Excisanin A suppresses proliferation by inhibiting hypoxia- inducible factor-1α expression in human hepatocellular carcinoma cells

Purpose: To investigate the effect of excisanin A on human hepatocellular carcinoma cells as well as to elucidate its mechanism of action. Methods: Molecular docking was used to determine the binding characteristics of excisanin A to HIF-1α protein. The transcriptional activation and viability of excisanin A were assessed using Luciferase reporter and MTT assay. The HIF-1α protein in the nucleus was assayed using western blot and immunofluorescence. HIF-1α and VEGF mRNA levels were evaluated using reverse-transcription polymerase chain reaction (RT-PCR). Cell proliferation was determined by flow cytometry, as well as by EdU and clonogenic assays. In vivo tumor growth was assessed in a murine xenograft model of SKHep1 cells. Results: Excisanin A inhibited HIF-1α transcriptional activation, as well as HIF-1α protein synthesis (p < 0.001). Excisanin A also reduced VEGF protein and mRNA expressions (p < 0.001). In addition, the compound inhibited the proliferation of hepatocellular carcinoma cells. and tumor growth in the xenograft tumor model. Conclusion: Excisanin A is a potent HIF-1α inhibitor, supporting its potential development for human hepatoma therapy.


INTRODUCTION
When oxygen levels are very low, HIF-1α transfers to the nucleus. Then, HIF-1α and HIF-1β begin the transcriptional program [4]. HIF-1α levels are closely related to VEGF expression involved in tumor angiogenesis and proliferation index [5]. As HIF-1α could promote tumor angiogenesis and growth, targeting HIF-1α may be a critical drug for cancer treatment. Many types of Isodon (Labiatae), a species of plant widely distributed throughout China, exhibit antiinflammatory and anti-bacterial activities. Isodon diterpenoids have been identified to possess intense antitumor activity and very low toxicity, thus receiving considerable attention from both phytochemical and biological fields [6]. In this research, we discovered that excisanin A inhibited HIF-1α protein. Moreover, excisanin A suppressed the HIF-1α downstream genes such as VEGF, which is very important for tumor growth. Excisanin A also inhibited tumor cell proliferation. Based on these results, we further demonstrated that in vivio, excisanin A showed significant antitumor activity, which elicited no apparent toxicity in experimental animals.

EXPERIMENTAL Cell lines and chemicals
Hep3B and SK-Hep1 cells (ATCC, USA) were routinely cultured in DMEM including 10% FBS and 1% penicillin/streptomycin. The hypoxic was kept in atmosphere at 1% O2, 5 % CO2 and 37 o C. Dimethyl sulfoxide, cycloheximide and MG132 were purchased from Sigma-Aldrich. Excisanin A (≥ 98%) was extracted from Isodon Macrocalyxin D, and the structure of excisanin A is displayed in Figure 1 A.

Luciferase reporter assay
pGL3-HRE-Luciferase plasmid and pRL-CMV were co-transfected into SK-Hep1 cells. Then, cells were incubated with Excisanin A and under hypoxia for 12 h. Determined luciferase activity with a luciferase assay kit (Promega, USA).

MTT assay
After cells adhered, they were treated with excisanin A for 24 h. Then, living cells changed MTT (Sigma-Aldrich) to formazan, which dissolved in DMSO, producing blue-purple color.

Immunofluorescence assay
Immunofluorescence assay was performed as described previously. Briefly, the cell nuclei were labeled with DAPI. The HIF-1α proteins appeared green and the nuclei appeared blue under confocal microscopy (Nikon, Japan) [6].

SK-Hep1 cells treated with Exisanin A and isolated
total RNA according to the manufacturer's instructions (Invitrogen, USA). GAPDH served as a housekeeping gene control. The bands were visualized by 3% agarose gel under UV light and photographed.

Flow cytometry analysis
Cell cycle was detected by propidium iodide staining, as described previously [8].

Clonogenic assay
Cells treated with excisanin A. Two weeks later, Colonies were fixed with 10 % formaldehyde and tinted with 1% crystal violet. The image was photographed with a camera.

EdU assay
Cells were plated in 96-well culture plates. Twenty-four hours later, cells were treated with excisanin A. Cells were incubated with EdU. Then, cells treated 1 × Apollo ® reaction cocktail and the cell nucleus was tinted with Hoechst 33342.

Tumor xenografts
All mouse protocols were approved by the Yanbian University Institutional Animal Care and Use Committee. 1 × 10 7 cells in PBS were injected into the subcutaneous. Five days later, BALB/C nude mice (n = 5/group, five-week, male, Vital River, China) were given excisanin A three times per week. The body weight and tumor was measured with a caliper for 40 days and it was calculated following equation: (length × (width) 2 )/2.

Immunohistochemical analysis
The paraffin-embedded sections of the tumor tissue were prepared for H&E staining and immunohistostaining. Histopathological changes and positive stained area was photographed by a photo microscope [2,9].

Statistics
Data are expressed as mean ± SD, and were compared using one-way ANOVA and Tukey's multiple comparison tests with the aid of SPSS software. P < 0.05 was considered statistically significant.

Excisanin A was identified as a HIF-1 inhibitor
We performed HIF-1α reporter assays, and found excisanin A suppressed hypoxia-induced reporter gene expression in a concentrationdependent manner (Figure 1

Excisanin A decreases HIF-1 protein levels
In Hep3B and SK-Hep1 cells, excisanin A significantly inhibited HIF-1 expression in a concentration-dependent manner in 1% O2 (Figure 3 A). And, excisanin A inhibited the accumulation of HIF-1α at each time point in 1% O2 (Figure 3 B). Next, immunofluorescence staining was performed. In Hep3B and SK-Hep1 cells, excisanin A (30 μM) nearly totally suppressed hypoxia-induced HIF-1α protein in the nucleus after 12 h of treatment (Figure 3 C).

Excisanin A inhibits HIF-1α protein synthesis
Excisanin A decreased HIF-1α protein levels in the presence of proteasome inhibitor (MG-132) (lanes 3 and 5 in Figure 4 A). The results indicated that excisanin A significantly inhibits HIF-1α protein synthesis. The use of the cycloheximide (CHX) inhibits protein synthesis. As displayed in Figure 4 B, while HIF-1α levels declined promptly in CHX treatment, excisanin A did not affect the HIF-1α degradation rate. Thus, excisanin A does not facilitate HIF-1α degradation. HIF-1α mRNA level was not altered with Excisanin A treatment in Hep3B and SK-Hep1 cells ( Figure 5 A and 5 B). These findings suggested that excisanin A inhibited HIF-1α protein expression but did not inhibited HIF-1α mRNA expression.

Excisanin A inhibits cell cycle progression in the G1 phase
Hep3B cells were treated with excisanin A resulted in an increase of G1 phase cells from 56.28 to 61.14, 67.65, and 71.84 % in the concentrations of 5, 10, and 30 μM excisanin A. Similarly, treatment of SK-Hep1 with excisanin A also increases the number of G1-phase cells ( Figure 6). In Figure 7, excisanin A inhibited cyclin D1 and c-Myc expression, thereby blocking cell cycle at the G1 phase.

Excisanin A inhibits the proliferation
EdU assay confirmed that excisanin A suppressed EdU-positive cells number, indicating that excisanin A suppressed Hep3B and SK-Hep1 cells proliferation in vitro (Figure 8 A). Clonogenicity of cells in excisanin A-treated groups were decreased with increasing concentration (Figure 8 B). Furthermore, MTT assay showed that excisanin A inhibits cell proliferation in both normoxia and hypoxic conditions (Figure 9 C and D).

Excisanin A inhibits tumor growth in a xenograft tumor model
As shown in Figure 10 A, excisanin A inhibits growth of hepatocellular carcinoma, while mice body weight was unchanged (Figure 10 B). After the last treatment, the tumor was harvested, and the representative tumor block was displayed in Figure 10 C. Consistent with above findings, excisanin A decreased HIF-1α protein in tumors (Figure 10 D).
Excisanin A inhibited the HIF-1α and VEGF expression in tumor (Figure 10 E). Taken together, our experiment confirmed that HIF-1α downregulation by excisanin A contributed to inhibit tumor growth and angiogenesis in tumor tissues.

DISCUSSION
Hypoxic conditions induce activation of the HIF-1α sub-unit, an important transcription factor for adaptation to hypoxic conditions within the tumor microenvironment. Thus, the development of HIF-1α inhibitors in cancer treatment is a huge challenge. In the process of trying to find anticancer agents from natural products, excisanin A, a natural ent-kaurane diterpenoid, was isolated from the traditional Chinese medicine Isodon MacrocalyxinD. Here, we found that excisanin A suppressed HIF-1α protein synthesis.
VEGF, a downstream target of HIF-1α, exerts multiple effects on tumor angiogenesis, including stimulating new blood and lymphatic vessel formation and increasing vascular permeability [12][13][14][15][16]. VEGF promotes the delivery of peripheral oxygen via stimulating angiogenesis, which involves the migration, proliferation and differentiation of the endothelial cell and the proteolysis of extracellular matrix. As expected, excisanin A decreased VEGF mRNA and protein levels.
We found that Excisanin A reduces cell proliferation by arresting the cell cycle and inhibits cyclin D1 and c-Myc protein level. Moreover, Figure 6C and 6D showed that the effects of excisanin A on cell proliferation are consistent in normoxia or hypoxic conditions. We examined HIF-1α and VEGF expression in the sections of tumor and found that excisanin A suppressed their expression in tumor tissues.

CONCLUSION
The findings of this study demonstrate that excisanin A suppresses HIF-1α protein synthesis in Hep3B and SK-Hep1 cells. Furthermore, excisanin A suppresses cancer cell proliferation by arresting cell cycle at the G1 phase. This mechanism may partly explain the anti-tumor mechanism of excisanin A, thus supporting its development as an anticancer drug.