검색
검색 팝업 닫기

Ex) Article Title, Author, Keywords

Article

Split Viewer

Original Research Article

DTT 2022; 1(1): 19-26

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.009

Copyright © The Pharmaceutical Society of Korea.

Rh1 Abolishes MCF-7 Cell Growth via Down-Regulation of ROS-Induced PKCδ/p38/ERK1/2 Signaling Pathway

Diem Thi Ngoc Huynh , Kyung-Sun Heo

College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon, Korea

Correspondence to:Kyung-Sun Heo, kheo@cnu.ac.kr

Received: April 13, 2022; Revised: May 12, 2022; Accepted: May 31, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Challenges in cancer treatment have led to the discovery of new potential agents for cancer therapy. The increase in protein kinase C (PKC) activity in malignant breast tissue leads to a more aggressive neoplastic phenotype in MCF-7 breast cancer cells. We previously reported that the ginsenoside Rh1 exerted promising anticancer effects on MCF-7 human breast cancer cells. However, whether this ginsenoside could abolish the detrimental impacts of PKC in phorbol myristate acetate (PMA)-treated MCF-7 cells remain unknown. In this study, we found that ginsenoside Rh1 significantly inhibited the proliferation of MCF-7 cells. Mechanistically, Rh1 inhibited the activation of p38 and extracellular signal‐regulated kinase (ERK)1/2 following PMA stimulation. Additionally, Rh1 enhanced reactive oxygen species (ROS) production in PMA-treated MCF-7 cells, as demonstrated by 2´7´-dichlorodihydrofluorescein diacetate and dihydroethidium staining. The inhibitory effects of Rh1 on cell viability and phosphorylation of p38 and ERK1/2 were rescued by the ROS scavenger, N-acetyl cysteine. Notably, a PKC activator, PMA increased PKCα and PKCδ activation at 30 min and 24 h, respectively, whereas Rh1 abolished PMA-induced these PKC activation in a dose-dependent manner. Interestingly, Rh1-induced ROS production was only involved in inhibition of PKCδ activation, but not PKCα activation. Altogether, Rh1 displayed an anti-growth effect on PMA-stimulated MCF-7 cells through inhibition of the ROS-mediated PKCδ/p38/ERK1/2 signaling pathway.

Keywordsbreast cancer, extracellular signal‐regulated kinase1/2, ginsenoside Rh1, protein kinase C, p38, reactive oxygen species

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).

  1. Aggarwal V, Tuli HS, Varol A, Thakral F, Yerer MB, Sak K, Varol M, Jain A, Khan MA, Sethi G (2019) Role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements. Biomolecules 9:735. doi: 10.3390/biom9110735.
    Pubmed KoreaMed CrossRef
  2. Al-Alem LF, McCord LA, Southard RC, Kilgore MW, Curry TE Jr (2013) Activation of the PKC pathway stimulates ovarian cancer cell proliferation, migration, and expression of MMP7 and MMP10. Biol Reprod 89:73. doi: 10.1095/biolreprod.112.102327.
    Pubmed KoreaMed CrossRef
  3. Cerda SR, Bissonnette M, Scaglione-Sewell B, Lyons MR, Khare S, Mustafi R, Brasitus TA (2001) PKC-delta inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells. Gastroenterology 120:1700-1712. doi: 10.1053/gast.2001.24843.
    Pubmed CrossRef
  4. Garg R, Benedetti LG, Abera MB, Wang H, Abba M, Kazanietz MG (2014) Protein kinase C and cancer: what we know and what we do not. Oncogene 33:5225-5237. doi: 10.1038/onc.2013.524.
    Pubmed KoreaMed CrossRef
  5. He S, Li Q, Huang Q, Cheng J (2022) Targeting protein kinase C for cancer therapy. Cancers (Basel) 14:1104. doi: 10.3390/cancers14051104.
    Pubmed KoreaMed CrossRef
  6. Huynh DTN, Baek N, Sim S, Myung CS, Heo KS (2020) Minor ginsenoside Rg2 and Rh1 attenuates LPS-induced acute liver and kidney damages via downregulating activation of TLR4-STAT1 and inflammatory cytokine production in macrophages. Int J Mol Sci 21:6656. doi: 10.3390/ijms21186656.
    Pubmed KoreaMed CrossRef
  7. Huynh DTN, Jin Y, Myung CS, Heo KS (2021) Ginsenoside Rh1 induces MCF-7 cell apoptosis and autophagic cell death through ROS-mediated Akt signaling. Cancers (Basel) 13:1892. doi: 10.3390/cancers13081892.
    Pubmed KoreaMed CrossRef
  8. Huynh DTN, Jin Y, Van Nguyen D, Myung CS, Heo KS (2022) Ginsenoside Rh1 inhibits angiotensin II-induced vascular smooth muscle cell migration and proliferation through suppression of the ROS-mediated ERK1/2/p90RSK/KLF4 signaling pathway. Antioxidants (Basel) 11:643. doi: 10.3390/antiox11040643.
    Pubmed KoreaMed CrossRef
  9. Jeon H, Jin Y, Myung CS, Heo KS (2021) Ginsenoside-Rg2 exerts anti-cancer effects through ROS-mediated AMPK activation associated mitochondrial damage and oxidation in MCF-7 cells. Arch Pharm Res 44:702-712. doi: 10.1007/s12272-021-01345-3.
    Pubmed CrossRef
  10. Jin Y, Huynh DTN, Myung CS, Heo KS (2021) Ginsenoside Rh1 prevents migration and invasion through mitochondrial ROS-mediated inhibition of STAT3/NF-κB signaling in MDA-MB-231 cells. Int J Mol Sci 22:10458. doi: 10.3390/ijms221910458.
    Pubmed KoreaMed CrossRef
  11. Kang JH (2014) Protein kinase C (PKC) isozymes and cancer. New J Sci 2014:231418. doi: 10.1155/2014/231418.
    CrossRef
  12. Keshamouni VG, Mattingly RR, Reddy KB (2002) Mechanism of 17-beta-estradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta. J Biol Chem 277:22558-22565. doi: 10.1074/jbc.M202351200.
    Pubmed CrossRef
  13. Lee S, Rauch J, Kolch W (2020) Targeting MAPK signaling in cancer: mechanisms of drug resistance and sensitivity. Int J Mol Sci 21:1102. doi: 10.3390/ijms21031102.
    Pubmed KoreaMed CrossRef
  14. Lee SO, Jeong YJ, Im HG, Kim CH, Chang YC, Lee IS (2007) Silibinin suppresses PMA-induced MMP-9 expression by blocking the AP-1 activation via MAPK signaling pathways in MCF-7 human breast carcinoma cells. Biochem Biophys Res Commun 354:165-171. doi: 10.1016/j.bbrc.2006.12.181.
    Pubmed CrossRef
  15. Lennicke C, Rahn J, Lichtenfels R, Wessjohann LA, Seliger B (2015) Hydrogen peroxide- production, fate and role in redox signaling of tumor cells. Cell Commun Signal 13:39. doi: 10.1186/s12964-015-0118-6.
    Pubmed KoreaMed CrossRef
  16. Liu JF, Crépin M, Liu JM, Barritault D, Ledoux D (2002) FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway. Biochem Biophys Res Commun 293:1174-1182. doi: 10.1016/S0006-291X(02)00350-9.
    Pubmed CrossRef
  17. Lyu X, Xu X, Song A, Guo J, Zhang Y, Zhang Y (2019) Ginsenoside Rh1 inhibits colorectal cancer cell migration and invasion in vitro and tumor growth in vivo. Oncol Lett 18:4160-4166. doi: 10.3892/ol.2019.10742.
    CrossRef
  18. McCracken MA, Miraglia LJ, McKay RA, Strobl JS (2003) Protein kinase C delta is a prosurvival factor in human breast tumor cell lines. Mol Cancer Ther 2:273-281.
  19. Nguyen TLL, Huynh DTN, Jin Y, Jeon H, Heo KS (2021) Protective effects of ginsenoside-Rg2 and -Rh1 on liver function through inhibiting TAK1 and STAT3-mediated inflammatory activity and Nrf2/ARE-mediated antioxidant signaling pathway. Arch Pharm Res 44:241-252. doi: 10.1007/s12272-020-01304-4.
    Pubmed CrossRef
  20. Park J, Lee GE, An HJ, Lee CJ, Cho ES, Kang HC, Lee JY, Lee HS, Choi JS, Kim DJ, Choi JS, Cho YY (2021) Kaempferol sensitizes cell proliferation inhibition in oxaliplatin-resistant colon cancer cells. Arch Pharm Res 44:1091-1108. doi: 10.1007/s12272-021-01358-y.
    Pubmed CrossRef
  21. Park SK, Hwang YS, Park KK, Park HJ, Seo JY, Chung WY (2009) Kalopanaxsaponin A inhibits PMA-induced invasion by reducing matrix metalloproteinase-9 via PI3K/Akt- and PKCdelta-mediated signaling in MCF-7 human breast cancer cells. Carcinogenesis 30:1225-1233. doi: 10.1093/carcin/bgp111.
    Pubmed CrossRef
  22. Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A (2020) ROS in cancer therapy: the bright side of the moon. Exp Mol Med 52:192-203. doi: 10.1038/s12276-020-0384-2.
    Pubmed KoreaMed CrossRef
  23. Silva C, Perestrelo R, Silva P, Tomás H, Câmara JS (2019) Breast cancer metabolomics: from analytical platforms to multivariate data analysis. A review. Metabolites 9:102. doi: 10.3390/metabo9050102.
    Pubmed KoreaMed CrossRef
  24. Siraj FM, Natarajan S, Kim YJ, Yang DC (2015) In silico screening of ginsenoside Rh1 with PPARγ and in vitro analysis on 3T3-L1 cell line. Mol Simul 41:1219-1226. doi: 10.1080/08927022.2014.970188.
    CrossRef
  25. Tam DNH, Truong DH, Nguyen TTH, Quynh LN, Tran L, Nguyen HD, Shamandy BE, Le TMH, Tran DK, Sayed D, Vu VV, Mizukami S, Hirayama K, Huy NT (2018) Ginsenoside Rh1: a systematic review of its pharmacological properties. Planta Med 84:139-152. doi: 10.1055/s-0043-124087.
    Pubmed CrossRef
  26. Ways DK, Kukoly CA, deVente J, Hooker JL, Bryant WO, Posekany KJ, Fletcher DJ, Cook PP, Parker PJ (1995) MCF-7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype. J Clin Invest 95:1906-1915. doi: 10.1172/JCI117872.
    Pubmed KoreaMed CrossRef
  27. Won HJ, Kim HI, Park T, Kim H, Jo K, Jeon H, Ha SJ, Hyun JM, Jeong A, Kim JS, Park YJ, Eo YH, Lee J (2019) Non-clinical pharmacokinetic behavior of ginsenosides. J Ginseng Res 43:354-360. doi: 10.1016/j.jgr.2018.06.001.
    Pubmed KoreaMed CrossRef
  28. Wu CH, Hsu FT, Chao TL, Lee YH, Kuo YC (2022) Revealing the suppressive role of protein kinase C delta and p38 mitogen-activated protein kinase (MAPK)/NF-κB axis associates with lenvatinib-inhibited progression in hepatocellular carcinoma in vitro and in vivo. Biomed Pharmacother 145:112437. doi: 10.1016/j.biopha.2021.112437.
    Pubmed CrossRef
  29. Yang J, Yuan D, Xing T, Su H, Zhang S, Wen J, Bai Q, Dang D (2016) Ginsenoside Rh2 inhibiting HCT116 colon cancer cell proliferation through blocking PDZ-binding kinase/T-LAK cell-originated protein kinase. J Ginseng Res 40:400-408. doi: 10.1016/j.jgr.2016.03.007.
    Pubmed KoreaMed CrossRef

Article

Original Research Article

DTT 2022; 1(1): 19-26

Published online July 31, 2022 https://doi.org/10.58502/DTT.22.009

Copyright © The Pharmaceutical Society of Korea.

Rh1 Abolishes MCF-7 Cell Growth via Down-Regulation of ROS-Induced PKCδ/p38/ERK1/2 Signaling Pathway

Diem Thi Ngoc Huynh , Kyung-Sun Heo

College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon, Korea

Correspondence to:Kyung-Sun Heo, kheo@cnu.ac.kr

Received: April 13, 2022; Revised: May 12, 2022; Accepted: May 31, 2022

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ((http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Challenges in cancer treatment have led to the discovery of new potential agents for cancer therapy. The increase in protein kinase C (PKC) activity in malignant breast tissue leads to a more aggressive neoplastic phenotype in MCF-7 breast cancer cells. We previously reported that the ginsenoside Rh1 exerted promising anticancer effects on MCF-7 human breast cancer cells. However, whether this ginsenoside could abolish the detrimental impacts of PKC in phorbol myristate acetate (PMA)-treated MCF-7 cells remain unknown. In this study, we found that ginsenoside Rh1 significantly inhibited the proliferation of MCF-7 cells. Mechanistically, Rh1 inhibited the activation of p38 and extracellular signal‐regulated kinase (ERK)1/2 following PMA stimulation. Additionally, Rh1 enhanced reactive oxygen species (ROS) production in PMA-treated MCF-7 cells, as demonstrated by 2´7´-dichlorodihydrofluorescein diacetate and dihydroethidium staining. The inhibitory effects of Rh1 on cell viability and phosphorylation of p38 and ERK1/2 were rescued by the ROS scavenger, N-acetyl cysteine. Notably, a PKC activator, PMA increased PKCα and PKCδ activation at 30 min and 24 h, respectively, whereas Rh1 abolished PMA-induced these PKC activation in a dose-dependent manner. Interestingly, Rh1-induced ROS production was only involved in inhibition of PKCδ activation, but not PKCα activation. Altogether, Rh1 displayed an anti-growth effect on PMA-stimulated MCF-7 cells through inhibition of the ROS-mediated PKCδ/p38/ERK1/2 signaling pathway.

Keywords: breast cancer, extracellular signal‐regulated kinase1/2, ginsenoside Rh1, protein kinase C, p38, reactive oxygen species

Introduction

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 and Methods

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.

Results

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.

Discussion

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.

Conflict of interest

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

Acknowledgements

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

Fig 1.

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.
Drug Targets and Therapeutics 2022; 1: 19-26https://doi.org/10.58502/DTT.22.009

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.
Drug Targets and Therapeutics 2022; 1: 19-26https://doi.org/10.58502/DTT.22.009

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.
Drug Targets and Therapeutics 2022; 1: 19-26https://doi.org/10.58502/DTT.22.009

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.
Drug Targets and Therapeutics 2022; 1: 19-26https://doi.org/10.58502/DTT.22.009

Fig 5.

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.
Drug Targets and Therapeutics 2022; 1: 19-26https://doi.org/10.58502/DTT.22.009

References

  1. Aggarwal V, Tuli HS, Varol A, Thakral F, Yerer MB, Sak K, Varol M, Jain A, Khan MA, Sethi G (2019) Role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements. Biomolecules 9:735. doi: 10.3390/biom9110735.
    Pubmed KoreaMed CrossRef
  2. Al-Alem LF, McCord LA, Southard RC, Kilgore MW, Curry TE Jr (2013) Activation of the PKC pathway stimulates ovarian cancer cell proliferation, migration, and expression of MMP7 and MMP10. Biol Reprod 89:73. doi: 10.1095/biolreprod.112.102327.
    Pubmed KoreaMed CrossRef
  3. Cerda SR, Bissonnette M, Scaglione-Sewell B, Lyons MR, Khare S, Mustafi R, Brasitus TA (2001) PKC-delta inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells. Gastroenterology 120:1700-1712. doi: 10.1053/gast.2001.24843.
    Pubmed CrossRef
  4. Garg R, Benedetti LG, Abera MB, Wang H, Abba M, Kazanietz MG (2014) Protein kinase C and cancer: what we know and what we do not. Oncogene 33:5225-5237. doi: 10.1038/onc.2013.524.
    Pubmed KoreaMed CrossRef
  5. He S, Li Q, Huang Q, Cheng J (2022) Targeting protein kinase C for cancer therapy. Cancers (Basel) 14:1104. doi: 10.3390/cancers14051104.
    Pubmed KoreaMed CrossRef
  6. Huynh DTN, Baek N, Sim S, Myung CS, Heo KS (2020) Minor ginsenoside Rg2 and Rh1 attenuates LPS-induced acute liver and kidney damages via downregulating activation of TLR4-STAT1 and inflammatory cytokine production in macrophages. Int J Mol Sci 21:6656. doi: 10.3390/ijms21186656.
    Pubmed KoreaMed CrossRef
  7. Huynh DTN, Jin Y, Myung CS, Heo KS (2021) Ginsenoside Rh1 induces MCF-7 cell apoptosis and autophagic cell death through ROS-mediated Akt signaling. Cancers (Basel) 13:1892. doi: 10.3390/cancers13081892.
    Pubmed KoreaMed CrossRef
  8. Huynh DTN, Jin Y, Van Nguyen D, Myung CS, Heo KS (2022) Ginsenoside Rh1 inhibits angiotensin II-induced vascular smooth muscle cell migration and proliferation through suppression of the ROS-mediated ERK1/2/p90RSK/KLF4 signaling pathway. Antioxidants (Basel) 11:643. doi: 10.3390/antiox11040643.
    Pubmed KoreaMed CrossRef
  9. Jeon H, Jin Y, Myung CS, Heo KS (2021) Ginsenoside-Rg2 exerts anti-cancer effects through ROS-mediated AMPK activation associated mitochondrial damage and oxidation in MCF-7 cells. Arch Pharm Res 44:702-712. doi: 10.1007/s12272-021-01345-3.
    Pubmed CrossRef
  10. Jin Y, Huynh DTN, Myung CS, Heo KS (2021) Ginsenoside Rh1 prevents migration and invasion through mitochondrial ROS-mediated inhibition of STAT3/NF-κB signaling in MDA-MB-231 cells. Int J Mol Sci 22:10458. doi: 10.3390/ijms221910458.
    Pubmed KoreaMed CrossRef
  11. Kang JH (2014) Protein kinase C (PKC) isozymes and cancer. New J Sci 2014:231418. doi: 10.1155/2014/231418.
    CrossRef
  12. Keshamouni VG, Mattingly RR, Reddy KB (2002) Mechanism of 17-beta-estradiol-induced Erk1/2 activation in breast cancer cells. A role for HER2 AND PKC-delta. J Biol Chem 277:22558-22565. doi: 10.1074/jbc.M202351200.
    Pubmed CrossRef
  13. Lee S, Rauch J, Kolch W (2020) Targeting MAPK signaling in cancer: mechanisms of drug resistance and sensitivity. Int J Mol Sci 21:1102. doi: 10.3390/ijms21031102.
    Pubmed KoreaMed CrossRef
  14. Lee SO, Jeong YJ, Im HG, Kim CH, Chang YC, Lee IS (2007) Silibinin suppresses PMA-induced MMP-9 expression by blocking the AP-1 activation via MAPK signaling pathways in MCF-7 human breast carcinoma cells. Biochem Biophys Res Commun 354:165-171. doi: 10.1016/j.bbrc.2006.12.181.
    Pubmed CrossRef
  15. Lennicke C, Rahn J, Lichtenfels R, Wessjohann LA, Seliger B (2015) Hydrogen peroxide- production, fate and role in redox signaling of tumor cells. Cell Commun Signal 13:39. doi: 10.1186/s12964-015-0118-6.
    Pubmed KoreaMed CrossRef
  16. Liu JF, Crépin M, Liu JM, Barritault D, Ledoux D (2002) FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway. Biochem Biophys Res Commun 293:1174-1182. doi: 10.1016/S0006-291X(02)00350-9.
    Pubmed CrossRef
  17. Lyu X, Xu X, Song A, Guo J, Zhang Y, Zhang Y (2019) Ginsenoside Rh1 inhibits colorectal cancer cell migration and invasion in vitro and tumor growth in vivo. Oncol Lett 18:4160-4166. doi: 10.3892/ol.2019.10742.
    CrossRef
  18. McCracken MA, Miraglia LJ, McKay RA, Strobl JS (2003) Protein kinase C delta is a prosurvival factor in human breast tumor cell lines. Mol Cancer Ther 2:273-281.
  19. Nguyen TLL, Huynh DTN, Jin Y, Jeon H, Heo KS (2021) Protective effects of ginsenoside-Rg2 and -Rh1 on liver function through inhibiting TAK1 and STAT3-mediated inflammatory activity and Nrf2/ARE-mediated antioxidant signaling pathway. Arch Pharm Res 44:241-252. doi: 10.1007/s12272-020-01304-4.
    Pubmed CrossRef
  20. Park J, Lee GE, An HJ, Lee CJ, Cho ES, Kang HC, Lee JY, Lee HS, Choi JS, Kim DJ, Choi JS, Cho YY (2021) Kaempferol sensitizes cell proliferation inhibition in oxaliplatin-resistant colon cancer cells. Arch Pharm Res 44:1091-1108. doi: 10.1007/s12272-021-01358-y.
    Pubmed CrossRef
  21. Park SK, Hwang YS, Park KK, Park HJ, Seo JY, Chung WY (2009) Kalopanaxsaponin A inhibits PMA-induced invasion by reducing matrix metalloproteinase-9 via PI3K/Akt- and PKCdelta-mediated signaling in MCF-7 human breast cancer cells. Carcinogenesis 30:1225-1233. doi: 10.1093/carcin/bgp111.
    Pubmed CrossRef
  22. Perillo B, Di Donato M, Pezone A, Di Zazzo E, Giovannelli P, Galasso G, Castoria G, Migliaccio A (2020) ROS in cancer therapy: the bright side of the moon. Exp Mol Med 52:192-203. doi: 10.1038/s12276-020-0384-2.
    Pubmed KoreaMed CrossRef
  23. Silva C, Perestrelo R, Silva P, Tomás H, Câmara JS (2019) Breast cancer metabolomics: from analytical platforms to multivariate data analysis. A review. Metabolites 9:102. doi: 10.3390/metabo9050102.
    Pubmed KoreaMed CrossRef
  24. Siraj FM, Natarajan S, Kim YJ, Yang DC (2015) In silico screening of ginsenoside Rh1 with PPARγ and in vitro analysis on 3T3-L1 cell line. Mol Simul 41:1219-1226. doi: 10.1080/08927022.2014.970188.
    CrossRef
  25. Tam DNH, Truong DH, Nguyen TTH, Quynh LN, Tran L, Nguyen HD, Shamandy BE, Le TMH, Tran DK, Sayed D, Vu VV, Mizukami S, Hirayama K, Huy NT (2018) Ginsenoside Rh1: a systematic review of its pharmacological properties. Planta Med 84:139-152. doi: 10.1055/s-0043-124087.
    Pubmed CrossRef
  26. Ways DK, Kukoly CA, deVente J, Hooker JL, Bryant WO, Posekany KJ, Fletcher DJ, Cook PP, Parker PJ (1995) MCF-7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype. J Clin Invest 95:1906-1915. doi: 10.1172/JCI117872.
    Pubmed KoreaMed CrossRef
  27. Won HJ, Kim HI, Park T, Kim H, Jo K, Jeon H, Ha SJ, Hyun JM, Jeong A, Kim JS, Park YJ, Eo YH, Lee J (2019) Non-clinical pharmacokinetic behavior of ginsenosides. J Ginseng Res 43:354-360. doi: 10.1016/j.jgr.2018.06.001.
    Pubmed KoreaMed CrossRef
  28. Wu CH, Hsu FT, Chao TL, Lee YH, Kuo YC (2022) Revealing the suppressive role of protein kinase C delta and p38 mitogen-activated protein kinase (MAPK)/NF-κB axis associates with lenvatinib-inhibited progression in hepatocellular carcinoma in vitro and in vivo. Biomed Pharmacother 145:112437. doi: 10.1016/j.biopha.2021.112437.
    Pubmed CrossRef
  29. Yang J, Yuan D, Xing T, Su H, Zhang S, Wen J, Bai Q, Dang D (2016) Ginsenoside Rh2 inhibiting HCT116 colon cancer cell proliferation through blocking PDZ-binding kinase/T-LAK cell-originated protein kinase. J Ginseng Res 40:400-408. doi: 10.1016/j.jgr.2016.03.007.
    Pubmed KoreaMed CrossRef