Resveratrol enhances ionizing radiation-induced premature senescence in lung cancer cells

Abstract

Radiotherapy is used in >50% of patients during the course of cancer treatment both as a curative modality and for palliation. However, radioresistance is a major obstacle to the success of radiation therapy and contributes significantly to tumor recurrence and treatment failure, highlighting the need for the development of novel radiosensitizers that can be used to overcome tumor radioresistance and, thus, improve the efficacy of radiotherapy. Previous studies indicated that resveratrol (RV) may sensitize tumor cells to chemotherapy and ionizing radiation (IR). However, the mechanisms by which RV increases the radiation sensitivity of cancer cells have not been well characterized. Here, we show that RV treatment enhances IR-induced cell killing in non-small cell lung cancer (NSCLC) cells through an apoptosis-independent mechanism. Further studies revealed that the percentage of senescence-associated β-galactosidase (SA-β-gal)-positive senescent cells was markedly higher in cells treated with IR in combination with RV compared with cells treated either with IR or RV alone, suggesting that RV treatment enhances IR-induced premature senescence in lung cancer cells. Comet assays demonstrate that RV and IR combined treatment causes more DNA double-strand breaks (DSBs) than IR or RV treatment alone. DCF-DA staining and flow cytometric analyses demonstrate that RV and IR combined treatment leads to a significant increase in ROS production in irradiated NSCLC cells. Furthermore, our investigation show that inhibition of ROS production by N-acetyl-cysteine attenuates RV-induced radiosensitization in lung cancer cells. Collectively, these results demonstrate that RV-induced radiosensitization is associated with significant increase of ROS production, DNA-DSBs and senescence induction in irradiated NSCLC cells, suggesting that RV treatment may sensitize lung cancer cells to radiotherapy via enhancing IR-induced premature senescence.

Figure 1.

RV sensitizes lung cancer cells to IR-induced tumor cell killing. (A) Representative images of clonogenic assays showing that IR inhibits the colony-forming capacity of cancer cells in a dose-dependent manner and that RV enhances the tumor suppressive effect of IR. (B) Clonogenic assays indicate that RV sensitizes A549 to IR-induced cell killing. (C) Clonogenic assays show that RV sensitizes H460 to IR-induced cell killing. (D) Western blot analyses were performed to determine the expression levels of cleaved caspase-3 and cleaved PARP in H460 cells. Actin was probed as a loading control. ^*p<0.05 vs. DMSO control; ^**p<0.01 vs. DMSO control.

Figure 2.

Treatment with RV increases IR-induced premature senescence in NSCLC cells. (A) SA-β-gal staining assays were performed to determine senescent cells in irradiated lung cancer cells. Representative microimages of SA-β-gal staining in H460 cells are presented. (B) SA-β-gal assays show that RV pretreatment increases IR-induced premature senescence in A549 cells. (C) SA-β-gal assays show that RV pretreatment augments IR-induced premature senescence in H460 cells. ^ap<0.01 vs. control; ^bp<0.01 vs. IR; ^cp<0.05 vs. control.

Figure 3.

Treatment with RV enhances IR-induced DNA damage in NSCLC cells. (A) Representative photomicrographs of comet assays showing that IR-induced DNA-DSBs in A549 (upper panel) and H460 cells (lower panel), respectively. (B) The percentage of tail DNA in A549 cells with different treatments was quantified. (C) The percentage of tail DNA in H460 cells with different treatments was quantified. ^ap<0.001 vs. control; ^bp<0.05 vs. IR.

Figure 4.

RV inhibits the phosphorylation of Akt and increases the accumulation of p-p53 and p-chk2. (A) Western blot analyses were performed to determine the expression levels of p-Akt, p-mTOR and p-S6K in H460 cell. (B) Western blot analyses were performed to determine the expression levels of DNA damage response protein p-chk2 and p-p53 in H460 cells. Actin was probed as a loading control.

Figure 5.

Effect of RV treatment on IR-induced ROS generation in lung cancer cells. (A) The levels of ROS were measured by DCF-DA staining and flow cytometric analyses at 24 h after 5 Gy of irradiation. Shown is a representative analysis of ROS production in lung cancer cells by flow cytometry. (B) The relative levels of ROS in A549 cells are presented as fold change compared to control cells. (C) The relative levels of ROS in H460 cells are shown as fold change compared to control cells. ^ap<0.01 vs. control; ^bp<0.05 vs. control; ^cp<0.05 vs. IR.

Figure 6.

Treatment with NAC attenuates RV-induced radiosensitization in lung cancer cells. (A) H460 cells were pretreated with NAC prior to IR exposures. Two hours after IR, neutral comet assays were performed to assess DNA-DSBs in lung cancer cells. (B) SA-β-gal staining was employed to determine senescence in H460 cells. ^ap<0.05 vs. control; ^bp<0.01 vs control; ^cp<0.01 vs IR.