Urther increased after re-treatment with everolimus. The finding that proteins involved
Urther increased after re-treatment with everolimus. The finding that proteins involved in mitotic control were further up-regulated after applying a therapeutic everolimus concentration is clinically relevant, since mitotic activity of tumor cells is often accelerated, once resistance has developed. In the present investigation the number of mitotic cells significantly increased when Cakires cells were exposed to low-dosed everolimus. This finding might, therefore, explain why RCC patients, treated with temsirolimus or everolimus often BAY1217389 web develop progressive disease [23]. The same progression has been observed in different gynecologic cancers as well as estrogen receptor-positive breast cancer and colorectal carcinoma, indicating a correlation between treatment resistance and enhanced aggressiveness characterized by accelerated tumor growth [24-26]. The functional relevance of cdk2 and cyclin A in tumor growth was verified by siRNA knock down, revealing significant growth inhibition after cdk2 and cyclin A loss. Cdk2 and cyclin A establish complexes in the S-phase and are required for entrance into the G2/Mphase. Indeed, low expression of cdk2 and cyclin A has been shown PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27321907 to be associated with cell cycle arrest and accumulation of cells in the S-phase [27]. Everolimus resistance has also been associated with a considerable increase in cdk2 in prostate cancer [7] and in RCC cells [6]. Thus, augmented cdk2 seems closely related to nonresponsiveness towards everolimus and deserves attention in overcoming resistance development. High levels of cyclin A have been associated with a worse outcome and have been proposed as a prognostic factor in breast cancer, as well [28-30]. Similarly, a cyclin A increase in RCC has been associated with elevated tumor size and poor survival [31]. In good accordance with the present findings regarding Cakires, cyclin A expression has been shown to be inversely correlated with the expression of the cell cycle negative regulator p27 in RCC [31]. It may, therefore, be concluded that resistance development towards everolimus is accompanied by elevated cdk2/cyclin A, driving tumor cells from the S- into the G2/M-Juengel et al. Molecular Cancer 2014, 13:152 http://www.molecular-cancer.com/content/13/1/Page 5 ofFigure 4 Cell growth analysis. Growth inhibitory efficacy of 1 nM or 1 mM VPA in Cakipar and Cakires cells treated for one (A) or two weeks (B). *Indicates significant difference to untreated controls, set at 100 . #Indicates significant difference between Cakipar + VPA and Cakires + VPA (SD 22 , n = 6).Figure 5 Western blot analysis of Cakires exposed to 1 mM VPA for one (1 wk) or two weeks (2 wks) showing cell cycle regulating proteins and target molecules of mTOR- (A) and HDAC-inhibitors (B). VPA untreated cells served as controls. -actin served as the internal control. The figure shows one representative from three separate experiments.Juengel et al. Molecular Cancer 2014, 13:152 http://www.molecular-cancer.com/content/13/1/Page 6 ofFigure 6 siRNA knock-down controls. siRNA blockade of cdk2 or cyclin A (A) and HDAC1 or HDAC2 (B) were confirmed by western blotting. Cakipar and Cakires cells were transfected with cdk2, cyclin A, HDAC1 or HDAC2 siRNA. Untreated cells and with control siRNA transfected cells served as controls. One representative from three experiments is shown.phase, leading to a more aggressive tumor phenotype with enhanced growth capacity. The HDAC-inhibitor VPA caused a significant decrease.