Icotinib hydrochloride enhances chemo- and radiosensitivity by inhibiting EGFR signaling and attenuating RAD51 expression and function in Hela S3 cells

Figures & data

Figure 1 Icotinib hydrochloride enhances cisplatin- or radiation-induced proliferation inhibition in Hela S3 cells.

Notes: (A and B) Viability of Hela S3 cells was measured by CCK-8 assay 24 hours after various doses of irradiation (A) or cisplatin (B) treatment following preincubation with different concentrations of IH for 2 hours. (C and D) Cell proliferation of Hela S3 cells in the following 72 hours after being treated with 5 μM IH plus 6 Gy radiation (C) or 2 μg/mL cisplatin (D). Error bars, SD. *P<0.05, **P<0.01, and ***P<0.001 by two-tailed Student’s t-test; n=3 independent cell cultures.
Abbreviations: Ctrl, control; IH, icotinib hydrochloride; IR, ionizing radiation; Cis, cisplatin; CCK-8, Cell Counting Kit-8; h, hours.

Figure 2 Effect of icotinib hydrochloride on irradiation- or cisplatin-induced cell cycle arrest.

Notes: (A and B) Cell cycle distribution was determined by flow cytometry in Hela S3 cells following different treatments. (C and D) Histogram plots show the percentage of cells distributing in S, G1 and G2 phases, respectively. Error bars, SD.
Abbreviations: Ctrl, control; IH, icotinib hydrochloride; IR, ionizing radiation; Cis, cisplatin.

Figure 3 Effect of icotinib hydrochloride on cell apoptosis following radiation or cisplatin treatment.

Notes: (A and B) Cell apoptosis was determined by flow cytometry in Hela S3 cells following different treatments. (C and D) The apoptotic rate is represented as the percentage of Annexin V-FITC-positive cells. Error bars, SD. *P<0.05, **P<0.01, and ***P<0.001 by two-tailed Student’s t-test; n=3 independent cell cultures.
Abbreviations: Ctrl, control; IH, icotinib hydrochloride; IR, ionizing radiation; Cis, cisplatin.

Figure 4 Icotinib hydrochloride inhibits EGFR signaling pathway in Hela S3 cells.

Notes: Total EGFR, p-EGFR, total AKT, p-AKT, total ERK and p-ERK were detected using Western blot assays in Hela S3 cells 2 hours after combination treatment of radiation (A) or cisplatin (B) with IH (top). Histograms indicate the relative protein expression levels in the cells by grayscale analysis (bottom). Error bars, SD. *P<0.05, **P<0.01, and ***P<0.001 by two-tailed Student’s t-test; n=3 independent cell cultures.
Abbreviations: Ctrl, control; IH, icotinib hydrochloride; IR, ionizing radiation; Cis, cisplatin.

Figure 5 Radiation and chemotherapy sensitization of icotinib hydrochloride by delaying DNA damage repair progress.

Notes: (A) Survival of Hela S3 cells after exposure to different treatments was measured by colony formation assay. (B and C) Immunofluorescence stain detecting different treatment-induced γ-H2AX foci in Hela S3 cells. (D) Quantification of the number of γ-H2AX foci-positive cells after treatment. Error bars, SD. **P<0.01 by two-tailed Student’s t-test; n=3 independent cell cultures.
Abbreviations: DMSO, dimethyl sulfoxide; IH, icotinib hydrochloride; IR, ionizing radiation; Cis, cisplatin.

Figure 6 Icotinib hydrochloride attenuates chemo- or radiotherapy-induced HR protein RAD51 upregulation and nuclear foci formation.

Notes: RAD51 mRNA levels at indicated time points after exposure to irradiation (A) or cisplatin (B) were detected using qRT-PCR. (C and D) RAD51 protein levels were examined by Western blotting at indicated time points post radiation (C) or cisplatin (D) treatment (top). Histograms indicate the relative protein expression levels in the cells by grayscale analysis (bottom). (E and F) Immunofluorescence stain detecting different treatment-induced RAD51 foci in Hela S3 cells. (G) Quantification of the number of RAD51 foci-positive cells after treatment.(H) RAD51 protein levels in nucleus and cytoplasm were determined separately using Western blot assays (top). Histograms indicate the relative RAD51 expression levels in the nucleus and cytoplasm by grayscale analysis (bottom). Error bars, SD. *P<0.05, **P<0.01, and ***P<0.001 by two-tailed Student’s t-test; n=3 independent cell cultures.
Abbreviations: DMSO, dimethyl sulfoxide; qRT-PCR, quantitative reverse transcription polymerase chain reaction; HR, homologous recombination; h, hours; min, minutes; ns, non-significant; Ctrl, control; IH, icotinib hydrochloride; IR, ionizing radiation; Cis, cisplatin; C, cytoplasmic fraction; N, nuclear fraction.

Figure S1 Chemo- and radiotherapy sensitization effects and mechanism of icotinib hydrochloride against SiHa cells.

Notes: (A) Proliferation of SiHa cells in the following 72 hours after being treated with 5 μM IH plus 6 Gy radiation or 2 μg/mL cisplatin. (B) Results from flow cytometry showing cell cycle distribution of SiHa cells following different treatments. (C) The apoptotic rate represented as the percentage of Annexin V-FITC-positive cells. (D) Colony formation assay showing survival of SiHa cells after exposure to different treatments. (E and F) Immunofluorescence stain detecting IH plus radiation (E) or cisplatin (F) induced γ-H2AX foci in SiHa cells. (G) Quantification of the number of γ-H2AX foci-positive cells after treatment. (H and I) Immunofluorescence stain detecting different treatment-induced RAD51 foci in SiHa cells. (J) Quantification of the number of RAD51 foci-positive cells after treatment. (K) RAD51 protein levels were examined with Western blotting 24 hours post radiation or cisplatin treatment (top). Histograms show the relative protein expression levels in the cells by grayscale analysis (bottom). (L) RAD51 protein levels in nucleus and cytoplasm were determined separately using Western blot assays 24 hours after different treatments (top). Histograms indicate the relative RAD51 expression levels in the nucleus and cytoplasm by grayscale analysis (bottom). Error bars, SD. *P<0.05 and **P<0.01 by two-tailed Student’s t-test; n=3 independent cell cultures.
Abbreviations: DMSO, dimethyl sulfoxide; Ctrl, control; IH, icotinib hydrochloride; IR, ionizing radiation; Cis, cisplatin; C, cytoplasmic fraction; N, nuclear fraction; h, hours.

Figure S2 Comparison of EGFR expression levels between Hela S3 and SiHa cells Notes: Results from qRT-PCR (A) and Western blotting (B) assays showing EGFR mRNA levels (A) and protein levels (B) in Hela S3 and SiHa cells. Error bars, SD. **P<0.01 by two-tailed Student’s t-test. All experiments were performed in triplicate.

Abbreviation: qRT-PCR, quantitative reverse transcription polymerase chain reaction.

Figure S3 Effects of IH on irradiation-induced nuclear translocation of EGFR and phosphorylation of DNA-PKcs and ATM.

Notes: Nuclear expressions of EGFR (A) and total DNA-PKcs, p-DNA-PKcs, total ATM, and p-ATM (B) in Hela S3 cells were examined using Western blotting 20 minutes after radiation with or without IH pretreatment. All experiments were performed in triplicate.
Abbreviations: IH, icotinib hydrochloride; IR, ionizing radiation; C, cytoplasmic fraction; N, nuclear fraction.