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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Anti-tumor effects and mechanisms of Astragalus membranaceus (AM) and its
specific immunopotentiation: Status and prospect
Shanshan Li
a,1
, Yi Sun
b,1
, Jin Huang
a
, Bin Wang
c
, Yinan Gong
a
, Yuxin Fang
a,d
, Yangyang Liu
a,d
,
Shenjun Wang
a,d
, Yi Guo
a,e
, Hong Wang
d
, Zhifang Xu
a,d,∗
, Yongming Guo
a,d,∗∗
a
Acupuncture Research Center, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China
b
Nephropathy and Rheumatology Department, Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin, 300250, China
c
Tianjin Medical University Cancer Institute of Hospital, National Clinical Research Center for Cancer, Tianjin, 300060, China
d
Acu-moxibustion and Tuina Department, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China
e
College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, 301617, China
ARTICLE INFO
Keywords:
Astragalus membranaceus
Tumor immune microenvironment
Host immunity
Organic immunity
Chemotherapy
ABSTRACT
With cancer deaths increasing, the initiation, pathophysiology and curative management of cancer is receiving
increasing attention. Traditional therapies such as surgery and chemoradiotherapy are often accompanied by
suppression of host immunity, which increase the risk of metastasis. Astragalus membranceus (AM) is commonly
utilized as one herbal medicine of traditional Chinese medicines (TCMs) with a variety of biological activities.
Studies have shown that the active ingredients of AM and AM-based TCMs, combined with chemotherapy, can
enhance anti-tumor efficacy in cancer patients, in addition to reduce complications and avoid side effects in-
duced by chemotherapy. By using various cancer models and cell lines, AM has been found to be capable of
shrinking or stabilizing tumors by direct anti-proliferation or pro-apoptosis effect on tumor cells. Further, AM
ameliorates immunosuppression by activating M1 macrophages and T cells tumor-kill function in tumor mi-
croenvironment (TME). AM is also found to improve systemic immunity which may help promoting efficacy of
chemotherapy and preventing metastasis. Thereby this review contributes to an understanding of AM as an
adjunctive therapy in the whole course of cancer treatment, at the same time providing useful information for
development of more effective anti-tumor medication. The combination of AM and immune checkpoint therapies
has a promising therapeutic prospect, and the observation of direct efficacy and mechanisms on tumor growth
and metastasis of AM combined with chemotherapies or other therapies require more in vivo validations and
further clinical investigation as well.
1. Introduction
With the rapid growth and aging of the global population, cancer
has become increasingly prominent as a leading cause of death (Bray
et al., 2018). It is predicted that the incidence of all cancer cases will
increase from 12.7 million new cases in 2008 to 22.2 million in 2030
(Bray et al., 2012). Therefore, cancer occurrence, pathophysiology and
therapeutic option development are receiving increased attention
worldwide, especially in low- and middle-income countries such as
China.
Traditional therapies such as surgery and chemoradiotherapy can
directly act on cancer cells while have several serious drawbacks.
Firstly, most cancer patients are diagnosed too late to perform surgery.
Even if there are surgical indications, a series of complications such as
bleeding, infection, lymphedema may occur after surgery. Secondly,
although chemoradiotherapy is still the main adjuvant therapy to sur-
gery or the preferred treatment for patients with advanced malignant
tumors, there are also many side effects and complications, such as
bone marrow suppression, impaired liver and kidney function, nausea
and vomiting or local radiotherapy damage. More importantly, several
regularly used chemotherapeutic drugs convert cancer cells into cancer
stem cells, thereby resulting in therapeutic resistance and accelerating
https://doi.org/10.1016/j.jep.2020.112797
Received 18 December 2019; Received in revised form 10 March 2020; Accepted 23 March 2020
∗
Corresponding author. Acu-moxibustion and Tuina Department, Tianjin University of Traditional Chinese medicine, No.10 Poyang Lake Road, Tuanbo new town,
Jinghai district, Tianjin, 301617, China.
∗∗
Corresponding author. Acu-moxibustion and Tuina Department, Tianjin University of Traditional Chinese medicine, No.10 Poyang Lake Road, Tuanbo new town,
Jinghai district, Tianjin, 301617, China.
E-mail addresses: xuzhifangmsn@hotmail.com (Z. Xu), guoymxr@163.com (Y. Guo).
1
Equal contribution to this work.
Journal of Ethnopharmacology 258 (2020) 112797
Available online 01 April 2020
0378-8741/ © 2020 Elsevier B.V. All rights reserved.
T
cancer cell metastasis by worsening host immunity (Martins-Neves
et al., 2016;Safa et al., 2015).
Accumulating evidence has confirmed that cancer cells reside in a
specialized microenvironment, or niche, namely the TME. Tumor cells
must recruit and reprogram the surrounding normal cells to serve as
contributors so that ensure their rapid proliferation, survival, local in-
vasion and remote metastasis (Casey et al., 2015). With tumor devel-
opment, there is a dynamic alteration on molecular and cellular pro-
cesses in TME involving the interactions between cancer cells and
immune cells. Given the effectiveness of T cells in mediating anti-tumor
immune responses, T cell-based immunotherapy is considered as an
important and promising therapeutic approach against cancer (Chen
and Mellman, 2017). However, the majority of patients treated with
immune monotherapies fail to achieve the desired therapeutic response.
The main reasons could be the severe immune-suppressive micro-
environment including impaired antigen presentation capability in
tumor sites that inhibit the proliferation, migration and survival of in-
filtrating T cells. How to improve the host immune status is therefore
very important in T cell-based immunotherapy (Nicolas-Boluda and
Donnadieu, 2019).
Astragalus membranaceus (AM, Huang qi in Chinese) is a plant belong
to the leguminous family. AM particularly its dry root Astragali Radix,is
a popular tonic in traditional Chinese medicine (TCM). It has been
found to have multiple biofunctions, such as immunomodulatory, anti-
hyperglycemic, anti-inflammatory, anti-oxidant and anti-viral activities
(Shao et al., 2004). Modern pharmacological evidence has shown that
AM and its active ingredients have strong anti-tumor activity and en-
hance host immune function. Thus, this review will summarize the
capability of AM to reduce the side effects and complications and in-
crease the anti-tumor efficacy caused by chemotherapy in cancer pa-
tients. Meanwhile, the mechanism evidences that AM directly shrinks
focus or stabilizes cancer state, enhancing the organic immunity to
improve the efficacy of chemotherapy and prevent metastasis in a
variety of pathways will be accumulated, providing a systematic review
and evaluation of the anti-tumor effects and mechanisms of AM. More
importantly, the evidence of AM being able to regulate the tumor im-
mune microenvironment (TIME) will be provided, highlighting the
multiple anti-tumor targets and signaling pathways of AM as an im-
munity enhancer, may act as a booster for checkpoint immunotherapy
and chemotherapy.
2. Method
We searched the PubMed database for studies published unlimited,
beginning from January 2000 to January 2020. The keywords included
[“Astragalus membranaceus”or “Huangqi”]or[“Astragalus”] and
[“tumor”or “cancer”or “immunity”or “immune”]. Language was
limited to English and Chinese. The filter process was firstly done by
search engine of the website which screened out 422 articles. We ex-
cluded 221 articles due to absence of abstract or being not fit of the
theme in abstract, by the authors and screened out 201 articles. We
then excluded 123 articles due to unavailability of the full text and
being not fit of the theme by the authors which screened out 78 articles.
Therefore, the number of basic, clinical and review article/meta-ana-
lysis were 40, 9 and 29 respectively. Flowchart of the search processes
is shown in Fig. 1.
3. General introduction of Astragalus membranaceus
AM was first recorded in the Han Dynasty's "Shen Nong's Herbs"
2000 years ago [Li et al., 2019a,b]. Chinese doctor also calls it “huang
qi”, but this refers mainly to the roots of AM, named “Astragali Radix”.
AM is commonly recognized as a tonic to treat patients with a defi-
ciency in vitality, which present as a lack of strength, anorexia, spon-
taneous sweating, edema, and abscesses. It can also induce urination
and promote the discharge of pus, plus the growth of new tissue
formulated as an ingredient of herbal mixtures in decoction. It has also
been used as a health food supplement as well as in Chinese ethnic
tonifying soups, gruel and tea in some Asian populations (Lin et al.,
2019;Psihogios et al., 2019).
AM is a leguminous plant, mainly distributed in the northeast, north
and northwest regions of China, Mongolia and North Korea. There are
over 2000 species of AM, like Astragalus membranaceus (Fisch.) Bge.
(Fam Leguminosae) and Astragalus membranaceus (Fisch.) Bge. Var.
mongholicus (Bge.), among which Hsiao are the most commonly ap-
plied. More than 200 compounds were identified from AM including
polysaccharides, saponins, flavonoids and some others, several of which
have been found with biological activities (Li et al., 2014;Qi et al.,
2008;Zhang et al., 2015). Because polysaccharides are large molecules
with complex chemical structures, their precise chemical analysis is
very limited (Jin et al., 2014;Xie et al., 2016), but it is widely used in
the clinic. Saponin components have been explicitly included in 161
types, and astragalosides I, II, and IV, and isoastragaloside I and II
making up more than 80% of the total. There are 63 flavonoids, among
them, isoflavones are the most important and have been studied a lot
(Fu et al., 2014;Song et al., 2007). In recent years, AM and its com-
monly used components, including astragalus polysaccharide (APS),
astragaloside IV and formoterol abstracted from AM flowers, have been
extensively studied for their remarkable anti-tumor activities (Burrell
and Swanton, 2014;Hanahan and Weinberg, 2011;Hong et al., 2017;
Schmetterer and Pickl, 2017;Skaggs et al., 2008). The commonly used
dosage forms are granula, capsule, oral liquid and injection (Dong et al.,
2010;Guo et al., 2012;Li et al., 2015).
4. Astragalus membranaceus reduces the side effects and improves
the therapeutic actions of chemotherapy in cancer patients
APS is more commonly used in a variety of tumors, including gastric
cancer and colon cancer. A systematic review based on 15 TCMs com-
bining with a commonly used drug for gastric cancer, showed that APS
injection could relieve clinical symptoms (odds ratio and 95% con-
fidence intervals, 3.06 (1.01, 8.99), achieving a higher performance
status, and was superior in reducing leucopenia and gastrointestinal
reaction to chemotherapy than using FOLFOX regimen single (Zhang
et al., 2017). Aiming at solving the problem of poor systemic treatment
and large toxic side effects of non-small cell lung cancer (NSCLC),
McCulloch et al. evaluated the evidence from 34 randomized control
trials (RCT) and found that AM components and AM-based TCM in-
cluding its combination with platinum-based chemotherapy could re-
duce the risk of death in 12 months ((risk ratio [RR] = 0.67; 95% CI,
0.52 to 0.87)), with improved tumor response data (RR = 1.34; 95% CI,
1.24 to 1.46) and reduced toxic reaction of chemotherapy (McCulloch
et al., 2006). Besides, Guo et al. reported after 3 cycles of treatment,
APS injection combined with vinorelbine and cisplatin (VC) could
Fig. 1. Flow chart of the search processes.
S. Li, et al. Journal of Ethnopharmacology 258 (2020) 112797
2
improve the quality of life (QOL), physiological function, fatigue,
nausea and vomiting, pain, loss of appetite and other symptoms in
patients with advanced NSCLC compared with the patients on VC alone
(Guo et al., 2012). Very recently, a phase II double-blind randomized
placebo-controlled trial showed that APS injection displayed less con-
current chemoradiotherapy (CCRT)-associated adverse events, QOL
fluctuation from the baseline, while a significant improvement in the
pain, appetite loss, and social eating behavior during CCRT in the
CCRT/placebo groups of patients with advanced head and neck squa-
mous cell carcinoma (Hsieh et al., 2020).
Besides AM and its extracts, the prescriptions by using AM as the
main component have also been proved beneficial for cancer patients.
For instance, Shenqi Fuzheng Injection (SFI) was approved as an in-
jectable Chinese medicine formula by the China Food and Drug
Administration (FDA) in the 1990s, which consists of two TCM's, AM
and Radix Codonopsis in a ratio of 1:1. In the past 30 years, SFI has been
reported to be effective in a variety of advanced cancer types. A sys-
tematic meta-analysis evaluated the efficacy of SFI in the treatment of
advanced gastric cancer involving 13 randomized controlled trials and
860 patients (Li et al., 2015). Results showed that chemotherapy
combined with SFI improved QOL, complete remission rate and partial
remission rate, and reduced adverse reactions such as nausea, vomiting,
oral mucositis and leukopenia (Li et al., 2015). In addition, SFI com-
bined with platinum-based chemotherapy improved the efficacy and
toxicity of advanced NSCLC and colorectal cancer (CRC) (Dong et al.,
2010).
In fact, many clinical trials of AM and AM-based TCM prescriptions
efficacy have been reported in Chinese journals, with capacity such as
improving QOL of cancer patients, alleviating the side effects of
radiotherapy and chemotherapy as well as improving the organic im-
munity. However, these articles are not collected by PUBMED data
base. To summarize, main components of AM and AM-based TCMs can
reduce the side effects and enhance the effect of chemotherapies, al-
though the direct curable actions on shrunk tumors has not been re-
ported yet. There are some limitations mentioned in the cited meta-
analysis, including methodological deficiencies, small sample sizes,
limited to East Asian patients, which would lead to potential risks of
bias (Cao et al., 2019). It is expected that more high-quality and large-
sample RCTs are carried out and reported in the high-impact journal, to
further verify the above conclusion.
5. The mechanisms of Astragalus membranaceus anti-tumor actions
Using tumor animal models or cell lines, AM has shown direct anti-
tumor activity in various tumor models such as NSCLC, liver cancer,
gastric cancer, breast cancer, and ovarian cancer. Based on the existing
literatures, it is speculated that AM mainly exerts its anti-tumor effects
by directly inhibiting the proliferation and promoting apoptosis of
tumor cells; increasing the efficacy of chemotherapies, potentially
preventing tumor cell metastasis and improving TME by enhancing
organic or local immunity.
5.1. The anti-proliferative and pro-apoptotic actions on cancer by
Astragalus membranaceus and related pathways
Studies haves shown that AM and its main components have the
capacity to inhibit the growth of many types of tumor tissues and cells
via several signaling pathways in vitro and in vivo as shown in
Table 1(Auyeung et al., 2016). Various components of AM can inhibit
the proliferation of tumor cells and promote their apoptosis. Among
them, Park et al. found that the ethyl acetate fraction of AM (EAM)
reduced the proliferation of NSCLC cells in a dose and time-dependent
manner with increased numbers of cells in a non-proliferative state. In
addition, EAM treated cells displayed chromatin condensation, an in-
creased annexin V-positive cell population accompanied by the up-
regulated expression of cleaved caspase-8 and -9 and the accumulation
of lysed poly ADP-ribose polymerase (PARP) via extracellular regulated
protein kinases (ERK) signaling pathway (Park and Park., 2018). It has
been recently found that APS was able to inhibit the proliferation of
human gastric cancer MGC-803 cells and promote mitochondria-de-
pendent apoptosis in a concentration and time-dependent manner. The
underlying mechanisms involved APS induction of intracellular reactive
oxygen species (ROS) accumulation and further promotion of the
apoptosis (Yu et al., 2019). Zhou et al. reported that four isoflavones of
AM extracts campanulin, ononin, calycosin, and formononetin could
inhibit proliferation and induce apoptosis of several breast cell lines
(MCF-7, SK-BR-3, MDA-MB-231) mediated through the PI3K/AKT/
mTOR pathways (Zhou et al., 2018). However, studies using antago-
nists of these signaling pathways need to be conducted to provide a firm
conclusion in this regard.
Formononetin is one of the major isoflavonoid constituents isolated
from AM and demonstrates diverse pharmacological benefits. Yang
et al. demonstrated that three cell lines of NSCLC and A549 treated with
formononetin showed inhibited cell growth in a time- and dose-de-
pendent manner with G1-phase cell cycle arrest and enhancement of
apoptosis in NSCLC mediated by the p53 signaling pathway (Yang et al.,
2014). Baicalein, another AM active ingredient, has shown its anti-
tumor ability in various cancers. It could inhibit the growth of human
nasopharyngeal carcinoma cells by inhibiting their cell proliferation
and inducing apoptosis. Baicalein could also effectively limit both
CNE1-and CNE2-transplanted tumors in nude mice with down-regula-
tion of Bcl-xl and Mcl-1 proteins and up-regulation of Bax and Bad (Guo
et al., 2019). All the evidences above indicates diverse components
abstracted from AM can inhibit the growth of tumor by targeting both
proliferation and apoptosis in several cancer types, but this direct anti-
tumor actions need clinical conformation.
5.2. The actions of Astragalus membranaceus in tumor-associated
environment
Tumor cells reside in TME which contains a variety of mesenchymal
cells, particularly fibroblasts, myofibroblasts, endothelial cells, peri-
cytes, mesenchymal stem cells, innervating nerves and immune cells, in
addition to secreted factor from these cells (Burrell and Swanton,
2014). Many "characteristics of cancer" are associated with TME, which
has the capacity to promote proliferation and inhibit apoptosis, en-
hance angiogenesis and create hypoxia and an immune-suppressive
environment to avoid immune detection and support cancer growth,
even invasion and metastasis (Hanahan and Weinberg, 2011). Hence,
the manipulation of the immune niche of TME has been used as an
approach to treat cancer and prevent its progression.
5.2.1. Alleviation of immunosuppressive status in tumor immune
microenvironments by Astragalus membranaceus
An increasing number of immune cell subpopulations and related
molecules have been observed in TME, which play an important role in
promoting and inhibiting the occurrence and progression of cancers
(Wu et al., 2019). There are two basic profiles in TME: a “hot”immune
inflamed profile which displays an increased distribution of CD8
+
tumor infiltrating lymphocytes (TILS), accompanied by cells derived
from bone marrow such as myeloid lineages with higher levels of in-
terferon and chemokines including CXCL9, CLCL10, CXCL11 and other
pro-inflammatory effector cytokines. In contrast, a “cold”non-inflamed
profile is characterized by immunosuppression, with high infiltratio of
myeloid derived suppressor cells, M2-phynotype macrophages and
regulatory T cells (Treg, CD4
+
CD25
+
Foxp3
+
dominant), with en-
hanced expression of IL-10, IL-4 and TGF-β(Hong et al., 2017;
Schmetterer and Pickl, 2017;Skaggs et al., 2008;Namdar et al., 2018;
Safarzadeh et al., 2018;Chanmee et al., 2014;Zhou et al., 2014).
As has been shown, macrophages can be divided into M1 and M2
phenotypes. As the forerunner in the defense against tumor cells, M1-
macrophages rapidly colonize and secrete cytokines that kill tumor
S. Li, et al. Journal of Ethnopharmacology 258 (2020) 112797
3
cells, as well as support pro-inflammatory cells by activating dendritic
cells and natural killer cells (Hong et al., 2017). However, advanced
tumors display severe immune-suppression, dominated by M2-macro-
phages which promote the progression of numerous cancers and predict
a poor prognosis for tumor-bearing animals and human patients (Jiang
et al., 2019). M2-macrophages polarization is induced by several im-
mune-suppressive cytokines including IL-4, IL-10, IL-13 and gluco-
corticoids, which can accelerate the metastasis and angiogenesis of
tumors (Xu et al., 2018). CD8
+
T cells are cytotoxic T lymphocytes
(CTLs) that kill tumor cells in TME, but are inhibited by Tregs and
immunosuppressive cytokines, such as IL-4, IL-10 and TGF-β. CTLs
however highly express PD-1, which can induce apoptosis by tumor
secreted PD-1 ligand (Huang et al., 2012). Tregs are key components of
tumor-acquired tolerance and inhibit most types of immune responses
(Chatila, 2005;Zhang et al., 2014).
According to TCM theory, Chinese herbal medicine with qi-toni-
fying effect, has the function of enhancing the defense ability of the
immune system. Until now, various pharmacological studies have
shown that AM has immunomodulatory properties, manifested by ac-
tivation of lymphocytes, natural killer cells and macrophages and up-
regulation of related cytokine production such as interferon and TNF-
α(Li et al., 2019a,b).
As shown in Table 2, lines of evidence have shown that AM com-
ponents could relieve the severe immunosuppressive condition in can-
cers or cancer-bearing organisms. For instance, it has demonstrated that
Arg-1 (a M2 macrophages marker) deficient mice displayed shrunken
tumor size compared to those of wild type mice (Xu et al., 2018), and an
ethanol extract of AM, Astragaloside IV (AS-IV) could inhibit more than
50% expression of Arg-1 and CD206, possibly by targeting the im-
munosuppressive activity of M2-macrophages (Jiang et al., 2019).
Further, AS-IV was found to inhibit M2 conditioned medium-induced
A549 and H1299 cell invasion, migration and angiogenesis. In vivo in-
vestigation confirmed that the tumor region contained a decreased
proportion of M2-macrophages in Lewis lung cancer-bearing animals in
the AS-IV treatment group, with inhibited AMPKαactivation in M2
macrophages, while silencing AMPKαpartially abolishes the effect of
AS-IV, suggesting that AS-IV reduces the growth, migration and an-
giogenesis of lung cancer by blocking the polarization of M2 macro-
phages via the AMPK signaling pathway (Xu et al., 2018).
It is encouraging that the anti-tumor activity of APS-activated
macrophages with up-regulating the concentration of nitric oxide (NO)
and TNF-α, which may be the upstream of tumor cell proliferation in-
hibition such as G1 cell cycle arrest, and modulation of apoptosis-re-
lated genes, directly preventing the growth of cancer cells (Li et al.,
2019a,b). Recent studies reported that APS increased the M1/M2
macrophage polarization ratio in NSCLC H441 and H1299 cells, with
concomitant marked inhibition of cell proliferation, clonogenicity and
tumor sphere formation (Bamodu et al., 2019). A hydrosoluble poly-
saccharide named RAP extracted from AM(Wei et al., 2016) has been
shown directly to induce the macrophage cell line RAW264.7 to pro-
duce the inflammatory cytokines TNF-α, IL-6 and iNOS, with were
blocked by TLR4 inhibitors. Further evidence supported that RAP could
activate TLR-4-related MAPKs, including phosphorylation of ERK and
p38 and induction of translocation NF-κB signaling, indicating RAP
activates macrophages via the TLR-4-MAPK-NF-κb pathway (Zhou
et al., 2017).
Regarding the effect of AM in T cell regulation, in ex-vivo tumor
mice using clinical sample from the NSCLC cohort showed that APS
inhibited tumor growth, promoted functional maturation of dendrite
cells with consequent enhancement of T cell-mediated anti-tumor im-
mune responses, synergistically enhanced the anti-M2-mediated anti-
tumor effect of cisplatin which could explain the clinical efficacy of AM
and chemotherapy. Meanwhile, the combination of AM and IL-2 was
able to enhance anti-tumor activity by increasing infiltrating IL-2 gen-
erated lymphokine-activated killer cells (LAK), although with lowered
side effects of recombinant IL-2 therapy, such as acute renal failure,
capillary leak syndrome, myocardial infarction and fluid retention
compared to those patients treated with IL-2 alone (Zou and Liu, 2003),
indicating that AM could potentially have beneficial effects in combi-
nation with several immune therapies (Guo et al., 2012). However,
more evidences of the potential to increase T cell activity in TME by AM
need further studied.
In recent years, immunotherapy has become an effective treatment
in a variety of cancers. Cytotoxic T-lymphocyte protein 4 (CTLA4) and
programmed cell death protein 1 (PD-1) inhibitors are currently the two
most important immunological checkpoints since they can induce ac-
tivation and clonal-proliferation of tumor-specific T cells in TME.
However, although antibodies against the immunoregulatory factors
CTLA4 and PD-L1/PD-1 have been clinically successful, only a small
percentage of patients have shown persistent responses. It has been
speculated that the main reasons for this were that the severe immune-
suppressive TME and impaired antigen presentation capability in tumor
sites inhibit the proliferation and survival of infiltrating T cells, and that
suppressive organic immunity inhibits the migration of more T cells
into tumor sites (Chen and Mellman, 2017;Wu et al., 2019). AM and its
major components are good immunopotentiators in organic and tumor
local immunity, providing potential candidates as an immunoadjuvant
checkpoint for the treatment of various cancers. This could perhaps be
in combination with other effective agents such as chemotherapy, to
further enhance each other's anti-tumor effects, and further studies of
these combination therapies should be carried out in the future.
Angiogenesis is the key to the development, progression, and
Table 1
Anti-proliferation and pro-apoptotic mechanisms of Astragalus membranaceus.
Refs. Component, medication,
dose
Tumor model Control Effect indicators Mechanism index
(Park and Park,
2018)
EAM
200 μg/ml
NSCLC cell: H1299, H460,
A549, H1975
DMSO Cell survival↓proliferation↓
apoptosis↑
Caspase-8↑, caspase -9↑, PARP↑
(Yu et al., 2019) APS4
200, 400 and 800 μg/ml
Gastric carcinoma cell:
MGC-803
NC-DMEM, positive
control- 5-FU;
Proliferation↓, apoptosis↑, the
number of cells in S phase↓
ROS↑, Bax/Bcl-2↑, caspase-9/3↑,
PARP↑
(Zhou et al., 2018)AM
25, 50 μg/ml
Brest cancer cell: MCF-7,
SK-BR-3, MDA-MB-231
NC-DMSO Proliferation↓percentage of cell
death↑
PI3K/AKT/mTOR: P-PI3K↓, P-G53Kβ↓,
p-Akt↓, P-mTOR↓
(Yang et al., 2014) Formononetin
100, 150 and 200 μM
NSCLC cell: A549, NCI-H23 NC-PBS Proliferation↓, apoptosis↑, the
number of cells in S phase↓
P53: phosphorylation at Ser15, Ser20
Proliferation: G1 phase: p21↑, cyclin
A↓, cyclin 1↓
Apoptosis: caspase-3↑, bax↑, bcl-2↓
(Guo et al., 2019) Baicalein, cell: 25, 50,
100 μM,
animal: 1, 2, and 3 mg/
kg,ip.
NPC cell: CNE1, CNE2
animal: CNE1 and CNE2-
bearing mice
PC-DDP Apoptotic rate↑, tumor size↓,
weight↓,
tumor inhibition rate↑
Apoptosis: Bcl-xl↓, Mcl-1↓, caspase 3↑,
caspase 8↑, Bax↑, Bad↑
EAM: ethyl acetate fraction of AM; APS4: a novel cold-water-soluble polysaccharide was isolated from Astragalus membranaceus; AM: isoflavones, campanulin,
ononin, calycosin and formononetin; Baicalein: an active ingredient separated from AM; NC: negative contorl; PC: positive control.
S. Li, et al. Journal of Ethnopharmacology 258 (2020) 112797
4
Table 2
Mechanism of Astragalus membranaceus on improving TIME and organic immunity.
Ref. Medication, dose, mode Tumor model Control Effect indicators Immune indicators & mechanism indexes
Enhance tumor immune microenvironment
(Xu et al., 2018) AS-IV cell: 40, 80 μM animal: 40 mg/kg,
intragastrically
Lung cancer cell: A549, H1299
animal: LLC-bearing mice
cell: NC-DMSO animal: NC-NS Tumor size↓, survival rate↑, tumor
vessel maturation↓, metastasis↓
IL-13↓, IL-4↓,M2↓, CD206↓, IL-10↓, TGF-
β↓, p-AMPKα↓
(Li et al., 2019a,b) APS cell: 50, 100, 200, 500 and 1000 μg/
mL
Breast cancer cell: MCF-7 NC- conditioned medium PC-5-FU (50 μg/
mL)
Proliferation↓, G1-phase↓, apoptosis↑Macrophages↓,NO↑, TNF-α↑, Bax/Bcl-2↑
(Bamodu et al.,
2019)
APS cell: 8 mg/ml animal: 3 mg/kg
biweekly for 16 weeks, ip.
NSCLC cell: H441, H1229, H1437
animal: LLC1 and H1437, THP-1 cells-
bearing mice
cell: NC-PBS
animal: NC-DMSO
Body weight↑, tumors size and
weight↓, metastasis
nodules↓
M1/M2↑, DCs↑, T cell↑, IL-6↓, IL-10↓, NF-
κB↓, CD11b↑, CD31↓
(Wei et al., 2016) RAP cell: 30, 100 μg/ml Carcinoma cell: carcinoma (4T1),
macrophage (RAW264.7)
NC-DMEM Cell viability↑NO↑, TNF-α↑, IL-6↑, iNOS↑
(Zhou et al., 2017) APS cell: 400, 100 μg/ml, animal:
500mg/kg/d, oral 25 days
Breast cancer cell: RAW264.7,
animal: EAC- bearing mice
cell: NC-DMEM, PC- LPS (100 ng/ml),
animal: NC-NS, PC-ADM (4mg/kg/d), LPS
(5 mg/kg)
Weight of tumor↑, organ indexes↑,
inhibition rate↑
IL-6↑, IL-Iβ↑, TNF-α↑
(Law et al., 2012) AST cell: 80 μg/ml animal:100 mg/kg,
once daily for oral 14 days
Colon cancer cell: HCT116, HT-29
animal: HCT116-bearing mice
NC-DMEM Tumor volume↓HIF-1α↓, bFGF↓, VEGF↓, PTEN↑, P-AKT↓,
P-mTOR↓, COX-2↓
Enhance the organic immunity to improve chemotherapy efficacy
(Yang et al., 2013) APS animal: 100, 200, 400 mg/kg, once
per day for 10 days intragastrically
Hepatocellular carcinoma animal: H22-
bearing mice
NC-NS, PC-5-FU (20 mg/kg) Tumor weight↓, tumor inhibitory↑Spleen and thymus indexes↑, macrophages
phagocytotic↑, IL-2↑, IL-12↑, TNF-α↑, IL-
10↓
Enhance the organic immunity to prevent tumor invasion and metastasis
(Zhang et al., 2014) APS cell: 100 ng/ml animal: 50 mg/kg Lung cancer cell: A549, PC9, B16F10,
LL2, animal: B16F10 and LL2-bearing
mice
Animal: NC-PBS Tumor growth↓, the survival↑, body
weight↑
Tcell↑, IDO↓, spleen and thymus indexes↑,
Cx43↑
(Zhang et al., 2018) Formononetin
80, 160, 240 μM
Ovarian cancer cell: A2780, SKOV3,
normal ovarian epithelial cell: IOSE80
NC -DMEM Proliferation↓, apoptosis↑, migration
and invasion↓
MMP-2/9↓, P-ERK↓, caspase3/9↑, Bax/Bcl-
2↑
AS-IV: Astragaloside IV;PG2(APS): Astragalus polysaccharides; RAP: a hyperbranched heteroglycan with average molecular weight of 1334 kDa; AST: Radix Astragali; PG2: extracts of Astragalus membranaceus;AMP:
Astragalus membranaceus polysaccharide; NC: negative contorl; PC: positive control.
S. Li, et al. Journal of Ethnopharmacology 258 (2020) 112797
5
metastasis of solid tumors (Li et al., 2018). Recent studies have found
that the tumor's vascular system is highly abnormal and dysfunctional,
and therefore, the ability of immune effector cells to penetrate solid
tumors is impaired. Normalization of tumor vasculature can improve
the infiltration of immune effector cells, thereby enhancing tumor-
killing or inhibiting capacity. At the same time, stimulating immune
cell function also contributes to the normalization of the tumor vessel
system (Huang et al., 2018). Therefore, it has been proposed that the
regulation of crosstalk between tumor vascular normalization and im-
mune reprogramming can enhance long-lasting anti-tumor immunity
(Law et al., 2012).
Vascular endothelial growth factor (VEGF) has been verified as the
most critical angiogenic factor in angiogenesis and vascular abnorm-
alities. Since VGEF is released by various tumor cells, it is overexpressed
in different human tumors and leads to tumor progression. Law et al.
found that total Astragalus saponins could down-regulate the levels of
VEGF protein in HCT 116 colon cancer cells in a time- and dose-de-
pendent manner, with the mechanisms of modulation of Akt/mTOR
signaling molecules, upregulate PTEN, decrease Akt phosphorylation
with subsequent activation of mTOR (Law et al., 2012). The anti-car-
cinogenic action of AS- IV was further illustrated in HCT 116 xeno-
grafted athymic nude mice, with inhibited tumor growth and serum
VEGF levels. Levels of p-Akt, p-mTOR, VEGF, VEGFR1 and VEGFR2 in
tumor tissues were decreased by AS- IV, indicating that AS-IV can exert
anti-tumor activity in colon cancer by regulating mTOR-VEGF signaling
(Ichinohe, 2001;Semenza, 2003). However, the role of AS-IV in im-
proving immune-vascular crosstalk requires further investigation.
5.2.2. Astragalus memeranaceus improves the chemotherapy efficacy and
prevent tumor invasion and metastasis via boosting organic immunity
5.2.2.1. Astragalus membranaceus improves the organic immunity. Tumor
cells can escape from immune surveillance due to the host immune
system including not only tumor local immunity but also organic
immunity failing to deal with tumor-associated antigens, which
promote tumor invasion and metastasis, or affect therapeutic
response (Zhang et al., 2014). Thus boosting the organic immunity is
important in anti-tumor therapy. Lines of evidences have demonstrated
that AM, as a tonic, can enhance the organic immunity in physiological
status and various disease. On the innate immune response, the aqueous
extract of AM induced the maturation, activation and migration of
monocytes in peripheral blood (Denzler et al., 2010). In addition, the
activity of macrophage migration into the peritoneal cavity and
macrophage phagocytic activity were found to be enhanced by AM
(Cho and Leung, 2007). In the meantime, AM could also affect the
acquired immune response. For instance, through in vitro and in vivo
investigations, Cho et al. found that AM exhibited mitogenic and co-
mitogenic potentials on mouse spleen cells and lymphocytes, as well as
increasing IL-2 receptor expression on splenic cells which may mediate
the proliferation of T cells. In terms of immune recovery activity, AM
restored the lymphocyte germination reaction of aged mice to the level
of normal young mice. Meanwhile, intraperitoneal injection of AM
significantly enhanced the antibody response of the mice to sheep
erythrocytes (Cho and Leung, 2007).
5.2.2.2. Astragalus membranaceus improves chemotherapy efficacy via
boosting organic immunity. Chemotherapy is considered to be an
important approach for treating malignant tumors, with
immunosuppression being one of the common clinical manifestations
in chemotherapy. Alleviating immunosuppression is one of the
important obstacles of chemotherapy to overcome. Lines of evidence
have confirmed that the immunomodulatory effects of AM are closely
related to its protective effects of immune organs by regulation of
lymphocytes, leukocytes and macrophages in chemotherapy-induced
immunosuppression (Huang et al., 2007;Li et al., 2008;Wang et al.,
2012;Zhang et al., 2009). In the cyclophosphamide-induced
immunosuppressive mice, the spleen index, peripheral blood
leukocyte and bone marrow cell counts showed accelerated recovery
and increased splenic natural killer cell activity, and peritoneal
macrophage phagocytosis was increased by AM. At the same time,
levels of IFN-γ, IL-12P70, IL-6 and IL-17 were also up-regulated by AM,
indicating that AM can enhance organic immunity in
immunosuppressive mice (Huang et al., 2007;Li et al., 2008;Wang
et al., 2012;Zhang et al., 2009). Astragalus oligosaccharide (AOS),
degraded from APS was found to alleviate cyclophosphamide-induced
immunosuppression mediated by stimulating the secretion of GM-CSF,
which promoted the differentiation of progenitor cells after
proliferation in bone marrow, spleen and thymus (Zhu et al., 2017).
Multiple clinical trials mentioned in Section 4have shown that AM
can reduce chemotherapy-related adverse reactions and improve effi-
cacy in cancer patients. In the mechanism studies as shown in Table 2,it
was found that APS could induce connexin 43 which is involved in
facilitating the passage of chemotherapeutic drugs to bystander tumor
cells; And decrease indoleamine 2, 3-dioxygenase, which can deplete
tryptophan, reduce the active T cell number and destroy immune sur-
veillance, providing the evidence that APS could be suitable combina-
tions with chemotherapies (Phacharapiyangkul et al., 2019). In the
meantime, SFI was designed to elucidate the in vivo immuno-enhance-
ment effects of SFI in immunosuppressed mice induced by cyclopho-
sphamide treatment. Results showed that SFI treatment accelerated
recovery dose-dependently of spleen index, peripheral white blood cell
and bone marrow cell counts, enhanced T cell and B cell proliferation
responses, as well as splenic nature killer cell activity and peritoneal
macrophage phagocytosis, and restored the level of interleukin-2 in the
serum, providing experimental evidences for supporting clinical effi-
cacy of combinational SFI chemotherapy in cancer patients (Wang
et al., 2012).
5.2.2.3. Astragalus membranaceus enhances the organic immunity so that
to prevent tumor invasion and metastasis. The vast majority of cancer-
related deaths are the result of metastasis due to the scarcity of current
therapeutic options for patients with advanced metastatic tumors.
During metastasis, the crosstalk between cancer cells and the immune
system dictates the fate of tumor progression. For example, monocyte
counts in peripheral blood play a premetastatic role, and patient-
derived monocytes showed a greater ability to promote cancer cell
invasion and angiogenesis compared to monocytes from healthy donors
(Chittezhath et al., 2014). On the one hand, immune evasion by tumors
is to establish an immunosuppressive environment that inhibits the
development of anti-tumor immune responses in both the local and
systemic immune environment. These tumor-educated myelocytes,
especially tumor-associated macrophages (TAMs) and tumor-
associated neutrophils (TANs), can inhibit the anti-tumor immune
response through the production of immunosuppressive cytokines, T-
cell expression of co-inhibitory molecules, the reduction of amino acids
which are vital for the functioning of T-cells, and the production of ROS
(Fleming et al., 2018). Similarly, TAM counts are related to poor
clinical outcomes in several types of cancer (Campbell et al., 2011;
Steidl et al., 2010), and higher neutrophil-to-lymphocyte ratios in
peripheral blood are associated with poor survival (Templeton et al.,
2014). The potential mechanism involved is that immunosuppressive
cells can migrate to the pre-metastatic niche and create a suitable
environment for tumor growth (Blomberg et al., 2018;Wang et al.,
2017;Wculek and Malanchi, 2015,2019), and then drive tumor cells
migration and extravasation to the pre-metastatic region far from
primary tumor site (Hiratsuka et al., 2002;Kowanetz et al., 2010;
Qian et al., 2011). Therefore, alleviating the systemic
immunosuppressive environment has become an attractive strategy
for combating metastasis. In view of the significant immunosuppression
and pro-metastatic role of tumor-infiltrating M2-macrophages, N2-
neutrophils, and Tregs, more researchers are exploring strategies for
recruiting, polarization and effector molecules. AM may be a potential
organic immune enhancer for preventing tumor metastasis (Blomberg
S. Li, et al. Journal of Ethnopharmacology 258 (2020) 112797
6
et al., 2018).
As mentioned above, chemotherapy contributes to induce organic
immunosuppressive status, while AM could alleviate such an im-
munosuppressive condition (Table 2). Zhou et al. also found that
TLR4
+/+
and MyD88
+/+
wild-type tumor-bearing mice treated with
APS displayed reduced tumor size, up-regulated levels of immune organ
indexes and increased circulatory levels of pro-inflammatory cytokines
including TNF-α, IL-1βand IL-6 (Zhou et al., 2017). Given that the
cytoplasmic portion of CD45 contains protein tyrosine phosphatase
activity and is critical for TCR-mediated T cell activation, AS-II was
found to enhance the proliferation of primary splenocytes and the de-
phosphorylation of Tyr505 in primary T cells mediated by CD45 PTPase
modulation. Furthermore, AS-II could induce Th1 polarization, which
manifested with significantly increased IL-2 and IFN-γtranscription and
secretion from primary splenocytes upon TCR stimulation. In vivo stu-
dies showed that oral administration of AS-II restored the proliferation
of splenic T cells and the production of IFN-γand IL-2 in CTX-induced
immunosuppressed mice. Taken together, these results show that AS-II
enhances T cell activation by regulating the activity of CD45 PTPase
(Wan et al., 2013).
AM could partially restore the organic immune function of tumor-
bearing mice and cyclophosphamide-treated mice as well (Choi et al.,
2014). Yang et al. used the H22 tumor-bearing mouse model to study
the in vivo therapeutic effect of APS against cancer. The results showed
that APS can not only enhance tumor deterioration, but also increase
body weight, spleen index and thymus index and the phagocytotic
ability of macrophages in mice with tumors. In addition, APS admin-
istration increased levels of IL-2, ILI-2 and TNF-α, indicating that APS
inhibits tumor growth at least to some extent by improving organic
immunity. In the in vitro setting of H22 in hepatoma cells treated with
ASP, it was observed that ASP could significantly inhibit tumor growth
as well as elevate serum cytokine levels (TNF-α, IL-2 and IFN-γ) and
activate immune cells (macrophages, lymphocytes and NK cells),
thereby inducing tumor cells apoptosis (Yang et al., 2013).
Few studies support that AM may prevent tumor-related metastasis
via boosting organic immunity. Wang et al. found APS combined with
cisplatin (DDP) could alleviate the pathological changes of the
recurrent tumor tissues and the metastasis-related protein expressions
of CD44, CD62P and osteopontin. following Lewis lung carcinoma
(LLC) surgery (Liu et al., 2018). Another study showed formononetin
suppressed the migration and invasion of ovarian cancer cells by using
in vitro wound healing and trans well chamber assay accompanied with
decreased expression of MMP-2/9 proteins and phosphorylation level of
ERK, indicating formononetin may have potential to prevent cancer
metastasis but further evidence should be provided by future in-
vestigation (Zhang et al., 2018).
6. Conclusion
AM is commonly used as a tonic in TCM with multiple bioactivities.
To conclude in Fig. 2, it has been demonstrated that AM and its active
constituents, combined with chemotherapies, are capable of dimin-
ishing the side effects and complications induced by chemotherapies
and enhancing the efficacy in cancer patients. Utilizing various cancer
models and cell lines, AM has been found to capable of shrinking or
stabilizing tumors by direct anti-proliferation or by pro-apoptosis of
tumor cells. Meanwhile, the crucial mechanism of AM immune reg-
ulation is to alleviate the immune-suppressive status by activating M1
macrophages via the TLR4/MyD88/NFκB signaling pathway and en-
hancing tumor-kill capacity of T cells in tumor environment. In addi-
tion, it was found that AM could also improve systemic immunity which
may help promoting efficacy of chemotherapy and preventing metas-
tasis. Thereby this review contributes to an understanding of AM as an
adjunctive therapy in the whole course of cancer treatment, with pro-
viding useful information for development of more effective anti-tumor
medication. However, due to the methodological quality of the current
clinical trials, it is prospected that rigorously designed RCTs are war-
ranted to generate a high level of clinical evidence. The combination of
AM and immune checkpoint therapies would has a promising prospect,
and observation of direct efficacy and mechanisms on tumor growth
and metastasis by AM combined with chemotherapies or immune
checkpoint therapies require more in vivo validations and further clin-
ical investigation as well.
Fig. 2. Anti-tumor effects and mechanisms of Astragalus membranaceus and its specific immunopotentiation.
S. Li, et al. Journal of Ethnopharmacology 258 (2020) 112797
7
Declaration of competing interest
The authors have no conflict of interests regarding this paper.
Acknowledgment
This study was financially supported by the National Natural
Science Foundation of China (NSFC) No. 81704146, 81273868,
81873369, and 81873368, the Tianjin Municipal Bureau of Labor and
Social Security No.2018015.
Abbreviation
AM Astragalus membranaceus
APS Astragalus polysaccharides
AS-IV Astragaloside IV
(CCRT) concurrent, chemoradiotherapy
CTLA4 cytotoxic T-lymphocyte protein 4
CTLs cytotoxic T lymphocytes
CRC colorectal cancer
EAM ethyl acetate fraction of AM
ERK extracellular regulated protein kinases
HCC hepatocellular carcinoma
NMA network meta-analysis
LAK lymphokine-activated killer
NSCLC non-small-cell lung cancer
NO Nitric oxide
QOL quality of life
PARP poly ADP-ribose polymerase
PD-1 programmed cell death protein 1
ROS reactive oxygen species
SFI Shenqi Fuzheng Injection
TAMs tumor-associated macrophages
TME tumor microenvironment
TANs tumor-associated neutrophils
TCMs traditional Chinese medicines
TIM tumor immune microenvironment
TILs tumor infiltrating lymphocytes
TLR4 Toll-like receptor 4
VEGF Vascular endothelial growth factor
VC vinorelbine and cisplatin
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jep.2020.112797.
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