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

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.

Bladder Cancer Metastasis Induced by Chronic Everolimus Application Can Be Counteracted by Sulforaphane In Vitro

“…Nevertheless, Akt deserves further investigation. A recent investigation shows that the influence of SFN on mTOR-Akt signaling in bladder cancer leads to a significant increase in Akt phosphorylation (pAkt) in tumor cells following chronic everolimus administration [ 23 ]. Surprisingly, enhanced pAkt was also seen in the presence of SFN alone, but not in the presence of the everolimus-SFN combination where pAkt was reduced, compared to the untreated controls.…”

Identification of Activated Protein Kinase Cα (PKCα) in the Urine of Orthotopic Bladder Cancer Xenograft Model as a Potential Biomarker for the Diagnosis of Bladder Cancer

“…Furthermore, PKCα is expressed at higher levels in bladder cancer tissues than in normal bladder tissues [29]. The bladder cancer cell lines used in this study represent different pathological grades, with KU-7 for grade 1, KU-1 for grade 2, T24 and UMUC-3 for grade 3, and TCCSUP for grade 4 [29][30][31]. However, activated PKCα was found in all the cell lines, and all the cell lysates phosphorylated a PKCα-specific peptide.…”

Sulforaphane Impact on Reactive Oxygen Species (ROS) in Bladder Carcinoma

“…The Akt-mTOR-pathway serves as a central regulator of cell growth and proliferation. In three bladder cancer cell lines (RT112, UMUC3 and TCCSUP), SFN treatment significantly suppressed the amount of phosphorylated Akt and phosphorylation of the mTOR subunit Rictor [ 144 ]. Reduction of Akt and mTOR phosphorylation, along with diminished p70S6k downstream signaling under SFN, has also been observed in HTB-9 cells [ 145 ], pointing to a common mechanism of SFN action.…”

“…The relevance of SFN as a cell-cycle inhibitor has furthermore been proven in terms of diminished expression of the cell-cycle-regulating proteins of the cyclin and CDK family. Accordingly, the CDK inhibitors, p19 and p21, are elevated under SFN [ 144 , 145 ]. The suppressive effect of SFN on Akt-mTOR signaling has also been seen with long-term treatment, in contrast to resistance induction evoked under chronic use of the established mTOR-inhibitors, everolimus and temsirolimus [ 144 , 146 ].…”

“…Accordingly, the CDK inhibitors, p19 and p21, are elevated under SFN [ 144 , 145 ]. The suppressive effect of SFN on Akt-mTOR signaling has also been seen with long-term treatment, in contrast to resistance induction evoked under chronic use of the established mTOR-inhibitors, everolimus and temsirolimus [ 144 , 146 ]. Further investigation into adding SFN to everolimus/temsirolimus treatment for reversion or prevention of drug resistance might, therefore, be warranted.…”