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

Abstract

Chronic treatment with the mTOR inhibitor, everolimus, fails long-term in preventing tumor growth and dissemination in cancer patients. Thus, patients experiencing treatment resistance seek complementary measures, hoping to improve therapeutic efficacy. This study investigated metastatic characteristics of bladder carcinoma cells exposed to everolimus combined with the isothiocyanate sulforaphane (SFN), which has been shown to exert cancer inhibiting properties. RT112, UMUC3, or TCCSUP bladder carcinoma cells were exposed short- (24 h) or long-term (8 weeks) to everolimus (0.5 nM) or SFN (2.5 µM), alone or in combination. Adhesion and chemotaxis along with profiling details of CD44 receptor variants (v) and integrin α and β subtypes were evaluated. The functional impact of CD44 and integrins was explored by blocking studies and siRNA knock-down. Long-term exposure to everolimus enhanced chemotactic activity, whereas long-term exposure to SFN or the SFN-everolimus combination diminished chemotaxis. CD44v4 and v7 increased on RT112 cells following exposure to SFN or SFN-everolimus. Up-regulation of the integrins α6, αV, and β1 and down-regulation of β4 that was present with everolimus alone could be prevented by combining SFN and everolimus. Down-regulation of αV, β1, and β4 reduced chemotactic activity, whereas knock-down of CD44 correlated with enhanced chemotaxis. SFN could, therefore, inhibit resistance-related tumor dissemination during everolimus-based bladder cancer treatment.

Sulforaphane Reduces Prostate Cancer Cell Growth and Proliferation In Vitro by Modulating the Cdk-Cyclin Axis and Expression of the CD44 Variants 4, 5, and 7

“…Accordingly, CD44v7 was significantly down-regulated in primary squamous cell carcinomas and was not detectable in the majority of metastasis-derived specimens [ 38 ]. Based on a recent publication, exposure of SFN to bladder cancer cells induced a significant increase of CD44v4 and v7 and diminished chemotaxis, whereby knock-down of CD44 correlated with enhanced chemotaxis [ 39 ]. This broad spectrum of effects of SFN on different tumor entities indeed shows that SFN exerts its influence through different molecular mechanisms, depending on the tumor type.…”

“…Valproic acid has also been shown to modify the adhesion behavior of PC3 and LNCaP cells differently, and has been traced back to differing integrin equipment [ 41 ]. The disparate mechanistic influence of SFN has also been verified on several bladder cancer cell lines, with the supposition that SFN acts on a set of integrin receptors that may differ according to the initial characteristic integrin composition of the particular cell type [ 39 ]. We did not investigate whether this may hold true in the present investigation.…”

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

“…In breast cancer, CD44v has been shown to inversely correlate with CSC signatures, and switching from CD44s to CD44v was essential for cells to undergo mesenchymal–epithelial transition [ 151 ]. An increased expression level of the CD44 variants v3–v7 on bladder cancer cells following SFN exposure was recently documented by Justin et al [ 149 ]. Hypothetically, CD44v elevation caused by SFN forces the acquisition of an epithelial phenotype and prevents adaptive plasticity of bladder cancer cells.…”

“…A connection between integrins and Akt may explain why SFN not only diminishes bladder cancer cell adhesion but migration as well. In fact, the process of migration regulation is accompanied by altered α and β expression levels [ 149 ]. Modification of redox homeostasis by inducing the production of radicals and mitochondrial depolarization has been associated with overexpression of α5 and β1 integrin subunits, providing cross-communication between integrin adhesion receptors and oxidative stress, presumably via ROS [ 150 ].…”

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“…However, the mechanism is not fully known, but there is two possibilities either N is translocated into the nucleus, leading to the activation of caspase-6 or N can be imported into the nucleus only if casapse-6 is involved during apoptotic processes 44 . Also, down-regulation of FAK (focal adhesion kinase) and fibronectin expression by SARS-CoV have been reported 40 .…”