Jiyi Bi,Zixun Go,Guogng Zhng,Yu Chen

a.School of Life Science and Biopharmaceutics,Shenyang Pharmaceutical University,Shenyang 110016,China;b.School of Traditional Chinese Materia Medica,Shenyang Pharmaceutical University,Shenyang 110016,China

Abstract Cancer is a serious threat to human life and a big problem in clinical treatment.Some natural active substances extracted from Traditional Chinese Medicine can effectively inhibit the growth of cancer cells,which is a new direction for finding more effective anticancer drugs.Osthole is a natural coumarin compound extracted from Traditional Chinese Medicines such as Cnidium monnieri,Angelica pubescens and Peucedanum praeruptorum Dunn.It has significant inhibitory activity against a variety of cancers.This paper summarizes the anticancer effects and molecular mechanisms of osthole in the treatment of cancers in recent years in order to provide references for further research.

Key words: osthole;anticancer effects;molecular mechanisms

1 Introduction

Cancer,a disease with a very high mortality,has become one of the major diseases that seriously threaten the physical and mental health of people all over the world.It was estimated that there were nearly 18.1 million new cancer cases and 9.6 million cancer deaths worldwide in 2018 [1].Cancer not only causes endless pain to patients,but also brings heavy economic burden to families and society.

Osthole,7-methoxy-8-(3-methyl-2-butenyl)coumarin (Fig.1),is an ingredient of Traditional Chinese Medicines such as

Cnidium monnieri

,

Angelica pubescens

and

Peucedanum praeruptorum

Dunn.Due to its significant and diverse pharmacological activities,including antitumor,neuroprotection,cardiovascular protection,enhance immunity,anti-osteoporosis and liver protection,it has received considerable attention recently and is becoming a promising natural lead compound for new drug development [2,3].In recent years,many studies at home and abroad have shown that osthole has good anticancer activity against gastric cancer [4-6],lung cancer [7-10],breast cancer [11-14],hepatocellular carcinoma [15],prostate cancer [16-18],renal cancer [19,20],colon cancer [4,21],cervical cancer [22],glioma [20],nasopharyngeal carcinoma [23],bladder cancer [24],and ovarian cancer [25].Its anticancer mechanism includes the proliferation,apoptosis,and migration of cancer cells.In this paper,the latest research progress on the anticancer effects and molecular mechanisms of osthole is reviewed,which will provide a theoretical basis for further research on osthole and the development of new anti-tumor drugs.The antitumor effects and molecular mechanisms of osthole are shown in Fig.2.

Fig.1 The structure of osthole

Fig.2 Antitumor effects and molecular mechanisms of osthole

2 Anticancer effects of osthole

2.1 Gastric cancer

Gastric cancer is one of the most aggressive malignant cancers in the world,especially in China.Li et al.confirmed that osthole could inhibit the growth of human stomach cancer cells MKN-45 and BGC-823

in vitro

,and the inhibiting effect of osthole

in vitro

was better than that of the ethanol extracts from

Angelica pubescens

,which indicated that the main antitumor constitution of

Angelica pubescens

was osthole [4].Xu et al.tested the effects of osthole on the proliferation of human gastric cancer cells SGC-7901 and HGC-27 [5].The results showed that osthole could inhibit gastric cancer cells proliferation by reducing PI3K/AKT to induce cell cycle arrest in G2/M phase.Yang et al.proved that osthole was successful in inhibiting the growth of N87 gastric cancer cells in a dose-dependent manner [6].The results also indicated that osthole caused cell cycle arrest in G2/M phase and finally resulted in caspase-3 dependent apoptosis in N87 cells.

2.2 Lung cancer

Lung cancer is the leading cause of cancerrelated deaths worldwide.Kao et al.demonstrated that osthole could inhibit the expression of NF-κBmediated matrix metalloproteinase (MMP)-9 and thus prevent the invasion and migration of lung cancer cells [7].The results indicated that osthole might be a potential agent to prevent the invasion and metastasis of lung cancer.Xu et al.found that osthole enhanced the anticancer effect of cisplatin in human lung cancer,and used western blot analysis to show that the combination effect of osthole and cisplatin was due to the regulation of Bcl-2 family proteins [8].Feng et al.studied the inhibitory effect and mechanisms of osthole on TGF-beta 1-induced epithelial-mesenchymal transition (EMT) in A549 cells,and confirmed that osthole inhibited TGF-beta 1-induced EMT,adhesion,migration,and invasion through inactivation of NF-kB-Snail pathways in A549 cells [9].Xu et al.demonstrated that osthole could inhibit proliferation of lung cancer cells and induce their apoptosis via IAP family proteins in a dose-dependent manner [10].

2.3 Breast cancer

Breast cancer is the most common malignancy in women.Yang et al.found that osthole was effective in inhibiting the migration and invasion of breast cancer cells through wound healing and transwell assays [11].Luciferase and zymography assays showed that osthole could effectively inhibit matrix metalloproteinase-2 promoter and enzyme activity,which might be one of the reasons why osthole inhibited migration and invasion.Wang et al.demonstrated that osthole inhibited the proliferation of human breast cancer cells MDA-MB 435 [12].The mitochondrion-mediated apoptotic pathway was involved in osthole-induced apoptosis,as indicated by PARP degradation after the activation of caspase-9 and caspase-3.G1 phase arrest was induced because of the up-regulation of p53 and p21 and down-regulation of Cdk2 and cyclin D1 expression.Park et al.demonstrated that osthole inhibited cellular proliferation and induced cell cycle arrest by modulating cell cycle regulatory genes in BT-474 and MCF-7 breast cancer cells [13].Dai et al.found that osthole inhibited the growth of a panel of triple-negative breast cancer (TNBC) cells and induced apoptosis in both cultured cells and TNBC xenografts [14].They used a high-throughput proteomics platform and identified signal transducer and activator of transcription 3 (STAT3) as a potential binding protein of osthole.They further demonstrated that osthole suppressed STAT3 in TNBC cells to inhibit growth and induce apoptosis.

2.4 Hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common tumor with high metastatic ability and recurrence rate.Lin et al.found that osthole inhibited HCC cell proliferation,induced cell cycle arrest,triggered DNA damage and suppressed migration in HCC cell lines [15].Furthermore,they demonstrated that osthole not only caused cell cycle G2/M phase arrest by down-regulating Cdc2 and cyclin B1 levels,but also induced DNA damage by increasing ERCC1 expression.In addition,osthole inhibited the migration of HCC cell lines by significantly downregulating MMP-2 and MMP-9 levels.Finally,they demonstrated that osthole inhibited EMT by increasing the expression of epithelial biomarkers E-cadherin and β-catenin and significantly decreasing mesenchymal N-cadherin and vimentin protein expression.These results suggested that osthole might have potential chemotherapeutic activity against HCC.

2.5 Prostate cancer

Prostate cancer is the second most common male cancer in the world and poses a serious threat to human health.Wen et al.demonstrated that osthole inhibited migration and invasion of androgenindependent prostate cancer (AIPC) cells,as well as epithelial-to-mesenchymal transition (EMT)

in vitro

and metastasis of AIPC

in vivo

[16].Qu et al.found that osthole induced LNCaP cells apoptosis in a dose-dependent manner and observed the apoptosis cells with a fluorescent electron microscope.Osthole reduced the expression level of apoptosis-related protein bcl-2,but increased Bax expression level in a concentration-dependent manner [17].Chen et al.demonstrated that osthole significantly increased the cytotoxicity of doxorubicin against p53-wildtype prostate cancer cell line LNCaP [18].Osthole significantly inhibited the expression of SIRT1 in the LNCaP cells.Transfection with SIRT1 plasmid reduced the cytotoxicity of osthole and doxorubicin co-treatment against LNCaP cells.Osthole combined with doxorubicin significantly induced the overexpression and acetylation of p53.Transfection with p53 siRNA significantly decreased the synergistic effect of osthole on the cytotoxicity of doxorubicintreated LNCaP cells.Osthole combined with doxorubicin significantly induced the release of cytochrome C into the cytoplasm from mitochondria,activated caspase-9 and its downstream molecule caspase-3,and led to cell apoptosis in LNCaP cells.

2.6 Renal cancer

Renal cell carcinoma (RCC) is a common urinary malignancy,accounting for 2%-3% of all malignant diseases.Liu et al.demonstrated that osthole had an inhibitory effect on RCC cells through dose-dependent inhibition of cell proliferation,induction of apoptosis,suppression of migration and invasion,and attenuation of EMT [19].This study supported the possibility that osthole might be a potential novel herbal agent against RCC.Min et al.revealed that osthole sensitized TNFrelated apoptosis-inducing ligand (TRAIL)-mediated apoptosis by down-regulating c-FLIP expression in human renal Caki cells [20].Therefore,osthole might be a promising sensitizer for TRAIL-resistant cancer cells.

2.7 Colon cancer

Colon cancer is one of the most common diseases in the world with high incidence and mortality.Li et al.found that osthole (3.75-120

μ

g/mL)inhibited the multiplication of human colon cancer cells LOVO and the inhibition was concentrationdependent [4].The ICof LOVO cells was 36.231

μ

g/mL.Huang et al.assessed osthole-induced cell death in two different human colon cancer cell lines,HCT116 and SW480 [21].The results showed that osthole activated the proapoptotic signaling pathways and reduced cell motility in the two human colon cancer cell lines.

2.8 Cervical cancer

Cervical cancer is the fourth most common cancer in women worldwide.Che et al.demonstrated that osthole dose-dependently reduced the viability,proliferation,migration and invasion of cervical cancer cells,but induced apoptosis [22].At the protein level,osthole affected the expression of epithelial-mesenchymal transition markers in cervical cancer cells,manifested as increased expression of E-cadherin and decreased expression of vimentin.Osthole treatment also sensitized cervical cancer cells to irradiation,increased DNA damage as assessed by the Comet assay,and inhibited nuclear factor-κB signaling.Therefore,osthole was an herbal agent with potential for adjuvant treatment of cervical cancer.

2.9 Other cancers

Min et al.found that osthole and TRAIL alone had no effect on apoptosis,but the combination treatment significantly induced apoptosis in U251MG (glioma) cells [20].Chen et al.confirmed that osthole effectively inhibited the proliferation of CNE2 (nasopharyngeal carcinoma) stem cells and increased their radiosensitivity,which might be related to the down-regulation of pGSK-3,β-catenin,and Cyclin D1 in tumor stem cells by osthole [23].Liu et al.revealed that osthole could reduce the expression of COX-2,VEGF and NF-kappaB and increase the activity of caspase-3,eventually blocking the growth and invasion of bladder cancer cells [24].Jiang et al.showed that osthole inhibited the proliferation and migration of two widely used ovarian cancer cell lines,A2780 and OV2008 cells,in a dose-dependent manner [25].In addition,osthole also showed good anticancer activity against pancreatic cancer [26],epidermal carcinoma [27,28],melanoma [28],osteosarcoma [29],vulvar squamous cell carcinoma [30],multiple myeloma [31],head and neck squamous cell carcinoma [32],leukemic [33],and esophageal squamous cell carcinoma [34].

3 Molecular mechanisms of osthole

3.1 Inhibit tumor cell proliferation

Cell proliferation refers to the process of cell division and regeneration,which is one of the important characteristics of cell life.One characteristic that distinguishes tumor cells from normal cells is the unlimited proliferation of tumor cells.Therefore,inhibiting this unlimited proliferation is the key to the treatment of malignant tumors.

Xu et al.examined gastric cancer cell viability using MTT assay,and found that osthole inhibited the cell viability of HGC27 and SGC-7901 cells in a dosage-dependent manner [5].After treated with 20,40 or 80

μ

M osthole for 48 h,the HGC27 and SGC-7901 cells exhibited obvious morphological features,such as shrinkage and distortion;besides,cells treated with osthole became rounded,consistent with the MTT assay.Park et al.performed proliferation assay on BT-474 and McF-7 cells to study the effect of osthole on breast cancer cells,and found that osthole reduced cell proliferation in a dose-dependent manner [13].Osthole reduced proliferation of BT-474 cells by 51% (

P

＜ 0.01) at 50

μ

M and 59%(

P

＜ 0.001) at 100

μ

M.Proliferation of MCF-7 cells was reduced by 54% (

P

＜ 0.01) after 50

μ

M osthole treatment and 70% (

P

＜ 0.001) after 100

μ

M osthole treatment.However,osthole did not affect the cell viability of MCF-12A normal breast epithelial cells.Liu et al.determined through CCK8 assay that osthole could suppress the viability and proliferation of both ACHN and 786-O cells in a dose-dependent manner [19].A colony formation assay was then performed to examine the proliferation of RCC cells in the presence of osthole.Osthole also inhibited colony formation of both ACHN and 786-O cells in a dose-dependent manner.Che et al.performed a cell viability MTT assay to assess the anti-cervical cancer activity of osthole,and found that osthole reduced the viability of the four cervical cancer cell lines in a dose-dependent manner [22].In addition,the tumor cell colony formation assay showed that osthole treatment dose-dependently inhibited the growth of cervical cancer cells.Morphologically,osthole treatment also reduced cell-to-cell contact between HeLa and SiHa,and the osthole-treated tumor cells had more filopodia and cell layers.

3.2 Induce tumor cell cycle arrest

The proliferation cycle of tumor cells generally needs to go through four stages:G1-S-G2-M.When cell proliferation stops at a certain stage due to certain factors,apoptosis of cells can be induced.Studies have shown that osthole can act on a certain part of the cell cycle,thus inhibiting cell proliferation and inducing cell apoptosis.Cell cycle arrest at any stage can inhibit tumor cell proliferation and induce tumor cell apoptosis.

Yang et al.confirmed that the anti-proliferation effect of osthole might function through induction of cell cycle arrest (G2/M phase) and apoptosis in head and neck squamous cell carcinoma (HNSCC) [32].Zhu et al.demonstrated that the inhibitory effect of osthole against the growth of esophageal squamous cell carcinoma (ESCC) cells was associated with accumulation of cell cycle progression in G2/M phase [34].Wang et al.found that osthole induced cell cycle arrest in G1/S phase in osteosarcoma cells lines,MG-63 and SAOS-2,and caused the increase of cells in G1 phase and the decrease of cells in S phase [37].Wang et al.demonstrated that osthole could effectively inhibit the proliferation of MDA-MB 435 cells,and the G1 phase population of osthole-treated cells increased significantly in a dose-dependent manner [12].

3.3 Promote tumor cell apoptosis

Apoptosis is the spontaneous and orderly death of cells strictly controlled by multiple genes.Studies have found that apoptosis defects in the continuous proliferation of tumor cells can avoid death.Therefore,inducing apoptosis of tumor cells can help treat cancer and maintain homeostasis,and is one of the important mechanisms of various drugs to inhibit tumor growth.

The Bcl-2 family members,including both proand anti-apoptotic proteins,are generally known to be involved in the mitochondria-associated apoptotic pathway,which regulates the apoptotic process.Pro-apoptotic Bcl-2 family members,such as Bax and Bak,activate apoptosis.In contrast,the antiapoptotic members,Bcl-2 and Bcl-XL,restrain apoptosis.Increases in Bax/Bcl-2 may lead to release of cytochrome C and subsequent activation of Caspase3 and Caspase9,which induce apoptosis by degrading intracellular substrates,including PARP.Yang et al.examined the effect of osthole on apoptosis of HNSCC cells,and found that osthole could also down-regulate the level of apoptosisrelated proteins,such as Bcl-2,Survivin and PARP1,while up-regulating the expression of Bax,Cleaved Caspase3/9 and Cleaved PARP1 [32].Therefore,osthole might induce apoptosis in HNSCC cells through the mitochondria-associated apoptotic pathway.Zhu et al.studied the pro-apoptotic mechanism of osthole on esophageal squamous cell carcinoma (ESCC) cells,and found that osthole treatment elicited the up-regulation of Cleaved Bax,Cleaved PARP1,Cleaved Caspase3 and Cleaved Caspase9,and the downregulation of PARP1 and Survivin [34].The results thus suggested that osthole could induce apoptosis of ESCC cells through the mitochondria-associated apoptotic pathway.

PI3K/AKT pathway is involved in the growth of various kinds of tumors.AKT is the major downstream target of PI3K and the phosphorylation of AKT is considered to be active.Actived AKT regulates many cellular functions,including proliferation,cell growth,cell cycle,apoptosis and protein synthesis.PTEN is a key negative regulator of the PI3K/AKT pathway and exerts inhibitory effect on cell growth and proliferation.Yang et al.demonstrated that osthole treatment significantly up-regulated the PTEN protein level and downregulated the phosphorylated AKT and PI3K protein levels [32].Meanwhile,the expression of total AKT remained constant.Therefore,PI3K/AKT pathway was suppressed.It suggested that osthole might have therapeutic applications in patients with HNSCC.Zhu et al.showed that treatment of ESCC cells with osthole increased the expression of PTEN and reduced the expression of PI3K and phosphorylated AKT,while total level of AKT protein remained constant [34].Therefore,the up-regulation of PTEN which in turn inhibits the activity of the PI3K/AKT pathway might represent a novel molecular effect of osthole in ESCC.

STAT3,a member of STAT transcription factors,mediates many aspects of immunity,proliferation,apoptosis and differentiation.STAT3 is phosphorylated in a number of human cancer cell lines and primary tumors.Constitutive activation of STAT3 is a point of convergence for malignant transformation at several levels,including transformation,proliferation,invasion,and metastasis.Dai et al.found that osthole induced apoptosis and cell cycle arrest in TNBC cells by inhibiting STAT3 phosphorylation and nuclear translocation [14].This inhibitory activity was partly reversed by STAT3 overexpression.Owing to this inhibitory activity on STAT3 phosphorylation,osthole prevented the proliferation of TNBC implanted in mice and induced apoptosis.

Targeting p53,a tumor suppressor,is one of the promising strategies for anticancer therapy,which induces apoptosis by regulating the expression of downstream pro-apoptotic factors.Huang et al.found that osthole could induce the apoptosis of human colon cancer cell lines,HCT116 and SW480,and the expression of p53 was significantly upregulated [21].

3.4 Inhibit tumor cell migration and invasion

The migration and invasion of tumor cells is a complex biological process,which is realized through the interaction of various growth factors,cytokines,enzyme systems,signaling pathways and EMT.Clinically,most tumor patients die from invasive and metastatic diseases rather than primary tumors,so the ability to inhibit the migration and invasion of tumor cells is also the key to the treatment of malignant tumors.

Liu et al.demonstrated that pre-treatment with osthole attenuated the migratory and invasive abilities of renal cell carcinoma (RCC) cells in a dose-dependent manner,as evidenced by the reduction in migrating cells in the Transwell assay and the decrease of wound closure ratio in the scratch assay [19].In addition,osthole down-regulated the expression of migration/invasion-related proteins,MMP-2 and MMP-9.Osthole significantly upregulated epithelial biomarkers (E-cadherin and beta-catenin) and down-regulated mesenchymal biomarkers (N-cadherin and vimentin).Furthermore,the results suggested that osthole suppressed the expression of epithelial-mesenchymal transition transcription factors,Smad-3,Snail-1,and Twist-1.

Che et al.demonstrated that osthole treatment significantly reduced the migration and invasion of cervical cancer cell [22].At the gene level,osthole up-regulated epithelial markers (E-cadherin and β-catenin) and down-regulated mesenchymal markers (N-cadherin and vimentin),MMP-2 and MMP-9,which was consistent with the hypothesis that osthole inhibited the EMT of cervical cancer cells.

Feng et al.found that osthole inhibited TGFb1-induced EMT,adhesion,invasion and migration in A549 cells possibly by down-regulating NF-kBSnail pathways [9].This finding shed new insights into the anti-metastatic effect of osthole.The experiment conducted by Wang et al.showed that osthole significantly inhibited the cell viability of MG63 and SAOS-2 in a dose-and time-dependent manner [35].Cell cycle was arrested and the ability of migration and invasion was obviously reduced when cells were exposed to osthole.

3.5 Reverse tumor cell resistance

Multi-drug resistance (MDR) refers to the phenomenon that tumor cells not only have resistance after long-term exposure to a certain chemotherapy drug,but also have cross-resistance to other chemotherapy drugs with different structures and mechanisms of action.MDR is closely related to the recurrence and metastasis of malignant tumors,and is one of the main reasons for the failure of chemotherapy in a large number of tumor patients.

Wang et al.revealed for the first time that osthole was a novel and potent modulator and paved the way for reversing MDR in human myelogenous leukemia K562/ADM cells [36].At the same time,the potential connection of P-gp-mediated MDR reversal and PI3K/Akt signaling cascade was identified,which might be the major mechanism involved in the reversal of MDR by osthole in K562/ADM cells.Wang et al.demonstrated that osthole could reverse adriamycin (ADM) resistance of human bladder cancer cells

in vitro

at a non-toxic concentration [37].The mechanism might be the inhibition of P-gp activity of T24/ADM cells by osthole.

3.6 Enhance the effectiveness of commonly used chemotherapy drugs

Chemotherapy for malignant tumor is one of the most commonly used methods in tumor drug therapy.Seeking effective and low-toxic antitumor drug synergies with wide range of targets is currently one of the hot spots to improve the efficacy of chemotherapy.Jarzab et al.found that the combination of osthole and cisplatin augmented their anti-cancer activities and yielded an additive type of pharmacologic interaction by means of isobolographic analysis [38].Xu et al.showed that the combination of osthole and cisplatin resulted in greater efficacy in growth inhibition and apoptosis induction than single agent treatment [8].The mechanism was related to the regulation of Bcl-2 family expression,with the expression of Bax up-regulated and the expression of Bcl-2 downregulated.

3.7 Inhibit angiogenesis

Tumor angiogenesis,a common feature of all solid tumors,plays an important role in tumor growth and metastasis.Therefore,antiangiogenic tumor biotherapy has attracted much attention from oncologists in recent years.Yao et al.showed that osthole could attenuate angiogenesis to inhibit the growth of HCC [39].Hu et al.found that the effect of osthole on anti-angiogenesis

in vitro

was better than the effect of alcohol extracts from

Angelica Pubescens

,which indicated that the main antiangiogenic component of

Angelica Pubescens

was osthole [40].Osthole effectively suppresses angiogenesis

in vitro

,most likely by inhibiting cell proliferation,

migration and tube-formation,inducing apoptosis and suppressing cell cycle of human umbilical vein endothelial cells (HUVECs).

4 Conclusion

Based on the existing studies,this paper reviews the anti-tumor effects and possible mechanisms of osthole.Osthole mainly plays an anti-tumor role by inhibiting tumor cell proliferation,inducing tumor cell cycle arrest,promoting tumor cell apoptosis,inhibiting tumor cell migration and invasion,reversing tumor cell resistance,enhancing the effectiveness of commonly used chemotherapy drugs and inhibiting angiogenesis.Based on these possible mechanisms,further research is needed to obtain more effective anti-tumor targets and to clarify the anti-tumor mechanism of osthole.Osthole is natural,safe and economical,which requires us to further explore whether it can be used in adjuvant therapy of tumors.

Acknowledgements

This work was financially supported by Liaoning Province Science and Technology Administration (2019-ZD-0456),and funded by the Jiangsu Provincial Project of Invigorating Health Care through Science,Technology and Education(wk018-002).