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\begin{document}

\title{Flavokawain A suppresses the vasculogenic mimicry of HCC by inhibiting
CXCL12 mediated EMT}



\author[1]{Ting Xiao}%
\author[1]{Jiali Bao}%
\author[1]{Rong Lin}%
\author[1]{Jiao Tian}%
\author[1]{Zihui Zhang}%
\author[1]{Yuxin Zhu}%
\author[1]{Yiming He}%
\author[1]{Dandi Gao}%
\author[1]{Ronghao Sun}%
\author[2]{Fubo Zhang}%
\author[1]{Yexin Cheng}%
\author[1]{Jiadelati Shaletanati}%
\author[1]{Honggang Zhou}%
\author[1]{Chunfeng Xie}%
\author[1]{Cheng Yang}%
\affil[1]{Nankai University}%
\affil[2]{Tianjin First Central Hospital}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Background and Purpose Hepatocellular carcinoma has high ability of
vascular invasion and metastasis. Vasculogenic mimicry (VM) is closely
related to the metastasis and recurrence of hepatocellular carcinoma
(HCC). According to previous research, Chloranthus henryi has anti-tumor
effect, but its molecular mechanism in the treatment of HCC has not yet
been stated. In our study, we aimed to investigate the effect of the
extract of Chloranthus henryi in HCC and its target and molecular
mechanism. Experimental Approach In this study, we isolated a chalcone
compound from Chloranthus henryi, compound 4, identified as flavokawain
A (FKA). We determined the anti-HCC effect of FKA by MTT and identified
the target of FKA by molecular docking and CETSA. Hepatoma cells
proliferation, migration, invasion, and VM formation were examined using
EDU, wound healing, transwell, vasculogenic mimicry, and IF. WB, RT-PCR,
and cell transfection were used to explore the mechanism of FKA on
hepatoma cells. Tissue section staining is mainly used to demonstrate
the effect of FKA on HCC in vivo. Key Results We confirmed that FKA can
directly interact with CXCL12 and HCC proliferation, migration,
invasion, and VM formation were all inhibited through reversing the EMT
progress in vitro and in vivo through the PI3K/Akt/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling
pathway. Additionally, by overexpressing and knocking down CXCL12, we
got the same results. Conclusion and Implications FKA attenuated
proliferation, invasion and metastatic and reversed EMT in HCC via
PI3K/Akt/HIF-1\selectlanguage{greek}α/\selectlanguage{english}NF-\selectlanguage{greek}κ\selectlanguage{english}B/Twist1 pathway by targeting CXCL12. This study
proposed that FKA may be a candidate drug and prospective strategy for
HCC therapy.%
\end{abstract}%



\sloppy


\textbf{Flavokawain A suppresses the vasculogenic mimicry of HCC by
inhibiting CXCL12 mediated EMT}

\textbf{Running title: FKA inhibits VM of HCC by targeting CXCL12}

Ting Xiao \textsuperscript{1\#}, Jiali Bao \textsuperscript{1,2\#}, Rong
Lin\textsuperscript{1\#}, Jiao Tian\textsuperscript{1}, Zihui
Zhang\textsuperscript{1}, Yuxin Zhu\textsuperscript{1,2}, Yiming
He\textsuperscript{1,2}, Dandi Gao\textsuperscript{1,2,} Ronghao
Sun\textsuperscript{1}, Fubo Zhang\textsuperscript{3}, Yexin
Cheng\textsuperscript{1}, Jiadelati\selectlanguage{ngerman}·Shaletanati \textsuperscript{1},
Honggang Zhou\textsuperscript{1,2*}, Chunfeng Xie\textsuperscript{1*},
Cheng Yang\textsuperscript{1,2*}

1 State Key Laboratory of Medicinal Biology, College of Pharmacy and
Tianjin Key Laboratory of Molecular Drug Research, Nankai University,
Tianjin 300353, China

2 Tianjin Key Laboratory of Molecular Drug Research, Tianjin
International Joint Academy of Biomedicine, Tianjin 300457, China

3 Organ Transplantation Center, Tianjin First Central Hospital, Tianjin
300192, China

\# These authors contributed equally to this work.

\textbf{Words:} 5128

\textbf{Acknowledgments and funding}

This study was supported by the Shenzhen Science and Technology Program
{[}Grant JCYJ20210324122006017{]}, the Fundamental Research Funds for
the Central Universities, Nankai University {[}Grant 735-63221434{]},
Tianjin Science and Technology Project {[}Grant 20JCYBJC00600{]}, and
the Foundation of Organ Fibrosis Druggability Joint Research Centre of
Nankai and Guokaixingcheng {[}Grant 735-F1040051{]}. This study also
supported by the 111 Project B20016.

\textbf{Conflict of interest statement}

The authors declare no competing financial interests.

* Corresponding author: Cheng Yang,
\textbf{email:}\emph{cheng.yang@nankai.edu.cn}

Chunfeng Xie, \textbf{email:}\emph{xiechunfeng@nankai.edu.cn}Honggang
Zhou, \textbf{email:} \emph{honggang.zhou@nankai.edu.cn}

\# Coauthors: Ting Xiao, \textbf{e-mail:} \emph{tingxiao@nankai.edu.cn}

Jiali Bao, \textbf{e-mail:} \emph{baojiali20@163.com}

Rong Lin, \textbf{e-mail:} \emph{linrong0827@163.com}

\textbf{Abstract}

\textbf{Background and Purpose}

Hepatocellular carcinoma has high ability of vascular invasion and
metastasis. Vasculogenic mimicry (VM) is closely related to the
metastasis and recurrence of hepatocellular carcinoma (HCC). According
to previous research, \emph{Chloranthus henryi} has anti-tumor effect,
but its molecular mechanism in the treatment of HCC has not yet been
stated. In our study, we aimed to investigate the effect of the extract
of \emph{Chloranthus henryi} in HCC and its target and molecular
mechanism.

\textbf{Experimental Approach}

In this study, we isolated a chalcone compound from \emph{Chloranthus
henryi} , compound \textbf{4} , identified as flavokawain A (FKA). We
determined the anti-HCC effect of FKA by MTT and identified the target
of FKA by molecular docking and CETSA. Hepatoma cells proliferation,
migration, invasion, and VM formation were examined using EDU, wound
healing, transwell, vasculogenic mimicry, and IF. WB, RT-PCR, and cell
transfection were used to explore the mechanism of FKA on hepatoma
cells. Tissue section staining is mainly used to demonstrate the effect
of FKA on HCC in vivo.

\textbf{Key Results}

We confirmed that FKA can directly interact with CXCL12 and HCC
proliferation, migration, invasion, and VM formation were all inhibited
through reversing the EMT progress in vitro and in vivo through the
PI3K/Akt/NF-\selectlanguage{greek}κ\selectlanguage{english}B signaling pathway. Additionally, by overexpressing and
knocking down CXCL12, we got the same results.

\textbf{Conclusion and Implications}

FKA attenuated proliferation, invasion and metastatic and reversed EMT
in HCC via PI3K/Akt/HIF-1\selectlanguage{greek}α/\selectlanguage{english}NF-\selectlanguage{greek}κ\selectlanguage{english}B/Twist1 pathway by targeting CXCL12.
This study proposed that FKA may be a candidate drug and prospective
strategy for HCC therapy.

\textbf{Graphical abstract:}

We summarized our findings, FKA inactivated PI3K/Akt/HIF-1\selectlanguage{greek}α \selectlanguage{english}pathway by
targeting CXCL12. Then, inhibiting the expression of Twist1 in the
nucleus, thereby suppressing the invasion, metastasis, VM and EMT of HCC
cells. Meanwhile, by blocking the excitation of NF-\selectlanguage{greek}κ\selectlanguage{english}B, the proliferation
of HCC cells was affected.

\textbf{Key words:} HCC; vasculogenic mimicry; EMT; CXCL12; Flavokawain
A

\textbf{Abbreviations:}

HCC: hepatocellular carcinoma; VM: vasculogenic mimicry; HBV: hepatitis
B virus; HCV: hepatitis C virus; CXCL12: stromal cell-derived factor-1;
CXCR4: CXC chemokine receptor 4; PI3K: phosphoinositide -3 kinase; Akt:
protein kinase B; HIF-1\selectlanguage{greek}α: \selectlanguage{english}hypoxia-inducible factor-1alpha; EMT:
epithelial-mesenchymal transition; FKA: flavokawain A; NF-\selectlanguage{greek}κ\selectlanguage{english}B: nuclear
transcription factor-kappa B; GO: Gene Ontology; KEGG: Kyoto
Encyclopedia of Genes and Genomes; PAS: periodic acid-Schiff; MTT:
methylthiazolyldiphenyl-tetrazolium bromide; IHC: immunehistochemistry.

\textbf{1. Introduction}

Hepatocellular carcinoma (HCC) is one of the most ordinary malignant
tumors in global, its incidence ranks among the top five in the world,
and is the principal cause of death in cancer (Sung et al., 2021; Yang
\& Heimbach, 2020). The tumorigenesis of HCC is involved multiple
etiological factors, including chronic virus infection (''Viral
Analysis: Early Detection of HCC,'' 2020), alcoholism (Pearson et al.,
2021), non-alcoholic steatohepatitis (NASH) (Pfister et al., 2021),
drug-induced liver injury and aflatoxin exposure (Llovet et al., 2021;
Zhu et al., 2021). Despite great break throughs has been made in cure,
HCC still has a low response rate to curative treatment, due to the
resistance to chemotherapy drugs and high recurrence rate (50\%-70\%)
(Zhang, Zhang, \& Chen, 2021). Therefore, it is particularly important
to investigate the etiopathogenesis of HCC and explore more effective
therapeutic target proteins.

As a prerennial herb, \emph{Chloranthus henryi} has been used in China
to improve blood circulation, eliminate swelling and pain (Yun, Nho,
Park, \& Yang, 2021). \emph{C.henryi} contained a variety of
sesquiterpenoids, diterpenoids, and chalcone chemicals that were
neuroprotective (Chen et al., 2020; Chen et al., 2021),
anti-inflammatory(Luo et al., 2016; Pan et al., 2017), antitumor(W. Wang
et al., 2014), and tyrosinase inhibitory effects(Lee et al.,
2009).\emph{C. henryi} whole herb as part of research on bioactive
constituents of medicinal plants or folk medicines whose chemical
constituents were used in investigated. One bran-new eudesmanolide
(\textbf{1} ) and five known compounds (\textbf{2} -\textbf{6} ) were
extracted and separated from \emph{C. henryi} . The anti-tumor screening
results showed that one chalcone derivative flavokawain A (FKA,
compound\textbf{4} ), exhibited the best inhibit effect on the activity
and migration of HCC cells. The role of FKA in HCC is still original,
and its mechanism and target remain unclear.

High expression of mesenchymal markers in patients with liver cancer
with worse prognosis. EMT is frequently participated in a series of
pathological processes of HCC progression, including invasion,
metastasis and vasculogenic mimicry (Jin et al., 2019; M. L. Wang et
al., 2017). Previous researches have shown that HIF-1\selectlanguage{greek}α\selectlanguage{english}is a strong
inducer of EMT by regulating the expression of Snail1 (Giannelli,
Koudelkova, Dituri, \& Mikulits, 2016), modulate the EMT through Twist1,
a regulator of the EMT (Y. Liu et al., 2014). However, it is not clear
whether FKA inhibits cell migration by inhibiting EMT in HCC. Our study
was designed to find the target of FKA and described its mechanism.

We identified that FKA can inhibit the PI3K/Akt/HIF-1\selectlanguage{greek}α/\selectlanguage{english}Twist1 pathway to
attenuate HCC cells migration, invasion, VM formation and EMT
progression by targeting CXCL12. And it can also influence proliferation
and apoptosis of HCC cells by stimulating NF-\selectlanguage{greek}κ\selectlanguage{english}B activation. Thus, we
provide new targets and new ideas for the study of HCC.

\textbf{2. Materials and methods}

\textbf{2.1 Plant material}

We collected \emph{C. henryi} whole plantfrom Jiuling Mountain, Jiangxi
province, China, in September 2018. Part of authors (C. X.) spread the
botanical identification, and a certificate specimen (No. 201809CH) was
reserved at College of Pharmacy, Nankai University, China.

\textbf{2.2 Cell lines and cell culture}

Tumor cellsSMMC-7721, huh7, PANC-1, HepG2, Hela and Hep3b were stored in
our laboratory and maintained in DMEM (Solarbio, Beijing, China) medium.
A549 cells were cultivated in RPMI-1640 medium. And 10\% (v/v) fetal
calf serum (Procell South America) was contained in all medium. All
cells were grown under standard conditions (37\selectlanguage{ngerman}°C, 95\% air atmosphere,
5\% CO\textsubscript{2}).

\textbf{2.3 Cell viability assay}

SMMC-7721 and HepG2 and other cells were centrifuged and grown in
96-well plates at a density of 5\selectlanguage{english} x\selectlanguage{english} 10\textsuperscript{5} cells/mL for 24
h, incubation temperature was 37\selectlanguage{english}, and the
CO\textsubscript{2}concentration was 5\%. Then, culture media with drugs
(10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M, FKA and 30 \selectlanguage{greek}μ\selectlanguage{english}M LIT-927) and without were added
as control. HepG2, SMMC-7721, Hela, PANC-1 and A549 were treated with
six extracts and cisplatin. After acting for 48 h, MTT that each well at
an ultimate concentration of 5 mg/mL was used to detect cell viability
at 37 for 4 h. Dissolved dark blue formazan crystals with dimethyl
sulfoxide at room temperature and measured the absorbance at 570 nm by a
microplate reader (Mutiskan FC, Thermo, USA). GraphPad Prism 9.0
software is used to calculate results. The experimental results were
repeated more than three times.

\textbf{2.4 Wound healing assay}

SMMC-7721, huh7 and HepG2 cells were centrifuged efficiently and then
spread into a 24-well plate. After cells reaching 100\% confluence, a
sterile pipette tips were used to make cell scratch wounds. Cells
cultured in a serum-free medium containing FKA or LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M) for 0
h, 24 h and 48 h. All experiments were performed with triplicate
samples. Images were collected under microscope (Nikon, Japan). Wound
migration distance was measured using Image J (NIH USA).

\textbf{2.5 Cellular thermal shift assay (CETSA)}

HepG2 cells that had converged to about 80 \% (10cm petri dish) were
treated with PBS or FKA (40 \selectlanguage{greek}μ\selectlanguage{english}g/mL) for four hours. Trypsin digested and
centrifuged at 300xg to collect cells. Wash with PBS or PBS (40 \selectlanguage{greek}μ\selectlanguage{english}g/mL
FKA), collect by centrifugation and resuspend. According to each tube
100 \selectlanguage{greek}μ\selectlanguage{english}L cell suspension is divided into RNA-free tube. Placed at
different temperatures (42, 44, 46, 49, 51, 54, 60 ) for 5min, back to
room temperature, frozen and thawed three times with liquid nitrogen,
and finally centrifuged at 20,000g for 20min at 4 \selectlanguage{ngerman}°C to collect the
supernatant. And Western blot analysis.

\textbf{2.6 IP-One HTRF® assay}

HepG2 cells were cultured in a 10 cm diameter dish for 24 h, then
collected and counted. According to the instructions of the IP-one Gq
kit (cisbio, Shanghai, China), HepG2 cells are laid in white 96-shallow
well microplates with 5 × 10\textsuperscript{4} cells / well. First,1×
Stimulation Buffer as blank control, the remaining wells were added 10
ng/mL CXCL12 protein (sino biological, Pennsylvania, USA). At the same
time, adding FKA (final drug concentration was 10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M)
and LIT-927 (final drug concentration was 30 \selectlanguage{greek}μ\selectlanguage{english}M) and culture in
incubator for 1.5 h. Then,3 \selectlanguage{greek}μ\selectlanguage{english}L of premixed fluorescent donor
(anti-IP1-Cryptate) and fluorescent acceptor (d2-labeled IP1) in lysis
buffer were added. Following an additional 1 h at 25, the white plate
was scanned by enzyme-labeled instrument. Calculate the ratio (Ratio =
Signal 665 nm/Signal 620 nm\selectlanguage{ngerman}×10\textsuperscript{4} ) of the acceptor and
donor emission signals for each individual well. Data were analyzed in
GraphPad Prism 9.0.

\textbf{2.7 RNA extraction and quantitative quantitative real-time PCR
(RT-PCR)}

By using Trizol (Solarbio) reagent to extract total RNA from SMMC-7721
and HepG2 cells, the concentration of total RNA was determined by a
microspectrophotometer (Thermo-nanodrop one, Amarica). The fastking gDNA
Dispelling RT Super Mix (TIANGEN, Beijing, China) was used to reversely
transcribe for synthesizing cDNA. Then, quantitive PCR was performed
depending unicon qPCR SYBR Green Master Mix (Yeason, Shanghai, China).

\textbf{2.8 Western blotting (WB) analysis}

The cells (SMMC-7721 and HepG2) were disclosed with FKA (10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M
and 40 \selectlanguage{greek}μ\selectlanguage{english}M) and LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M) for 24 h. Cells were lysed by RIPA
buffer, and protein were collected and their concentrations were
measured through a BCA kit (Solarbio, Beijing, China). Equivalent
protein samples were separated by 8\%-12\% gels and transferred onto
polyvinylidene fluoride membranes (Merck, Darmstadt, Germany). After
blocked in 5\% nonfat milk for 1 h, the membranes were incubated with
corresponding primary antibodies (1:1000) overnight in 4 and secondary
antibodies (1:10000) 2 h in atmospheric temperature. Then
immunoreactions were detected with ECL reagent (Affinity).
Quantification of band intensities was analyzed with ImageJ. The
antibodies used include: CXCL12 (cat.AF5166), E-cadherin (cat.AF0131),
VE-cadherin (cat.DF7514), Vimentin (cat.AF7013), Snail1 (cat.AF6032),
Akt (cat.AF6261), p-Akt (cat.AF0016), PI3K (cat.AF6241), p-PI3K
(cat.AF3241), Twist1 (cat.AF4009), NF-\selectlanguage{greek}κ\selectlanguage{english}B (cat.AF5006), HIF-1\selectlanguage{greek}α
\selectlanguage{english}(cat.AF1009), GAPDH (cat.AF7021).

\textbf{2.9 EDU(5-Bromo-2-Deoxyuridine) detection of cell proliferation}

EDU can be inserted into a DNA molecule being replicated during cell
proliferation, which is used to mark the increment number of SMMC-7721
and HepG2 cells. Cells were cultured for 24 h in petri dish with FKA (10
\selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M) and LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M). Adding with EDU (Solarbio,
Beijing, China) working solution, cells in each group were fixed, and
incubated in a dark place for 30 min with Click Additive Solution. Laser
scanning confocal microscope (SP800) was used to observe the staining.

\textbf{2.10 Transwell assay}

A 24-well transwell plate, carrying 8.0-\selectlanguage{greek}μ\selectlanguage{english}m pore membranes, was used to
test cell invasive ability. Mix the Matrigel with DMEM medium in 1: 1
and add to the upper chamber at 37\selectlanguage{ngerman}°C for 30 min. The matrigel and
membrane were hydrated with 0.1\% FBS medium for 30min. And then, cells
(5×10\textsuperscript{4}/ well) were collected and suspended in the
0.1\% FBS medium, and adding complete medium (500 \selectlanguage{greek}μ\selectlanguage{english}L/well) to the lower
chamber as an attractant. After cultured for 24 h at 37\selectlanguage{ngerman}°C, the cotton
swabs were used to remove cells that remaining in the upper-chamber. And
fixed 15 min with 4\% formalin. Next, stained with the crystal violet
solution. Capture invasive cells through inverted fluorescence
microscope.

\textbf{2.11 Vasculogenic mimicry assay}

50 \selectlanguage{greek}μ\selectlanguage{english}L of precooled matrigel (ABW\selectlanguage{ngerman}\textsuperscript{®} Matrigengel,
Shanghai, China) and FBS-free medium (1:1) was moved into each well of a
96-well plate and converged for 20 min at 37°C. Each well was added in
SMMC-7721 and HepG2 (1×10\textsuperscript{4}) in 100 \selectlanguage{greek}μ\selectlanguage{english}L of medicated
medium and incubated for 24 h at 37\selectlanguage{ngerman}°C, 5\%CO\textsubscript{2}.The tube
counts were recorded through an inverted microscope. Each condition was
assessed in triplicate.

\textbf{2.12 Flow cytometry analysis}

Cells were seeded in 6-well plated and exposed to the FKA (10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M
and 40 \selectlanguage{greek}μ\selectlanguage{english}M) and LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M). Then collected and stained with Annexin
V-FITC and propidium iodide successively. A flow cytometer (FAC Scan; BD
Biosciences) was used to gather the signals. The results were analyzed
with Flow jo 7.6.1.

\textbf{2.13 Immunofluorescence (IF) assay}

Collected cells and grown on glass slides in 24-well plates for 24 h.
After co-culture with FKA (10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M) and LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M),
After fixed by 4\% paraformaldehyde, cells were permeabilized using
0.2\% Triton X-100 and -interrupted to prevent nonspecific antibody
using 5\% bovine serum albumin. Then, the slides were covered overnight
at 4\selectlanguage{ngerman}°C by the primary antibodies E-cadherin (cat.BF0219,1:100),
VE-cadherin (cat.DF7514,1:100), Vimentin (cat.BF8006,1:100), Snail1
(cat.AF6032,1:100). Cells were incubated with TRITC-conjugated or
FITC-conjugated secondary antibody at room temperature. Samples were
then washed in PBS containing 0.1\% Tween-20 and nuclei were
counterstained with DAPI (Beyotime, Shanghai, China), and digital images
were acquired using a Leica SP8 confocal microscope.

\textbf{2.14 Construction of CXCL12 overexpression and knockdown cell
lines}

For construction of CXCL12-overexpressing huh7 cell lines, EX-Z1991-M03
were purchased from Genecopoeia and transfected into huh7 cells for 24
h, cultured in serum-free medium in advance. After replaced with fresh
medium (10\% FBS) and added 0.3\selectlanguage{english} mg/mL neomycin, cells were continued to
cultivate in medium for 48h. shCXCL12 (GeneCopoeia, Guangzhou, China)
was transfected into HepG2 cells using Liposome transfection reagent
(Yeason, Shanghai, China) according to the manufacturer's directions for
24 h. The un-transfected cells were screened for puromycin (1 \selectlanguage{greek}μ\selectlanguage{english}g/mL).

\textbf{2.15 Animal and H\&E staining analysis}

Female mice (BABL/c nude and 5-6 weeks) were selected in animal
experiments, and all mice were fed in specific pathogen-free (SPF)
barriers. HepG2 cells (1 \selectlanguage{ngerman}×\selectlanguage{english} 10\textsuperscript{6}/mouse) were used to
build the cancer modes by subcutaneous injection on the left front leg
of nude mice. The mice were randomly divided into two groups. As
follows:(1) normal saline (0.9\% NaCl) group or model group; (2) FKA
group (30\selectlanguage{english} mg/kg/day, 60\selectlanguage{english} mg/kg/day and 120\selectlanguage{english} mg/kg/day). The tumor volume
(V = LxW\textsuperscript{2}/2) and body weight of the nude mice were
organized and recorded daily within 28 days. After euthanized, the
xenograft tumors from each mouse were weighed and analyzed. The lung
tissues and tumor of each mouse was fixed in 10\% formalin. Then, tissue
samples were embedded, sectioned and stained with H\&E.

\textbf{2.16 Immunoprecipitation and PAS-CD31 staining}

After deparaffinization with xylene and hydration with graded alcoholic,
sections were immersed in citric acid antigen retrieval solution and
incubated with heating in a microwave oven. According to the
instructions of the ready-to-use immunohistochemical kit (Maxim Biotech,
Shanghai, China), sections were incubated with the primary antibodies
overnight at 4degC and the biotinylated secondary antibody at room
temperature. After washing, the sections were colored with Glycogen
Periodic Acid Schiff (PAS) Stain Kit (Solarbio, Beijing, China) and
counterstained with haematoxylin.

\textbf{2.17 Statistical analysis}

All data were obtained by repeating the experiment three times and were
finally displayed as mean +- standard deviation (SD). GraphPad Prism 9.0
was used to run the statistics, and \emph{t} -test was used to calculate
the significant difference between the control group and the
experimental group. And \emph{P} values~\textless{}~0.05 were considered
to differ significantly. (* \emph{P} \selectlanguage{english} \textless{}\selectlanguage{english} 0.05; **\emph{P}
\selectlanguage{english} \textless{}\selectlanguage{english} 0.01; *** \selectlanguage{english}\emph{P } \textless{}\selectlanguage{english} 0.001).

\textbf{3. Results}

\textbf{3.1 Isolation and identification of chemical components
from\emph{C. henryi}}

By methanol extraction of the whole plants of \emph{C. Henryi} , we
found that a new eudesmanolide (1\emph{R} , 2\emph{S} , 5\emph{R} ,
8\emph{S} , 10\emph{R} )-1,
2-dihydroxy-eudesma-1,3,7(11)-trien-8,12-olide (\textbf{1} ) along with
five known compounds 5,8\selectlanguage{greek}β\selectlanguage{english}H-eudesma-1,3,7(11)-trien-8,12-olide
(\textbf{2} ) (Blay, Cardona, Garcia, Pedro, \& Sanchez, 1996),
4\selectlanguage{greek}β-\selectlanguage{english}hydroxy-8,12-epoxyeudesma-7,11-diene-1,6-dione (\textbf{3} ) (Wu, He,
Wu, \& Pan, 2008), flavokawain A (\textbf{4} ) (Seidel, Bailleul, \&
Waterman, 2000), 2'-hydroxy-4,3',4',6'-tetramethoxy-chalcone (\textbf{5}
) (Huang et al., 2021), and 13-epitorulosol (\textbf{6} ) (Su, Fang, \&
Cheng, 1994) in the soluble layer of EtOAc (Fig. 1). Compound\textbf{2}
that was synthesized previously by Blay et al (Blay et al., 1996) was
isolated for the first time from natural source. The structure of
compound \textbf{1} was characterized as shown in Supporting
information.

\textbf{3.2 FKA inhibits cell viability and migration of HCC}

To detect the effects of compounds \textbf{1} -\textbf{6} (20 \selectlanguage{greek}μ\selectlanguage{english}M) on
cell viability of HCC cells (SMMC-7721, HepG2), lung cancer cell(A549),
cervical cancer cells (Hela), and human pancreatic cancer cell (PANC-1),
MTT was carried out. As showed in Figure 2A, cells exposed to compounds
for 48 hours, compared with cisplatin, compound 4 had the most potent
inhibitory effect. Next, to further explore the inhibitory effects of
six compounds on hepatocellular carcinoma cells, SMMC-7721 cells were
exposed to different compounds for 48 h. The results (Fig. 2B) showed
that compound \textbf{4} significantly inhibited the viability of
SMMC-7721 cells and positively correlated with drug concentration. In
addition, the effects of compound \textbf{1} -\textbf{6} on the
migration of SMMC-7721 cells were also studied and the results of 48 h
wound healing indicated that the compound \textbf{4} had the best
inhibitory effect on cell migration (Fig. 2C, 2D).

\textbf{3.3 FKA dampens themigration, invasion and VM formation of
SMMC-7721 and HepG2 cells by targeting CXCL12}

To uncover the target of FKA, we predicted potential targets for FKA
through website (\emph{https://sea.bkslab.org/}). The result showed that
CXCL12 is the most potential target of FKA. Next, by molecular docking
we found that FKA bound to Arg12/Phe13/Pro10/Cys50 of CXCL12 (Fig. 3A).
And docking scores were calculated using the auto dock software with a
score of 6.45. Then we performed CETSA experiments. Compared with the
PBS group, FKA protected CXCL12 from degradation with increasing
temperature (Fig. 3B).

Several studies have shown that CXCR4, as a receptor for CXCL12, is a
GPCR. And previous studies have reported that the activated CXCL12 binds
to CXCR4 (chemokine receptor 4) and thus mediates tumor growth,
metastasis and angiogenesis (Morein, Erlichman, \& Ben-Baruch, 2020;
Mortezaee, 2020; Zhou, Cao, Li, \& Zhao, 2018). So, we tested the
inhibitory effect of FKA on CXCR4 activation with an IP one assay kit.
As shown in Figure 3C, FKA significantly inhibited CXCR4 activation
induced by CXCL12 in a dose-dependent manner. By western blot (Fig. 3D),
we determined the expression level of CXCL12 in different HCC cells, and
finally decided to evaluate the antitumor activity of FKA with SMMC-7721
and HepG2 cells, which are high expressed with CXCL12. Then, to assess
the cytotoxicity of FKA on SMMC-7721 cells, as well as HepG2 cells
proliferation, 48 h MTT assay was conducted. After treatment with FKA
(10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M), the viability of cells (SMMC-7721 and HepG2)
was higher than 80\%, and showed lower drug toxicity compared with
LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M). (Fig. 3E). Next, EDU staining exhibited that
proliferation of the SMMC-7721 cells and HepG2 cells significantly
decreased after treatment with FKA with concentration dependence (Fig.
3F). In addition, the results of flow cytometry demonstrated that the
extent of cells (SMMC-7721 and HepG2) apoptosis were enhanced as the
concentration of FKA increased when compared with the blank group (Fig.
3G), but the effect was not obvious. To explore more about the role of
FKA targeting CXCL12 protein, wound healing, transwell, and cells 3D
culture assays were performed. The result of wound healing indicated
that FKA could effectively restrain the migration of HCC cells, while
treated with 40 \selectlanguage{greek}μ\selectlanguage{english}M of FKA showed significant inhibition of SMMC-7721
cells and HepG2 cells migration (\emph{p} \textless{} 0.0001) (Fig. 4A,
4B). Transwell method also was implemented to evaluate the effect of FKA
on cells invasion ability. The amounts of cells perforated the membrane
of SMMC-7721 cells and HepG2 cells disposed with FKA were obviously
lower than that of the control group (Fig. 4C). The results of 3D
culture assay in Figure 3D showed that the number of tubes in the two
cells decreased significantly as the concentration of FKA increase. At
the same time, the LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M) also showed a certain inhibitory
effect. These results illustrated that FKA could restrain HCC cells
migration, invasion and VM formation, and the suppression ratio was
proportional to drug concentration.

\textbf{3.4 FKA} \textbf{suppresses EMT of HCC cells by targeting
CXCL12}

Multiple studies have shown that enhanced cell migration and invasion
ability were associated with the occurrence of EMT. We next examined the
impact of FKA targeting CXCL12 on EMT progression. In the experiment,
epithelial protein biomarkers (E-cadherin), mesenchymal protein
biomarkers (VE-cadherin and Vimentin) and EMT transcription factor
(Snail1) were evaluated in HCC cells (SMMC-7721 and HepG2) using WB
analysis. The results indicated that FKA reduced the expression of
VE-cadherin, Vimentin and Snail1 proteins in cells, but the expression
of E-cadherin increased (Fig. 5A-D). In addition, the results of RT-PCR
assay showed that the mRNA expression level of VE-cadherin, Vimentin and
Snail1 were reduced, while E-cadherin was increased by different
concentrations of FKA (Fig. 5E). Notably, consistent with the above
results, the consequences of immunofluorescence (Fig. 5F) showed that
diverse concentrations of FKA up-regulation expression of E-cadherin and
down-regulation expression of VE-cadherin, Vimentin and Snail1.On the
whole, these results suggested that FKA inhibits EMT progression of HCC
cells after acting on CXCL12.

\textbf{3.5 FKA induces transcriptomic changes in HCC cells}

To further clarify the effect of FKA on HCC cells, genome-wide changes
that occurred after impact of FKA were characterized by RNA-seq. After
HepG2 cells were treated with FKA (40 \selectlanguage{greek}μ\selectlanguage{english}M) for 24 h and collected,
RNA-seq analysis was performed. The heat map and PCA were used to
identify the differential gene expression (Fig. 6A, 6G). The volcano
plot and Venn analyses showed that 1062 rose and 603 declined genes were
quantified (Fig. 6B-D). Then, KEGG and GO analyses were executed based
on RNA-Seq data (Fig. 6E, 6F). We found that these differential genes
were enriched in calcium signaling pathway, HIF-1\selectlanguage{greek}α \selectlanguage{english}signaling pathway,
NF-kappa B signaling pathway, p53 pathway and some inflammatory response
pathways. Moreover, the relationship between CXCL12/CXCR4 and HIF-1\selectlanguage{greek}α,
\selectlanguage{english}NF-\selectlanguage{greek}κ\selectlanguage{english}B, Akt, caspase protein and proliferation protein Ki67 was confirmed
by protein interaction analysis (\emph{https://cn.string-db.org/})
(Fig.6H).

\selectlanguage{greek}\textbf{3.6 FKA inhibits EMT process via the inactivation of the
PI3K/Akt/NF-κB signal transduction pathway}\selectlanguage{english}

Previous researches have shown that obstructing CXCL12/CXCR4 pathway can
restrain the migration, invasion and EMT progress in lung cancer and
breast cancer(H. Z. Li et al., 2015) (Gr\selectlanguage{ngerman}ündker et al., 2015; Xia et al.,
2018) and PI3K/Akt /NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway can mediate EMT in a variety of cancer
cells (He et al., 2017; Yu, Qi, Xiaoxiang, Xu, \& Liu, 2017). Through
our studies, we concluded that inhibition of CXCL12/CXCR4 axis by FKA
attenuated EMT in HCC cells. According to the results of omics analysis,
to determine whether FKA affected the PI3K/Akt/ NF-\selectlanguage{greek}κ\selectlanguage{english}B pathway by
targeting CXCL12, we verified it by WB experiment. We found that FKA
inhibited the phosphorylation levels of PI3K and Akt without affecting
their background expression levels of SMMC-7721 cells (Fig. 7A, 7B) and
HepG2 cells (Fig. 7C, 7D) in a dose-dependent manner. Meanwhile, FKA
also reduced the expression of EMT-related transcription factors HIF-1\selectlanguage{greek}α,
\selectlanguage{english}NF-\selectlanguage{greek}κ\selectlanguage{english}B and Twist1.

\textbf{3.7 FKA suppresses HCC tumor growth, metastasis and vasculogenic
mimicry formation in \emph{vivo}.}

To verify whether FKA inhibited proliferation and metastasis of HCC
in\emph{vivo}, we established mice models according to the method
described above. We randomly divided the animals into four groups: Model
(physiological saline), FKA (30 mg/kg/d), FKA (60 mg/kg/d) and FKA
(120 mg/kg/d). The results showed that FKA could significantly inhibit
tumor growth and had little influence on the bodyweight of mice (Fig.
8A-C and Fig. 9A-C). Next, the lung tissue samples of mice were stained
with H\&E. The results indicated that compared with the model group, FKA
could reduce the number of nodules in lung, which confirmed FKA could
inhibit the lung metastasis in mice (Fig. 8D and Fig. 9D). Then, the
vasculogenic mimicry of tumor tissue was investigated by PAS/CD31
staining. The results showed that FKA effectively shrunk the formation
quantities of VM and MV inside the tumor tissues (Fig. 8E and Fig. 9E).
Meanwhile, IHC staining was used to detect the expression levels of
E-cadherin, VE-cadherin, Vimentin, p-Akt and Twist1in tumor tissues. As
shown in Figure 8F and Figure 9F, the expression level of E-cadherin
increased, the expression levels of VE-cadherin, Vimentin and Twist1
decreased, and the phosphorylation level of Akt reduced (Fig. 8F and
Fig. 9F). Taken together, we concluded that FKA suppressed EMT
progression of liver cancer and affects PI3K/Akt/Twist1 pathway
in\emph{vivo}.

\textbf{3.8 FKA inhibits HCCgrowth} \textbf{, invasion and VM formation
in \emph{vitro} and in \emph{vivo} by targeting CXCL12.}

In order to further confirmed that FKA inhibits the migration, invasion
and EMT process of HCC cells by acting on CXCL12, we detected the
expression level of EMT-related marker proteins and the phosphorylation
levels of Akt and PI3K in huh7 cells with over expressing of CXCL12 by
western blot assay. The level of E-cadherin in CXCL12 overexpressed huh7
cells was declined, while the levels of VE-cadherin, Vimentin and Twist1
increased, and the phosphorylation levels of Akt and PI3K were
up-regulated. However, after adding FKA (40 \selectlanguage{greek}μ\selectlanguage{english}M), a reverse adjustment
occurred (Fig. 10A, 10C). Surprisingly, when the expression of CXCL12
was knocked down by shRNA in HepG2 cells, the results revealed that the
protein level of E-cadherin was increased, while the levels of
VE-cadherin, Vimentin as well as Twist1 were decreased, and the
phosphorylation of Akt and PI3K were also down-regulated. And FKA made
this adjustment effect more obvious (Fig. 10B, 10D). Similarly, findings
from the wound healing and transwell assays gave evidence of that
overexpression of CXCL12 enhanced the migration and invasion of huh7
cells, while knockdown of CXCL12 produced the opposite results in HepG2
cells. Addition of 40 \selectlanguage{greek}μ\selectlanguage{english}M FKA to the huh7 or HepG2 cells reversed the
effects of overexpression and knockdown of CXCL12 expression in HCC
cells (Fig. 10E, 10F). These results indicated that CXCL12 was crucial
in FKA regulating EMT of hepatoma carcinoma cell. In our research, we
established the subcutaneous tumor-bearing mice model to confirm the
effect of CXCL12 in HCC tumor. Compared with HepG2 group, knocked down
the expression of CXCL12 significantly inhibited HCC tumor growth, and
FKA (60mg/kg) treatment for shCXCL12 mice didn't significantly reduce
the volume of the tumor (Fig.11A-C). By H\&E staining of mice lung
tissue sections (Fig11D, 11E) and PAS/CD31 staining of mice tumor tissue
sections (Fig11F, 11G), the results showed that the number of nodules in
lung and the counts of VM and MV inside the tumor tissue with knockdown
of CXCL12 and administered with FKA was significantly reduced. In brief,
these data suggested that interfering with CXCL12 expression not only
inhibits HCC growth, invasion and VM formation, but also weakened the
inhibitory effect of FKA.

\textbf{Discussion}

Natural products from plants have been the go-to source of drugs
especially for anticancer agents (Harvey, Clark, Mackay, \& Johnston,
2010; Thomford et al., 2018). Previous studies have highlighted the
ability of codonolactone from \emph{C.Henryi} to inhibit breast cancer
invasion and migration (W. Wang et al., 2014). In our study, a chalcone
compound flavokawain A (FKA) from \emph{chloranthus henryi} was found
for the first time that it had a certain therapeutic effect on liver
cancer. In addition, it is reported that FKA has induces apoptosis in
breast and bladder cancer (Abu et al., 2014; Zi \& Simoneau, 2005).
However, the anti-tumor mechanism and target of FKA on liver cancer is
not fully elucidated yet.

Through the target prediction, we found that CXCL12 may be the target of
FKA, and then we verified it by molecular docking and CETSA experiments.
CXCL12, also known as stromal-derived factor 1 alpha (SDF1\selectlanguage{greek}α\selectlanguage{english}), is a
chemokine(X. B. Wang et al., 2020). Chemokines maintain tumor cell
growth through autocrine or paracrine mechanisms, induce angiogenesis
and promote tumor escape by escaping immune
surveillance(Vandercappellen, Van Damme, \& Struyf, 2008). CXCL12 is
mainly expressed in hypoxia and angiogenic environment during tumor or
autoimmune disease (Janssens, Struyf, \& Proost, 2018; Kryczek et al.,
2005), and with the increasing of hypoxia-inducible factor 1\selectlanguage{greek}α \selectlanguage{english}(HIF-1\selectlanguage{greek}α\selectlanguage{english})
(Y. Q. Liu, Bi, Zhang, Wang, \& Ding, 2020) and cancer-associated
fibroblasts (CAFs), the CXCL12 expression was enhanced (Lerman et al.,
2010; Orimo et al., 2005). After CXCL12 specific binding to CXCR4 (a G-
protein-coupled receptor, GPCR), CXCR4 exerts its activity through a
heterotrimeric G-protein, which is divided into activated subunits.
These subunits activate diverse pathways, remarkably calcium release and
cell migration, PI3K/Akt and cell proliferation (Domanska et al., 2013;
Ghanem et al., 2014), and inducing tumor cells to invade and proliferate
and increasing tumor related angiogenesis (Q. Guo et al., 2015; Teicher
\& Fricker, 2010; D. Wang et al., 2020). In our study, we found that FKA
could significantly inhibit the proliferation, invasion, migration and
VM of SMMC-7721 and HepG2 with high expression of CXCL12 by targeting
CXCL12.

It has reported that EMT is relevant to tumor development, invasion and
metastasis (Brabletz, Kalluri, Nieto, \& Weinberg, 2018; Mittal, 2018;
Pastushenko \& Blanpain, 2019). Most experiments have shown that EMT is
characterized by increasing of mesenchymal markers (Vimentin and
N-cadherin), and by a significant down-regulation of epithelial markers
(E-cadherin) (Paolillo \& Schinelli, 2019). This corresponds to our
experimental results. By reports, EMT is associated with PI3K/Akt
pathway in many cancer cells, and including liver cancer (D. Guo et al.,
2020; Liang et al., 2021; Ma, Lou, \& Jiang, 2020; Yu et al., 2017).
Activation of this pathway promoted tumor proliferation, angiogenesis,
and survival in response to a range of signals, including G
protein-coupled receptor signaling (Noorolyai, Shajari, Baghbani,
Sadreddini, \& Baradaran, 2019). Then, we found that the phosphorylation
of PI3K and Akt was restrained after HCC cells were exposed to FKA.
NF-kappa B, as a normally downstream of the PI3K/Akt pathway, is
activated by the degradation of the I-\selectlanguage{greek}κ\selectlanguage{english}B kinase (IKK), resulting in I-\selectlanguage{greek}κ\selectlanguage{english}B
degradation to enhance NF-kB activity (Ho et al., 2021; Y. R. Li et al.,
2017), which in turn affects EMT progress. The protein level of HIF-1\selectlanguage{greek}α
\selectlanguage{english}was regulated by the PI3K/Akt pathway (Noorolyai et al., 2019), the
phosphorylation level of PI3K and Akt were inhibited, and the expression
of HIF-1\selectlanguage{greek}α \selectlanguage{english}was also down-regulated. In this study, we discovered that
Twist1 is indeed involved in FKA-adjust EMT. And inactivation of HIF-1\selectlanguage{greek}α
\selectlanguage{english}was associated with downregulation of Twist1. Through transcriptome
analysis, we found that FKA can significantly affect PI3K/Akt, NF-\selectlanguage{greek}κ\selectlanguage{english}B and
HIF-1\selectlanguage{greek}α \selectlanguage{english}signal pathway. Therefore, in this experiment, PI3K/Akt pathway,
Twist1 and NF-\selectlanguage{greek}κ\selectlanguage{english}B were combined to study the mechanism of FKA in the
treatment of HCC.

Similarly, we obtained the same results in huh7 cells overexpressing
CXCL12 and HepG2 cells knocking down CXCL12. FKA could reverse the EMT
by inhibiting PI3K/Akt phosphorylation and Twist1 expression, thereby
restrain the invasion and migration of hepatocellular carcinoma cells.
Besides, we also found that FKA could inhibit HCC tumor growth, lung
transfer and angiogenesis by targeting CXCL12 and suppressing the
downstream PI3K/Akt/Twist1 pathway and EMT in \emph{vivo} .

\textbf{Conclusions}

In this research, we found the therapeutic effect of FKA on HCC and the
target of FKA in the treatment of tumors. FKA inhibits proliferation,
invasion, metastasis, VM and EMT of hepatocellular carcinoma by
inhibiting PI3K/Akt/NF-\selectlanguage{greek}κ\selectlanguage{english}B/HIF-1\selectlanguage{greek}α/\selectlanguage{english}Twist1 pathway. Overall, our report
will hopefully support that flavokawain A as a potential clinical
targeted drug for HCC treatment.

\textbf{Author contributions}

Cheng Yang, Chunfeng Xie, and Honggang Zhou conceived and designed the
experiments. Ting Xiao, Jiali Bao and Rong Lin performed all the
experiments. Jiali Bao, Tian Jiao, Zihui Zhang, Yuxin Zhu, Yiming He,
Dandi Gao, Ronghao Sun, Fubo Zhang, Yexin Cheng and
Jiadelati\selectlanguage{ngerman}·Shaletanati analyzed the data. Jiali Bao and Ting Xiao wrote
the manuscript. All authors read and approved the final manuscript.

\textbf{Institutional Review Board Statement}

All animal care and experimental procedures conformed to guidelines
approved by the Institutional Animal Care and Use Committee (IACUC) of
Nankai University (Permit No. SYXK 2014-0003).

\textbf{Informed Consent Statement}

Informed consent was obtained from all subjects involved in the study.

\textbf{Data Availability Statement}

The data presented in this study are available in the insert article or
in the supplementary material.

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\textbf{Figure Legends:}

\textbf{Figure 1. Structures of compounds 1-6 isolated
from\emph{chloranthus henryi}.}

\textbf{Figure 2. Effect of compound 1-6 on tumor cell activity and
migration.} (A) Cell viability was examined by MTT assay. MTT analysis
of six plant extracts (compound \textbf{1-6} ) on Hela, HepG2, A549,
SMMC-7721 and PANC-1 cells. Cisplatin as a positive control. (B) MTT
analysis of plant extracts on SMMC-7721 cells at different
concentrations (7.8125 \selectlanguage{greek}μ\selectlanguage{english}g/mL, 15.625 \selectlanguage{greek}μ\selectlanguage{english}g/mL, 31.25 \selectlanguage{greek}μ\selectlanguage{english}g/mL, 62.5 \selectlanguage{greek}μ\selectlanguage{english}g/mL and
125 \selectlanguage{greek}μ\selectlanguage{english}g/mL) for 48 h. (C) Six plant extracts (20 \selectlanguage{greek}μ\selectlanguage{english}M) inhibited the wound
healing of SMMC-7721 cells. (D) Distance of SMMC-7721 cells migration. *
\emph{p} \textless{} 0.05, ** \emph{p} \textless{}0.01, *** \emph{p}
\textless{}0.005, ****\emph{p} \textless{}0.0001

\textbf{Figure 3. Affection of FKA on proliferation and apoptosis of
hepatocellular carcinoma cells after acting on CXCL12.} (A)The docking
stick model of flavokawain A combining with the Arg12/Phe13/Pro10/Cys50
domain of CXCL12. Molecular docking score achieved 6.45. (B) Cellular
thermal shift assay (CETSA): qualitative and quantitative analysis of
the binding strength of FKA to CXCL12 in HepG2 cells at different
temperatures. PBS as a negative control. (C) The HTRF ratio after the
co-action of CXCL12 and LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M) or FKA (10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M)
was detected to evaluate the accumulation of IP-One. (E) MTT analysis of
survival rate of SMMC-7721 cells and HepG2 cells treated by LIT-927 (30
\selectlanguage{greek}μ\selectlanguage{english}M) and FKA (10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M). (F) SMMC-7721 cells and HepG2
cells exposed to LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M) and FKA (10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M) for
1.5 h for immunofluorescence analysis. **** \emph{p} \textless{}0.0001
(G) SMMC-7721 cells and HepG2 cells were incubated with LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M)
and FKA (10\selectlanguage{greek}μ\selectlanguage{english}M, 20\selectlanguage{greek}μ\selectlanguage{english}M and 40\selectlanguage{greek}μ\selectlanguage{english}M), and the subjected to flow cytometry to
assess the apoptosis levels.

\textbf{Figure 4. Inhibition of FKA on migration, invasion and VM
formation of hepatocellular carcinoma cells after acting on CXCL12.} FKA
(10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M) and LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M) attenuated the HCC cells
including SMMC-7721 (A) and HepG2 (B) wound healing after 24 h, 48h.
Left image: representative pictures of five groups in scratch analysis.
Right image: quantitative show of the result of scratch analysis. (C)
FKA (10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40 \selectlanguage{greek}μ\selectlanguage{english}M) and LIT-927 (30 \selectlanguage{greek}μ\selectlanguage{english}M) attenuated SMMC-7721
cells and HepG2 cells invasion after 24 h. Left image: representative
pictures of five groups in transwell test. Right image: quantitative
show of the result of transwell test. (D) The angiogenesis ability of
FKA and LIT-927 were evaluated in SMMC-7721 cells and HepG2 cells by
tube formation assays. Data was expressed as the means \selectlanguage{ngerman}± SD, n = 3. *
\emph{p} \textless{} 0.05, ** \emph{p} \textless{}0.01, *** \emph{p}
\textless{}0.002, ****\emph{p} \textless{}0.0001

\textbf{Figure 5. Inhibition of FKA on EMT progression in hepatocellular
carcinoma cells.} (A, B, C, D) SMMC-7721 cells (A, C) and HepG2 cells
(B, D) were treated successively with either FKA (10 \selectlanguage{greek}μ\selectlanguage{english}M, 20 \selectlanguage{greek}μ\selectlanguage{english}M and 40
\selectlanguage{greek}μ\selectlanguage{english}M) or LIT-927 (30 \selectlanguage{greek}µ\selectlanguage{english}M) for 24 hours, and then assessed by western blot
(A, B) and qRT-PCR (C, D). (E) Typical immunofluorescence images of
E-cadherin, snail1, Vimentin and VE-cadherin expression in HCC cells
(200\selectlanguage{ngerman}×) (Left panel: SMMC-7721 cells, Right panel: HepG2 cells). Data
represent mean\selectlanguage{english} +-\selectlanguage{english} SD of three separate experiments. *\selectlanguage{english}\emph{p }
\textless{}\selectlanguage{english} 0.05, ** \selectlanguage{english}\emph{p } \textless{}\selectlanguage{english} 0.01, ***\selectlanguage{english}\emph{p }
\textless{}\selectlanguage{english} 0.002, **** \emph{p} \textless{}0.0001

\textbf{Figure 6. Seq-RNA analysis of FKA-treated HepG2 cells after 24
h.} (A) Analysis of gene difference between FKA-treated and non-treated
groups by thermograph. (B) Cross plots show the number of co-expression
genes between FKA-treated and control groups in HepG2 cells. (C, D) Venn
analysis (C) and Volcano plot (D): The control group was used as a
reference, the number of high and low expression genes in HepG2 cells
treated with FKA. (E, F) The analysis of FKA affecting signaling
pathways by Kyoto Encyclopedia of Genes and Genomes (KEGG). (F) Gene
ontology (GO) analysis the expressed difference of proteins related to
pathways. (G) Principal Component Analysis (PCA) 3D plot shows the
principal component differences between three control samples and three
FKA-treated samples. (F) Schematic representation of the mechanisms of
flavokawain A on proliferation, migration, VM and EMT progression of
hepatocellular carcinoma cells.

\textbf{Figure 7. FKA restrains EMT in HCC via inactivation of
PI3K/Akt/NF-} \selectlanguage{greek}\textbf{κB/Twist1 signaling.}\selectlanguage{english} (A, C) Protein levels of
phosphorylated Akt, PI3K and the expression level of NF-\selectlanguage{greek}κ\selectlanguage{english}B, Twist1 in
SMMC-7721 cells (A) and HepG2 cells (C). (B, D) Quantification assay of
the western blotting data from three independent experiments was
displayed. And compared with the blank control group. * \emph{p}
 \textless{} 0.05, **\emph{p}  \textless{} 0.01, *** \emph{p}
 \textless{} 0.002, **** \emph{p}\textless{}0.0001

\textbf{Figure 8. FKA weakens tumor growth, metastasis and VM formation
of SMMC-7721 xenograft tumor.} (A) Image of tumor tissue in FKA (30
mg/kg/d, 60 mg/kg/d and 120 mg/kg/d) treated or untreated group. (B)
Statistics and comparison of tumor volume between treated group and
un-treated group (0.9\% saline). (C) Variations of body weight of mice
during administration. (D) Typical pictures of lung tissue sections of
mice after H\&E stained and the number of lung metastasis area in four
groups. (E) Tumor sections were double-colored with PAS and CD31. VM
(red arrows) was identified as PAS positive (the polysaccharides in the
tumor sections appear pink) and CD31 negative, and MV (green arrows) was
affirmed as CD31 positive (the endothelial tissue was stained brown) and
PAS positive. (100\selectlanguage{ngerman}×). Right panel: Number of the VM or MV. (F)
Immunohistochemical staining analysis of E-cadherin, VE-cadherin,
Vimentin, Twist1 and Akt phosphorylation expression; 100×. Data are
expressed as the means\selectlanguage{english} +-\selectlanguage{english} SD, n = 3. * \selectlanguage{english}\emph{p } \textless{}\selectlanguage{english} 0.05,
**\selectlanguage{english}\emph{p } \textless{}\selectlanguage{english} 0.01, *** \emph{p} \selectlanguage{english} \textless{}\selectlanguage{english} 0.002, ****
\emph{p}\textless{}0.0001

\textbf{Figure 9. FKA weakens tumor growth, metastasis and VM formation
of HepG2 xenograft tumor.} (A) Image of tumor tissue in FKA (30 mg/kg/d,
60 mg/kg/d and 120 mg/kg/d) treated or untreated group. (B) Statistics
and comparison of tumor volume between treated group and un-treated
group (0.9\% saline). (C) Variations of body weight of mice during
administration. (D) Typical pictures of lung tissue sections of mice
after H\&E stained and the number of lung metastasis area in four
groups. (E) Tumor sections were double-colored with PAS and CD31. VM
(red arrows) was identified as PAS positive (the polysaccharides in the
tumor sections appear pink) and CD31 negative, and MV (green arrows) was
affirmed as CD31 positive (the endothelial tissue was stained brown) and
PAS positive. (100x). Right panel: Number of the VM or MV. (F)
Immunohistochemical staining analysis of E-cadherin, VE-cadherin,
Vimentin, Twist1 and Akt phosphorylation expression; 100x. Data are
expressed as the means\selectlanguage{english} +-\selectlanguage{english} SD, n = 3. * \selectlanguage{english}\emph{p } \textless{}\selectlanguage{english} 0.05,
**\selectlanguage{english}\emph{p } \textless{}\selectlanguage{english} 0.01, *** \emph{p} \selectlanguage{english} \textless{}\selectlanguage{english} 0.002, ****
\emph{p}\textless{}0.0001

\textbf{Figure 10. Affections of FKA on silencing and overexpression of
CXCL12 in} \textbf{hepatoma carcinoma cells.} (A, B) E-cadherin,
VE-cadherin, Vimentin, Twist1 levels and the phosphorylation levels of
Akt and PI3K were analyzed in CXCL12 silenced (sh-CXCL12) HepG2 cells
(A) and overexpressed (OE) huh7 cells (B), respectively. (C, D)
Quantitative representation of WB data in CXCL12 silenced (sh-CXCL12)
HepG2 cells (C) and overexpressed (OE) huh7 cells (D). GAPDH was used as
an internal reference to normalize each protein expression level. ***
\emph{p} \selectlanguage{english} \textless{}\selectlanguage{english} 0.001, **** \emph{p} \textless{}0.0001 (E, F) The
wound-healing assay that the effect of FKA on HepG2-shCXCL12 cells (E)
and huh7-CXCL12 cells (F) after 48 h. Left image: Representative
pictures of four groups in scratch assay. Right image: Quantification of
the result of scratch analysis. Compared with control group. ** \emph{p}
\selectlanguage{english} \textless{}\selectlanguage{english} 0.01, ***\emph{p} \selectlanguage{english} \textless{}\selectlanguage{english} 0.002, **** \emph{p}
\textless{}0.0001

\textbf{Figure 11: Knockdown of CXCL12 expression attenuated tumor
growth, metastasis, and VM formation in HepG2 xenograft tumors
in\emph{vivo}. (} A) Image of tumor tissue in Model (HepG2), shCXCL12
control group and FKA (60 mg/kg/d) treated. (B) Statistics and
comparison of tumor volume between model group, shCXCL12 control group
and FKA-treated group. (C) Variations of body weight of mice during
administration. (D) Typical pictures of lung tissue sections of mice
after H\&E stained and (E) the number of lung metastasis area in three
groups. (F) Tumor sections were double-colored with PAS and CD31. VM
(red arrows) was identified as PAS positive and CD31 negative, and MV
(green arrows) was affirmed as CD31 positive and PAS positive. (100x).
(G) The number of VM and MV in three groups. ** p\selectlanguage{english} \textless{}\selectlanguage{english} 0.01, ***
p\selectlanguage{english} \textless{}\selectlanguage{english} 0.001\selectlanguage{english}
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