iNOS Promotes the Development of Osteosarcoma via Wnt/β-Catenin Pathway

Inducible nitric oxide synthase (iNOS), accompanied with protumor and antitumor activity, has been studied in multiple cancers. However, the role of iNOS expression in osteosarcoma (OS) is far from being fully understood. In present work, iNOS levels were detected in OS tissues and cell lines. Colony formation assay, Transwell assay, and fow cytometer were used to assess proliferation, migration, invasion, and apoptosis abilities in vitro after iNOS inhibition. Western blotting determined the expressions of iNOS, MMP2, MMP9, C-MYC, Ki67, PCNA, and β-catenin. Mice transfected with OS cells were to evaluate tumor formation. IHC assay was to evaluate the expressions of iNOS and β-catenin in mice. The results showed that iNOS was upregulated in both OS tissues and cells compared with that in matched normal tissues or cells. And we found that proliferation, migration, and invasion numbers of OS cells were decreased, and apoptosis numbers of OS cells were increased after iNOS inhibition. MMP2, MMP9, C-MYC, Ki67, and PCNA levels were also reduced in OS cells treated with iNOS inhibition. Else, iNOS inhibition would suppress β-catenin expression in OS cells to regulate MMP2, MMP9, C-MYC, Ki67, and PCNA expressions. In addition, tumor formation, iNOS expression, and β-catenin expression were inhibited in mice transplanted with iNOS knockout OS cells. These results indicated that iNOS might be a potential therapeutic target for OS.


Introduction
Osteosarcoma (OS) is the most common primary malignant bone cancer, which occurs frequently in children and adolescents [1]. Epidemiological data reports that there is around 5.2 for children aged 0-19 years per millions of people every year [2]. And the treatment of surgery in combination with chemotherapy increases the survival rate to 60%-75% [3]. However, the existence of highly metastatic potential remains to be major cause of death [4]. Therefore, it is necessary for OC to develop new therapeutic target.
Inducible nitric oxide synthase (iNOS, NOS2) is one kind of nitric oxide synthases that catalyze L-arginine to produce nitric oxide (NO). Its expression is associated with inflammation and malignant diseases [5]. Studies show that iNOS is only nitric oxide synthase (NOS) to participate in tumor progression [6]. And iNOS is highly expressed in tumors such as breast, colon, pancreatic, colon, prostate, ovarian, gastric, bladder, brain, lung cancer, hepatocellular carcinoma, leukemia, head and neck squamous carcinoma, glioblastoma, and melanoma [5][6][7][8]. The expression of iNOS is associated with aggressiveness, high histological grade, early recurrences, and poor prognosis of tumor [8][9][10]. However, the function of iNOS in osteosarcoma development isn't clear yet.
Based on above findings, we hypothesize that iNOS plays a regulatory role in the development of OS via Wnt/βcatenin pathway and it might be a promising therapeutic target for OS.       Journal of Immunology Research

Materials and Methods
The qRT-PCR data were normalized using the 2 −ΔΔ Ct method. The expression of mRNA was normalized to GAPDH.
2.5. Colony Formation. The proliferation ability of 143B and Saos2 cells was detected by clone formation assay. The cells were incubated in six-well plates at 37°C in 5% CO 2 for 2 weeks. Then, the cells were fixed with 4% paraformaldehyde and stained them with 0.1% Ritz-jimsa dye. The clones were imaged and counted.

Western
Blotting. According to the manufacturer's recommendations, protein from cells or tissues was extracted using RIPA buffer (Nacalai Tesque). Equal amounts of protein were separated by SDS-acrylamide gel and transferred into a PVDF membrane (Millipore). After blocking, the membrane was incubated with the following primary human monoclonal antibodies: anti-GAPDH, anti-iNOS, anti-MMP2, anti-MMP9, anti-C-MYC, anti-Ki67, anti-PCNA, and anti-β-catenin. Then, the blots were washed with TBS-Tween 20 buffer and incubated with the horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. The signal was visualized by enhanced chemiluminescence (ECL).

Ectopic Tumor Growth.
The four-week-old male BALB/c nude mice were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). 143B and Saos2 cells transfected with sh-iNOS were injected into the backs of nude mice. All mice were sacrificed 20 days after coculture of the tumor cells; then, tumor weights were measured. Tumor volumes were calculated by using the formula: volume = 0:4 × width 2 × length.
2.11. Immunohistochemistry (IHC) Assay. The mice tumor tissues were collected and spliced into 5 μm thickness. Then, the expressions of iNOS and β-catenin were determined by IHC assay.

Statistical Analysis.
Results were represented as mean ± sd. Significance values were compared using Student's t -test using GraphPad Prism. p < 0:05 was considered statistically significant. All experiments were done in triplicate.

The iNOS Is Overexpressed in OS.
To explore the role of iNOS in the development of OS, we firstly detected the iNOS expression of 45 OS tissues and 45 normal tissues. It was found that iNOS levels in OS tissues were significantly higher than those in normal tissues, as shown in Figures 1(a) and  1(b). Additionally, iNOS in human osteosarcoma cell lines Saos2 and 143B was significantly upregulated than that in osteoblast cell line hFOB1.19, as shown in Figures 1(c) and 1(d).

iNOS Knockdown Inhibits Cell Proliferation, Migration, and Invasion and Promotes Cell Apoptosis in OS.
Then, colony formation assay and BrdU assay were used to testify the function of iNOS on the OS cell proliferation. The results showed that iNOS knockdown or iNOS inhibitor 1400 W suppressed the proliferation of Saos2 and 143B cells, as shown in Figures 2(a) and 2(b). Else, Transwell assay showed that iNOS knockdown or iNOS inhibitor 1400 W suppressed cell migration and invasion of Saos2 and 143B cells, as shown in Figure 2(c). Apoptosis assay showed that iNOS knockdown or iNOS inhibitor 1400 W induced the apoptosis of Saos2 and 143B cells in Figure 2(d). It was also observed that the expressions of MMP2, MMP9, C-MYC, Ki67, and PCNA in Saos2 and 143B cells were higher than those in hFOB1.19 cells. However, Saos2 and 143B cells treated with iNOS knockdown would significantly suppress the expressions of MMP2, MMP9, C-MYC, Ki67, and PCNA in OS cells, as shown in Figures 2(e) and 2(f).

Effect of iNOS on Wnt/β-Catenin Pathway of OS Cells.
Subsequently, the signaling pathway of iNOS acting on the OS progression was explored. Firstly, we demonstrated that the expression of β-catenin was significantly reduced in OS cells treated with the inhibitor of Wnt/β-catenin pathway IWP-1, but the expression of iNOS was not significantly

Effect of iNOS on Tumorigenicity of OS Cells In Vivo.
Mice were supplied with OS cells treated with sh-iNOS or sh-NC, and in vivo experiments showed that iNOS inhibition would suppress tumor growth in OS, as shown in Figure 4(a). The tumor volume and tumor weight were significantly reduced after iNOS knockdown, as shown in Figures 4(b) and 4(c). Also, it showed that iNOS levels were downregulated in tumor treated with sh-iNOS than those in tumor treated with sh-NC, as shown in Figure 4(d). Western blotting showed that the expression of β-catenin was decreased in OS tumor tissues treated with sh-iNOS in Figure 4(e). IHC assay also proved that the expressions of iNOS and βcatenin were reduced in OS tumor tissues transplanted with sh-iNOS than those in OS tumor tissues transplanted with sh-NC.

Discussion
The studies of iNOS function in OS pathogenesis are still limited. Therefore, we detected mRNA and protein expression of iNOS in tumor tissues and matched adjacent tissues. We found that iNOS level was significantly higher in OS tissues or cells than that in normal tissues or cells. These findings are in agreement with previous studies in tumors. Granados-Principal et al. found that iNOS was highly expressed in triple-negative breast cancer [8]. Benkhelifa et al. observed that significantly increased iNOS mRNA expression in metastatic tissues of colorectal cancer compared to primary tumors and unaffected mucosa [19]. Pereira et al. showed that iNOS expressions were significantly increased in cancer-associated fibroblasts in pancreatic ductal adenocarcinoma [20]. Further studies showed that the downregulation of iNOS inhibited proliferation and metastasis of OS cells and inhibited apoptosis of OS cells. In human intrahepatic cholangiocarcinoma, iNOS knockdown and iNOS inhibitor (1400 W) suppressed cell proliferation, invasion, and migration [21]. iNOS system played a dominant role in increasing proliferation, migration, and invasion of glioblastoma cells surviving a photochallenge [22]. And we noticed that the function of iNOS in OS was relative with the Wnt/β-catenin pathway. Previous studies reported that NOS activity modulates βcatenin posttranslational modifications to promote endothelial cell survival [23]. NO acted a mediator in colorectal cancer via Wnt/β-catenin pathway, which was closely associated with cancer initiation and metastasis [24]. All findings indicate that iNOS might be an important regulator in OS development.
This work makes a preliminary exploration for the role of iNOS in OS. However, there are some limitations here. The study size in this research is small. And whether iNOS regulates β-catenin through nitric oxide is unclear. Else, the effects of iNOS inhibitor can be applied to clinical treatment for OS patients' needs to explore.
In summary, our data demonstrates that iNOS may be a useful biomarker to assess tumor progression for OS. iNOS inhibition plays a negative role in tumor proliferation,