Combination of rapamycin and SAHA enhanced radiosensitization by inducing autophagy and acetylation in NSCLC

Abstract

Radiotherapy plays an essential role in the treatment of non-small-cell lung cancer (NSCLC). However, cancer cells' resistance to ionizing radiation (IR) is the primary reason for radiotherapy failure leading to tumor relapse and metastasis. DNA double-strand breaks (DSB) repair after IR is the primary mechanism of radiotherapy resistance. In this study, we investigated the effects of autophagy-inducing agent, Rapamycin (RAPA), combined with the histone deacetylase inhibitor (HDACi), Suberoylanilide Hydroxamic Acid (SAHA), on the radiosensitivity of A549 and SK-MES-1 cells, and examined the combination effects on DNA damage repair, and determined the level of autophagy and acetylation in A549 cells. We also investigated the combination treatment effect on the growth of A549 xenografts after radiotherapy, and the level of DNA damage, autophagy, and acetylation. Our results showed that RAPA combined with SAHA significantly increased the inhibitory effect of radiotherapy compared with the single treatment group. The combined treatment increased the expression of DNA damage protein γ-H2AX and decreased DNA damage repair protein expression. RAPA combined with SAHA was induced mainly by regulating acetylation levels and autophagy. The effect of combined treatment to increase radiotherapy sensitivity will be weakened by inhibiting the level of autophagy. Besides, the combined treatment also showed a significantly inhibited tumor growth in the A549 xenograft model. In conclusion, these results identify a potential therapeutic strategy of RAPA combined with SAHA as a radiosensitizer to decreased DSB repair and enhanced DNA damage by inducing acetylation levels and autophagy for NSCLC.

Keywords: DNA damage; autophagy; histone deacetylase inhibitor; radiation; rapamycin.

Figure 1

Cytotoxic effects of combination treatment with RAPA or/and SAHA in NSCLC cells. Cell viabilities were assessed by the cell counting kit-8 (CCK-8). (A, B) Concentration-dependent effects of RAPA (A) and SAHA (B) on the viability of two NSCLC cells (A549, SK-MES-1) for 24h. *p<0.05, RAPA or SAHA versus control. (C, D) Time-dependent effects of RAPA (100nmol/L) or/and SAHA (2.5μmol/L) on the viability of A549 (C) and SK-MES-1 (D) cells. *p<0.05, combined treatment versus single treatment, ^#p<0.05, 48h or 72h versus 24h.

Figure 2

Cytotoxic effects of combination treatment with RAPA or/and SAHA in irradiated NSCLC cells. Survival fractions were assessed by the colony formation assay. (A, B) The radiation dose-response survival curves after 24h of treatment with RAPA (100nmol/L) or/and SAHA (2.5μmol/L) in two NSCLC cells A549 (A), SK-MES-1 (B). (C, D) Colony formation assay (C) and flow cytometry analysis of the cell cycle (D) in two NSCLC cells (A549, SK-MES-1) resulting from IR (4Gy) after treatment with RAPA (100nmol/L) or/and SAHA (2.5μmol/L) for 24h.

Figure 3

Effects of combination treatment with RAPA and SAHA on DNA damage and repair after IR in NSCLC cells. (A, B) the protein level of γ-H2AX was determined by western blot analysis. Two NSCLC cells (A549, SK-MES-1) were treated with RAPA (100nmol/L) or/and SAHA (2.5μmol/L) for 24h and were subsequently exposed to IR (4Gy), the γ-H2AX protein was tested at 1h and 24h after IR. *p<0.05, 1h after IR versus control; ^#p<0.05, 24h versus 1h after IR; ⁺p<0.05, combined treatment versus single treatment 24h after IR. (C–F) the protein (C) and mRNA level of Rad51 (D), Ku80 (E), Ku70 (F) were determined by western blot and RT-qPCR analysis. Two NSCLC cells (A549, SK-MES-1) were treated with RAPA (100nmol/L) or/and SAHA (2.5μmol/L) for 24h and were subsequently exposed to IR (4Gy) and were tested after 4h. *p<0.05, compared with control, ^#p<0.05, compared with IR.

Figure 4

Effects of combination treatment with RAPA and SAHA on autophagy and acetylation in NSCLC cells. (A–C) RAPA, SAHA, and IR induce cellular autophagy. A549 cells were treated with RAPA (100nmol/L) or/and SAHA (2.5μmol/L) for 24h or were exposed to IR (4Gy) and were tested after 4h. (A) The ultrastructures of autophagosomes in A549 cells were observed under a transmission electron microscope (TEM). Black arrows and rectangles indicate intracellular autophagosomes. (B) The distribution of LC3 dots in A549 cells was observed by using an immunofluorescence confocal microscope. Quantitative data were calculating the number of LC3 dots per cell. *p<0.05, compared with control, ^#p<0.05, compared with combined treatment. (C) The level of autophagy-related protein (LC3 I/II, p62, Atg5) was determined by western blot analysis. (D) SAHA induces acetylation of A549 cells. A549 cells were treated with SAHA (2.5μmol/L) and/or RAPA (100nmol/L) for 24h. The level of acetylation of histone H3 was determined by western blot analysis.

Figure 5

Effects of combination treatment with RAPA and SAHA on IR after down-regulate the level of autophagy in NSCLC cells. A549 cells were infected by a lentivirus delivered Atg5 shRNA for 24h. (A, B) The relative protein and mRNA expression levels of Atg5 were determined by western blot and RT-qPCR analysis. (C) Quantitative data were calculating the number of LC3 dots per A549 cell by using an immunofluorescence confocal microscope. (D) Survival fractions of A459 cells were assessed by the colony formation assay. (E) γ-H2AX protein was determined by western blot analysis. A549 cells were treated with RAPA (100nmol/L) and SAHA (2.5μmol/L) for 24h and were subsequently exposed to IR (4Gy) for 1h and 24h after transfected with or without Atg5 shRNA for 24h. (F) Colony formation assay in A549 cells resulting from IR after treatment with different concentrations of 3-MA or without 3-MA. A549 cells were pretreated with 3-MA for 1h before RAPA and SAHA treatment.

Figure 6

Effects of combination treatment with RAPA and SAHA on A549 xenografts model after receiving IR. (A) Bodyweight in A549 cell xenografts measured every 4 days. (B, C) Specimens and tumor weight of A549 cell xenograft after different treatments for 28 days. (D) Tumor volume of A549 cell xenograft in nude mice measured every 4 days. (E) Immunohistochemical staining of γ-H2AX, LC3 and Ac-H3 in A549 cell xenografts (×200).