Astragaloside in cancer chemoprevention and therapy

Abstract

Tumor chemoprevention and treatment are two approaches aimed at improving the survival of patients with cancers. An ideal anti-tumor drug is that which not only kills tumor cells but also alleviates tumor-causing risk factors, such as precancerous lesions, and prevents tumor recurrence. Chinese herbal monomers are considered to be ideal treatment agents due to their multi-target effects. Astragaloside has been shown to possess tumor chemoprevention, direct anti-tumor, and chemotherapeutic drug sensitization effects. In this paper, we review the effects of astragaloside on tumor prevention and treatment and provide directions for further research.

Introduction

According to the cancer statistics from Chen et al^[1], there will be approximately 4,820,000 and 2,370,000 new cancer cases, and 3,210,000 and 640,000 cancer deaths in China and the USA in 2020, respectively. Radical surgery has good prognostic outcomes in patients with early solid malignant tumors. However, some patients develop postoperative recurrence and metastasis, which leaves the postoperative prognosis unsatisfactory. Systemic tumor therapy including cytotoxic chemotherapy and molecular targeted therapy can destroy residual tumor cells after surgery and prevent postoperative recurrence. However, the response rate of systematic treatment is low, and there are also problems with high toxicity, adverse reactions, and drug resistance.^[2] Therefore, finding new treatment approaches to suppress tumor cell growth, metastasis, and recurrence, and increase efficacy of chemotherapy, has become a major focus in cancer research.

It has long been recognized that tumor recurrence may be driven by proliferation of residual tumor cells as well as persistent presence of tumor causative factors (such as precancerous lesions). Michael Sporn introduced the concept of chemoprevention in 1976. Chemoprevention is defined by the National Cancer Institute (NCI) and other institutions as the use of natural, synthetic, or biological substances to stop, slow, or reverse cancer development.

An increasing number of studies have demonstrated that herbal monomers can be used as antitumor and tumor chemoprevention agents, adjuvant drugs for systemic therapy, and the prevention of tumor recurrence after surgery.

Astragalus mongholicus Bunge, a traditional restorative Chinese medicine, exhibits pharmacological effects such as tonifying qi, fixing the surface of the body, promoting water retention and swelling, detoxifying, and draining pus. Therefore, it can be used to treat cardiovascular, kidney, skin, hepatitis, and other autoimmune diseases. Technological improvements in extraction methods have led to identification of more active ingredients in Astragalus mongholicus Bunge. Currently, the known components^[3] of Astragalus mongholicus Bunge include saponins, polysaccharides, flavonoids, phytosterols, cannabis oil, and fatty acids. The concentration of triterpene saponins, the main active components of astragalus, ranges from 0.5 mg/g to 3.5 mg/g. Several pharmacological components of triterpene saponins, such as astragalosides, acetylastragaloside isoastragaloside, and astramembrannin, have been identified.^[4] Among them, astragalosides I (ASI), astragalosides II (ASII), astragalosides IV (ASIV), and isoastragaloside I and II are the main components of astragalosides, accounting for 80% of the total saponins in this compound. ASIV is often used as a qualitative and quantitative indicator of astragalus.^[5] Notably, ASI and ASII are acetylated derivatives of ASIV and they are distinguished by existence of β-D-xylose, and the number and position of acetyl groups attached at C-3. Studies have shown that the difference in acetylation is accompanied by instability, and factors such as alkalinity can lead to the loss of acetyl groups from the final stable product ASIV.^[6]

ASIV (chemical formula: C₄₁H₆₈O₁₄; molecular weight: 784 kD) is the main active component of astragalus with high stability.So, it is widely used in the field of tumor research. In comparison, few studies have investigated the anti-tumor mechanism of ASII (chemical formula: C₄₃H₇₀O₁₅ [Supplementary Figure 1, https://links.lww.com/CM9/B507]).

Astragaloside as a Tumor Chemoprevention Agent

Chemoprevention involves inhibition, reversal, or delay of tumorigenesis/cancer development using therapeutic agents. With the depth of the understanding of chemoprevention, research on chemopreventive drugs has focused on three aspects: non-toxic, inexpensive, and available for oral administration.^[7] Herbal extracts can meet these aspects and have promising application potential as chemoprevention agents with the advantages of being multi-level, multi-target, and coordinated intervention effects.^[7] A review of recent studies investigating the use of astragaloside as a chemopreventive drug in preneoplastic diseases showed that astragaloside has preventive effects in viral hepatitis B, hepatic fibrosis, and gastrointestinal mucosal lesions [Figure 1].

Figure 1: Chemopreventive function of astragaloside. HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; IL: Interleukin; MPO: Myeloperoxidase; NF-κB: Nuclear transcription factor-kappa B; SOD: Super oxide dismutase; TGF-β1: Transforming growth factor-β; TNF-α: Tumor necrosis factor-α.

Viral hepatitis

The current treatment approaches for hepatitis B virus (HBV) include the use of guanine nucleotide analogs for long-term control of the level of HBV DNA. Although this treatment can effectively eliminate the level of HBV DNA within in a short period of time, clinical applications have revealed that some HBV patients develop drug resistance. Moreover, nucleoside and nucleotide analogs are associated with side effects such as nephrotoxicity, bone toxicity, and muscle toxicity. Astragalus mongholicus Bunge has early antiviral effects, and ASIV has been found to inhibit the growth of Coxsackie B3 virus.^[8] The anti-HBV efficacy of ASIV has been studied in recent years. In a comparative clinical trial of ASIV and lamivudine, Zhang et al^[9] found that in the HepG2.2.15 cell line, a hepatocellular carcinoma (HCC) cell line infected with HBV, 5 mg/L of lamivudine as a control had the strongest inhibition of HBV DNA, and the inhibition of hepatitis B surface antigen (HBsAg) at day 6 was 74.6% in the ASIV group and 14.4% in the lamivudine group. The same concentration of ASIV caused 75.1% inhibition of HBeAg at day 9 whereas the lamivudine caused 25.4% inhibition. Of note, ASIV had no significant toxic effects on HepG2.2.15. In a similar comparative trial of lamivudine and ASIV by Wang et al,^[10] ASIV treatment at a dose of 100 mg/kg caused 23.6% inhibition of HBsAg on day 9 in duck HBV (DHBV)-infected ducklings, which was slightly higher than that of lamivudine group as a control. ASIV treatment also suppressed DHBV DNA replication in ducks, suggesting that ASIV has potential anti-HBV effects.

The mechanisms by which astragalosides inhibit HBV have not been evaluated. But we could get inspiration from the mechanism of astragaloside inhibition of other viruses. Zhang et al^[11] demonstrated that ASIV can stimulate the formation of autophagosomes in H1N1-infected cells, which then alleviate inflammatory response in infected cells and inhibit viral replication. Shang et al^[12] argued that ASIV inhibited viral proliferation and induced apoptosis in infected viral cells by inducing adenovirus 3-infected cells' responses. We look forward to further research to investigate the exact mechanisms by which ASIV inhibits HBV.

Hepatic fibrosis

Hepatic fibrosis is a common chronic liver lesion. Hepatic fibrosis causes 80–90% of HCC cases, which make hepatic fibrosis become significant precancerous lesions of HCC. Astragaloside has been repeatedly found to slow down the hepatic fibrosis process, thereby preventing hepatic cancer development and recurrence.

Colchicine (Col) is a commonly used drug for the treatment of hepatic fibrosis. Liu et al^[13] found that 4.0 mg/kg of astragaloside could achieve similar effects to 0.1 mg/kg of Col in inhibiting fibrosis of hepatic fibrosis rat models. The hepatic fibrosis related factors were significantly reduced in both groups. Further pathological staging indicated that two out of 10 mice in the ASIV (4.0 mg/kg) group did not develop hepatic fibrosis and no samples showed fibrosis stage IV, while one out of 10 mice in the Col control group did not develop hepatic fibrosis, suggesting that ASIV can inhibit hepatic fibrosis.

Several studies have investigated the mechanisms by which ASIV prevents hepatic fibrosis. Inflammatory factors are among the major causes of hepatic fibrosis. Mechanistically, inflammatory factors can directly activate hepatic fibrosis effector cells such as hepatic stellate cells (HSCs).^[14] This process involves multiple signaling pathways, among which transforming growth factor-β (TGF-β) plays a major role. It has been shown that TGF-β is upregulated in response to liver injury, which triggers the secretion of extracellular matrix and collagen, thereby exacerbating hepatic fibrosis.^[14]

In an ex vivo model of CCL4-induced hepatic fibrosis mice, isolated liver tissues treated with total astragaloside containing astragaloside I–IV and soy saponin decreased the concentrations of tumor necrosis factor-α (TNF-α) and TGF-β1 through a mechanism involving inhibition of the release of TGF-β1 in activated Kuffer cells.^[15] Wang et al^[16] showed that ASIV can inhibit the expression of protease-activated receptor-2 (PAR2) to suppress expression levels of TGF-β. This slows down the process of hepatic fibrosis. In addition, Smad protein families downstream of the TGF-β pathway participate in activation of HSCs. Smad3 can directly activate Smad3-dependent fibrotic genes and markers such as α-SMA, E-cadherin, and tissue inhibitor of metalloproteinases-1 (TIMP1) to inhibit matrix degradation. Recent studies have shown that the TGF-β1/Smad^[17] signaling pathway is a potential target for the treatment of hepatic fibrosis. In vitro experiments by Liu et al^[13] and Yuan et al^[17] demonstrated that ASIV significantly inhibited the TGF-β1/Smad signaling pathway in serum and hepatocytes, and additionally the production of collagen through the TGF-β1/Smad pathway appeared to be significantly reduced in the ASIV-treated group, and hepatic fibrosis was inhibited.

Increasing evidence has demonstrated that oxidative stress is a major player in hepatic fibrogenesis.^[18] Glutathione is antioxidant that can inhibit oxidative stress by downregulating reactive oxygen species (ROS) levels.^[19] Experiments by Li et al^[20] showed that ASIV treatment increased the expression of nuclear factor E2-related factor (Nrf2), which promotes glutathione production in the liver, and decreased the levels of the ROS, resulting in the inhibition of HSC activation, further inhibiting hepatic fibrosis. In the study, different concentrations of ASIV suppressed the release of active TGF-β from HSCs, but the inhibition of TGF-β did not show statistical significance (P > 0.05), and so it cannot be excluded that other cytokines may contribute to the inhibition of HSC by ASIV.

The results discussed above demonstrate that ASIV can downregulate TGF-β1/Smad and upregulate Nrf2 expression levels to prevent the progression of hepatic fibrosis. However, inconsistent findings have been reported. In the bile duct-ligated rats treated with ASIV, Zhao et al^[21] found that ASIV induced accumulation of Nrf2 in the nucleus of hepatic cells and HSCs, synthesized antioxidant enzymes through negative regulation of glycogen synthase kinase-3β, and scavenged ROS, finally resulting in synergistic alleviation of hepatic fibrosis. But ASIV treatment did not have a significant effect on the expression of Smad. Even though it is controversial whether ASIV inhibits hepatic fibrosis through the TGF-β1/Smad signaling pathway, many studies have reported that ASIV can directly or indirectly inhibit the secretion of inflammatory factors that activate HSC and extracellular matrix, thereby slowing down fibrosis process.

Gastric mucosal lesions

Persistent stimulation of gastric mucosa by stress, non-steroidal anti-inflammatory drugs (NSAIDS), Helicobacter pylori (H. pylori), ethanol, and other factors predispose it to gastric cancer. ASIV exerts multifaceted protective effects on gastric mucosa.^[22] After ASIV processing, expression of BCL2-associated X protein (Bax) and levels of superoxide dismutase (SOD) were reduced in gastric mucosa, and lipid peroxidation, apoptosis, and inflammatory factors in the mitochondria were effectively inhibited. In a precancerous lesions of gastric carcinoma (PLGC) model established by Cai et al,^[23] ASIV activated p53 to increase Ambra1 and Beclin1 expression, leading to upregulation of autophagic activity. The expression of elevated Ambra1 and Beclin1 can induce apoptosis and autophagy in PLGC cells and gastric cancer cells and protect the gastric mucosa from gastric precancerous lesions (GPL) and gastric cancer cell invasion.

Aerobic glycolysis is the characteristic metabolic mode of tumors, characterized by efficient glucose uptake and lactate production independent of oxygen levels. It has been demonstrated that H. pylori infection dysregulates glycolysis in the gastric mucosa, and suppressing glycolysis can improve the prognosis of patients with H. pylori infection. ASIV has been shown to inhibit glycolysis in GPL. Specifically, ASIV can inhibit the expression of lactate dehydrogenase A (LDHA), the rate-limiting enzyme of glycolysis, by upregulating microRNA-34a, which is a negative regulator of pyruvate to lactate conversion during glycolysis. In addition, ASIV can increase the expression of glucose transporter 1/4 and inhibit glycolysis by inhibiting the p53.^[24] Apart from that, GPL releases lactate via lactate transporters MCT1 and MCT4 to maintain glycolic process in tumors. ASIV can attenuate the expressions of MCT1 and MCT4, further improving the intestinal metaplasia and dysplasia in PLGC rat. Taken together, these results demonstrate that ASIV can correct impaired anaerobic cellular metabolism, and reverse precancerous cells or induce their death,^[23,24] preventing the further progression of PLGC.

Intestinal mucosal disease

UC is a high risk factor for colon cancer. The study by Qiao et al^[25] on UC showed that ASII treatment decreased the expression level of IL-6, TNF-α, IL-1β, NO, and myeloperoxidase (MPO) in mouse colonic tissues, and downregulated the expression levels of p-p65 and p-IκB proteins, which in return inhibit UC development. It was also reported that ASIV ameliorated inflammation and inhibited UC progression by suppressing the release of inflammatory factors, thereby downregulating NF-κB signaling in vivo and in vitro.^[26] It is distinct that astragaloside has a function in the treatment of UC. Moreover, ASII has been shown to repair the epithelial barrier after intestinal injury by promoting L-arginine (L-Arg) uptake and activating the mTOR pathway.^[27] L-Arg regulates protein synthesis and its uptake is an important process in colonic epithelial cell recovery, which can be enhanced by ASII. Treatment with ASII upregulated L-Arg to promote phosphorylation of p70S6K and 4E-BP1 in the mTOR pathway. The phosphorylated proteins, p70S6K and 4E-BP1, increase intestinal protein synthesis and epithelial repair.

Antitumor Mechanisms of Astragaloside

Astragaloside, the major anti-cancer component of Astragalus mongholicus Bunge [Fabaceae], has been found to regulate tumor cell apoptosis, proliferation, invasion, and metastasis [Figure 2 and Table 1].

Figure 2: Anti-tumor mechanism of astragaloside. Astragaloside can inhibit tumor progression by regulating tumor apoptosis, proliferation, differentiation, invasion, tumor microenvironments, and angiogenesis in various tumors. Upregulation is labeled with red and downregulation is labeled with blue. FGF2: Fibroblast growth factor 2; FVII: Factor VII; GSK-3β: Glycogen synthase kinase-3β; HGF: Hepatocyte growth factor; IL: Interleukin; MMP: Matrix metalloproteinases; Nrf2: Nuclear factor E2-related factor; NOS2: Nitric oxide synthase 2; TF: Transcription factors; TGF-β: Transforming growth factor-β; VEGF: Vascular endothelial growth factor.

Table 1 - Antitumor mechanisms of astragaloside.

Classification	Effect	Target	Cell lines	Refernce
Apoptosis	Promotion of apoptosis	Caspase-3	SK-Hep1, Hep3B	[45]
			MGC-803	[46]
			HCC827, A549, NCI-H1299	[47]
			SW962	[48]
		β-catenin	SMMC-7721, Huh-7	[49]
		Bip, CHOP, Caspase-12	DU-145	[50]
Proliferation	Inhibition of cell cycle	CyclinD1	U251	[51]
			HCT116, FHC, SW620	[52]
	Prevention of angiogenesis	FGF2, MMP-2, VEGF, HGF	HepG2	[53]
		TF, FVII
		miRNA-122
		miRNA-221
		TGF-β, IL-4	A549, H1299	[54]
		TGF-β, IL-10	CT26	[55]
Invasion and metastasis	Supperession of invasion and metastasis	Akt	Huh7, MHCC97-H	[56]
			BGC-823, MKN-74	[57]
			SiHa	[58]
		TGF-β	A549	[59]
		miRNA-134	SW-480	[60]
		IncRNA-ATB	SMMC-7721, Huh-7	[49]
		Vav3	HepG2	[61]
		MMP	MDA-MB-231	[62]
			U251	[63]
Tumor microenvironment	Regulation of GCAF	TIMP2	GCAF	[64]
	Regulation of oxidative stress	NrF2	MCF-7	[65]
			Huh-7	[66]
	M1-like TAMs increase	IL-12, NOS2	CT26	[55]
	M2 -like TAMs decrease	Arg1,MRc1	A549	[54]
		AMPK
		IL-4, IL-13	SKOV3	[67]
	Enhanced CD8+ T cells	IDO	3LL-luc	[68]

AMPK: AMP-activated protein kinase; Arg1: Arginase 1; BIP: Binding immunoglobulin protein; CHOP: C/EBP homologous protein; FGF2: Fibroblast growth factor 2; FVII: Factor VII; GCAF: Giant cell angiofibroma; HGF: Hepatocyte growth factor; IL-4/10/12/13: Interleukin-4/10/12/13; MMP-2: Matrix metallopeptidase 2; NOS2: Nitric oxide synthase2; Nrf-2: Nuclear factor E2-related factor; TAM: Tumor-associated macrophages; TF: Transcription factors; TGF-β: Transforming growth factor-β; TIMP2: Tissue inhibitors of metalloproteinase 2; VEGF: Vascular endothelial growth factor; IDO: Indoleamine 2,3-dioxygenase.

Apoptosis

Tumor cells exhibit an enhanced anti-apoptotic capacity as a mechanism to sustain their survival. Several studies have attempted to develop agents based on herbal monomers that can induce apoptosis in tumor cells.

The BCL-2, an anti-apoptotic protein highly expressed in various tumors, can inhibit the release of cytochrome C and prevent BAX-induced endogenous apoptosis. Treatment with ASIV activated apoptosis of tumor cells by downregulating BCL-2 levels and upregulating BAX levels,^[29] a phenomenon that has been observed in a variety of tumor cells including liver cancer,^[28] gastric cancer,^[29] and non-small cell lung cancer.^[30] However, the mechanism by which astragaloside causes apoptosis in tumors has not been fully elucidated. β-Catenin is one of the classical molecules that regulate apoptosis.^[48] A study by Cui et al.^[49] demonstrated that ASIV promoted the apoptosis of HCC cells by upregulating microRNA-150-5p, which in turn reduced the expression of β-catenin and BCL-2, and increased the expression of the apoptosis-related protein, BAX.ASIV can also modulate apoptosis in tumor cells through TGF-β. In vulvar squamous cell carcinoma (VSCC),^[31] ASIV increased the expression level of pro-apoptotic protein BAX and cleaved-caspase 3 protein via the TGF-β1/Smad4 signaling pathway, thereby inducing apoptosis of VSCC cells. In the prostate cancer cell line DU-145.^[33]

In conclusion, although astragaloside has been found to induce apoptosis in a variety of tumors, the mechanisms involved remain unclear. This is probably due to the fact that molecular targets of astragaloside on the apoptotic pathway have not been clearly established, and the effects of astragaloside on apoptosis vary across tumors.

Astragaloside inhibits tumor cell proliferation

Effects of astragaloside on the cell cycle

Abnormal regulation of cell cycle may lead to enhanced proliferation of tumor cells. This implies that agents targeting the cell cycle are potential therapeutic agents for tumor therapy. The activated complex promotes the transition of immature cancer cells from G1 to S phase and achieves enhanced proliferative capacity. Wang et al^[35] found by flow cytometry that ASIV may induce colon cancer cells proliferation inhibition due to G0/G1 cell cycle arrest. Further studies have shown that ASIV inhibits NF-κB, downregulating the expression level of B7-H3 and upregulating miR-29c levels. The inactivated NF-κB decreases the cycle protein D1 and formation of cyclinD-CDK4 complex leading to cell cycle arrest. Apart from regulating cyclinD1 via B7-H3, it has been found that^[34] ASIV can also stop cell cycle progression by inhibiting the TGF-β-mediated Wnt/β-catenin signaling in glioma cells, which in turn reduces expression of downstream cyclinD1 protein. These results indicate that cyclinD1 may mediate the effects of astragaloside on the cell cycle of tumor cells.

Astragaloside inhibits tumor angiogenesis

Tumor angiogenesis is regulated by various factors such as angiogenic factors, macrophage polarization, fibroblast growth factor, angiogenin, and oncogenes.^[50] Zhang et al^[36] found that ASIV and curcumin administered alone and as combinations significantly inhibited tumor growth in an ex vivo study. Moreover, these treatments downregulated expression level of angiogenic factors associated with tumor angiogenesis such as VEGF, FGF 2, Matrix metallopeptidase 2 (MMP-2), and hepatocyte growth factor, as well as thrombosis-related factors TF and FVII. These demonstrated that tumor angiogenesis was inhibited.

In addition, ASIV inhibited tumor angiogenesis by affecting macrophage polarization in the tumor microenvironment. In the hypoxic tumor environment, M2-type macrophages release angiogenic factors BFGF and VEGF and express MMP-2, MMP-7, and cyclooxygenase-2, all of which promote tumor angiogenesis. A previous study indicated that ASIV induced the polarization of M2 macrophages to M1 macrophages in colon cancer^[38] and lung cancer^[37] cells. In lung cancer,^[37] ASIV inhibited the AMP-activated protein kinase (AMPK) pathway by reducing the expression level of inflammatory factors IL-4 and TGF-β, inducing M2 macrophages' polarization into M1 macrophages. These findings suggest that ASIV could regulate tumor microenvironment and angiogenic factors to inhibit tumor angiogenesis.

Astragaloside inhibits cancer cell invasion and metastasis

Effect of astragaloside on epithelial–mesenchymal transition (EMT)

EMT is one of the main mechanisms of tumor metastasis. During EMT, the adhesion characteristics of epithelial basement membrane and adjacent cells are lost, and the cytoskeleton and polarity are altered to form a more invasive and metastatic mesenchymal phenotype. This allows tumor cells to leave the parenchyma and enter the circulatory system. The transition of the cell adhesion factor E-cadherin to the mesenchymal marker N-cadherin is a hallmark of EMT onset. Previous studies have found that ASIV can inhibit EMT in non-tumor cells. ASIV increases expression of proteins associated with SIRT1/NF-κB/p65 pathway to inhibit glucose-induced EMT in glomerular podocytes.^[51] ASIV can also increase Smad7 expression to downregulate TGF-β1-mediated EMT in mesothelial cells.^[52] Therefore, it has been postulated that ASIV may also inhibit EMT in tumor cells. Activated Akt can phosphorylate EMT transcription factors, Snail and Twist, as well as inhibit E-cadherin expression. ASIV inhibits HCC cell invasion and metastasis in vitro, which is achieved by limiting Akt activity and enhancing E-cadherin expression. AS-IV treatment resulted in a decrease in the phosphorylated forms of Akt that inhibited the EMT in HCC cells.^[39] A study by Zhu and Wen^[40] found that ASIV reversed the conversion of E-cadherin to N-cadherin and inhibited EMT in gastric cancer cells by suppressing TGF-β-induced activation of the PI3K/Akt/NF-κB.

ASIV has also been reported to activate microRNA-134 to downregulate the EMT regulatory protein cREB1, and therefore inhibited the EMT of colorectal cancer cell. ASIV treatment also inhibited the expression of the oncogenic gene IncRNA-ATB, thereby suppressing EMT through the IL-11/STAT3 signaling pathway, which in turn inhibited migration and cell viability of HCC cells.^[32]

Effects of astragaloside on Vav proteins

To elucidate on the mechanisms of ASIV in inhibition of HCC invasion and metastasis, Qi et al.^[42] subjected ASIV-treated and untreated HepG2 cells to mass spectrometry. They found that after ASIV treatment, Vav3.1 showed a 1.7-fold decrease in signal intensity. Vav3.1 is one of the isoforms of Vav. Vav, a guanine nucleotide exchange factor for the Rho family of GTPases, is involved in cell activities, such as cell motility and building the cytoskeleton. As a proto-oncogene, overexpressions of Vav3 promotes cell invasion as well as metastasis.^[53] This suggests that ASIV can inhibit Vav3 to suppress tumor metastasis.

Effects of astragaloside on matrix metalloproteinases

Matrix metalloproteinases (MMP) are zinc-dependent endopeptidases that induce extracellular matrix degradation, allowing cells to cross tissue barriers to promote tumor cell invasion. Among them, MMP-2 and MMP-9 are important in tumor metastasis. The MAPK/ERK pathway regulates MMP secretion. Activated MAPK can phosphorylate ERK and upregulate MMP expressions. In breast cancer^[43] and glioma,^[44] ASIV suppresses MMP-2 and MMP-9 expressions by inhibiting MAPK pathway activity and reducing phosphorylated ERK levels to increase surface adhesion and reduce tumor metastasis. Besides, protein kinase C-α (PKC-α) mediates elevations of MMP levels through the ERK1/2/NF-κB signaling pathway.^[54] However, ASIV can inhibit PKC-α translocation from the cytoplasm to the membrane, thereby reducing the amount of PKC-α on the cell membrane and suppressing MMPs expressions, which in turn inhibits lung cancer metastasis. These findings imply that ASIV inhibits MMPs by suppressing ERK phosphorylations; however, the upstream factors regulating phosphorylated ERK are not single, and it would be more meaningful to clarify the specific mechanisms of phosphorylated ERK changes after exposure of tumor cells to astragaloside.

Tumor microenvironment regulation

The antioxidant properties of astragaloside have been extensively studied. Astragaloside suppresses malondialdehyde levels and elevates glutathione peroxidase (GSH-Px) as well as SOD levels to scavenge for ROS.^[55] ROS is involved in tumorigenesis and tumor development, but few studies have been reported on the downregulation of ROS levels by astragaloside in tumor cells.^[56]

ROS are associated with tumor progression and formation of the tumor microenvironment. As described above, Nrf2 is an important factor that inhibits ROS levels. Zhang et al^[45] found that ASIV could upregulate Nrf2 in breast cancer cells by inhibiting the PI3K/Akt/mTOR signaling pathway. They also detected a significant decrease in ROS levels in ASIV-treated breast cancer cells, leading to an increase in apoptosis. They further knockdown Nrf2 using small interfering RNA, thereby reversing the antitumor effects of ASIV, with a significant increase in the volume of small interferingNrf2-treated tumor tissues and active tumor cell proliferation. However, the authors did not elucidate on ASIV-associated mechanisms of ROS reduction. Similarly, ASIV-treated HCC cells (Huh-7) exhibited elevated expressions and promoted nuclear translocation of the Nrf2 protein, which in turn reduced ROS production and inhibited oxidative stress in Huh-7 cells.^[46] These findings imply that Nrf2 is involved in anti-oxidative mechanisms of astragaloside in tumor cells, which forms the basis for translational clinical applications of astragaloside in regulating oxidative stress in the field of cancer therapy.

In the tumor microenvironment, tumor-associated macrophages (TAM)^[37,38] can regulate inflammatory reactions. Activated M1 macrophages have a certain role in non-specific immunity against tumors, while activated M2 macrophages are involved in tumor progression and tissue repair. In astragaloside-treated colon cancer cells,^[38] expressions of M2-associated genes (Arg1 and Mrc1) as well as the M2 cell surface marker (CD206) were markedly downregulated; meanwhile, protein expressions of M1-associated genes (IL12 and NOS2) were increased, implying astragaloside induced the conversion of M2 to M1 macrophages. In ovarian cancer,^[47] ASIV inhibited IL-4/IL-13-induced transcriptions of CD14+/CD206+ cell ratio and M2 macrophage markers (CD206 and CCL24) in THP-1 macrophages. This inhibition was also seen in lung cancer cells. Xu et al^[37] found that AS-IV blocks macrophage M2 polarization partly through the AMPK signaling pathway, reducing the pro-tumor activities of macrophage M2 in angiogenesis.

Some progress has been made in the study of immune checkpoint-related inhibitors, but TAM affects its treatment efficacy, and increasing the dose produces adverse effects. ASIV may be of value in addressing these issues, and programmed death 1 (PD-1) combined with ASIV is currently being applied experimentally in colon cancer,^[38] and in promoting the conversion of M2 to M1. Combination therapy has been shown to reverse the tumor immunosuppressive microenvironment^[57–60] and increase cytotoxic T lymphocyte infiltrations. The combination of ASIV with the PD-1 antibody was associated with better efficacies, relative to single use. Thus, the immunomodulatory abilities of ASIV may enhance cell sensitivity to various immunotherapies, such as PD-1, which may reduce adverse effects due to dose accumulation.

Astragaloside Improves the Efficacy of Chemotherapies

Astragaloside increased chemotherapeutic sensitivity

Multidrug resistance in cancer cells, which involves multiple mechanisms, is a major impediment to chemotherapeutic efficacy. Various studies are evaluating the roles of cellular autophagy in tumor drug resistance. Moderate autophagy can degrade chemotherapeutic drug-damaged cellular components in tumor cells and maintain tumor cell survival. Inhibition of autophagy can restore tumor cell sensitivity to chemotherapy. Beclin1 is a key factor in the development of autophagy. Cisplatin increases Beclin1 expressions to induce autophagy and drug resistance in tumor cells. In studies involving non-small cell carcinoma cell lines (A549 and H1299), ASIV downregulated endoplasmic reticulum stress-related protein (GRP78), protein kinase-like endoplasmic reticulum kinase (PERK), and Beclin1 expressions, destabilized the drug-resistant environment of tumor cells in terms of both inhibition of endoplasmic reticulum stress and autophagy, and increased tumor cell sensitivity to cisplatin. Although the involved mechanisms were not established, these findings provide a new basis for the cisplatin-sensitizing effects of ASIV. Huang et al^[61,62] found that ASII promoted the activation of mTOR by suppressing the phosphorylation levels ERK1/2 and p38 in the MAPK pathway, thereby reducing the levels of Beclin1 and autophagy-associated protein (LC3-II), inhibiting autophagy-induced drug resistance, and ultimately reversing tumor cell resistance to 5-fluorouracil. In HCC and gastric cancer cells, Yang et al^[63] found that ASII activated the PI3K/Akt/mTOR pathway, reduced cisplatin-induced tumor autophagic levels, and increased gastric cancer cell sensitivity to cisplatin.

Other mechanisms through which astragaloside affects chemotherapeutic resistance have been discovered continuously. ASIV significantly downregulated NOTCH3 levels in cisplatin-induced intestinal cancer cells,^[64] while forced expressions of NOTCH3 reversed the sensitizing effects of ASIV, suggesting that ASIV combination enhances cisplatin sensitivity by inhibiting NOTCH3 expressions.The P-glycoprotein (P-gp), which is encoded by multidrug resistance gene MDR1, acts as a drug efflux pump that expels chemotherapeutic drugs from tumor cells, leading to chemo drug resistance.^[65] It was found that ASII can downregulate MDR1 mRNA levels in HCC cells,^[61] inhibit the transcription as well as translation of P-gp, and suppress the transport activity of P-gp through substrate competition, thereby reversing P-gp-induced multidrug resistance in HCC and increasing chemosensitivity to 5-fluorouracil.

Inducing excessive ROS production in tumor cells is an effective strategy to enhance chemotherapy sensitivity. Caveolin-1 (Cav-1), a structural protein of plasma membrane invagination, has an effect on regulating oxidative stress.^[66,67] ASIV has been shown to inhibit Cav-1 expressions in breast cancer cells and activate the nitric oxide synthase (eNOS), which is inhibited by Cav-1, thereby increasing intracellular NO levels. The accumulation of NO increased oxidative damage in breast cancer cells and their sensitivity to paclitaxel.^[68]

Due to multiple mechanisms involved in tumor chemotherapy resistance, the multi-target advantage of astragaloside may reverse tumor cell drug resistance. Thus, elucidation of its mechanisms in reversing drug resistance will lay the foundation for its clinical applications. In addition, a few researches on astragaloside enhanced with molecularly targeted drugs have been retrieved. Since there is a partially common mechanism of resistance of tumor cells to chemotherapeutic drugs and molecularly targeted drugs, elucidating its mechanisms for reversing resistance to chemotherapeutic drugs may provide new ideas for conducting basic research on the efficacy of astragaloside combined with molecular targeting drugs.

Astragaloside reduces chemotherapeutic side effects

Cisplatin is associated with up to 40 specific toxicities, including nephrotoxicity, ototoxicity, and neurotoxicity. The toxic effects have been correlated with oxidative stress and inflammatory responses. Since astragaloside has good anti-inflammatory and free radical scavenging effects, it has a great potential for reducing toxic applications of platinum drugs. Xiong et al^[69] found that ASIV significantly suppressed NO levels in the cochlea of cisplatin-treated guinea pigs, suggesting that ASIV can inhibit NO synthase, which has the ability to damage the cochlea. In addition, cisplatin combined with ASIV reduced outer hair cell damage and ABR thresholds. Through scanning electron microscopy, it was established that stereocilia in the first and second rows of cisplatin/saline group were significantly distorted and even disappeared. Mean loss was significantly higher than that in the ASIV/cisplatin group. These results suggest that the antioxidant effects of ASIV mitigated the ototoxicity of cisplatin.

Both excess ROS-mediated oxidative stress and TNF-α-mediated pro-inflammatory responses play a role in cisplatin damage to renal tubular epithelial cells.^[70] In HK-2 cells and in mice, ASIV activated Nrf2/HO-1 pathway and promoted the upregulation of target antioxidant enzymes after cisplatin treatment, resulting in the decrease in ROS levels. ASIV-activated Nrf2 pathway also inhibited NF-κB activation to reduce the expression of its downstream inflammatory factors (TNF-α and IL-1β). By the two methods mentioned above, ASIV prevents acute kidney injury during cisplatin treatment. In contrast, ASIV protects against cisplatin-induced acute liver and kidney injury in rats through inhibiting the expression of NLRP3. NLRP3 functions to mediate inflammation and aggravate cisplatin toxicity.^[71]

In bleomycin-induced pulmonary fibrosis,^[72] ASIV dose-dependently reduced ROS levels, increased SOD levels, and suppressed bleomycin-induced elevations of TNF-α and IL-1β, thereby attenuating lung fibroblast overproliferation and extracellular matrix production.

Toxic reactions of chemotherapeutic drugs are highly associated with inflammatory reactions as well as oxidative stress, both of which can be controlled by ASIV to enhance drug efficacy. Even though a few studies have proven this, given the antioxidant and anti-inflammatory efficacy of ASIV in diabetes^[73] and cardiovascular diseases, its applications in oncology are promising.

Conclusion and Perspectives

Currently, most of the studies have been conducted on ASIV and a few on ASII. ASII has immunomodulation, antitumor, antioxidant, and anti-inflammatory properties,^[74] while ASIV has anti-aging, metabolic modulation, antioxidant, anti-asthma, anti-fibrosis, immunomodulation, anti-vascular related disease, neuroprotective and cognitive pathological change, and antitumor properties.^[75] In terms of their antitumor mechanisms, we found that ASIV regulates tumor apoptosis, the cell cycle, tumor angiogenesis, EMT, tumor microenvironment, and chemotherapeutic sensitization, while ASII was only established to have chemotherapy sensitization functions. However, the anti-aging, metabolism-regulating, and immunomodulatory roles of ASII and ASIV in non-tumor cells suggest more anti-tumor mechanisms.

Various studies have reported on the safe doses of ASII and ASIV in vivo. Hong et al^[76] found that when the astragalus extract was used as an immune adjuvant in combination with a vaccine, a weight loss of 5–10% occurred with 500 μg of ASII, while the same dose of ASIV showed almost no weight loss. In animal experiments, oral administration of ASIV for 14 weeks was not associated with any side effects, suggesting that ASIV has a greater safety range. However, in rat models treated with the astragalus extract mixture, Song et al^[77] did not find any adverse effects with oral administrations of up to 4000 mg∙kg^-1∙day^-1. More studies are needed to determine safe doses of ASII and ASIV that can be used in humans. In terms of bioavailability, the absolute bioavailability of orally administered ASIV was found to be 7.4%,^[78] which was attributed to poor intestinal permeability, high molecular weight, low lipophilicity, and its paracellular transport.^[79] In a comparative pharmacokinetic study, ASII was associated with a rapid absorption rate and a higher extraction volume, compared to ASIV (4.09 mg/mL vs. 0.95 mg/mL), whereas the maximum plasma concentration of astragaloside II was much lower than that of astragaloside IV. Moreover, ASII had low biotransformation rates,^[80] and thus, there is a need to establish suitable approaches for improving the bioavailability of ASII and ASIV. Specific intestinal Lactobacillus bacteria can improve astragaloside bioavailability, and intestinal microbial metabolism is an important mechanism for efficient astragaloside absorption.^[81] Currently, nanomaterials are being studied for potential applications in drug development. As drug carriers, protein nanoparticles from licorice rhizomes can penetrate cell membranes and dissolve insoluble astragaloside by encapsulation.^[82] Studies have attempted to embed astragaloside into nanoparticles for gradual release to promote wound healing. These studies revealed strong cell fusion and low toxicity in MDCK, HepG2, L-02, and other cells. Moreover, when encapsulated into nanoparticles to achieve slow release, astragaloside promotes wound healing,^[83] thereby laying a good foundation for improved absorption and efficacy of astragaloside. Nanocarrier-targeted release of astragaloside in tumors has the ability to improve its efficacy and clinical applications.

The mechanisms involved in chemoprevention of tumors are complex and varied. For liver cancer, chemoprevention can be divided into primary (prevention of HCC development) and secondary chemoprevention (prevention of HCC recurrence).^[84] The risk factors contributing to the recurrence of HCC are complex. The recurrence of HCC tumor within 2 years after local treatment or surgical resection may be due to the presence of microscopic metastases in the liver, whereas the recurrence of HCC tumor over 2 years may be as a result of new cancer caused by the still existing risk factors such as chronic HBV and HCV infection, liver cirrhosis, and nonalcoholic fatty liver disease. Therefore, the effect of secondary prevention targeting a single factor may not be satisfactory, and the targeting should involve multiple components. A review of literature on the application of chemoprevention agents in HCC revealed that astragaloside may be a promising agent for secondary chemoprevention of HCC. First, for microscopic lesions of HCC that exist after local or surgical treatment, astragaloside may reduce the risk of tumor recurrence by inducing apoptosis and inhibiting the tumor microenvironment. For risk factors that persist after local or surgical treatment, astragaloside can reduce the risk of new cancer by inhibiting HBV replication and delaying the progression of cirrhosis. The application of astragaloside in secondary chemoprevention may also be extended to other tumors such as gastric cancer and colon cancer. Further studies are needed to test this possibility.

Funding

This work was supported by grants from the Natural Science Foundation of China (Nos. 81902484, 82002809), China Postdoctoral Science Foundation (No. 2020M670864), Jilin Province young health scientist training program (No. 2020Q017), Medical and Health Talents Project of Jilin Province (Nos. 2020SCZT039, 2020SCZT097), Youth Support Project of Jilin Association for Science and Technology (No. 202028) and Jilin Province Science and Technology Development Plan(No. 20210204028YY).

Conflicts of interest

None.