Prolonged sulforaphane treatment does not enhance tumorigenesis in oncogenic K-ras and xenograft mouse models of lung cancer

Abstract

Background: Sulforaphane (SFN), an activator of nuclear factor erythroid-2 related factor 2 (Nrf2), is a promising chemopreventive agent which is undergoing clinical trial for several diseases. Studies have indicated that there is gain of Nrf2 function in lung cancer and other solid tumors because of mutations in the inhibitor Kelch-like ECH-associated protein 1 (Keap1). More recently, several oncogenes have been shown to activate Nrf2 signaling as the main prosurvival pathway mediating ROS detoxification, senescence evasion, and neoplastic transformation. Thus, it is important to determine if there is any risk of enhanced lung tumorigenesis associated with prolonged administration of SFN using mouse models of cancer.

Materials and methods: We evaluated the effect of prolonged SFN treatment on oncogenic K-ras (K-ras(LSL-G12D))-driven lung tumorigenesis. One week post mutant-K-ras expression, mice were treated with SFN (0.5 mg, 5 d/wk) for 3 months by means of a nebulizer. Fourteen weeks after mutant K-ras expression (K-ras(LSL-G12D)), mice were sacrificed, and lung sections were screened for neoplastic foci. Expression of Nrf2-dependent genes was measured using real time RT-PCR. We also determined the effect of prolonged SFN treatment on the growth of preclinical xenograft models using human A549 (with mutant K-ras and Keap1 allele) and H1975 [with mutant epidermal growth factor receptor (EGFR) allele] nonsmall cell lung cancer cells.

Results: Systemic SFN administration did not promote the growth of K-ras(LSL-G12D)-induced lung tumors and had no significant effect on the growth of A549 and H1975 established tumor xenografts in nude mice. Interestingly, localized delivery of SFN significantly attenuated the growth of A549 tumors in nude mice, suggesting an Nrf2-independent antitumorigenic activity of SFN.

Conclusions: Our results demonstrate that prolonged SFN treatment does not promote lung tumorigenesis in various mouse models of lung cancer.

Keywords: EGFR; K-ras; Keap1; Nrf2; lung cancer; sulforaphane.

Figure 1

SFN does not promote oncogenic K-ras-driven lung tumorigenesis. (a) Schematic of the experimental design. (b) Bar graph showing relative number of neoplastic lesions (hyperplasia, adenoma, adenocarcinoma) and percentage of the area of the lung covered by the tumor cells. (c) Real-time RT-PCR data showing induction of Nrf2-dependent gene expression in the lungs of the SFN-treated cohort of mice. P-values are calculated using t test

Figure 2

Systemic delivery of SFN does not promote growth of Keap1 and K-ras mutant, A549, lung cancer xenografts. (a) Schematic of the experiment. (b) Graph showing relative growth of A549 cells in nude mice. Tumor volume is not significantly different between the three cohorts of mice (ANOVA). (c) Tumor weights are not significantly different between the three groups. (d) Total body weights are not significantly different between the three groups. (e) Real-time RT-PCR showing expression of the Nrf2-dependent gene, NQO1, in the liver and tumor tissues.*P < 0.05 (t test).

Figure 3

Systemic delivery of SFN does not promote growth of EGFR mutant (H1975) lung cancer xenografts. (a) Schematic of the experiment. (b) Graph showing relative growth of H1975 tumors. Tumor volume is not significantly different between the two cohorts of mice (t test). (c) Tumor weights (day 30) are not significantly different between the two groups (t test). (d) Total body weights are not significantly different between the two groups. (e) Real-time PCR showing expression of the Nrf2-dependent gene, NQO1, in the liver and tumor tissues.*P < 0.05 (t test)

Figure 4

Localized delivery of SFN inhibits the growth of A549 lung cancer xenografts. (a) Schematic of the experiment. (b) Graph showing relative growth of A549 tumors treated with 1000 nmol of SFN. *P < 0.05 (t test). (c) Tumor weights were significantly lower in the SFN-treated group. *P < 0.05 (t test). (d) Body weight did not change significantly in response to SFN treatment. (e–g) Relative tumor growth, average tumor weight, and body weight in the cohort of mice treated with 500 nmol of SFN. (h–j) Relative expression of Nrf2-dependent genes in the liver and tumor tissues. *P < 0.05.