Ginsenoside Rg5 induces apoptosis and autophagy via the inhibition of the PI3K/Akt pathway against breast cancer in a mouse model

Yannan Liu ab and Daidi Fan *ab
aShaanxi Key Laboratory of Degradable Biomedical Materials, School of Chemical Engineering, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, China. E-mail: fandaidi@nwu.edu.cn
bShaanxi R&D Center of Biomaterials and Fermentation Engineering, School of Chemical Engineering, Northwest University, 229 North Taibai Road, Xi'an, Shaanxi 710069, China

Received 6th June 2018 , Accepted 18th August 2018

First published on 21st August 2018


Breast cancer is the most frequently diagnosed cancer and has become the main cause of cancer-related death among women worldwide. Traditional chemotherapy for breast cancer has serious side effects for patients, such as the first-line drug docetaxel. Ginsenoside Rg5, a rare ginsenoside and the main ingredient extracted from fine black ginseng, has been proved to have anti-breast cancer efficacy in vitro. Here, the in vivo anti-breast cancer efficacy, side effects and potential molecular mechanisms of Rg5 were investigated on a BALB/c nude mouse model of human breast cancer. The tumor growth inhibition rate of high dose Rg5 (20 mg kg−1) was 71.4 ± 9.4%, similar to that of the positive control docetaxel (72.0 ± 9.1%). Compared to docetaxel, Rg5 showed fewer side effects in the treatment of breast cancer. Treatment with Rg5 induced apoptosis and autophagy in breast cancer tissues. Rg5 was proved to induce caspase-dependent apoptosis via the activation of the extrinsic death receptor and intrinsic mitochondrial signaling pathways. The autophagy induction was related to the formation of an autophagosome and accumulation of LC3BII, P62 and critical Atg proteins. Further studies showed that Rg5 in a dose-dependent manner induced apoptosis and autophagy through the inhibition of the PI3K/Akt signaling pathway as indicated by the reduced phosphorylation level of PI3K and Akt. Taken together, Rg5 could be a novel and promising clinical antitumor drug targeting breast cancer.


1. Introduction

Breast cancer, a hormone-responsive cancer that accounts for about one fourth of all female cancers, has become the leading cause of cancer-related death among women in the world.1 Recently, the American Cancer Society (ACS) reported that an estimated 249[thin space (1/6-em)]260 new cases of invasive breast cancer have be diagnosed, resulting in 40[thin space (1/6-em)]890 deaths in 2016 in the US.2 In addition, there is approximately 12.6% lifetime risk for the occurrence of invasive breast cancer among women in the US.3 Currently, docetaxel (DTX), a vital chemotherapeutic agent belonging to the taxane class, is considered as the first-line drug for the treatment of breast cancer.4 However, the therapeutic application of docetaxel is blocked by its poor aqueous solubility, chemotherapeutic resistance in breast cancer cells, side effects and tumor recrudescence.5,6 Therefore, it is urgent to develop novel anti-tumor drugs with less side effects to satisfy the therapeutic demand of breast cancer patients.

Cell death is an effective strategy for controlling cancer progression and several chemotherapeutics use this mechanism of action to treat cancer.7 Two main types of programmed cell death exist: apoptosis (type I) and autophagy (type II).7,8 Apoptosis is a gene regulated phenomenon and is morphologically characterized by cell shrinkage, cell membrane blebbing, chromatin condensation, DNA fragmentation, and nuclear fragmentation.9,10 Apoptosis plays an obvious role in chemotherapies against various types of cancers.9 There are two pathways of apoptosis, one of which is the extrinsic death receptor pathway, related to the activation of caspase-8; the other is the intrinsic mitochondrial pathway, related to the activation of caspase-9. The expression and translocation of the Bcl-2 family proteins and activation of caspase-3 are essential for apoptosis.11,12 Autophagy is a genetically programmed, evolutionarily conserved process that degrades robust cellular proteins and organelles, including the endoplasmic reticulum, Golgi apparatus and mitochondria, and makes them become vacuoles called autophagosomes.4,13,14 Activated autophagy promotes autophagosome formation, increase in LC3-II expression and decrease in P62 expression, which contribute to autophagic cell death.8,15 Moreover, autophagy was reported to relate to the anticancer action of chemotherapeutic drugs for breast cancer.16–18 Recent studies have demonstrated that lots of chemotherapeutics known to induce apoptosis also activate autophagy.19

The PI3K/Akt signaling pathway is a crucial signal transduction pathway which has been reported to play an important role in inhibiting cell apoptosis and increasing cell proliferation.20–22 The activation of phosphatidylinositol 3-kinase (PI3K)/Akt is an important step in promoting cell survival and suppressing cell death.23 The PI3K/Akt pathway has been implicated in a variety of human malignant tumors.21,22 PI3K and Akt are important mediators of carcinogenesis via phosphorylation.24 The classic autophagic pathway acts downstream of the mammalian target of rapamycin (mTOR) kinase,25–27 which was reported to be related to the regulation of autophagy in mammalian cells.24,28 Research has also demonstrated that inhibition of the PI3K/Akt pathway would result in the suppression of mTOR and induction of cancer cell apoptosis by inducing autophagy.29,30 The PI3k/Akt/mTOR signaling pathway is an important regulator in autophagy activation.31 Moreover, Bad can contact the PI3K/Akt pathway directly with the apoptotic pathway, which is considered a center of pro-apoptotic and anti-apoptotic regulatory cascades.32

Ginsenosides are the major component of ginseng, which have been used for therapy against many types of cancers.33,34 Ginsenoside Rk1 has been reported to show anti-tumor activity in human hepatocellular carcinoma cells through the inhibition of telomerase activity and induction.35 Ginsenoside Rg3 inhibits the growth of lung cancer cells via the PI3K-Akt signaling pathway.20 Ginsenoside Rh2 significantly inhibits tumor growth in H22 tumor-bearing mice.36 Ginsenoside compound K significantly inhibits the growth of breast cancer.37 In our research group, we have also conducted deep research in ginsenosides. Ginsenoside Rk3 obviously suppresses the growth of non-small cell lung cancer.38 Ginsenoside Rh4 shows a remarkable anti-cancer effect on colorectal cancer by inducing apoptosis autophagy through the ROS/JNK/p53 pathway.39 This gives us a reference for study on the antitumor activity of ginsenosides. In addition, Ginsenoside Rg5, a rare ginsenoside (Fig. 1A), which is a main component extracted from fine black ginseng, induced apoptosis and DNA damage in human cervical cancer cells.40 It also exhibited strong anti-breast cancer activities in vitro.41 However, the defined molecular mechanisms underlying the Rg5-regulated suppression of human breast cancer and side effects in vivo have not been studied.


image file: c8fo01122b-f1.tif
Fig. 1 Rg5 significantly inhibits the growth of the human breast cancer xenograft in vivo. MCF-7 cells were inoculated subcutaneously in the left flank of BALB/c nude mice. Intraperitoneal administration of the solvent or Rg5 (10 and 20 mg kg−1 d−1) or docetaxel (10 mg kg−1 3d−1) was started when the tumor volume reached around 150 mm3. After 30 days of treatment, all mice were killed. (A) The chemical structure of Ginsenoside Rg5. (B) Representative image and (C) tumor weight of the human breast cancer xenograft from the control group, Rg5-treated groups and docetaxel-treated group. (D) Tumor volume and (E) body weight were measured every three days. (F) H&E staining of the tumor specimens. Scale bars = 100 μm. (G) The apoptotic and autophagic status of the tumor tissues was assessed by the TEM assay. Scale bars = 10 μm. The data are presented as mean ± SD of three independent experiments. P < 0.05 was considered to indicate statistical significance. Different letters indicate significant differences between each group.

In this study, we investigate the inhibitory effects of Rg5 on tumor growth in human breast cancer in vivo. The potential effect of Rg5 in the induction of caspase-dependent apoptosis and autophagy via the inhibition of the PI3K/Akt signaling pathway in human breast cancer tissues will also be verified. Besides, the first-line anticancer drug docetaxel will be used as the positive control. The in vivo anti-breast cancer effect and the side effects of Rg5 as well as docetaxel will be comprehensively studied.

2. Materials and methods

2.1 Experimental materials

Ginsenoside Rg5 (purity > 95%) was purchased from Chengdu Puruifa Technology Co., Ltd (Sichuan, China). RPMI 1640 and penicillin/streptomycin were purchased from HyClone (LA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Israel).

Antibodies against cleaved caspase-3, cleaved caspase-9, cleaved PARP, AKT, phospho-AKT, mTOR, phospho-mTOR and phospho-Bad were purchased from AbSci (Maryland, USA). Cleaved caspase-8, Fas, cytochrome-c, Bax, Bcl-2, Atg5, Atg7, Atg12, Beclin-1, LC3B, P62, PI3K, Bad, α-SMA and COX-2 were purchased from Proteintech Group Inc. (Chicago, IL, USA). β-Actin was purchased from Abcam (Cambridge, UK). Phospho-PI3K was purchased from Abbkine (California, USA).

2.2 Human breast cancer xenograft nude mice model

Four-week-old female BALB-c nude mice (13 ± 2 g) were purchased from Hunan SJA Lab Animal Co., Ltd (Hunan, China). They were maintained under specific pathogen free (SPF) conditions and provided with experimental rats maintain feed purchased from Chengdu Dashuo Experimental Animal Co., Ltd (Sichuan, China) and Milli-Q water. Human breast cancer cells (MCF-7) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines were cultured in RPIM 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin in a 5% CO2 atmosphere incubator at 37 °C.

After acclimation for one week, the mice were inoculated with MCF-7 cells (1 × 107 cells per mouse) in the left flank. When the tumors reached a size of 150 mm3, the mice were randomly assigned to four groups (n = 5): (1) control group: mice were injected intraperitoneally (i.p.) with saline every day; (2) two Rg5 groups: mice were injected i.p. with 10 mg kg−1 and 20 mg kg−1 Rg5 every day, respectively; and (3) positive control group (docetaxel group): mice were injected i.p. with 10 mg kg−1 docetaxel every three days. The body weight and tumor size were measured every three days, and the tumor volume was calculated using the standard formula: length × width2/2. After 30 days of treatment, the mice were sacrificed, tumor weights were measured and the tumors were stored in liquid nitrogen or fixed in formalin or glutaraldehyde for further analysis. All animal experiments were performed in compliance with the Animal Ethics Procedures and Guidelines of the People's Republic of China and approved by the Northwest University Animal Ethics Committee.

2.3 Transmission electronic microscopy (TEM)

The tumor tissues were fixed in 2.5% glutaraldehyde in PBS (pH 7.8) for 24 h at 4 °C. After dehydration with a graded ethyl alcohol series, the tumor tissues were embedded in epoxy resin and sectioned. Ultrathin sections (50 nm) were cut using an ultramicrotome, and double stained with uranyl acetate and lead citrate. Finally, the sections were observed via TEM (JEM-1230, Japan Electron Optics Laboratory, Japan).

2.4 Histopathology and immunohistochemistry

The tumor tissues and primary organs, including heart, lung, spleen, kidney and liver sections, were fixed in 10% buffered formalin and embedded in paraffin, and 5 μm sections were stained with hematoxylin and eosin (H&E). The tumor tissues were immunostained with cleaved caspase-3 (1[thin space (1/6-em)]:[thin space (1/6-em)]100), cleaved caspase-8 (1[thin space (1/6-em)]:[thin space (1/6-em)]200), cleaved caspase-9 (1[thin space (1/6-em)]:[thin space (1/6-em)]200), cleaved PARP (1[thin space (1/6-em)]:[thin space (1/6-em)]100), Fas (1[thin space (1/6-em)]:[thin space (1/6-em)]100), cytochrome-c (1[thin space (1/6-em)]:[thin space (1/6-em)]100), Bax (1[thin space (1/6-em)]:[thin space (1/6-em)]50), Bcl-2 (1[thin space (1/6-em)]:[thin space (1/6-em)]100), Atg-5 (1[thin space (1/6-em)]:[thin space (1/6-em)]100), Atg-7 (1[thin space (1/6-em)]:[thin space (1/6-em)]100), Atg-12 (1[thin space (1/6-em)]:[thin space (1/6-em)]100), Beclin-1 (1[thin space (1/6-em)]:[thin space (1/6-em)]100), LC3B (1[thin space (1/6-em)]:[thin space (1/6-em)]100), P62 (1[thin space (1/6-em)]:[thin space (1/6-em)]50), PI3K (1[thin space (1/6-em)]:[thin space (1/6-em)]200), phospho-PI3K (1[thin space (1/6-em)]:[thin space (1/6-em)]50), AKT (1[thin space (1/6-em)]:[thin space (1/6-em)]50), phospho-AKT (1[thin space (1/6-em)]:[thin space (1/6-em)]50), mTOR (1[thin space (1/6-em)]:[thin space (1/6-em)]50), phospho-mTOR (1[thin space (1/6-em)]:[thin space (1/6-em)]50), Bad (1[thin space (1/6-em)]:[thin space (1/6-em)]200) and phospho-Bad (1[thin space (1/6-em)]:[thin space (1/6-em)]100). The liver tissues were immunostained with α-SMA (1[thin space (1/6-em)]:[thin space (1/6-em)]100) and COX-2 (1[thin space (1/6-em)]:[thin space (1/6-em)]200). IHC signals were visualized with DAB; hematoxylin was used as a counterstain. The images were captured using a Nikon TE 2000 fluorescence microscope (Nikon, Japan).

2.5 Hemogram assay and measurement of biochemical parameters

Blood samples were collected from each group on the 30th day. Peripheral blood was drawn from the retro-orbital venous plexus and then collected in tubes containing ethylenediaminetetraacetic acid. White blood cell (WBC) count, lymphocyte (LYM) count and granulocyte (GRAN) count in each sample were determined by using an automatic hematology analyser (HC2200, Meiyilinm, China).

Serum samples were obtained from each group on the 30th day. Two important markers of liver function, ALT and AST, and two key markers of renal function, BUN and CRE, were determined using commercial kits according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China).

2.6 Western blot assay

Tumor tissues were extracted in ice-cold RIPA buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were centrifuged at 11[thin space (1/6-em)]000g for 20 min at 4 °C, and the total protein concentration was determined using the BCA Protein Assay Kit (Solarbio, Beijing, China). The proteins were separated by using SDS-PAGE gels and transferred to PVDF membranes. After blocking with TBST containing 5% skimmed milk for 2 h, the PVDF membranes were incubated with cleaved caspase-3 (1[thin space (1/6-em)]:[thin space (1/6-em)]1000), cleaved caspase-8 (1[thin space (1/6-em)]:[thin space (1/6-em)]4000), cleaved caspase-9 (1[thin space (1/6-em)]:[thin space (1/6-em)]1000), cleaved PARP (1[thin space (1/6-em)]:[thin space (1/6-em)]1000), Fas (1[thin space (1/6-em)]:[thin space (1/6-em)]500), cytochrome-c (1[thin space (1/6-em)]:[thin space (1/6-em)]500), Bax (1[thin space (1/6-em)]:[thin space (1/6-em)]4000), Bcl-2 (1[thin space (1/6-em)]:[thin space (1/6-em)]1000), Atg-5 (1[thin space (1/6-em)]:[thin space (1/6-em)]500), Atg-7 (1[thin space (1/6-em)]:[thin space (1/6-em)]500), Atg-12 (1[thin space (1/6-em)]:[thin space (1/6-em)]500), Beclin-1 (1[thin space (1/6-em)]:[thin space (1/6-em)]100), LC3B (1[thin space (1/6-em)]:[thin space (1/6-em)]500), P62 (1[thin space (1/6-em)]:[thin space (1/6-em)]2000), PI3K (1[thin space (1/6-em)]:[thin space (1/6-em)]500), phospho-PI3K (1[thin space (1/6-em)]:[thin space (1/6-em)]500), AKT (1[thin space (1/6-em)]:[thin space (1/6-em)]500), phospho-AKT (1[thin space (1/6-em)]:[thin space (1/6-em)]500), mTOR (1[thin space (1/6-em)]:[thin space (1/6-em)]500), phospho-mTOR (1[thin space (1/6-em)]:[thin space (1/6-em)]500), Bad (1[thin space (1/6-em)]:[thin space (1/6-em)]500) and phospho-Bad (1[thin space (1/6-em)]:[thin space (1/6-em)]500) at 4 °C overnight. Then, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h and detected using a enhanced chemiluminescence (ECL) substrate (Merck Millipore, Massachusetts, USA) with a Gel Image system (Tanon 5200, Shanghai, China). Band intensities were quantified using a Gel Image system (Tanon 5200, Shanghai, China).

2.7 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the frozen tumor tissue using TRIzol reagent (Ambion, MA, USA) according to the manufacturer's instruction. The quality of total RNA was determined by using a NanoDrop 1000 spectrophotometer (Thermo Scientific). cDNA was obtained from total RNA by using a reverse-transcription kit (Thermo Scientific). Gene expression levels were analyzed by qRT-PCR using SYBR green mix and CFX (BioRad). The cDNA was amplified as follows: 95 °C for 10 min, and 40 cycles of amplification at 95 °C for 10 s, 60 °C for 10 s, and 72 °C for 15 s. The data were calculated using the 2−ΔΔCT method. The expression of β-actin was used as an internal control for the quantitative real-time PCR experiments. The primers used are shown in Table 1.
Table 1 Sequences of primers used in the quantitative real-time reverse-transcription-polymerase chain reaction
Genes Forward primer (5′–3′) Reverse primer (5′–3′)
β-Actin ttccagccttgcttcctg tacttgcgcttgggagga
Fas tcatggccagaagtgcaa ttggggccactaggtgaa
Cytochrome-c ggcaggagaatcgcttga ccccaacttggcaacatc
Bax cccccgagaggtcttttt tcccggaggaagtccaat
Bcl-2 aatgtgcccagcctcttg tctgttgcccaactgcaa
Atg5 tttgcacaagaggctggtc tgcaaaggcctgacactg
Atg7 ggaccggctccagaaaat agcaatgacggcaggaag
Atg12 agaaaagcacgcccactg ggaaactgcagcggaaga
LC3B atgttgccacctcccaaa tccagcacgagttcacga
P62 agcgggcatcagtttgag gccctcctttccgatgat


2.8 Statistical analysis

The experimental data are expressed as mean values ± standard deviations (SD), which are obtained from three samples independently. One-way analysis of variance (ANOVA) followed by Duncan's test was performed using SPSS version 19.0 software (SPSS Inc., IL, USA). Statistical significance was considered at P < 0.05.

3. Results

3.1 Rg5 inhibited the growth of breast cancer in vivo

The in vivo effect of Rg5 on breast cancer was determined via subcutaneous administration in a tumor-transplanted mouse model. The morphological changes of the tumor are shown in Fig. 1B, and the growth of breast cancer was obviously inhibited by the Rg5 group and the docetaxel group. After 30 days of administration, Rg5 at doses of 10 and 20 mg kg−1 and docetaxel at a dose of 20 mg kg−1 significantly decreased the tumor weights by 38.2 ± 12.3%, 71.4 ± 9.4% and 72.0 ± 9.1%, respectively (Fig. 1C). The tumor volumes and body weights of the mice in each group were measured every three days after injection. The tumor volume increased more slowly in the 10 and 20 mg kg−1 Rg5 and docetaxel groups than in the control group (P < 0.05; Fig. 1D). It is worth noting that the mean body weight was not significantly different in the 10 mg kg−1 Rg5 group (19.97 ± 0.62 g), the 20 mg kg−1 Rg5 group (19.01 ± 0.64 g) and the control group (21.04 ± 0.81 g) (P > 0.05), whereas the weight of the docetaxel group (15.21 ± 0.61 g) was significantly lower than that of the control group (P < 0.05); the body weight loss of the docetaxel group reached 27.7 ± 2.9% (Fig. 1E), suggesting that compared to docetaxel, Rg5 exhibited few side effects.

H&E staining and TEM assay demonstrated more dead cells and a much larger apoptosis proportion could be found in the Rg5 group and the docetaxel group in the tumor tissues (Fig. 1F and G). In H&E staining, the tumor cells in the control group were arranged tightly and disordered, and had deep staining and less intercellular substance, whereas the tumor cells in the Rg5 group and the docetaxel group were loosely arranged with lighter staining, partial degeneration and necrosis (Fig. 1F). From TEM, we could observe that the tumor cells in the control group appeared large and had irregular nuclei. In the group treated with low dose Rg5 (10 mg kg−1), there was cytoplasmic vacuolation, partially. However, there were large areas of nuclear condensation and cytoplasmic vacuolation in the high dose Rg5 (20 mg kg−1) group and the docetaxel group (Fig. 1G). Taken together, these results strongly suggested that Rg5 inhibited the growth of breast cancer in vivo.

3.2 Rg5 had no damage on the normal function of the vital organs and immune cells in vivo

To analyze whether Rg5 affected the normal functions of the vital organs and cellular immunity in vivo, firstly, we assayed the immune cell content in the peripheral blood in the normal group (the normal feeding nude mice with no tumor inoculation), the control group, the low dose Rg5 group, the high dose group and the docetaxel group, respectively. The WBC, LYM and GRAN contents in the peripheral blood from the normal group, the control group, the low dose Rg5 group and the high dose group showed no significant difference (P > 0.05; Fig. 2A). However, the WBC, LYM and GRAN contents in the peripheral blood from the docetaxel group obviously decreased compared to the normal group (P < 0.05; Fig. 2A), suggesting that docetaxel could damage the immune function in nude mice. However, Rg5 had no damage on the immune system.
image file: c8fo01122b-f2.tif
Fig. 2 The effect of Rg5 and docetaxel on the normal functions of the vital organs and immune cells in vivo. (A) Hemogram assay reveals the white blood cell (WBC), lymphocyte (LYM) and granulocyte (GRAN) counts of the peripheral blood from each group. (B) The levels of the serum biochemical markers ALT, AST, BUN and CRE from each group. (C) H&E staining of major organs from each group, including the heart, liver, spleen, lung and kidney. Immunohistochemical staining of liver from each group. Scale bars = 100 μm. The data are presented as mean ± SD of three independent experiments. P < 0.05 was considered to indicate statistical significance. Different letters indicate significant differences between each group.

There was no significant difference in the serum levels of two transaminases (ALT and AST) among the normal group, the control group, the low dose Rg5 group and the high dose group (P > 0.05; Fig. 2B). Compared to the normal group, the serum levels of these two transaminases were obviously elevated in the docetaxel group (P < 0.05; Fig. 2B), suggesting that docetaxel damaged the liver function. However, Rg5 had no damage on the liver function. Furthermore, the serum levels of BUN and CRE showed no significant difference among the normal group, the control group, the low dose Rg5 group, the high dose group and the docetaxel group (P > 0.05; Fig. 2B), indicating that Rg5 and docetaxel had no effect on the renal function.

In addition, we analyzed the pathological pattern of major organs, including the heart, lung, spleen, kidney and liver. As shown in Fig. 2C, H&E staining indicated that the low dose and high dose Rg5 groups did not affect the normal functions of the vital organs compared to the normal group. However, docetaxel led to some eosinophilic cell necrosis in the liver tissue, which is revealed by the arrow (Fig. 2C). Furthermore, COX-2 and α-SMA were the two proteins associated with liver injury. Increased expression of Cox-2 and α-SMA proteins could lead to the occurrence of liver fibrosis. As shown in Fig. 5B, immunohistochemistry analysis of liver tissue showed that the mean areas that stained positively for COX-2 and α-SMA in the docetaxel group were both larger than that in the normal group, suggesting that docetaxel could cause liver injury. Otherwise, there was no significant difference in the mean area that stained positively for COX-2 and α-SMA between the Rg5 group and the normal group. Taken together, these results demonstrated that Rg5 had no effect on the normal functions of the vital organs and immune cells in vivo.

3.3 Rg5 induced caspase-dependent apoptosis in vivo via the extrinsic and intrinsic pathways

To determine whether apoptosis was responsible for Rg5 inhibited breast cancer in vivo, we performed western blot, qRT-PCR and immunohistochemistry assay. Caspases are a family of cytoplasmic cysteine endoproteases, which play an important role in regulating apoptosis. The activation of caspases represents the propagation of death signals via the extrinsic and intrinsic pathways.11,42 Therefore, we attempted to determine which pathway was involved. To clarify the involvement of the extrinsic pathway in the Rg5-induced apoptosis of breast cancer tissues, we examined whether Rg5 affects the expression of the membrane death receptor and adaptor proteins in breast cancer tissues. Western blotting results indicated that Rg5 and docetaxel all markedly increased the relative protein expression level of Fas and cleaved caspase-8 (P < 0.05; Fig. 3A and B). What's more, Rg5 increased the expression levels of the two proteins in a dose-dependent manner (Fig. 3A and B). These findings demonstrated that extrinsic death receptor signaling could lead to Rg5-induced apoptosis in vivo. In addition, the release of cytochrome-c from the mitochondria to the cytoplasm and the activation of the proteins Bax and Bcl-2 play important roles in the intrinsic pathway of the apoptosis pathway.43 As shown in Fig. 2A and B, the high dose Rg5 group significantly increased the expression levels of the pro-apoptotic proteins Bax and cytochrome-c, and suppressed the expression of the anti-apoptotic protein Bcl-2, which was similar to the results in the docetaxel group compared to the control group (P < 0.05). Rg5 increased the protein expression levels of Bax and cytochrome-c and decreased the protein expression level of Bcl-2 in a dose-dependent manner (Fig. 2A and B). The Bax/Bcl-2 ratio values in the breast cancer tissues treated with Rg5 at 10 and 20 mg kg−1 and docetaxel were significantly increased by 57.0 ± 11.7%, 285.7 ± 19.2%, and 299.4 ± 30.0%, respectively, compared to the control group (P < 0.05; Fig. 3C). Rg5 also obviously promoted the cleavage of downstream protein caspase-9 and increased the protein expression level of cleaved caspase-9 in a concentration-dependent manner (Fig. 3A and B). These results showed that Rg5 induced apoptosis in vivo also through the intrinsic pathway. Moreover, Fig. 2A and B show that Rg5 significantly promotes the activation of downstream apoptosis proteins caspase-3 and PARP and increased the protein expression levels of cleaved caspase-3 and cleaved PARP in a concentration-dependent manner (Fig. 3A and B).
image file: c8fo01122b-f3.tif
Fig. 3 Rg5 induces the apoptosis of human breast cancer in vivo. (A) Western blotting assay for detecting the expression of apoptosis-associated proteins in the tumor tissues from each group. β-Actin was used as an endogenous reference. (B) The histogram represents the statistical analysis of the relative expression level of apoptosis-associated proteins. (C) The histogram represents the statistical analysis of the protein expression level of Bax/Bcl-2. (D) Real-time qRT-PCR assay was performed to measure the relative mRNA expressions of Fas, cytochrome-c, Bax and Bcl-2 in the tumor tissues from each group. (E) Immunohistochemical staining was performed to measure the expression of apoptosis-associated proteins in the tumor tissues from each group. Scale bars = 100 μm. The data are presented as mean ± SD of three independent experiments. P < 0.05 was considered to indicate statistical significance. Different letters indicate significant differences between each group.

In addition, the data of the apoptotic proteins were consistent with the results of our qRT-PCR analysis of apoptosis-related genes. Rg5 was also able to promote the cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, cleaved PARP, Fas, cytochrome-c and Bax expressions and suppress Bcl-2 expression at the mRNA level (P < 0.05; Fig. 3D). Furthermore, through a series of immunohistochemistry experiments, we observed that compared to the control group, the mean areas that stained positively for cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, cleaved PARP, Fas, cytochrome-c and Bax expression in the high dose Rg5 group and the docetaxel group were larger than those in the control group. The mean areas that stained positively for Bcl-2 in the high dose Rg5 group and the docetaxel group were smaller than those in the control group (Fig. 3E). The immunohistochemistry data were therefore in good agreement with the qRT-PCR and western blot data. Taken together, these results implied that Rg5 induced caspase-dependent apoptosis in vivo via the extrinsic and intrinsic pathways.

3.4 Rg5 induced autophagy in vivo with the regulation of critical Atg proteins

We then determined whether Rg5 could induce autophagy in vivo. Autophagic cell death (type II programmed cell death) is also related to cancer development, which is different from apoptosis (type I programmed cell death).44 TEM was used to directly demonstrate autophagosome formation. Fig. 1F shows that the high dose Rg5 and docetaxel treated groups had more autophagosomes in the cytoplasm than were observed in the control group, which is shown by the arrow. LC3 is a critical protein in autophagy. During autophagy, LC3 is cleaved into an intermediate (referred to as LC3-II) that relates to the autophagosome formation.14 P62, which is also a typical autophagic substrate, is widely used as an indicator of autophagic degradation.45,46Fig. 4A and B show that in the high dose Rg5 and docetaxel groups, the protein levels of LC3B-II were both outstandingly increased and the protein levels of P62 were both significantly decreased compared with the control group (P < 0.05). Rg5 increased the protein expression level of LC3B-II and decreased the protein expression level of P62 in a dose-dependent manner (Fig. 4A and B). The LC3B-II/LC3B-I ratio values in the breast cancer tissues were significantly increased when treated with Rg5 at 10 and 20 mg kg−1 and docetaxel by 87.9 ± 13.7%, 141.4 ± 9.4% and 140.0 ± 4.4%, respectively, compared to the control group (P < 0.05; Fig. 4C). These data were consistent with the results of our western blot analysis of autophagy marker proteins, including Atg-5, Atg-7 and Atg-12. As shown in Fig. 4A and B, the expression levels of Atg-5, Atg-7 and Atg-12 were significantly greater in the high dose Rg5 group (vs. the control group). Rg5 increased the protein expression levels of Atg-5, Atg-7 and Atg-12 in a concentration-dependent manner (Fig. 4A and B). Otherwise, in contrast to the docetaxel group, the protein levels of Atg-5, Atg-7 and Atg-12 were significantly increased in the high dose Rg5 group (P < 0.05, Fig. 4A and B), suggesting that the high dose Rg5 group was more effective on the autophagy proteins than the docetaxel group.
image file: c8fo01122b-f4.tif
Fig. 4 Rg5 induces autophagy in human breast cancer in vivo. (A) Western blotting assay for detecting the expression of autophagy-related proteins in the tumor tissues from each group. β-Actin was used as an endogenous reference. (B) The histogram represents the statistical analysis of the relative expression level of autophagy-related proteins. (C) The histogram represents the statistical analysis of the protein expression level of LC3-II/LC3-I. (D) Real-time qRT-PCR assay was performed to measure the relative mRNA expressions of autophagy-related proteins in the tumor tissues from each group. (E) Immunohistochemical staining was performed to measure the expression of autophagy-related proteins in the tumor tissues from each group. Scale bars = 100 μm. The data are presented as mean ± SD of three independent experiments. P < 0.05 was considered to indicate statistical significance. Different letters indicate significant differences between each group.

In addition, the data of the autophagy proteins were consistent with the results of our qRT-PCR analysis of autophagy-related genes. As shown in Fig. 4D, Rg5 was able to promote LC3B, Atg-5, Atg-7 and Atg-12 expressions and suppress p62 expression at the mRNA level. Furthermore, we evaluated the effect of Rg5 on tumor tissue autophagy by immunohistochemistry assay. Consistently, a similar tendency was observed by IHC staining of LC3B-II, Atg-5, Atg-7, Atg-12 and P62. Compared to the control group, the number of autophagic cells with positive staining of LC3B-II, Atg-5, Atg-7 and Atg-12 was much higher in the high dose Rg5 group, and the number of autophagic cells with positive staining of P62 was much less (Fig. 4D). Collectively, all the above results suggest that Rg5 obviously induced autophagy in vivo, and Rg5 was more effective on autophagy in vivo than docetaxel.

3.5 Rg5 inhibited the PI3K/Akt signaling pathway in vivo

To further explore the upstream pathway of Rg5 on apoptosis and autophagy in vivo, the level of PI3K, Akt, mTOR and Bad and their phosphorylated forms was measured by western blot and immunohistochemistry analysis. As shown in Fig. 5A and B, treatment of breast cancer tissues with high dose Rg5 and docetaxel all decreased the phosphorylation level of the PI3K protein at Tyr607 compared to the control group (P < 0.05). Notably, exposure of cancer tissues to high dose Rg5 did not significantly affect the level of PI3K (P > 0.05). The docetaxel group showed the same result (Fig. 5B). However, the ratio of the p-PI3K level to that of PI3K in the tumor tissue was significantly increased by Rg5 at 10 mg kg−1 and 20 mg kg−1 in a concentration-dependent manner, compared to the control group. The p-PI3K/PI3K ratio was decreased from 1.39 ± 0.12 at the basal level to 1.15 ± 0.07, 0.74 ± 0.03 and 0.69 ± 0.08, when the tissues were treated with low dose Rg5, high dose Rg5 and docetaxel, respectively (Fig. 5C). Moreover, immunohistochemistry analysis of the tumor tissue demonstrated a similar result; the mean areas that stained positively for p-PI3K in the high dose Rg5 group and the docetaxel group were both smaller than that in the control group (Fig. 5D).
image file: c8fo01122b-f5.tif
Fig. 5 Rg5 mediate the PI3K/Akt signaling pathway in vivo. (A) Western blotting assay reveals the expression of the PI3K/Akt pathway proteins (PI3K, Akt, mTOR and Bad) in the tumor tissues from each group. β-Actin was used as an endogenous reference. (B) The histogram represents the statistical analysis of the relative expression level of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, Bad and p-Bad. (C) The histogram represents the statistical analysis of the protein expression level of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR. (D) Immunohistochemical staining was performed to measure the expression of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, Bad and p-Bad in the tumor tissues from each group. Scale bars = 100 μm. The data are presented as mean ± SD of three independent experiments. P < 0.05 was considered to indicate statistical significance. Different letters indicate significant differences between each group.

Rg5 significantly decreased the phosphorylation of Akt at Ser473 in the tumor tissue in a concentration-dependent manner (Fig. 5A and B). With increasing concentrations of Rg5, the phosphorylation level of Akt at Ser473 was significantly decreased, indicating a clear dose dependence of Akt Ser473 phosphorylation inhibition by Rg5 (Fig. 5B). In addition, compared to the control group, the tumor tissue treated with 10 mg kg−1 and 20 mg kg−1 Rg5 and docetaxel did not significantly affect the expression of the Akt protein (P > 0.05; Fig. 5B). However, the ratio of p-Akt level to that of Akt in the tumor tissue was significantly increased by Rg5 at 10 mg kg−1 and 20 mg kg−1 in a concentration-dependent manner, compared to the control group. The p-Akt/Akt ratio was decreased from 1.69 ± 0.15 at the basal level to 1.52 ± 0.03, 1.28 ± 0.03 and 1.29 ± 0.04 when the tissues were treated with low dose Rg5, high dose Rg5 and docetaxel, respectively (Fig. 5C). Consistently, immunohistochemistry analysis of the tumor tissue demonstrated the same result, the mean areas that stained positively for p-Akt in the high dose Rg5 group and the docetaxel group were both smaller than that in the control group (Fig. 5D).

mTOR plays an important role in autophagic cell death and homeostasis.25 mTOR is phosphorylated at Ser2448 through the PI3K/Akt signaling pathway.47 In this study, we have observed that Rg5 treatment obviously reduced the phosphorylation level of mTOR at Ser2448 in the tumor tissue in a concentration-dependent manner (Fig. 5A and B). In addition, compared to the control group, the tumor tissue treated with 10 mg kg−1 and 20 mg kg−1 Rg5 and docetaxel did not significantly affect the expression level of the mTOR protein (P > 0.05; Fig. 5B). However, the ratio of the p-mTOR level to that of mTOR in the tumor tissue was significantly increased by Rg5 at 10 mg kg−1 and 20 mg kg−1 in a dose-dependent manner, compared to the control group. The p-mTOR/mTOR ratio was decreased from 1.82 ± 0.11 at the basal level to 1.28 ± 0.08, 1.06 ± 0.10 and 1.29 ± 0.09, when the tumor tissues were administered with low dose Rg5, high dose Rg5 and docetaxel, respectively (Fig. 5C). Furthermore, immunohistochemistry analysis of the tumor tissue demonstrated the same result; the mean areas that stained positively for p-mTOR in the high dose Rg5 group and the docetaxel group were both smaller than that in the control group (Fig. 5D).

The pro-apoptotic protein Bad can be suppressed by phosphorylation at either Ser112 or Ser136. Akt specifically phosphorylates Bad at Ser136 (bad1). As shown in Fig. 5A and B, treatment of breast cancer tissues with high dose Rg5 and docetaxel all significantly increased the expression level of Bad and decreased the phosphorylation level of Bad at Ser136 compared to the control group (P < 0.05). Rg5 elevated the expression level of Bad and reduced the phosphorylation level of Bad at Ser136 in a concentration-dependent manner (Fig. 5B). Consistently, immunohistochemistry analysis of the tumor tissue demonstrated the same result; the mean areas that stained positively for Bad in the high dose Rg5 group and the docetaxel group were both larger and the mean areas that stained positively for p-Bad were both smaller compared to those in the control group (Fig. 5D).

4. Discussion

Previous studies have found that Rg5, one of the main components of Panax ginseng, has strong anti-breast cancer activities in vitro.41 Here, we studied and demonstrated the defined molecular mechanisms underlying the Rg5-regulated suppression of breast cancer and biological safety in vivo. In a xenograft nude mice model, we found that Rg5 at doses of 10 and 20 mg kg−1 significantly inhibited tumor growth (38.2 ± 12.3% and 71.4 ± 9.4%). In contrast, treatment with docetaxel led to an approximately 72.0 ± 9.1% reduction in tumor growth. H&E staining and TEM assay also demonstrated that the high dose Rg5 group and the docetaxel group significantly inhibited the growth of the tumor compared to the control group. However, the body weight of the mice administered with docetaxel was markedly lower. There was no significant difference in body weight between the Rg5 treatment group and the control group. Therefore, Rg5 showed high anti-breast cancer activity in vivo.

The first-line anticancer drug docetaxel is one of the semi-synthetic analogues of paclitaxel, which is used to treat multiple cancers.48 However, docetaxel has dose side effects in the treatment of breast cancer.49,50 Previous reported demonstrated that docetaxel can cause liver injury and bone marrow depression.4,48 Consistently, our serum biochemical parameter assay and H&E and IHC staining results for liver tissues treated with docetaxel showed liver injury. Besides, compared to the normal group, the results revealed that docetaxel obviously decreased the content of the immune cells, including WBC, LYM and GRAN, suggesting that docetaxel had a high toxicity. In contrast, for the Rg5 treatment group, H&E and IHC staining indicated that the low dose and high dose Rg5 groups did not affect the normal functions of the vital organs, including the heart, liver, spleen, lung and kidney. Serum biochemical parameter assays indicated that Rg5 had no damage on the liver function and renal function. The hemogram assay demonstrated that Rg5 did not reduce the content of the immune cells, suggesting that Rg5 showed no immune damage. Therefore, these results suggested that Rg5 showed less side effects in the treatment of breast cancer.

Then, we studied the defined molecular mechanisms underlying the Rg5-regulated suppression of human breast cancer. Type I cell death apoptosis and type II cell death autophagy are two primary morphologically distinctive modes of programmed cell death.25 Apoptosis can be caused by multisignal pathways, which is a conventional pathway inducing cells to go through a highly regulated form of cell death in response to pro-apoptotic stimuli.9,51 Apoptosis is executed by members of the caspase family of cysteine proteases and activated by two main pathways, which are the extrinsic death receptor pathway and the intrinsic mitochondrial pathway, respectively.52,53 The extrinsic pathway is mediated by the stimulation of the trans-membrane death receptor family, such as Fas (Apo-1/CD95). The Fas antigen (Fas) is a 43–52 kDa type I cell surface glycoprotein,54 which transports external apoptotic signals to the death machinery, leading to the activation of caspase-8, which indicates an extrinsic apoptosis pathway.55 Our results showed that Rg5 significantly increased the protein expression levels of Fas and cleaved caspase-8, which indicated that Rg5 initiated the extrinsic death receptor signaling pathway in the tumor tissue. The Bcl-2 protein families are the important initiator of the intrinsic mitochondrial apoptotic pathway. This family includes both the pro-apoptotic proteins Bax and Bad and the anti-apoptotic protein Bcl-2.56 The Bcl-2 protein is a critical member of the Bcl-2 family and is a widely studied modulator of programmed cell death, which can promote cell survival in many systems.57 Furthermore, Bax is an opposition partner of Bcl-2; its overexpression promotes the increase of the Bcl-2 protein expression level.56 The activation of Bax promotes the release of cytochrome-c from the mitochondria into the cytoplasm, and then activates the caspase cascade.58 Caspase-9 is the main initiating caspase for the intrinsic pathway to cell death.59 Cytochrome-c in the cytoplasm forms a multi-protein complex known as an apoptosome binding to Apaf 1. This complex activates caspase-9 and leads to mitochondrial apoptotic cell death.11 Our studies demonstrated that Rg5 obviously increased the protein expression levels of Bax, cytochrome-c and cleaved caspase-9 and decreased the protein expression level of Bcl-2, which indicated that Rg5 initiated the intrinsic mitochondrial apoptotic pathway in the tumor tissue. Caspase-3 is known to be related to propagate the caspase cascade, and be activated at the execution phase of programmed cell death.60 The extrinsic and intrinsic apoptotic pathways are related and both trigger the activation of caspase-3 and in turn cleave intracellular substrates including PARP (DNA repair proteins), which ultimately resulted in DNA damage and apoptosis.11 Our results showed that Rg5 significantly promoted the cleavage of caspase-3 and PARP. All the above results demonstrated that Rg5 induced caspase-dependent apoptosis in vivo via both the extrinsic death receptor and intrinsic mitochondrial signaling pathways.

Besides apoptosis, autophagy also plays an important role in determining cellular fate.9 Autophagy is a cellular process characterized by the formation of an autophagosome which is a double membrane structure containing cytoplasmic material.61 In this study, we observed the formation of autophagosomes with the TEM assay. Previous reports have shown that the process of autophagosome formation is regulated by proteins encoded by autophagy interrelated genes (Atg).14 LC3B-II, generated from lipidated LC3B-I, is related to autolysosome formation and is a crucial marker for autophagy.62 As a typical negative regulation protein of autophagy, P62 can promote the movement of ubiquitinated substrates to the autophagosomes.63 Impairments in autophagy are usually related to lots of accumulation of the SQSTM1/P62 protein.64 Our results suggested that Rg5 promoted the conversion of LC3B-I into LC3B-II, increased the protein expression level of LC3B-II/LC3B-I, and induced autophagosome formation. In addition, Rg5 significantly reduced the protein expression level of P62 and activated autophagy. The Atg-5, Atg-7 and Atg-12 proteins play a positive regulation role in autophagic cell death.61 Our results suggested that Rg5 obviously promoted the expression of Atg-5, Atg-7 and Atg-12 at both the protein and mRNA levels. Besides, our studies demonstrated that high dose Rg5 was more effective in increasing the positive regulation autophagy protein expression and decreasing the negative regulation autophagy protein expression compared to the docetaxel group, suggesting that Rg5 promoted the occurrence of autophagy in vivo compared to docetaxel. Therefore, these results suggested that Rg5 induced autophagy in vivo, and Rg5 was more effective on autophagy in vivo than docetaxel.

Next, we explored the upstream pathways. The PI3K/Akt pathway is critical for the cell signaling pathway and regulates lots of cellular functions, such as survival, proliferation and differentiation.65 PI3K activates the serine/threonine kinase Akt, which results in the phosphorylation and activation of the serine/threonine kinase mTOR via a cascade of regulators.25,66 Previous studies have shown that the PI3K/Akt signaling pathway can negatively regulate autophagy by promoting the phosphorylation of mTOR.67 Our results demonstrated that Rg5 significantly decreased the phosphorylation level of PI3K, Akt and mTOR in a concentration-dependent manner. Besides, the obvious decreased values of p-PI3K/PI3K, p-Akt/Akt and p-mTOR/mTOR indicated that Rg5 inhibited the PI3K/Akt/mTOR pathway. Therefore, these results imply that Rg5 induced autophagy via the inhibition of the PI3K/Akt/mTOR pathway.

Akt is the main downstream target of PI3K and the phosphorylation of Akt is considered to be active type.68 The phosphorylation of Akt is a significant link in the cell apoptosis process.56 Activated Akt phosphorylates the serine residues of Bad at Ser136 and weakens its actions.23 Bad is a vital pro-apoptotic protein of the Bcl-2 family,58 which can form heterodimers with Bcl-2, therefore attenuating their anti-apoptotic activity.32 The phosphorylated Bad binds with the 14-3-3 protein, which prevents Bad from binding with Bcl-2 at the mitochondrial membrane.23 Thus, the phosphorylation of Bad is critical for the inhibition of apoptosis. Previous reports have demonstrated that the PI3K/Akt pathway inhibits mitochondria-mediated apoptosis in papillary thyroid carcinoma (PTC) cells by promoting the phosphorylation of Bad.69 Our results showed that Rg5 obviously promoted the decrease of Akt phosphorylation and Bad phosphorylation, which indicated that Rg5 could induce tumor tissue apoptosis via the PI3K/Akt/Bad cascade.

5. Conclusion

In summary, our results verified that Ginsenoside Rg5 could exhibit strong antitumor effects against human breast cancer in vivo. Compared to the first-line anticancer drug docetaxel, Rg5 showed almost no liver injury or immune damage in the treatment of human breast cancer. The potential mechanism of Rg5 in inducing caspase-dependent apoptosis and autophagy was via the inhibition of the PI3K/Akt signaling pathway in human breast cancer in vivo (Fig. 6). These findings revealed the efficacy and multiple anticancer molecular mechanisms of Rg5 and recommended Rg5 as a prospect for improving the current chemotherapeutic strategy for the treatment of human breast cancer.
image file: c8fo01122b-f6.tif
Fig. 6 Schematic representation of the hypothesized molecular mechanism underlying the anti-breast cancer activity of Rg5 in vivo. The PI3K/Akt signaling pathway is involved in Rg5-induced apoptosis and autophagy.

Abbreviations

Rg5Ginsenoside Rg5
WBCWhite blood cell
LYMLymphocyte
GRANGranulocytes
ALTAlanine aminotransferase
ASTAspartate aminotransferase
BUNBlood urea nitrogen
CRECreatinine
PI3KPhosphoinositide 3-kinase
mTORMammalian target of rapamycin
Cleaved cas.3Cleaved caspase-3
Cleaved cas.8Cleaved caspase-8
Cleaved cas.9Cleaved caspase-9

Conflicts of interest

No potential conflict of interest was reported by the authors.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21476182, 21776228 and 21776227), the Shaanxi Key Laboratory of Degradable Biomedical Materials Program (2015SZSj-42, 2014SZS07-P05 and 2016SZSj-35), and the Shaanxi R&D Center of Biomaterials and Fermentation Engineering Program (2015HBGC-04).

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