SIRT6 overexpression induces apoptosis of nasopharyngeal carcinoma by inhibiting NF-κB signaling

Abstract

Background: Previous reports show that SIRT6 serves as a critical modulator of the development of multiple malignancies as well as other disorders. However, its role in nasopharyngeal carcinoma (NPC) is unknown. Thus, we elucidated the effects of SIRT6 on the survival of NPC cells, and modulation of cell death.

Methods: We found that expression of SIRT6 is downregulated in ten human NPC specimens as well as in the human NPC cell lines, 5-8 F and CNE1, as compared with that in healthy tissues and normal nasopharyngeal NP69 cells. The MTT assay and colony formation assay revealed that upregulation of SIRT6 impaired the proliferation, as well as the survival of 5-8 F and CNE1 cells. The TUNEL assay, annexin V-FITC/propidium iodide, and flow cytometry were performed to detect apoptosis. The results revealed that the expression of SIRT6 resulted in increased apoptosis.

Results: Western blotting results showed that SIRT6 overexpression decreased anti-apoptotic Bcl-2 levels, whereas it promoted an increase in pro-apoptotic Bax and cleaved caspase-3 levels. Moreover, NF-κB levels were markedly reduced in cells expressing SIRT6, whereas they were increased in cells transfected with shRNA-SIRT6. Recovery of NF-κB expression was found to counter the suppressive influence of SIRT6 on NPC cell survival, whereas, NF-κB knockdown increased apoptosis of NPC cells.

Conclusion: Thus, the findings of our study offer insight into the biological and molecular mechanisms underlying the development of NPC and may lead to the development of new and innovative strategies for the treatment of NPC.

Keywords: NF-κB; SIRT6; apoptosis; nasopharyngeal carcinoma.

Figure 1

Expression of SIRT6 in NPC cells or tissue specimens. Notes: (A, B) Expression of SIRT6 in NPC, 5-8 F, and CNE1 cells, as well as nasopharynx cells NP69, was detected using Western blotting. (C, D) Subcellular localization of SIRT6 in NPC cell lines 5-8 F and CNE1 was detected using immunofluorescence; [anti-SIRT6 antibody (red) and nuclear DNA (blue)]. (E, F) Expression of SIRT6 in 5-8 F and CNE1 cells determined using qRT-PCR. (G) Expression of SIRT6 in specimens obtained from NPC patients (n=10) vs normal healthy patients (n=10). Data are represented as mean ± SD. *P<0.05. Abbreviation: NPC, nasopharyngeal carcinoma.

Figure 2

SIRT6 overexpression affected viability of NPC cells. Notes: (A, B) Western blotting and (C, D) qRT-PCR were performed to evaluate SIRT6 expression in 5-8 F and CNE1 cells following infection with adenovirus-SIRT6 (AD-SIRT6) or AD-NC. (E, F) MTT assay was used to examine the proliferation of 5-8 F and CNE1 cells 12–72 hours post-culture. (G, H) Soft agar colony formation assay for the 5-8 F and CNE1 cells infected with AD-SIRT6, AD-NC, and that for uninfected cells. (I, J) The number of colonies formed in each group. Data are shown as mean ± SD. **P<0.01, ***P<0.001. Abbreviations: AD, adenovirus; NC, negative control; NPC, nasopharyngeal carcinoma.

Figure 3

Overexpression of SIRT6 enhanced apoptosis of NPC cells. Notes: (A) TUNEL assay was performed in each group of 5-8 F cells expressing SIRT6 or NCs. Magnification, 400×. The number of TUNEL-positive 5-8 F cells is displayed in the lower panel. (B) Annexin V-FITC/PI staining and flow cytometry were performed to evaluate the number of apoptotic cells. Early apoptotic cells are shown in the upper right quadrant of each plot, and later apoptotic cells are displayed in the lower right quadrant of each plot. Analysis of the apoptotic rate of 5-8 F cells in all groups is shown in the lower panel. (C) Expression levels of SIRT6-regulated Bcl-2 and Bax, total caspase-3, and cleavage of caspase-3 in the Hep-2 cells. (D) Relative density of cleaved caspase-3 bands. (E, F) qRT-PCR was performed to assess the influence of SIRT6 on mRNA levels of Bcl-2 and Bax. Data are shown as mean ± SD. *P<0.05, **P<0.01. Abbreviations: AD, adenovirus; NC, negative control; NPC, nasopharyngeal carcinoma; PI, propidium iodide.

Figure 4

SIRT6 regulated NF-κB expression in NPC cells. Notes: (A, B) Expression of NF-κB in NPC, 5-8 F, and CNE1 cells, as well as nasopharynx cells NP69, was detected using Western blotting. (C, D) 5-8 F and CNE1 cells were infected with AD-SIRT6 or AD-NC. Protein expression of SIRT6 and NF-κB was detected using Western blotting. (E, F) 5-8 F and CNE1 cells were transfected with shRNA-SIRT6 and shRNA-NC to knock down SIRT6 expression. Protein expression of SIRT6 and NF-κB was then analyzed using Western blotting. (G, H) Subcellular localization of NF-κB in NPC cells transfected with shRNA-SIRT6 or not, was detected using immunofluorescence; [anti-NF-κB antibody (red) and nuclear DNA (blue)]. Data are shown as mean ± SD. Abbreviations: AD, adenovirus; CTRL, control; NC, negative control; NPC, nasopharyngeal carcinoma.

Figure 5

NF-κB overexpression restored cell viability impaired by SIRT6. Notes: 5-8 F cells were first infected with AD-SIRT6, and then transfected with the NF-κB-expressing vector to elevate NF-κB levels. (A) Protein expression of SIRT6, NF-κB, Bcl-2 and Bax in each group was detected using Western blotting. (B, C) qRT-PCR was performed to assess the influence of NF-κB on mRNA expression of Bcl-2 and Bax. (D) Annexin V-FITC/PI staining and flow cytometry were performed to evaluate the number of apoptotic cells. Early apoptotic cells are shown in the upper right quadrant of each plot, and later apoptotic cells are displayed in the lower right quadrant of plot. Analysis of the apoptotic rate of 5-8 F cells in all groups is shown in the lower panel. Data are shown as mean ± SD. *P<0.05, ***P<0.001. Abbreviations: AD, adenovirus; NC, negative control; PI, propidium iodide.

Figure 6

NF-κB knockdown had a similar effect on cell viability as that of SIRT6 overexpression. Notes: 5-8 F cells were transfected with shRNA-SIRT6, or co-transfected with shRNA-SIRT6 and shRNA-NF-κB. (A) Protein expression of SIRT6, NF-κB, Bcl-2, and Bax in each group was assessed using Western blotting. (B, C) qRT-PCR was performed to examine the mRNA expression of Bcl-2 and Bax. (D) Annexin V-FITC/PI staining and flow cytometry were performed to count the number of apoptotic cells. Early apoptotic cells are shown in the upper right quadrant of each plot, and later apoptotic cells are displayed in the lower right quadrant of plot. Analysis of the apoptotic rate of 5-8 F cells in all groups is shown in the lower panel. Data are shown as mean ± SD. *P<0.05, ***P<0.001. Abbreviations: NC, negative control; PI, propidium iodide.