Down-Regulation of Nucleolar and Spindle-Associated Protein 1 (NUSAP1) Expression Suppresses Tumor and Cell Proliferation and Enhances Anti-Tumor Effect of Paclitaxel in Oral Squamous Cell Carcinoma

Abstract

Background: Nucleolar and spindle-associated protein 1 (NUSAP1) is an important mitotic regulator. In addition to its crucial function in mitosis, NUSAP1 has recently received attention due to the interesting roles in carcinogenesis. The aim of this study was to reveal functional mechanisms of NUSAP1 in oral squamous cell carcinoma (OSCC).

Methods: mRNA and protein expression levels of NUSAP1 in 9 OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and immunoblotting analyses. The correlation between the NUSAP1 expression profile and the clinicopathological factors was evaluated by immunohistochemistry (IHC) in clinical OSCC samples (n = 70). The NUSAP1 knockdown cells were established with short hairpin RNA (shRNA) in OSCC cells, and functional assays were performed using these cells. In addition to the evaluation of cellular proliferation and cell cycle, we also investigated the potential role of NUSAP1 in paclitaxel (PTX)-induced cellular responses.

Results: mRNA and protein expression of NUSAP1 were significantly up-regulated in OSCC-derived cells compared with human normal oral keratinocytes (P < 0.05). IHC revealed that NUSAP-1 expression is closely associated with primary advanced T stage (P<0.05). Suppression of NUSAP1 expression levels led to significant (P < 0.05) inhibition of cellular proliferation. Furthermore, apoptosis induced by PTX was enhanced in NUSAP1 knockdown OSCC cells.

Conclusions: NUSAP1 may be a crucial biomarker for OSCC. Moreover, down-regulated NUSAP1 expression suppresses tumor proliferation and also enhances anti-tumor effect of PTX by activating apoptotic pathways. Thus, the present study strongly suggests that regulating NUSAP1 expression should contribute to the therapy for OSCC.

Fig 1. Evaluation of NUSAP1 expression in OSCC-derived cell lines.

a. Quantification of NUSAP1 mRNA expression levels in OSCC-derived cell lines by qRT-PCR analysis. mRNA expression levels are normalized to GAPDH. Significant (p < 0.05, Mann-Whitney’s U test*) up-regulation of NUSAP1 mRNA is seen in nine OSCC-derived cell lines (HSC-2, HSC-3, HSC-4, KOSC2, Ca9-22, Sa3, HO-1-N-1, HO-1-u-1, and KON) compared with the HNOKs. Data are expressed as the means ± SEM of triplicate results. b. Representative immunoblotting data of NUSAP1 in OSCC-derived cell lines and HNOKs show that NUSAP1 protein expression is up-regulated in OSCC-derived cell lines compared with the HNOKs. The molecular weight of the NUSAP1 is 49 kDa. Densitometric NUSAP1 protein data are normalized to GAPDH protein levels. The values are expressed as a percentage of the HNOKs.

Fig 2. Evaluation of NUSAP1 protein expression in primary OSCCs.

Representative IHC results for NUSAP1 in a normal oral tissue and b primary OSCC (original magnification, x400. Scale bars, 50 μm). Strong NUSAP1 immunoreactivity is detected in primary OSCCs, whereas normal oral tissues show almost negative immunostaining. c The state of NUSAP1 protein expression in the normal counterparts and primary OSCCs (n = 70). The NUSAP1 IHC scores for normal oral tissues and OSCCs range from 0 to 38.1 (median 12.8) and 40.2 to 170.3 (median 136.3), respectively. NUSAP1 protein expression levels in OSCCs are significantly (p < 0.001, Mann-Whitney’s U test) higher than that in normal oral tissues.

Fig 3. Establishment of NUSAP1 knockdown cells.

To obtain stable NUSAP1 knockdown transfectants, we performed transfection of the shNUSAP1 and Mock vectors into OSCC cell lines (HSC-3 and Sa3). a,b Expression of NUSAP1 mRNA in shNUSAP1 and Mock cells. c,d Immunoblotting analysis of NUSAP1 protein in shNUSAP1 and Mock cells. The NUSAP1 mRNA and protein are significantly down-regulated in shNUSAP1 cells (HSC-3 and Sa3).

Fig 4. Functional analyses of NUSAP1 knockdown cells.

Reduced cellular growth was observed in a shNUSAP1 HSC-3 and b shNUSAP1 Sa3 cells. To determine the effect of shNUSAP1 on cellular proliferation, shNUSAP1 and Mock cells are seeded in 6-well plates at a density of 1×10⁴ viable cells/well. The shNUSAP1 HSC-3 and shNUSAP1 Sa3 cells show a significant (p < 0.05, Mann-Whitney’s U test*) decrease in cellular growth compared with Mock cells. The results are expressed as the means ± SEM of values from three assays. The asterisks indicate significant (p < 0.05, Mann-Whitney U test*) differences between the shNUSAP1 and Mock cells. c shNUSAP1- and Mock-transfected cells were counted on 4 consecutive days by a hemocytometer.

Fig 5. shNUSAP1 promotes G2 arrest.

Flow cytometric analysis was performed to investigate cell-cycle progression in the shNUSAP1- and shMock-transfected cells after synchronization at the G2/M phase to treatment with nocodazole. a-d The percentage of cells at the G2 phase in the shNUSAP1-transfected cells (HSC-3- and Sa3-derived transfectants) is increased markedly compared with the shMock- transfected cells.

Fig 6. Apoptosis of shNUSAP1 determined by flow cytometry.

a HSC-3 cells after dual staining with Annexin V-FITC and propidium iodide (PI). Annexin V-FITC/PI staining is monitored overtime, after 48 hours in HSC-3 and Sa3 cells exposed to PTX at concentrations corresponding to IC50 (100nM). Representative dot plots of three independent experiments are given, presenting intact cells in the lower-left quadrant, FITC(-)/PI(-); early apoptotic cells in the lower-right quadrant, FITC(+)/PI(-); late apoptotic or necrotic cells in the upper-right quadrant, FITC(+)/PI(+); and necrotic cells in the upper-left quadrant, FITC(-)/PI(+). b Total apoptosis rate was analyzed from 3 times experiment.