Norcantharidin nctd

Norcantharidin (NCTD, CAS number 5442-12-6) Sigma^®, Livonia, MI, USA; pentoxifylline (PTX, CAS number 6493-05-6) Sigma^®, USA, dissolved in sterile saline solution; and their combination were administered intraperitoneally (i.p.), forming seven groups, as follows: Group 1—control, sterile saline solution; Group 2—PTX 60 mg/kg; Group 3—PTX 30 mg/kg; Group 4—NCTD 3 mg/kg; Group 5—PTX 60 mg/kg + NCTD 3 mg/kg; Group 6—PTX 60 mg + NCTD 0.75 mg/kg; and Group 7—NCTD 0.75 mg/kg. The treatment was administered by being interspersed in five doses, as shown in Figure 7. The test doses were established based on the previous results from our work group for PTX [10 (link)], and the doses considered safe for NCTD without systemic toxic effects were selected based on the study by Martínez-Razo et al. [21 (link)].

The MTT reagent (Amresco, USA) can be metabolized by active cells to formazan, an insoluble purple dye that can be measured in a spectrophotometer, and was used according to the manufacturer's instructions. Cells were seeded in quadruplicates in 96-well plates (1,600 per well). After 24-h adhesion time, the cells were treated with the compounds chosen, and anticancer product norcantharidin (NCTD) (Sigma, USA) in positive group (Zimu and Zhenxiao, 2015 (link)). 0, 24, 48, and 72 h later, 100 μl of MTT reagent were added to each well and the cells were again incubated for 4 h at 37°C and 5% CO₂. After incubation and 10 minutes shaking, the absorbance was measured on a Genios Microplate Reader (Spectra Max190). All experiments were performed in triplicates.