In addition, our published data showed that the IC50 values of cisplatin, which is the most effective and widely used chemotherapeutic agent, were 11.91 g/mL (35.71 M) for A2780/CP70 and 10.10 g/mL (30.30 M) for OVCAR-3, respectively [19]. TSE1 was selected as the experimental drug. The purpose is to detect the population of ALDH+ cells that were accumulated in two ovarian cancer cell lines and determine if those cells have certain stem cell characteristics, then investigate the effect of TSE1 on the ALDH+ cells. 2. Results 2.1. Expression of ALDH in Both Tumor and Sphere Cells According to the basic principle of serum-free culture, the differentiated mature tumor cells cannot adhere to the wall and go to apoptosis in the serum-free state, whereas undifferentiated CSCs within total tumor cells can grow and experience multidifferentiation into both tumor and CSCs to form a spherical aggregate state, thus differentiating from each other. The spheres derived from A2780/CP70 and OVCAR-3 cells appeared and completely formed within seven days (one week). It can be seen in Figure 1a that two ovarian cancer cells exhibit spindle or oval-shaped single cell distribution in the adherent culture, but, in the serum-free culture state, both cells showed Bax inhibitor peptide P5 different degrees of spherical dense multicellular aggregation and were able to float in the culture fluid. The results indicated the presence of CSCs in both ovarian cancer cells. An ALDEFLUOR Stem Cell Identification Kit was used to examine the proportion of ALDH+ cells in the tumor and different culture algebraic suspension cells. The experimental results showed (Figure 1b) that ALDH+ cells ratio was 1.05%, 5.75%, 12.20% and 29.50% in tumor cells and sphere cells with 1-week, 2-weeks and 3-weeks in A2780/CP70, and 1.25%, 2.75%, 7.20%, 24.95% in OVCAR-3, respectively. Our results indicated that there was indeed a very small amount of ALDH+ cells in both ovarian cancer cells, which was less than 2.0%, while the proportion of ALDH+ cells in two cell lines showed a significant increase trend in a time-dependent manner. In addition, the increasing trend of the A2780/CP70 cell line and the proportion of ALDH+ cells were higher than that of OVCAR-3. The proportion of ALDH+ cells in both of the suspension spheres after three generations of culture exceeded 20%, indicating that the serum-free suspension culture method can significantly enrich CSCs, and is a simple and effective enrichment method. Open in a separate window Figure 1 The population of ALDH of both tumor and sphere cells cultured in serum-free medium with different weeks from A2780/CP70 and OVCAR-3 cell lines. (a) morphological photographs of ovarian tumor and sphere cells (3-weeks) for both two cell lines (200); (b) ALDH ratio after culturing in serum-free medium could accumulate in a time-dependent manner. Data was expressed as percent of ALDH+ cells and shown as mean SD (= 3), * = 0.05, a significant difference compared with zero-time control. 2.2. Sphere Cells Exhibits Stemness Bax inhibitor peptide P5 Properties The single cell sphere formation ability experimental results showed (Figure 2a) that the average number of suspended spheres after one week culturing of tumor cells (0 generation) was only about 10. From the first generation to the third generation of suspension cells, the average number of suspended spheres increased significantly after one Bax inhibitor peptide P5 week of culture, indicating that the proportion of ALDH+ cells was positively correlated with the single cell pelleting ability. Observing the number of spheres of different generations of cells in different cultures on the same day, the same rule was found, and, in Ntrk1 particular, the ability of the Bax inhibitor peptide P5 third generation cells was significantly increased. It was confirmed that ALDH+ cells have stronger single-cell spherule ability, and.