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. 2020 Apr;19(4):2989-2995.
doi: 10.3892/ol.2020.11355. Epub 2020 Jan 27.

NOS1 Expression Promotes Proliferation and Invasion and Enhances Chemoresistance in Ovarian Cancer

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Free PMC article

NOS1 Expression Promotes Proliferation and Invasion and Enhances Chemoresistance in Ovarian Cancer

Zhiwei Zou et al. Oncol Lett. .
Free PMC article

Abstract

Nitric oxide (NO), an important chemical messenger, serves a dual role in tumor progression. Nitric oxide synthase isoform 1 (NOS1) was observed to be increasingly expressed in various types of cancer, and its expression has been associated with tumor progression. However, the level of NOS1 expression and the associated functions of NOS1 in human ovarian cancer remain undefined. Using gene expression profiles of ovarian cancer from the Gene Expression Omnibus (GEO) database, the present study revealed that NOS1 was increasingly expressed in ovarian cancer tissues. The present study investigated the level of NOS1 expression and its effects on in vitro cell function, including proliferation, migration and invasion as well as chemoresistance to cispatin (DDP) treatment in OVCAR3 cells. Reverse transcription-quantitative polymerase chain reaction demonstrated that the level of NOS1 mRNA expression varied in different ovarian cancer lines. However, immunoblotting indicated that the level of NOS1 protein expression was constitutively high in ovarian cancer cell lines. Treatment with NOS inhibitor NG-nitro-L-arginine methyl ester or transfection with NOS1 short hairpin RNA significantly inhibited cell proliferation, migration and invasion compared with the control, whereas the sensitivity of OVCAR3 cells to DDP treatment was increased. The results of the present study indicated that NOS1 promoted the function of ovarian cancer cells, including proliferation, invasion and chemoresistance, providing a potential target for ovarian cancer therapeutic.

Keywords: chemoresistance; invasion; migration; nitric oxide synthase isoform 1; ovarian cancer; proliferation.

Figures

Figure 1.
Figure 1.
Analysis of gene expression profiles of NOS isoforms in ovarian cancer tissues. Expression data was downloaded from Gene Expression Omnibus (accession no. GSE14407). The relative expression level of mRNA of three NOS isoforms was higher in ovarian cancer tissues compared with normal ovarian tissues. However, only the alteration of NOS1 expression was statistically significant (P<0.05). NOS, nitric oxide synthase.
Figure 2.
Figure 2.
Analysis of mRNA and protein expression of NOS isoforms, and NO production in ovarian cancer cell lines. (A) mRNA expression of NOS isoforms were analyzed by reverse transcription-quantitate polymerase chain reaction, and expression varied in OVCAR3, SKOV3 and ES-2 cells. The levels of NOS mRNA expression were normalized to GAPDH expression. (B) The proteins of the three NOS isoforms were all detected by western blotting in SKOV3 and OVCAR3 cells. (C) The levels of NO expression in the culture medium of OVCAR3 cells was determined by Griess assay and was less than half of the concentration released by cells treated with 50 µM DETA-NONOate (50 nM NO). Error bars represent the mean ± standard deviation of triplicate experiments. *P<0.05 and ***P<0.001. All compared with control. CON, control; DETA, DETA-NONOate; L-NAME, NG-nitro-L-arginine methyl ester; NOS, nitric oxide synthase; NO, nitric oxide.
Figure 3.
Figure 3.
NOS1 expression promotes proliferation in OVCAR3 cells. L-NAME inhibited proliferation of OVCAR3 cells as determined by (A) MTT and (B) colony formation assays. *P<0.05 vs. control. The shRNA assay revealed that the levels of mRNA (C and D) protein expression of NOS1 were markedly decreased compared with the corresponding control in OVCAR3 cells. *P<0.05. NOS1 knockdown inhibited proliferation of OVCAR3 cells as determined by (E) MTT and (F) colony formation assays. Bars represent the mean ± standard deviation of three independent tests. *P<0.05. sh, short hairpin; CON, control; NOS, nitric oxide synthase; OD, optical density; L-NAME, NG-nitro-L-arginine methyl ester.
Figure 4.
Figure 4.
NOS1 promotes invasion and migration of OVCAR3 cells. (A) The migratory ability of OVCAR3 cells was inhibited by L-NAME (1 mM; 24 h) as determined by scratch wound assay. (B) L-NAME (1 mM) inhibited invasive ability of OVCAR3 cells as determined by invasion assays. NOS1 knockdown inhibited (C) migratory and (D) invasive abilities of OVCAR3 cells as determined by scratch-wound assay and invasion assays, respectively. Data is presented as the mean ± standard deviation of three independent tests (×40 magnification). *P<0.05 and **P<0.01. CON, control; L-NAME, NG-nitro-L-arginine methyl ester; NOS, nitric oxide synthase.
Figure 5.
Figure 5.
NOS1 activity inhibition or shRNA-mediated NOS1 knockdown increases the sensitivity of OVCAR3 cells to DDP treatment. (A) Cytotoxic curve, as determined by MTT assay, indicated the IC50 of cisplatin for OVCAR3 cells was 5 µM. (B) MTT assay indicated that the cytotoxicity of OVCAR3 cells to DDP treatment was increased by treatment with NOS inhibitors. (C) MTT assay demonstrated that NOS1 knockdown also decreased the viability of OVCAR3 cells in response to DDP treatment. Data is presented as the mean ± standard deviation of three independent tests. *P<0.05 and **P<0.01. DDP, cisplatin; shRNA, short hairpin RNA; L-NAME, NG-nitro-L-arginine methyl ester; NOS, nitric oxide synthase.

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