Cholera toxin, a typical protein kinase A activator, induces G1 phase growth arrest in human bladder transitional cell carcinoma cells via inhibiting the c-Raf/MEK/ERK signaling pathway

Abstract

The biotoxin cholera toxin has been demonstrated to have anti-tumor activity in numerous types of cancer, including glioma. However, the role of cholera toxin in the tumorigenesis of transitional cell carcinoma (TCC), the most common malignant tumor of the bladder, remains to be elucidated. To address this, in the present study, two TCC cell lines, T24 and UM-UC-3, were treated with cholera toxin [protein kinase A (PKA) activator] and KT5720 (PKA inhibitor). Cell survival and proliferation, cell cycle alterations and apoptosis were analyzed using Hoechst staining, the MTT assay, fluorescence microscopy and flow cytometry. Western blot analysis was used to detect the expression of proteins involved in cell cycle regulation. The results revealed that cholera toxin significantly induced G1 arrest and downregulated the expression of cyclin D1 and cyclin-dependent kinase 4/6 in the TCC cell lines, and this was rescued by KT5720. Furthermore, it was demonstrated that cholera toxin downregulated the activation of the c-Raf/Mek/Erk cascade, an important mediator of tumor cell proliferation, via the PKA-dependent c-Raf phosphorylation at Ser-43. Furthermore, inhibition of Mek activity with UO126 mimicked the effects of cholera toxin. In conclusion, these results confirmed that cholera toxin specifically inhibited proliferation and induced G1 phase arrest in human bladder TCC cells. This effect was due to PKA-dependent inactivation of the c-Raf/Mek/Erk pathway. This suggested that cholera toxin may be a viable therapeutic treatment against tumorigenesis and proliferation in bladder cancer.

Heterogeneous Family of Cyclomodulins: Smart Weapons That Allow Bacteria to Hijack the Eukaryotic Cell Cycle and Promote Infections

“…Moreover, using two human bladder cell lines, T24 and UM-UC-3, it was demonstrated that CTX inactivates the c-Raf/Mek/Erk cascade, via the PKA-dependent c-Raf phosphorylation at Ser-43. This results in the downregulation of the expression of the regulatory molecules (cyclin D1, Cdk4, and Cdk6), followed by the G1 phase arrest (Figure 3; Zheng et al, 2014). …”

The prognostic significance of DAPK1 in bladder cancer

“…Mutation of Braf gene in bladder cancer is an infrequent event [34], however, Ras mutation, which act upstream of Braf, or Ras pathway has been shown to play an important role in bladder cancer tumorigenesis [35], while MEK inhibitor, PD-0325901, has been shown to suppress bladder tumor growth in a patient-derived xenograft model [36] and the inhibition of Raf/MEK/ERK pathway through Cholera toxin also resulted in growth arrest in cell line model [37]. In this study, we found that vemurafenib, a Braf inhibitor, and trametinib, a MEK inhibitor, both could reverse DAPK1-associated gene expression signature, by connectivity mapping.…”

Inhibitory effect of Au@Pt-NSs on proliferation, migration, and invasion of EJ bladder carcinoma cells: involvement of cell cycle regulators, signaling pathways, and transcription factor-mediated MMP-9 expression

“…4,5 Thus, through signal induction experiments, we investigated whether Au@Pt-NSs influenced the phosphorylation of ERK1/2, JNK1/2, p38, or AKT. Both EJ and HUCs were treated with Au@Pt-NSs at different concentrations (0, 0.1, 0.3, and 0.5 μM) for different periods (1,6,12, and 24 h) followed by immunoblotting.…”

“…Bladder cancer cells react to chemotherapeutic reagents through the activation or inhibition of key signaling pathways, including MAPKs and PI-3K/AKT. [4][5][6][7] These molecular cues are transmitted to the nucleus by several steps of a signaling cascade, which redistributes the association of regulators of the cell cycle, including cyclin-dependent kinases (CDKs) and their binding partners, at certain cell cycle phases: G1, S, and G 2 /M. [8][9][10] Transition from G1 phase to S phase is determined by interactions among key regulators, including cyclin D/E and CDK4/6, CDK inhibitors (p21WAF1 and p27KIP1), and retinoblastoma protein (pRB).…”