Since the early 1980s, naturally occurring antioxidants and vitamins have been investigated and promoted as chemopreventive agents. Antioxidants have been popular as chemopreventive agents because there is strong scientific evidence supporting reduced incidence of epithelial cancers associated with high dietary intake of fruits and vegetables. Because of this evidence, major clinical trials were launched to test β-carotene, alone or in combination with vitamin E or vitamin A.[41
] The largest trials for chemoprevention of lung cancers in high-risk smoker populations were ATBC, the alpha tocopherol (vitamin E), beta-carotene trial in Finland, and CARET, the beta-carotene and retinol efficacy trial smokers in the United States.[42
] The results were devastatingly similar, with excess lung cancer incidence and mortality rates in the active treatment arm, compared with those in the placebo arm of each trial.[41
] The unexpected and unwanted outcomes from the CARET and ATBC trials prompted us to reconsider issues related to both the effectiveness and the safety of micronutrient supplementation and careful evaluation of chemopreventive agents in preclinical cancer models.
SFN derived from broccoli sprouts is currently under clinical evaluation in diseases in which oxidative stress and inflammation play important roles in disease pathogenesis, namely, pulmonary diseases, such as chronic obstructive pulmonary disease, asthma, and cystic fibrosis, and cardiovascular disease, and protection against radiation dermatitis. In addition to these, SFN is being evaluated as a potential chemopreventive agent in melanoma and breast cancer and as an anticancer agent in patients with recurrent prostate cancer. At present, there are 17 clinical trials listed at www.clinicaltrials.gov that are using SFN or an enriched broccoli sprout preparation for treatment of a variety of diseases. The molecular targets of SFN vary depending on cancer stage and target tissue. Although SFN is known to modulate several signaling pathways, it is a potent inducer of Nrf2-Keap1 signaling, and the anticarcinogenic and anti-inflammatory activities of SFN are mediated primarily by Nrf2-dependent induction of phase-II detoxification and antioxidant enzymes.[13
Results from recent studies suggest that SFN offers protection against tumor development and progression during the post-initiation phase by modulating diverse cellular processes that inhibit the growth of transformed cells.[14
] The ability of SFN to induce reactive oxygen species production, apoptosis, and cell cycle arrest is associated with regulation of many molecules, including the BCL-2 family of proteins, caspases, p21, cyclins, cyclin-dependent kinases, and histone deacetylases and nuclear factor-kappa-B pathways.[14
] SFN also suppresses angiogenesis and metastasis by downregulating hypoxia-inducible factor 1 alpha, matrix metallopeptidase 2, matrix metallopeptidase 9
, and vascular endothelial growth factor
in various preclinical models of cancer[14
] Lastly, SFN inhibits self-renewal of cancer stem cells.[24
However, in the past few years, loss of function mutations in Keap1 and activating mutations in Nrf2 leading to gain of Nrf2 have been reported in lung cancer.[31
] In addition to lung cancer, gain of Nrf2 function has been reported in other cancers such as prostate, gallbladder, esophagus, breast, skin, and ovarian cancer. In addition to mutations, results from recent studies have demonstrated that the oncogenes K-ras, B-raf, and Myc upregulate Nrf2 signaling to evade senescence and apoptosis and promote tumorigenesis.[44
A comprehensive profiling of SFN bioavailability and tissue distribution kinetics revealed a dose-dependent increase in tissue concentrations after oral administration of SFN in mice.[45
] SFN accumulation peaked at 2 h in lung, liver, kidney, brain, and plasma, but in small intestine, colon, and prostate, the highest concentrations were recorded at 6 h. Maximum SFN accumulation was detected in small intestine. Furthermore, tissue concentrations of SFN metabolites varied as much as 100-fold between different tissues suggesting that peak plasma concentrations do not always align precisely with target tissues.[45
] Thus, route of administration may have a critical impact on SFN tissue distribution and accumulation. In this study, we compared three routes of SFN administration; (a) direct delivery to the target organ (lung) using nebulizer, (b) localized delivery to tumor bearing area (subcutaneous injection), and (c) systemic delivery with minimal risk of potential side effects due to systemic distribution (intraperitoneal injection).
To evaluate the effect of prolonged SFN treatment on growth of lung cancer xenografts, we selected two models: (1) A549 cells harboring mutant K-ras oncogene and mutant Keap1 gene[31
] and (2) H1975 cells harboring mutant EGFR oncogene but wild-type Keap1 and Nrf2. Prolonged systemic SFN treatment did not promote the growth of these tumors in vivo
. On the contrary, localized delivery of SFN significantly abrogated the growth of Keap1 mutant A549 tumors in vivo
suggesting that the tumor suppression effects of SFN are mediated by non-Nrf2-dependent signaling mechanisms. Importantly, significant tumor growth inhibition and a trend toward reduction with localized and systemic route of SFN administration, respectively, suggests that localized delivery of SFN probably results in better target tissue distribution and accumulation as compared to the systemic route.
Next, we investigated whether prolonged SFN administration affects oncogenic K-ras (K-rasG12D
)-driven lung tumorigenesis. A study by DeNicola et al
] showed that oncogenic K-ras signaling upregulates Nrf2 expression and its transcriptional network.[36
] Interestingly, although SFN administration via aerosol inhalation only modestly affected the Nrf2 signaling, we still noticed a trend toward reduced tumor burden in SFN treated cohort of mice. This raises a possibility that alternative route of administration resulting in localized accumulation and robust induction of Nrf2 signaling may significantly inhibit K-ras mediated lung tumor growth. In summary, SFN treatment does not accelerate K-ras-mediated lung tumorigenesis. However, one limitation of our study is that we only evaluated the effect of SFN on K-rasG12D
-mediated lung tumorigenesis post oncogenic K-ras activation and tumor foci formation. It remains to be investigated if SFN administration prior to cre-recombinase mediated oncogenic K-rasLSL-G12D
activation may impact the overall tumor burden.