Data are expressed as the
mean ± standard error of the mean. For statistical comparison, results were analyzed using analysis of variance and Student’s Tanespimycin price t test. A p value < 0.05 was considered statistically significant. All statistical tests were carried out using the computer program STATISTICA version 4.5 (StatSoft Inc., Tulsa, OK, USA). To understand the mode of action of the antiproliferative and proapoptotic activities of G-Rp1, we first examined whether G-Rp1 was able to block the proliferation of LoVo colorectal cancer cells. As shown in Fig. 2A, G-Rp1 dose-dependently suppressed up to 70% of the proliferation of LoVo cells at 60μM. Although the antiproliferative activity of G-Rp1 in colorectal cancer cells is weaker than in human breast cancer cells [9], the inhibition of LoVo cell proliferation by G-Rp1 indicates
that this compound may have common antiproliferative activity regardless of the cell type. Indeed, PI staining strongly implied that the G-Rp1-induced antiproliferative activity was due to the induction of proapoptotic activity by this compound. Thus, G-Rp1 treatment dose- and time-dependently enhanced DNA fragmentation as assessed by PI staining (Fig. 2B), similar to that observed in previous studies [9] and [20]. Unlike previous approaches that have examined apoptosis-inducing mechanisms of G-Rp1 [20], this study used proteomic analysis to determine the mode of action of G-Rp1. As Fig. 3A depicts, many proteins bands could be detected in LoVo cells using 2-DE. After preparing whole cell lysates see more with G-Rp1-treated LoVo cells, the blotting patterns between these samples were compared.
As shown in Fig. 3A, most band patterns Orotidine 5′-phosphate decarboxylase appeared similar, although several bands (indicated with white arrows in Fig. 3A) were strikingly increased in G-Rp1-treated cells. To determine which bands showed higher expression patterns, we further analyzed the biochemical properties of these bands using proteomic analysis. As Fig. 3B indicates, the bands were revealed to be Apo-A1; a major component of high-density lipoprotein that regulates reverse cholesterol transport by modulating the levels of cholesterol and phospholipids in cells [21], and helps control inflammatory responses and oxidative stress [22]. The induction level of Apo-A1 in G-Rp1-treated LoVo cells was also confirmed by immunoblotting analysis of other cancer cells such as SNU-407, DLD-1, SNU-638, AGS, KPL-4, and SK-BR-3. Thus, Fig. 4 clearly indicates that the protein level of Apo-A1 was strikingly enhanced with G-Rp1 treatment, suggesting its involvement in the mechanism of action of G-Rp1. To evaluate further the regulatory mechanism of G-Rp1-mediated apoptosis, small-interfering (si)RNA for Apo-A1 was introduced into the G-Rp1-treated LoVo cells. As shown in Fig.