Gallic Acid Impedes Non-Small Cell Lung Cancer Progression via Suppression of EGFR-Dependent CARM1-PELP1 Complex

Abstract

Background: Non-small cell lung cancer (NSCLC) is a common cause of cancer-related deaths. This study identified the regulatory pattern of gallic acid in NSCLC.

Methods: Human NSCLC cells were treated with different doses of gallic acid, after which, MTT assay and flow cytometry were performed to determine the survival and apoptotic rate of human NSCLC cells. Then, co-immunoprecipitation assay was performed to analyze the relationships between gallic acid, epidermal growth factor receptor (EGFR), and CARM1-PELP1. Next, we analyzed whether PELP1, CARM1 and EGFR were associated with the effects of gallic acid on NSCLC cells by conducting rescue experiments. The expression pattern of phosphorylated EGFR, EGFR, Ki67, as well as Fas, FasL and Caspase 3 proteins in cancer cells or xenografts was measured by Western blot analysis. Lastly, the role of gallic acid in the tumor growth was assessed in nude mice.

Results: The ideal dose of gallic acid that presented good suppressive effect on NSCLC cells were 30 μM, 50 μM and 75 μM, respectively. Gallic acid played an inhibiting role in the activation of EGFR, which further reduced the formation of CARM1-PELP1 complex, ultimately repressed the proliferation and elevated apoptosis of NSCLC cells. Meanwhile, CARM1 repression led to decreased growth, proliferation and migration abilities of NSCLC cells. Animal experiments confirmed that gallic acid contributed to the inhibition of tumor growth in vivo.

Conclusion: To sum up, gallic acid could potentially prevent NSCLC progression via inhibition of EGFR activation and impairment of the binding of CARM1 to PELP1, highlighting a novel therapy to dampen NSCLC progression.

Keywords: CARM1; CARM1-PELP1 complex; EGFR; PELP1; gallic acid; non-small cell lung cancer.

Figure 1

Gallic acid inhibited the development of NSCLC and repressed EGFR activation. (A) The viability of A549 and NCI-H1299 cells detected by MTT assay. (B) The apoptotic rate of A549 and NCI-H1299 cells detected by flow cytometry. (C) Western blot analysis of phosphorylated EGFR, EGFR, Ki67, Fas, FasL and Cleaved-caspase 3 proteins in A549 and NCI-H1299 cells. *p < 0.05 vs. the control cells (0 μM of gallic acid). The above results were all measurement data and expressed as mean ± standard deviation. Comparisons among multiple groups were analyzed using one-way ANOVA, followed by a Tukey’s multiple comparisons post hoc test. The cell experiment was independently repeated three times.

Figure 2

The binding of CARM1 and PELP1 was enhanced by EGFR activation. (A) The binding of CARM1 to PELP1 detected by Co-IP assay. (B) Western blot analysis of EGFR protein in EGF-treated A549 cells. (C) The binding of CARM1 to PELP1 in EGF-treated A549 cells detected by Co-IP assay. *p < 0.05 vs. cells treated with DMSO. The above results were all measurement data and expressed as mean ± standard deviation. Comparisons between two groups were analyzed using the unpaired t-test. Comparisons among multiple groups were analyzed using one-way ANOVA, followed by a Tukey’s multiple comparisons post hoc test. The cell experiment was independently repeated three times.

Figure 3

CARM1 positively regulates the carcinogenic activity of PELP1. (A) Cell viability of A549 and NCI-H1299 cells detected by MTT assay. *p < 0.05 vs. the control cells (0 μM of gallic acid). (B) Colony formation abilities of A549 and NCI-H1299 cells assessed by clonogenic assay. *p < 0.05 vs. cells treated by oe-NC plasmid and DMSO, ^#p < 0.05 vs. cells treated by oe-PELP1 plasmid and DMSO. (C) Migration ability of A549 and NCI-H1299 cells assessed by QCM Colorimetric Cell Migration Assay. *p < 0.05 vs. cells treated by oe-NC plasmid and DMSO, ^#p < 0.05 vs. cells treated by oe-PELP1 plasmid and DMSO. (D) Western blot analysis of phosphorylated EGFR, EGFR, Ki67, Fas, FasL and Cleaved-caspase 3 proteins in A549 and NCI-H1299 cells. *p < 0.05 vs. cells treated by oe-NC plasmid and DMSO, ^#p < 0.05 vs. cells treated by oe-PELP1 plasmid and DMSO. The above results were all measurement data and expressed as mean ± standard deviation. Comparisons among multiple groups were analyzed using one-way ANOVA, followed by a Tukey’s multiple comparisons post hoc test. The cell experiment was independently repeated three times.

Figure 4

Higher dosage of Gallic acid resulted in weaker binding of CARM1 to PELP1. The binding of CARM1 to PELP1 was detected in the A549 and NCI-H1299 cells treated with Gallic acid at different dosages by Co-IP experiment.

Figure 5

The growth of NSCLC xenografts was suppressed by gallic acid. (A and C) Representative images of xenograft tumors and quantitative analysis of body weight and tumor weight after treatment of 10 μg/kg, 20 μg/kg, and 40 μg/kg of gallic acid. (B) Volume data of the transplanted tumors at different time points after treatment with 10 μg/kg, 20 μg/kg, and 40 μg/kg of gallic acid. (D) Western blot analysis of phosphorylated EGFR, EGFR, Ki67, Fas, FasL and Cleaved caspase 3 proteins in transplanted tumors. (E) The binding of CARM1 to PELP1 in nude mice treated with 10 μg/kg, 20 μg/kg, and 40 μg/kg of gallic acid detected by Co-IP assay. The above results were all measurement data and expressed as mean ± standard deviation. Data comparison in panel B was performed by repeated measurement ANOVA, followed by Bonferroni’s post hoc test. Data in panel C and D were analyzed using one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test. *p < 0.05 vs. the control mice (without treatment of gallic acid). Comparisons between two groups were analyzed using an unpaired t-test, comparisons among multiple groups were analyzed using one-way ANOVA, followed by a Tukey’s multiple comparisons post hoc test. Comparisons at different time points were performed by repeated-measures ANOVA, followed by Bonferroni’s post hoc test, n = 10.

Figure 6

A mechanism map depicting the role of gallic acid in the progression of NSCLC via EGFR-CARM1-PELP1 axis. Gallic acid disrupts the activation of EGFR to inhibit the binding of CARM1 to PELP1, whereby inhibiting the carcinogenic activity of PELP1 and repressing the development of NSCLC.