Gemcitabine gemzar

Human colorectal cancer cell lines LoVo and Caco-2 were obtained from ATCC (Manassas, VA, USA). LoVo cells were grown in F-12K with 10% FBS, and Caco-2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS. Both cells were maintained in a humidified incubator at 37°C and 5% CO₂. Compound A was synthesized at laboratories in Takeda California as a selective and potent MEK allosteric site inhibitor (IC₅₀ of 38 nM against purified MEK1), which was orally bioavailable and efficacious in tumor xenograft models, as previously reported.^{22 (link)} PD0325901 was obtained from SelleckChem (Houston, TX, USA), Gemcitabine (GEMZAR) was obtained from Eli Lilly (Indianapolis, IN, USA), and Bicalutamide (Casodex tablets) was obtained from AstraZeneca (London, UK).

Oridonin was obtained from the Beijing Institute of Biological Products (Beijing, China). The purity of oridonin was measured by HPLC and determined to be 99.4%. Oridonin was dissolved in dimethyl sulfoxide (DMSO) to make a stock solution at 10 mmol/L concentration and stored at −20 °C. The DMSO concentration was kept below 0.1% in all cell culture experiments and did not exert any detectable effects on cell growth or cell death. Gemcitabine (Gemzar; Eli Lilly and Company) was stored at 4 °C and dissolved in sterile PBS at 0.2 mmol/L concentration on the day of use. The Cell Counting Kit-8 (CCK-8) was obtained from Abcam. Annexin V-FITC/PI apoptosis detection kit was purchased from Biological Development Co. Ltd. The RNA extraction kit was purchased from Life Technologies. cDNA first-strand synthesis kit was obtained from Fermentas. The 2×Taq PCR MasterMix was purchased from TIANGEN and the mitochondrial/cytosol fractionation kit was purchased from BioVision, CA, USA. Fetal bovine serum (FBS), trypsin containing EDTA, Roswell Park Memorial Institute-1640 (RPMI-1640), Ribonuclease A (RNase A), propidium iodide (PI), DMSO, antibodies against Bax, Smac, X-linked inhibitor of apoptosis protein (XIAP), caspase-9, cleaved caspase-3, β-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-rabbit and goat-anti-mouse) were all purchased from Sigma.