Suppression of oral cancer by induction of cell cycle arrest and apoptosis using Juniperus communis extract

Abstract

The oral cancer incidence rate is slowly increasing and is now the fifth leading cause of cancer-related death due to its high metastasis and recurrence rate. Juniperus communis is used as a traditional Chinese medicine and has been proven to have anti-cancer activity against neuroblastomas. In the present study, we further investigated the anti-cancer mechanisms of J. communis extract (JCo) on oral cancer and evaluated the synergistic effects of JCo combined with 5-fluorouracil (5-FU). We found that JCo inhibited oral cancer cell growth, and that JCo might be less cytotoxic to normal cells than to cancer cells. After JCo treatment, cell cycle arrest was observed at the G0/G1 phase through modulation of p53/p21 and Rb signaling. JCo also caused an increase in the sub-G1 phase and cell apoptosis via the intrinsic and extrinsic apoptosis pathways. JCo combined with 5-FU presented a synergistic effect to reduce cell viability. In conclusion, JCo inhibited oral cancer cell growth by inducing cell cycle arrest and activating cell apoptosis, and JCo significantly synergized with 5-FU. JCo might have the potential to be an adjuvant and a new therapeutic drug for oral cancer treatment.

Keywords: 5-Fluorouracil (5-FU); Juniperus communis; Oral cancer; cell apoptosis; cell cycle; synergistic effect.

Figure 1. Anti-proliferative activity of JCo on the three cell lines

OECM-1, SVEC and MDCK cells were treated with JCo (0–200 μg/ml) or 5-FU (0–20 μg/ml) for 24, 48 and 72 h, and the cell viability was determined using the MTT assay. The data are shown as mean ± SD.

Figure 2. Flow cytometric analysis of cell cycle in OECM-1 cells

OECM-1 cells were treated with JCo for the allocated time period, stained with PI, and analyzed using flow cytometry. (A) Cell cycle distribution, (B and C) cell cycle statistical data. *, #: It was significant difference increasing or decreasing between treating and non-treating groups (P<0.05).

Figure 3. Effects of JCo on the expression of cell cycle-related proteins

The cells were treated with JCo (60 μg/ml) for the allocated time period and then lysed by western blotting to detect protein expression. *, #: It was significant difference increasing or decreasing between treating and non-treating groups (P<0.05).

Figure 4. Apoptotic effects of JCo in OECM-1 cells

After JCo exposure, cells were collected and observed. (A) The morphology of OECM-1 cells. (B) The sub-G₁ phase of the cell population. *: It was significant difference increasing between control and treating groups (P<0.05). (C) The cells were stained with PI (red) and TUNEL (green) reagents; white arrows indicate apoptotic bodies; red arrows indicate anoikis; blue arrows indicate chromatin condensation; yellow arrows indicate DNA fragments.

Figure 5. Effects of JCo on the expression of cell apoptosis pathway proteins

The OECM-1 cell line was treated with JCo (60 μg/ml) for the allocated time periods and then lysed by Western blotting to detect protein expression. *, #: It was significant difference increasing or decreasing between treating and non-treating groups (P<0.05).

Figure 6. JCo combined with 5-FU

The cells were plated and subjected to a combination of JCo and 5-FU. After 24 and 48 h incubation, removing the drugs, adding MTT solution and dissolving with DMSO, the cell viability was measured using the MTT assay. (A) The cell viability of JCo combined with 5-FU. (B) The CI values of JCo combined with 5-FU in 24 and 48 h. Results are shown as mean ± SD. Data were compared for each drug separately and taken together using the Student’s t-test. Significant differences (#P < 0.05) were observed in the two drugs concerning cytotoxicity.