Isothiocyanates induce oxidative stress and suppress the metastasis potential of human non-small cell lung cancer cells

Abstract

Background: Isothiocyanates are natural compounds found in consumable cruciferous vegetables. They have been shown to inhibit chemical carcinogenesis by a wide variety of chemical carcinogens in animal models. Recent studies have also shown that isothiocyanates have antitumor activity, inhibiting the growth of several types of cultured human cancer cells. Our previous study showed that PEITC inhibited human leukemia cells growth by inducing apoptosis. However, the effect of isothiocyanates on lung cancer cell metastasis has not been studied. In the present study, we investigated the inhibitory effects of BITC and PEITC on metastatic potential of highly metastatic human lung cancer L9981 cells.

Methods: Cell migration and invasion were measured by wound healing assay and transwell chemotaxis assay. Expression of metastasis-related genes was assessed by quantitative RT-PCR and Western blotting. The mechanisms of action were evaluated by flow cytometry, reporter assay and Western blotting.

Results: Our data showed that both BITC and PEITC inhibited L9981 cell growth in a dose-dependent manner, the IC50 values were 5.0 and 9.7 microM, respectively. Cell migrations were reduced to 8.1% and 16.5% of control, respectively; and cell invasions were reduced to 2.7% and 7.3% of control, respectively. Metastasis-related genes MMP-2, Twist and beta-catenin were also modulated. BITC and PEITC inhibited cell survival signaling molecules Akt and NFkappaB activation. Moreover, BITC and PEITC increased ROS generation and caused GSH depletion. Pretreatment with NAC blocked BITC and PEITC induced ROS elevation and NFkappaB inhibition.

Conclusion: Our results indicated that BITC and PEITC suppress lung cancer cell metastasis potential by modulation of metastasis-related gene expression, inhibition of Akt/NFkappaB pathway. Induction of oxidative stress may play an important role.

Figure 1

Structures of BITC and PEITC.

Figure 2

Effects of BITC and PEITC on growth of highly and low metastatic cells. L9981 and NL9980 cells were treated with 1 - 40 μM of BITC or PEITC for 48 h, and were collected and counted by a Vi-CELL Cell Viability Analyzer. Values represent the mean ± SD from three independent measurements.

Figure 3

Effect of BITC and PEITC on L9981 cells migration. Wound healing assays were performed to assess cell migration. Cells were treated or untreated with 5 μM of BITC or 10 μM of PEITC for 24 and 30 h. Representative photographs of treated and untreated cells are presented (×40 magnification).

Figure 4

Effect of BITC and PEITC on L9981 cells migration. Wound healing assays were performed to assess cell migration. Cells were treated or untreated with 5 μM of BITC or 10 μM of PEITC for 24 and 30 h. Number of cells migrated at 24 and 30 h time point are presented. Values represent the mean ± SD of three independent experiments (*** P < 0.001).

Figure 5

Effect of BITC and PEITC on L9981 cells invasion. Invasion Chamber Assays were performed to assess the effect on cell invasion. Cells were treated or untreated with 5 μM of BITC or 10 μM of PEITC for 24 h. (A) Representative photographs of treated and untreated cells are presented (×200 magnification). (B) Number of cells invaded at 24 h time point. Values represent the mean ± SD of three independent experiments (*** P < 0.001).

Figure 6

Effect of BITC and PEITC on metastasis-related gene expression. (A) L9981 cells were treated with 5 μM of BITC or 10 μM of PEITC for 4 h. MMP-2, Twist and β-catenin mRNA expressions were detected by real-time PCR. (B) L9981 cells were treated with 5 - 20 μM of BITC or PEITC for 24 h. MMP-2 and Twist protein expression were detected by Western blotting analyses (* P < 0.05, ** P < 0.01, *** P < 0.001). Similar results were obtained in three independent experiments.

Figure 7

Effect of BITC and PEITC on ROS generation. L9981 cells were treated with 5 μM of BITC or 10 μM of PEITC for 4 h, then reacted with DCFH-DA. ROS levels were detected by flow cytometry. For NAC protection, cells were pretreated with NAC (1 mM) for 1 h.

Figure 8

Effect of BITC and PEITC on intracellular GSH levels. L9981 cells were treated with 5 or 10 μM of BITC or PEITC for 3 - 24 h, intracellular total GSH levels were detected at indicated time points by microplate reader. Values represent the mean ± SD of three independent experiments.

Figure 9

Effect of BITC and PEITC on Akt activation. L9981 cells were treated with 5 - 20 μM of BITC or PEITC for 24 h. Cell lysates were prepared, p-Akt and total Akt were detected by Western blotting analyses. Similar results were obtained in three independent experiments.

Figure 10

Effect of BITC and PEITC on NFκB transcriptional activation. L9981 cells were transfected with pNFκB-luc, and treated with 5 - 20 μM of BITC or PEITC for 18 h. NFκB activation was detected by luciferase reporter assay. For NAC protection, cells were pretreated with NAC (1 mM) for 1 h. Values represent the mean ± SD of three independent experiments (* P < 0.05, ** P < 0.01, *** P < 0.001).