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Inhibition of glioma growth by flavokawain B is mediated through

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DOI: 10.1080/15548627.2018.1501133

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 Autophagy

ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: http://www.tandfonline.com/loi/kaup20

Inhibition of glioma growth by flavokawain B is

mediated through endoplasmic reticulum stress
induced autophagy

Jiwei Wang, Qichao Qi, Wenjing Zhou, Zichao Feng, Bin Huang, Anjing
Chen, Di Zhang, Wenjie Li, Qing Zhang, Zheng Jiang, Rolf Bjerkvig, Lars
Prestegarden, Frits Thorsen, Xinyu Wang, Xingang Li & Jian Wang

To cite this article: Jiwei Wang, Qichao Qi, Wenjing Zhou, Zichao Feng, Bin Huang, Anjing
Chen, Di Zhang, Wenjie Li, Qing Zhang, Zheng Jiang, Rolf Bjerkvig, Lars Prestegarden, Frits
Thorsen, Xinyu Wang, Xingang Li & Jian Wang (2018): Inhibition of glioma growth by flavokawain
B is mediated through endoplasmic reticulum stress induced autophagy, Autophagy, DOI:
10.1080/15548627.2018.1501133

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AUTOPHAGY
https://doi.org/10.1080/15548627.2018.1501133

RESEARCH PAPER - TRANSLATIONAL

Inhibition of glioma growth by flavokawain B is mediated through endoplasmic

reticulum stress induced autophagy
Jiwei Wanga, Qichao Qia, Wenjing Zhoua, Zichao Fenga, Bin Huanga, Anjing Chena, Di Zhanga, Wenjie Lia, Qing Zhanga,
Zheng Jianga, Rolf Bjerkvigb, Lars Prestegardenb, Frits Thorsenb,c, Xinyu Wanga, Xingang Lia, and Jian Wanga,b
a
Department of Neurosurgery, Qilu Hospital of Shandong University and Brain Science Research Institute, Shandong University, Key Laboratory of
Brain Functional Remodeling, Jinan, Shandong, P.R. China; bKristian Gerhard Jebsen Brain Tumour Research Centre, Department of Biomedicine,
University of Bergen, Bergen, Norway; cThe Molecular Imaging Center, Department of Biomedicine, University of Bergen, Bergen, Norway

ABSTRACT ARTICLE HISTORY

Flavokawain B (FKB), a natural kava chalcone, displays potent antitumor activity in various types of Received 6 April 2017
cancer. The mechanism of action, however, remains unclear. Here, we evaluated the efficacy of FKB in Revised 18 June 2018
the treatment of human glioblastoma multiforme (GBM) as well as the molecular basis for its inhibitory Accepted 10 July 2018
effects in cancer. Approximately 60% of GBM cells became senescent after treatment with FKB as KEYWORDS
assessed in the senescence-associated (SA)-GLB1/SA-β-galactosidase assay. The cellular process of Apoptosis; autophagy; ER
autophagy potentially contributed to the establishment of senescence. Transmission electron micro- stress; flavokawain B;
scopy revealed the formation of autophagic vesicles under FKB treatment, and MAP1LC3B (microtubule senescence
associated protein 1 light chain 3 beta)-II was increased. Transfection of ATG5 or ATG7 small interfering
RNAs (siRNAs) inhibited FKB-induced autophagy in U251 cells. Western blot revealed that molecular
components of the endoplasmic reticulum stress pathway were activated, including ATF4 (activating
transcription factor 4) and DDIT3 (DNA damage inducible transcript 3), while levels of TRIB3 (tribbles
pseudokinase 3) increased. In addition, based on the phosphorylation status, the AKT-MTOR-RPS6KB1
pathway was inhibited, which induced autophagy in GBM cells. Inhibition of autophagy by autophagy
inhibitors 3-methyladenine and chloroquine or knockdown of ATG5 or ATG7 caused FKB-treated U251
cells to switch from senescence to apoptosis. Finally, knockdown of ATG5 or treatment with chloroquine
in combination with FKB, significantly inhibited tumor growth in vivo. Our results demonstrated that FKB
induced protective autophagy through the ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway in
GBM cells, indicating that the combination treatment of FKB with autophagy inhibitors may potentially
be an effective therapeutic strategy for GBM.
Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; AKT: AKT serine/threonine kinase;
ATF4: activating transcription factor 4; ATG: autophagy related; CASP3: caspase 3; CCK-8: cell counting kit-
8; CDKN1A: cyclin-dependent kinase inhibitor 1A; CQ: chloroquine; DDIT3: DNA damage inducible transcript
3; DMEM: Dulbecco’s modified Eagle’s medium; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3:
eukaryotic translation initiation factor 2 alpha kinase 3; ER: endoplasmic reticulum; FKB: flavokawain B;
GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBM: glioblastoma multiforme; GFP: green fluores-
cent protein; HSPA5: heat shock protein family A (Hsp70) member 5; MAP1LC3B: microtubule associated
protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; PARP1: poly(ADP-ribose)
polymerase; 1RPS6KB1: ribosomal protein S6 kinase B1; SA-GLB1: senescence-associated galactosidase
beta 1; siRNA: short interfering RNA; SQSTM1: sequestosome 1; TEM: transmission electron microscopy;
TRIB3: tribbles pseudokinase 3; TUNEL: deoxynucleotidyl transferase-mediated dUTP nick-end labeling

Introduction Although cancers, such as GBM, may be intrinsically resis-

tant to therapy, the ability to engage survival mechanisms in
Glioblastoma multiforme (GBM) is a highly aggressive
response to treatment potentially further reduces efficacy of
malignant primary human brain tumor. Tumors are char-
any promising agent [3–5] Autophagy is one such conserved
acterized by a high proliferation rate and chemoresistance
cellular pathway. The process removes dysfunctional or
[1]. Despite advances in combination treatments consisting
damaged organelles through lysosomal degradation and
of radiation and chemotherapy following surgical resection,
recycles the products for cellular metabolic needs [6].
the 5-year survival rate of WHO grade IV glioblastoma
Autophagy is thus essential for maintaining homeostasis and
remains at less than 5% [1,2]. Although significant
mediates resistance to anticancer therapies such as radiation,
advances have been made in our understanding of the
chemotherapy and some targeted therapies [7]. Increasing
molecular status of this tumor type, novel efficacious ther-
evidence supports that treatment with autophagy inhibitors
apeutic avenues are critically needed.
such as bafilomycin A1 and chloroquine (CQ) potentiates the

CONTACT Xingang Li lixg@sdu.edu.cn; Jian Wang jian.wang@uib.no

Supplemental data for this article can be accessed here
© 2018 Informa UK Limited, trading as Taylor & Francis Group
2 J. WANG ET AL.

effects of different cancer treatments [8,9]. These studies have human GBM cell populations in vitro and in vivo. While we
led to the initiation of multiple clinical trials combining che- report that that FKB does inhibit GBM cell growth largely
motherapeutic agents and autophagy inhibitors for various through the processes of senescence and autophagy, we were
cancer types [10]. However, the role of autophagy in cancer able to promote apoptosis by combining treatment with
is still controversial as it may suppress tumors during cancer autophagy inhibitors. These results support the strategy of
development but promote cell survival during cancer progres- combination therapy of FKB and autophagy inhibitors in the
sion [11]. Thus, the specific role of autophagy seems to be treatment of human GBM.
highly cell type and context dependent.
Natural products have received recent interest in the discov-
ery of novel anticancer therapeutic agents as they have long been Results
used as alternative remedies for a variety of diseases, including
FKB inhibits proliferation of GBM cells in vitro
cancer, with relatively few side effects [12,13]. Flavokawain B
(FKB), a natural kava chalcone, has displayed anticancer activity To begin to determine whether FKB might be effective against
in various types of cancer, such as osteosarcoma, lung cancer, GBM, FKB treatment was first evaluated in U251, U87, T98,
leiomyosarcoma, and prostate cancer [14–17]. The cancer spe- and P3 cells in vitro, using the cell viability assay CCK-8
cific cytotoxic activity of FKB has been mainly attributed to (Figure 1A). Cells were treated with differing concentrations
induction of cell cycle arrest and apoptosis characterized by the of FKB in vitro, and viability was assessed at 12, 24, and 48 h.
generation of intracellular reactive oxygen species and the upre- Decreases in cell viability (~ 50%) relative to untreated cells
gulation of BCL2L11, a proapoptotic molecule [18,19]. were statistically significant at 48 h in 3 μg/mL FKB for all cell
The role that FKB plays in cell death in GBM cells and lines.
whether it induces autophagy remain largely unclear. Here, Quantification of EdU incorporation also revealed a statis-
we investigated the chemotherapeutic potential of FKB in tically significant decrease in proliferation for U251, U87, and

Figure 1. FKB inhibits proliferation of GBM cells. (A) Graphic representation of results from CCK-8 assays to determine cell viability of U251, U87, T98, and P3 cells
treated with different concentrations of FKB for 24, 48 and 72 h. Data points are the percentage (%; OD450 treated/OD450 untreated) relative to untreated cells at
that time point. (B) Fluorescence images of EdU incorporation in U251, U87, and T98 cells treated with FKB or DMSO for 48 h. Cells were stained with Apollo 567 (red)
to detect EdU and DAPI (blue) to highlight nuclei, and images were superimposed (scale bars: 50 μm). (C) Graphic representation of cell number and EdU content of
U251, U87, and T98 cells treated with different concentrations of FKB for 48 h. The percentage of EdU+ cells (EdU+/DAPI+ × 100%) was determined in 4 random fields
per sample. All data are expressed as the mean ± SD of values from experiments performed in triplicate. *P < 0.05 and ** P < 0.01 compared to controls.
 AUTOPHAGY 3

T98 cells lines after exposure to FKB at 3 μg/mL for 48 h Alternatively, we examined whether FKB triggered senes-
(~ 45% vs ~ 15%, untreated vs treated cells). These results cence in GBM cells. Three different assays were used. First,
indicated that FKB potently arrested proliferation in GBM distribution of the cell cycle as determined by flow cytometry
cells and in a dose-dependent manner (Figure 1B and 1C). revealed that GBM cells were primarily arrested at the G2/M
phase after FKB treatment in all 3 cell lines (~ 12% vs ~ 35%,
untreated vs treated cells; Figure 2A). Second, FKB treatment
led to an increase in nuclei positive for phosphorylated
FKB induces cellular senescence in GBM cells in vitro (Ser139) H2AFX (commonly termed γH2AFX) which detects
Chemotherapeutic agents lead to decreases in cancer cell a cause of senescence, DNA double-strand breaks (Figure 2B
proliferation through a variety of biological processes, includ- and 2C and Figure S2). Finally, accumulation of senescent SA-
ing apoptosis and cellular senescence. GBM cells have been GLB1 positive cells in response to FKB was statistically signifi-
shown to undergo apoptosis or cellular senescence in response cant in all cell lines (increases of ~ 10% to 20% relative to
to ionizing radiation, for example, which is mediated by the untreated cells; Figure 2D, 2E and S3). These results indicated
PTEN tumor suppressor protein [20]. In the case of FKB, that FKB induced cellular senescence in GBM cell lines in vitro.
studies have shown that the molecule induces apoptosis in a
variety of cancer cell types [15,16,21]. We therefore investi-
gated first whether FKB induced apoptosis in GBM cells. FKB induces autophagy in GBM cells in vitro
Increases in the percentage of apoptotic cells after FKB treat- Autophagy has been reported to facilitate cellular senescence
ment were not statistically significant in PTEN-mutated cell [22,23]. To determine whether FKB induced autophagy in
lines, U251, U87, T98, and P3, or the PTEN wild-type cell GBM cells, transmission electron microscopy (TEM) was per-
lines, LN18 and LN229 (Figure S1). These results indicated formed to detect autophagic vesicles. TEM analysis demon-
that the inhibitory effect of FKB observed in these cell lines strated an increased production of autophagosomes in U251,
was not through induction of apoptosis. U87, and T98 cells under treatment with FKB (3 μg/mL for

Figure 2. FKB induces cellular senescence in GBM cells. (A) Graphic representation of cell cycle distribution analyzed by flow cytometry in U251, U87, and T98 cells
treated with FKB (3 μg/mL) or DMSO (vehicle control) for 48 h. (B) Immunofluorescence staining for γH2AFX (green) used to detect DNA damage in U251 cells treated
with FKB (3 μg/mL) or DMSO for 48 h. Cell nuclei were counterstained with DAPI (blue) (scale bars: 10 μm). (C) Statistical results of the percentage of γH2AFX-positive
cells determined in 4 random fields per sample. (D) SA-GLB1 staining to detect cellular senescence U251, U87, and T98 cells treated with FKB (3 μg/mL) or DMSO for
48 h. Images were taken under bright-field microscopy (scale bars: 50 μm). (E) Statistic results of the percentage of SA-GLB1-positive cells determined in 4 random
fields per sample. All data are expressed as the mean ± SD of values from experiments performed in triplicate. * P < 0.05, ** P < 0.01 and *** P < 0.001 compared to
controls.
4 J. WANG ET AL.

Figure 3. FKB induces autophagy in GBM cells. (A) TEM images of U251, U87, and T98 cells treated with FKB (3 μg/mL) or DMSO for 48 h. Arrows highlight
autophagosomes (arrows). The scale bar in the original image represents 0.8 μm and the scale bar in the enlarged image represents 0.3 μm. (B) Western blot analysis
to detect protein levels of MAP1LC3B, SQSTM1, and GAPDH (control for loading) in U251, U87, and T98 cells treated with FKB (3 μg/mL) for the indicated times or the
indicated concentration of FKB for 48 h. Data are representative of 3 independent experiments. The numbers below the blots correspond to relative quantification by
densitometry compared with the reference point set to 1. (C) Fluorescence images of GFP-MAP1LC3B puncta in U251 cells treated with FKB (3 μg/mL) for the
indicated times or the indicated concentration of FKB for 48 h. GFP-MAP1LC3B puncta were quantified (scale bars: 50 μm) .

48 h; Figure 3A and Figure S4A). Western blot analysis of Levels of SQSTM1, a protein that is degraded through
lysates prepared from treated cells also indicated that FKB autophagy, decreased over time and with increasing concen-
induced the formation of autophagosomes. MAP1LC3B-II, a trations of FKB (Figure 3B). These results further supported
marker for autophagy, increased in a dose-dependent and time- the idea that autophagy was enhanced in FKB-treated cells.
dependent manner in GBM cells (Figure 3B). Finally, an Autophagic flux was used to further evaluate FKB induced
increase in vesicle formation in real time was observed under autophagy. We cotreated U251 cells with FKB (3 μg/mL) and
fluorescence microscopy when cells were transduced with a autophagy inhibitors, 3-MA or chloroquine (CQ), which block
construct expressing GFP tagged MAP1LC3B. In transduced upstream and downstream steps of the process, respectively.
GBM cells, the percentage of GFP-MAP1LC3B-positive cells Western blot analysis demonstrated that coincubation of cells
increased in a dose-dependent and time-dependent manner with FKB and 3-MA (10 mM) for 48 h led to decreased FKB-
under FKB treatment (Figure 3C and Figures S4B and S4C). induced MAP1LC3B-II formation (Figure 4A). FKB-induced
 AUTOPHAGY 5

Figure 4. FKB induces autophagy flux in GBM cells. Western blot analysis performed on lysates (20 µg) to detect levels of MAP1LC3B and GAPDH in U251 cells
pretreated with (A) 3-MA (10 mM) or (B) CQ (3 μM) for 1 h, followed by exposure to FKB (3 μg/mL) or DMSO for another 48 h. Western blot analysis performed to
detect levels of ATG5 or ATG7 and MAP1LC3B in U251 cells transfected with siRNAs. Numbers below the dots correspond to relative quantification by densitometry
compared with the reference point set to 1. (C) ATG5 (#1 and #2) or (D) ATG7 (#1 and #2). After 48 h, cells were treated with FKB (3 μg/mL) or DMSO and then
harvested after another 48 h. Data are representative of 3 independent experiments. (E) Fluorescence images of GFP-MAP1LC3B puncta in U251 cells pretreated with
autophagy inhibitors (3-MA or CQ) or transfected with ATG5 or ATG7 siRNAs followed by exposure to FKB (3 μg/mL) or DMSO for another 48 h (scale bars: 50 μm). (F)
Graphical representation of the quantification of GFP-MAP1LC3B puncta. All data are expressed as the mean ± SD of values from experiments performed in triplicate.
* P < 0.05, ** P < 0.01 and *** P < 0.001 compared to controls.

MAP1LC3B puncta were also reduced (Figure 4E and 4F). In Finally, we transfected U251 cells with ATG5 or ATG7
contrast, co-incubation of cells with FKB and CQ (3 μM) still siRNAs to inhibit protein expression of ATG5 or ATG7 in
led to increased conversion of MAP1LC3B-II (Figure 4B) and the presence of FKB. Western blot analysis and fluorescence
accumulation of MAP1LC3B puncta (Figure 4E and 4F). microscopy demonstrated that knockdown of ATG5 or ATG7
6 J. WANG ET AL.

led to decreased MAP1LC3B-II formation (Figure 4C and 4D) relative to control-knockdown cells (Figure 5C). Activation of
and number of MAP1LC3B puncta, respectively (Figure 4E the ER stress signaling was, however, partially restored in
and 4F). Taken together, these data indicated that FKB knockdown cells when treated simultaneously with an
induced autophagy in U251, U87, and T98 cell lines in vitro. ER stress antagonist, 4-phenylbutyrate (4-PBA, 1 mM;
Figure 5D). These results demonstrated that FKB induced
autophagy through activation of the ATF-DDIT3 ER stress
FKB induces autophagy through ER stress-dependent signaling pathway in GBM cells.
upregulation of ATF4 and DDIT3 in GBM cells
Growing evidence indicates that endoplasmic reticulum (ER)
FKB induces autophagy through the ATF4-DDIT3-TRIB3-
stress signaling is closely linked to autophagy [24,25]. We
AKT-MTOR-RPS6KB1 signaling pathway in GBM cells
therefore investigated whether FKB induced ER stress in
U251 GBM cells by examining protein levels of classic ER One of the key molecules negatively regulating autophagy is
signaling protein markers, including HSPA5, p-EIF2AK3, MTOR (mechanistic target of rapamycin kinase); autophagy
p-EIF2A, ATF4 and DDIT3, by western blot. U251 cells were activity is inhibited through phosphorylation catalyzed by the
treated with FKB as indicated (Figure 5A). Increased dose and protein kinase AKT, an upstream regulator of MTOR com-
treatment time with FKB induced HSPA5, p-EIF2AK3, plex 1 [26,27]. We therefore investigated the phosphorylation
p-EIF2A, ATF4 and DDIT3 in U251 cells (Figure 5A). These status of MTOR in cells under FKB treatment. Western blot
results indicated that FKB activated ER stress in GBM cells. analysis revealed that p-MTOR was decreased in FKB-treated
To determine whether FKB induces autophagy through the U251, U87, and T98 cells. FKB treatment also led to decreased
ER stress-mediated ATF4-DDIT3 pathway, ATF4 siRNAs phosphorylation of AKT and RPS6KB1, an MTOR substrate
were transfected into U251 cells and the protein levels of (Figure 6A). These results indicated that FKB induced autop-
DDIT3 and MAP1LC3B-II were measured by western blot. hagy through inhibition of AKT-MTOR-RPS6KB1 signaling
Levels of DDIT3 and MAP1LC3B-II did not increase in in U251, U87, and T98 cell lines.
ATF4-knockdown U251 cells, despite treatment with FKB, Recent studies have demonstrated that DDIT3 may inhi-
to the levels in control-knockdown cells (Figure 5B). U251 bit AKT activation through upregulation of TRIB3 expres-
cells transfected with DDIT3 siRNA also exhibited weak sion [28,29]. We, therefore, examined levels of TRIB3
increases in MAP1LC3B-II protein levels after FKB treatment protein in treated cells. Upregulation of TRIB3 occurred

Figure 5. FKB induces autophagy through ER stress-dependent upregulation of ATF4 and DDIT3 in GBM cells. Western blot analysis performed on lysates (20 µg) for
(A) HSPA5, p-EIF2AK3, p-EIF2A, ATF4, DDIT3, and GAPDH in U251 cells treated with indicated concentration of FKB for 48 h or FKB (3 μg/mL) for the indicated times;
(B) ATF4, DDIT3, MAP1LC3B, and GAPDH in U251 cells transfected with control or ATF4 siRNAs for 48 h followed by exposure to FKB (3 μg/mL) or DMSO for another
48 h; (C) DDIT3, MAP1LC3B, and GAPDH in U251 cells transfected with control or DDIT3 siRNAs for 48 h followed by exposure to FKB (3 μg/mL) or DMSO for another
48 h; and (D) ATF4, DDIT3, MAP1LC3B, and GAPDH in U251 cells pretreated with 4-PBA (1 mM) for 30 min followed by exposure to FKB (3 μg/mL) or DMSO for
another 48 h. Numbers below the blots correspond to relative quantification by densitometry compared with the reference point set to 1. Data are representative of
3 independent experiments.
 AUTOPHAGY 7

Figure 6. FKB induces autophagy through the ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway in GBM cells. Western blot analysis performed on lysates
(20 µg) to detect levels of (A) p-AKT, AKT, p-MTOR, MTOR, p-RPS6KB1, RPS6KB1, and GAPDH in U251, U87, and T98 cells treated with the concentration of FKB
indicated for 48 h; (B) TRIB3 and GAPDH in U251, U87, and T98 incubated in the indicated concentration of FKB for 48 h; (C) ATF4, DDIT3, TRIB3, MAP1LC3B, and
GAPDH in U251 cells transfected with control or ATF4 siRNAs for 48 h followed by exposure to DMSO or FKB (3 μg/mL) for another 48 h; and (D) TRIB3, p-AKT, AKT,
p-MTOR, MTOR, p-RPS6KB1, RPS6KB1, MAP1LC3B, and GAPDH in U251 cells transfected with control or TRIB3 siRNAs for 48 h followed by exposure to DMSO or FKB (3
μg/mL) for another 48 h. Numbers below the blots correspond to relative quantification by densitometry compared with the reference point set to 1. Data are
representative of 3 independent experiments.

in a dose-dependent manner in U251, U87, and T98 cell examined in cells after treatment by western blot. TRIB3
lines (Figure 6B). To investigate whether the upregulation levels and MAP1LC3B-II did not increase in response to
of TRIB3 was induced through the ATF4-DDIT3 pathway, FKB in ATF4-knockdown cells as in control-knockdown
U251 cells were transfected with ATF4 siRNA. Autophagy cells. These results indicated that ATF4 is required for
flux and protein levels of ATF4, DDIT3, and TRIB3 were upregulation of TRIB3 in response to FKB (Figure 6C and
8 J. WANG ET AL.

Figure S5). We next investigated whether FKB mediated the following treatment groups: Control (n = 5); FKB (n = 5);
inhibition of AKT-MTOR-RPS6KB1 occurred through CQ (n = 5); FKB + CQ (n = 5). Phosphate-buffered saline
increased TRIB3. U251 cells were transfected with TRIB3 (PBS) alone (control), FKB (50 mg/kg/day), CQ (25 mg/kg/
siRNA, and levels of TRIB3, p-AKT, p-MTOR, p-RPS6KB1 day) or FKB (50 mg/kg/day) plus CQ (25 mg/kg/day) was
and MAP1LC3B-II were assessed after FKB treatment (3 intraperitoneally injected every other day starting on day 3
μg/mL for 48). In TRIB3-knockdown cells, the expression following implantation. Tumor growth was monitored over
of p-AKT, p-MTOR, and p-RPS6KB1 recovered after FKB time using bioluminescence values. Our results demonstrated
treatment (Figure 6D and Figure S5), indicating the ATF4- that FKB monotherapy significantly reduced tumor growth
DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling pathway (~ 35 × 108 vs ~ 20 × 108 photons/s, control vs FKB-treated;
played a critical role in FKB-induced autophagy in GBM Figure 9A and 9B). Although no statistically significant differ-
cells. ences were found between the CQ and control arms, FKB
combined with CQ treatment was more effective compared to
FKB alone (~ 20 × 108 vs ~ 15 × 108 photons/s, FKB-treated vs
Autophagy plays a protective role in fkb-treated GBM
FKB + CQ-treated; Figure 9A and 9B). Intriguingly, in con-
cells
trast to the profound effects of combination treatment in vitro
To determine whether FKB-induced autophagy protects (Figures 7 and 8), the growth inhibition induced with FKB
tumor cells, cell viability was assessed in the presence of + CQ on GBM in vivo was not significant compared to FKB
inhibitors of autophagy, 3-MA or CQ. Cell viability was first treatment alone (Figure 9A). This phenomenon may be due to
assessed using the CCK-8 assay. We found that autophagy the bystander effects of CQ [30]. In addition, the optimal dose
inhibitors or knockdown of ATG5 or ATG7 led to further and schedule of this multi-active drug with respect to che-
decreases in cell viability of U251 cells after exposure to FKB motherapy need to be experimentally determined [31].
(Figure 7A and Figure S6). EdU assays also revealed that Kaplan-Meier analysis of the survival data demonstrated a
inhibition of autophagy led to further increases in the inhibi- statistically significant difference between control and FKB-
tion of proliferation in FKB treated U251 cells (~ 18% vs treated (P = 0.03) or FKB + CQ-treated (P = 0.005) mice
~ 7%, autophagy-uninhibited vs autophagy-inhibited cells (~ 28 days vs > 30 days, control vs FKB-treated and FKB
with FKB treatment; Figure 7B and 7C). + CQ-treated; Figure 9C).
Immunohistochemistry was performed on tissue sections
from FKB + CQ-treated animals to examine autophagy and
Inhibition of autophagy switches fkb-induced senescence
proliferation. The marker for autophagy, MAP1LC3B, was
to apoptosis in GBM cells
increased in tumor cells (Figure 9D and S8A), while MKI67/
Based on the fact that FKB induced senescence and protective Ki67, a marker for proliferation was decreased (Figure 9E and
autophagy, we hypothesized that more cells would undergo senes- S8B). Furthermore, senescence (SA-GLB1 and CDKN1A/p21
cence under the combination treatment of FKB with inhibition of [cyclin dependent kinase inhibitor 1A]) was decreased
autophagy. GLB1 staining, however, demonstrated that FKB- (Figure S9), while apoptosis (TUNEL) was significantly
induced senescence was blocked by either 3-MA or CQ, or knock- increased in xenografts from animals treated with FKB
down of ATG5 or ATG7 (Figure 8A and 8B). Inhibition of + CQ (Figure 9F and S8C).
autophagy by either 3-MA or CQ, or knockdown of ATG5 or Luciferase-stable U251 cells were then infected with a lenti-
ATG7 also attenuated accumulation of FKB-treated GBM cells in virus expressing sh-ATG5. The efficiency of shRNA-mediated
the G2/M phase (Figure S7). These results indicated that activation knockdown of ATG5 was confirmed by western blot
of autophagy promoted FKB-induced senescence in U251 cells. (Figure S10A). Consistent with results for CQ-treated GBM
We next investigated whether inhibition of autophagy cells, infection with sh-ATG5 markedly enhanced FKB inhibi-
induced apoptosis in FKB-treated GBM cells. Increases in tion of tumor growth (~ 15 × 108 vs ~ 5 × 108 photons/s, FKB
apoptosis were indeed observed in FKB-treated U251 cells in + sh-NC vs FKB + sh-ATG5; Figure 10A and 10B). Survival time
early (ANXA5+ PI−) and late (ANXA5+ PI+) stages in the under treatment with FKB was also prolonged in mice with sh-
presence of 3-MA or CQ, or with knockdown of ATG5 or ATG5 expressing xenografts (Kaplan-Meier analysis, P = 0.04;
ATG7 (Figure 8C). Cleavage of PARP1 and CASP3, protein median survival, 27 vs > 30 days; Figure 10C).
markers for apoptosis, were significantly increased in FKB Immunohistochemistry performed on tissue sections from
treated U251 cells under pharmacological or genetic inhibi- xenografts demonstrated that sh-ATG5 attenuated accumula-
tion of autophagy, as assessed by western blot (Figure 8D). tion of MAP1LC3B protein in mice under FKB treatment
Taken together, our results demonstrated that FKB in combi- (Figure 10D and S10B), whereas MKI67 was markedly
nation with inhibition of autophagy led to an increase in cells decreased (20% vs 10%, FKB + sh-NC vs FKB + sh-ATG5;
undergoing apoptosis rather than senescence in vitro. Figure 10E and Figure S10C). Finally, senescence (SA-GLB1
and CDKN1A) was decreased (Figure S11), while apoptosis
(TUNEL) was significantly increased in sh-ATG5-expressing
FKB inhibits growth of GBM cells in vivo
xenografts from FKB treated animals (~ 5% vs 20%, control vs
To determine the potential therapeutic efficacy of FKB, tumor FKB + sh-ATG5; Figure 9F and Figure S10C). These data
growth in response to treatment was investigated in an intra- demonstrated that FKB targeted GBM cells both in vitro and
cranial tumor model. Athymic nude mice (n = 20) were in vivo, and that combined treatment with an inhibitor of
implanted with luciferase-stable U251 cells and assigned to autophagy enhanced FKB-induced tumor growth inhibition.
 AUTOPHAGY 9

Figure 7. Inhibition of autophagy enhances FKB-induced inhibition of proliferation in GBM cells. (A) CCK-8 assay to assess cell viability of U251 cells pretreated with
autophagy inhibitors (3-MA or CQ) or transfected with ATG5 or ATG7 siRNAs, followed by exposure to FKB (3 μg/mL) or DMSO for another 48 h. (B and C) EdU assays.
U251 cells pretreated with autophagy inhibitors (3-MA or CQ) or transfected with ATG5 or ATG7 siRNAs, followed by exposure to FKB (3 μg/mL) or DMSO for another
48 h. Cells were stained with Apollo 567 (red) to detect EdU and DAPI (blue) to highlight nuclei, and images were superimposed (scale bars: 50 μm). (B).
Representative images of EdU+ cells stained with DAPI taken under fluorescence microscopy. (C). Graphic representation of percentage of EdU+ cells ([EdU+/DAPI+]
× 100%) as determined in 4 random fields per sample from treated cells as indicated. All data are expressed as the mean ± SD of values from experiments performed
in triplicate. *P < 0.05, ** P < 0.01, and *** P < 0.001 compared to controls. #P < 0.05, and ### P < 0.001 compared between the 2 treatments indicated.

Discussion trials, and has been used in combination with several

chemo- and radiotherapies [33]. Here, we have demonstrated
Multiple studies have provided compelling evidence that con- that FKB, a natural kava chalcone, induced autophagy in
current inhibition of autophagy may improve the outcomes of human glioma cell lines in vitro. This induction of autophagy
cancer therapy [8,32]. CQ, for example, is an autophagy furthermore contributed to the establishment of FKB-induced
inhibitor that is currently in phase I and phase II clinical senescence in GBM cells. Inhibition of autophagy blocked
10 J. WANG ET AL.

Figure 8. Inhibition of autophagy switches FKB-induced senescence to apoptosis in GBM cells. (A and B) SA-GLB1 staining (blue) to detect cellular
senescence in U251 cells pretreated with autophagy inhibitors (3-MA or CQ) or transfected with ATG5 or ATG7 siRNAs followed by treatment with FKB
(3 μg/mL) or DMSO (control) for another 48 h. Images were acquired under bright-field microscopy. Statistic results of SA-GLB1-positive cells and total cell
number (%) were determined in 4 random fields per sample (scale bars: 50 μm). (C) Flow cytometric analysis of ANXA5 and PI staining for the
determination of apoptosis in U251 cells pretreated with autophagy inhibitors (3-MA or CQ) or transfected with ATG5 or ATG7 siRNAs with subsequent
exposure to FKB (3 μg/mL) or DMSO for 48 h. (D) Western blot analysis of lysates (20 µg) prepared from U251 cells pretreated with autophagy inhibitors
(3-MA or CQ) or transfected with ATG5 or ATG7 siRNAs and subsequently treated with FKB (3 μg/mL) or DMSO for 48 h. Membranes were incubated with
antibodies against PARP1, cleaved PARP1, CASP3, cleaved CASP3, and GAPDH (protein loading control). All data are expressed as the mean ± SD of values
from experiments performed in triplicate. * P < 0.05 and ** P < 0.01 compared to controls.
 AUTOPHAGY 11

Figure 9. FKB inhibits tumor growth in an orthotopic model for GBM in mice. (A) U251 cells expressing luciferase were orthotopically implanted into athymic nude
mice, and tumor growth was monitored using the IVIS-200 imaging system for detection of bioluminescence. Bioluminescent signals were measured at days 7, 14,
21, and 28 after implantation. (B) Bioluminescence values plotted as a function of time in days to assess tumor growth (days 7, 14, 21 and 28). (C) Overall survival was
determined by Kaplan-Meier survival curves, and a log-rank test was used to assess the statistical significance of the differences. (D) Images of immunohistochemical
staining for MAP1LC3B and (E) MKI67 in tumors from each group as indicated (scale bars: 50 μm). (F) Images of TUNEL assays (under fluorescence microscopy)
performed on sections from tumors in each group as indicated (scale bars: 50 μm). All data are expressed as the mean ± SD of values from experiments performed in
triplicate. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared between the 2 treatments.

FKB-induced senescence and resulted in the cells undergoing autophagy displayed classic molecular features of the process.
apoptosis. Finally, combination therapy in an in vivo brain MAP1LC3B positive autophagosomes accumulated in human
tumor model in mice led to improved survival. Our results glioma cell lines followed by reduced SQSTM1 protein levels.
therefore provide a basis for the development of alternative To further understand the signaling pathways mediating the
strategies, such as combination therapy with autophagy inhi- response, we also investigated a possible association with ER
bitors, to improve the efficacy of FKB in GBM patients. stress signaling which has been previously linked to the induc-
Autophagy is yet another of the myriad and unexpected tion of autophagy [25]. We demonstrated that FKB upregu-
mechanisms cancer usurps to survive the dynamic change lated ER stress-related proteins and ATF4-DDIT3 signaling.
occurring in the tumor microenvironment, including expo- These results highlighted this pathway as a crucial mediator of
sure to chemo- or radiotherapies [34]. We were able to take a FKB-induced autophagy in GBM cells. We were able to iden-
closer look at this phenomenon when we discovered that FKB tify additional molecular regulators by examining the phos-
induced autophagy in human glioma cell lines. FKB-induced phorylation status of MTOR, as well as critical upstream and
12 J. WANG ET AL.

Figure 10. Knockdown of ATG5 enhances FKB inhibition of tumor growth in an orthotopic model for GBM in mice. (A) Luciferase-stable U251 cells infected with
lentiviruses expressing sh-NC or sh-ATG5 were orthotopically implanted into athymic nude mice, and tumor growth was monitored using the IVIS-200 imaging
system for detection of bioluminescence. Bioluminescent signals were measured at days 7, 14, 21, and 28 after implantation. (B) Bioluminescence values plotted as a
function of time in days to assess tumor growth (days 7, 14, 21 and 28). (C) Overall survival was determined by Kaplan-Meier survival curves, and a log-rank test was
used to assess the statistical significance of the differences. (D) Images of immunohistochemical staining for MAP1LC3B and (E) MKI67 in tumors from each group as
indicated (scale bars: 50 μm). (F) Images of TUNEL assays (under fluorescence microscopy) performed on sections from tumors in each group as indicated (scale bars:
50 μm). All data are expressed as the mean ± SD of values from experiments performed in triplicate. *P < 0.05, ** P < 0.01 and *** P < 0.001 compared between the
2 treatments.

downstream components, AKT and the substrate RPS6KB1. and senescence remains poorly defined [36]. Autophagy does
TRIB3, which is activated by DDIT3, was also induced after appear to play a key role in the establishment of cellular senes-
FKB treatment and led to reduced activation of AKT. We now cence in response to cancer therapy. Acute treatment with
have a working molecular model to further investigate the temozolomide for example has been shown to induce DNA
efficacy of FKB in the treatment of GBM (Figure 11). FKB damage in GBM cells and to produce a transient induction of
induces ER stress which upregulates ATF4-DDIT3-TRIB3. autophagy followed by senescence [37]. Inhibition of autophagy,
Increased activity in these proteins inhibits AKT-MTOR- however, only delays, but does not fully abrogate senescence
RPS6KB1 signaling which leads to increased autophagy flux. [38]. In this study, we found that FKB induced senescence in
While crosstalk between apoptosis and autophagy is well- GBM cells, indicating that while FKB inhibits proliferation of
established [35], the relationship between autophagy, apoptosis, GBM cells, it does not necessarily lead to effective elimination of
 AUTOPHAGY 13

Sirna transfections
Gene-specific siRNAs and negative control siRNA were
synthesized by GenePharma (Shanghai, China) and were
transfected into U251 cells for 48 h using Lipofectamine
2000 (Thermo Fisher Scientific, 11,668–027) according to
the manufacturer’s protocol. The following siRNA sequences
were used to target the RNAs indicated: ATG5#1 and
ATG5#2, 5ʹ-CCT TTG GCC TAA GAA GAA A-3ʹ and 5ʹ-
CAT CTG AGC TAC CCG GAT A-3ʹ; ATG7 #1 and ATG7
#2, 5ʹ-GGA GTC ACA GCT CTT CCT T-3ʹ and 5ʹ-CAG CTA
TTG GAA CAC TGT A-3ʹ; ATF4, 5ʹ-TCC CTC AGT GCA
TAA AGG A-3ʹ; DDIT3, 5ʹ-GCC TGG TAT GAG GAC CTG
C-3ʹ; TRIB3, 5ʹ-CGA GCU CGA AGU GGG CCC C-3ʹ; non-
Figure 11. A hypothetical model for the signaling pathway and the role of
FKB-induced autophagy in GBM cells. FKB induces autophagy through the targeting control siRNA, 5ʹ- UUC UCC GAA CGU GUC
ER stress-dependent ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 signaling path- ACG A-3ʹ.
way in GBM cells. Briefly, FKB upregulates ER stress-related proteins and
ATF4-DDIT3-TRIB3 signaling. Increased activity in these proteins inactivates
the phosphorylation status of AKT and its critical downstream components,
MTOR and RPS6KB1, thus leading to increased autophagy flux. Shrna transfections
Short hairpin (sh)-ATG5 (5ʹ-CCA GAT ATT CTG GAA TGG
them. Here, we demonstrated that introducing inhibition of AAA-3ʹ) were ligated in the lentiviral vector of pLenti-
autophagy to FKB-treated GBM cells induced apoptosis rather shRNA-ZsGreen1 with a puromycin resistant region
than senescence. Thus, the combination treatment is possibly (GeneBio, GY-sh001; Shanghai, China). Luciferase-stable
more effective in actually killing GBM cells. Taken together, U251 glioma cells were plated and infected with lentiviruses
FKB-induced protective autophagy may assist GBM cells in expressing sh-ATG5 for 24 h, according to the manufacturer’s
evading apoptosis and remaining in a senescent state, which protocol. Western blotting was performed to validate knock-
effectively promotes cell survival. Therefore, we speculate that down efficiency, and cells were split for different assays.
the status of autophagy determines a cell’s fate to undergo
senescence or apoptosis. Cell viability and proliferation assays
In summary, our data indicates that FKB inhibits malignant
behavior of GBM cells and induces cytoprotective autophagy Cell viability was assessed with the cell counting kit-8 (CCK-8;
through the ATF4-DDIT3-TRIB3-AKT-MTOR-RPS6KB1 sig- Dojindo, CK04-500). Cells (1.0 × 104 cells/well) were seeded
naling pathway. FKB thus warrants further investigation as a into 96-well plates and incubated at 37°C overnight. FKB
natural bioactive molecule with cancer killing potential. We (Abcam, ab141933), 3-MA (Sigma-Aldrich, M9281), or CQ
predict that combination treatment with FKB and pharmaco- (Sigma-Aldrich, C6628) were dissolved in DMSO (Sigma-
logical autophagy inhibitors will be an effective therapeutic Aldrich, D2650) and diluted to working concentrations in
strategy in the treatment of GBM. culture medium. After the desired treatment, cells were incu-
bated for an additional 4 h at 37°C with 10 μL of CCK-8 in
100 μL of serum-free DMEM. The absorbance at 450 nm was
measured using a microplate reader (Bio-Rad, model 680;
Materials and methods
Hercules, CA, USA). Proliferation was assessed using the
Ethics statement EdU incorporation assay according to the manufacturer’s
protocol (Ribobio, C103103). Briefly, EdU was incorporated
All animal procedures were approved by the Institutional
into proliferating cells and detected through a catalyzed reac-
Animal Care and Use Committee (IACUC) of Shandong
tion with a fluorescently labeled azide. Labeled cells were
University (Jinan, China).
examined under fluorescence microscopy and quantified.

Cell lines and cultures Cell cycle and senescence associated-glb1/β-

galactosidase (SA-GLB1) assays
Human glioma cell lines U251 and U87 were purchased from
the Chinese Academy of Sciences Cell Bank (Shanghai, China; Cells were harvested, incubated with propidium iodide (BD
TCHu58, TCHu138). Human fibroblast glioblastoma cell line Biosciences, 550,825), and subjected to flow cytometry (BD
T98, primary human GBM biopsy xenograft propagated Biosciences, AccuriTM C6; San Jose, CA, USA) for cell cycle
tumor cells, P3, and luciferase-stable U251 glioma cells were analysis. Cellular senescence was evaluated using an SA-GLB1
kindly provided by Prof. Rolf Bjerkvig (University of Bergen). assay. Briefly, cells were fixed with glutaraldehyde and incu-
Cells were cultured in Dulbecco modified Eagle medium bated with X-gal staining solution (Cell Signaling Technology,
(DMEM; Thermo Fisher Scientific, SH30022.01B) supplemen- 9860) overnight at 37°C. SA-GLB1-positive cells were exam-
ted with 10% fetal bovine serum (GE Healthcare Life Sciences, ined under bright-field microscopy and quantified. For in
10,082,147) in 5% CO2 in a humidified incubator at 37°C. vivo, frozen sections were immersed overnight in X-gal
14 J. WANG ET AL.

staining solution and counterstained with nuclear fast red. Apoptosis assay
Nuclei of glioma cells were then counted by nuclear fast red
Cells were harvested, resuspended in binding buffer, and incu-
staining. The percentage of SA-GLB1-positive cells (ratio:
bated with ANXA5-FITC antibody (BD Biosciences, 556,547)
[SA-GLB1+ cells/all nuclei] × 100%) was determined using
according to the manufacturer’s instructions. Apoptotic cells
images taken for 3 random fields per sample (n = 3 samples)
were detected by flow cytometry (BD Biosciences, AccuriTM
under bright-field microscopy.
C6; San Jose, CA, USA), and the results were analyzed using
the software Flowjo (Tree Star; Ashland, OR, USA).
Immunofluorescence staining
Cells were fixed with 4% paraformaldehyde in PBS (Beyotime Intracranial xenograft model and drug therapy
Biotechnology, ST476), permeabilized with 0.5% Triton X-100
(Beyotime Biotechnology, ST795) in PBS, and incubated with Athymic mice (male; 4 weeks old; 20 to 30 g) were provided
rabbit anti-phospho-histone H2AFX (Ser139)/γH2AFX anti- by Shanghai SLAC Laboratory Animal Co., Ltd (Shanghai,
body (1:200; Cell Signaling Technology, 9718S) in 5% bovine China). The mice were anesthetized with chloral hydrate
serum albumin (BOSTER, AR0004) in PBS overnight. and secured on a stereotactic frame. A longitudinal incision
Primary antibody was detected with FITC-conjugated anti- was made in the scalp and a 1 mm-diameter hole was drilled
rabbit IgG (Abcam, 150,077). Cells were incubated in the dark 2.5 mm lateral to the bregma. Luciferase-stable U251 glioma
with DAPI to stain nuclei. Slides were examined under fluor- cells (2 × 105) in 20 μL of serum-free DMEM were implanted
escence microscopy, and images were acquired using a CCD 2.5 mm into the right striatum using a Hamilton syringe.
(charge-coupled device) digital camera (Olympus, DP71; Mice were monitored by bioluminescence imaging every
Waltham, MA, USA). week. Briefly, mice were injected with 100 mg luciferin
(Caliper, 122,796), simultaneously anesthetized with isoflur-
ane, and subsequently imaged with a cooled charge-coupled
Transmission electron microscopy (TEM) device camera (IVIS-200, Xenogen; Alameda, CA, USA).
Bioluminescence values of tumors were quantitated using
Cells were fixed with 4% glutaraldehyde and postfixed with the Living Image 2.5 software package (Xenogen). Mice were
1% OsO4 in 0.1 M cacodylate buffer for 2 h. The samples were euthanized after 30 days and perfused with 4% paraformalde-
then stained with 1% Millipore-filtered uranyl acetate, dehy- hyde in PBS. Brains were coronally sectioned for immunohis-
drated in increasing concentrations of ethanol, and infiltrated tochemistry and TUNEL assays.
and embedded in epoxy resin (ZXBR, Spon 812). Electron
photomicrographs were taken of ultrastructures of GBM cells
with a transmission electron microscope (JEM-1200EX II, Immunohistochemistry
JEOL; Tokyo, Japan).
Paraffin-embedded samples were sectioned (4 µm) and mounted
on microscopic slides. Heat-induced epitope retrieval was per-
Western blot analysis formed in 10 mmol/L citric acid buffer at pH 7.2 in a microwave.
Sections were incubated with the primary antibody at 4°C over-
Cell lysates (20 µg protein) were subjected to western blot analy-
night (MAP1LC3B, 1:200, Cell Signaling Technology, 2775;
sis, according to previously described protocols [39]. Membranes
MKI67/Ki67, 1:200, Cell Signaling Technology, 9027; CDKN1A/
were incubated with the following antibodies from Cell Signaling
p21, 1:50, Cell Signaling Technology, 2947), rinsed with PBS, and
Technology: SQSTM1 (5114), MAP1LC3B (2775), ATG5 (2630),
incubated with horseradish peroxidase-linked goat anti-rabbit
ATG7 (2631), HSPA5 (3177), EIF2A (5324), p-EIF2A (3398),
secondary antibody (ZSGB-BIO, PV-9000). Visualization was
p-EIF2AK3 (3179), AKT (9272), p-AKT (4060), MTOR (2972),
achieved using diaminobenzidine (ZSGB-BIO, ZLI-9033) as the
p-MTOR (2974), RPS6KB1 (9202), p-RPS6KB1 (9204), CASP3
substrate, and slides were counterstained with Mayer hematoxylin
(9662), PARP1 (9542), GAPDH (5174). Additional antibodies
(Beyotime Biotechnology, C0107).
were ATF4 (Santa Cruz Biotechnology, sc-200), TRIB3 (Abcam,
ab73547) and DDIT3 (Abcam, ab11419). Band density was mea-
sured (ImageJ software) and normalized to GAPDH. TUNEL assay
TUNEL assays were performed using the In Situ Cell Death
Cell transfection and GFP-LC3 dot assay Detection Kit following the manufacturer’s protocol (Roche,
GFP-LC3B (pBABEpuro, 22,405)-expressing vectors were 11,684,795,910). After labeling, images were acquired under
obtained from Addgene and deposited by EndoFree Plasmid fluorescence microscopy with a DP71 CCD digital camera.
Maxi Kit (QIAGEN, 12,362). Lentiviral supernatants were
prepared according to the manufacturer’s instructions and
Statistical analysis
provided by GenePharma. Lentiviral infections were carried
out accordingly. Cells displaying ≥ 3 GFP-LC3B dots after Three independent experiments were performed, and results
FKB treatment were considered to be autophagic and were were expressed as the mean ± the standard deviation (SD). Data
counted. Images were acquired with a CCD digital camera were compared using paired Student t tests in GraphPad Prism 5
(Olympus, DP71; Waltham, MA, USA). software (San Diego, CA, USA). P-values determined from
 AUTOPHAGY 15

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Disclosure statement drial and JNK pathway [Research Support, Non-U.S. Gov’t].
No potential conflict of interest was reported by the authors. Biotechnol Lett. 2012 Oct;34(10):1781–1788. Epub 2012 Jun 23.
[15] Eskander RN, Randall LM, Sakai T, et al. Flavokawain B, a novel,
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2012 Aug;38(8):1086–1094. Epub 2012 Apr 30.
This work was supported by Natural Science Foundation of China Grant [16] Ji T, Lin C, Krill LS, et al. Flavokawain B, a kava chalcone, inhibits
(81572487, 81402060 and 81472353), the Special Foundation for Taishan growth of human osteosarcoma cells through G2/M cell cycle
Scholars (No. ts20110814, tshw201502056 and tsqn20161067), Department arrest and apoptosis. Mol Cancer. 2013 Jun 10;(12):55.
of Science & Technology of Shandong Province (2015ZDXX0801A01 and DOI:10.1186/1476-4598-12-55.
2014kjhm0101), Shandong Province Natural Science Foundation [17] Li X, Liu Z, Xu X, et al. Kava components down-regulate expres-
(ZR2014HM074), Shandong Provincial Outstanding Medical Academic sion of AR and AR splice variants and reduce growth in patient-
Professional Program, the Fundamental Research Funds of Shandong derived prostate cancer xenografts in mice [Research Support, N.I.
University (2016JC019), the University of Bergen and the K.G. Jebsen Brain H., Extramural]. PLoS One. 2012;7(2):e31213. Epub 2012 Feb 9.
Tumour Research Centre; National Natural Science Foundation of China [18] Hseu YC, Lee MS, Wu CR, et al. The chalcone flavokawain B
(NSFC) [81572487]; National Natural Science Foundation of China (NSFC) induces G2/M cell-cycle arrest and apoptosis in human oral
[81472353]; National Natural Science Foundation of China (NSFC) carcinoma HSC-3 cells through the intracellular ROS generation
[81402060]; Shandong Province Natural Science Fundation [ZR2014HM074]. and downregulation of the Akt/p38 MAPK signaling pathway
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