Vorinostat

The HCT116 and SW620 colorectal carcinoma cell lines were originally purchased from American Type Culture Collection (Manassas, VA, USA), and the identities of our laboratory's versions were confirmed by short tandem repeat analysis (Table S1). The LoVo-92 colorectal carcinoma cell line was kindly provided by Dr. Paul Noordhuis (VU Medical Centre, Amsterdam, The Netherlands) [17] (link). The cell lines were cultured as previously described [8] (link), [17] (link). Xenografts were established by subcutaneous injections of HCT116 or SW620 cell suspensions (2×10⁶ cells) bilaterally on the flanks of locally bred female BALB/c nude (nu/nu) or Athymic Nude-Foxn1^nu mice, 6–8 weeks of age. Vorinostat (Cayman Chemical, Ann Arbor, MI, USA; 100 mg/kg, dissolved in dimethyl sulfoxide to a concentration of 100 mg/ml immediately before use) or vehicle was given by intraperitoneal injection 13 days (HCT116) or 20 days (SW620) after establishment of xenografts. Three and 12 hours after administration, the tumors were extirpated, snap-frozen in liquid nitrogen, and stored at −70°C. The xenografts were sectioned using a cryostat microtome prior to RNA extraction using TRIzol® Reagent (Invitrogen Dynal AS, Oslo, Norway). RNA concentration was assessed using the RNA/DNA calculator Gene Quent II (Pharmacia Biotech, Piscataway, NJ, USA).

Cells were treated with a single drug or a combination of two drugs for 4 days. The MYC inhibitor 10058-F4 was purchased from Cayman Chemical (Ann Arbor, MI, USA). The BCL6 inhibitors 79-6 and FX1 were from Merck KGaA (Darmstadt, Germany) and Selleck Chemical (Houston, TX, USA), respectively. The BET domain inhibitor JQ1 was obtained from Merck KGaA, the HDAC inhibitor vorinostat was from Cayman Chemical, and the PLK1 inhibitor volasertib was from ChemScene (Monmouth Junction, NJ, USA). All reagents were dissolved in DMSO. DMSO concentration in the control medium was the same as that used to make up the highest concentration of drugs in growth medium for the same set of experiments.

For in vitro studies, tadalafil (Euroasian Chemicals Private Ltd., Mumbai, India) and vorinostat (Cayman Chemical Company, Ann Arbor, MI, USA) were dissolved in DMSO at 10 mM and to final concentrations in medium cell. For in vivo studies, tadalafil (Cialis, Eli Lilly & Company) was administered in vivo by oral gavage at a dose of 1 mg/kg and was prepared as previously described [17 (link)]. vorinostat was administered intraperitoneally (i.p.) at a dose of 12.5 mg/kg and was dissolved in 10 % DMSO, 10 % Tween-20 and 90 % saline solution.