Figure 4. Induction of apoptosis and in vitro cytotoxicity by VTD in human cancer cell lines with different statuses of p53 and mot-2.

A–D. Cells were treated with VTD for 24, 48, and 72 hours. After incubation, dead cells were washed off, and the remaining attached cells were calculated by counting in five random fields using the Axiovert 200 M inverted microscope and AxioVision software (cell detachment assay). The % of cells attached was calculated as % of control cells. VTD significantly decreases the % of cells attached in a dose-dependent manner in cancer cells with WT-p53 (A: HCT-116, B: LoVo and C: U2OS) but not with mutant p53 (D: SW480). The data in Panel D suggest Veratridine might promote cell proliferation. However, a strict statistical analysis rejects any significant changes in SW-480 cells. The data is shown as mean ± SEM of three independent experiments (n = 3, *p < 0.05). E–K. Cells were plated for 5 days, and the colonies of cells were treated with different concentration of VTD. The colonies of viable cells were stained with crystal violet dye and absorbance, as an index of measurement of colony forming units, was read at 562 nm (clonogenic survival assay). VTD induced a significant decrease in cell viability in a p53- and differentiation grade manner in HCT-116 poorly differentiated cells (E), HCT-116 p53^+/− (F), HCT-116 p53^−/− (G), LoVo well-differentiated cells (H), SW-480 (K), and two non-colon cancer cells: HepG2 (I) and U2OS (J) The data is shown as mean ± SEM of three independent experiments (n = 3, *p < 0.05). L. Determination of endogenous levels of mot-2, UBXN2A, HSC70, and p53 proteins in various cancer cell lines using WB. M. HCT-116 cells were treated with VTD (10 and 30 μM) and cytoplasmic and nuclear fractions were subjected to WB. HSC70 and Orc-2 antibodies were used as cytoplasmic and nuclear markers, respectively. VTD increases p53 in both cytoplasm and nucleus compartments in a dose-dependent manner.