Novel Anti-Cancer Properties of Gold
Lotion in Human and Animal Cancer
Cells via Autophagy and Apoptosis
Chi-Chen Lin, Michiko Suzawa & Manashi Bagchi
Institute of Biomedical Sciences, National Chung Hsing
University, Taiwan; Miyauchi Citrus Research Center Ltd.,
Japan; Dr. Herbs LLC, Concord, CA
Formulated Extract From Multiple
Citrus Peels
Gold lotion (GL), a
formulated product made
from the peels of six citrus
fruits (navel oranges,
Citrus hassaku, Citrus
limon, Citrus natsudaidai,
Citrus miyauchi iyo and
Satsuma), was initially
marketed as cosmetics in
Japan to protect skin from
ultraviolet (UV) light
irradiation
Chemical Analysis of GL
Abundant
existence of
Flavonoids:0.45
mg/ml
&
Polymethoxylated
Flavonoids
(PMFs): 0.1
mg/ml
Toxicology & Safety Evaluation
Broad spectrum safety studies were conducted by
Huntington Research Center in UK demonstrated safety
(Reports from Miyauchi Citrus Research Center)
Acute Oral Toxicity Study: > 5,000 mg/kg body
weight in male and female Sprague Dawley rats
Primary Eye Irritation Study: No irritation was
observed in New Zealand white Albino rabbits
Delayed Hypersensitivity in Albino Guinea Pigs:
No evidence of hypersensitivity was observed
Phototoxic Potential: No phototoxicity of GL was
observed in animals when exposed to UV lights
over a span of 4 hrs following treatment with GL
Anti-Cancer Effects of GL
No carcinogenicity was observed in mice when
they were treated for 30 days with 15 cc of GL
No carcinogenicity was observed in dogs when
they were fed 20 cc of GL over a period of 90
days
Consumer reports indicate that topical and oral
treatment of GL demonstrated anti-cancer
properties for melanoma, prostate, lung and liver
cancers.
Miyauchi, 2013, US Patent No. 8,425,952
Anti-Cancer Effect of GL
• Oral administration of GL protects mice against azoxymethane-induced
aberrant crypt foci (ACF) in colonic tissues of mice by decreasing
expression of genes associated with inflammation, such as iNOS and
COX-2 in colonic tissues of mice.
Effective suppression of azoxymethane-induced aberrant crypt
foci formation in mice with citrus peel flavonoids. Mol Nutr Food Res.
2013, Mar;57(3):551-5
• Topical application of GL prevents 7,12-dimethybenez[a]- anthracene
(DMBA)/TPA-induced skin inflammation and tumor formation.
Inhibition of citrus flavonoids on 12-O-tetradecanoylphorbol 13-acetateinduced skin inflammation and tumorigenesis in mice. Food Science and
Human Wellness, 2012; 1(1):65-73.
• Lai et al. also demonstrated that potent anti-cancer effects of GL in
human prostate xenograft tumors in nude mice
Potent anti-cancer effects of citrus peel flavonoids in human prostate
xenograft tumors. Food Funct. 2013 Jun;4(6):944-9.
Materials & Methods
 Cell viability was performed using MTT assay
 Flow cytometry analysis: Cells were stained with acridine
orange and analyzed on a FACS Calibur Flow Cytometer and
the % of cells in each phase of cell cycle was analyzed with
Win MDI software
 V/PI Staining: Biovision V-FITC apoptosis kit was used for
apoptosis assay. The cells were evaluated immediately by
flow cytometry after staining.
 Characterization of Autophagy: Biochemical marker of
autophagy is LC3, which was monitored here by GFP-LC3
puncta and LC3 lipidation on Western blot.
 Accumulation of GFP-LC3 was also visualized by using
fluorescence microscope as bright fluorescent puncta.
Present Investigation
Previous studies demonstrated the anticancer
activites of GL in vivo, however, the underlying
molecular mechanisms of the antitumor activity of
GL is still not understood.
Also, there is no report on the antitumor effects of
GL against human lung cancer cells.
Hence, the anti-cancer efficacy of GL was
evaluated on human NSCLC and other cancer cell
lines.
The possible molecular mechanisms responsible
for its anticancer activity were also investigated.
GL Reduced Cell Viability in Human Lung Cancer cells
PC-9
120
24 h
48 h
72 h
80
***
60
***
40
***
***
***
***
***
***
80
***
60
***
***
40
***
20
20
0
0
0
5
10
15
20
25
0
5
10
GL (%)
60
***
***
***
40
***
***
80
***
***
0
15
GL (%)
20
25
***
40
0
10
***
***
20
5
***
60
20
0
25
24 h
48 h
72 h
100
Cell Viability (%)
***
20
CL1-5
120
24 h
48 h
72 h
100
80
15
GL (%)
A549
120
Cell Viability (%)
24 h
48 h
72 h
100
Cell Viability (%)
100
Cell Viability (%)
H322M
120
0
5
10
15
GL (%)
20
25
GL Induced G2/M arrest and Sub-G1 in A549 Lung Cancer
Control
GL 20 %
24 hr
24 hr
Percent of cells
80
**
60
Control
GL 20%
40
20
ns
*
**
G
2/
M
S
1
G
Su
bG
1
0
48 hr
48 hr
Percent of cells
80
**
60
** *
40
20
Control
GL 20%
**
2/
M
G
S
1
G
Su
bG
1
0
72 hr
**
Control
GL 20%
60
** **
40
20
**
2/
M
G
S
G
1
0
Su
bG
1
72 hr
Percent of cells
80
GL Induced G2/M arrest and Sub-G1 in CL1-5 Lung Cancer
Control
GL 20 %
24 hr
***
Percent of cells
60
24 hr
*
40
20
ns
***
Control
GL 20%
G
2/
M
S
G
Su
bG
1
1
0
48 hr
***
48 hr
Percent of cells
60
Control
GL 20%
40
20
***
*
*
2/
M
G
S
G
Su
bG
1
1
0
72 hr
***
60
40
***
Control
GL 20%
*
*
20
2/
M
G
S
1
G
1
0
Su
bG
72 hr
Percent of cells
80
GL Induced Apoptosis in CL1-5 Lung Cancer
24 hr
GL 20 %
40
Annexin V+ %
Control
CL1-5
**
30
**
20
**
10
48 hr
72 hr
hr
72
hr
48
24
hr
0
Control
20% GL
GL Induced Late-Autophagy in CL1-5 Lung Cancer
24 hr
0%
2.0%
5.4%
40
**
20
0
48
hr
0.%
0.1%
Control
20% GL
**
60
24
AVO +
0.0%
80
0.0%
hr
0.0%
72
0%
CL1-5
Annexin V+ %
0.0%
GL 20 %
hr
Control
48 hr
0.0%
24 hr
10.6%
0
0.1%
LC3
0.0%
44.2%
14.4%
72 hr
b-actin
0.0%
0.6%
20
%
72 hr
48 hr
0
20
%
0
20 (Gold lotion)
%
GL Induced LC-3 Puncta in CL1-5 Lung Cancer
Control
GL 20 %
24 hr
GL 20%
48 hr
72 hr
GL inhibited CL1-5-luciferase Tumor Growth in vivo
Day 28
CL1-5-luc xenograft
days
Daymice-42
48
Control GL 200 ul
Control GL 200 ul
Control
Control
GL
Averag
e (x103)
24.0 ±
15.5
5.2 ±
2.0
Max
45.3 ±
22.6
10.4 ±
1.6
GL
GL inhibits growth of CL1-5-lucifease in nude mice. Representative tumors from the control and
GL-treated groups (day 28, 48). Mean of tumor volume measured at the day 48 after implant.
GL induced Apoptosis and Autophagy in CL1-5 xenograft
Control
ki67
Caspase-3
LC3b
GL 200 ul
Major Flavonoids in 20% GL
Concentration
GL
20 %
1. Nobiletin
10.16 mg/ml
2. Sinesetin
4.26 mg/ml
3. (3,5,6,7,8,3′,4′-Hepta-methoxyflavone
3.84 mg/ml
4. Tangeretin
2.12 mg/ml
5. 3,5,6,7,3′,4′-Hexamethoxy-flavone
0.62 mg/ml
6. (5,6,7,4′-Tetramethoxy-flavone
0.22 mg/ml
7. Naringin
50.72 mg/ml
8. Hesperidin
20.94 mg/ml
Summary & Conclusions
• Present studies in conjunction with earlier studies
demonstrated that GL, enriched in flavonoids
having a high percentage of polymethoxy
flavones, has broad spectrum safety as well as
anti-cancer, anti-angiogenic and tumor shrinking
properties by inducing apoptosis and cell death in
various cell culture system.
• The therapeutic mechanism of GL may be due to
its anti-inflammatory and anti-proliferative
efficacy towards cancer cells and induction of
apoptosis.
Acknowledgements
• Yutaka Miyauchi
• Michiko Suzawa
•
(Miyauchi Citrus Citrus Research Center,Ltd.
(Japan)
• Shimming Li, PhD
•
College of Life Sciences, Huanggang Normal
University, Hubei, China)
• Chi Tang Ho, PhD
•
(Department of Food Science, Rutgers
University, USA)
• Min Hsiung Pan, PhD
•
(Department of Food Science, National
Taiwan University, Taiwan)