Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Apr 17;9(4):e95337.
doi: 10.1371/journal.pone.0095337. eCollection 2014.

Imiquimod induces apoptosis of squamous cell carcinoma (SCC) cells via regulation of A20

Kyung-Cheol Sohn  1 , Zheng Jun Li  1 , Dae-Kyoung Choi  1 , Tiejun Zhang  2 , Jae Woo Lim  1 , In-Kyu Chang  1 , Gang Min Hur  2 , Myung Im  1 , Young Lee  1 , Young-Joon Seo  1 , Jeung-Hoon Lee  1 , Chang Deok Kim  1
Affiliations

Imiquimod induces apoptosis of squamous cell carcinoma (SCC) cells via regulation of A20

Kyung-Cheol Sohn et al. PLoS One. .

Abstract

Imiquimod, a nucleoside analogue of the imidazoquinoline family, is being used to treat various cutaneous cancers including squamous cell carcinoma (SCC). Imiquimod activates anti-tumor immunity via Toll-like receptor 7 (TLR7) in macrophage and other immune cells. Imiquimod can also affect tumor cells directly, regardless of its impact on immune system. In this study, we demonstrated that imiquimod induced apoptosis of SCC cells (SCC12) and A20 was involved in this process. When A20 was overexpressed, imiquimod-induced apoptosis was markedly inhibited. Conversely, knockdown of A20 potentiated imiquimod-induced apoptosis. Interestingly, A20 counteracted activation of c-Jun N-terminal kinase (JNK), suggesting that A20-regulated JNK activity was possible mechanism underlying imiquimod-induced apoptosis of SCC12 cells. Finally, imiquimod-induced apoptosis of SCC12 cells was taken place in a TLR7-independent manner. Our data provide new insight into the mechanism underlying imiquimod effect in cutaneous cancer treatment.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Expression of A20 in skin cells.
(A) Expression of A20 was detected using Western blot analysis. High expression of A20 is observed in squamous carcinoma cell line SCC12 and SCC13. (B) Expression of A20 was detected in skin tissues by immunohistochemistry. In normal skin, A20 expression is increased in upper layers of epidermis. In SCC, high expression of A20 is detected in cancer lesions. In negative control, primary antibody was omitted.
Figure 2
Figure 2. Imiquimod-induced apoptosis of SCC12 cells.
(A) Cells were treated with imiquimod at the indicated concentrations for 24 h. Cell viability was determined by MTT assay. Data are expressed as percentage of control (0 µg/ml imiquimod). The mean values ± SD are averages of triplicate measurements. (B) To determine whether imiquimod induces apoptosis of SCC12 cells, cleavage of PARP, a prominent feature of the apoptotic execution phase, was checked by Western blot. Anti-actin antibody was used as a loading control. Imiquimod induces cleavage of PARP in a dose-dependent manner. (C) Cells were treated with imiquimod at the indicated concentrations and/or for the indicated time points. Expression of A20 was detected by Western blot. Imiquimod induces down-regulation of A20 in a dose- and time-dependent manner. (D) Cells were pretreated with MG132 then treated with imiquimod (150 µg/ml) for the indicated time points. MG132 blocks imiquimod-driven A20 down-regulation.
Figure 3
Figure 3. Effect of A20 overexpression on imiquimod-induced apoptosis of SCC12 cells.
(A) Cells were transduced with 10 multiplicity of infection (MOI) of adenovirus expressing GFP-tagged A20 (Ad/GFP-A20) or control adenovirus (Ad/GFP) for 6 h. Cells were replenished with fresh medium, and incubated for a further 2 d. Then, cells were treated with imiquimod (150 µg/ml) for 16 h. Apoptosis was determined by flow cytometry. Annexin V high and propidium iodide (PI) dim cells (bold box) represent apoptotic cells. Imiquimod-induced apoptosis is markedly reduced in GFP-A20 overexpressed group (Ad/GFP-A20) compared to GFP overexpressed control group (Ad/GFP). (B) Cleavages of PARP and caspase-3 were detected by Western blot. In GFP-A20 overexpressed cells, imiquimod-driven PARP and caspase-3 cleavages are reduced compared to control group. (C) After adenoviral transduction, cells were treated with imiquimod (150 µg/ml) for the indicated time points, and phosphorylation of MAPKs was detected by Western blot. Imiquimod induces phosphorylation of JNK, which is inhibited by overexpression of GFP-A20.
Figure 4
Figure 4. Effect of A20 knockdown on imiquimod-induced apoptosis of SCC12 cells.
(A) The stable SCC12 cells expressing shRNA were established, and phosphorylation of MAPKs was detected by Western blot. In A20-knockdowned cells (shRNA-A20 (#1), shRNA-A20 (#2)), phosphorylation of JNK is increased compared to control cells (shRNA-Scr). (B) Cells were treated with imiquimod (150 µg/ml) for 16 h and apoptosis was determined by flow cytometry. Bold boxes represent apoptotic cells. Imiquimod-induced apoptosis is markedly potentiated in A20-knockdowned cells (shRNA-A20 (#2)) compared to control cells (shRNA-Scr).
Figure 5
Figure 5. Effect of TLR7 knockdown on imiquimod-induced apoptosis of SCC12 cells.
(A) Cells were transduced with 10 multiplicity of infection (MOI) of adenovirus expressing miR-TLR7 or control adenovirus (miR-Scr) for 6 h. Cells were replenished with fresh medium, and incubated for a further 2 d. Endogenous expression of TLR7 is markedly decreased by miR-TLR7. (B) After adenoviral transduction, cells were treated with imiquimod (150 µg/ml) for 16 h and apoptosis was determined by flow cytometry. Bold boxes represent apoptotic cells. There is no difference in apoptotic cell populations between TLR7-knockdowned group (miR-TLR7) and control group (miR-Scr). (C) Cleavages of PARP was detected by Western blot. TLR7 knockdown does not affect imiquimod-driven PARP cleavage.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alam M, Ratner D (2001) Cutaneous squamous-cell carcinoma. N Engl J Med 344: 975–983. - PubMed
    1. Brash DE, Rudolph JA, Simon JA, Lin A, McKenna GJ, et al. (1991) A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma. Proc Natl Acad Sci USA 88: 10124–10128. - PMC - PubMed
    1. Kolev V, Mandinova A, Guinea-Viniegra J, Hu B, Lefort K, et al. (2008) EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer. Nat Cell Biol 10: 902–911. - PMC - PubMed
    1. Zhao L, Li W, Marshall C, Griffin T, Hanson M, et al. (2009) Srcasm inhibits Fyn-induced cutaneous carcinogenesis with modulation of Notch1 and p53. Cancer Res 69: 9439–9447. - PMC - PubMed
    1. van Hogerlinden M, Rozell BL, Ahrlund-Richter L, Toftgard R (1999) Squamous cell carcinomas and increased apoptosis in skin with inhibited Rel/nuclear factor-κB signaling. Cancer Res 59: 3299–3303. - PubMed

Publication types

MeSH terms

Grants and funding

This study was supported by a grant from the National Research Foundation of Korea (NRF-2010-0023545). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.