC-97L cells treated with the indicated doses of pimozide. The images (a) and statistical results (b) are shown. C. Sphere formation assay of CD133-positive and negative MHCC-97L cells treated with pimozide. D. Transwell migration assay of CD133-positive MHCC-97L cells treated with 10 μM pimozide for 24h. Images are shown at a magnification of 100×, and the statistical results are shown. The data are summarized from 3 independent experiments, *p < 0.05, **p < 0.01, compared to the control.
Figure 6
Figure 6. Pimozide suppresses the activity of STAT3 signaling and reverses cancer stem-like cell phenotypes induced by IL-6 addition in HCC cells
A. MHCC-97L and Hep 3B cells were incubated with the different doses of pimozide for 48h before subjected to RT-PCR to analyse the expression of the STAT3 downstream genes c-Myc, Bcl-xL and Mcl-1. B. Western blot analysis of the expression of phosphorylated STAT3 (p-STAT3 Y705), STAT3 and c-Myc. C. HEK293T cells were transfected with STAT3 reporter and pRL-TK renilla luciferase reporter plasmids. After 24 hours of transfection, the cells were treated with different doses of pimozide for another 48h. Dual luciferase assay was performed to detect the relative luciferase activity. MHCC-97L cells were treated with 20 ng/ml IL-6, 10 μM pimozide or a combination as indicated for 12 h and subjected to western blot analysis (D), colony (E) and sphere (F) formation assays, and transwell migration assay (G) Relative representative figures are shown. The data are summarized from 3 independent experiments, *p < 0.05, **p < 0.01.
Figure 7
Figure 7. Pimozide reduces the tumor burden in a nude mice xenograft model
Pimozide was administered to mice previously injected with MHCC-97L cells. A. The tumour volume was measured and analysed at the indicated time points. Points, mean; bars, SE, **p < 0.01. B. Representative tumors removed from mice of each group are shown. C. Body weights were determined for the animals in each treatment group. Points, mean; bars, SE.
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References

    1. Boguski MS, Mandl KD, Sukhatme VP. Drug discovery. Repurposing with a difference. Science. 2009;324(5933):1394–1395. - PubMed
    1. Carley DW. Drug repurposing: identify, develop and commercialize new uses for existing or abandoned drugs. Part I. IDrugs. 2005;8:306–309. - PubMed
    1. Carley DW. Drug repurposing: identify, develop and commercialize new uses for existing or abandoned drugs. Part II. IDrugs. 2005;8:310–313. - PubMed
    1. Blatt J, Corey SJ. Drug repurposing in pediatrics and pediatric hematology oncology. Drug Discov Today. 2013;18:4–10. - PubMed
    1. Kasznicki J, Sliwinska A, Drzewoski J. Metformin in cancer prevention and therapy. Ann Transl Med. 2014;2:57. - PMC - PubMed

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