Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway

doi:10.1016/j.ajur.2021.12.007

Asian Journal of Urology, 2023, 10 (1): 50-57 doi: 10.1016/j.ajur.2021.12.007

Original Articles

Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway

Hua Gong^a,1,Kang Chen^b,1,Lan Zhou^b,Yongchao Jin^b,Weihua Chen^b,*(

)

^a Department of Urology, Zhoupu Hospital, Medicine and Health Sciences, Shanghai University, Shanghai, China
^b Department of Urology, Shanghai East Hospital, School of Medicine, Tongji University, Shanghai, China

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Abstract

Objective: Deleted in liver cancer 1 (DLC1) is a GTPase-activating protein that is reported as a suppressor in certain human cancers. However, the detailed biological function of DLC1 is still unclear in human prostate cancer (PCa). In the present study, we aimed to explore the function of DLC1 in PCa cells.

Methods: Silencing and overexpression of DLC1 were induced in an androgen-sensitive PCa cell line (LNCaP) using RNA interference and lentiviral vector transduction. The Cell Counting Kit-8 assay was performed to determine cell proliferation. The cell cycle was examined by performing a propidium iodide staining assay.

Results:Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of LNCaP cells. Moreover, DLC1 expression was negatively correlated with Rho-associated protein kinase (ROCK) expression in LNCaP cells. Importantly, this study showed that the ROCK inhibitor Y27632 restored the function of DLC1 in LNCaP cells and reduced the tumorigenicity of LNCaP cells in vivo.

Conclusion: Our results indicated that DLC1 overexpression markedly suppressed the proliferation and cell cycle progression of PCa cells and negatively correlated with ROCK expression in PCa cells and tissue.

Key words： Cell cycle Deleted in liver cancer 1 Proliferation Prostate cancer Rho-associated protein kinase

Received: 25 May 2021 Available online: 20 January 2023

Corresponding Authors: Weihua Chen E-mail: drchenweihua@tongji.edu.cn

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Cite this article:

Hua Gong,Kang Chen,Lan Zhou, et al. Deleted in liver cancer 1 suppresses the growth of prostate cancer cells through inhibiting Rho-associated protein kinase pathway[J]. Asian Journal of Urology, 2023, 10(1): 50-57.

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http://www.ajurology.com/EN/10.1016/j.ajur.2021.12.007 OR http://www.ajurology.com/EN/Y2023/V10/I1/50

DLC1 was downregulated in PCa tissue and PCa cells. (A) The mRNA levels of DLC1, ROCK1, and ROCK2 were examined in PCa tissues (n=25 in each group). (B) The relative mRNA levels of DLC1, ROCK1, and ROCK2 in LNCaP and RWPE-1 cells were presented, respectively. (C) The relative protein levels of DLC1, ROCK1, and ROCK2 in LNCaP and RWPE-1 cells were presented, respectively. (D) DLC1 mRNA was negatively correlated with ROCK1 mRNA or ROCK2 mRNA in PCa tissue, n=25 for each group. DLC1, deleted in liver cancer 1; PCa, prostate cancer; ROCK, Rho-associated protein kinase; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase. ?? p<0.01, ??? p<0.001.

Overexpression and knockdown of DLC1 in LNCaP cells. (A) The mRNA levels of DLC1 in oeDLC1-transfected cells were examined using qRT-PCR. (B) The protein levels of DLC1 in oeDLC1-transfected cells were examined using Western blot. (C) The relative mRNA levels of DLC1 in siDLC1-transfected cells were examined using qRT-PCR. (D) The relative protein levels of DLC1 in siDLC1-transfected cells were examined using Western blot. DLC1, deleted in liver cancer 1; PCa, prostate cancer; siNC, negative control siRNA; oeNC, negative control of overexpression; oeDLC1, overexpression of DLC1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, quantitative real-time real-time polymerase chain reaction. ??? p<0.001.

DLC1 overexpression suppressed the proliferation and cell cycle of LNCaP cells. (A) Cell proliferation was detected 12 h, 24 h, 48 h, and 72 h after transfection with NC (both siNC and oeNC), oeDLC1, and siDLC1.(B) Colony formation assay was performed in cells as indicated above. (C) Cell cycle profiles of cells transfected with NC, oeDLC1, or siDLC1 were examined by using flow cytometry. (D) The protein levels of ROCK1, ROCK2, β-catenin, cyclin D1, and cyclin B in different transfected cells. DLC1, deleted in liver cancer 1; PCa, prostate cancer; ROCK, Rho-associated protein kinase; OD450, optical density 450; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NC, negative control; oeDLC1, overexpression of DLC1; siDLC1, DLC1 siRNA; S, synthesis phase; G1, gap phase 1; G2, gap phase 2. ? p<0.05; ?? p<0.01; ??? p<0.001.

The ROCK inhibitor Y27632 rescued the function of DLC1 in LNCaP cells. (A) Cell proliferation was detected at 12 h, 24 h, 48 h, and 72 h in cells transfected with siNC, siDLC1, or siNC and treated with Y27632. (B) Colony formation assay was performed in cells as indicated above. (C) Y27632 reduced the effect of siDLC1 in PCa cell cycle regulation. (D) Western blot was used to examine the protein levels of β-catenin, cyclin D1, and cyclin B in cells as indicate above. ROCK, Rho-associated protein kinase; PCa, prostate cancer; siNC, negative control siRNA; DLC1, deleted in liver cancer 1; siDLC1, DLC1 siRNA; G1, Gap phase 1; G2, Gap phase 2; S, synthesis phase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ? p<0.05; ?? p<0.01; ?? p<0.001.

The ROCK inhibitor Y27632 inhibited the tumorigenicity of LNCaP cells that transfecting with siDLC1 in vivo. (A and B) The tumor volume and weight in nude mice injected with PCa cells transfected with siNC or siDLC1, with or without Y27632 treatment (n=6 in each group). (C) The relative protein levels of DLC1, ROCK1, ROCK2, β-catenin, and cyclin D1 in different tumors as indicated. ROCK, Rho-associated protein kinase; PCa, prostate cancer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DLC1, deleted in liver cancer 1; siDLC1, DLC1 siRNA; siNC, negative control siRNA. ??? p<0.001.

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