Allyl isothiocyanate induces replication-associated DNA damage response in NSCLC cells and sensitizes to ionizing radiation

Abstract

Allyl isothiocyanate (AITC), a constituent of many cruciferous vegetables exhibits significant anticancer activities in many cancer models. Our studies provide novel insights into AITC-induced anticancer mechanisms in human A549 and H1299 non-small cell lung cancer (NSCLC) cells. AITC exposure induced replication stress in NSCLC cells as evidenced by γH2AX and FANCD2 foci, ATM/ATR-mediated checkpoint responses and S and G2/M cell cycle arrest. Furthermore, AITC-induced FANCD2 foci displayed co-localization with BrdU foci, indicating stalled or collapsed replication forks in these cells. Although PITC (phenyl isothiocyanate) exhibited concentration-dependent cytotoxic effects, treatment was less effective compared to AITC. Previously, agents that induce cell cycle arrest in S and G2/M phases were shown to sensitize tumor cells to radiation. Similar to these observations, combination therapy involving AITC followed by radiation treatment exhibited increased DDR and cell killing in NSCLC cells compared to single agent treatment. Combination index (CI) analysis revealed synergistic effects at multiple doses of AITC and radiation, resulting in CI values of less than 0.7 at Fa of 0.5 (50% reduction in survival). Collectively, these studies identify an important anticancer mechanism displayed by AITC, and suggest that the combination of AITC and radiation could be an effective therapy for NSCLC.

Keywords: DNA damage response; dietary isothiocyanates; non-small cell lung cancer; radiosensitization; replication stress.

Figure 1. AITC and PITC exhibit cytotoxic effects to NSCLC cells

Clonogenic survival assays show AITC and PITC inhibits survival of A549 (A) and H1299 (B) cells in a dose dependent manner. Cytotoxic effects of AITC and PITC are specific to NSCLC cells at the concentrations of 5 and 10 μM (C and D respectively). Cells were exposed to the indicated concentrations of ITCs for three days and cell viability was assessed using Tryphan blue exclusion assay. Data presented are an average of triplicates and ± SD presented as error bars (*P < 0.01, **P < 0.001).

Figure 2. AITC-induces slow progression through S-phase and leads to G2/M arrest

H1299 cells were exposed to 20 μM AITC or PITC for 6 (top panel) and 24 hours (bottom panel) and cell cycle profiles were assessed by flow cytometry (A). Data presented in (B) and (C) are average values from three independent experiments for 6 and 24 hours respectively. The error bars presents ± SD.

Figure 3. AITC-induces replication-stress mediated DDR in NSCLC cells

H1299 cells were treated with 20 μM AITC or DMSO for six hours and assessed for formation of γH2AX (A) and FANCD2 foci (B). Number of cells positive for γH2AX foci (C) and FANCD2 foci (D) were presented in histograms. To assess the co-localization of FANCD2 foci with replication forks, cells were labeled with BrdU for 20 min. At least individual 10 fields were counted and ± SD presented as error bars (**P < 0.001).

Figure 4. AITC exposure induces replication associated DNA damage and activates cell cycle checkpoints in A549 cells

Exponentially growing A549 cells (A) were exposed to 20 μM AITC or PITC and cell lysates were prepared after indicated times. The normalized proteins were resolved on SDS-PAGE and blotted for different DDR proteins. Quantitation of p-ATM (B) and pChk1 (C) proteins are shown as bar diagram. Data presented are an average values from three independent experiments and ± SD presented as error bars.

Figure 5. AITC exposure induces replication associated DNA damage and activates cell cycle checkpoints in H1299 cells

Exponentially growing H1299 cells were exposed to either 20 μM AITC or 20 μM PITC and cell lysates were prepared after 6 and 24 hours of drug treatment. The normalized proteins were resolved on SDS-PAGE and blotted for different DDR proteins (A). Quantitation of p-ATM (B) and pChk1 (C) proteins are shown as bar diagram. Data presented are an average of three independent experiments and ± SD presented as error bars.

Figure 6. AITC is a potent inhibitor of NSCLC cells cell migration

A549 cells were used for scratch assay and cell migration was measured for 24 hours after exposed to 10 μM AITC or PITC for 24 hours. Images of the scratch assays are shown before and after treatment with the ITCs treatments (A) The percent of cell migration quantified from three independent experiments. The average values were presented in the histogram and the error bars indicates ± SD (B).

Figure 7. AITC-induces apoptosis in NSCLC cells

A549 (top panel) and H1299 (bottom panel) cells were exposed to AITC for 24 or 48 hours and cells were co-stained with PI and Annexin V antibody and analyzed by flow cytometry. The data presented in (B) and (C) are the average values of three independent experiments for 24 and 48 hours respectively. The error bars represents ± SD (**P < 0.001).

Figure 8. AITC pretreatment sensitizes NSCLC cells to radiation treatment

Clonogenic survival assays were performed after pretreating A549 and H1299 cells with 5 μM AITC and exposed them to different doses of ionizing radiation. After 10 days colonies were counted and plotted as percent survival fraction for A549 (A) and H1299 cells (B). Pretreatment with AITC enhances radiation induced DDR in A549 cells (C). The data presented in (A) and (B) are mean of three independent experiments and the error bars indicates ± SD

Figure 9. AITC and radiation combination therapy exhibits synergistic therapeutic activity in NSCLC cells

The Isobologram curve showing the synergistic effect of AITC with Radiation in A549 cell line (A) and H1299 cell lines (B) at different effective doses (ED). The combination index plots for AITC and radiation combination therapy shows synergy all the Fractions affected (Fa) for both A549 (C) and H1299 (D) cells. Considering CI > 1, antagonistic; CI = 1, additive and CI < 1, synergistic.