Rotundic Acid Induces DNA Damage and Cell Death in Hepatocellular Carcinoma Through AKT/mTOR and MAPK Pathways

Abstract

Hepatocellular carcinoma (HCC) is the fourth largest cause of cancer-related deaths worldwide with limited therapeutic interventions. Renewed interest in natural products as drug leads has resulted in a paradigm shift toward the rapid screening of medicinal plants for the discovery of new chemical entities. Rotundic acid (RA), a plant-derived triterpenoid, has been anecdotally reported to possess anti-inflammatory and cardio-protective abilities. The present study highlights the anti-cancer efficacy of RA on HCC in vitro and in vivo. The inhibitory effects of RA on HCC cell viability was determined by MTT. Soft agar colony formation and clonogenic assays also showed that RA inhibited HCC cell proliferation. Flow cytometry, confocal, and western blot results further indicated that RA induced cell cycle arrest, DNA damage, and apoptosis by modulating the AKT/mTOR and MAPK pathways. Besides the suppression of migration and invasion, tube formation and VEGF-ELISA revealed the anti-angiogenic abilities of RA on HCC. Moreover, RA also inhibited tumor growth in a HepG2 xenograft mouse model. To our best knowledge, this is the first extensive study of the anticancer activity of RA on HCC. The results demonstrate that RA could be a potential drug candidate for HCC treatment.

Keywords: DNA damage; anti-angiogenesis; apoptosis; hepatocellular carcinoma; rotundic acid.

Figure 1

Inhibitory effects of rotundic acid (RA) on HCC and endothelial cell growth. MTT assays were performed to determine the effects of RA on HCC and endothelial cell growth. RA inhibited the cell viability and proliferation of (A) HepG2 cells, (B) SMMC-7721 cells in a time and dose-dependent manner. The dose survival curves of (C) HepG2 cells, (D) SMMC-7721 cells, (E) HUVEC's, and (F) LO2 cells after RA treatment. RA exhibited no significant toxicity on the normal hepatic LO2 cells. Data are represented as mean ± SD (n = 3; ^{$$, ##}, **p ≤ 0.01 and ^#, *p ≤ 0.05 vs. control).

Figure 2

RA restricts the clonogenic properties of HCC cells in vitro. Clonogenic/Colony formation assay was performed to study the long-term effects of RA on the survival and proliferation of HCC cells. RA inhibited the colony forming ability of (A,B) HepG2 and (C,D) SMMC-7721 cells in a concentration-dependent manner. Data are represented as mean ± SD (n = 3, and **p ≤ 0.01, *p ≤ 0.05 vs. control).

Figure 3

RA attenuates extracellular matrix-independent growth of HCC cells. RA treatment limited the anchorage-independent colony forming ability of (A,B) HepG2 and (C,D) SMMC-7721 cells in a dose-dependent manner. Data are represented as mean ± SD (n = 3, magnification = 40×, scale bar = 200 μm and **p ≤ 0.01, *p ≤ 0.05 vs. control).

Figure 4

RA restricts the migration and invasion of HepG2 cells by inhibiting MMP-2/MMP-9 secretion. (A,C) RA inhibited the migration of HepG2 cells in a dose-dependent manner. (B,D) RA treatment weakened the ability of HepG2 cells to invade through the basement membrane in a concentration-dependent manner. RA restricted the secretion of matrix metalloproteinases (E) MMP-2 and (F) MMP-9 from HepG2 cells in a concentration-dependent manner. Data are expressed as mean ± SD. Images for migration and invasion assays were taken at 100 and 200× magnifications and the scale bars are 50 and 20 μm, respectively (n = 3 or more; ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 vs. control).

Figure 5

Inhibitory effects of RA on the migration and invasion of SMMC-7721 cells. SMMC-7721 cells were treated with various concentrations of RA and subjected to migration and invasion assays. RA treatment prevented the (A,B) wound closure and (C,D) extracellular matrix invasion of SMMC-7721 cells in a concentration-dependent manner (n = 3 or more; **p ≤ 0.01, *p ≤ 0.05 vs. control).

Figure 6

RA induces S-phase cell cycle arrest and apoptosis in HepG2 hepatocellular carcinoma cells. (A,B) RA treatment resulted in S-phase cell cycle arrest in HepG2 cells. (C,D) Morphological changes in the cell nucleus and nuclear fragmentation were observed upon RA treatment. (E,F) The pro-apoptotic effects of RA on HepG2 cells were determined by Annexin V-FITC/PI staining. (G) Western Blot analysis of apoptosis-related proteins like PARP, caspase-3, Bax, and Bcl2 indicated that RA induced apoptosis in HepG2 cells in a concentration-dependent manner. Densitometric analysis of (H) Bax/Bcl2 ratio, (I) cleaved caspase 3, and (J) cleaved PARP obtained in WB. Confocal images were taken at 400× magnification and the scale bar is 20 μm (n = 3; **p ≤ 0.01, *p ≤ 0.05 vs. control).

Figure 7

RA inhibits VEGF secretion and endothelial cell-mediated angiogenesis in HCC. (A,B) HUVECs grown in the conditioned medium of RA-treated HepG2 cells produced shortened and severely broken 3D-tubular networks on the basement membrane matrix in a dose-dependent manner. (C) RA treatment abated VEGF release from HepG2 cells. Moreover, RA treatment also impeded the (D) migration and (E) invasion of HUVEC cells in a concentration-dependent manner. (F) RA modulated the expressions of AKT/mTOR and MAPK pathway molecules in the endothelial cells (n = 3 or more, 100× magnification, scale bar = 50 μm, **p ≤ 0.01 and *p ≤ 0.05 vs. control).

Figure 8

RA modulates AKT/mTOR and MAPK signaling pathways. Representative immunoblots showing the expression levels of AKT/mTOR and MAPK pathway related proteins in RA-treated HepG2 cells in an (A) concentration-dependent and (C) time-dependent manner. Densitometric analysis of AKT/mTOR and MAPK pathway related proteins in RA-treated HepG2 cells in an (B) concentration-dependent and (D) time-dependent manner (n = 3; ***p ≤ 0.001, **p ≤ 0.01 and *p ≤ 0.05 vs. control/0 hr., respectively).

Figure 9

RA abrogates in vivo tumor formation in Balb/c nude mice. (A) Schematic representation of the experimental protocol. (B) Tumor-bearing control and RA-treated mice. (C) Mice were euthanized after 60 days; tumors were extracted, weighed, and photographed. Significant reduction of (D) tumor volumes and (E) tumor weights were observed in the RA-treated group vs. the control group. (F) RA did not yield any adverse effects on the mice body weights (**p ≤ 0.01, *p ≤ 0.05). Western blot of tumor tissue lysates indicated that RA. (G) attenuated the expressions of Ki-67, CD-31, and induced apoptosis in tumor cells. (H) RA modulated the AKT/mTOR and MAPK pathways in vivo (n = 3).