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Clinical Trial
. 2016 Jul;9(7):547-57.
doi: 10.1158/1940-6207.CAPR-15-0290. Epub 2016 Jun 23.

Prevention of Carcinogen-Induced Oral Cancer by Sulforaphane

Affiliations
Free PMC article
Clinical Trial

Prevention of Carcinogen-Induced Oral Cancer by Sulforaphane

Julie E Bauman et al. Cancer Prev Res (Phila). .
Free PMC article

Abstract

Chronic exposure to carcinogens represents the major risk factor for head and neck squamous cell carcinoma (HNSCC). Beverages derived from broccoli sprout extracts (BSE) that are rich in glucoraphanin and its bioactive metabolite sulforaphane promote detoxication of airborne pollutants in humans. Herein, we investigated the potential chemopreventive activity of sulforaphane using in vitro models of normal and malignant mucosal epithelial cells and an in vivo model of murine oral cancer resulting from the carcinogen 4-nitroquinoline-1-oxide (4NQO). Sulforaphane treatment of Het-1A, a normal mucosal epithelial cell line, and 4 HNSCC cell lines led to dose- and time-dependent induction of NRF2 and the NRF2 target genes NQO1 and GCLC, known mediators of carcinogen detoxication. Sulforaphane also promoted NRF2-independent dephosphorylation/inactivation of pSTAT3, a key oncogenic factor in HNSCC. Compared with vehicle, sulforaphane significantly reduced the incidence and size of 4NQO-induced tongue tumors in mice. A pilot clinical trial in 10 healthy volunteers evaluated the bioavailability and pharmacodynamic activity of three different BSE regimens, based upon urinary sulforaphane metabolites and NQO1 transcripts in buccal scrapings, respectively. Ingestion of sulforaphane-rich BSE demonstrated the greatest, most consistent bioavailability. Mucosal bioactivity, defined as 2-fold or greater upregulation of NQO1 mRNA, was observed in 6 of 9 evaluable participants ingesting glucoraphanin-rich BSE; 3 of 6 ingesting sulforaphane-rich BSE; and 3 of 9 after topical-only exposure to sulforaphane-rich BSE. Together, our findings demonstrate preclinical chemopreventive activity of sulforaphane against carcinogen-induced oral cancer, and support further mechanistic and clinical investigation of sulforaphane as a chemopreventive agent against tobacco-related HNSCC. Cancer Prev Res; 9(7); 547-57. ©2016 AACR.

Conflict of interest statement

Disclosure of Potential Conflicts of Interest

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Sulforaphane elevates dose- and time-dependent expression of NRF2 in Het-1A cells and HNSCC cell lines. A, Het-1A, a normal mucosal epithelial cell line, or the HNSCC cell lines UMSCC-22A and UMSCC-1 were left untreated, or were treated for 6 hours with vehicle (0.1% DMSO) or 10 μM sulforaphane (SF). Whole cell lysates were subjected to anti-NRF2 immunoblotting. Blots were re-probed with anti-β-actin to demonstrate equal protein loading. B, Het-1A were treated for 24 hours with varying concentrations of SF, followed by immunoblotting for NRF2 or β-actin. C, Het-1A were treated with 10 μM SF for the indicated number of hours, then subjected to immunoblotting. D and E, UMSCC-22A were treated and analyzed as in Panels B and C, respectively. All experiments were performed a minimum of three times, with similar results.
Figure 1
Figure 1
Sulforaphane elevates dose- and time-dependent expression of NRF2 in Het-1A cells and HNSCC cell lines. A, Het-1A, a normal mucosal epithelial cell line, or the HNSCC cell lines UMSCC-22A and UMSCC-1 were left untreated, or were treated for 6 hours with vehicle (0.1% DMSO) or 10 μM sulforaphane (SF). Whole cell lysates were subjected to anti-NRF2 immunoblotting. Blots were re-probed with anti-β-actin to demonstrate equal protein loading. B, Het-1A were treated for 24 hours with varying concentrations of SF, followed by immunoblotting for NRF2 or β-actin. C, Het-1A were treated with 10 μM SF for the indicated number of hours, then subjected to immunoblotting. D and E, UMSCC-22A were treated and analyzed as in Panels B and C, respectively. All experiments were performed a minimum of three times, with similar results.
Figure 1
Figure 1
Sulforaphane elevates dose- and time-dependent expression of NRF2 in Het-1A cells and HNSCC cell lines. A, Het-1A, a normal mucosal epithelial cell line, or the HNSCC cell lines UMSCC-22A and UMSCC-1 were left untreated, or were treated for 6 hours with vehicle (0.1% DMSO) or 10 μM sulforaphane (SF). Whole cell lysates were subjected to anti-NRF2 immunoblotting. Blots were re-probed with anti-β-actin to demonstrate equal protein loading. B, Het-1A were treated for 24 hours with varying concentrations of SF, followed by immunoblotting for NRF2 or β-actin. C, Het-1A were treated with 10 μM SF for the indicated number of hours, then subjected to immunoblotting. D and E, UMSCC-22A were treated and analyzed as in Panels B and C, respectively. All experiments were performed a minimum of three times, with similar results.
Figure 2
Figure 2
Induction of NRF2 target genes by sulforaphane. Het-1A, UMSCC-22A, and UMSCC-1 were left untreated, or treated for 4 hours with vehicle or 10 μM SF. Following treatment, RNA was purified and subjected to qPCR for NQO1 or GCLC, or GAPDH as internal control. Columns represent means and error bars standard deviations. Analysis was performed by one-way ANOVA with Tukey’s adjustment for multiple comparisons.
Figure 3
Figure 3
Sulforaphane induces rapid dephosphorylation of STAT3. A and B, Het-1A were treated for 12 hours with the indicated concentrations of SF (A), or were treated with 10 μM SF for the indicated times (B), followed by immunoblotting for phospho-Tyr705 STAT3 (pSTAT3), total STAT3, or β-actin. C and D, UMSCC-22A were treated and analyzed as in Panels A and B. E, Het-1A, UMSCC-22A, and UMSCC-1 were left untreated, or treated for 24 hours with DMSO control or 10 μM SF, followed by immunoblotting for anti- and pro-apoptotic Bcl-2 family members. Experiments were performed three times with similar results.
Figure 3
Figure 3
Sulforaphane induces rapid dephosphorylation of STAT3. A and B, Het-1A were treated for 12 hours with the indicated concentrations of SF (A), or were treated with 10 μM SF for the indicated times (B), followed by immunoblotting for phospho-Tyr705 STAT3 (pSTAT3), total STAT3, or β-actin. C and D, UMSCC-22A were treated and analyzed as in Panels A and B. E, Het-1A, UMSCC-22A, and UMSCC-1 were left untreated, or treated for 24 hours with DMSO control or 10 μM SF, followed by immunoblotting for anti- and pro-apoptotic Bcl-2 family members. Experiments were performed three times with similar results.
Figure 3
Figure 3
Sulforaphane induces rapid dephosphorylation of STAT3. A and B, Het-1A were treated for 12 hours with the indicated concentrations of SF (A), or were treated with 10 μM SF for the indicated times (B), followed by immunoblotting for phospho-Tyr705 STAT3 (pSTAT3), total STAT3, or β-actin. C and D, UMSCC-22A were treated and analyzed as in Panels A and B. E, Het-1A, UMSCC-22A, and UMSCC-1 were left untreated, or treated for 24 hours with DMSO control or 10 μM SF, followed by immunoblotting for anti- and pro-apoptotic Bcl-2 family members. Experiments were performed three times with similar results.
Figure 4
Figure 4
Role of NRF2 in sulforaphane-induced STAT3 inactivation and induction of apoptosis. A, Het-1A and UMSCC-22A were transfected for 6 hours with non-specific siRNA or NRF2 siRNA. Following transfection, cells were allowed to recover overnight before treatment for 12 hours with 0.1% DMSO or 10 μM SF. Cells were then subjected to immunoblotting for NRF2, pSTAT3, total STAT3, or β-actin. Similar results were seen in three independent experiments. B, UMSCC-22A transfected with non-specific siRNA or NRF2 siRNA as in Panel A were left untreated, or were treated for 24 hours with 0.1% DMSO or 10 μM SF, then analyzed by flow cytometry for Annexin V/PI staining. Numbers indicate the percentage of Annexin V-positive cells (p=0.001 by 2-way ANOVA when comparing SF-treated NRF2 siRNA versus SF-treated non-specific siRNA).
Figure 4
Figure 4
Role of NRF2 in sulforaphane-induced STAT3 inactivation and induction of apoptosis. A, Het-1A and UMSCC-22A were transfected for 6 hours with non-specific siRNA or NRF2 siRNA. Following transfection, cells were allowed to recover overnight before treatment for 12 hours with 0.1% DMSO or 10 μM SF. Cells were then subjected to immunoblotting for NRF2, pSTAT3, total STAT3, or β-actin. Similar results were seen in three independent experiments. B, UMSCC-22A transfected with non-specific siRNA or NRF2 siRNA as in Panel A were left untreated, or were treated for 24 hours with 0.1% DMSO or 10 μM SF, then analyzed by flow cytometry for Annexin V/PI staining. Numbers indicate the percentage of Annexin V-positive cells (p=0.001 by 2-way ANOVA when comparing SF-treated NRF2 siRNA versus SF-treated non-specific siRNA).
Figure 5
Figure 5
Sulforaphane reduces the incidence and size of tongue tumors in 4NQO-treated mice. A, Wild-type C57BL/6 mice (n=17/group) were treated for 16 weeks with 4NQO (100 μM in drinking water) plus vehicle (thrice weekly via oral gavage), or with 4NQO plus SF (6 μmole thrice weekly via oral gavage). After the 16-weeks, treatments were discontinued and mice given regular tap water for an additional 8 weeks. The number of tongue tumors in each mouse was counted, with significantly fewer in sulforaphane-treated mice (p=0.012 by Poisson ANOVA). B, Tongues from mice exhibiting tumors were fixed, embedded in paraffin, sectioned, and stained with hematoxylin/eosin. The stained sections were evaluated by a pathologist blinded to the treatment groups. The number of mice exhibiting normal tongue tissue only, squamous hyperplasia, dysplasia, or invasive SCC were scored. C, The total tumor volume per mouse (open circles) was significantly lower in sulforaphane-treated mice (p=0.005 by Welch’s two-sample t-test). Bold bars represent the median tumor volume (9.55 vs. 0.90 mm3), and boxes represent the 25th and 75th percentiles.
Figure 5
Figure 5
Sulforaphane reduces the incidence and size of tongue tumors in 4NQO-treated mice. A, Wild-type C57BL/6 mice (n=17/group) were treated for 16 weeks with 4NQO (100 μM in drinking water) plus vehicle (thrice weekly via oral gavage), or with 4NQO plus SF (6 μmole thrice weekly via oral gavage). After the 16-weeks, treatments were discontinued and mice given regular tap water for an additional 8 weeks. The number of tongue tumors in each mouse was counted, with significantly fewer in sulforaphane-treated mice (p=0.012 by Poisson ANOVA). B, Tongues from mice exhibiting tumors were fixed, embedded in paraffin, sectioned, and stained with hematoxylin/eosin. The stained sections were evaluated by a pathologist blinded to the treatment groups. The number of mice exhibiting normal tongue tissue only, squamous hyperplasia, dysplasia, or invasive SCC were scored. C, The total tumor volume per mouse (open circles) was significantly lower in sulforaphane-treated mice (p=0.005 by Welch’s two-sample t-test). Bold bars represent the median tumor volume (9.55 vs. 0.90 mm3), and boxes represent the 25th and 75th percentiles.
Figure 5
Figure 5
Sulforaphane reduces the incidence and size of tongue tumors in 4NQO-treated mice. A, Wild-type C57BL/6 mice (n=17/group) were treated for 16 weeks with 4NQO (100 μM in drinking water) plus vehicle (thrice weekly via oral gavage), or with 4NQO plus SF (6 μmole thrice weekly via oral gavage). After the 16-weeks, treatments were discontinued and mice given regular tap water for an additional 8 weeks. The number of tongue tumors in each mouse was counted, with significantly fewer in sulforaphane-treated mice (p=0.012 by Poisson ANOVA). B, Tongues from mice exhibiting tumors were fixed, embedded in paraffin, sectioned, and stained with hematoxylin/eosin. The stained sections were evaluated by a pathologist blinded to the treatment groups. The number of mice exhibiting normal tongue tissue only, squamous hyperplasia, dysplasia, or invasive SCC were scored. C, The total tumor volume per mouse (open circles) was significantly lower in sulforaphane-treated mice (p=0.005 by Welch’s two-sample t-test). Bold bars represent the median tumor volume (9.55 vs. 0.90 mm3), and boxes represent the 25th and 75th percentiles.
Figure 6
Figure 6
Broccoli sprout extracts are bioavailable and bioactive in the oral mucosa of healthy volunteers. A, Overnight urine was collected at baseline, and following the final BSE dose in each regimen. Regimen 1, 600 μmol of glucoraphanin-rich BSE/day; Regimen 2, 150 μmol of sulforaphane-rich BSE/day; Regimen 3, 150 μmol of sulforaphane-rich BSE was swished, gargled and expectorated for 6 minutes daily. Sulforaphane and sulforaphane-N-acetylcysteine were quantified by isotope dilution mass spectrometry, and normalized to urine creatinine. Sulforaphane-rich BSE was significantly more bioavailable than either glucoraphanin-rich BSE or topical sulforaphane-rich BSE (p=0.0013 by mixed-effects ANOVA). Bars represent 90% confidence intervals. B, Buccal cells were collected at baseline, and days 3–5 of each BSE regimen. mRNA transcripts for NQO1 were measured by qPCR. Three participants had adequate mRNA at every regimen time point and are displayed. The protocol definition of mucosal bioactivity, ≥2-fold upregulation of NQO1 transcripts, is shown for glucoraphanin-rich BSE in participants #2 and #8, for sulforaphane-rich BSE in participants #7 and #8, and for topical sulforaphane-rich BSE in none. C, A mixed-effects ANOVA was conducted to explore the effect of regimen and day on NQO1 qPCR. Fold change in ΔCT is presented by participant (gray lines), regimen, and day. Bold lines represent the means estimated from the mixed-effects ANOVA.
Figure 6
Figure 6
Broccoli sprout extracts are bioavailable and bioactive in the oral mucosa of healthy volunteers. A, Overnight urine was collected at baseline, and following the final BSE dose in each regimen. Regimen 1, 600 μmol of glucoraphanin-rich BSE/day; Regimen 2, 150 μmol of sulforaphane-rich BSE/day; Regimen 3, 150 μmol of sulforaphane-rich BSE was swished, gargled and expectorated for 6 minutes daily. Sulforaphane and sulforaphane-N-acetylcysteine were quantified by isotope dilution mass spectrometry, and normalized to urine creatinine. Sulforaphane-rich BSE was significantly more bioavailable than either glucoraphanin-rich BSE or topical sulforaphane-rich BSE (p=0.0013 by mixed-effects ANOVA). Bars represent 90% confidence intervals. B, Buccal cells were collected at baseline, and days 3–5 of each BSE regimen. mRNA transcripts for NQO1 were measured by qPCR. Three participants had adequate mRNA at every regimen time point and are displayed. The protocol definition of mucosal bioactivity, ≥2-fold upregulation of NQO1 transcripts, is shown for glucoraphanin-rich BSE in participants #2 and #8, for sulforaphane-rich BSE in participants #7 and #8, and for topical sulforaphane-rich BSE in none. C, A mixed-effects ANOVA was conducted to explore the effect of regimen and day on NQO1 qPCR. Fold change in ΔCT is presented by participant (gray lines), regimen, and day. Bold lines represent the means estimated from the mixed-effects ANOVA.
Figure 6
Figure 6
Broccoli sprout extracts are bioavailable and bioactive in the oral mucosa of healthy volunteers. A, Overnight urine was collected at baseline, and following the final BSE dose in each regimen. Regimen 1, 600 μmol of glucoraphanin-rich BSE/day; Regimen 2, 150 μmol of sulforaphane-rich BSE/day; Regimen 3, 150 μmol of sulforaphane-rich BSE was swished, gargled and expectorated for 6 minutes daily. Sulforaphane and sulforaphane-N-acetylcysteine were quantified by isotope dilution mass spectrometry, and normalized to urine creatinine. Sulforaphane-rich BSE was significantly more bioavailable than either glucoraphanin-rich BSE or topical sulforaphane-rich BSE (p=0.0013 by mixed-effects ANOVA). Bars represent 90% confidence intervals. B, Buccal cells were collected at baseline, and days 3–5 of each BSE regimen. mRNA transcripts for NQO1 were measured by qPCR. Three participants had adequate mRNA at every regimen time point and are displayed. The protocol definition of mucosal bioactivity, ≥2-fold upregulation of NQO1 transcripts, is shown for glucoraphanin-rich BSE in participants #2 and #8, for sulforaphane-rich BSE in participants #7 and #8, and for topical sulforaphane-rich BSE in none. C, A mixed-effects ANOVA was conducted to explore the effect of regimen and day on NQO1 qPCR. Fold change in ΔCT is presented by participant (gray lines), regimen, and day. Bold lines represent the means estimated from the mixed-effects ANOVA.

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