Antiproliferative Effects of Methanolic Extracts of Cryptocarya concinna Hance Roots on Oral Cancer Ca9-22 and CAL 27 Cell Lines Involving Apoptosis, ROS Induction, and Mitochondrial Depolarization

Cryptocarya-derived natural products were reported to have several biological effects such as the antiproliferation of some cancers. The possible antioral cancer effect of Cryptocarya-derived substances was little addressed as yet. In this study, we firstly used the methanolic extracts of C. concinna Hance roots (MECCrt) to evaluate its potential function in antioral cancer bioactivity. We found that MECCrt significantly reduced cell viability of two oral cancer Ca9-22 and CAL 27 cell lines in dose-responsive manners (P < 0.01). The percentages of sub-G1 phase and annexin V-positive of MECCrt-treated Ca9-22 and CAL 27 cell lines significantly accumulated (P < 0.01) in a dose-responsive manner as evidenced by flow cytometry. These apoptotic effects were associated with the findings that intracellular ROS generation was induced in MECCrt-treated Ca9-22 and CAL 27 cell lines in dose-responsive and time-dependent manners (P < 0.01). In a dose-responsive manner, MECCrt also significantly reduced the mitochondrial membrane potential in these two cell lines (P < 0.01–0.05). In conclusion, we demonstrated that MECCrt may have antiproliferative potential against oral cancer cells involving apoptosis, ROS generation, and mitochondria membrane depolarization.


Introduction
Oral squamous cell carcinoma (OSCC) is a type of cancer that frequently occurs in oral cavity. Although it is comparatively easy to clinically inspect by a dentist or to detect by some OSCC tumor markers [1,2], this carcinoma is usually ignored by patients especially for the early stage. Subsequently, OSCC is frequently diagnosed at advanced stages which then lead to high mortality [3]. Therefore, the drug development of antioral cancer is still necessary and remains to be a challenge.
Natural products have improved the drug discovery for anticancer therapy [4]. For example, some anticancer
These two cell lines were humidly incubated at 37 ∘ C with 5% CO 2 in the humid atmosphere.
C. concinna was identified by one of the authors (Ih-Sheng Chen) and its roots were collected at Mudan, Pingtung County, Taiwan, in May 2004. A voucher specimen (Chen 6153) has been deposited in the Herbarium of the School of Pharmacy, College of Pharmacy, Kaohsiung Medical University. The dried roots of C. concinna were processed by slicing and cold methanol-extraction for three times at room temperature. Finally, the solution was evaporated under reduced pressure to yield the methanolic extract (MECCrt). MECCrt was stored at −20 ∘ C and dissolved in dimethyl sulfoxide (DMSO) before treatment.

Cell
Viability. Cell viability was measured by the CellTiter 96 AQueous one solution cell proliferation assay (MTS) (Promega Corporation, Madison, WI, USA) as previously described [11]. Ca9-22 and CAL 27 cell lines were seeded at a density of 1 × 10 5 and 2 × 10 5 cells per well in a 6-well plate, respectively. After plating for 24 h, these cells were incubated with different concentrations of MECCrt for 24 h and finally subjected to a MTS assay applying an ELISA reader at 490 nm.

Cell Cycle Progression and Sub-G1 Population.
Propidium iodide (PI, Sigma, St. Louis, MO, USA) was added to stain the cellular DNA content [29]. In brief, 3 × 10 5 cells per well in 6 well plates were plated for 24 h and then treated with vehicle (DMSO; 1 L/2 mL culture medium) as a control or 5, 10, 15, 20, and 25 g/mL of MECCrt for 24 h. After exposure termination, cells were centrifuged, washed twice with PBS, fixed overnight with 70% ethanol, and centrifuged. Subsequently, the cell pellets were resuspended in 50 g/mL PI reagent and stand for 30 min at 37 ∘ C in darkness. Cell cycle distribution was evaluated by a flow cytometer (BD Accuri C6; Becton-Dickinson, Mansfield, MA, USA) and a BD Accuri C6 Software (version 1.0.264).

Apoptosis.
To validate apoptosis in MECCrt-treated oral cancer cells, annexin V (Strong Biotect Corporation, Taipei, Taiwan) [30]/PI (Sigma, St Louis, MO, USA) method was used [31]. Briefly, 3 × 10 5 cells per well in 6 well plates were plated for 24 h and then treated with vehicle or indicated concentrations of MECCrt for 24 h. Subsequently, apoptotic cells were stained for 30 min with 100 L binding buffer containing 2 L of annexin-V-fluorescein isothiocyanate (FITC) stock (0.25 g/ L) and 2 L of PI stock (1 mg/mL). Finally, it was suspended with 400 L PBS for analysis of a flow cytometer (BD Accuri C6; Becton-Dickinson) and its software.

Intracellular ROS.
The dye 2 ,7 -dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect ROS by its fluorescence change [7]. Cells at the density of 3 × 10 5 in 2 mL medium per well in 6 well plates were plated for 24 h. centrifugation, cell pellets were resuspended in 1 mL PBS before analyzing by a flow cytometer (BD Accuri C6; Becton-Dickinson) and its software.
2.6. Mitochondrial Membrane Potential. MitoProbe DiOC 2 (3) assay kit (Invitrogen, Eugene, OR, USA) was applied to analyze mitochondrial membrane potential (MMP) as described previously [10]. Briefly, 3 × 10 5 cells in 2 mL medium per well in 6 well plates were plated for 24 h. After MECCrt treatment, 10 L of 10 M DiOC 2 (3) was added per well and incubated in a cell culture incubator for 20 min. After being harvested, cells were washed and resuspended in 1 mL PBS for analysis using a flow cytometer (BD Accuri C6; Becton-Dickinson) and its software.

Statistical
Analysis. The significance of differences was determined by Student's -test compared with the test data with the vehicle controls. Data are expressed as means ± SDs.

Apoptosis of MECCrt-Treated Two Oral Cancer Cell Lines.
To validate the possible outcome of apoptosis in MECCrtinduced sub-G1 accumulation of these two oral cancer cells, annexin V/PI profiles of flow cytometry were generated (Figure 3(a)). In Figure 3 (Figures 4(c) and 4(d)).  0 g/mL 5 g/mL 10 g/mL  0 g/mL 5 g/mL 10 g/mL       Moreover, the anticancer effect for trunk bark of C. infectoria-derived methanol extracts was reported to be cytotoxic to KB cells [24]. KB cells were regarded as oral epidermal carcinoma, however, it was recently validated to have marker chromosomes and DNA finger printings of human cervical cancer HeLa cells (http://www.ncbi.nlm.nih.gov/mesh?Db=mesh&term= KB+Cells) [32]. Accordingly, the anticancer effect of oral cancer by the bioactive compounds from Cryptocarya plant remains unclear. Conversely, we here demonstrate the antioral cancer effect of methanolic extracts of a Cryptocarya species for the first time, using two OSCC cell lines Ca9-22 and CAL 27.

Discussion
In several anticancer drugs [6,7,10,11,[33][34][35][36], ROS generation is one of the common strategies to inhibit cancer cell proliferation. ROS plays a vital role in early stages of apoptosis [37] and leads to MMP depolarization [38,39]. Escaping apoptosis is demonstrated to be involved in the drug resistance of cancer cells [40,41]. To enhance apoptotic induction of anticancer drugs may interfere the drug resistance if there. In the present study, we observed that apoptosis was inducible by MECCrt in two OSCC cell lines as it was demonstrated by sub-G1 monitoring and annexin V/PI assay. We also found that MECCrt significantly induced the ROS level and reduced the MMP level in two oral cancer cell lines in dose-responsive ways. These findings suggest that oxidative stress may be involved in the MECCrt-induced antiproliferative effect in two oral cancer Ca9-22 and CAL 27 cell lines. However, the role of oxidative stress in MECCrt need to be further examined by the ROS scavenger such as N-acetylcysteine [42] to confirm if raised ROS has played a critical role in the process of apoptosis. Furthermore, the ROS may generate nonapoptotic effect like autophagy described in literature [43,44]. Therefore, it was warranted to further investigate the role of autophagy in MECCrt-treated oral cancer cell lines in future.

Conclusions
We demonstrated the antiproliferative and apoptotic effects of MECCrt through ROS generation and mitochondrial depolarization in two OSCC cell lines. Therefore, these results suggest that MECCrt has anticancer potential for oral cancer therapy.