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. 2020 Jun 4;21(11):4026.
doi: 10.3390/ijms21114026.

Chronic Sulforaphane Administration Inhibits Resistance to the mTOR-Inhibitor Everolimus in Bladder Cancer Cells

Saira Justin  1 , Jochen Rutz  1 , Sebastian Maxeiner  1 , Felix K-H Chun  1 , Eva Juengel  1   2 , Roman A Blaheta  1
Affiliations

Chronic Sulforaphane Administration Inhibits Resistance to the mTOR-Inhibitor Everolimus in Bladder Cancer Cells

Saira Justin et al. Int J Mol Sci. .

Abstract

Progressive bladder cancer growth is associated with abnormal activation of the mammalian target of the rapamycin (mTOR) pathway, but treatment with an mTOR inhibitor has not been as effective as expected. Rather, resistance develops under chronic drug use, prompting many patients to lower their relapse risk by turning to natural, plant-derived products. The present study was designed to evaluate whether the natural compound, sulforaphane (SFN), combined with the mTOR inhibitor everolimus, could block the growth and proliferation of bladder cancer cells in the short- and long-term. The bladder cancer cell lines RT112, UMUC3, and TCCSUP were exposed short- (24 h) or long-term (8 weeks) to everolimus (0.5 nM) or SFN (2.5 µM) alone or in combination. Cell growth, proliferation, apoptosis, cell cycle progression, and cell cycle regulating proteins were evaluated. siRNA blockade was used to investigate the functional impact of the proteins. Short-term application of SFN and/or everolimus resulted in significant tumor growth suppression, with additive inhibition on clonogenic tumor growth. Long-term everolimus treatment resulted in resistance development characterized by continued growth, and was associated with elevated Akt-mTOR signaling and cyclin-dependent kinase (CDK)1 phosphorylation and down-regulation of p19 and p27. In contrast, SFN alone or SFN+everolimus reduced cell growth and proliferation. Akt and Rictor signaling remained low, and p19 and p27 expressions were high under combined drug treatment. Long-term exposure to SFN+everolimus also induced acetylation of the H3 and H4 histones. Phosphorylation of CDK1 was diminished, whereby down-regulation of CDK1 and its binding partner, Cyclin B, inhibited tumor growth. In conclusion, the addition of SFN to the long-term everolimus application inhibits resistance development in bladder cancer cells in vitro. Therefore, sulforaphane may hold potential for treating bladder carcinoma in patients with resistance to an mTOR inhibitor.

Keywords: bladder cancer; drug resistance; everolimus; growth; mTOR; proliferation; sulforaphane.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cell number in response to everolimus (Ever- 5, 1, or 0.5 nM), sulforaphane (Sulf- 5, 2.5 µM), or (Ever + Sulf) under short-term application in bladder cancer cell lines RT112, UMUC3, and TCCSUP. Untreated cells served as controls. Cell number was evaluated after 24 (100%), 48, and 72 h by the MTT assay. Bars indicate standard deviation. Experiments were repeated five times. * indicates significant difference to untreated controls, p ≤ 0.05.
Figure 2
Figure 2
Evaluation of clonogenic growth (A) and BrdU incorporation (B) under short-term application of 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S). Control cells (C) remained untreated. RT112 clones were counted at day 8 and TCCSUP at day 10 following incubation. UMUC3 cells did not form clones (n.c.- not counted). The BrdU assay was carried out with synchronized cells with untreated control cells set at 100%. * indicates significant difference to untreated controls. # indicates significant difference between the mono and the combined applications.
Figure 3
Figure 3
Cell cycle analysis—short-term treatment of synchronized cells with 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus+2.5 µM sulforaphane (E + S). Untreated cells served as controls (C). Percentage of RT112, UMUC3, or TCCSUP cells in G0/G1, S and G2/M-phase is indicated. Inter-assay variation <10%, intra-assay variation <40%.
Figure 4
Figure 4
Protein profile of cell cycle regulating proteins (Akt, CDKs, Cyclins) after short-term exposure to 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S) in synchronized RT112 tumor cells. Controls (C) received cell culture medium alone. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown here. The ratio of protein intensity/β-actin intensity was calculated and expressed as a percentage of the controls, set to 100%. * indicates significant difference to controls, p ≤ 0.05.
Figure 5
Figure 5
Protein profile of cell cycle regulating proteins (Rictor, Raptor, histone acetylation, p19, p27) after short-term exposure to 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S) in synchronized RT112 tumor cells. Controls (C) received cell culture medium alone. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. * indicates significant difference to controls, p ≤ 0.05.
Figure 6
Figure 6
Growth of RT112, UMUC3, and TCCSUP cells exposed to 0.5 nM everolimus (Ever) or 2.5 µM sulforaphane (Sulf) or 0.5 nM everolimus + 2.5 µM sulforaphane (Ever + Sulf). Control cells (Control) remained unexposed. Cells were incubated in 96-well-plates for 24, 48, and 72 h. Cell number was set to 100% after 24 h incubation. Experiments were repeated five times. * indicates significant difference to untreated controls. # indicates significant difference between the mono and combined applications.
Figure 7
Figure 7
Evaluation of clonogenic growth (A) and BrdU incorporation (B) under the long-term application of 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S). Control cells (C) remained untreated. RT112 clones were counted at day 8 and TCCSUP at day 10 following incubation. UMUC3 cells did not form clones (n.c.- not counted). The BrdU assay was carried out with synchronized cells with untreated control cells set at 100%. * indicates significant difference to untreated controls. # indicates significant difference between the monotherapy and the combination therapy.
Figure 8
Figure 8
Cell cycle analysis—long-term treatment of synchronized RT112, UMUC3, and TCCSUP cells with 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S). Untreated cells served as the controls (C). Percentage of cells in the G0/G1-, S-, and G2/M-phase is indicated. Inter-assay variation <10%, intra-assay variation <40%.
Figure 9
Figure 9
Protein profile of cell cycle regulating proteins (Akt, CDKs, Cyclins) after long-term exposure to 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S) to synchronized RT112 tumor cells. Controls (C) received cell culture medium alone. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. The right panel shows the results of the pixel density analysis. The ratio of protein intensity/β-actin intensity was calculated and expressed as a percentage of the controls, set to 100%. * indicates significant difference to controls, p ≤ 0.05.
Figure 10
Figure 10
Protein profile of cell cycle regulating proteins (Rictor, Raptor, histone acetylation, p19, p27) after the long-term application of 0.5 nM everolimus (E) or 2.5 µM sulforaphane (S) or 0.5 nM everolimus + 2.5 µM sulforaphane (E + S) to synchronized RT112 tumor cells. Controls (C) received cell culture medium alone. One representative of three separate experiments is shown. Each protein analysis was accompanied by a β-actin loading control. One representative internal control is shown. The ratio of protein intensity/β-actin intensity is expressed as percentage of controls, set to 100%. * indicates significant difference to controls, p ≤ 0.05.
Figure 11
Figure 11
Tumor cell growth after functional blocking with small interfering RNA (siRNA) targeting CDK1/cyclin B (A), CDK2/Cyclin A (B), and p19 (C) of RT112 cells. Controls remained untreated. medium-C = cells treated with culture medium alone, siRNA-C = cells treated with unspecific siRNA, siRNA = cells treated with specific siRNA). β-actin served as the loading control. Protein data are shown on the left, growth response on the right side of the figure. One representative of three separate experiments is shown. * indicates significant difference to control.
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