Apigenin Induced Apoptosis by Downregulating Sulfiredoxin Expression in Cutaneous Squamous Cell Carcinoma


. 2022 Aug 4;2022:8172866.


doi: 10.1155/2022/8172866.


eCollection 2022.

Affiliations

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Wenhua Wang et al.


Oxid Med Cell Longev.


.

Abstract

Cutaneous squamous cell carcinoma (cSCC) is the second carcinoma in nonmelanoma skin cancer (NMSC). Sulfiredoxin (Srx) is an antioxidant protein with a role in maintaining redox homeostasis. And Srx has an oncogenic role in skin tumorigenesis. In the current study, we found that apigenin, as a natural flavonoid, downregulated the expression of Srx protein in cSCC cell lines. Apigenin also inhibited the ability of cell proliferation and migration and induced apoptosis in cSCC cell lines. Our results also showed that apigenin induced apoptosis via the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, as well as downregulated Srx expression in cSCC cell lines. Importantly, the effect of downregulation Srx by apigenin has been rescued with the inhibitor of the MAPK signaling pathway intervention. And induced apoptosis by apigenin was partially attenuated by the addition of MAPK inhibitor, Binimetinib. Our research revealed that apigenin induced apoptosis by downregulation of Srx expression through regulating the MAPK signaling pathway in cSCC cells, thus providing evidence of its applicability as a potentially effective therapeutic agent for cSCC treatment.

Conflict of interest statement

The authors declare no conflict of interest.

Figures


Figure 1



Figure 1

Apigenin can downregulate Srx expression in TPA-induced JB6 and A431 cSCC cells. (a) JB6 was treated with control (DMSO), TPA (20 nM), or apigenin (40 or 80 μM) for different time points (6 h-48 h). Srx expression was detected by western blotting (left). Western blot was served to analyze the expression of Srx while TPA-induced JB6 cells were incubated with apigenin (80 μM) for different times (6 h-48 h) (right). (b) Human SCC A431 cells were treated with apigenin at different times, and the expression of Srx was detected by WB. (c) The mRNA level of Srx in TPA-induced JB6 and A431 cells was conducted to measure after incubation with 80 μM apigenin for 6 h and 12 h. (d) Representative images of immunofluorescence staining of Srx in TPA-induced JB6 treated with control (DMSO) or 80 μM apigenin for 6 h-48 h (left). Quantitative analysis of Srx means fluorescence intensity (MFI) (right) (mean values ± SEM, n = 3). Significant differences were evaluated using a one-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001vs. control. For JB6 cells, TPA-induced sample as control.


Figure 2



Figure 2

Apigenin can attenuate cell proliferation and migration in cSCC cells. (a) CCK-8 assay was implied to analyze the cell viability after the treatment of TPA-induced JB6 and A431 cells with different concentrations of apigenin (up to 80 μM) as time gone (mean values ± SEM, n = 6). ∗∗∗∗p < 0.0001vs. control by ANOVA. (b) Representative images of colony formation assay in TPA-induced JB6 treated with control (DMSO) or 80 μM apigenin. (c) Typical pictures (left) and quantitative analysis (right) of wound healing assay in TPA-induced JB6 (up) and A431 (down) cells. Cells were treated with control (DMSO) or 80 μM apigenin for indicated time points. ns: no statistical significance; ∗∗∗∗p < 0.0001vs. control by Student’s unpaired t-test.


Figure 3



Figure 3

Apigenin induced apoptosis in cSCC cells. (a) Flow cytometry was used to analyze the apoptosis cells. Cells were stained with annexin V and PI to quantify the percentage of apoptotic cells. TPA-induced JB6 cells (upper) or A431 cells (lower) were treated with control (DMSO) or 80 μM apigenin for different times. (b) A concrete percentage of apoptosis cells in TPA-induced JB6 and A431 cells were evaluated using a one-way ANOVA (mean values ± SEM, n = 3) ∗p < 0.05, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001vs. control (TPA-induced sample as control for JB6 cells). (c) TPA-induced JB6 cells were incubated with control (DMSO) or 80 μM apigenin for 6 h-48 h. Western blot was served to analyze the expression of apoptosis-associated proteins (left). The bar graphs on the right showed the intensity of the protein band from each treatment relative to the housekeeping protein (β-actin). Valued represent the means ± SEM. Significant difference was designed by ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001vs. control (only TPA-induced sample). (d) The apoptosis-associated proteins were detected while 80 μM apigenin was used for treatment for different time-points in A431 cells. The bar graphs showed the intensity quantification of the protein band relative to the housekeeping protein. Significant difference from control by ANOVA, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.


Figure 4



Figure 4

Apigenin activated the MAPK signaling pathway in cSCC in vitro. (a, b) Variation of the MAPK signaling pathway with the treatment of apigenin in TPA-induced JB6 (a) and A431 cells (b). The cells were treated with 80 μM apigenin for different time points up to 24 h. Western blot was applied to analyze the expression of MAPK pathway-associated proteins, includingp38, ERK1/2 and JNK compared with GAPDH (left). The bar graph on the right showed the intensity of the phosphorylation protein band from each treatment relative to the total protein. Valued represent the means ± SEM. Significant difference was designed by ANOVA, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001vs. control (TPA-induced sample as control for JB6 cells). (c) TPA-induced JB6 cell was incubated with control (DMSO, TPA-alone, and apigenin-alone) or apigenin (40 or 80 μM) for indicted time points. Western blot was served to analyze the expression of Nrf2. β-Actin was used as the reference for the loading quantity of protein sample. The bar graph indicated the density quantification of the Nrf2 band relative to β-actin. ∗p < 0.05 and ∗∗∗p < 0.001vs. TPA-induced control by ANOVA.


Figure 5



Figure 5

Apigenin induced cell apoptosis and inhibited the expression of Srx via regulating the MAPK signaling pathway in cSCC. (a, b) TPA-induced JB6 cells (a) and A431 cells (b) were treated with the combination of apigenin and inhibitor of MEK1/2, Binimetinib (5 or 10 nM), for 8 h. Western blotting was used to analyze the expression of Srx, p-Erk, and the apoptosis-related proteins BAX and Bcl2. Representative images are shown on the left. The bar graph on the right indicated the intensity quantification of the protein band relative to GAPDH or total protein (ERK). Significant difference was designed by ANOVA, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001vs. control (TPA-induced sample as control for JB6 cells). (c) Real-time PCR was conducted to quantify the mRNA expression of Srx in cSCC cell lines while cells were treated for indicated compounds. ∗∗p < 0.01 and ∗∗∗p < 0.001 vs control by ANOVA analysis. (d) Representative images of cell apoptosis analysis by flow cytometry after 5 nM Binimetinib (MEK1/2 inhibitor) treatment for 24 h, respectively, in TPA-induced JB6 (up) and A431 (down). The percentage of apoptosis cells after apigenin treatment with or without Binimetinib is shown in the bar graph. ∗∗∗∗p < 0.0001vs. control by ANOVA analysis. (e, f) TPA-induced JB6 (e) and A431 (f) were incubated with or without the MEK1/2 inhibitor (5 nM Binimetinib) for 24 h in the presence of 80 μM apigenin. Whole-cell lysates were subjected to western blotting to detect the apoptosis-associated proteins caspase 3, caspase 8, and PARP. The bar graph showed the intensity quantification of the protein bands from each treatment. Significant difference was designed by ANOVA, ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001vs. the apigenin-alone group.


Figure 6



Figure 6

The summarization of the effect of apigenin in cSCC cell lines. After treatment with apigenin, the MAPK signaling pathway was activated gradually through the form of phosphorylation, especially the ERK1/2 pathway. Then, Phospho-MAPK from the cytoplasm to nucleus may generally downregulate the expression of Srx by inhibiting the expression of Nrf2. Then, apigenin might induce cell apoptosis in cSCC cells.

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