Figure 2.

Pretubulysin (PT) and tubulysin A (Tub A) abrogate cell proliferation, long-term survival, anchorage-independent growth and induce anoikis. (a) T24 and MDA-MB-231 cells were treated with PT or Tub A for 72 h. As shown by crystal violet staining, PT inhibits cell proliferation similar to Tub A. (b) PT and Tub A impede clonogenic survival of cancer cells. T24 and MDA-MB-231 cells were treated with PT/Tub A for 4 h, detached, freshly seeded at very low density (5000 cells/6-well plate) and grown for 7 days. Taxol (Tax, 50 nM) was used as positive control. Representative pictures of wells are shown on the right. (c) PT inhibits anchorage-independent growth of T24 and MDA-MB-231 cells. After treatment with PT/Tub A for 4 h, cells were seeded in very low cell number in soft agar, allowed to grow for several days and stained with MTT. Number of colonies was counted. Treatment with taxol was used as positive control. (d) Increasing concentrations of PT dose-dependently induce anoikis in MDA-MB-231 breast cancer cells after incubation for 48 h. Bars represent the mean±S.E.M. of three independent experiments performed in triplicate, *P<0.05 (one-way analysis of variance (ANOVA), Dunnett's test). Ctrl, control; NS, nonsignificant