Sunitinib

Resistance to sunitinib was induced by chronic exposure to ascending sunitinib (free Base, Massachusetts LC Laboratories, Woburn, MA, USA) concentrations from 0.1–1 µM until the cells survived and adapted to the highest dosage. sunitinib resistance in the RCC cells occurred in average 10 weeks after starting application. Thereafter, they were maintained with 1 µM sunitinib applied three times a week. The IC50 (half-maximal inhibitory concentration) of sunitinib was investigated to verify drug resistance. After starving chronically sunitinib-treated RCC cells for 3 days 0.1–100 µM sunitinib was applied for 72 h. Therapy-sensitive (parental) RCC subcell lines served as controls. RCC cells were designated as sunitinib-resistant when the IC50 under 72 h sunitinib application was approximately doubled.
Artesunate (ART) (Sigma-Aldrich, Darmstadt, Germany) was applied at a concentration of 1–100 μM. Controls (parental and sunitinib-resistant) remained ART-untreated. The IC50 of ART in parental and sunitinib-resistant RCC cells was evaluated analog to sunitinib using the 72 h growth data at a concentration of 1–100 µM ART. To evaluate possible toxic effects of sunitinib and/or ART, cell viability was determined in parallel to experimentation by testing aliquoted cells with trypan blue (Sigma-Aldrich, Darmstadt, Germany). Only viable cells were employed.

Temsirolimus (PZ0020) and Sunitinib (PZ0012) were purchased from Sigma-Aldrich. Ki16425 (355025-24-0) and Y27632 (257-00511) were from Wako Chemicals, and SecinH3 was from Santa Cruz (853625-60-2). Other chemicals were purchased from Wako Chemicals, unless otherwise indicated. Since Y27632 did not inhibit cell invasiveness in our experiments, which used renal cancer cells, we confirmed the activity of Y27632 by its inhibition of the amoeboid-type invasion of HT1080 cells, as described previously22 (link).

Sunitinib (Sutent^®) was a product of Pfizer (New York, NY, USA). Sunitinib was dissolved in sterile DMSO (Sigma-Aldrich, D8418-50ML) and further diluted in a culture medium. A maximal concentration of 0.1% DMSO in culture medium specific to each cell line or the co-cultures was allowed for any of the screened conditions and was used as a control (CTRL). Panobinostat, vorinostat, axitinib, and pictilisib were purchased, stored, and dissolved as previously described [37 (link),46 (link)]. The PVAP combination containing panobinostat (10 nM), vorinostat (0.1 µM), axitinib (0.02 µM), and pictilisib (2 μM) was freshly prepared before each experiment as previously reported [37 (link),46 (link)]. For further information, see Table S2.