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. 2015 Jun 12:9:3017-30.
doi: 10.2147/DDDT.S84448. eCollection 2015.

Oleanolic acid induces mitochondrial-dependent apoptosis and G0/G1 phase arrest in gallbladder cancer cells

Huai-Feng Li  1 , Xu-An Wang  1 , Shan-Shan Xiang  1 , Yun-Ping Hu  1 , Lin Jiang  1 , Yi-Jun Shu  1 , Mao-Lan Li  1 , Xiang-Song Wu  1 , Fei Zhang  1 , Yuan-Yuan Ye  1 , Hao Weng  1 , Run-Fa Bao  1 , Yang Cao  1 , Wei Lu  1 , Qian Dong  1 , Ying-Bin Liu  1
Affiliations

Oleanolic acid induces mitochondrial-dependent apoptosis and G0/G1 phase arrest in gallbladder cancer cells

Huai-Feng Li et al. Drug Des Devel Ther. .

Abstract

Oleanolic acid (OA), a naturally occurring triterpenoid, exhibits potential antitumor activity in many tumor cell lines. Gallbladder carcinoma is the most common malignancy of the biliary tract, and is a highly aggressive tumor with an extremely poor prognosis. Unfortunately, the effects of OA on gallbladder carcinoma are unknown. In this study, we investigated the effects of OA on gallbladder cancer cells and the underlying mechanism. The results showed that OA inhibits proliferation of gallbladder cancer cells in a dose-dependent and time-dependent manner on MTT and colony formation assay. A flow cytometry assay revealed apoptosis and G0/G1 phase arrest in GBC-SD and NOZ cells. Western blot analysis and a mitochondrial membrane potential assay demonstrated that OA functions through the mitochondrial apoptosis pathway. Moreover, this drug inhibited tumor growth in nude mice carrying subcutaneous NOZ tumor xenografts. These data suggest that OA inhibits proliferation of gallbladder cancer cells by regulating apoptosis and the cell cycle process. Thus, OA may be a promising drug for adjuvant chemotherapy in gallbladder carcinoma.

Keywords: apoptosis; cell cycle arrest; gallbladder carcinoma; mitochondrial pathway; oleanolic acid.

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Figures

Figure 1
Figure 1
Chemical structure of oleanolic acid.
Figure 2
Figure 2
Oleanolic acid inhibits proliferation and viability of gallbladder cancer cells. Notes: (A) Cell viability and IC50 were measured by MTT assay. (B) GBC-SD and NOZ cells were treated with various concentrations of oleanolic acid (0, 3, 6, and 9 μmol/L) for 48 hours and then allowed to form colonies in fresh medium without oleanolic acid for 14 days. (C) Detailed information on colony formation is shown. All data are presented as the mean ± standard deviation and are from three independent experiments. *P<0.05, **P<0.01 versus the control group.
Figure 3
Figure 3
Oleanolic acid induces apoptosis in gallbladder cancer cells. Notes: (A) GBC-SD and NOZ cells treated with OA (0, 30, 60, and 90 μmol/L) for 48 hours were stained with Annexin V-FITC/PI and analyzed by flow cytometry. (B) The percentage of apoptotic cells is presented as the mean ± standard deviation. (C) Changes in apoptotic nuclear morphology were observed by Hoechst 33342 staining and visualized by fluorescent microscopy. The results shown are representative of data from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus the control group. Abbreviations: FITC, fluorescein isothiocyanate; OA, oleanolic acid; PI, propidium iodide.
Figure 3
Figure 3
Oleanolic acid induces apoptosis in gallbladder cancer cells. Notes: (A) GBC-SD and NOZ cells treated with OA (0, 30, 60, and 90 μmol/L) for 48 hours were stained with Annexin V-FITC/PI and analyzed by flow cytometry. (B) The percentage of apoptotic cells is presented as the mean ± standard deviation. (C) Changes in apoptotic nuclear morphology were observed by Hoechst 33342 staining and visualized by fluorescent microscopy. The results shown are representative of data from three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus the control group. Abbreviations: FITC, fluorescein isothiocyanate; OA, oleanolic acid; PI, propidium iodide.
Figure 4
Figure 4
Oleanolic acid decreases the mitochondrial membrane potential in gallbladder cancer cells. Notes: (A) GBC-SD and NOZ cells treated with OA (0, 30, 60, and 90 μmol/L) for 48 hours were treated with Rhodamine 123, retention of which was measured by flow cytometry. (B) Corresponding histogram showing percentage cell survival, with data expressed as the mean ± standard deviation. The results shown are representative of data from three independent experiments. *P<0.05, **P<0.01 versus the control group. Abbreviations: OA, oleanolic acid; FITC, fluorescein isothiocyanate.
Figure 5
Figure 5
Oleanolic acid induced apoptosis via regulation of members of the caspase family and Bcl-2 family in gallbladder cancer cells. Notes: (A) GBC-SD and NOZ cells were treated with OA (0, 30, 60, and 90 μmol/L) for 48 hours. Expression levels of Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, cleaved PARP, and cytochrome C were detected by Western blot analysis, and GAPDH was used as a loading control. The band densities of Bcl-2 and Bax are listed under the band, and the band density of control group is designated as 1.0. (B) Relative ratio of Bcl-2 to Bax. *P<0.05, **P<0.01 versus the control group. Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OA, oleanolic acid.
Figure 6
Figure 6
Oleanolic acid induces G0/G1 phase arrest by regulating expression of cell cycle-related proteins in gallbladder cancer cells. Notes: (A) Cell cycle phases of OA-treated cells were evaluated by flow cytometry. (B) Data are expressed as the mean ± standard deviation (n=3). (C) Expression levels of cyclin D1 and CDK4 were measured by Western blot analysis, and GAPDH was used as a loading control. Results are representative of three independent experiments. *P<0.05 versus the control group. Abbreviations: OA, oleanolic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 6
Figure 6
Oleanolic acid induces G0/G1 phase arrest by regulating expression of cell cycle-related proteins in gallbladder cancer cells. Notes: (A) Cell cycle phases of OA-treated cells were evaluated by flow cytometry. (B) Data are expressed as the mean ± standard deviation (n=3). (C) Expression levels of cyclin D1 and CDK4 were measured by Western blot analysis, and GAPDH was used as a loading control. Results are representative of three independent experiments. *P<0.05 versus the control group. Abbreviations: OA, oleanolic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure 7
Figure 7
Oleanolic acid exhibits an anticancer effect in vivo. Notes: (A) NOZ cells were subcutaneously injected into the right flank of nude mice. The mice were then administered 0.2 mL of vehicle (10% dimethyl sulfoxide and 90% phosphate-buffered saline) or OA (75, 150 mg/kg) intraperitoneally every 2 days for up to 15 days. Photographs of five representative mice (n=10) from each group are presented to show the sizes of the resulting tumors. (B, C) Tumors were excised from the animals and weighed. (D) Hematoxylineosin staining and IHC staining of Ki-67 in xenograft tumors. (E) Scoring of Ki-67 IHC staining. **P<0.01 versus the control group. Abbreviations: OA, oleanolic acid; HE, hematoxylineosin; IHC, immunohistochemistry.
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