A498 cells were derived from Cell Lines Service (Heidelberg, Germany). phase. RCC cells became resistant to sunitinib after 8?weeks, demonstrated by accelerated cell growth along with enhanced cdk1, cdk2, loss of p27, activation of Akt, Rictor and Raptor. Switching to sorafenib only slightly reduced growth of the sunitinib resistant RCC cells and molecular analysis indicated unique cross-resistance. In contrast, full response was accomplished when the malignancy cells were treated with RAD001. p19 and p27 strongly improved, phosphorylated Akt, Rictor and Raptor decreased and the tumour cells accumulated in G0/G1. It is concluded that an mTOR-inhibitor for second-line therapy could be the strategy of choice after first-line sunitinib failure. RAD001 in a second line establishing. RCC cells, which have been driven to sunitinib-resistance were treated with sorafenib or RAD001 for different time periods and the biological as well as the molecular reactions were investigated. Our data point to distinct differences between the sorafenib and the RAD001 centered regimen. Sorafenib only slightly counteracted resistance effects caused by Mc-Val-Cit-PAB-Cl sunitinib and only moderately diminished RCC tumour growth, compared to its influence on sunitinib-sensitive cells. In contrast, RAD001 evoked a strong response of the sunitinib-resistant RCC cells, which was similar to the one seen in sunitinib-sensitive cells. Molecular analysis exposed cross-resistance between sunitinib and sorafenib, which might be responsible for the limited effect observed with second collection sorafenib treatment. Materials and methods Cell tradition Kidney carcinoma Caki-1 and KTC-26 cells were purchased from LGC Promochem (Wesel, Germany). A498 cells were derived from Cell Lines Services (Heidelberg, Germany). Tumour cells were cultivated and subcultured in RPMI 1640 medium (Seromed, Berlin, Germany) supplemented with 10% FCS, 100?IU/ml penicillin and 100?g/ml streptomycin (all Gibco/Invitrogen, Karlsruhe, Germany) at 37C inside a humidified, 5% CO2 incubator. Medicines RAD001 (provided by Novartis Pharma AG, Basel, Switzerland) was dissolved in DMSO (Merck, Darmstadt, Germany) as 10?mM stock solution and stored in aliquots at ?20C. Prior to the experiments, RAD001 was diluted in cell tradition medium to a final concentration of 5?nM. Sunitinib and sorafenib were from LC Laboratories, Woburn, MA, USA, and used at a final concentration of 1 1?M (sunitinib) or 5?M (sorafenib). Renal cell carcinoma cell lines were treated twice a week with sunitinib over a period of 8?weeks. Subsequently, sunitinib was replaced by sorafenib or RAD001 for a further period of 8?weeks. Both sorafenib and RAD001 were applied twice a week. Control cells received cell tradition medium only or sunitinib for a total of 16?weeks. Additionally, new cells, not pre-treated with sunitinib, were exposed to sorafenib or RAD001 to investigate the maximum effect of RAD001 and sorafenib. The strategy of chronic drug treatment with a constant, instead of an increasing dose was based on an earlier study, whereby this protocol proved to initiate resistance 6. Cell viability was determined by trypan blue (Gibco/Invitrogen, Karlsruhe, Germany) 1?day time and 8?weeks after sunitinib software, and 1?day time and 8?weeks after sorafenib or RAD001 software. Cell viability was also controlled at every cell passage. For all further checks, tumour cells were subjected to the assays listed below 1?day time and 8?weeks after sunitinib software, and 1?day time and 8?weeks after sorafenib or RAD001 software. Apoptosis To detect apoptosis the manifestation of Annexin V/propidium iodide (PI) was evaluated using the Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, Heidelberg, Germany). Tumour cells were washed twice with PBS, and then incubated with 5?l of Annexin V-FITC and 5?l of PI in the dark for 15?min. at RT. Cells were analysed on a FACScalibur (BD Biosciences, Heidelberg, Germany). The percentage of apoptotic cells (early and late) in each quadrant was determined using CellQuest software (BD Biosciences). Caspase-3, Bcl-2 and Bax manifestation were additionally evaluated by Western blotting using the following antibodies: Anti-caspase-3 (#9662; Cell Signalling-Millipore, Darmstadt, Germany), Anti-Bcl-2 (clone N-19), Anti-Bax (clone N-20, both Santa Cruz, Heidelberg, Germany). Measurement of tumour cell growth Cell growth was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Caki-1 cells (50?l, 1??105 cells/ml) were seeded onto 96-well Mc-Val-Cit-PAB-Cl cells tradition plates. After 24, 48 Mc-Val-Cit-PAB-Cl and 72?hrs, 10?l Rabbit polyclonal to ZNF264 MTT (0.5?mg/ml) were added for an additional 4?hrs. Thereafter, cells were lysed inside a buffer comprising 10% SDS in 0.01?M HCl. The plates were incubated over night at 37C, 5% CO2. Absorbance at 550?nm was determined for each well using a microplate ELISA reader. A standard curve was run in parallel to determine the cell number, assuming that mitochondrial activity was the same in all the cell cultures. Each experiment was carried out in triplicate. After subtracting background Mc-Val-Cit-PAB-Cl absorbance, results were indicated as mean cell number..