Despite the advances in diagnosis and treatment, breast cancer (BC) remains a common cause of cancer-related deaths in women worldwide (Silva et al. 2019). Therefore, discovering new potential candidates for breast cancer therapy is highly essential.

Elevated protein kinase C (PKC) activity in malignant breast tissue has been reported to result in a more aggressive neoplastic phenotype in MCF-7 breast cancer cells, which is characterized by increased proliferation, anchorage-independent growth, morphological alterations with loss of an epithelioid appearance, and increased tumorigenicity in nude mice (Ways et al. 1995).

PKC isozymes include three groups: classical PKCs (cPKCs) that constitute PKCα, PKCβ (PKCβI and PKCβII), and PKCγ; novel PKCs (nPKCs) that consist of PKCδ, PKCε, PKCη, and PKCθ; and atypical PKCs (aPKCs) ζ and ι (λ). cPKCs and nPKCs are activated by diacylglycerol (DAG), a secondary messenger signaling lipid (Garg et al. 2014). Interestingly, phorbol ester can mimic the actions of DAG (Garg et al. 2014). Particularly, phorbol myristate acetate (PMA), widely used as a PKC activator, promotes the proliferation of cancer cells such as ovarian cancer and BC cells (Park et al. 2009; Al-Alem et al. 2013). PMA induces activation of mitogen-activated protein kinases (MAPKs) in BC cells (Lee et al. 2007).

MAPK pathway plays a vital role in the regulation of cell growth and survival (Lee et al. 2020). Dysregulated MAPK signaling can result in increased or uncontrolled cell proliferation and resistance to apoptosis (Lee et al. 2020). Therefore, targeting the MAPK pathway is a potential strategy for cancer therapy.

Reactive oxygen species (ROS) are a group of highly reactive molecules such as hydroxyl (HO^•) andsuperoxide (O₂^•) free radicals, and hydrogen peroxide (H₂O₂) (Perillo et al. 2020). Moderate ROS levels are important to regulate cell proliferation and differentiation (Perillo et al. 2020). However, excessive ROS production can promote cell death (Perillo et al. 2020). A number of data have reported that various anticancer agents induced cytotoxicity in cancer cells by increasing ROS production (Aggarwal et al. 2019).

Ginsenosides are bioactive compounds extracted from the root of Panax ginseng Meyer that exert a variety of pharmacological effects such as anti-inflammatory, anticancer, and antidiabetic (Yang et al. 2016). Particularly, ginsenoside Rh1 is a metabolite of various major ginsenosides, including Rg1 and Re, in the gastrointestinal tract (Won et al. 2019). Hence, the biological effects of this ginsenoside are worth investigating. Ginsenoside-Rh1 displays a cytotoxic effect on several cancer cell lines such as mouse lymphoid neoplasm (P388), human lung carcinoma (A549), human cervix uterine adenocarcinoma (HeLa), and colorectal cancer cell lines (Tam et al. 2018; Lyu et al. 2019).

Previously, we reported that ginsenoside Rh1 exerts a potential anticancer effects on BC cells by inducing cell cycle arrest, apoptosis, and autophagy (Huynh et al. 2021). However, whether this ginsenoside can suppress cell growth under increased PKC activity remains unclear. Therefore, this study aimed to investigate the effect of ginsenoside Rh1 on PMA-stimulated MCF-7 cells and the underlying mechanisms.

Materials

Rabbit anti-phospho-ERK1/2, rabbit anti-ERK1/2, rabbit anti-phospho-p38, rabbit anti-p38, rabbit anti-phospho-PKCδ antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse anti-β-tubulin, mouse anti-phospho-PKCα, and rabbit anti-PKCα were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). Rabbit anti-GAPDH (#LF-PA0018) was purchased from AbFrontier (Seoul, Korea). Anti-mouse IgG (#T5393), crystal violet (#C0775), 2’,7’-dichlorodihydrofluorescein diacetate (DCF-DA), dihydroethidium (DHE), sulforhodamine B sodium salt (SRB, #S1402), trichloroacetic acid (TCA, #T6399), and Rh1 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate buffered saline (PBS, #EBA-1105) was purchased from Elpisbio (Daejeon, South Korea). Dulbecco’s modified Eagle’s medium (DMEM, #11963-092), Roswell Park Memorial Institute Medium (RPMI; 11875093)-1640, and fetal bovine serum (FBS, #10082147) were purchased from Gibco (Carlsbad, CA, USA).

Cell culture

Human mammary carcinoma cell lines MCF-7 (AHTB-22TM) and T47D (HTB-133TM) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). MCF-7 cells were maintained in DMEM containing 10% FBS, 100 U/mL penicillin, and streptomycin. T47D cells were cultured in RPMI-1640 supplemented with 10% FBS and 100-U/mL penicillin and streptomycin. The cells were incubated at 37℃, in humidified atmosphere 5% CO₂ (HERAcell 150i, Thermo Electron Corp., Waltham, MA, USA).

Sulforhodamine B (SRB) assay

Cells were seeded at a concentration of 1.9 × 10⁴ cells/well in 96-well plates and incubated with various concentrations of Rh1 for 24 and 48 h. At the end of the treatment, the cells were fixed with cold 10% (wt/vol) TCA at 4℃ for 1 h. The plates were then washed and air-dried at room temperature (RT) before stained with 0,057% SRB for 30 min at RT. The plates were quickly rinsed four times with 1% acetic acid and air-dried at RT. The dye was solubilized with 10 mM Tris base solution (pH 10.5). Finally, absorbance was measured at 510 nm in a microplate reader.

Western blot

After the treatment, MCF-7 cells were washed with PBS, and total cell lysates were prepared using 2X sodium dodecyl sulfate (SDS) lysis buffer, including 1 M Tris-HCl (pH 7.4), 25% Glycerol, 10% SDS, 5% 2-mercaptoethanol and 1% bromphenol blue. The protein extracts were then resolved by SDS-PAGE and analyzed indicated proteins as previously described (Huynh et al. 2022).

Colony formation assay

MCF-7 cells were seeded into the 12-well plates at a density of 300 cells per well. After 2 h for attachment, cells were treated with various concentrations of Rh1 for 10 days, and the medium was replaced every 2 days. Finally, 4% formaldehyde was used to fix the cells for 10 min, and then 0.5% crystal violet was used to stain the colonies.

Measurement of intracellular ROS levels

After the treatment, MCF-7 cells were stained with 10 µM DCF-DA or DHE in PBS for 30 min at 37℃. Afterward, the cells were washed and maintained in PBS. Fluorescence was monitored using a fluorescence microscope.

Statistical analysis

GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. Data were analyzed by one-way analysis of variance (ANOVA), followed by a Turkey’s multiple comparison test. At least three independent replications were carried out. The difference was considered significant when the p value was < 0.05. All results were represented as the mean ± SD.

Rh1 exerts an anti-growth effect on BC cells

To investigate the inhibitory effect of ginsenoside Rh1 on the growth of BC cells, MCF-7 and T47D cells were treated with different concentrations of Rh1 (25, 50, and 100 µM) for 24 and 48 h, and a SRB assay was then performed to analyze the cell viability. The results showed that Rh1 decreased the cell viability in both the cell types, with a stronger inhibitory effect on MCF-7 cells (Fig. 1A, B). Therefore, MCF-7 cells were used for further experiments. The inhibitory effect of Rh1 on MCF-7 cells was confirmed using colony formation assay. The cells were treated with different concentrations of Rh1 for 10 days, and the medium was replaced every two days. Colony formation was significantly decreased by treatment with Rh1 in a dose-dependent manner; in particular, 100 µM Rh1 almost suppressed reproductive ability of the cells (Fig. 1C, D).

Figure 1. Anti-growth effect of Rh1 on BC cells. (A, B) MCF-7 and T47D cells were treated with Rh1 (0, 25, 50, and 100 µM) for 24 and 48 h. Cell viability was evaluated using an SRB assay. (C, D) Colony formation was assayed on a 12-well plate for 10 days, and then the cells were fixed with 4% formaldehyde and stained with 0.5% crystal violet. Representative images (B) and quantified bar graph (C) indicate colony formation of MCF-7 cells. (A, B) Data are expressed as the mean ± SD (n = 3), *p < 0.05, **p < 0.01 and ***p < 0.001 as compared with control. SRB: sulforhodamine B.

Rh1 inhibits the activation of PMA-induced p38 and ERK1/2 in MCF-7 cells

Since the MAPK signaling pathway plays a crucial role in the growth and survival of cancer cells (Park et al. 2021), the effect of Rh1 on p38 and ERK1/2 activation in PMA-treated MCF-7 cells was examined. MCF-7 cells were pretreated with various concentrations of Rh1 (5, 25, and 50 µM) before exposure to PMA for 30 min and 24 h. Western blot analysis showed that PMA dramatically increased phosphorylation of ERK1/2 and p38 after 30 min, which remained constant for 24 h, whereas Rh1 significantly suppressed these effects (Fig. 2).

Figure 2. Rh1 inhibits activation of PMA-induced p38 and ERK1/2 in MCF-7 cells. (A-C) MCF-7 cells were pretreated with Rh1 at various concentrations (5, 25, and 50 µM) for 1 h, followed by treatment with PMA (50 ng/mL) for 30 min. (D-F) The cells were pretreated with Rh1 at various concentrations (5, 25, and 50 µM) for 3 h, before the exposure to PMA (50 ng/mL) for 24 h. The whole cell lysates were then used to analyze the kinase activity of p38 and ERK1/2. The relative quantification of protein levels was analyzed using Image J software. Results are represented as means ± SD (n = 3), *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the control, ^#p < 0.05 and ^##p < 0.01 compared with the PMA-treated sample.

Rh1 enhances ROS production in PMA-stimulated MCF-7 cells

Since ROS have been widely reported to induce cytotoxicity in cancer cells (Jeon et al. 2021), the effects of Rh1 on ROS generation were evaluated. MCF-7 cells were pretreated with various concentrations of Rh1 (5, 25, and 50 µM) for 3 h, followed by the treatment with PMA 50 ng/mL for 24 h. Treatment with PMA resulted in a slight increase in ROS levels in MCF-7 cells. Meanwhile, pre-treatment with Rh1 before PMA treatment significantly enhanced ROS production in MCF-7 cells, which was observed in both DCF-DA and DHE staining (Fig. 3).

Figure 3. Rh1 enhances ROS production in PMA-stimulated MCF-7 cells. MCF-7 cells were pretreated with Rh1 (5, 25, and 50 µM) for 3 h and then stimulated with PMA (50 ng/mL) for 24 h. The intracellular ROS levels were monitored by DCF-DA and DHE staining.

Rh1-induced ROS production is involved in the down-regulation of p38 and ERK1/2 activation in PMA-stimulated MCF-7 cells

To clarify whether the inhibitory effect of Rh1 on MAPK phosphorylation is involved in its ability to increase ROS production, the cells were incubated with N-acetyl cysteine (NAC), a ROS scavenger, in the presence or absence of Rh1, before the exposure to PMA for 30 min. Western blot analysis revealed that the inhibitory effect of Rh1 on p38 and ERK1/2 activation was rescued by NAC pre-treatment (Fig. 4).

Figure 4. Rh1-induced ROS production is involved in down-regulation of p38 and ERK1/2 activation in PMA-stimulated MCF-7 cells. MCF-7 cells were pretreated with 25 µM Rh1 in the presence or absence of NAC for 1 h, before 30 min of exposure to 50 ng/ml PMA. The whole cell lysates were then used to analyze the kinase activity of p38 and ERK1/2. The relative quantification of protein levels was analyzed using Image J software. Results are represented as means ± SD (n = 3). ***p < 0.001 compared with the control, ^#p < 0.05 and ^##p < 0.01 compared with the PMA-treated sample; ^$p < 0.05 and ^$$p < 0.01 compared with the corresponding sample.

Rh1-induced ROS production inhibits PKCδ activation in PMA-stimulated MCF-7 cells

PKC has been known to be an upstream effector of MAPK (Lee et al. 2007; Park et al. 2009; Nguyen et al. 2021). Therefore, the effects of Rh1 on phosphorylation of PKCα and PKCδ were investigated in PMA-stimulated MCF-7 cells. The results showed that Rh1 remarkably inactivated the phosphorylation of PKCδ after the incubation with PMA for both intervals of 30 min and 24 h (Fig. 5A, B). Meanwhile, the inhibitory effect of Rh1 on PKCα activation was marginal (Fig. 5A, B. Consistently, the PMA-induced activation of PKCδ, but not PKCα, inhibited by Rh1 treatment, was recovered by the inhibition of ROS production (Fig. 5C). Furthermore, the role of ROS on cell proliferation was also confirmed using the SRB assay. Pre-incubation with Rh1 inhibited the increase in cell viability induced by PMA, which was recovered by pre-treatment with NAC (Fig. 5D).

Figure 5. Rh1-induced ROS production inhibits PKCδ activation in PMA-stimulated MCF-7 cells. (A) MCF-7 cells were pretreated with Rh1 at various concentrations (5, 25, and 50 µM) for 1 h, followed by treatment with PMA (50 ng/mL) for 30 min. (B) The cells were pretreated with Rh1 at various concentrations (5, 25, and 50 µM) for 3 h, before the exposure to PMA (50 ng/mL) for 24 h. (C) MCF-7 cells were pretreated with 25 µM Rh1 in the presence or absence of NAC for 1 h, before 30 min of exposure to 50 ng/mL PMA. The whole cell lysates were then used to analyze the phosphorylation of PKCα and PKCδ. (D) The cells were pretreated with 25 µM Rh1 in the presence or absence of NAC for 3 h, followed by the treatment with 50 ng/mL PMA for 48 h. Cell viability was evaluated using an SRB assay. Results are represented as means ± SD (n = 3). ***p < 0.001 compared with the control, ^###p < 0.001 compared with the PMA-treated sample, and ^$$$p < 0.001 compared with the corresponding sample.

PKC isozymes are involved in various signaling pathways and affect a variety of biological functions, including the survival, proliferation, migration, invasion, and apoptosis of cancer cells (Park et al. 2009; Kang 2014). PKC plays a critical role in the development and progression of cancer (He et al. 2022). We previously reported that ginsenoside Rh1 exerts a potential anticancer activity in MCF-7 human breast cancer cells by inducing cell apoptosis, cell cycle arrest, and autophagy. In the current study, we investigated whether this ginsenoside could abolish the impact of PKC on MCF-7 cells to inhibit cell proliferation induced by the PKC activator, PMA, and determined the underlying mechanism.

Although several findings suggest that PKCδ is involved in cell apoptosis and growth inhibition (Cerda et al. 2001), a number of studies have revealed that PKCδ promotes cell growth and metastasis in BC (Cerda et al. 2001; Keshamouni et al. 2002; Liu et al. 2002; McCracken et al. 2003). In particular, the roles of PKC in cancer progression are associated with its downstream MAPK p38 and ERK1/2 pathways (Keshamouni et al. 2002; Wu et al. 2022). In this study, Rh1 pre-treatment inhibited PKCδ activation in MCF-7 cells treated with PMA (Fig. 5A, B). Consistently, Rh1 pre-treatment suppressed the increase in cell proliferation induced by the PKC activator, PMA (Fig. 5D). Mechanistically, Rh1 deactivated the PMA-induced phosphorylation of MAPK p38 and ERK1/2 in MCF-7 cells (Fig. 2).

ROS, which function as regulators of various critical signaling pathways in the cells, play different roles in cancer cells (Perillo et al. 2020). Moderate levels of ROS are necessary for cell survival and proliferation (Perillo et al. 2020). However, the overproduction of ROS can cause cell death, which is a common mechanism of various anticancer agents (Perillo et al. 2020). Here, PMA maintained the normal levels of ROS for cell proliferation, whereas Rh1 pre-treatment enhanced ROS generation in MCF-7 cells, compared to PMA-treated cells. Meanwhile, inhibition of ROS production using a ROS scavenger, NAC, reversed the inhibitory effects of Rh1 on activation of p38, ERK1/2, and PKCδ as well as on cell proliferation (Fig. 4 and 5). These results indicated the role of ROS production in the anticancer effect of Rh1 on PMA-treated MCF-7 cells. Furthermore, ginsenoside Rh1 treatment seemed to exert different effects depending on the types of ROS. Fig. 4 showed that treatment with Rh1 increased the levels of hydrogen peroxide more than those of superoxide in PMA-treated MCF-7 cells, as revealed by DCF-DA and DHE staining. ROS are derived from different sources including NADPH oxidases (NOXs), mitochondria, nitric oxide synthase, lipoxygenases, cyclo‐oxygenases, xanthine oxidase and cytochrome P450 enzymes (Perillo et al. 2020). NOXs are major sources of hydrogen peroxide (Lennicke et al. 2015). Our previous findings reported that inhibition of NOXs using diphenyleneiodonium or apocynin recovered the inhibitory effect of ginsenoside Rh1 on cell viability of MCF-7 cells (Huynh et al. 2021). The difference in ROS levels increased by Rh1 treatment may result from the different ROS sources targeted by the ginsenoside in the cells. Mitochondria are also a significant source of ROS, particularly superoxides (Jin et al. 2021). Whether ginsenoside Rh1 targets the mitochondria in MCF-7 cells to promote ROS generation is worth investigating in future studies.

Notably, although ginsenoside Rh1 exerts a potential cytotoxic effect on cancer cells, its low toxicity has been observed in normal cells and vascular cells in vitro and in animal models (Siraj et al. 2015; Huynh et al. 2020; Nguyen et al. 2021; Huynh et al. 2022). Therefore, this ginsenoside can be a promising candidate for BC treatment.

In conclusion, ginsenoside Rh1 exerts an anti-growth effect on MCF-7 cells by inactivating the ROS-mediated PCKδ/p38/ERK1/2 signaling pathway.

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

This research was funded by Basic Research Lab grant from National Research Foundation of Korea (2017R1A4A 1015860).