Diosgenin increased DDX3 expression in hepatocellular carcinoma

Abstract

Liver cancer, one of the most common malignant tumors occurred worldwide, has emerged as a main health trouble and accounts for leading cancer-related death. Diosgenin is provided as an important material in the pharmaceutical industry, and is used to manage various medical troubles such as cancer because of its multiple bioactivities. DEAD box polypeptide 3 (DDX3) is involved in cancer biogenesis and modulates cancer progression. However, the role of DDX3 in human hepatocellular carcinoma (HCC) has not been fully understood. In the present study, we investigated the anti-tumor effects of diosgenin on HCC cells and whether DDX3 is involved in its antitumor activity. We observed that diosgenin dramatically inhibited cell proliferation, triggered apoptotic cell death, induced G2/M phase arrest, suppressed cell migration and invasion abilities. Moreover, the expression of DDX3 was measured and the results showed that DDX3 was significantly up-regulated upon diosgenin exposure. All together, our data indicated that diosgenin shows a cytotoxic effect on HCC cells and has potential therapeutic values for HCC patients.

Keywords: DDX3; diosgenin; hepatocellular carcinoma; invasion; proliferation.

Figure 1

Diosgenin inhibited human hepatocellular carcinoma (HCC) cells growth. HepG2 and SMMC-7721 cells (5×10³ cells/well) were seeded in 96-well plates and treated with 0-100 µM of diosgenin. After exposed to diosgenin for 24 h, 48 h and 72 h, MTT assay was carried out to detect the cell viabilities. *P<0.05, compared to the control groups treated with DMSO.

Figure 2

Diosgenin induced apoptotic cell death in HCC cells. HepG2 and SMMC-7721 cells were inoculated to 6-well plates at a density of 1×10⁵ cells/well and were exposed to 0, 50 and 75 µM of diosgenin for 48 h. Annexin V-FITC/PI staining and flow cytometry assay were performed to detect the apoptotic cell death.

Figure 3

Diosgenin induced cell cycle arrest in HCC cells. Diosgenin-induced HCC cell cycle arrest was measured by PI staining and flow cytometry. Cells were harvested with trypsin and washed twice with PBS. The collected cells were fixed with 70% ice-cold ethanol and maintained at 4°C. After washed twice with PBS, cells were suspended with PBS containing 100 μg/ml RNase and 50 mg/ml PI at 37°C for 30 min in the dark. Cell cycle stage was measured using flow cytometer.

Figure 4

Inhibitory effects of diosgenin on HCC cell migration. (A) HepG2 and SMMC-7721 cells were incubated in 6-well plates till grew to around 90% confluence. Rectangular lesions were made on the cell monolayer by using sterile tips. Cells were washed with PBS and debris and detached cells were carefully sucked. After 20 h, cells that migrated into the wound area were photographed using an inverted microscope. (B) Quantitative results are illustrated for (A). *P<0.05, **P<0.01 vs control.

Figure 5

Diosgenin suppressed HCC cell invasion. Left panel: The inhibitory effect of diosgenin on HCC cell invasion was detected by Transwell chambers assay. 24-well Transwell chambers with Matrigel coating were subjected to cell invasion assay for HepG2 and SMMC-7721 cells. The cells were inoculated into the upper chamber of the insert in the 24-well plate. In the lower chamber, 500 μl complete DMEM was added. After 20 hours, the cells were allowed to invade the lower chamber containing FBS. The non-invasive cells remaining on the upper surface of the membrane were removed. The invasive cells attached to the lower surface of the filter membrane were dyed with Calcein-AM for 10 min and then counted under a light microscope. Right panel: Quantitative results of left panel. *P<0.05, **P<0.01 vs control.

Figure 6

Diosgenin up-regulated DDX3 expression. A. mRNA level of DDX3 was measured by q-PCR in diosgenin treated HepG2 and SMMC-7721 cells using power SYBR Green PCR Master Mix on an ABI Prism 7500 Fast Real-Time PCR system. The relative expression of DDX3 was calculated. GAPDH was used as an endogenous reference. B. The expression of DDX3, Cyclin D1, p21, Notch-1, E-cadherin, and β-catenin was detected by Western blotting analysis in diosgenin treated HepG2 and SMMC-7721 cells. *P<0.05, **P<0.01 vs control.