Notoginsenoside R1 attenuates breast cancer progression by targeting CCND2 and YBX3.

BACKGROUND: Breast cancer (BC) is a common malignancy with highly female incidence. So far the function of notoginsenoside R1 (NGR1), the extract from Panax notoginseng, has not been clearly elucidated in BC.
METHODS: Optimal culture concentration and time of NGR1 were investigated by cell counting kit-8 assay. Cell proliferation ability was measured by colony formation assays. Transwell assay was used to detect the effect of NGR1 on cell migration and invasion. The apoptosis rate of cells between each group was measured by TUNEL assay.
RESULTS: NGR1 treatment has an inhibitory effect on proliferation, migration, invasion, and angiogenesis and a stimulating effect on cell cycle arrest and apoptosis of Michigan Cancer Foundation-7 (MCF-7) cells. The 50% growth inhibitory concentration for MCF-7 cells at 24 h was 148.9 mmol/L. The proportions of MCF-7 cells arrested in the G0/G1 phase were 36.94±6.78%, 45.06±5.60%, and 59.46±5.60% in the control group, 75, and 150 mmol/L groups, respectively. Furthermore, we revealed that NGR1 treatment attenuates BC progression by targeted downregulating CCND2 and YBX3 genes. Additionally, YBX3 activates phosphatidylinositol 3-phosphate kinase (PI3K)/protein kinase B (Akt) signaling pathway by activating kirsten rat sarcoma viral oncogene, which is an activator of the PI3K/Akt signaling pathway.
CONCLUSION: These results suggest that NGR1 can act as an efficacious drug candidate that targets the YBX3/PI3K/Akt axis in patients with BC.

Introduction

The incidence of breast cancer (BC), the most common neoplasm in women, is rapidly increasing worldwide, with approximately 1.7 million new cases each year.[ Nevertheless, for women with established diagnoses, survival rates have improved, but the median survival is dramatically low.[ Most metastatic or relapsed breast tumors are incurable, and substantial improvement in survival rates still remains a researchers’ dream.[ BC carcinogenesis is a complex biological process, and the mechanism of BC occurrence and metastasis, even therapy, remains unclear and requires deep exploration.[

Notoginsenoside R1 (NGR1), the main effective component isolated from Panax notoginseng, is one of the most valuable traditional Chinese medicines and was once used in ancient China to treat cardiovascular diseases. It has been well characterized by various studies and has many pharmacological effects, including anti-inflammatory, antioxidant, anti-apoptotic, and anti-tumor effects.[ Regarding its anti-tumor functions, recently, investigators determined that P. notoginseng suppressed the metastasis of colorectal cancer (CRC) by inhibiting migration, invasion, and adhesion abilities.[ In addition, NGR1, one of the most important components of P. notoginseng, also significantly suppressed the metastasis of CRC through the regulation of matrix metalloproteinase-9, integrin-1, E-selectin, and intercellular cell adhesion molecule-1 expression. Factors of NGR1 influencing cancer prevention and treatment have also been explored in colon cancer, leukemia, and melanoma.[ However, previous studies have not addressed the effects of NGR1 on BC, and the related regulatory mechanisms remain unclear.

Migration and invasion play significant roles in tumor metastasis.[ With initial cancer tissue growth, the original cancer cells must separate from the primary tumor mass by losing their cell-cell contact and must invade the stroma. This process is called an invasion. Another specific ability of tumor cells is to produce substances that promote angiogenesis, which provide nutrition for tumor cell growth and development. Apoptosis refers to programmed cell death that occurs in normal cells without any outside attack. An increasing number of people are studying effective ways to induce or promote cancer cell apoptosis to reduce cancer.[

In this study, we reported that NGR1 suppressed the progression of BC by inhibiting the migration, invasion, and angiogenesis of MCF-7 and MDA-MB-231 cells and promoting the apoptosis of BC cells. Furthermore, mechanistic investigations showed that NGR1 influenced BC cells by downregulating CCND2 and YBX3. However, both CCND2 and YBX3 are recognized as oncogenes that have previously been observed in colon cancer, lung cancer, and ovarian cancer.[ In this study, we found that YBX3 acts as an oncogene by activating the KRAS/ phosphatidylinositol 3-phosphate kinase (PI3K)/protein kinase B (Akt) signaling pathway in MCF-7 cells. Thus, we indicated that NGR1 attenuates BC progression by targeting CCND2 and YBX3; of note, YBX3 is an activator of the KRAS/PI3K/AKT signaling pathway.

Methods

Bioinformatics analysis

Microarray datasets from the Gene Expression Omnibus (GEO) database were used to test differential expression genes after NGR1 (MedChemExpress, New Jersey, USA) treatment in BC cells. Raw microarray data were downloaded from GEO including GSE85871. Then, the raw microarray data were normalized and z-score-transformed using RMAExpress (http://www.rmaexpress.bmbolstad.com/).

Cell culture

MCF-7 cells, MDA-MB-231 cells, and human umbilical vein endothelial cells (HUVECs) were obtained from European Collection of Authenticated Cell Cultures (Shanghai, China). Cells were cultured in Dulbecco's modification of Eagle's medium (Gibco, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco). All cells were incubated at 37°C in a 5% CO2 humidified incubator.

Cell transfection

Small interfering RNA, si-YBX3, and si-NC were purchased from RiboBio (Guangzhou, China). The cells were seeded into six-well plates. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used to transfect MCF-7 cells according to the manufacturer's instructions.

Cell viability assay

Cells were digested by trypsin and uniformly seeded in a 96-well plate. Each group contained three duplicate wells. The wells on the edges of the 96-well plate were filled with phosphate buffer saline (PBS), and the plate was placed in a 37°C, 5% CO2 incubator. A total of 5 × 103 cells were inoculated in each well. The medium was aspirated, and the cells were treated with a medium containing the specified concentrations of the substance for the specified durations. The cell counting kit-8 solution was added at 10 μL/well. The 96-well plate was incubated in a 37°C incubator for 1 h. The optical density of each well at 490 nm was measured with a microplate reader. The optical density values were averaged, and the experiment was repeated at least 3 times.

Colony formation assay

A total of 1000 cells were plated in a six-well plate and mixed well with 3 mL of medium containing 10% FBS. Cells were maintained in an incubator at 37°C and 5% CO2 for 1 week. Cells in the wells were fixed using paraformaldehyde for 30 min and were then stained with 0.1% crystal violet. Clusters containing >30 cells were counted as a single colony.

Cell migration and invasion assays

Cells were harvested, resuspended in serum-free medium, and seeded in the upper chamber of a Transwell insert containing a membrane filter (Corning, NY, USA). Medium containing 10% FBS was added to the lower chamber. The cells were incubated for 24 h. After treatment with paraformaldehyde, cells were stained with 0.1% crystal violet. Finally, cells were imaged and counted using an Olympus microscope (Tokyo, Japan).

Endothelial tube formation assay

Matrigel (Corning) (10 μL) was added to each well of a 96-well plate and allowed to solidify (at 37°C for 30 min). HUVECs were resuspended in supernatant collected from each group. Then, 300 μL of supernatant containing 4 × 104 HUVECs was added to each well and incubated at 37°C. After 8 h, tube formation was observed under a microscope.

Chromatin immunoprecipitation-seq (ChIP-seq)

To identify the promoter that binds to YBX3, a ChIP assay was performed using the EZ-Magna ChIP kit (Millipore, Shanghai, China) according to the manufacturer's protocol. In brief, cells were fixed with 4% paraformaldehyde and incubated with glycine for 10 min to generate DNA-protein cross-links. Then, the cells were lysed with Cell Lysis Buffer and Nuclear Lysis Buffer and sonicated to generate chromatin fragments of 400 to 800 bp. ChIP-qPCR and ChIP-seq of MCF-7 cells were performed using an anti-YBX3 antibody (Invitrogen). The primers are as follows: F-5′-GCCACTGATGATCTGGGAGT-3′; R-3′-GTGAAAGACAGGGAAGGGGA-5′.

Western blotting analysis

The nuclear protein fraction and total protein were prepared with an isolation kit (KeyGen Biotech, Nanjing, China). Western blotting was performed with antibodies against CCND2, YBX3, cleaved caspase-3, and cleaved caspase-9 (Abcam Cambridge, MA, USA); glyceraldehyde-3-phosphate dehydrogenase and Bax (ProteinTech, Wuhan, China); and P-PI3K, P-AKT, and KRAS (Affinity, Biosciences OH, USA). Protein quantification was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA) and normalized to glyceraldehyde-3-phosphate dehydrogenase values.

Flow cytometric analysis of the cell cycle

The cell cycle was analyzed using flow cytometry. MCF-7 cells after treatment were digested with trypsin, washed twice with cold PBS, and centrifuged at 1000 r/min for 5 min at room temperature. Then, the cells were fixed in 70% pre-cooled ethanol at 4°C overnight, washed, and resuspended in cold PBS after fixation. For cell cycle analysis, cells were incubated in PBS containing 100 μg/mL ribonuclease for 20 min and stained with 1 mg/mL propidium iodide (Sigma, St. Louis, MO, USA) solution for 30 min at 4°C in the dark. Data were acquired using a FACScan flow cytometer (BD, Franklin Lakes, NJ, USA).

TUNEL assay

The TUNEL assay for apoptotic cell detection was performed using an in situ apoptosis detection kit (Roche, Branchburg, NJ, USA) in tumor tissue sections. Apoptotic cells nine random fields per slide were identified and analyzed under a light microscope.

Statistical analysis

The results are presented as the mean ± standard deviation from three independent experiments performed in triplicate. The P values were calculated using Student's t test or one-way analysis of variance. A P value of <0.05 was considered to indicate a statistically significant result. Statistical analyses were performed using GraphPad Prism 6.0 statistical software (GraphPad Software Inc., La Jolla, CA, USA).

Results

NGR1 treatment inhibits the expression of CCND2 and YBX3

The chemical structure of NGR1 is shown in Figure 1A (Based on https://www.medchemexpress.cn/Notoginsenoside-R1.html?src=bd-product). To investigate the impact of NGR1 treatment on BC genetic alterations, we screened the GEO database (GSE85871) and found that 141 genes were downregulated and 114 genes were upregulated in the treatment group compared with the control group [Figure 1B]. The top ten down-regulated genes after NGR1 treatment in BC cells are shown in Table 1. Western blotting and quantitative real time polymerase chain reaction tests were used to verify the results of the GEO analysis. As shown in Figure 1C, there were clear trends of decreasing expression of CCND2 and YBX3 proteins in BC cells with NGR1 treatment in both dose- and time-dependent manners (F = 441.1, P < 0.0001). Together, these results indicated that treatment with NGR1 exerts an obvious inhibitory effect on the expression of CCND2 and YBX3 in BC cells. In addition, both CCND2 and YBX3 are oncogenes in many kinds of cancers, and it can be speculated that NGR1 exerts a cancer suppressive effect by down-regulating the expression of multiple oncogenes.

Figure 1

Identification of notoginsenoside R1 targets in MCF-7 cells. (A) The chemical structure of notoginsenoside R1. (B) Differentially expressed genes were detected using the GEO database (GES85871). (C) R1 treatment significantly decreased the expression of CCND2 and YBX3 in MCF-7 cells in a dose- and time-dependent manner.∗P<0.05.

Table 1

Top ten down-regulated genes were elucidated by GSE85871.

ID	P value	t	B	logFC	Gene.symbol	Gene.title
205804_s_at	0.0002372	−19.03	0.66188	−3.431	TRAF3IP3	TRAF3 interacting protein 3
220489_s_at	0.0004291	−15.73	0.42691	−3.448	SERINC2	Serine incorporator 2
206841_at	0.0007268	−13.28	0.16337	−3.451	PDE6H	Phosphodiesterase 6H
206433_s_at	0.0009893	−12.02	−0.01652	−3.451	SPOCK3	Sparc/osteonectin (testican) 3
215500_at	0.0032183	−8.18	−0.88305	−3.534	SNX29	Sorting nexin 29
205587_at	0.0006082	−14.06	0.25858	−3.617	FGFR1OP	FGFR1 oncogene partner
213319_s_at	0.0019963	−9.56	−0.49887	−3.676	YBX3	Y-box binding protein 3
200953_s_at	0.0009744	−12.08	−0.00722	−3.802	CCND2	Cyclin D2
207930_at	0.0004448	−15.55	0.41069	−3.838	LCN1	Lipocalin 1
211617_at	0.0001866	−20.55	0.74019	−3.854	ALDOAP2	Aldolase, fructose-bisphosphate A pseudogene 2

NGR1 treatment inhibits the migration, invasion, and angiogenesis of MCF-7 and MDA-MB-231 cells

Two sets of assays examined the impact of NGR1 on the proliferation of MCF-7 and MDA-MB-231 cells with different NGR1 doses and different exposure times. BC cells were treated with different concentrations of NGR1 for 24, 48, and 72 h, and cell counting kit-8 assays were then performed. The results are shown in Figure 2A. With the increase in the dose or the prolonged action of NGR1, the cell proliferation ability decreased rapidly. The 50% growth inhibitory concentration for MCF-7 cells at 24 h was 148.9 μmol/L. Colony formation assays were performed to analyze the long-term effect of NGR1 treatment on the proliferation of MCF-7 and MDA-MB-231 cells. The results indicated the same conclusion that NGR1 inhibited the proliferation ability of BC cells [Figure 2B] (F = 62.2, P < 0.0001). To evaluate whether NGR1 affects cell migration, invasion, and angiogenesis ability, Transwell assays with and without Matrigel and capillary tube formation assays were performed. The results shown in Figure 2C and 2D show that NGR1 treatment obviously weakened the BC cell migration, invasion, and angiogenesis abilities as the dose increased. Collectively, these data suggest that NGR1 treatment can attenuate BC progression in vitro by altering proliferation, migration, invasion, and angiogenesis.

Figure 2

Notoginsenoside R1 suppresses the proliferation, migration, invasiveness, and angiogenic capacity of BC cells. (A) BC cells after treatment with R1 (0, 50, 100, 150, or 200 μmol/L) for 24, 48, or 72 h. R1 suppressed the proliferation of breast cancer cells in a dose- and time-dependent manner. (B) Photomicrographs of crystal violet-stained colonies of BC cells growing in six-well plates for 7 days after R1 treatment. R1 inhibited colony formation in a breast cancer cell line in a dose-dependent manner. (C) The cells were treated with R1 for 24 h. The images show that R1 inhibited the invasion and migration of MCF-7 cells across the Transwell inserts (F = 62.2, P < 0.0001). (D) Capillary tube formation (CTF) assays. All data represent the mean ± standard error of the mean of values from three biological replicates (F = 80.11, P < 0.0001). ∗P < 0.05. BC: Breast cancer.

NGR1 regulates the cell cycle and apoptosis in MCF-7 cells

To assess the influence of NGR1 on the cell cycle, flow cytometry was used. The proportions of MCF-7 cells arrested in the G1 phase were 36.94% ± 6.78%, 45.06% ± 5.60%, and 59.46% ± 5.60% in the control group, 75, and 150 μmol/L groups, respectively [Figure 3A]. To evaluate the possible mechanism of apoptosis induced by NGR1, the main proteins of the mitochondrial pathway were examined by western blotting. The results showed that NGR1 induced an increase in the protein levels of Bax, cleaved caspase-3, and cleaved caspase-9 [Figure 3B]. Apoptotic MCF-7 cells were also detected by TUNEL staining after treatment with NGR1. All of the nuclei were stained blue by 4,6-diamino-2-phenyl indole, while apoptotic nuclei were stained green in the TUNEL assays [Figure 3C]. Significant changes were detected between groups, and a higher percentage of apoptosis events occurred in the NGR1 high-dose group compared with the low-dose group and control group.

Figure 3

Notoginsenoside R1 regulates the MCF-7 cell cycle and apoptosis. (A) FACS analysis showing significant decreases in MCF-7 and MDA-MB-231 cells in the S phase after notoginsenoside R1 treatment (F = 90.66, P < 0.0001). (B, C) The effect of notoginsenoside R1 on cell apoptosis was measured by TUNEL and Western blotting assays. The error bars in all graphs represent SD, and each experiment was repeated three times (F = 70.89, P < 0.0001). ∗P < 0.05. SD: Standard deviation.

NGR1 inhibits the KRAS/PI3K/Akt pathway via YBX3

Previous results have demonstrated that NGR1 regulates the cell cycle by downregulating CCND2. Further experiments should focus on the molecular regulatory mechanism of YBX3 in BC. To study the further regulatory mechanism of YBX3 in MCF-7 cells, we performed a ChIP-seq to the YBX3 gene in MCF-7 cells. A pie chart was generated to illustrate how peaks were distributed over important genomic features [Figure 4A]. Motif centrality analysis of YBX3 binding regions was performed using CentriMo [Figure 4B]. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of YBX3 ChIP-seq promoters with the top five enrichment scores is shown in Figure 4C; the PI3K/Akt pathway was the most enriched. Thus, YBX3 might up-regulate the PI3K/Akt pathway, and ChIP experiments verified that YBX3 plays such a role by binding the promoter regions of KRAS [Figure 4D]. Quantitative real time polymerase chain reaction also demonstrated that the expression of KRAS was regulated by YBX3 [Figure 4E]. To connect the above conclusions to form an axis, we performed western blotting assays which indicated that NGR1 inhibited the KRAS/PI3K/Akt pathway by decreasing YBX3 [Figure 4F]. It was shown that P-PI3K and P-AKT, the key proteins in the PI3K/Akt pathway, were down-regulated with increasing NGR1 concentration. Meanwhile, the expression of KRAS was also suppressed by NGR1 treatment. Furthermore, cell functional experiments were performed to detect the effect of NGR1 on BC cells after YBX3 knockdown, such as colony formation and Transwell assays [Figure 5A–C]. The results showed NGR1 had little impact on BC cells after YBX3 knockdown, which means NGR1 could regulate BC progression by targeting YBX3 directly.

Figure 4

Notoginsenoside R1 inhibits the KRAS/PI3K/Akt pathway via YBX3. (A) Pie diagram illustrates how peaks are distributed over important genomic features. (B) Motif centrality analysis of YBX3 binding regions was performed using CentriMo. (C) KEGG pathway enrichment analysis of YBX3 ChIP-seq promoters with the top five enrichment scores. (D) The ChIP assay results confirmed the occupancy of YBX3 in the KRAS promoter (F = 75.36, P < 0.0001). (E) The expression of KRAS was down-regulated after siYBX3 transfection. (F) The expression of KRAS/p-PI3K/p-Akt was measured by Western blotting after notoginsenoside R1 treatment. ChIP-seq: Chromatin immunoprecipitation-seq; KEGG: Kyoto Encyclopedia of Genes and Genomes ∗P < 0.001.

Figure 5

Notoginsenoside R1 inhibits BC aggressiveness by targeting YBX3. Colony formation (A), Transwell (B), and tube formation assays (C) were used to detect the effect of NGR1 on BC cells after YBX3 knockdown. Cells were stained with 0.1% crystal violet (original magnification × 400). BC: Breast cancer. ∗P < 0.001. n.s.: Not significant.

Discussion

In this study, we demonstrated that treatment with NGR1 significantly attenuated the growth of MCF-7 and MDA-MB-231 cells in a dose- and time-dependent manner. Next, we examined the effect of NGR1 on tumor-promoting activities including proliferation, invasion, and angiogenesis. As expected, NGR1 played a significant role in suppressing the tumorigenesis of MCF-7 and MDA-MB-231 cells in vitro. Additionally, NGR1 treatment induced cell cycle arrest and apoptosis in MCF-7 cells via the KRAS/PI3K/Akt pathway. Furthermore, the data from the sequencing models of the control group and NGR1-treated group revealed the genes specifically downregulated by NGR1 treatment. Western blotting also indicated the down-regulation of CCND2 and YBX3 by NGR1 treatment in a dose- and time-dependent manner in MCF-7 cells. Thus, we illustrated that NGR1 may affect the growth or progression of MCF-7 cells by changing the expression of the CCND2 and YBX3 genes. Furthermore, KEGG enrichment analysis showed that the PI3K/Akt pathway was the pathway most closely related to YBX3 in MCF-7 cells. ChIP assays further demonstrated that YBX3 activated the PI3K/Akt pathway by combining it with KRAS. Consequently, these findings indicated an anti-cancer role of NGR1 in MCF-7 cells and revealed a potential molecular mechanism by which the YBX3/KRAS/PI3K/Akt axis functions in MCF-7 cells, as its inhibition has been associated with proliferation, migration, invasion, and angiogenesis in MCF-7 cells.

The main components of P. notoginseng are saponins. Additionally, three major types of saponins in P. notoginseng are ginsenosides (eg, Rb1, Rg1, Rg3), notoginsenosides (eg, R1, R2, R3), and jiaogulan glucosides (eg, R6). NGR1 is the most abundant notoginsenoside in P. notoginseng.[ Previous anti-cancer studies have indicated that ginsenosides Rb1 suppresses phorbol myristate acetate-induced invasion and migration in MCF-7 cells.[ However, we are the first to report that NGR1 significantly suppresses BC metastasis and to clarify its molecular mechanisms in MCF-7 cells. Moreover, our results are consistent with those observed in other human malignancies, such as colon cancer and liver cancer.[ One of the related findings is that NGR1 inhibits the migration, invasion, and adhesion of HCT-116 cells[ by regulating MMP-9, integrin-1, E-selectin, and intercellular cell adhesion molecule-1 expression. Moreover, ginsenoside Rg1 also suppresses transforming growth factor β1-induced invasion and migration in HepG2 liver cancer cells.[ The above studies indicate that saponins can reduce the tumorigenesis of many kinds of cancers. However, the effect of NGR1 on BC has not been studied. We are the first to show that NGR1 inhibits the tumorigenesis of MCF-7 cells and promotes cell cycle arrest and apoptosis of MCF-7 cells.

The YBX3 gene is located on chromosome 12 at locus p13.1 and encodes two spliced mRNA isoforms, which act as tumor promotors.[CCND2 is another tumor promotor that plays a significant role in accelerating cell growth and cell migration, yet the inhibition of CCND2 triggers cell apoptosis.[ In this study, we demonstrated that NGR1 treatment causes a decrease in the expression of CCND2 in a time- and dose-dependent manner in MCF-7 cells. However, the detailed mechanism of CCND2 in BC was not explored in this study. However, it is clear that CCND2, an oncogene in many cancers, plays a role in MCF-7 cells. On the other hand, we clarified the mechanisms of YBX3 in MCF-7 cells. With ChIP-seq analysis, KEGG pathway analysis, and other detection methods, we predicted and verified the mechanism of YBX3 in MCF-7 cells. This is the first report that YBX3 works by combining with KRAS to activate the PI3K/Akt signaling pathway, and KRAS is an established activator of the PI3K/Akt signaling pathway.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 81260576), the Major Program of Science and Technology Foundation of Guizhou Province (QKHJZ[2014]2003), the Major Science and Technology Projects of Guizhou Province (QKHJZ[2015]2002-2-5), and the Science and Technology Foundation of Guizhou Province (QKHJY[2011]3007).

Conflicts of interest

None.