Twenty gram of the fresh ginger root was grinded. Then, 100ml of absolute ethanol solvent (Merck, Germany) was added into the flask together with the ginger root. The process was conducted at 78.4^oC for 12 hours, with 5-6 heat cycles in a heating mantle (MTOP, Republic of Korea) for 1 hour.

100 g of the fresh ginger root was grinded and placed in a flask. Then, 500 ml of absolute ethanol was placed into the Ultrasonic Cleaner machine (JPS, CA, USA). The ultrasound process was conducted at a temperature of 15^oC for 30 minutes. The mixture after the ultrasound was filtered. The process was continued by refining 2-3 times with Whatman filter paper (Sigma Aldrich, MO, USA) to remove residue completely.

100 g of the fresh ginger root was soaked in 500 ml of absolute ethanol in Becher for 72 hours. After 72 hours, the whole mixture was filtered to remove residue.

Human hepatocarcinoma cell line HepG2 was purchased from ATCC (Manassas, VA, USA). The cell line was grown in DMEM/F12 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO, USA) and 5% antibiotic (Thermo Fisher Scientific). HepG2 cells were put into a 5% CO₂ humidified atmosphere at 37˚C. All the experiments were performed in triplicate.

The HepG2 cell line was seeded in a Hanging drop 96-well plate (3D Biomatrix, Ann Arbor, MI, USA) with DMEM/F12 medium supplemented with 10% FBS and 5% antibiotic. The cell density was 5000 cells per well (in 50 μl volume). The cells tended to cluster together and form spheroids after three days.

Cells were labeled with 3 μl of Annexin V-FITC (Miltenyi Biotec, Germany) and 3 μl of Propidium Iodide (PI) (Miltenyi Biotec) in 500 μl of binding buffer for 15 min to detect apoptotic and necrotic cell death using a FACSCalibur Flow Cytometer (BD, Franklin Lake, NJ, USA). Data were analyzed by Cellquest software (BD).

After 2 days for 2D culture and 3 days for 3D culture, the cells in 96-well plates were treated with the ginger root extract by Soxhlet, Sonication, and Maceration. The concentration of these extracts was diluted 2-fold serially from 2000 µg/ml to 15 µg/ml. Following 48h of incubation, the cells were evaluated for viability by staining with Alamar Blue and reading the measurements through a DTX 880 machine (Beckman Coulter, CA, USA).

To detect cell death after treatment with the extracts, cells were stained with 10 μl of PI. After that, the cells were incubated in a 5% CO₂ humidified atmosphere at 37˚C. Cell images were captured after 1 h by a Zeiss Axio Imager fluorescence microscope (Zeiss, Germany).

The raw data from the cytotoxicity assay on the 2D and 3D culture cells were calculated for IC₅₀ values. Accordingly, based on the untreated group (negative control) with 100% live cells, the calculation would detect the concentration of 50% of cells. The experiments were repeated three times and processed statistically with 95% confidence intervals. Statistical analysis was done using the GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).

HepG2 cells were treated with 3 kinds of ginger root extracts (from Maceration (G1), Soxhlet (G3), and Sonication (G5) methods) in 2D cell culture conditions to select for the extract with the highest cytotoxicity on HepG2 cells. The results showed that Soxhlet extract had the strongest cytotoxic activity on HepG2 with an IC₅₀ of 83.3 ± 0.9189 µg/ml, while the IC₅₀ values of the extract from methods of Maceration and Sonication were 159 ± 7.6 µg/ml and 284 ± 5.116 µg/ml, respectively. These results showed that the ginger root extract from Soxhlet showed the stronger antitumor cytotoxicity against HepG2.

Figure 2

After the treatment of HepG2 with ginger root extract in 2D conditions (Figure 1), we continuously used ginger root extract to treat HepG2 in 3D conditions (Figure 2). The results showed no significant differences in IC₅₀ between the ginger root extracts from the different preparation methods; the IC₅₀ of G3 extract was 228 ± 33.52 µg/ml, while IC₅₀ of G1 was 341 ± 3.93 µg/ml and of G5 was 603.7 ± 56.33 µg/ml. The data show that G3 has more potent cytotoxicity compared with G1 and G5.

As aforementioned, from the results of IC₅₀ of ginger root extract on 2D and 3D culture conditions, it turned out that G3 (the ginger root extract by Soxhlet method) had the strongest anti-toxicity on HepG2 in both 2D and 3D culture conditions. Therefore, we selected G3 to evaluate its ability to induce apoptosis of HepG2 cells. We treated HepG2 cells with G3 extract at the concentrations of 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml, or 0 µg/ml (control). The results demonstrated G3 extract induced apoptosis in a dose-dependent manner. At the concentration of 200 µg/ml, almost all cells underwent apoptosis, with 78.77% in late apoptosis.

To perform Annexin-V staining assay, cells were stained with PI and Hoechst 33342 and observed under microscopy. The results showed that at the concentration of 200 µg/ml (Figure 3D), 400 µg/ml (Figure 3E), and 800 µg/ml (Figure 3F), almost all cells were stained with PI (red color) (Figure 4). At the high microscope magnification, the nuclei of cells treated with G3 extract at 200 µg/ml were condensed and disintegrated (Figure 5). These kinds of features indicate that the cells underwent apoptosis.

Figure 3

Figure 4

Figure 5

We also evaluated how the G3 extract impacted HepG2 spheroids in 3D culture conditions. After culturing HepG2 cells in 3D and following spheroid development, the spheroids were treated with G3 extract and compared with G1 and G5 extracts (Figure 6). The HepG2 spheroids showed to break apart and to stain with PI at the concentration 250 µg/ml of Ginger extract, while untreated spheroids still remained the initial shape. At the concentrations of 500 and 1000 µg/ml, the G1, G2, and G3 extracts all had an impact on the HepG2 spheroids. These results demonstrate that G3 has strong activity on HepG2 spheroids.

We then selected the extract G3 to evaluate its ability to induce apoptosis by flow cytometry. We observed that even in untreated spheroids, there was about 30% of cells which underwent apoptosis (Figure 7). This was likely due to the core of HepG2 spheroids, where there is a lack of oxygen and nutrients. However, after spheroids were treated with G3 extract, the apoptotic populations increased to 40% at the IC₅₀/2; 77.04% at IC₅₀; almost all cells were in late apoptosis at IC₅₀ x 2 and IC₅₀ x 4. These results demonstrated the impact of G3 extract on HepG2 spheroids. Together with the ability to break the HepG2 spheroids apart, the G3 extract induced the HepG2 cells in spheroids to undergo apoptosis.

Figure 7

Phytochemical extracts from fruits and vegetables are increasingly being shown to exert potent antioxidant and anti-proliferative effects. It is widely becoming appreciated that chemo-preventative agents offer superior potential in the long term over chemotherapeutic agents. Life style and dietary habits have been identified as major risk factors, particularly in cancer growth and progression.

Ginger root is extensively used in the form of a fresh paste or dried powder to flavor food and beverages. Recently, a study showed that treatment with 2 mg/ml of ginger with 31 mg/ml of gelam honey inhibited the growth of most HT29 cells and induced apoptosis in a dose-dependent manner, with the combined treatment yielding the highest apoptosis rate. This was caused by the downregulation of gene expression of Akt, mTOR, and Raptor, while cytochrome C and caspase 3 genes where shown to be upregulated26 Recently, ginger root extract also showed anti-leukemia and anti-drug resistant effects27.

In the present study, we show that ginger extracted by Soxhlet has stronger cytotoxic activity on HepG2 cells than extracts from Maceration and Sonication. The ginger extract could induce apoptosis of HepG2 cell lines cultured in both 2D and 3D conditions. Sonication and Maceration methods were not strong enough to break down the cell wall of the ginger plant to release certain active agents, but the Soxhlet method was capable of that. Recently, some studies have shown that ginger polysaccharides induced cell cycle arrest and apoptosis of HepG2 through up-regulation of Bax, caspase-3, and p53, and down-regulation of Bcl-228. We observed the typical apoptotic morphological changes in HepG2 cells, including cell shrinkage and detachment, and nuclear condensation and fragmentation in 2D culture conditions of HepG2. This was in accordance with a study by Elkady et al.29 which demonstrated that ginger extract induced the upregulation of p53 and p21, which inducing down-regulation of cyclin D1 and cyclin-dependent kinanse-4 expression, resulting in G2/M2 arrest.

We firstly demonstrated the anti-tumor effect of ginger extract in 3D conditions. The extract induced HepG2 spheroids to break part from the original round shape. We also found that ginger extract induced cells in the HepG2 spheroids to undergo apoptosis. This might have caused the cells to lose their cell-to-cell contact, resulting in the disassembly of the HepG2 spheroids.

6-shogaol is a major active constituent of dietary ginger that has been demonstrated to inhibit cell proliferation in osteosarcoma30, lung cancer31, and colon cancer32, through increasing apoptosis pathways which lead to a decrease of expression levels of anti-apoptotic proteins (e.g. survivin and Bcl-2) and increasing pro-apoptotic proteins (e.g. Bax). In HepG2 cell lines, 6-shogaol and 6-gingerol (two active components in ginger) showed anti-invasion and anti-metastasis properties in vitro33.

Taken together, ginger extract possesses constituents that are able to block proliferation and induce the death of HepG2 cells in both 2D and 3D culture conditions.

Ginger has a long history of use in the diet and medicine of humans. This study contributes further evidence of the potency of ginger root extract. We show that ginger possesses potent cytotoxicity on HepG2 cells grown in 2D or 3D, suggesting its potential benefit as an anticancer agent in disease treatment. Some studies on the constituents of ginger have further documented its ability to induce apoptosis in vitro. Further studies of ginger root extract, and its active constituents, in vivo using animal models, are needed to ascertain its anticancer potency prior to its application for human disease.

G1: ginger root extract by Maceration method

G3: ginger root extract by Soxhlet method

G5: ginger root extract by Ultrasonic method

NFκB: nuclear factor kB

TNF- α: tumor necrosis factor alpha

IL: interleukin

VEGF: vascular endothelial growth factor

We have no conflict of interest to declare.

Sinh Nguyen, Phuc Van Pham conceived and planned the experiments. Phuc Hong Vo, Thao Duy Nguyen, Nghia Minh Do, Bao Huu Le, Duong Thuy Dinh performed the experiments. Kiet Dinh Truong, Sinh Nguyen analysed the data. Sinh Nguyen wrote the manuscript. Phuc Pham and Kiet Dinh Truong revised the manuscript.

This work was supported by the Vietnam National University, Ho Chi Minh City, Vietnam, under grant A2015-18-01.

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