Figure 1.
Structures of oleanolic acid and OEOA.
Oleanolic acid, 3β-hydroxy-olea-12-en-28-oic acid, MW: 456.71; OEOA, olean-12-eno[2,3-c] [1], [2], [5]oxadiazol-28-oic acid, MW: 480.69.
Figure 2.
OEOA inhibited cell proliferation in leukemia cell lines.
Different cell lines were treated with various concentrations of OEOA or OA for 2 days. Cell growth was measured by MTT assay: (A) K562, (B) HEL, (C) Jurket (D) HEKneo, and (E) HepG2, MCF-7 and HeLa cells. Data are mean ± SEM of three independent experiments (* p<0.05).
Figure 3.
OEOA did not induce cell death in K562 and HEL cells.
K562 (A & C) and HEL (B & D) cells were treated with OEOA (1 µM) for 6 days. Cell viability was measured by trypan blue exclusion as described in Materials and Methods. Data are mean ± SEM of three independent experiments (* p<0.05).
Figure 4.
OEOA attenuated phosphorylation of Rb protein in K562 and Jurket cells.
(A) K562 and Jurket cells were treated with OEOA (0.1–10 µM) for 2 days, and the cell lysates were subjected to Western blot analysis for p-Rb and Rb. Actin served as an equal loading control. Histograms in (B) show the relative expression of p-Rb (normalized to actin) as compared to the vehicle-treated cells. Results were representative blots from three separate experiments, (* p<0.05).
Figure 5.
OEOA induced G1 cell cycle arrest in K562 cells.
(A) Cells were incubated with OEOA (1 or 10 µM) for 24 h. The distribution of cell cycle was examined by PI staining method. The table summarized the distribution of cells in OEOA-treated or control cells. Data represented mean ± SEM of three independent experiments (* p<0.05). (B) K562 cells were cultured in the presence of OEOA (1 or 10 µM) for 2 days. Total proteins were collected for Western blot analysis to detect the expression of p27, Cdk4, Cdk6, Cyclin D1, Cyclin E and RAMP. Actin served as an equal loading control. Histograms on the right show the relative expression of various proteins (normalized to actin) as compared to the control cells. Results were representative blots from three separate experiments, (* p<0.05).
Figure 6.
OEOA promoted erythroid differentiation in K562 cells.
K562 cells were treated with OEOA (0.1–10 µM) for 2 days. Total RNA was reverse transcribed and subjected to real time-PCR analysis with primers specific to γ-globin (A) and cd41b (B), respectively. hprt1 served as an internal housekeeping gene control. Data were expressed as fold change to the control cells as mean ± SEM of three independent experiments (* p<0.05). (C) K562 cells were treated with OEOA (0.1–10 µM) for 2 days. Western blot analysis of Bcr-Abl and Erk1/2 was performed. Actin served as an equal loading control. Histograms on the right show the relative expression of various proteins (normalized to actin) as compared to the control cells. Results were representative blots from three separate experiments, (* p<0.05).
Figure 7.
OEOA was retained for a longer period in mouse blood plasma than OA.
MS-MS product ion mass spectra of OEOA (A) and OA (B). (C) K562 cells were treated with OA or OEOA (1 µM) for 6 h. Culture media and cell lysates were collected at indicated time points for HPLC-MS/MS analysis. Data was represented as mean ± SEM in cell lysate compared to total exposure, n = 2 (* p<0.05). (D) Concentrations of OA and OEOA in mouse plasma after intraperitoneal injection. Blood was collected from mice at different time points after single administration of OA and OEOA. Data was represented as mean ± SEM, n = 2 animals per time point (* p<0.05).