A Novel Caffeoylquinic Acid from Lonicera japonica Exerts Cytotoxic Activity by Blocking the YAP-CTGF Signaling Pathway in Hepatocellular Carcinoma

We have purified a novel caffeoylquinic acid named 3,4-di-O-caffeoylquinic acid isobutyl ester from the flower buds of Lonicera japonica Thunb., Caprifoliaceae. However, the biological function of 3,4-di-O-caffeoylquinic acid isobutyl ester is still unknown. Here, we found that 3,4-di-O-caffeoylquinic acid isobutyl ester effectively inhibited the proliferation and migration of hepatocellular carcinoma cells, and it displayed less toxicity to a normal liver cell line. Mechanistic studies showed that 3,4-di-O-caffeoylquinic acid isobutyl ester diminished the expression of YAP at the mRNA level. Overexpression of YAP significantly rescued HepG2 cells from the 3,4-di-O-caffeoylquinic acid isobutyl ester–induced suppression of proliferation and migration. Furthermore, the YAP downstream target gene CTGF was significantly repressed upon 3,4-di-O-caffeoylquinic acid isobutyl ester treatment, which was ameliorated by YAP overexpression. In addition, 3,4-di-O-caffeoylquinic acid isobutyl ester decreased the expression of β-catenin as well as CDK4/6. Collectively, 3,4-di-O-caffeoylquinic acid isobutyl ester exerts antihepatocellular carcinoma activity by inhibiting the YAP-CTGF pathway which controls the proliferation and migration of hepatocellular carcinoma cells. The Wnt/β-catenin pathway might be another pathway by which 3,4-di-O-caffeoylquinic acid isobutyl ester exerts antihepatocellular carcinoma activity. As a novel natural compound, 3,4-di-O-caffeoylquinic acid isobutyl ester might be a promising agent for hepatocellular carcinoma therapy.


Introduction
Hepatocellular carcinoma (HCC), which accounts for the majority of primary liver cancers, is a malignant digestive system tumor (Singal et al. 2020). Hepatocellular carcinoma usually occurs in the context of oxidative stress and inflammation, caused by chronic hepatitis B or C virus (HBV or HCV), alcoholic and nonalcoholic steatohepatitis, and aflatoxin-mediated toxicity (Shariff et al. 2009;Petrick et al. 2020). Hepatocellular carcinoma has the sixth highest incidence rate (Villanueva 2019) and is the fourth largest cause of cancer-related death worldwide (Bray et al. 2018). At present, the first-line drugs sorafenib and lenvatinib as well as the second-line drug regorafenib 1 3 are being used for advanced HCC (Villanueva 2019). However, the survival benefit of sorafenib and regorafenib only obtained an additional 2-3 months of overall survival (Llovet et al. 2008) (Cheng et al. 2009). Lenvatinib was noninferior to sorafenib in overall survival in untreated advanced HCC (Kudo et al. 2018). In addition, many patients with liver cancer do not tolerate drug toxicity, and some patients may acquire adaptive drug resistance (Llovet et al. 2018;Yau et al. 2011). Therefore, there is an urgent need to develop less toxic and more effective potential drugs for HCC treatment.
The Hippo/YAP pathway, a cellular signal transduction pathway with high conservation, plays an important role in controlling organ size and tumorigenesis. Core components of the Hippo pathway include Mst1/2, Lats1/2, and yes-associated protein (YAP) which is the ultimate effector of the Hippo pathway. The phosphorylation of YAP at Ser127 leads to cytoplasmic retention of YAP and suppression of its transcriptional activity (Callus et al. 2006;Yu et al. 2012;Ma et al. 2019). YAP participates in signal transduction and regulates the transcription of genes including CYR61 and CTGF (Zhao et al. 2010;Wang et al. 2016). Increased expression of YAP has been detected in HCC (Cho et al. 2021). Moreover, high expression of YAP was positively correlated with the tumor volume of HCC (Wu et al. 2016). YAP is a potential therapeutic target in HCC development.
The Wnt/β-catenin pathway is a key molecular mechanism involved in embryonic development and tissue homeostasis (Krishnamurthy and Kurzrock 2018). Aberrant activation of the Wnt/β-catenin pathway is one of the important reasons for the occurrence and development of a variety of cancers (Zhang and Wang 2020). β-Catenin is the ultimate downstream effector of the canonical Wnt signaling pathway. Cytoplasmic retention and expression are tightly controlled by a multiprotein destruction complex composed of adenomatous polyposis coli (APC), AXIN, and glycogen synthase kinase-3β (GSK3β). This complex phosphorylates β-catenin at Ser37 and Thr41. The phosphorylation of β-catenin leads to its ubiquitination and proteasome-mediated degradation (Nong et al. 2021). β-Catenin regulates the expression of target oncogenes including c-MYC and CCND1 by combining with lymphoid enhancer/transcription factor (lef/tcf) (Katoh 2018). Aberrant β-catenin activation has been detected in HCC in both humans and rodents (Cassano et al. 2017). β-Catenin expression is inversely correlated with survival in HCC tumor tissues (Huo et al. 2021). Targeting β-catenin might be an effective strategy for anti-HCC therapy.
Chinese herbal medicines have long been applied in the clinic because of their advantages of low toxicity and targeting of multiple factors and pathways (Li et al. 2022). The flower bud of Lonicera japonica Thunb., Caprifoliaceae, is a traditional herbal medicine with various biological activities, including antihepatoma and anti-HBV activities (Ge et al. 2019). However, the underlying mechanism remains to be investigated. In our previous study, we purified a novel caffeoylquinic acid named 3,4-di-O-caffeoylquinic acid isobutyl ester (3,4-CQIE) from L. japonica flower buds. 3,4-CQIE (1) is a pale-yellow amorphous powder. The formula of 3,4-CQIE is C 29 H 31 O 12 and its molecular weight is 571.08. Preliminary studies showed that it had potential anti-HCC activity. However, its molecular mechanisms need to be further explored. In the present study, we further investigated the anti-HCC activity of 3,4-CQIE and the underlying molecular mechanism.

Isolation of 3,4-CQIE
3,4-CQIE (1) was isolated from L. japonica flower buds, which were collected in May 2017 from Pingyi County, Linyi City, Shandong Province. The plants were identified by Dr. XB Zeng of Shenzhen People's Hospital, and a voucher specimen (No. 20170930) was deposited at the Center Lab of Longhua Branch, Shenzhen People's Hospital, Second Clinical Medical College of Jinan University, Shenzhen, China. The extraction and isolation procedure of 3,4-CQIE was described in detail in our previous patent (application number CN202111148950.5).

HPLC of 3,4-CQIE
High-performance liquid chromatography (HPLC) analysis was carried out on an Essentia LC-16 HPLC system (Shimadzu, Tokyo, Japan) equipped with a diode-array detector. All separations were performed using a YMC-Pack ODS-A column (4.6 mm × 250 mm, 5 μm) with a flow rate of 1 ml/min. The mobile phase was eluted with 50% methanol in water. The injection volume of the test sample was set at 10 μl each time, and the UV spectra were recorded at 330 nm. The isolation of 3,4-CQIE in this study has been patented in China (application number CN202111148950.5).

Wound-Healing Assay
The first step in the scratch wound-healing assay was to place the culture insert into 24-well plates, and 70 μl suspension, including 1 × 10 5 cells, was placed in the culture insert for 12 h to form a confluent monolayer. The culture inserts were then removed.

3
The cells were washed once with PBS to remove cell debris and cultured with DMEM containing 5% serum with 3,4-CQIE (0, 100, and 120 μM) for HepG2 cells, or DMEM without serum with 3,4-CQIE (0, 100, and 120 μM) for Huh7 cells. The migration distances of the cells were observed under a light microscope when the cells were cultured at 37 °C for 0, 24, and 48 h.

RT-qPCR
Total RNA was extracted using an RNA Rapid extraction kit (Aidlab, RN07) according to the manufacturer's protocol. cDNA was prepared with a reverse transcription kit (Vazyme, R211-02) using a Thermal Cycler (Bio-Rad Laboratories). The following cycling parameters were used: 1 cycle of 25 °C for 5 min, 50 °C for 15 min, and 85 °C for 2 min. RT-qPCR was performed using the One-Step SYBR PrimeScript Plus RT-PCR kit (Vazyme, Q711-02-AA). PCR primers were custom synthesized by Guangzhou IGE Technology (Guangzhou, China). Briefly, the primers, qPCR mix, and cDNA templates were mixed for the PCRs. RT-qPCR analysis was performed in a LightCycler® 480 Real-Time PCR System (Roche, Switzerland). The relative mRNA expression levels of the target genes were standardized with the housekeeping gene β-actin, the value of which was set as 100%. All of the reactions were run in triplicate, and the data were analyzed according to the comparative Ct (2-ddCt) method. All primers used in this study are listed in Table 1.

Immunofluorescence Assay
Cells were grown on coverslips. After the cells were treated with 3,4-CQIE, the cell culture was discarded, and the coverslips were fixed with 4% paraformaldehyde at room temperature to fix for 10 min and then washed with phosphate-buffered saline three times. The coverslips were then incubated with staining buffer (1% BSA and 0.1% Triton in PBS) for 10 min and washed with PBS three times. The coverslips were incubated with primary antibodies for YAP (1:100) and CTGF (1:100), and incubated with secondary antibodies for Alexa Fluor 594 (1:1000) or Alexa Fluor 488(1:1000) in the dark for 1 h. Finally, the coverslips were stained with 0.5 μg/ ml DAPI for 10 min and visualized using a Leica TCS SP8 confocal microscope (Leica, Germany).

Transient Transfection
For transient transfection, HepG2 cells were seeded in six-well plates or twenty-well plates. The GFP-YAP and control plasmids were transfected into a HepG2 cell using Lipofectamine® 3000 Transfection Kit reagent following the manufacturer's protocol. Western blotting assays were performed to detect transfection efficiency after 48 h. The transfected cells were then used for CCK8 and a scratch wound-healing assay after transfection for 24 h.

Statistical Analysis
The experimental data are expressed as mean ± standard deviation (x ± s). Comparisons between treatments were  TCT TCC TGA TGG ATG GGA AC  TTG GGT CTA GCC AAG AGG TG  CTGF  CTT GTC TGA TCG TTC AAA GC  CAA TCT GTT TTG ACG GAC TG  CDK4  ACA GTT CGT GAG GTG GCT TT  TAC CTT GAT CTC CCG GTC AG  CDK6  GAG GCA CCT GGA GAC CTT C  TGG TTT CTC TGT CTG TTC GTG  CCND1 TGG TGA ACA AGC TCA AGT GG CTC TGG CAT TTT GGA GAG GA β-Catenin GGA AGA GTC CGG AGG AGA TG CTG GCT GTC AGG TTT GAT CC performed using ANOVA, followed by Tukey's test. Statistical significance was set at p < 0.05, and software such as Graphpad, Adobe Photoshop, and ImageJ is used for graphing.

Inhibition of Migration
Increased migratory ability is one of the most prominent features of HCC cells (Weiler et al. 2020). To determine whether 3,4-CQIE affects cell migration, we conducted a wound-healing assay and discovered that the gaps between the cells were larger with 3,4-CQIE treatment than in the control group ( Fig. 2A-D). Altogether, these data imply that 3,4-CQIE suppresses HCC cell migration.

Inhibition of YAP
To further explore the molecular mechanism of 3,4-CQIE against HCC, we examined the expression of key proteins of the Hippo pathway, including YAP, TAZ, and CTGF. The results showed that YAP and CTGF showed a downwards trend with increasing 3,4-CQIE concentrations (Fig. 3A, B). Therefore, we further carried out an immunofluorescence assay to explore the nuclear localization of YAP upon 3,4-CQIE treatment. The results showed an overall decrease in YAP staining intensity in 3,4-CQIE (120 μM)-treated HepG2 cells (Fig. 3C). In addition, the RT-PCR experiment showed that the mRNA level of YAP was decreased after 3,4-CQIE treatment (Fig. 3D). Moreover, the proteasome inhibitor MG132 could not rescue HCC cells from 3,4-CQIE-induced YAP degradation (Fig. 3E), indicating that YAP might be downregulated by 3,4-CQIE treatment at the mRNA level.

Overexpression of YAP
To further confirm the effect of YAP in HCC cells and elucidate how 3,4-CQIE regulates the proliferation and migration of HCC cells, we performed overexpression experiments by transfecting HepG2 cells with GFP-YAP plasmid and treating them with 3,4-CQIE. We first performed a western blot assay to confirm that cells transfected with the YAP plasmid had stronger expression of YAP than cells transfected with an empty vector (Fig. 4A). To detect the effect of YAP on 3,4-CQIEinduced proliferation arrest, we performed a CCK8 assay. The results showed that overexpression of YAP rescued the 3,4-CQIE-induced decrease in cell viability in HepG2 cells (Fig. 4B). The results suggested that YAP was involved in the anti-growth effect of 3,4-CQIE. Subsequently, wound-healing assays were conducted to assess the role of YAP in 3,4-CQIE-induced migration suppression. The results demonstrated that YAP overexpression significantly reduced the 3,4-CQIE-induced suppression of migration in HepG2 cells (Fig. 4C, D). Collectively, YAP plays a critical role in cell proliferation and migration in response to 3,4-CQIE. Fig. 1 3,4-CQIE (1) inhibits HCC cell growth. A and E HepG2 cells were exposed to 3,4-CQIE at the indicated doses for 24 or 48 h, and the dose-escalation effects of 3,4-CQIE were assessed by CCK8 assay. B and F Huh7 cells were exposed to 3,4-CQIE at the indicated doses for 24 or 48 h, and the dose-escalation effects of 3,4-CQIE were assessed by CCK8 assay. C and G THLE-3 cells were exposed to 3,4-CQIE at the indicated doses for 24 or 48 h, and the dose-escalation effects of 3,4-CQIE were assessed by CCK8 assay. D and H The inhibitory intensities of 3,4-CQIE were expressed as IC 50 for 24 and 48 h. I and M HepG2 cells were exposed to lenvatinib at the indicated doses for 24 or 48 h, and the dose-escalation effects of lenvatinib were assessed by CCK8 assay. J and N Huh7 cells were exposed to lenvatinib at the indicated doses for 24 or 48 h, and the dose-escalation effects of lenvatinib were assessed by CCK8 assay. K and O THLE-3 cells were exposed to lenvatinib at the indicated doses for 24 or 48 h, and the dose-escalation effects of lenvatinib were assessed by CCK8 assay. L and P The inhibitory intensities of lenvatinib were expressed as IC 50 for 24 and 48 h. Data are representative of three experiments with similar results. means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group

Inhibition of the YAP/CTGF Pathway
Acting as a transcription factor, YAP increases the expression of the target oncogene CTGF. Since 3,4-CQIE can significantly inhibit the expression of YAP, we wondered whether 3,4-CQIE might also regulate CTGF. RT-PCR was performed, and the results showed that 3,4-CQIE treatment significantly inhibited the mRNA levels of CTGF (Fig. 5A). Immunofluorescence assays then showed that YAP overexpression rescued the 3,4-CQIE-induced CTGF decrease (Fig. 5B). The results suggested that 3,4-CQIE repressed the expression of CTGF by inhibiting the expression of YAP.

Inhibition of β-Catenin
YAP overexpression failed to fully restore the 3,4-CQIEinduced decrease in cell viability, suggesting that the anti-HCC effect of 3,4-CQIE may have other mechanisms. The Wnt pathway plays an important role in HCC progression. β-Catenin is the core component of the Wnt pathway. CCND1 is the downstream target of β-catenin. CCND1 has been reported to activate cyclin-dependent kinases (CDK4/6), leading to phosphorylation of retinoblastoma (Rb) and resulting in promotion of cell cycle progression (Weinberg 1995). Here, we explored whether 3,4-CQIE treatment could affect the Wnt pathway. The results showed that 3,4-CQIE markedly reduced the expression of β-catenin and its downstream target gene CCND1 as well as CDK4/6 in a concentration-dependent manner ( Fig. 6A-F). Moreover, 3,4-CQIE repressed the mRNA levels of β-catenin, CCND1, and CDK4/6 ( Fig. 6G-J) in a concentrationdependent manner. Collectively, another way for 3,4-CQIE to exert its anti-HCC effect may be dependent on inhibiting β-catenin expression as well as cell cycle-related proteins.

Discussion
Accumulating evidence indicates that the overexpression of YAP plays a critical role in the proliferation and migration of HCC cells (Cho et al. 2021). Therefore, we examined the mRNA and protein levels of YAP in 3,4-CQIE-treated HepG2 cells and found that YAP expression was downregulated by 3,4-CQIE treatment at the mRNA level but not at the protein level. We also found that 3,4-CQIE decreased the transcription of CTGF, which has been reported as a YAP downstream target gene. In our study, YAP overexpression restored the 3,4-CQIE-induced repression of CTGF. CTGF has been reported to promote the proliferation and migration of HCC cells. These results suggest that 3,4-CQIE might exert anti-HCC activity by inhibiting the YAP-CTGF pathway. A HepG2 cells were exposed to 3,4-CQIE at the indicated doses for 48 h, and cell lysates were immunoblotted with the indicated antibodies. β-Actin was used as an internal control in western blotting. B ImageJ statistical analysis was performed simultaneously. C HepG2 cells were exposed to the indicated dose of 3,4-CQIE for 48 h, and the expression of YAP was examined by confocal microscopy. The scale bars represent 20 µm.  Means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group YAP overexpression could not completely restore the 3,4-CQIE-induced decrease in cell viability, indicating that the anti-HCC effect of 3,4-CQIE could also be exerted indirectly through other proteins. The Wnt/β-catenin pathway plays an important role in HCC progression (Perugorria et al. 2019). In addition, there are β-catenin-acquired mutations in approximately 20 to 40% of HCC patients, and FDA-approved drugs targeting β-catenin have not yet been used in the clinic (Rao et al. 2017). In the present study, we found that 3,4-CQIE could induce β-catenin degradation and repress the expression of CCND1, which has been reported as one of the β-catenin downstream target genes. Collectively, on the one hand, 3,4-CQIE suppressed the proliferation and migration of HCC cells by inhibiting the Hippo/YAP pathway; on the other hand, 3,4-CQIE might induce cell cycle arrest in HCC cells by inhibiting the Wnt/β-catenin pathway. These results suggested that both the Hippo/YAP and Wnt/βcatenin pathways were most likely involved in the 3,4-CQIEinduced suppression of HCC cells and implied that 3,4-CQIE might be a promising agent for HCC therapy.

Conclusion
3,4-CQIE is a novel natural caffeoylquinic acid purified from L. japonica flower buds. In this study, we found that 3,4-CQIE could significantly inhibit the viability and migration of HCC cells but had much less effect on normal liver cells. Further research revealed that 3,4-CQIE treatment led to a decrease in YAP and its target gene CTGF. In addition, 3,4-CQIE decreased the expression of β-catenin protein and its downstream gene CCND1, which promoted cell cycle progression. Collectively, we identified that a caffeoylquinic acid, 3,4-CQIE, suppressed HCC cell growth in vitro by inhibiting YAP and β-Catenin mRNA expression. Notably, 3,4-CQIE might be valuable as a potential drug for HCC therapy.

Patents
The isolation of 3,4-CQIE in this paper has been patented in China (application number CN202111148950.5.).