Fig. 6. VPA induces cell differentiation and apoptosis in carcinoma cell lines and inhibits tumor growth and metastasis formation in the rat. (A) F9 teratocarcinoma cells were treated for 48 h with TSA (30 nM) or VPA (1 mM). The appearance of AP-2 protein as a specific marker of the VPA-induced type of F9 cell differentiation was as described (Werling et al., 2001). One out of two experiments with similar results is shown. (B) Thymidine incorporation into cultures of HT-29 colonic cancer or MT-450 breast carcinoma cells was tested as a parameter for cell proliferation. Cells were cultured for 72 h in the absence or presence of the indicated concentrations of VPA prior to analysis of [³H]thymidine incorporation. The graph shows the means ± SD from quadruple determinations. Similar results were obtained in two additional independent experiments. (C) The appearance of apoptotic cells in VPA-treated cultures of MT-450 cells was analyzed by staining of cell surface-exposed phosphatidylserine by FITC-conjugated annexin V after exclusion of necrotic cells by means of propidium iodide uptake (lower right quadrant of the graphs). Similar results were obtained in a second experiment. (D) Subcutaneous tumor growth and lung metastasis of MT-450 breast cancer cells were analyzed in rats treated with VPA or saline, respectively. Tumor volume was determined at day 21 (onset of VPA treatment) and day 43 (termination of experiment). Lung metastasis visible from the organ surface was scored. Scores: 0, no metastasis; 1, <50 metastases or all metastatic nodules <2 mm in diameter (lower frames); 2, many metastases >2 mm in diameter (upper right frame); 3, most of the lung’s surface consists of metastatic nodules (upper left frame). Values are means ± SD and significance of differences was calculated by Student t-test. (E) Pictures of two preparations each representative for control or VPA-treated rats are shown. The original height of the frames is 25 mm.