Longikaurin A, a natural ent-kaurane, induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC.

Figure 1

LK-A inhibits cells proliferation and induces G2/M arrest in SMMC-7721 and HepG2 cells. (a) Chemical structure of LK-A. (b) HCC cells proliferation was assessed by PrestoBlue. Cells were treated with 0, 2, 4, 6, 8 and 10 μM of LK-A for 24, 36 and 48 h. At 36 h, IC₅₀ value for each cell lines determined from PrestoBlue results are as follows: SMMC-7721=2.75 μM, HepG2=5.13 μM, BEL-7402=6.83 μM, Huh7=7.12 μM and LO2=9.69 μM. (c) SMMC-7721 and HepG2 cells colonied formation with or without LK-A treatment. (d) LK-A caused a G2/M arrest. Cells were treated with DMSO and varying concentrations of LK-A (0, 1.5, 3.0, 4.5 μM) for 36 h. The cell cycle distributions were analyzed by flow cytometry. Histograms display the percentage of cell cycle distribution. Cell Number, peak value of phase; Columns, means; bars, S.D. (n=3). *P<0.05, **P<0.01 and ***P<0.001, significantly different compared with control by t-test

Figure 2

Evidence that LK-A induced apoptosis. (a) Cell morphological alterations and nuclear changes associated with SMMC-7721 and HepG2 cells after LK-A treatment were assessed by staining with Hoechst 33342 and visualized by fluorescence microscopy. (b) FACS analysis via Annexin V/PI staining was used to identify apoptosis induced by LK-A. The percentage of cell cycle distribution was shown as the mean±S.D. from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. (c) Cells were treated with or without various concentrations of LK-A for 36 h. Caspase-3, -8, and PARP levels were determined by western blot

Figure 3

LK-A induces SMMC-7721 G2/M phase arrest though downing regulation of Skp2. (a) The fold change in cell cycle-related genes after LK-A treated for 36 h. (b) SMMC-7721 cells were treated with or without LK-A for 36 h. The expression of cell cycle-related proteins were measured by western blot. (c) SMMC-7721 cells were transfected with pcDNA-SKP2 and pcDNA3.1 (+), respectively. After treatment with 3 μM LK-A for 36 h, protein lysates were prepared and analyzed by western blot. (d) After transfection and treatment with 3 μM LK-A 36 h, the cell cycle distribution of SMMC-7721 cells were determined by flow cytometry. Cell Number, peak value of phase. The percentage of cell cycle distribution was shown as the mean±S.D. from three independent experiments. **P<0.01; ns, not significant

Figure 4

The accumulation of ROS production induced by LK-A is required for cell apoptosis and necessary for activating JNK/c-Jun pathway. (a) Effect of LK-A on ROS generation. SMMC-7721 cells were loaded with DCFH/DA for 30 min and then treatment with LK-A for 3 h. The mean DCF fluorescence was measured by flow cytometry. (b) Cells were pre-incubated for 1 h in the presence or absence of NAC (10 mM), and then LK-A (3 μM) and incubated for 36 h. Induction of apoptosis was determined by flow cytometry. The data shown represent means±S.D. of three independent experiments. ***P<0.001 versus NAC-treated control group. (c) Effects of NAC on the expression of Caspase-3, -8 and PARP. Protein lysates were prepared from SMMC-7721 cells after treatment with 3 μM LK-A for 36 h in the presence or absence of NAC and analyzed by western blot. (d) Effects of LK-A on ERK, JNK and p38 MAP kinase activation in SMMC-7721 cells. SMMC-7721 cells were treated with various concentrations (0, 1.5, 3.0, 4.5 μM) of LK-A for 36 h and analyzed by western blot. (e) Cells were pre-incubated for 1 h in the presence or absence of SP600125 (5 μM), and 3 μM LK-A was added for an additional 36 h. The percentage of cell apoptosis was shown as the mean±S.D. from three independent experiments. ***P<0.001 compared with SP-treated control group, SP, SP600125. (f) Cells were pre-incubated for 1 h in the presence or absence of SP600125 (5 μM), and then treated with 3 μM LK-A for 36 h, followed by western blot analysis of apoptosis-related proteins. (g) Cells were pre-incubated for 1 h in the presence or absence of NAC (10 mM), and then treated with 3 μM LK-A for 36 h, followed by western blot analysis of JNK/p-JNK and c-Jun expression

Figure 5

LK-A inhibits liver cancer tumor xenograft growth in vivo. (a) SMMC-7721 cells were subcutaneously inoculated into the right flank of nude mice. The mice were randomly divided into four groups (n=6) and treated intraperitoneally with LK-A (3 mg/kg or 6 mg/kg), 5-Fu (10 mg/kg) and DMSO (dissolved in sodium chloride, control) every 3 days for 4 weeks. The resulting tumors were excised from the animals after treatment. (b) LK-A treatment resulted in significantly lower tumor weight compared with controls. **P<0.01, ***P<0.001. (c and d) Tumor volumes and body weights were measured every 3 days for 4 weeks. (e) The cleaved Caspase-3, Skp2 and Ki-67 expression in tumor xenograft tissues was examined using immunohistochemistry

Figure 6

No obviously organ-related toxicity was found. Non-tumor-bearing mice were intraperitoneally treated with LK-A (6 mg/kg) or DMSO (dissolved in sodium chloride, control) every 3 days for 4 weeks. After the experiment, animals were sacrificed and the organs were fixed in formalin overnight and processed for paraffin embedding. The paraffin-embedded blocks were sectioned and stained by hematoxylin and eosin