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

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

doi:10.3390/ijms21114026

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.

Highlights

Bladder cancer is the ninth most common malignant ailment and the fourteenth most common cause of cancer death worldwide
The PI3K Akt-mammalian target of the rapamycin (mTOR) pathway is crucial for regulating cellular growth, proliferation, survival, and motility
A drug dosage of 0.5 nM everolimus and 2.5 μM for SFN was set for further experiments

Summary

Introduction

Bladder cancer is the ninth most common malignant ailment and the fourteenth most common cause of cancer death worldwide. The PI3K (phosphoinositide-3 kinase) Akt (serine/threonine kinase)-mTOR (mammalian target of rapamycin) pathway is crucial for regulating cellular growth, proliferation, survival, and motility. In up to 40% of bladder cancers, mTOR pathway activation is closely involved with tumor progression [2]. Targeting the Akt-mTOR pathway is an attractive strategy to treat advanced urothelial carcinoma, and several mTOR inhibitors are clinically applied. Treatment of bladder cancer with these inhibitors has not been as effective as expected. The potential benefit of the mTOR-inhibitors, temsirolimus and everolimus, has been demonstrated only for a subset of bladder cancer patients, and use of these drugs has been associated with severe, not well-tolerated, adverse events [3,4]

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