Abstract
Paeoniflorin (PF) is an important pharmacological component of some Chinese traditional herbal formulas, such as Bai Shao, Chi Shao, and Dan Pi, which have been clinically used for centuries. Although many experimental studies have explored a wide range of pharmacological properties of PF, including anticancer, anti-inflammatory, antioxidant, immunoregulatory, and prevention of insulin resistance, there is no review to describe these reported effects systematically, especially the antitumor effect and the underlying mechanisms. In this review, we summarize the recent progress on the anticancer profiles both in vitro and in vivo of PF. Moreover, we highlight the integrated molecular mechanisms of PF and contemplate its future prospects as a potential anticancer drug.
Ethnopharmacological products have always been a major aspect in the prevention and treatment of cancer and other diseases around the world, especially in Asia and Africa. 1 -3 Their multiple pharmacological properties have not only provided a theoretical basis for the mechanism study of natural compounds but also have shown great potential for drug discovery. 4,5 The exploration of anticancer agents from natural sources started with the discovery of vinblastine and vincristine in 1950, 6,7 and from then on, more and more ingredients with anticancer activity have been isolated from plants, such as taxanes, anthracyclines, and podophyllotoxin. 8
Paeoniflorin (PF, Figure 1), an important pharmacologically active component derived from Bai Shao, Chi Shao, and other plants, 9 -13 is abundant in the fibrous root, bark-free root, and root bark of Paeonia lactiflora. 14 Recently, PF has drawn great interest from researchers because of its variety of pharmacological activities including anti-inflammatory, antioxidant, immunoregulatory, prevention of insulin resistance, as well as liver and nerve protective effects. 15 -20 In addition, numerous studies are emerging which suggest that PF may have potent effects on a wide range of cancers. 21 -24 Although many experimental studies have explored plenty of pharmacological properties of PF, there is lack of review that systematically describes these reported effects, especially the antitumor effect and its underlying mechanisms. In this review, we summarize the recent progress on the anticancer profiles both in vitro (Table 1) and in vivo of PF. Moreover, we highlight the integrated molecular mechanisms (Table 1) of PF and contemplate its future prospects as a potential anticancer drug.
(a) Plant sources of paeoniflorin. (b) Synthesis of paeoniflorin.
Anticancer Effects and Mechanisms of Paeoniflorin on Various Cancer Cell Lines.
APO-1, apoptosis-1; EMT, epithelial-to-mesenchymal transition; ERK, extracellular signal-regulated kinase; CCK-8, Cell Counting Kit-8; MAPK, mitogen-activated protein kinase; MDR, multi-drug resistance; MMP, matrix metalloproteinase; MTT, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; WAF1, wild-type p53-activated fragment 1; XTT, (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide).
Sources of PF
Plant Sources
Paeonia lactiflora and Paeonia suffruticosa are the most frequently reported plants which contain PF. 9,13 Paeoniae Radix Alba (Baishao in Chinese) and Paeoniae Radix Rubra (Chishao in Chinese), the dried root of P. lactiflora Pall., are the main ingredients in some Chinese traditional herbal formulas which have been clinically used for the treatment of liver diseases 41 and blood-stasis syndrome 42 for centuries. Dan Pi (literally, Tree Peony Bark), another traditional Chinese medicine material, derived from the root cortex of P. suffruticosa, is clinically used for the treatment of various diseases, such as macula and menstrual disorders. 43 In addition, PF is also found in Cimicifuga foetida. 44 The plant sources of PF are summarized in Figure 1(a).
It is known that plenty of factors can affect the content of secondary metabolites in plants, including the location, age, and growth conditions. The content of PF in different parts of P. lactiflora Pall. ranges from 0.16% to 6.91%. The content in the dry stem (0.16%) is less than that of the root (6.89%). 45 When collected in May, the content of PF in roots was less than 3.05%, whereas in August it was 4.72%. 46 Furthermore, the amount of PF increased with age, with yields of 3.26%, 3.30%, 3.36%, and 3.41% separately from 1- to 4-year-old plants. 47 In addition, proper levels of Mn, Fe, Zn, and Cu in the fertilizer facilitate the content of PF. 48 The content of PF in fleshy roots was not significantly different between wild and bud asexually cultivated plants. 49
Moutan cortex is derived from P. suffruticosa Andr., the content of PF of which ranged from 1.34% to 3.69%. 50 Other contents reported were 1.62%, 51 1.48%, and 0.71%. 52
Synthesis
PF can also be prepared synthetically (Figure 1(b)). 53 -55 To some extent, chemical synthesis is more targeted and more productive, but there are also many problems, such as many by-products and cumbersome procedures. Therefore, more efficient synthesis and/or isolation methods of PF should be further explored.
Anticancer Activity In Vitro and In Vivo
Abundant research has shown the anticancer effects of PF and provides evidence for it being a promising agent in cancer treatment. PF showed strong cytotoxic potential against human lung cancer A549 cells with an IC50 value of 11.4 µM. 24 In addition, PF displayed cytotoxicity on multidrug resistant gastric cancer cell line SGC7901/VCR, 40 but on drug-resistant CEM/ADR5000 cells, the IC50 value of PF was above 800 µM, suggesting that it had only slight toxicity on CEM/ADR5000 cells. 56 In addition, the inhibitory effect of PF on various human colorectal cancer cell lines, including HCT116, SW480, HT-29, and Caco-2 cells, is also slight, with IC50 values ranging from 0.82 to 13.34 mM. 23,56,57 Recently, PF was proved to display a cell growth inhibitory effect on cervical cancer Hela, 27 glioma U87 and U251 cells, 58 and osteosarcoma HOS and Saos-2 cells. 25
PF has also demonstrated an anticancer effect in vivo. Wang used a U87-luciferace orthotopic xenograft mouse model to prove the in vivo effect of PF on glioblastoma and found that tumor volumes were markedly decreased and mouse survival rates were enhanced after intraperitoneal injection with PF (400 mg kg−1 day−1). 32 Similarly, orally feeding with PF (1 g kg−1 day−1) also decreased glioblastoma growth in a U87 xenograft mouse model. 38 A RT4 cell xenograft nude mice model was established to determine if PF could inhibit bladder tumor growth in vivo, the results showed that intraperitoneal administration of PF (100 mg kg−1 day−1) reduced the weight and volumes of the tumor tissue. 31 Additionally, by oral gavage feeding of PF at 1 g kg−1 day−1, PF reduced the tumor volume by 30.8% and had a stronger growth inhibitory effect and lower blood toxicity than that of docetaxel. 57 In summary, these studies indicated that PF has a variety of antitumor activities.
The Anticancer Mechanisms of PF
Cell Cycle Arrest
It is reported that PF displays an inhibitory effect on A549 cells by blocking cell cycle progression at G0/G1 phase, which may be correlated with p53-independent induction of p21/wild-type p53-activated fragment 1. 24 PF also blocks cell cycle progression at G0/G1 phase in colorectal cancer cells and downregulates FoxM1 to inhibit cell growth. 26 Besides, PF induces G2/M phase cell cycle arrest in osteosarcoma HOS and Saos-2 cell lines. The expression levels of cell cycle regulators such as cyclin B1, p-CDK1, CDK1, and p21 significantly increased after PF treatment. 25 Interestingly, PF treatment for 12 hours significantly caused G2/M phase arrest in HT29 cells, but later a strong G1 phase arrest was detected at 24 and 48 hours. Further research demonstrated that cell cycle arrest may be mediated by p53 upregulation and 14-3-3ζ downregulation. 57
Apoptosis
Caspase and Bcl-2 protein family
Caspase and the Bcl-2 protein family are key regulators of apoptosis. Caspase activation is found in a great number of cancer cells after PF treatment. For instance, PF significantly increases caspase-3 activation in HepG2, SMMC-7721, 59 Hela, 27 and MGC-803 cells. 33 PF was also reported to upregulate both cleave caspase-3 and -9 in prolactinoma MMQ cells in a concentration-dependent manner. 60 Activation of caspase-3 and -9 was also detected in PF-treated BXPC-3, 29 SKO-007, 35 and HT29 cells. 57 Yang et al found that PF increased the activities of caspase-3, caspase-8, and caspase-9, thus inducing apoptosis in bladder carcinoma RT4 cells. 31 It is believed that the upregulation of pro-apoptosis factor Bax and the downregulation of anti-apoptosis factor Bcl-2 lead to cell apoptosis. As shown in some research, PF increases the Bax-to-Bcl-2 ratio in HepG2, SMMC-7721, 59 HeLa, 27 RT4, 31 MMQ, 60 HOS, and Saos-2 cells. 25
Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway
ERK, a subfamily of MAPK, is deeply involved in the occurrence and development of tumors. The MAPK/ERK pathway is reported to mediate cell proliferation, differentiation, and signal transduction. 61 When exposed to stressors, the MAPK/ERK pathway contributes to apoptosis by affecting the activity of the apoptosis-related Bcl-2 family of proteins. 62 Yang indicated that the inhibition of ERK by PF significantly contributes to pancreatic cancer cell apoptosis. 29 This result is supported by other research demonstrating that PF facilitates HL-60 cell apoptosis by blocking the Fos-Jun heterodimer-binding site of AP-1, which is a downstream target gene of the MAPK family. 21 However, PF exerts a growth inhibitory effect by activating the MAPK pathway in endometrial cancer cells. 30 How to explain the contradictory outcome and what role MAPK/ERK actually plays in cancer needs further investigation.
Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway
NF-κB is of great significance in tumorigenesis and tumor progression. The constitutive activation of NF-κB, which was proved to mediate the expression of apoptosis-related genes such as Bcl-2, has been found in many types of cancers. 63 Wu tested whether PF displays cytotoxicity through NF-κB pathway inhibition and found PF suppresses NF-κB activation in a dose- and time-dependent manner. Further investigation proved that the inhibition of phosphorylation and degradation of IκBα by PF contributes to NF-κB inhibition. Other research indicated that the inhibition of NF-κB by PF enhances 5-fluorouracil-induced gastric carcinoma cell apoptosis. 64 However, Zhang et al found that PF inhibits endometrial cancer cell growth by activating the NF-κB signaling pathway, indicating that NF-κB plays a dual role in PF-induced cell death and its effect depends on cell type and cell circumstance. 30
STAT3 pathway
STAT3, an important oncogene modulating cancer cell proliferation, invasion, metastasis, apoptosis, and immune escape, was proved to regulate the expression of different types of apoptosis-related genes such as Bcl-2 and Bax. 65,66 Targeting STAT3 to inhibit its phosphorylation may be a strategy to combat tumor occurrence and development. 67 A recent study by Zheng demonstrated that PF significantly inhibits the expression of PI3K, Akt, p-Akt, and p-STAT3 in MGC-803 cells. This effect may result from the upregulation of microRNA-124. The role of STAT3 is confirmed by the experimental result that the overexpression of STAT3 abrogates the inhibitory effect of PF on MGC-803 cells. 33 Similarly, research by Nie also revealed the inhibitory effect of PF on STAT3 and its downstream molecules in glioma cells U87 and U251. Besides, they further illuminated that the degradation of STAT3 is mainly through the ubiquitin-proteasome pathway. 68 In addition, PF also exerts its growth inhibitory effect on bladder carcinoma RT4 cells via suppressing STAT3 phosphorylation in vivo and in vitro. 31
Matrix metalloproteinases (MMPs)
MMP-2 and -9 play vital roles, not only in tumor invasion and metastasis, which is related to the degree of cancer malignancy, but also in cancer cell death. It is reported that the downregulation of MMP-9 and ERK signaling participates in PF-induced pancreatic cancer cell apoptosis. 29 Li demonstrated that PF accelerates glioma cell apoptosis via MMP-9 downregulation, which may be attributed to the upregulation of microRNA-16. 34 Similar to this study, PF promoted the apoptosis of multiple myeloma through MMP-2 downregulation, which is regulated by an increased level of microRNA-29b. 35
Other signaling pathways
Hu demonstrated that the downregulation of PGE2 receptor subtype EP2 induced by PF contributes to HepG2 and SMMC-7721 cell apoptosis. 59 Wang found that PF effectively suppresses TLR4 and its downstream molecules both in vivo and in vitro, and the overexpression of TLR4 abolishes the antiproliferation effect of PF, indicating that targeting TLR4 may be a novel mechanism for PF-induced cell death. This study also focused on the way of TLR4 degradation and revealed that PF promotes Triad3A to conjugate with TLR4, which leads to Triad3A-dependent ubiquitin degradation of TLR4. 32 Besides, PF-induced Notch-1 suppression plays a part in breast cancer cell apoptosis, which was indicated by an article describing that the overexpression of Notch-1 reverses PF-induced cell death and the knockdown of Notch-1 enhances the inhibitory effect of PF. 36 Global analysis of gene expression showed that HTRA3 is the most increased gene upon PF treatment in Capan-1 cells; further investigation demonstrated that HTRA3 increases the expression level of Bax, thus inducing pancreatic cancer cell apoptosis. 22 A novel study revealed that by inhibiting prolactin, PF induces prolactinoma cell apoptosis through the mitochondria-dependent pathway, which is related to phosphorylated P53 upregulation. 60 Moreover, PF-induced apoptosis of A549 cells is due to activation of the Fas/Fas ligand apoptotic system, reflected in the increased expression of Fas/apoptosis-1 and its 2 ligands. 24
PF is reported to have a cytotoxic effect on HL-60 and other leukemia cell lines through the inhibition of Fos-Jun-DNA complex formation. 21 However, in human leukemia U937 cells, Lim found that PF had no inhibitory effect although gene expression profiling demonstrated that PF changes the expression of apoptosis-related genes. Using a comparative global transcription analysis, PF was observed to activate the c-Jun N-terminal kinase (JNK) pathway less when compared with anisomycin treatment, but activate the fibroblast growth factor (FGF) signaling and MAPK pathway, which may be the explanation of this phenomenon. 28
Inhibition of Migration and Invasion
Epithelial-to-mesenchymal transition (EMT), a process of losing cell-to-cell adhesion, has been reported to play an essential role in tumor invasion and metastasis, which have always been a tough problem in cancer cure. Activation of EMT promotes cancer cell migration and invasion, so seeking compounds that can inhibit EMT is an urgent task nowadays. PF is proved to suppress migration and invasion in glioblastoma through the inhibition of transforming growth factor-β(TGF-β)-induced EMT, while the expression level of EMT markers such as snail, vimentin, N-cadherin, and MMP-2 and -9 reduces after PF treatment. These results were also confirmed in vivo using a U87 xenograft mouse model. 38 Lu demonstrated that PF significantly inhibits invasion, metastasis, and adhesion of HepG2 and Bel-7402 cells; this effect is mediated by the downregulation of MMP-9 and ERK and the upregulation of E-cadherin. 37 Besides, PF inhibits invasion in breast cancer MCF-7 and MDA-MB-231 cells through suppressing the Notch-1 signaling pathway. 36 Another study focused on hypoxia-induced EMT in breast cancer cells and found that PF restrains hypoxia-induced EMT in MDA-MB-231 cells by inhibiting HIF-1α expression, which is modulated by the PI3K/Akt signaling pathway. 39 HDAC2 is proved to participate in EMT of colorectal cancer cells. PF potently inhibits cell migration and invasion through the suppression of HDAC2-induced EMT, the downregulation of vimentin, and the upregulation of E-cadherin. Besides, the anticolorectal cancer effect is also verified in the tumor xenograft model. 23 Other research demonstrated that PF could reduce metastasis of Lewis lung cancer cell xenografts partly through inhibiting the alternative activation of macrophages. 69
Combat Multi-Drug Resistance (MDR)
During chronic treatment, various types of cancer cells gain acquired resistance to chemotherapeutic drugs, leading to treatment failure and high mortality in cancer therapy, so finding agents that can sensitize multi-drug resistance (MDR) cancer cells to chemotherapeutic drugs may be a strategy to combat multi-drug resistance. PF is reported to modulate MDR and sensitize SGC7901/VCR cells to VCR-induced apoptosis via the inactivation of NF-κB and its target genes MDR1, BCL-XL, and BCL-2. 40 In addition, PF increases the cytotoxicity of doxorubicin in CEM/ADR5000 and Caco-2 cells with a reversal ratio 2.32 and 1.50, respectively. This reversal effect is partly through inhibiting ABC transporters in CEM/ADR5000, but in Caco-2 cells, PF does not inhibit ATP-binding cassette (ABC) transporters, and the downregulation of AhR, CYP1A1, and GSTP1 may contribute to the reversal effect. 56
Discussion
In summary, PF is the main active ingredient of many Chinese herbal medicines, such as Bai Shao, Chi Shao, and Dan Pi. It has been demonstrated that PF has a wide spectrum of biological activities. Specially, it has significant and potent anticancer effects on various types of cancer, including leukemia, glioma, pancreatic, breast, and colorectal. Overall, the anticancer effect of PF is related to cell apoptosis, cell cycle arrest, EMT inhibition, and MDR reversal. It is reported that MMP-2, MMP-9, MAPK/ERK, JNK, STAT3, PI3K/Akt, NF-κB, Notch-1, and other signaling pathways may be involved in PF-induced cell death, but the target of PF remains unknown. Moreover, in different cancer cells, the antitumor mechanisms of PF are also different. How PF affects cancer cells may depend on a specific receptor located in the cell membrane, but the downstream regulators are almost the same, such as the caspase family in apoptosis, and MMP-2, MMP-9, and E-cadherin in EMT. Although PF displays a potent anticancer effect, the molecular target and signaling pathway have not been fully elucidated. Further research needs to be carried out in order to clarify this question.
Some research also revealed the growth inhibitory effect of Paeoniae Radix extract (PRE), which includes PF, gallic acid, benzoic acid, paeonol, and other active components. 70 Lee reported that PRE induces hepatoma cell apoptosis in a p53-independent pathway. 71 The antibladder cancer effect of Radix Paeoniae Rubra extract was proved in vitro and in vivo. 72 In addition, WU reported that the extract of Paeoniae Radix and Astragalus membranaceus (PAE) displayed an inhibitory effect on proliferation, migration, and invasion of hepatoma cells HepG2 and SMMC-7721, providing evidence for PAE as a promising agent for HCC treatment. 73 It is generally recognized that traditional Chinese medicines have multiple targets distinguished from monomeric compounds, so whether PF displays a stronger anticancer effect than the complex mixture PRE and their different mechanisms needs further determination.
Combination drug therapy is widely applied in clinical practice. As mentioned above, PF enhances 5-fluorouracil-induced apoptosis in human gastric carcinoma cells. 64 In addition, the anticancer effect of Erlotinib can also be reinforced by PF, which directly suppresses ErbB3 phosphorylation, indicating that Erlotinib in combination with PF is more effective than its use alone. 74 We believe more and more combination drug strategies between PF and other agents will be created in the future.
Although the therapeutic potential of PF looks promising in vitro and in vivo preclinical studies, several hurdles still exist in the current stage. Toxicity is an important issue with PF. It is worth synthesizing derivatives of PF with more selectivity and efficacy, and reducing the unexpected side effects. Additionally, there is little literature related to the pharmacokinetic profiles of PF, including absorption, distribution, metabolism, and excretion. Therefore, further pharmacokinetic and clinical studies are mandated to define the efficacy and safety of PF in cancer and other diseases.
Footnotes
Acknowledgments
The pictures of plants and Chinese medicines used in this article were obtained online: https://image.baidu.com. The databases used in this article were TCMSP: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform http://lsp.nwu.edu.cn/tcmsp.php and SymMap: http://www.symmap.org/; 
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Science Foundation of China (81803790).