Am J Cancer Res 2020;10(2):581-594
www.ajcr.us /ISSN:2156-6976/ajcr0107151

Original Article
Potent and broad anticancer activities of leaf
extracts from Melia azedarach L. of thesubtropical Okinawa islands
Kuniaki Nerome1, Taku Ito-Kureha2, Tiziana Paganini2, Takao Fukuda3, Yasuhiro Igarashi3, Hiroto Ashitomi4,
Shinya Ikematsu4, Tadashi Yamamoto2
The Institute of Biological Resources, 893-2, Nakayama, Nago-shi, Okinawa 905-0004, Japan; 2Cell Signal
Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Okinawa 904-0495, Japan;
3
Biotechnology Research Center and Department of Biotechnology, Faculty of Engineering, Toyama Prefectural
University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan; 4National Institute of Technology, Okinawa College,
Henoko 905, Nago-shi, Okinawa 905-2192, Japan
1

Received August 6, 2019; Accepted January 23, 2020; Epub February 1, 2020; Published February 15, 2020
Abstract: Plant extracts have been traditionally used for various therapeutic applications. By conducting an initial
screening of several subtropical plants, in this study, we evaluated the anticancer activities of Melia azedarach L.
The extract from Melia azedarach L. leaves (MLE) show high cytotoxic effects on cancer cells and in vivo mouse anddog tumor models. During the initial screening, MLE showed strong antiproliferative activity against HT-29 colon,
A549 lung, and MKN1 gastric cancer cells. In subsequent tests, using 39 human tumor cell lines, we confirmed thepotent anticancer activities of MLE. The anticancer activity of MLE was also confirmed in vivo. MLE markedly inhibited the growth of transplanted gastric MKN1 cancer xenografts in mice. To elucidate the mechanism underlying the
anticancer effects of MLE, MLE-treated MKN1 cells were observed using an electron microscope; MLE treatment
induced autophagy. Furthermore, western blot analysis of proteins in lysates of MLE-treated cells revealed induction of light chain 3 (LC3)-II autophagosomal proteins. Thus, MLE appeared to suppress MKN1 cell proliferation by
inducing autophagy. In addition, in the mouse macrophage cell line J774A.1, MLE treatment induced TNF-α production, which might play a role in tumor growth suppression in vivo. We also performed a preclinical evaluation of
MLE treatment on dogs with various cancers in veterinary hospitals. Dogs with various types of cancers showed amean recovery of 76% when treated with MLE. Finally, we tried to identify the active substances present in MLE. All
the active fractions obtained by reverse-phase chromatography contained azedarachin B-related moieties, such as
3-deacetyl-12-hydroxy-amoorastatin, 12-hydroxy-amoorastatin, and 12-hydroxyamoorastaton. In conclusion, MLE
contains substances with promising anticancer effects, suggesting their future use as safe and effective anticancer
agents.
Keywords: Cancer, anticancer therapeutics, plant extracts, Melia azedarach, autophagy inhibitors

Introduction
The development of new anticancer agents isof great importance to public health. Recent
progress in molecular oncology has led to thedevelopment of molecular-targeting technologies for cancer therapy, and various chemicaland biological therapeutics have proven effective in improving patient survival [1, 2]. Typicalexamples include imatinib, gefitinib, and erlotinib, which inhibit the kinase activities of oncogenic receptor kinases. Despite the progress inanti-cancer therapeutics, cancer is the second
leading cause of mortality globally. The World

Health Organization estimates that, globally, 14
million people are suffering from cancer andcancer caused 9.6 million deaths in 2018.
Japan is no exception and the number of cancer-related deaths in Japan increased from295,864 in 2000 to 353,000 in 2010, an
increase of ~1% per year [3].
Plant extracts have been used as traditional
remedies and are sources of several anticancer
drugs distributed worldwide [4]. Among the firstdrugs from plants used for the treatment ofcancer are microtubule-targeting agents, taxanes, and vinca alkaloids, which include pacli-

Anticancer activities of Melia azedarach L. leaf extracts
taxel, vinblastine, and vincristine [5-7].
Paclitaxel was originally isolated from the barkof the Pacific Yew, and vinblastine and vincristine were extracted from the Madagascar periwinkle [7, 8]. Inhibitors of DNA topoisomerase I,
such as topotecan and irinotecan (CPT-11), aresynthetic analogs of the cytotoxic alkaloid,
camptothecin (CPT), isolated from the bark andstems of the Chinese Happy Tree [9]. Several
synthetic analogs of CPT have better aqueous
solubility, lower toxicity, and greater antitumor
efficacy than the parent drug, among which irinotecan and topotecan are approved for clinical use [9, 10].
In this study, we purified and characterized
potential anticancer compounds extracted
from the leaf of the Melia tree (Melia azedarachL.) collected from the mainland of the Okinawa
region. By exploring over 2,000 subtropicalplants, we successfully discovered several
plants that contain compounds with remarkable anticancer activity against three humantumor cell lines, colon HT-29, lung A549, andgastric MKN1. Among these plants, we chose
the Melia tree for further studies on the basisof the strong anticancer activity of its compounds in vitro and in vivo.
Materials and methods
Preparation of MLE
The Melia leaves, obtained from Nago Municipality (Okinawa, Japan), were washed anddried at 65°C overnight. The samples were
powdered and suspended in nine volumes of
distilled water. The suspension was incubatedat 60°C for 3 h and centrifuged at 9400 × g for
30 min. After centrifugation, the supernatant
was dried by evaporation, and the dried material was suspended in distilled water at a concentration of 10.5 mg/ml; this suspension(crude extract) was tested for toxicity as
described in the following section. The suspension was filtered through a 10-kDa cutoff membrane filter (Pellicon 2 Mini Filters; Millipore
Corporation, Germany), and the filtrate wasused as a 10.5 mg/ml sample of M. azedarachleaf extracts (MLE).
Toxicity test of the crude extract
To determine the fraction with most toxicity, thecrude extract was filtered through various molecular weight cut-off membrane filters (Vivaspin 20: Sartorius); VS2091 (3,000 Da cut-off),
582

VS2011 (5,000 Da cut-off), VS2001 (10,000
Da cut-off), VS2041 (100,000 Da cut-off), andVS2051 (300,000 Da cut-off). The toxicity ofthe six filtrate samples was tested in mice;
4-week-old female ddY (SPF) mice (n = 5; JapanSLC Co., Ltd.) were intraperitoneally administered 0.5 ml of the samples per day, diluted to
1.5 mg/ml. On day 170, the mice were euthanized by administering an excess of pentobarbital via intraperitoneal injection. The livers,
spleens, kidneys, hearts, and lungs were thenexcised and weighed. The organs were fixed in
10% formalin (060-03845: Wako Pure Chemical Industries Ltd.) and sliced into 4-μm-thicksections, followed by hematoxylin and eosin(H&E) staining. All animal experiments werecarried out in specific pathogen-free (SPF) conditions in accordance with the Fundamental
Rules for Animal Experiments and the Guidelines for Animal Experiments Performed at
The Institute of Biological Resources, publishedby the Animal Welfare and Animal Care Committee, including the Animal Ethics Committeeof the Institute of Biological Resources(Okinawa, Japan).
Cell culture
The colon (HT-29), lung (A549), and gastric(MKN1) cancer cell lines (used for biologicalassays of MLE) were obtained from the Divisionof Molecular Pharmacology of Cancer Chemotherapy Center of the Japanese Foundation forCancer Research (Tokyo, Japan). The HT-29(JCRB 1383), A549 (JCRB 0076), and MKN1(JCRB 0252) cancer cell lines (used for molecular mechanism analysis of MLE) were purchased from the Japanese Collection of Research
Bioresources (JCRB) Cell Bank. The cells were
cultured in Roswell Park Memorial Institute
1640 (RPMI 1640) medium (Gibco; Life Technologies) supplemented with 10% fetal bovine
serum (FBS_F9423: Sigma-Aldrich). IMR90
and J774A.1 cells were purchased from theAmerican Type Culture Collection and cultured
in Dulbecco’s modified Eagle’s medium (DMEM)
containing 10% FBS.
Measurements of cell growth inhibition
Cell growth inhibitory capacity of the plant
extracts was measured as described previously[11-13]. Briefly, 10,000 cells were seeded into
each well of 96-well plates in RPMI 1640 with
5% fetal bovine serum and allowed to attachovernight. The extracts were prepared in a
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Anticancer activities of Melia azedarach L. leaf extracts
series of dilutions (10 -1-10 -8) in RPMI 1640
medium, and 100 µl of the extract was added
to each well and incubated for 2 days. Subsequently, cell growth was determined according
to the sulforhodamine B assay [14] as follows:
the cells were washed five times with 1% acetic
acid and incubated with 50 µl of 0.4% sulforhodamine B (in 1% acetic acid; Wako Pure Chemical Industries Ltd., Osaka, Japan). Then, 150µl of 10 mM unbuffered Tris reagent (pH 10.5;
Wako Pure Chemical Industries Ltd.) was added
to each well, and the absorbance was measured at 525 nm using a standard plate reader.
The concentration of test samples inhibiting
50% of the cell growth (GI50) was determinedusing the results of the above experiment.
Cell cycle analysis
MKN1 cells were cultured at an initial density of
2 × 105 cells/well in the presence or absence ofMLE (105, 10.5, and 1.05 µg/ml) or mitomycin
C (1 mg/ml, 139-18711: Wako Pure Chemical
Industries Ltd.). At 1 and 2 days after initial
seeding, the cells were ethanol-fixed andwashed with PBS and incubated with 0.5 mg/
ml RNase A (Sigma, R4875) for 30 min at 37°C.
Cells were then stained with propidium iodide(Wako, 169-26281) and subjected to flow
cytometry using a FACS Calibur (Japan BD
Biosciences, Tokyo, Japan). FlowJo V10.4.2 (BDBiosciences) was used to analyze the results.
Immunoblot analysis
Cells were incubated with lysis buffer (50 mM
Tris-HCl [pH 7.5], 150 mM sodium chloride, 1
mM ethylenediaminetetraacetic acid, 1% NP40, and protease inhibitor cocktail [cOmplete
Mini, Roche]). Proteins in the cell lysate wereseparated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred
to polyvinylidene difluoride membranes (Immobilon P, Merck Millipore). The membranes werethen incubated with primary antibodies, andthe immunoreactive proteins were visualizedusing anti-rabbit or anti-mouse IgG conjugatedto horseradish peroxidase (RPN1231-2ML: GE
Healthcare), followed by detection using an
Enhanced Chemiluminescence (ECL) Detection
System with film (GE Healthcare). The primary
antibodies used were as follows: Anti-LC3 (lightchain 3, PM036) was purchased from Medical& Biological Laboratories Co., Ltd. Anti-cleaved
caspase 3 (Asp 175, clone 5alE, #9664S), anti583

cleaved poly-ADP ribose polymerase (PARP)(Asp 214, human-specific, #9541S), and antiglyceraldehyde 3-phosphate dehydrogenase(GAPDH, rabbit mAb 14C10, #2118L) were purchased from Cell Signaling Technology Japan,
K.K. (Tokyo, Japan).
Tumor formation assay
MKN1 cells (106 cells) were subcutaneously
injected into BALB/c Slc-nu/nu (SPF) female
mice (5 weeks old, Japan SLC, Inc.). Whentumor sizes reached 50-150 mm3, the mice
were orally administered 0.5 ml MLE (1.5 mg/ml) every 4 days over a 3-week period. Tumor
growth and body weight were monitored daily.
Tumor volumes were determined using thestandard formula: V = 4πab2/3 (V: tumor volume, 2*a: the major axis, 2*b: the minor axis).
Transmission electron microscopy (TEM)
Cells were washed, fixed in 2.5% glutaraldehyde for 30 min at 25°C, treated with 2% osmium tetroxide, dehydrated in a graded series of
ethanol baths, infiltrated, and embedded inresin. Then, 50-nm-thick sections were cut
using a Leica EM UC6 Ultramicrotome (Leica
Microsystems), followed by staining with 4%
uranyl acetate and 2% lead citrate, and visualization by TEM using a JEOL JEM-1230R electron microscope (JEOL, Ltd.) at an accelerating
voltage of 100 kV.
Structure analysis of constituents in MLE
MLE (2 g/100 mL) was fractionated by C18
reversed-phase chromatography with 0.1% formic acid/acetonitrile (ACN) using a six-step gradient (20, 30, 40, 50, 60, and 70% ACN (v/v)).
Six fractions were obtained (AF28, AF37, AF46,
AF55, AF64, and AF73). Each fraction was
evaporated and dried to obtain 2044, 53.6, 56,
9.1, and 3.3 mg of AF28, AF37, AF46, AF55,
and AF64, respectively. No significant amountof material was recovered from the AF73 fraction. The fractions with anti-proliferative activity
were subjected to high-performance liquid
chromatography (Cosmosil AR-II, 20 × 250 mm)
using a 0.1% formic acid/methanol (MeOH) gradient (MeOH concentration: 50% for 0-5 min,
50-55% for 5-9 min, 55% for 9-15 min, 55-70%
for 15-22 min, and 70% for 22-27 min) to yieldcompounds with possible biological activities.
The structures of the isolated compounds were

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Anticancer activities of Melia azedarach L. leaf extracts
Table 1. Comparison of anticancer activity between MLE and Mitomycin C
Initial screening of anticancer compounds using three tumor cell lines
TumorsCell lines GI50* of crude extract from Melia tree leaf (μg/0.1 ml) GI50* of Mitomycin C (μg/0.1 ml)
Lung cancer
A549
1.50
0.29
Colon cancer
HT-29
0.31
0.31
Gastric cancer MKN1
1.30
1.42
*GI50: concentration inhibiting 50% of cell growth.

analyzed using nuclear magnetic resonance(NMR) spectroscopy and mass spectrometry(MS).
Statistical analysis
Statistically significant differences between
groups were determined using Student’s t-test,
Wilcoxon test, or a SAS mixed procedure. Pvalues less than 0.05 were considered statistically significant. Values are expressed as themeans of triplicate samples ± the standard
error of the mean.
Preclinical evaluation of MLE in canine cancers in veterinary hospitals
Dogs were orally administered, once or twice aday, with 0.5, 1.0, and 1.5 ml MLE for dogs with< 5, 15-20, and > 20 kg body weight, respectively. Thirty-three dogs with various cancers
from 15 veterinary hospitals in Niigata (2 Hospitals), Fukushima (1), Tokyo (2), Saitama (1),
Shizuoka (2), Aichi (2), Gifu (1), Osaka (1), andOkinawa (3) prefectures were tested. Dogs
were divided into test (n = 25) and placebo(only glycerin, n = 8) groups. Dogs previously
treated with anticancer or immune compounds
were excluded from this study. The following
factors were evaluated: appetite, locomotor
activity, body weight, tumor size, metastasis,and recurrence. The investigation was undertaken over a 6.5-month period. The test andplacebo groups were clinically treated in accordance with the general guidelines and the
Research Council guide for the care and use ofanimals of each hospital.
Results
Initial screening of anticancer compounds
from subtropical plants
We searched for anticancer substances from
more than 2,000 subtropical plants collectedin Okinawa, and at least 10 plants were foundto possess anticancer compounds. Particularly,584

crude extract from Melia tree leaves appeared
to show high anticancer activity against colon(HT-29), lung (A549), and gastric (MKN1) cancer cells (Table 1). The concentrations of Melia
leaf extract inhibiting 50% (GI50) of HT-29,
A549, and MKN1 cell growth were 0.31, 1.50,
and 1.30 µg/0.1 ml, respectively. These GI50
values were, except for that of A549 cells, principally similar to those of mitomycin C, which
was used as the control anticancer drug. The
crude extract of Melia tree leaves, however,
unexpectedly appeared to contain toxic substances against mice. Therefore, the identification of toxic fractions was essential for the
extract to be used for cancer treatment.
Identification of highly toxic components fromthe crude extract
When the crude extract of Melia tree leaf was
intraperitoneally administered to mice, withvarying doses, all 10 mice died within 2 h; pathological analysis revealed that their livers werelargely swollen, and centrilobular necrosis andcongestion was observed (Table 2). Splenicand myocardial necrosis were also observed.
Although the livers did not show hemorrhage,
hemorrhagic spots were observed in the heart(data not shown). However, mice that were orally administered the crude extract survived(Table 3). These results suggested that thecrude extract contains substances that are
toxic for mice when administered peritoneally.
To find the toxic fraction in the extract, theextract was filtered through various molecularweight cut off membrane filters, and the filtrates were tested for their toxicity against
mice. Intraperitoneal administration of the
300-kDa cut-off filtrate, which contained only
components of the crude extract under 300
kDa, resulted in 80% death of the treated mice
(Table 3). The histopathological features inmice killed by the 300-kDa-cut-off filtrate weresimilar to those in mice killed by the crude
extract, although splenic necrosis was observed
only in mice administered the unfiltered crude
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Anticancer activities of Melia azedarach L. leaf extracts
Table 2. Pathological changes in extract-treated mice
Administered extract
Massive necrosis in the liver
Centrilobular necrosis in the liver
Congestion in the liver
Hemorrhage in the liver
Splenic necrosis
Myocardial necrosis

Unfiltered
++
+++
++
++

Cut-off molecular weights of the membrane filters
300 kDa 100 kDa 10 kDa 5 kDa 3 kDa Control (PBS)
++
+++
+
-

+++: Severe, ++: Moderate, +: Mild, -: No particular; PBS: Phosphate buffered saline.

Table 3. The mortality rate in mice administered various extracts of Melia tree leaf
Route of administration

Unfiltered

Intraperitoneal administration
Oral administration

*5/5
0/5

Cut-off molecular weights of the membrane filters
300 kDa 100 kDa 10 kDa 5 kDa
3 kDa
4/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5
0/5

Control (PBS)
0/5
0/5*Number of dead mice/number of mice in group; PBS: Phosphate buffered saline.

Table 4. Evaluation of anticancer activity against nine tumors consisting of 39 tumor cell lines
TumorsCell lines

GI50*(μg/0.1 ml)

TumorsBreast Cancer

HBC-4

1.50

Lung Cancer

BSY-1

0.11

HBC-5

1.50

NCI-H522

0.97

MCF-7

1.50

NCI-H460

1.50

MDA-MB-231

0.90

A549

1.50

Brain Cancer

Colon Cancer

TumorsCell lines

GI50*(μg/0.1 ml)

NCI-H23

1.50

Kidney Cancer

RXF-631L

0.64

NCI-H226

0.90

ACHN

1.50

DMS273

0.24

DMS114

1.28

LOX-IMVI

0.88

U251

1.50

St.4

0.49

SF-268

1.50

MKN1

1.50

SF-295

0.44

MKN7

1.50

SF-539

1.50

MKN28

1.50

SNB-75

1.50

MKN45

0.52

SNB-78

1.50

HCC2998

0.82

KM-12

Skin Cancer

GI50*(μg/0.1 ml)

Cell lines

Ovarian Cancer

OVCAR-3

0.88

1.34

OVCAR-4

1.28
1.50

HT-29

0.14

OVCAR-5

HCT-151.50

OVCAR-8

1.16

HCT-116

0.41

SK-OV-3

1.50

Gastric Cancer

Prostate Cancer

MKN74

0.77

DU-145

1.50

PC-3

1.50

*GI50: concentration inhibiting 50% of cell growth.

extract (Table 2). The toxicity was thus eliminated from unfiltered crude extract if the components over 100 kDa were removed. Deathand histological abnormalities were not observed in mice intraperitoneally administered filtrates filtered through 100-, 10-, 5-, and 3-kDa
cut-off membrane filters. Therefore, in all further experiments, only the 100-kDa cut-off filtrate was used as the MLE solution.
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Growth inhibition activity of MLE against various cancer cell lines
To characterize the anticancer activity of MLE,
the growth inhibitory effect of MLE against 39
cancer cell lines was examined (Table 4). MLE
showed anticancer activity against a panel of
39 cancer cells with GI50 ranging from 0.11
µg/0.1 ml against the BSY-1 breast cancer cell
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Anticancer activities of Melia azedarach L. leaf extracts

Figure 1. MLE suppresses tumor growth in the mouse xenograft model. Three mice (BALB/c Slc-nu/nu) were injected with 106 MKN1 cells (A-C) or HT-29 cells (D) subcutaneously and were orally administered with 0.5 ml of MLE
solution (1.5 µg/ml) or PBS every 2 days. The administration started when tumors reached a volume of 50-150mm3. Average tumor volume was plotted with standard error (SE) against days after the administration. The tumor
growth period between PBS- and MLE-administered mice were analyzed using SAS mixed procedure, and P value
was determined (A). Mice were euthanized at 21 days after the administration, and the photographs of euthanized
mice are shown; red arrows indicated tumors (B). Tumors of MLE-administered mice were histopathologically examined after H&E staining (C). The infiltrated mononuclear-like cells (indicated by arrow) were also examined withelectron microscopy (inset in C). The thin sections of tumors were also Giemsa stained, and the M phase cells wereanalyzed (D).

line to 1.5 µg/0.1 ml in many other cancer celllines. The anticancer activities of MLE against
the 39 cancer cell lines showed significant differences, even among cell lines from the same
cancers. As previously reported [13, 15], we
also established a panel of these 39 cancer
cell lines and developed a database of theirchemosensitivities, in which various drugs were
profiled for their “fingerprints” patterns of differential activity against these cell lines [15]. A
significant correlation between a drug’s fingerprint and its mode of action was observed.
Based on the fingerprint database, the mechanism of anticancer activity of MLE was found to
involve vacuolar-type H+ ATPase inhibition
because the fingerprint pattern of MLE was
586highly similar to that of bafilomycin, which is awell-known vacuolar-type H+ ATPase inhibitor.
MLE efficiently prevented tumor growth in vivo
To determine whether the strong anticancer
activities observed against various cancer celllines can influence the tumor growth in an invivo mouse model, we transplanted MKN1 gastric cancer cells into nude mice (Figure 1). Afterthe size of the tumors reached 50-150 mm3,
the oral administration of MLE was started. The
administration significantly reduced the growthof the tumor. In PBS-treated mice, the tumor
size increased from 100% on the first day of theadministration to 200% and 300% on days 7
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Anticancer activities of Melia azedarach L. leaf extracts

Figure 2. Activation of macrophages by MLE. J774A.1
mouse macrophage cells were treated with MLE,
PBS, or LPS (Lipopolysaccharide from E. coli O55
served as a positive control) for 24 h. Cell culture
supernatants were collected, and TNF-α levels were
measured using ELISA (R&D Systems). Two different
lots of MLE (LOT 28 and LOT 28) were compared. Statistically significant differences between group meanvalues were determined using the Wilcoxon test.

tumors. Cell-division images were more often
observed in MLE-treated tumors than in control-treated tumors (Figure 1D). For quantitative analysis, we examined the frequency of
metaphasic cells in the proliferating cell population. Proliferating cells were defined as Ki67positive cells. The number of proliferating cells
and metaphasic cells in five fields were counted
under a microscope at 200x magnification. The
percentage of metaphasic cells among the proliferating cells was about 2 times higher intumors from PBS-treated mice than that intumors from MLE-treated mice: 10.5% in PBStreated mice and 4.7% in MLE-treated mice.
To examine the possibility of apoptosis induction by MLE treatment, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)
staining of thin tumor sections was attempted.
In MLE-treated gastric cancer tissue, muchhigher TUNEL-positive cells were observed thanin PBS-treated ones (data not shown).

and 12, respectively. In contrast, tumors in
MLE-administered mice were approximately
two-fold smaller than those in PBS-treatedmice at 12 days after the start of the administration. The result indicated that MLE treatment
efficiently suppressed tumor growth (Figure1A). We observed the effect of MLE administration until day 21. The difference in the appearance of tumors between MLE-administeredmice and PBS-administered mice was significant (Figure 1B). Similar results were obtainedin the mice transplanted with HT-29 colon cancer cells (data not shown).

Activation of macrophages by MLE

Histopathological analysis of tumors from
MLE-administered mice

To elucidate the tumor suppression activity ofMLE, we investigated the effect of MLE on
MKN1 cells in vitro. The number of MKN1 cells,
treated with either 105 or 10.5 µg/ml of MLE,
significantly decreased as compared to thosetreated with PBS (Figure 3A), which was consistent with the observed antitumor activity. In
contrast, MLE hardly affected the proliferationof human normal fibroblast cells (IMR90; Figure
3B). Unexpectedly, we observed that the number of trypan blue positive MKN1 cells was
unaltered for 2 days in the presence or absenceof MLE (Figure 3C), suggesting that MLE did notaffect the viability of MKN1 cells during theexperimental period. Thus, MLE efficiently prevented tumor growth in vivo and in vitro while it
did not directly cause cell death but suppressed
cell proliferation.

Histopathological analysis of MKN1 cell-transplanted mice demonstrated the difference
between the tumors from MLE-administered
and PBS-administered mice. In H&E-stained
sections of the tumors from MLE-administeredmice, infiltrates of mononuclear-like cells wereobserved at the peripheral tumor sites. The
dead gastric cancer MKN1 cells were enclosedwith infiltrated cells, which showed structurescharacteristic of NK cells (Figure 1C).
HT-29 colon cancer cells were also analyzed.
The observation of Giemsa-stained tumor slices revealed differences between images of M
phase cells in MLE-treated and PBS-treated587

The presence of infiltrated cells in tumors from
MLE-administered mice suggested that MLE
might activate immunological cells. To examine
the possibility, J774A.1 mouse macrophagecells were treated with MLE, and the released
tumor necrosis factor (TNF)-α was measured
by ELISA. As expected, MLE markedly induced
the release of TNF-α, an important antitumor
cytokine, from the macrophages (Figure 2).
Suppression of MKN1 cell proliferation by MLE

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Anticancer activities of Melia azedarach L. leaf extracts

Figure 3. Effect of MLE on cellular proliferation in vitro. MKN1 cells (A) and IMR90 cells (B) were cultured in thepresence or absence of MLE or mitomycin C at an initial density of 2 × 105 or 105 cells/well, respectively. Viable
cells were counted 1 and 2 days after seeding. The MLE- or PBS-treated cells were further cultured for 2 days,
and live and dead cells were counted using trypan blue staining (C). (D) Cell cycle analysis of MKN1 cells. Cell

588

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Anticancer activities of Melia azedarach L. leaf extracts
cycle progression was analyzed using flow cytometry after treatment with MLE for 3 days. Data is representativeof three independent experiments. The percentage of SubG1 phase cells from the replicates is also represented.
(E) Immunoblot analysis of cleaved caspase 3 and cleaved PARP1. GAPDH serves as a loading control. Data arerepresentative of two independent experiments. Bands were quantified by densitometry (right end). Statisticallysignificant differences between group mean values were determined using the Student’s t-test (***P < 0.001, **P< 0.01, and *P < 0.05).

To elucidate the mechanisms of the MLEinduced suppression, we first performed cell
cycle analyses. Cell cycle progression was
retarded, and the percentage of sub G1 fraction appeared to be increased by MLE treatment (Figure 3D). We examined the involvement of apoptosis in the retardation of the cell
cycle. However, western blot analysis revealed
no increase in cleaved caspase 3 and cleaved
PARP1 expression in MLE-treated cells (Figure3E) in contrast to the cells treated with mitomycin C, which is well-known to induce apoptosis.
The result suggested that MLE affected cellproliferation by mechanisms other than apoptosis.
MLE induces autophagy
We then examined the intracellular structure of
MLE-treated MKN1 cells using TEM. We observed autophagosome-like structures (Figure
4A). In addition, autophagy markers, LC3 I and
LC3 II, were significantly expressed in MLEtreated MKN1 cells (Figure 4B and 4C). These
data suggest that cell growth suppression by
MLE was due to autophagy of one of themechanisms.
Preclinical evaluation of MLE treatment efficacy for canine cancer
To evaluate the efficacy of MLE treatment inhigher vertebrates, we performed preclinicalevaluations in 33 dogs with various cancers,
including breast, liver, osteosarcoma, bladder,melanoma, mastocytoma, chromaffinoma, andthyroid carcinoma in 15 veterinary hospitals(Figure 5B). Dogs were divided into two groups(MLE: n = 25 and placebo: n = 8). We found thatcancer progression was significantly ameliorated by MLE treatment in 76% of the dogs, compared to that in the placebo group (Figure 5A).
Importantly, no abnormal and adverse reactions were observed in dogs. Thus, it could beconcluded that MLE efficiently prevents cancer
progression in dogs. These results suggested

589

that MLE administration may be useful to treat
cancer in humans and dogs. Furthermore, 14major canine cancers, including hepatocellular
carcinoma, osteosarcoma, and multiple myeloma, appeared to be markedly ameliorated(Figure 5B).
Identification of chemical compounds with
anti-proliferative activity
To identify the constituents in MLE, a six-step
gradient (20%, 30%, 40%, 50%, 60%, and 70%
ACN v/v as eluate) separation process using C18
reverse-phase chromatography was carriedout. We isolated five fractions (AF28, AF37,
AF46, AF55, and AF64) and evaluated theirantiproliferative effects. We found that thecomponents of AF46, AF55, and AF64 had antiproliferative effects, whereas the componentsof AF28 and AF37 barely showed any effects(Figure 6A).
We next determined the structures of the compounds in MLE using NMR and MS. AF46, AF55,
and AF64 all contained azedarachin B-related
moieties (Figure 6B). AF46 contained 12-hydroxyamoorastatone and 12-hydoroxyamoorastatin; AF55 contained 12-O-acetylazedarachin B,
meliatoxin B2, and toosendanin; and AF64 contained azedarachin B, 12-diacetyltrichilin, 3diacetyltrichilin H, neoazedarachin B, and 7, 14
epoxyazedarachin B.
Discussion
In this study, we demonstrated the definite anticancer activity in vitro and in vivo of an extractfrom M. azedarach L. (Melia tree), found in thesubtropical Okinawa. Plant extracts have been
adopted for the treatment of various diseases[16, 17]. Particularly, limonoids have been
investigated for possible use as medicinal remedies in humans and animals [5]. In this study,
we showed that extracts of M. azedarach L.
leaves contain compounds that are cytotoxic tocancer cells but not to normal cells (Tables 1,
4; Figure 3A, 3B). Similar cytotoxic activity waspreviously reported for the extracts from the

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Anticancer activities of Melia azedarach L. leaf extracts

Figure 4. MLE induces autophagy in MKN1 cells. A. Representative images of MLE-treated MKN1 cells acquired bytransmission electron microscopy. Autophagosomes highlighted in the square region are indicated via a zoomed-in
image. Scale bars: 2 μm and 0.5 μm (zoomed). B. Immunoblot analysis of LC3. GAPDH serves as a loading control. Data is representative of three independent experiments. Bands were quantified by densitometry (right end).
C. FACS analysis of LC3-expressing cells and fold change of Median fluorescence intensity (MFI). Cells that wereserum-starved for 4 h were used as positive controls. Data from two independent experiments are represented.
Statistically significant differences between group mean values were determined using the Student’s t-test (***P< 0.001, **P < 0.01, and *P < 0.05).

bark, roots, and seeds of M. azedarach L. [1822]. However, their underlying mechanism ofactivity remains elusive. We prepared theextract from leaves instead of bark, roots, orseeds of M. azedarach L. and showed that theextract induced autophagy in MKN1 gastriccancer cells (Figure 4). In contrast, toosendanin, which was identified in fraction AF55 in
our study, was reported to induce apoptosis in590

U937 cancer cells [23]. In contrast, apoptosis
induction was not detected in MLE-treated
MKN1 cells (Figure 3E), but an increase in thenumber of TUNEL-positive cells was observedin tumors from MLE-treated mice (data notshown). These results suggest that MLE might
inhibit cell growth depending on the type ofcancer cells. Therefore, it was necessary to
examine the effect of MLE on other cancer cell
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Anticancer activities of Melia azedarach L. leaf extracts
with anticancer drugs [27-30].
Accordingly, the development ofpotential anticancer drugs with
autophagy-inducing activity may
be an effective strategy in cancer therapy [31].
An important observation--made in a study that addressed
the mechanism of tamoxifen
resistance--was that althoughtamoxifen induced autophagy[32], whether the autophagy
was cytotoxic or rendered surviving cells tamoxifen-resistant
was unclear [33, 34]. Eventually,tamoxifen-induced autophagy
was shown to be cytotoxic tocancer cells due to autophagic
degradation of cellular k-Ras
protein [35]. Cells that lackk-Ras lose viability. In addition,
in response to stress, c-Jun
N-terminal kinase (JNK) was
activated, phosphorylating Bcell lymphoma-2 (Bcl2) or BIM to
dissociate Beclin from Bcl2,
which led to the formation of anactive Beclin1-VPS34 complex
responsible for autophagy induction [36]. In summary, celluFigure 5. Effects of MLE on various cancers in dogs. A. Comparative eflar stress can induce cell growthficacy of MLE against canine cancers; yellow, amelioration; red, completearrest or death. Some antitumor
response; light blue, aggravation; dark blue, severe aggravation. B. Classification of therapeutic efficacy of MLE on different canine cancers. The
drugs that inhibit the mammalidegree of MLE efficacy is shown on the left. Cases of treatment efficacy
an target of rapamycin complex
and MLE therapeutic efficacy on a large number of canine cancers are
1 (mTORC1), a negative regulaalso described in the center and the right of the table.
tor of the autophagic pathway,
induce autophagy [37, 38]. Clatypes, which might reveal different mecharification of the intracellular mechanism bynisms of cell death, such as apoptosis, necropwhich MLE induces autophagy would help guidetosis, or autophagy. Since various canine canits therapeutic use.
cers were ameliorated by MLE treatment, difData showing that autophagy is regulated byferent cytotoxic activities might have affectedcytokines in various biological contexts havedifferent cancers. Since we did not observe
been accumulating [39, 40]. For instance,
severe adverse effects in MLE-treated dogs,
TNF-α could play a role in stimulating autophaMLE might be useful as oral cancer therapeugy, although the underlying mechanism has nottics without remarkable side effects.
been well established. The mechanisms might
A few studies have suggested that autophagy
differ among cell types [41-44]. Conversely,
induction is an effective strategy for cancer
autophagy affects the transcription of cytokine
treatment [24]. However, various recently pubgenes and secretion of cytokines [45, 46]. The
lished studies indicate that autophagy inducautophagy inhibitor 3-methyladenine suppresstion helps the survival of adjacent cancer cells
es TNF-α transcription and secretion [47, 48].
following chemotherapy [25, 26], and autophaAlthough we observed increased levels of TNFgy inhibitors have been used in combination
α in MLE-treated macrophages in vitro, it is591

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Anticancer activities of Melia azedarach L. leaf extracts

Figure 6. Structures of the constituents with antiproliferative activity in
MLE. A. The antiproliferative activities of the five MLE fractions from the C18
column against HT 20 cells were evaluated as described in Materials andMethods (measurements of cell growth inhibition). B. Structures of the identified compounds in the fractions; AF46, AF55, and AF64, which showedstrong antiproliferative effects.

unclear whether TNF-α induction by MLE is
associated with a reduction of tumor growth invivo. Further investigation of crosstalk between
immunological cells and cancer cells would elucidate the mechanism of MLE-induced retardation of cancer development.
In this study, we not only identified MLE as an
autophagy-inducing agent but also found that
MLE treatment reduced the growth of multiple
cancers in clinical tests with dogs and cats.
Further studies should be conducted to examine whether cells that underwent autophagy following MLE treatment were killed. In addition,
the development of therapeutic reagentsagainst human cancers derived from MLE
should be explored.

592

Disclosure of conflict of interest
None.

Address correspondence to: Dr. Kuniaki Nerome,
The Institute of Biological Resources, 893-2, Nakayama, Nago City, Okinawa 905-0004, Japan. Tel:
+81-980-54-3376; Fax: +81-980-54-3457; E-mail:rnerome_ibr@train.ocn.ne.jp

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Acknowledgements
We thank Dr. T. Yoshikawa and Mr. S. Mukai(Institute of Biological Resources, Okinawa,
Japan) for their skillful technical help and general support. We thank Ms. N. Ohmine (Okinawa Institute of Science and Technology

Graduate University, Okinawa,
Japan: OIST) and Dr. O. Elisseeva (Institute of Physicaland Chemical Research, Kanagawa, Japan: RIKEN) for
their technical support. We
also thank Dr. K. Kuroda (Nihon
University School of Medicine,Tokyo, Japan) for useful comments on the manuscript. We
are grateful to the Kyoritsu
Seiyaku Corporation (Tokyo,
Japan) for their cooperation in
the preclinical evaluation of
MLE in veterinary hospitals.
We also appreciate the Okinawa Institute of Science andTechnology Graduate Schoolfor providing partial funding forthis work. Authors appreciate
Dr. R. Nerome (Institute of
Biological Resources, Okinawa,
Japan) for her generous supports for preparing the manuscript and figures.

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