Li-Yuan Bai a,c
, Jing-Ru Weng
b,*
, Jing-Lan Hu b
, Dasheng Wang e
, Aaron M. Sargeant f
, and Chang-Fang Chiu c,d a
College of Medicine, b
Department of Biological Science and Technology, China Medical
University, Taichung, 40402 Taiwan,
c
Division of Hematology and Oncology,
Department of Internal Medicine; d
Cancer Center; China Medical University Hospital,
Taichung, 40447 Taiwan; e
Division of Medicinal Chemistry, College of Pharmacy, The
Ohio State University, Columbus, OH 43210 USA; f
Charles River Laboratories,
Preclinical Services, Spencerville, Ohio, 45887 USA
Running Title: Antitumor effects of G15 in oral cancer cells
*To whom correspondence should be address.
Jing-Ru Weng, Ph.D.
Tel: (886)-4-22053366 ext.2511; Fax: (886)-4-22071507
91 Hsueh-Shih Road, Taichung 404, Taiwan
E-mail: columnster@gmail.com
1

Abstract
As GPR30 has been implicated in mediating cancer cell proliferation, this study aimed to examine the antitumor effect of the GPR30 antagonist G15 in human oral squamous cell carcinoma (OSCC). G15 induced dose-dependent cytotoxicity, apoptosis and G2/M cell cycle arrest in a panel of OSCC cells. The results showed that G15 could inhibit the growth of the oral cancer cells with IC
50
value 11.2

M for SCC4, 15.6

M for
SCC9, and 7.8

M for HSC-3, respectively. Flow cytometric analysis and Comet assay indicated that G15 suppressed the viability of SCC4 and HSC-3 cells by inducing apoptosis and G2/M arrest. In addition, G15 down regulated the expression of Akt, cell cycle-related proteins, and mitogen-activated protein kinases, but increased the levels of
LC3B-II and the accumulation of autophagosomes. Inhibition of autophagy by chloroquine does not affect the G15-induced apoptosis in SCC4 cells. Mechanistic evidence indicated that the antiproliferative effect was mediated through the downregulation of cdc2, cdc25C and NF-κB expression. Taken together, our findings suggest the potential of G15 in treating OSCC.
Keywords: GPR30; G15; oral cancer; autophagy; apoptosis
2

1. Introduction
Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer accounting for approximately 3% of all malignancies and 500,000 newly diagnosed cancers every year worldwide. Although the mechanisms underlying the development of
OSCC are not fully understood, there are increased risks for OSCC associated with tobacco use, alcohol, and betel quid chewing. In addition to surgery, chemotherapeutic agents including 5-fluorouracil, taxane, ifosgamide, and methotrexate are the main
treatment approaches for this disease [1]; however, patients eventually succumb to the
tumors once resistance to these agents develops. High incidence and mortality in the face of limited treatment modalities highlight the urgency to develop novel strategies for patients with OSCC .
G-protein-coupled receptor (GPR)30 is a novel estrogen receptor at the plasma membrane proposed as a candidate for triggering a broad range biological activities initiated by endogenous estrogens or dietary phytoestrogen. Documented effects of
GPR30 include stimulation of adenylyl cyclase, mobilization of intracellular calcium stores, and activation of mitogen-activated protein kinase (MAPK) and phosphoinositide
3-kinase (PI3K) signaling pathways [2, 3]. GPR30 expression identified in ovarian, breast,
endometrial, and other cancers shows the potential clinical relevance of targeting this
receptor in cancer patients [4-6]. In OSCC, recent findings suggesting the importance of
estrogen signaling and the value of GPR30 inhibition include 1) high-level expression of estrogen receptor

in tumor cells of human primary OSCC tissues and various OSCC
cultured cell lines [7], 2) superior and apoptotic activity of tamoxifen/cisplatin
3

combination therapy in OSCC cell lines [8], and 3) the correlation of GPR30 overexpression with the development of oral cancers [9].
G15 is a tetrahydro-3H-cyclopenta[c]quinoline analog that inhibits GPR30 with high affinity over ER α/β. While it has been reported to be active against proliferation of uterine epithelial cells stimulated by estrogen in vivo
[10], the activity of G15 in cancer
has been largely unexplored. The aim of this study was to evaluate the effects of G15 on the modulation of cell death, cell cycle arrest, apoptosis and autophagy in OSCC cells.
Our data indicated that G15 induced cell death and G2/M arrest and reduced the expression of G2/M-checkpoint regulators (cdc2 and cdc25c) irrespective of relative
GPR30 expression levels in different oral cancer cells. G15 also induced autophagy in
OSCC cells which appeared to occur independent of the apoptotic effects based on the inability of chloroquine to reverse G15-induced cytotoxicity.
2. Materials and methods
2.1. Reagents, antibodies, and plasmids
G15 ( 4-(6-bromo-benzo[1,3]dioxo-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]- quinoline )
was synthesized as described previously [10]. All agents were dissolved in
DMSO, diluted in culture medium, and added to cells at a final DMSO concentration of
0.1%. Antibodies against the following biomarkers were obtained from Cell Signaling
Technologies (Danvers, MA): Akt, p-
473
Ser Akt, p-
308
Ser Akt, Rel A, cyclin B1, cyclin E,
Cdc2, Cdc25c, LC3B, IKK

/

, p38, p-
180
Thr/
182
Tyr p38, JNK, p-
183
Thr/
185
Tyr JNK, ERK, p-
202
Thr/
204
Tyr ERK, p-
176/180
Ser IKK

/

, PARP, caspase-9 and caspase-3. Procaspase-8
4

and GPR30 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). β-actin was obtained from Sigma-Aldrich (St. Louis, MO). The GFP-LC3 plasmid was kindly provided by Professor Ching-Shih Chen (The Ohio State University). The enhanced chemiluminescence system for detection of immunoblotted proteins was from GE
Healthcare (Little Chalfont, Buckinghamshire, UK). Other chemicals and reagents were obtained from Sigma-Aldrich unless otherwise noted.
2.2. Cell Culture
OSCC cell lines SCC4 and SCC9 were kindly provided by Professor Susan R.
Mallery (The Ohio State University). The HSC-3 cell line was obtained from Japanese
Collection of Research Bioresources (Tokyo, Japan). MCF-7 (ER

/

/GPR30

) human breast cancer cells were purchased from American Type Tissue Collection (Manassas,
VA). All cells were cultured in DMEM/F12 (Invitrogen, Carlsbad, CA) and supplemented with 10 % heat-inactivated fetal bovine serum (FBS; Gibco, Grand Island, NY) and penicillin (100 U/ml)/streptomycin (100
 g/ml) (Invitrogen). All cell types were cultured at 37 o C in an atmosphere of 5% CO
2
.
2.3. Cell Viability Analysis
Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay in 6 to 12 replicates as described previously [11]. In brief,
cells were seeded at 5x10 3 cells per well in 96-well flat-bottomed plates; then, 24 h later, cells were treated with G15 or DMSO at the concentrations indicated in the individual figures. At the end of the treatment, the medium was removed, replaced by 200 µL
5

DMEM/F12 containing 0.5 mg/mL of MTT and cells were incubated in the CO
2
incubator at 37°C for 2 h. Supernatants were aspirated from the wells, and the reduced MTT dye was solubilized in 200 µL/well DMSO. Absorbance at 570 nm was determined using a plate reader.
2.4. Immunoblotting
Western blot analysis was performed as reported previously [11]. Briefly, treated
cells were washed with PBS, resuspended in SDS sample buffer, sonicated for 5 sec, and then boiled for 5 min. After brief centrifugation, equivalent amounts of proteins from the soluble fractions of cell lysates were resolved in 10% SDS-polyacrylamide gels on a
Minigel apparatus, and transferred to a nitrocellulose membrane using a semidry transfer cell. The transblotted membranes were washed thrice with TBS containing 0.05% Tween
20 (TBST). After blocking with TBST containing 5% nonfat milk for 120 min, the membranes were incubated with the appropriate primary antibodies at 1:500 dilution (with the exception of anti–β-actin antibody, 1:2,000) in TBST–5% low fat milk at 4°C overnight, and then washed thrice with TBST. Membranes were probed with goat anti-rabbit or anti-mouse IgG-horseradish peroxidase conjugates (1:2,500) for 90 min at room temperature, and washed thrice with TBST. The immunoblots were visualized by enhanced chemiluminescence.
2.5. Comet assay
Drug-treated or etoposide-treated cells (2 × 10 5 ) at the indicated concentrations were pelleted and resuspended in ice-cold PBS, and were mixed with 1.5% low-melting point
6

agarose. This mixture was loaded onto a fully frosted slide precoated with 0.7% agarose, and a coverslip was then applied to the slide. The slides were submerged in prechilled lysis solution (1% Triton X-100, 2.5 M NaCl, and 10 mM EDTA, pH 10.5) for 1 h at 4 o
C. After the slides had been soaked with prechilled unwinding and electrophoresis buffer (0.3 M
NaOH and 1 mM EDTA) for 20 min, they were subjected to electrophoresis for 30 min at
0.5 V/cm (20 mA). After electrophoresis, the slides were stained with propidium iodide
(PI) (2.5
 g/mL), and nuclei images were visualized and captured at 200× magnification by a fluorescence microscope.
2.6. Cell cycle and apoptosis analysis
Oral cancer cells (2×10
5
/3 mL) were treated with the indicated concentrations of G15 or DMSO for 48 h. After being washed twice with ice-cold phosphate-buffered saline
(PBS), cells were fixed in 70% cold ethanol for 4 h at 4°C. For cell cycle analysis, cells were stained with propidium iodide and analyzed by the multicycler software. For apoptosis evaluation, cells were stained with 4,6-diamidino-2-phenylindole (DAPI) and analyzed using BD FACSAria flow cytometer (Becton Dickinson, Germany).
For assessment of apoptosis, cells were stained with Annexin V-FITC and propidium iodide according to the vendor’s protocols (BD Pharmingen, San Diego).
2.7. Fluorescence Staining for Confocal Imaging
SCC4 cells (2 x 10
5
/3 mL) were plated on cover slips in each well of a six-well plate.
The cells were transfected with 1
 g GFP-LC3 plasmid, followed by the indicated concentrations of G15. Cells were fixed in 2% paraformaldehyde for 30 min at room
7

temperature, and permeabilized with 0.1% Triton X-100 for 20 min. Cells were washed with PBS and then covered before undergoing fluorescent microscopic examination.
2.8. Transient transfection
The plasmid (GFP-LC3) was transiently transfected into SCC4 cells with Fugene
HD reagent from Roche. After 24 h, the cells were treated with G15 for 1 h and subjected to confocal imaging.
2.9. Statistical analysis
All experiments were performed in three replicates. Statistical significance was determined with Student’s t test comparison between two groups of data sets. Differences between groups were considered significant at P < 0.05.
3. Results
3.1. Constitutive Expression of GPR30, ER

and ER

in Oral Cancer Cell Lines
The baseline expression of GPR30 was determined in a panel of human oral cancer cell lines including SCC4, SCC9, and HSC-3 cells, and MCF-7 (ER

/

/GPR30

) breast cancer cells were used as a positive control. Assays included the expression of
GPR30 and the classical nuclear estrogen receptors (ER

and ER

). All three oral cell lines expressed low levels of ER

(Arrowhead in Fig. 1A), and only SCC4 expressed modest levels of ER

(Fig. 1A). GPR30 expression was high in HSC-3 cells and low in
8

SCC4 and SCC9 cells. While ER

levels were similar among the different oral cancer cell lines, the expression analysis showed variability in the relative expression of ER
 and GPR30.
3.2. G15 inhibits growth in multiple oral cancer cell lines
SCC4, SCC9, and HSC-3 cells were used to investigate the antiproliferative effects of G15 in OSCC. Dose-dependent inhibition of cell proliferation occurred in all three cell lines regardless of GPR30, ER

, and ER

status (Fig. 1A). The concentrations of G15 to inhibit cell growth by 50% after 48 h were 11.2, 15.6, and 7.8

M in SCC4, SCC9, and
HSC-3 cells, respectively (Fig. 1B and Table 1). The in vitro efficacy of G15 in inhibiting the proliferation of SCC4 and HSC-3 cells was also examined by direct counting of drug-treated cells (Fig. 1 C). As the IC
50
values at 24 h were higher than those at 48 h (Fig.
1B), we treated cells with DMSO or G15 for 48 h in the subsequent experiments. SCC4 and HSC-3 cells were selected for use in additional experiments in light of the lower IC
50 values and contrasting expression of GPR30 in these cells.
3.3. G15 induces G2/M cell cycle arrest
To determine whether G15 regulates cell cycle progression in oral cancer cells, cells were treated for 48 h with 10-20

M of G15, and were stained with propidium iodide, followed by FACS analysis. Treatment of cells with various concentrations of G15 resulted in dose-dependent increases in the percentage of cells in G2/M phases (Fig. 2A
9

and 2B). Further, we examined the effects of G15 on proteins that were involved in controlling the G2/M phase transition. G15 did not affect the levels of cyclin B1, whereas the levels of cyclin E were increased in both SCC4 and HSC-3 cells (Fig. 2C). G15 treatment also decreased cdc2 and cdc25c levels in both cell lines after 48 h (Fig. 2C).
3.4. G15 induces apoptosis through activation of the caspase cascade and DNA damage
Annexin V/PI staining indicated that the treatment of SCC4 and HSC-3 cells with
G15 led to a dose-dependent increase in the proportion of apoptotic cells (Fig. 3A), suggesting that G15-induced cell death was, at least in part, attributable to apoptosis. We then investigated the apoptotic changes induced by G15 in oral cancer cells. G15 induced dose-dependent increases in the proteolytic cleavage of PARP, caspase-3 and caspase-9, and decreased the expression of procaspase-8, indicative of the involvement of both intrinsic and extrinsic apoptosis pathways (Fig. 3B). The effect of G15 on apoptosis induction was further confirmed by the Comet assay which showed that G15 at 15

M for 2 h (Fig. 3C), which indicated DNA strand breaks in SCC4 and HSC-3 cells. Cells treated with etoposide at 30 µM were used as a positive control.
3.5. G15 reduces Akt phosphorylation and NF-

B signaling
Previous data showed that GPR30 activates several signaling cascades, such as ERK,
PI3 kinase and phospholipase C pathways [12]. Accordingly, we evaluated whether G15
exerts activity through GPR30 in oral cancer cells. As shown, G15 inhibited ERK and
10

p38 phosphorylation in both oral cancer cell lines, but failed to inhibit JNK phosphorylation in HSC-3 cells (Fig. 4). Also, G15 decreased phosphorylated Akt levels at both phosphorylation sites and decreased the phosphorylation of I

B

, which releases
NF-

B upon stimulation by upstream p-IKK

/

. Notably, as activation of NF-

B is preceded by phosphorylation and proteolytic degradation of IkB

, G15 inhibited IkB
 phosphorylation and degradation in these cells. We also measured Bcl-2 expression since
GPR30 has been shown to upregulate genes involved in survival, including Bcl-2 [3], and
found down regulation of Bcl-2 in both oral cancer cell lines (Fig. 4).
3.6. G15 induces autophagy
An insoluble form of GPR37, a member of the G protein-coupled receptor family along with GPR30, has been reported to accumulate in the brains of Parkinson’s disease
patients in association with macroautophagy [13]. To investigate whether G15
influences autophagy, and if these effects contribute to its antitumor activity, we carried out a series of autophagy experiments in the oral cancer cells. G15 induced the formation of autophagosomes as confirmed by the conversion of cytosolic LC3B-I to autophagosomal membrane-bound LC3B-II (Fig. 5A). A greater increase in LC3B-II accumulation was noted with greater treatment duration in the time dependency analysis
(Fig. 5B). To further establish the effect of G15 on autophagy induction, an increase of
GFP-LC3 puncta representing autophagic vacuoles was formed in the cytoplasm (Fig.
5C).
11

3.7. Inhibition of autophagy does not reverse G15-induced cytotoxicity
Autophagy is thought to be a cytoprotective process in starving cells. However,
excess autophagy can induce type II programmed cell death (autophagic cell death) [14].
To study the role of autophagy in G15-induced cytotoxicity, cells were co-treated with
3-MA (a class III PI3K inhibitor that blocks autophagosome formation) or bafilomycin
A1 (a vacuolar-type H + -ATPase inhibitor that blocks autophagosome-lysosome fusion) or chloroquine (CQ; a late stage autophagy inhibitor) and G15 for 48 h and subjected to
MTT assay. The G15-induced reduction in cell viability wasn’t rescued by 3-MA or bafilomycin A1 or CQ (Fig. 6A). Notably, autophagy and apoptosis may be triggered in an independent or mutually exclusive manner, and these two phenomena jointly
determine cell fate [15]. Although apoptosis was confirmed by Annexin V-FITC/PI
double-staining and the cleavage of caspase-3, -9, and PARP (Fig. 3), G15-induced apoptosis were not affected by CQ, the apoptotic cells were determined by flow cytometry (Fig. 6B). Furthermore, we examined the levels of LC3B-II and PARP expression using Western blot. As shown in Fig. 6C, G15 in the presence of CQ significantly induced LC3B-II accumulation compare with G15 alone treatment. The levels of PARP cleaveage in co-treated cells compared with G15 alone treated cells did not showed obvious change (Fig. 6C). These results indicate that G15 simultaneously induces autophagy and apoptosis, and suggest that these two events occur independently.
4. Discussion
12

GPR30 is recognized as a promising target for cancer treatment because it is expressed in many cancers and triggers a broad range of rapid estrogen activity. Here, we report that G15, a GPR30 antagonist, leads to growth inhibition of oral cancer cells in association with apoptosis and autophagy. Furthermore, we found that the antiproliferative effects of G15 were not dependent on the relative expression levels of
GPR30 in the oral cancer cells studied (SCC4, SCC9, and HSC-3). It seems that G15 inhibits the cell growth through GPR30-dependent and -independent signaling in oral cancer cells. Fig. 7 demonstrates our proposed model by which the various biomarkers modulated by G15 are thought to contribute to the inhibition of oral cancer cell growth, including broad effects on the Akt pathway, cell cycle, and autophagy induction.
It is well recognized that Akt activation is a significant prognostic indicator for
OSCC [16] and that activation of NF-

B promotes oral cancer invasion [17]. Many
signaling events downstream of the Akt-NF-

B pathway, such as phosphorylating inactivation of GSK3

by Akt activation [18], and activation of IKK

[19] have also been
shown to promote the development of oral cancer. Moreover, recent evidence indicates that chronic exposure of oral fibroblasts and keratinocytes to subtoxic betel nut extracts leads to activation of Akt and NF-

B [20, 21], suggesting a mechanistic link between the
Akt-NF-

B signaling axis and betel quid chewing/smoking-induced oral carcinogenesis.
The cellular effects of GPR30 activation also include calcium mobilization [22], phosphatidylinositol-3-OH kinase (PI3K) activation [22], and protein kinase C

activation [23]. G15 therefore has the potential to modulate several clinically relevant
targets, which provides the rationale for its possible future development as a chemopreventive agent for the large betel quid-chewing population. In addition to NF-

B
13

and Akt pathways, MAPK has received increasing attention as a target for cancer prevention and therapy. The MAPK pathway consists of a three-tiered kinase core where an MAP3K activates an MAP2K that activates an MAPK (ERK, JNK, and p38), resulting in the activation of NF-

B, cell growth, and cell survival [24]. The previous reports
revealed that GPR30 induces the stimulation of the MAPKs and promotes the
proliferation and invasion in breast cancer cells and endometrial carcinoma [25, 26]. Our
results demonstrate that all MAPKs were downregulated by G15 in SCC4 cells, strongly suggesting that MAPK signaling is among the signaling pathways mediated by G15.
Autophagy is believed to play an important role in tumor development [27] with a
late stage influence on cancer promotion and an early stage influence on cancer
suppression [28]. In the late stage, autophagy has been found to promote tumorigenesis
by helping tumor cells locate the central area of a mass to survive hypoxia and nutrient
deprivation [28, 29].
In the early stage, in contrast, autophagy inhibits tumor cell growth.
Compared to human normal oral keratinocytes, the expression of autophagy-related
16-like 1 (ATG16L1) was found to be upregulated in
OSCC-derived cell lines [30].
We found that G15 increased the expression of both LC3B-I and LC3B-II in SCC4 cells. To confirm that G15 treatment was indeed associated with autophagy induction in the present study, GFP-LC3 puncta were monitored by confocal analysis to document autophagosome formation. Furthermore, to evaluate a pro-survival or pro-death effect of autophagy and a cross-talk between apoptosis and autophagy, CQ, a late stage autophagy inhibitor, was co-administered with G15. The apoptotic effect of G15 was not affected by CQ treatment.
14

In summary, the GPR30 antagonist G15 exerts anti-proliferative effects by inducing
G2/M phase cell cycle arrest and apoptosis and leads to autophagy in oral cancer cells.
The correlation of GPR30 overexpression with oral cancer development [9] provides
rationale for investigating GPR30 antagonists for the treatment of oral cancer; however, our study shows that GPR30 expression level is not a primary factor in G15 cytotoxicity in OSCC. Our work provides novel evidence of autophagy induced subsequent to G15 treatment that requires further study. Although inhibition of autophagy failed to enhance
G15’s apoptotic effect, autophagy as a protective mechanism against G15-induced apoptosis in OSCC cells could not be ruled out. Collectively, these results suggest value in pursuing G15 as a potential new approach in the treatment and prevention of human
OSCC.
Conflict of interest statement
The authors declare no competing financial interests.
Acknowledgements
This work was supported by grants from the Taiwan Department of Health, China
Medical University Hospital Cancer Research of Excellence (DOH102-TD-C-111-005) and National Science Council grant (NSC 101-2320-B-039-029-MY2).
15

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Figures legends
Fig. 1. Differential expression of GPR30, ER

and ER

on OSCC and the antiproliferative effects of G15 in three oral cancer cell lines (SCC4, SCC9, and HSC-3).
(A) A representative western blot analysis showing GPR30, ER

and ER

expression in cultured SCC4, SCC9, HSC-3, and MCF-7 cell lines. (B) Effect of G15 at the indicated concentrations on the viability of oral cancer cells. Cells were treated with G15 in 5%
FBS-supplemented DMEM/F12 medium in 96-well plates at 24 h or 48 h, and cell viability was assessed by MTT assays. Points, mean; bars, SD (n = 6). * P < 0.05, ** P <
0.005 compared to the control group. (C) Dose-dependent antiproliferative effect of G15 at the indicated concentrations in OSCC cells. Cells were seeded onto six-well plates
(200,000 per well) and exposed to the test agent at the indicated concentrations in 5%
FBS-supplemented DMEM/F12 medium. At different time intervals, cells were harvested and counted using a Coulter counter. Values were obtained in triplicate.
Fig. 2.
G15 induced cell cycle arrest in SCC4 and HSC-3 cells. (A) Cell cycle analysis showed increased sub-G1 and G2/M phases in cells treated with G15. (B) The percentage of cell cycle phase in response to each treatment is presented as a histogram. * P < 0.05,
** P < 0.005 compared to the control group. (C) Dose-dependent effects of G15 on the expression of cell cycle-related proteins. Cells were treated with G15 in 5%
FBS-supplemented DMEM/F12 medium for 48h, and cell lysates were immunoblotted as described in Material and Methods. The values in percentage or fold denote the relative intensity of protein bands of drug-treated samples to that of the respective DMSO
19

vehicle-treated control after being normalized to the respective internal reference (total respective protein or
 –actin). Each value represents the average of two independent experiments.
Fig. 3.
Evidence of apoptosis for G15-induced cell death. (A) Annexin V-FITC/ propidium iodide staining. (B) Dose-dependent effect of G15 on caspase-3, procaspase-8, and caspase-9 activation, and PARP cleavage in oral cancer cells after 48 h exposure in
5% FBS-supplemented DMEM/F12 medium. (C) Effects of G15 for 2 h on apoptosis assessed by determining the chromosomal DNA integrity using the Comet assay. Cells exposed to etoposide at 30 µM were used as a positive control.
Fig. 4.
Dose-dependent effects of G15 on the phosphorylation of Akt, ERKs, JNK, p38,
IKK

/

, and I

B, and the expression of NF-

B and GPR30 in SCC4 and HSC-3 cells.
Cells were treated with G15 in 5% FBS-supplemented DMEM/F12 medium for 48h, and cell lysates were immunoblotted as described in Material and Methods. The values in percentage or fold denote the relative intensity of protein bands of drug-treated samples to that of the respective DMSO vehicle-treated control after being normalized to the respective internal reference (total respective protein or
 –actin). Each value represents the average of two independent experiments.
Fig. 5. G15 induced autophagy. (A) The expression of LC3B-II in oral cancer cells.
SCC4 and HSC-3 cells were treated with G15 at the indicated concentrations for 48h. (B)
G15 at 15

M induced LC3B-II accumulation in SCC4 cells in a time-dependent manner.
20

(C) SCC4 cells expressing GFP-LC3 were treated with G15 at the indicated concentrations for 1 h and then fixed by 2.0% paraformaldehyde for confocal analysis.
Fig. 6. Effects of autophagic inhibitors on G15-induced autophagy and apoptosis. (A)
SCC4 cells were treated with 15

M G15 alone or in combination with 10

M 3-MA or
10 nM bafilomycin A1 or 10

M chloroquine (CQ) for 48 h. The cytotoxicity was assessed by MTT assay. Points, mean; bars, SD (n = 6). (B) SCC4 cells were treated with
15

M G15 alone or in combination with 10

M CQ for 48 h, and then Annexin
V-FITC/PI double-staining analysis was performed. (C) Western blot analyses of LC3B and PARP expressions in cells co-treated with 15

M G15 with CQ (10

M) in SCC4 cells.
Fig. 7.
Diagram depicting the pleiotropic effects of G15 on multiple signaling targets that regulate cell cycle, apoptosis, and autophagy.
21