Sirtuin-3 (SIRT3), a novel potential therapeutic target for oral cancer

Abstract

Background: Several sirtuin family members (SIRT1-7), which are evolutionarily conserved NAD-dependent deacetylases, play an important role in carcinogenesis. However, their role in oral cancer has not yet been investigated. Therefore, the objective of this study was to investigate whether sirtuins play a role in oral cancer carcinogenesis.

Methods: The expression levels of all sirtuins in several oral squamous cell carcinoma (OSCC) cell lines were compared with normal human oral keratinocytes and observed that SIRT3 was highly expressed. Therefore, tissue microarrays were used to evaluate the clinical relevance of this overexpression. SIRT3 down-regulation in OSCC cell proliferation and survival was investigated and analyzed by using cell-proliferation and cell-viability assays. Ionizing radiation and cisplatin were used to investigate whether SIRT3 down-regulation could increase the sensitivity of OSCC to both treatments. To further assess the in vivo role of SIRT3 in OSCC carcinogenesis, a floor-of-mouth oral cancer murine model was used to study the effect of SIRT3 down-regulation on OSCC tumor growth in immunodeficient mice.

Results: The current results demonstrated for the first time that SIRT3 is overexpressed in OSCC in vitro and in vivo compared with other sirtuins. Down-regulation of SIRT3 inhibited OSCC cell growth and proliferation and increased OSCC cell sensitivity to radiation and cisplatin treatments in vitro. SIRT3 down-regulation also reduced tumor burden in vivo.

Conclusions: The current investigation revealed a novel role for SIRT3 in oral cancer carcinogenesis as a promoter of cell proliferation and survival, thus implicating SIRT3 as a new potential therapeutic target to treat oral cancer.

Figure 1

Sirtuin-3 (SIRT3) is overexpressed in oral squamous cell carcinoma (OSCC). (A) Immunoblots reveal the levels of sirtuins (SIRT1-7) in the OSCC cell lines HSC-3, UM-SCC-1, and UM-SCC-17B and in normal human oral keratinocytes (K). β-Actin served as loading control. (B) These representative samples show (Top) SIRT3 expression levels in OSCC (tongue) and (Bottom) in normal tissues. Immunoglobulin G (IgG) served as a negative control. Scale bars = 200 μm for low-magnification photomicrographs (left column); 50 μm for high-magnification photomicrographs (middle and right columns). (C) These charts illustrate the percentage of normal and OSCC tissue specimens that expressed SIRT3 (Top) in all samples and (Bottom) in tongue samples, as determined by immunohistochemical staining. Note that SIRT3 staining intensities were designated as low (L) or high (H) from the data provided in Table 1.

Figure 2

The sirtuin inhibitors sirtinol and nicotinamide (NAM) inhibit cell growth and proliferation and induce apoptosis. (A) Shown are (Top) phase-contrast images and (Bottom) histograms that reveal the morphology of cell growth and cell proliferation (%) of the HSC-3, UM-SCC-1, and UM-SCC-17B oral squamous cell carcinoma (OSCC) cell lines after treatment with sirtuin inhibitors sirtinol (50 μM) or NAM (40 mM) for 16 hours and 24 hours, respectively (original magnification, ×100). (B) Cell death-detection ELISA assays were used to measure DNA fragmentation in cells after treatment with (Top) sirtinol and (Bottom) NAM as indicated. **P ≤ .01; ***P ≤ .001; OD, optical density.

Figure 3

Sirtuin-3 (SIRT3) down-regulation inhibits cell growth and proliferation and promotes apoptosis in oral squamous cell carcinoma (OSCC) cells. (A) (Top) Immunoblots reveal the transfection efficiency of SIRT3 in the UM-SCC-1 and UM-SCC-17B OSCC cell lines 36 hours after transfection with SIRT3 small interfering RNA (siSIRT3) or nontargeting control (siControl) (150 nM). β-Actin served as loading control. (Middle) Cells were transfected as indicated and were cultured for 1 week, then stained with crystal violet, and photographed. (Bottom) Numbers of colonies are presented as the percentage of colonies obtained relative to controls. (B) Cell death-detection ELISA assays were used to measure DNA fragmentation in the cells after transfection. **P ≤ .01; ***P ≤ .001; OD, optical density.

Figure 4

Sirtuin-3 (SIRT3) down-regulation enhances the sensitivity of oral squamous cell carcinoma (OSCC) to radiation and cisplatin-induced cytotoxicity. OSCC cells (UM-SCC-1 and UM-SCC-17B) were either untransfected or transfected with small interfering SIRT3 (siSIRT3) (150 nM) and were treated with either (A) ionizing radiation (2.5 grays) or (B) cisplatin (20 μM) for 24 hours, and cytotoxicity was determined by using the QUANT Cell Proliferation Assay Kit (Invitrogen). **P ≤ .01; ***P ≤ .001.

Figure 5

Sirtuin-3 (SIRT3) down-regulation reduces oral squamous cell carcinoma (OSCC) tumor burden in vivo. (A) These immunoblots show SIRT3 expression levels in 4 clones from UM-SCC-17B cell lines that were stably transfected with scrambled-short hairpin RNA (Scr-shRNA) or SIRT3-shRNA after 10 days of selection using Puromycin. β-Actin served as loading control. (B) Mice were injected with OSCC cells that stably expressed Scr-shRNA or SIRT3-shRNA. Images show (Top) the superficial growth of tumors in the head and neck region of the mice, (Middle) dissected tumors in situ, and (Bottom) dissected and isolated tumors from the 2 groups.