Ginsenoside (GS), a ginseng saponin, is a name derived from glycosides of ginseng, distinguishing it from the saponins of other plants. So far, about 200 GSs have been reported, and they show multifaceted pharmacological activities due to their unique steroidal structure (Ratan et al., 2021). GS is divided into major GS and minor GS according to their amount, and natural ginseng contains more than 90% of major GS such as Rb1, Rb2, Rc, Re, and Rf (Christensen, 2009). However, minor GS such as Rg2, Rg3, Rh1, Rh2, F1, F2, and CK are found in small amounts in natural ginseng (Kim, 2018). Major GS is not easily absorbed into the body as it is combined with polymer components, but is converted to minor GS, which is an easy-to-ingest form, by hydrolysis by intestinal microorganisms and gastrointestinal metabolism. Minor GS has a higher absorption rate in the body and has better pharmacological activity than Major GS (Fukami et al., 2019; Jin et al., 2019; Yang et al., 2020).

Ginseng is divided into three types: fresh ginseng, white ginseng, and red ginseng. Among them, Rg3, Rh1, Rh2, and Rs4, which are unique components of red ginseng, are generated during the heat treatment process of red ginseng, and the minor GS content present in trace amounts is increased (Park, 2019). Rh2 has high biological activities such as learning and memory (Hou et al., 2013), restoring immune deficiency (Qian et al., 2019), anti-inflammatory, and antioxidant (Choi et al., 2013). The antitumor activity of Rh2 has been reported in lung cancer (Chen et al., 2019), non-small cell lung cancer (Li et al., 2018), liver cancer (Chen et al., 2019), gastric cancer (Qian et al., 2016), colon cancer (Yang et al., 2016), colorectal cancer (Han et al., 2016), breast cancer (Lee et al., 2018), prostate cancer (Tong-Lin et al., 2018), cervical cancer (Shi et al., 2018; Kim et al., 2020), pancreatic cancer (Tang, 2013), leukemia (Xia et al., 2017), and ovarian cancer (Kim and Choi, 2016).

Cancer is a major public health problem worldwide. In the case of Korea, the number of new cancer patients in 2019 was 255,000, an increase of 3.6% from 246,000 in 2018, and is increasing every year from 218,000 in 2015. As of 2019, the ranking of cancer incidence for men was lung cancer, stomach cancer, colorectal cancer, prostate cancer, liver cancer, and thyroid cancer, and for women, breast cancer, thyroid cancer, colorectal cancer, stomach cancer, lung cancer, and liver cancer were in that order. The incidence of gastric cancer, colorectal cancer, liver cancer, and cervical cancer has decreased over the past decade, but breast cancer, prostate cancer, and thyroid cancer are on the rise (Ministry of Health and Welfare of South Korea, 2021). However, the unexpected COVID-19 pandemic that recently began in 2019 has had negatively impacted cancer diagnosis and treatment.

Rh2

Rh2 was prepared in a freeze-dried powder state by treating ginseng with recombinant β-glucosidase according to the method of Lim (2011). The chemical formula is C36H62O8, and the molecular weight is 622.87 g/mol (Zhang et al., 2021). Rh2 powder was dissolved in DMSO to make a 100 mM solution first, and then diluted again to make 20, 40, 60, 80 mM solutions and stored at -20℃ until use.

Cell culture and GS treatments

The cell lines used in this study were purchased from the Korean cell line bank (KCLB) in a frozen state, and the types of cancer cells and the corresponding media are shown in Table 1.

Table 1. Cancer cell lines and cell culture medium used in this study.

Cell line	KCLB No.	Cancer origin	Culture medium (Biowest)
Du145	30081	Prostate	High glucose DMEM
HCT116	10247	Colon; Colorectal	High glucose DMEM
Huh-7	60104	Liver	High glucose DMEM
MCF-7	30022	Breast	RPMI 1640
MDA-MB-231	30026	Breast	RPMI 1640
SK-N-MC	30010	Brain	High glucose DMEM

Cells of each cell line were cultured with the appropriate cell culture medium supplemented with 10% (v/v) fetal bovine serum (FBS, Biowest) and 1% (v/v) penicillin/streptomycin (100 unit/mL, Welgene) at 37℃ in a humidified 5% CO₂ incubator for 24-72 hours. The seeding density according to the cell culture container is shown in Table 2. When the cells were proliferated to an appropriate concentration, each concentration of Rh2 was treated by 1 µL per 1 mL of culture medium so that the final concentrations were 0, 20, 40, 60, 80, 100 µM.

Table 2. Cell culture container and cell seeding density for each experiment purpose.

	Seeding density	Experiment
T-25 cell culture flask	0.7 × 106	qPCR
6-well plate	0.3 × 106	Cell migration assay
24-well plate	0.05 × 106	Apoptosis/necrosis analysis
96-well plate	0.01 × 106	Cell viability

Cell viability and IC₅₀ assay

After cell seeding, the old medium of each well of the 96-well plate cultured for 48 hours was replaced with a new medium containing Rh2 at a concentration of 0-100 µM, and cultured for another 24 hours. Then, the existing medium was removed, and 100 µL of the culture medium containing 10 µL of EZ-Cytox (DoGen) solution was added to each well and incubated at 37℃, 5% CO₂ for 2 hours. After that, it was put into an ELISA reader (DiaTeK) and optical density (OD) values were measured at 450 nm.

Cell migration assay

After cell seeding in a 6-well plate, when the cells proliferated to 80-90%, a 100 µL-sized white tip was used to scratch a single line in the center of the cell monolayer to make a wound, and the images were taken with a phase-contrast inverted microscope. Then, after replacing the existing medium with new culture medium containing Rh2, the wounds were observed under a microscope and images were taken every day while culturing for 48 hours at 37℃ and 5% CO₂ to compare the changes.

Florescence analysis of apoptosis/necrosis

When the cells seeded in the 24-well plate proliferate up to 60-70%, the old medium were replaced with fresh culture medium containing Rh2 at the IC₅₀ concentration corresponding to each cancer cell, and then cultured at 37°C, 5% CO₂ condition. After 24 hours, all of the culture medium was removed from the cells and washed twice with phosphate buffered solution (PBS) and assay buffer. Then a buffer solution containing Apopxin Green Indicator (abcam) and 7-AAD (abcam) was added to the cells, followed by fluorescence staining at room temperature for 30 minutes. After washing twice again with assay buffer, the cells were observed under bright field, green, red, and blue fluorescent lights using an inverted microscope. The cell images were merged with the Image J program and presented as a result.

Statistical analysis

Each experiment was repeated three or more times and the values obtained were presented as mean ± SE and analyzed with the student t-test static between the Rh2 treated and the untreated control group at p < 0.05.

Cell viability

0, 20, 40, 60, 80, and 100 µL of Rh2 were treated to 6 types of cancer cells, their absorbances were measured at 450 nm, and cell viability was calculated based on the absorbance of the untreated control group and presented as a graph. Also, based on this, the IC₅₀ concentration of Rh representing 50% cell proliferation inhibition was calculated and displayed.

First, in the prostate cancer cell line, Du145, treated with various concentrations of Rh2, there was no difference in cell viability between the control and Rh2 groups when treated with 40 µM. However, the viability decreased sharply to 43.1% in the 60 µM treatment, and few cells survived in the 80 µM treatment. The IC₅₀ concentration of Rh2 for Du145 cells was confirmed to be 57.50 µM (Fig. 1). Tong-Lin et al. (2018) showed that the cell viability of Du145 was about 50% when treated with 100 µM of Rh2, and it can be seen that the IC₅₀ concentration of Rh2 for Du145 cells is twice as high as that of this study.

Figure 1.Cell viability of Du145, HCT116 and Huh-7 exposed to various concentrations of Rh2 and IC₅₀ concentration of Rh2 for each cancer cell.

In the colorectal cancer cell line, HCT116, the cell viability decreased as the Rh2 concentration increased, and the cell viability decreased to less than half at 40 µM and 60 µM. Therefore, the IC₅₀ concentration of Rh2 for HCT116 cells was 44.28 µM (Fig. 1). Although the IC₅₀ concentration of Rh2 is slightly lower than that of Du145 cells, it differs from Du145 cells in that cell viability gradually decreases as the concentration of Rh2 increases. Even when exposed to 100 µM of Rh2, the cell viability of HCT116 is maintained at 30%, indicating that complete inhibition of cell proliferation is hard. In study of Yang et al. (2016), HCT116 cells was treated with 0, 25, 50, 100, 150, and 200 µM of Rh2, and it was found that cell proliferation was inhibited by more than 80% in the Rh2 200 µM treatment group, twice the highest concentration used in this experiment.

In the Liver cancer cell line, Huh-7, the cell viability decreased sharply to 35.3% at the lowest treatment concentration of 20 µM Rh2 in the experiment. Accordingly, the IC₅₀ concentration was calculated to be 13.39 µM (Fig. 1). Huh-7 had the lowest IC₅₀ concentration of Rh2 among the 6 cancer cell lines used in this study.

The cell viability was compared after the two types of breast cancer cell lines were treated with Rh2 by concentration. The cell viability of MCF-7, a hormone-dependent breast cancer cell line, was inhibited by only about 27% after treatment with 60 µM Rh2. In 80 µM treatment, the cell viability decreased sharply to 10.3% compared to the control group. The IC₅₀ concentration of Rh2 against MCF-7 cells was 67.48 µM, which was the highest among the 6 cancer cells used in the study (Fig. 2). Lee et al. (2018) reported that cell viability decreased only in the 20 µM treated group among MCF-7 cells treated with 20 µM and 50 µM. This study and Lee et al. (2018) show that cell proliferation of MCF-7 cells is inhibited only when they are exposed to a certain concentration or more of Rh2.

Figure 2.Cell viability of MCF-7, MDA-MB-231 and SK-N-MC exposed to various concentrations of Rh2 and IC₅₀ concentration of Rh2 for each cancer cell.

On the other hand, MDA-MB-231, a triple-negative breast cancer cell line, was sensitive to Rh2 treatment, and the cell viability was reduced even at a low concentration of 20 µM, and hardly proliferated at 40 µM. The IC₅₀ concentration of Rh2 against MDA-MB-231 cells was 27.00 µM, which was the second lowest after the IC₅₀ concentration of Rh2 against Huh-7. Even in the same breast cancer cells, the treatment effect of Rh2 was much better than that of MCF-7 (Fig. 2). Peng et al. (2022) reported that when MCF-7 and MDA-MB-231 cells were treated with 0-80 µM of Rh2, IC₅₀ concentrations were 40-63 µM and 33-58 µM, respectively, similar to the results of this experiment.

The effect of Rh2 on the neuroblastoma cell line, SK-N-MC, has rarely been reported. In the SK-N-MC cells exposed to Rh2, the cell viability was significantly decreased to 22.2% at 40 µM, and thereafter, the viability did not decrease any more even when the concentration was increased. The IC₅₀ concentration of Rh2 for SK-N-MC was 32.4 µM (Fig. 2).

Most of the cancer cells exposed to Rh2, except for colorectal cancer cells, HCT116, showed a sharp decrease in cell viability at a certain concentration or higher, suggesting the importance of determining the therapeutic concentration. The cancer cells with the lowest IC₅₀ concentration were liver cancer cells, Huh-7 (13.39 µM), and the cells with the highest IC₅₀ concentration were breast cancer cells, MCF-7 (67.48 µM). In addition, breast cancer cell, MDA-MB-231 (27.00 µM), Neuroblastoma, SK-N-MC (32.40 µM), colorectal cancer cell, HCT116 (44.28 µM), prostate cancer cell, and Du145 (57.50 µM) showed the lowest IC₅₀ concentration in that order. Independent of the IC₅₀ concentration, even at the highest concentration of 100 µM, HCT116, SK-N-MC, and Huh-7 cells maintained a cell viability of 20 to 30% or more, so it was not easy to completely suppress cell survival. However, in Du145, MCF-7, and MDA-MB-231 cells, cell proliferation was sufficiently inhibited by treatment with an appropriate concentration of Rh2.

Cell migration assay

Cell migration assay is also called wound healing assay. After making a wound on the cell monolayer, the ability of cells to migrate and proliferate can be determined by observing whether the wound is filled over time. In addition, this is an experiment conducted for the purpose of finding out whether the treated cancer cell proliferation inhibitor works effectively to suppress this ability of cancer cells. This study was conducted to investigate whether the ginsenoside Rh2 is effective in inhibiting the migration of each cancer cell and whether the effective concentration is correlated with the IC₅₀ concentration based on the cell viability.

Fig. 3-5 are micrographs taken at 0 hour and 48 hours after wounding 6 types of cancer cells and treating Rh2 by concentration. In all cancer cells, wound closure occurred more than 90% in the group not treated with Rh2, (-) Rh2, indicating that the cancer cells migrated to the wound. However, when the concentration of Rh2 is increased, the wound did not close and became intact or rather enlarged. The Rh2 concentrations for which images are not presented are because the cells fell off from the bottom of the cell culture plate, it was impossible to photograph.

Figure 3.Cell migration assay results of Du145 and HCT116 cells treated with various concentrations of Rh2 (200×).

Figure 4.Cell migration assay results of breast cancer cells treated with various concentrations of Rh2 (200×).

Figure 5.Cell migration assay results of Huh-7 and SK-N-MC cells treated with various concentrations of Rh2 (200×).

Except for MCF-7 cells, cell migration of Du145, HCT116, MDA-MB-231, Huh-7, and SK-N-MC was inhibited at 40 µM of Rh2. The effect seems to have started from the previous concentration, between 20 µM and 40 µM. In particular, in Du145, MDA-MB-231, and SK-N-MC cells, significant cell detachment occurred even in the 60 µM treatment group, so that the wound shape could not be maintained. However, in MCF-7 cells, cell migration was inhibited from 60 µM and wound closure did not proceed. MCF-7, which had the highest IC₅₀ concentration, and HCT116 and Huh-7, which showed a cell viability of 20 to 30% even at 100 µM, were confirmed to have higher concentrations at which complete cell detachment was confirmed than other cancer cells. Table 3 shows the comparison of the IC₅₀ concentration of Rh2 and the cell migration inhibition starting concentration for each cancer cell.

Table 3. IC₅₀ and cell migration inhibition concentration of Rh2 according to cancer cell type.

Cell line	Concentration (µM) of Rh2		Result
	IC50	Cell migration inhibition
Du145	57.50 µM	20-40 µM	Fig. 3.3
HCT116	44.28 µM	20-40 µM	Fig. 3.3
Huh-7	13.39 µM	20-40 µM	Fig. 3.5
MCF-7	67.48 µM	40-60 µM	Fig. 3.4
MDA-MB-231	27.00 µM	20-40 µM	Fig. 3.4
SK-N-MC	32.40 µM	20-40 µM	Fig. 3.5

Apoptosis and necrosis analysis

In order to analyze apoptosis or necrosis of cancer cells, even if Rh2 is treated, the cells must proliferate to a certain level or more. Therefore, each cancer cell was treated with a concentration one level lower than the IC₅₀ concentration of Rh2 and observed. For example, the IC₅₀ concentration of Du145 is 57.50 µM and cell migration inhibition seems to have started between 20 µM and 40 µM, so the treatment concentration was determined to be 30 µM. Based on the same standards, HCT116 and MCF-7 were treated with 40 µM, and MDA-MB-231 was treated with 30 µM. In this experiment, only 4 types of cancer cells were used, but Huh-7 and SK-N-MC cells were excluded because most of the cells were separated during the fluorescence staining and washing process and analysis was impossible.

The image of the apoptosis/necrosis fluorescence analysis is shown in Fig. 6-9. In Du145, HCT116, MCF-7, and MDA-MB-231 cells, almost no necrosis (red fluorescence) was observed and only apoptosis (green fluorescence) was observed, suggesting that Rh2 effectively and significantly induces the programmed cell death, apoptosis, of various cancer cells.

Figure 6.Apoptosis and necrosis images of Rh2-treated Du145 cells through fluorescence staining analysis. Apoptotic cells and necrotic cells were stained with Apopxin Green indicator and 7-AAD, respectively (400×).

Figure 7.Apoptosis and necrosis images of Rh2-treated HCT116 cells through fluorescence staining analysis. Apoptotic cells and necrotic cells were stained with Apopxin Green indicator and 7-AAD, respectively (400×).

Figure 8.Apoptosis and necrosis images of Rh2-treated MCF-7 cells through fluorescence staining. Apoptotic cells and nectrotic cells were stained with Apopxin Green indicator and 7-AAD, respectively (400×).

Figure 9.Apoptosis and necrosis images of Rh2-treated MDA-MB-231 cells through fluorescence staining. Apoptotic cells and necrotic cells were stained with Apopxin Green indicator and 7-AAD, respectively (400×).

In this study, 6 types of cancer cells were treated with Rh2, among minor GS known to have high absorption and excellent pharmacological action, and their anticancer activity was analyzed. The cancer cell lines used in the study were Prostate cancer cell line, Du145, Colorectal cancer cell line, HCT116, Liver cancer cell line, Huh-7, Breast cancer cell lines, MCF-7 and MDA-MB-231, Brain cancer cell line, SK-N-MC. For all of these carcinomas, it is important to find natural products that can replace the side effects of existing cytotoxic anticancer drugs and induce programmed cell death, that is, apoptosis, in tumorigenesis control and treatment.

As a result of analyzing the cell viability of cancer cells after treatment with Rh2 at concentrations of 0, 20, 40, 60, 80, and 100 µL, it can be divided into a cancer cell group in which the cell viability sharply decreases above a certain concentration of Rh2 and a cancer cell group in which cell viability decreases in inverse proportion as the concentration of Rh2 increases. In addition, a cancer cell group in which cell proliferation was not inhibited by 20 to 30% or less was confirmed even at 100 µL, the highest concentration of Rh2 treatment. Based on these characteristics of cells and the IC₅₀ concentration of Rh2 for each cancer cell, Rh2 was effective in inhibiting the cell proliferation of Du145, MCF-7 and MDA-MB-231. As a result of the migration assay of each cancer cell treated with Rh2, inhibition of wound closure was observed between 40-60 µL of Rh2 in MCF-7 and between 20-40 µL in the remaining cells. In apoptosis and necrosis analysis by fluorescence staining after Rh2 treatment at the IC₅₀ concentration for each cancer cell, only apoptosis was observed in all cancer cells. In conclusion, ginsenoside Rh2 is effective on all 6 types of cancer cells, and especially has a stronger efficacy on prostate cancer cells and TNBC breast cancer cells. Several pathways to apoptotic events on various cancers have been widely studied and published with very detailed interconnected paths. In future studies, Further research will be expected for more clear impact on apoptotic events along with different compounds of ginsenosides and their bio-transformed compounds.

We appreciate Dr. W.T. Im’s lab for providing ginsenosides Rh2 as a raw material for this study.

Conceptualization, S.E.K., G.J.J.; data curation, S.E.K., M-H.L.; formal analysis, S.E.K., H-M.J., W-T.I, J.L.; funding acquisition, G.J.J.; investigation, S.E.K., M-H.L.; methodology, S.E.K., M-H.L., J.L.; project administration, G.J.J.; resources, S.E.K., G.J.J., W-T.I.; supervision, G.J.J.; roles/writing - original draft, S.E.K., M-H.L., H-M.J., J.L.; writing - review & editing, S-H.K., G.J.J.

This work was supported by the Technology development Program (S2860036) funded by the Ministry of SMEs and Startups (MSS, Korea).

Institutional Animal Care and Use Committee of Yonsei University (No. YWC-P120).

Not applicable.

Not applicable.

Not applicable.

No potential conflict of interest relevant to this article was reported.