Antiproliferative, cell-cycle dysregulation effects of novel asiatic acid derivatives on human non-small cell lung cancer cells

Article abstract of DOI:10.1248/cpb.c13-00328

Asiatic acid (AA) is a pentacyclic triterpene in Centella asiatica known to inhibit proliferation and induce apoptosis in several tumor cell lines. In the current study, we synthesized five AA derivatives and examined their inhibitory activities on growth in non-small cell lung cancer cell lines, A549 and PC9/G. Four derivatives were found to have stronger cell growth inhibitory activity than AA. Among them, compound A-3 showed the most significant antiproliferative effects on tumor. Growth of A549 and PC9/G cells was inhibited by A-3 in a dose- and time-dependent manner. To determine the cellular gene expression changes in A549 and PC9/G cells treated with A-3, Affymetrix GeneChip Human Genome U133 Plus 2.0 Array were used to screen transcriptome differences. Expression levels of 1121 genes in A549 and 1873 genes in PC9/G were significantly altered upon treatment with 10 μM A-3 after 48 h, with 357 overlapping genes. The signaling pathways molecules involved in the antiproliferative and cell cycle dysregulation effects of A-3 identified using microarray were further validated via Western blot analyses. The results collectively indicate that A-3 induces inhibition of cell proliferation via downregulation of the Ras/Raf/MEK/ERK pathway and cell cycle arrest at G1/S and G2/M.

Full text of DOI:10.1248/cpb.c13-00328

October 2013
Regular Article
Chem. Pharm. Bull. 61(10) 1015–1023 (2013)
1015
Antiproliferative, Cell-Cycle Dysregulation Effects of Novel Asiatic Acid
Derivatives on Human Non-small Cell Lung Cancer Cells
,b,d
,a
Liang Wang,a,# Jie Xu,b,# Chunhui Zhao,* Longxuan Zhao,c,d and Bin Feng*
b
a Department of Biotechnology, Dalian Medical University; Dalian 116044, China: School of Life Sciences, Liaoning
c
Normal University; School of Chemistry and Chemical Engineering, Liaoning Normal University; Dalian 116029,
d
China: and Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery; Dalian 116029, China.
Received April 26, 2013; accepted July 23, 2013; advance publication released online August 6, 2013
Asiatic acid (AA) is a pentacyclic triterpene in Centella asiatica known to inhibit proliferation and in-
duce apoptosis in several tumor cell lines. In the current study, we synthesized five AA derivatives and ex-
amined their inhibitory activities on growth in non-small cell lung cancer cell lines, A549 and PC9/G. Four
derivatives were found to have stronger cell growth inhibitory activity than AA. Among them, compound
A-3 showed the most significant antiproliferative effects on tumor. Growth of A549 and PC9/G cells was in-
hibited by A-3 in a dose- and time-dependent manner. To determine the cellular gene expression changes in
A549 and PC9/G cells treated with A-3, Affymetrix GeneChip® Human Genome U133 Plus 2.0 Array were
used to screen transcriptome differences. Expression levels of 1121 genes in A549 and 1873 genes in PC9/G
were significantly altered upon treatment with 10µM A-3 after 48h, with 357 overlapping genes. The signal-
ing pathways molecules involved in the antiproliferative and cell cycle dysregulation effects of A-3 identified
using microarray were further validated via Western blot analyses. The results collectively indicate that A-3
induces inhibition of cell proliferation via downregulation of the Ras/Raf/MEK/ERK pathway and cell cycle
arrest at G1/S and G2/M.
Key words asiatic acid derivative; non-small cell lung cancer; microarray analysis; antiproliferation
Lung cancer, the most common cancer in terms of both lated with the basic skeleton of pentacyclic triterpene, and the
incidence and mortality worldwide, was reported to have modification of C-2, C-3, C-23, and C-28 of AA can improved
recorded 1.61 million new cases and 1.38 million deaths in its biological activities.15,16) Dicarboxylic acid hemiesters of
2008. Nearly 70% of all the new cases of lung cancer in the pentacyclic triterpenoid such as ursolic acid, oleanolic acid,
world occur in the developed countries, with the highest rates and betulinic acid showed more potent inhibitory activity on
in Europe and North America.1) Non-small cell lung cancer human immunodeficiency virus (HIV)-protease.17) Optically
(NSCLC) types, including adenocarcinoma, squamous cell active D-amino acids are widely used in the pharmaceutical
carcinoma and large cell carcinoma, account for more than industry as intermediates for the synthesis of semisynthetic
80% of total pulmonary malignancies.2) Although drugs tar- antibiotics and pesticides. Among them, D-phenylglycine was
geting epidermal growth factor receptor (EGFR) mutations, used to synthesize drugs such as aspoxicillin, cefbuperazone,
such as gefitinib (Iressa®) and erlotinib (Tarceva®), have been and cefpiramide.18) In the current study, these functional
approved for treatment of advanced NSCLC, their efficacy is groups were modified to generate derivatives while maintain-
limited, and more than 50% of these patients are not suitable ing the ursane skeleton. The carboxylic group at C-28 was
for erlotinib and gefitinib treatment.3,4) Furthermore, almost all converted to methyl ester with retention of stereochemistry,
patients initially sensitive to gefitinib develop resistance to the which may significantly influence its physical properties or
drug.5–7) Therefore, it is essential to identify novel anticancer receptor interactions. Butanedioic acid or D-phenylglycine
drugs or reagents against NSCLC.
was introduced into the hydroxyl group at the C-2 position,
Asiatic acid (AA) is a pentacyclic triterpenoid derived from the dihydroxyl group at C-3 and C-23 was protected with ace-
the tropical medicinal plant, Centella asiatica (Apiaceae).8) tonide using 2,2-dimethoxy propane. We herein investigated
AA has been shown to reduce inflammation, inhibit tumor the cell growth inhibition activities of AA derivatives in the
cell proliferation and induce apoptosis through a mitochon- human lung cancer cell lines, A549 (intermediate-sensitive)19)
dria-dependent pathway.9) The anti-cancer efficacy of AA is and PC9/G (acquired resistance to gefitinib), and clarified the
attributed to its ability to inhibit transcription factors (nuclear mechanism of the effects.
factor-κB (NF-κB)) and kinases (p38 mitogen activated pro-
tein (MAP) and extracellular signal-regulated kinase (ERK))
Experimental
Reagents and Apparatus All the reagents and chemicals
in a variety of tumor cells.9–12) AA induces apoptotic cell
death in human hepatoma and malignant glioma cells through were of analytical grade or chemically pure, and obtained
enhancing intracellular calcium release.13,14) The compound from commercial suppliers. The synthetic pathways are pre-
contains five functional groups in its structure, specifically, sented in Fig. 1B. Target compounds were purified on a silica
three hydroxyl groups at C-2, C-3, and C-23, an olefinic group gel column (200–300 mesh, Qingdao Marine Chemical Fac-
at C-12, and carboxylic group at C-28, as presented in Fig. tory, China) with petroleum ether–ethyl acetate (or acetone)
1A. Based on previous reports that AA activities were re- as eluents. Their structures were confirmed using nuclear
magnetic resonance (NMR) on a Bruker 400MHz spectrom-
eter (Ettlingen, Germany) and infrared radiation (IR) spectros-
copy on a Shimadzu IRPrestige-21 FT-IR spectrophotometer
The authors declare no conflict of interest.
# These authors contributed equally to this work.
© 2013 The Pharmaceutical Society of Japan
*To whom correspondence should be addressed. e-mail: binfeng70@hotmail.com; chunhuiz@hotmail.com

1016
Vol. 61, No. 10
Fig. 1. Chemical Structures and Scheme
(A) Chemical structure of AA. (B) Reagents and conditions: a) (CH3)2C(OCH3)2, p-TsOH, DMF, reflux, rt; b) CH3I, K2CO3, DMF, rt; c) butanedioic anhydride, EDCI,
DMAP, CH2Cl2, rt; d) Fmoc-D-Phg-OH, EDCI, DMAP, CH2Cl2, rt; e) HNEt2, CH2Cl2, rt.
(Kyoto, Japan). Mass spectra were recorded on QTOF-Micro m/z 488.0 [M−H]−.
of Waters Micromass (Milford, MA, U.S.A.). Melting points
2α-Hydroxy-3β,23-isopropylidenedioxyurs-12-ene-28-
were determined in capillary tubes on X-5 melting point ap- oic Acid (A-1) A solution of AA (15g, 30.7mmol) and
paratus (Beijing Tech Instrument Co., Ltd., China) and left p-toluenesulfonic acid (catalytic amount, 50mg) in dry di-
uncorrected.
methyl formamide (50mL) was added 2,2-dimethoxypropane
2α, 3β, 23-Trihydroxyurs-12-ene-28-oic Acid (AA) A so- (4.5mL, 36.8mmol) and stirred. The reaction mixture was
lution of asiaticoside (36.34g, 37.81mmol) in methanol (700mL) stirred at room temperature for 5h. After neutralization with
was added 5M-NaOH solution (40mL) and stirred. The 5% NaOH to pH 7–8, the reaction mixture was diluted with
reaction mixture was refluxed for overnight. After completion ethyl acetate (250mL) and washed with water (100mL×3) and
of the reaction by checking on the TLC (n-buthanol–etha- saturated NaCl solution (80mL). The organic layer was dried
nol–water–ammonia water=6:4:1:0.5, v/v/v/v, disappearance over anhydrous magnesium sulfate. Filtration and evaporation
of the spot Rf=0.15), the reaction mixture was concentrated of solvent at reduced pressure gave light yellow solid, which
under reduced pressure to half of its volume and neutralized was purified by silica gel chromatography with an elution of
with 2M-HCl to pH 5–6. The resulting slurry was filtered and CH2Cl2–MeOH (20:1, v/v) to yield a white solid (14g, 86.2%),
washed with large portion of water (300mL×3). Filtrate was mp 156.4–157.8°C; 1H-NMR (400MHz, CDCl3) δ: 5.24 (t,
dried in drying-oven (110°C) to give 17g (92%) of product as 1H, J=3.6Hz, H-12), 3.80–3.76 (m, 1H, H-2), 3.50 (d, 1H,
1
a white solid, mp 242.0–244.0°C. H-NMR (400MHz, CDCl3) J=10.8Hz, H-23), 3.46 (d, 1H, J=10.8Hz, H-23), 3.32 (d, 1H,
δ: 5.23 (t, 1H, J=3.6Hz, H-12), 3.71–3.67 (m, 1H, H-2), 3.51 J=9.6Hz, H-3), 1.45, 1.44, 1.09, 1.06, 1.04, 0.75 (s, each 3H),
(d, 1H, J=11.2Hz, H-23), 3.49 (d, 1H, J=11.2Hz, H-23), 2.24 0.95 (d, 3H, J=6.0Hz), 0.85 (d, 3H, J=6.5Hz); IR νmax (KBr,
(d, 1H, J=11.2Hz, H-18), 1.24, 1.03, 0.96, 0.71 (s, each 3H), cm−1): 3400, 2932, 1706.
0.92 (d, 3H, J=5.2Hz), 0.82 (d, 3H, J=6.0Hz); 13C-NMR
Methyl
2α-Hydroxy-3β,23-isopropylidenedioxyurs-12-
(100MHz, CDCl3) δ: 179.81 (C-8), 140.25 (C-13), 126.71 ene-28-oate (A-2) A solution of A-1 (16g, 30.26mmol)
(C-12), 78.36 (C–OH), 69.78 (C–OH), 66.32 (C–OH), 54.91, and anhydrous potassium carbonate (10.45g, 75.64mmol) in
49.56, 49.36, 49.15, 48.94, 48.72, 47.53, 44.27, 43.63, 40.65, dry dimethyl formamide (100mL) was added methyl iodide
39.17, 38.58, 33.93, 32.28, 29.57, 25.80, 24.63, 24.30, 21.88, (3.77mL, 60.52mmol) and stirred. The mixture was stirred
19.26, 17.94, 17.85, 14.08; IR νmax (KBr, cm−1): 3422, 2926, at room temperature for 6h. The reaction mixture was di-
2873, 1696; electrospray ionization-mass spectra (ESI-MS): luted with ethyl acetate (500mL) and washed with water

October 2013
1017
(200mL×3) and saturated NaCl solution (80mL). The organic (m, 2H, –CH2–O), 4.15 (t, J=7.0Hz, 1H, fluorenyl H-9′),
layer was dried over anhydrous magnesium sulfate. Filtration 3.53 (s, 3H, –OCH3), 3.39 (d, J=10.7Hz, 1H, H-3), 3.29 (t,
and evaporation of solvent at reduced pressure gave light yel- J=10.4Hz, 2H, H-23), 2.16 (d, J=10.9Hz, 1H, H-18), 1.01
low solid, which was purified by silica gel chromatography (s, 9H, =C(CH3)2, –CH3), 1.12, 0.92, 0.65 (each, 3H), 0.88
with a gradient elution of ethyl acetate–petrol ether (1:4, (d, J=5.3Hz, 1H, H-29), 0.79 (d, J=6.5Hz, 1H, H-30); IR
v/v) to afford a white solid (15.1g, 92%), mp 213.2–213.9°C; νmax (KBr, cm−1): 3436, 2924, 2851, 1719, 1501, 1449, 1380,
1H-NMR (400MHz, CDCl3) δ: 5.18 (t, 1H, J=3.6Hz, H-12), 1198; ESI-MS: m/z 898.4 [M−H]−.
3.74–3.67 (m, 1H, H-2), 3.53 (s, 3H, –OCH3), 3.44 (d, 1H,
Methyl
2α-O-Phenyglycylurs-3β,23-isopropylidenedi-
J=10.7Hz, H-23), 3.39 (d, 1H, J=10.8Hz, H-23), 3.25 (d, 1H, oxyurs-12-ene-28-oate (A-5) A solution of A-4 (420mg,
J=9.6Hz, H-3), 2.53 (s, 1H, H-9), 2.17 (d, 1H, J=11.2Hz, 0.47mmol) in dry dichloromethane (8mL) was added diethyl-
H-18), 1.38, 1.37, 1.04, 1.00, 0.97, 0.66 (s, each 3H), 0.87 (d, amine (10mL) and stirred. The reaction mixture was stirred
3H, J=6.2Hz), 0.79 (d, 3H, J=6.5Hz); IR νmax (KBr, cm−1): at room temperature for 2h. Evaporation of solvent at reduced
3584, 3444, 2940, 2868, 1732.
pressure gave yellow solid, which was purified by silica gel
Methyl 2α-O-Carboxypropionyl-3β,23-isopropylidene- chromatography with a gradient elution of ethyl acetate–pet-
dioxyurs-12-ene-28-oate (A-3) A solution of A-2 (2.3g, rol ether (1:2, v/v) to afford a white solid (0.20g, 62%), mp
1
4.24mmol) and 4-dimethylaminopyridine (0.622g, 5.09mmol) 81.2–81.7°C; H-NMR (400MHz, CDCl3) δ: 7.3–7.2 (m, 5H,
in dry methylene chloride (20mL) was added butanedioic an- Ph-H), 5.18 (t, J=3.5Hz, 1H, H-12), 5.1–5.04 (m, 1H, H-2),
hydride (1.70g, 16.96mmol) and stirred. The mixture was re- 4.46 (s, 1H, Ph-CH), 3.53 (s, 3H, –OCH3), 3.39 (d, J=10.7Hz,
fluxed for 8h. The reaction mixture was diluted with methyl- 1H, H-3), 3.27 (dd, J1=14.9Hz, J2=14.3Hz, 2H, H-23), 1.01 (s,
ene chloride (100mL) and washed with water (50mL×3) and 9H, =C(CH3)2, –CH3), 1.13, 0.94, 0.65 (s, each 3H), 0.88 (d,
saturated NaCl solution (50mL). The organic layer was dried J=6.2Hz, 1H, H-29), 0.79 (d, J=4.3Hz, 1H, H-30); 13C-NMR
over anhydrous magnesium sulfate. Filtration and evaporation (100MHz, CDCl3) δ: 177.95, 138.22, 128.52, 127.75, 127.14,
of solvent at reduced pressure gave light yellow solid, which 125.03, 99.18, 79.02, 76.70, 72.56, 69.22, 58.88, 52.83, 51.50,
was purified by silica gel chromatography with a gradient elu- 51.02, 48.04, 47.60, 44.76, 42.05, 39.57, 39.06, 38.85, 38.38,
tion of ethyl acetate–petrol ether (1:4, v/v) to afford a white 37.41, 36.57, 32.32, 30.64, 29.40, 27.97, 24.16, 23.63, 23.20,
1
solid (2.49g, 91.7%), mp 106.2–107.1°C; H-NMR (400MHz, 21.19, 18.54, 17.70, 17.41, 16.98, 16.76, 13.52; IR νmax (KBr,
CDCl3) δ: 5.18 (t, 1H, J=3.6Hz, H-12), 4.99–4.91 (m, 1H, cm−1): 3443, 3424, 2936, 2869, 1734, 1455, 1380, 1367, 1198;
H-2), 3.60 (d, 1H, J=10.6Hz, H-23), 3.53 (s, 3H, –OCH3), 3.40 ESI-MS: m/z 676.3 [M+H ] +.
(d, 1H, J=10.6Hz, H-23), 2.63–2.49 (m, 4H, –CH2CH2–), 2.17
Cell Lines and Cultures Human NSCLC cells, A549 and
(d, 1H, J=11.4Hz, H-18), 1.45, 1.44, 1.04, 1.02, 1.01, 0.66 (s, PC9 were obtained from the Cell Culture Center of Chinese
each 3H), 0.87 (d, 3H, J=6.3Hz, H-29), 0.78 (d, J=6.4Hz, Academy of Medical Sciences (CAMS; Beijing, P.R. China),
H-30); 13C-NMR (100MHz, CDCl3) δ: 177.99, 176.41, 171.65, and maintained in RPMI 1640 (Hyclone) supplemented with
138.18, 125.10, 99.42, 78.92, 72.67, 69.32, 52.83, 51.52, 51.13, 10% fetal bovine serum (FBS), penicillin (100U/mL) and
48.05, 47.60, 44.64, 42.06, 39.58, 39.06, 38.86, 38.35, 37.50, streptomycin (0.1mg/mL). Cells were incubated at 37°C in
36.59, 32.35, 30.64, 29.70, 29.23, 28.83, 27.98, 24.17, 23.66, a humidified atmosphere with 5% CO2. Gefitinib-resistant
23.23, 21.20, 19.20, 17.66, 17.46, 17.01, 16.78, 13.53; IR νmax PC9/G cells were established using a previously reported
(KBr, cm−1): 3320, 2942, 2570, 1710, 1449, 1382, 1180; ESI- method20) and treated with 0.05µM gefitinib (Iressa®, ZD1839;
MS: m/z 641.8 [M−H]−.
AstraZeneca, Wilmington, DE, U.S.A.) to maintain cell re-
Methyl 2α-O-(N-Fluorenonemethyoxycarbonyl)-pheny- sistance. A subclone of the gefitinib-resistant cell line was
glycylurs-3β,23-isopropylidenedioxyurs-12-ene-28-oate obtained by limited dilution, and sensitivity to gefitinib de-
(A-4) A solution of A-2 (530mg, 0.98mmol) and Fmoc-D- termined with Cell Counting Kit-8 (CCK-8) (Dojindo, Japan).
phenylglycine (550mg, 1.46mmol) in dry dichlorometh- PC9/G cells showed more than a 100-fold higher IC50 for gefi-
ane (8mL) was added 4-dimethylamiopryidine (0.18g, tinib than the parent cells.
1.46mmol) and stirred. Dropwise the solution of 1-ethyl-3-(3-
EGFR Mutational Analysis Genomic DNA of PC9 and
dimethyllaminopropyl)carbodiimide hydrochloride (560mg, PC9/G cells were isolated using a MiniBEST universal ge-
1.46mmol) in dry dichloromethane (8mL) to reaction mixture nomic DNA extraction Kit (TaKaRa Biotechnology (Dalian)
at 0°C. Stirring the reaction mixture for 5min at 0°C. Keep Co., Ltd., China). EGFR exons 19 and 20 mutation analyses
stirring the reaction mixture for 3h at room temperature. The were performed via polymerase chain reaction (PCR) using
reaction mixture was diluted with dichloromethane (50mL) LA Taq DNA polymerase (TaKaRa). The primers for Exon 19
and washed with water (30mL×3) and saturated NaCl solution were 5′-CCACAGGACTTTATAACAGGC-3′ and 5′-GGC
(10mL). The organic layer was dried over anhydrous magne- CAGTGCTGTCTCTAAGG-3′, respectively. The primers for
sium sulfate. Filtration and evaporation of solvent at reduced Exon 20 were 5′-CTCCCACTGCATCTGTCACTTCAC-3′
pressure gave light yellow solid, which was purified by silica and 5′-ATGCAGATGGGACAGGCACTG-3′, respectively.
gel chromatography with a gradient elution of ethyl acetate– The PCR products were analyzed by agarose gel electrophore-
petrol ether (1:5, v/v) to afford a white solid 0.47g, 54.03%, sis and purified by MiniBEST DNA Fragment Purification Kit
mp 88.2–88.5°C; 1H-NMR (400MHz, CDCl3) δ: 7.69 (d, (TaKaRa) and sequenced (performed by TaKaRa Biotechnol-
J=7.5Hz, 2H, fluorenyl H-4, H-5), 7.51 (d, J=6.9Hz, 2H, fluo- ogy).
renyl H-1′, H-8′), 7.32 (d, J=7.4Hz, 2H, fluorenyl H-3′, H-6′),
Cell Viability Assay The proliferative activities of A549
7.26 (d, J=5.2Hz, 2H, fluorenyl H-2′, H-7′), 7.34–7.21 (m, 5H, and PC9/G cells under different treatments were assessed
Ph-H), 5.82 (d, J=7.2Hz, 1H, Ph-CH), 5.26 (d, J=7.4Hz, 1H, using the CCK-8 assay. Cells were inoculated in 96-well plates
–NH), 5.18 (s, 1H, H-12), 5.10–5.08 (m, 1H, H-2), 4.34–4.30 in complete medium and cultured for 24h, and media replaced

1018
Vol. 61, No. 10
with RPMI-1640, 10% or 1% FBS, with or without AA de- thetic pathways are presented in Fig. 1B. AA was treated with
rivatives. All compounds were dissolved in dimethyl sulfoxide 2,2-dimethoxypropane in the presence of p-toluenesulfonic
(DMSO) at final concentrations less than 0.1%, prior to addi- acid in N,N-dimethylformamide (DMF) to generate acetonide
tion to cell culture assays. Following incubation at 37°C for (A-1) with a 86.2% yield. A-1 was subjected to methyl iodide
12, 24, 48 or 72h, 10µL CCK-8 was added to each well, and treatment in the presence of K2CO3 in DMF to afford methyl
the plates incubated for 1–2h. A microplate reader (Multiskan ester (A-2) (92% yield), and A-2 treated with butanedioic
Ascen, Thermo Fisher Scientific, Waltham, MA, U.S.A.) was anhydride in the presence of 4-(dimethylamino)pyridine in
employed to determine optical density (OD) at 450nm, and CH2Cl2 to generate A-3 (91.7% yield). A-2 was treated with
values compared with that of the control group. The per- Fmoc-D-phenylglycine in the presence of 4-(dimethylamino)-
centage of cell viability resulting from each AA derivative pyridine in CH2Cl2 to generate A-4 with a 54% yield, which,
was calculated as follows: (OD450 treated cells−OD450 blank control)/ in turn, was treated with diethylamine in CH2Cl2 to produce
(OD450 control−OD450 blank control)×100%. More than three inde- A-5 with a 62% yield. Target compounds were purified on
pendent experiments were performed.
a silica gel column with petroleum ether–ethyl acetate (or
Microarray Analysis Cellular RNA from A549 and acetone) as eluents and their structures confirmed using NMR
PC9/G cells was extracted using TRIzol (Invitrogen), and and IR spectroscopy. All compounds were dissolved in DMSO
quality controlled as directed with the Affymetrix expression at final concentrations less than 0.1%, prior to addition to cell
technical manual. RNA (50ng) was used to produce biotin-la- culture assays.
beled cRNA, which was hybridized to Affymetrix GeneChip®
Sequence of EGFR Exons 19 and 20 in PC9/G Cells
Human Genome U133 Plus 2.0 Array. Array washing, scan- Multinucleotide in-frame deletions in exon 19 that eliminate
ning and probe quantification protocols were carried out ac- five amino acids (ELREA) are associated with increased
cording to the manufacturer’s instructions using Affymetrix sensitivity of lung adenocarcinomas to the selective EGFR
GeneChip Operating Software (GCOS) (http://www.affyme- kinase inhibitors, gefitinib (Iressa) and erlotinib (Tarceva).7,21)
trix.com). For each array, GCOS output was imported as CEL A second-site point mutation that substitutes methionine for
files into Partek Genomic Suite software (Agilent), and gene threonine at position 790 (T790M) is associated with approxi-
expression data quantified with the RMA (Robust Multichip mately half of case of acquired resistance to the EGFR kinase
Averaging) algorithm, normalized and corrected for multiple inhibitors.21) To determine whether PC9/G cells that acquired
testing with the Benjamini and Hochberg method for detection resistance to the gefitinib display specific genetic alterations,
of differentially expressed genes.
we analyzed the sequence of the EGFR exon 19 but found no
Western Blot Analysis Total proteins were extracted differences in their sequences between PC9 and PC9/G cells.
from cells using RIPA buffer (Sigma). After treatment with A deletion mutation (E746-A750), similar to that described
the AA derivative, A-3, for 48h, A549 and PC9/G cells were previously,7,22) was identified based on alignment of sequenc-
washed with ice-cold phosphate buffered saline (PBS) solu- ing data and GenBank homo sapiens genome EGFR sequence
tion and lysed in lysis buffer at 0°C for 20min. Cell lysates (GenBank accession number NC_000007). There is no muta-
were scraped and centrifuged at 14000rpm for 30min at 4°C, tion at the site in Exon 20 (position 790). The mechanism of
and the supernatant collected. Equivalent amounts of proteins drug resistance is thought to be multifactorial.20,21)
were subjected to sodium dodecyl sulfate-polyacrylamide gel
Antiproliferative Effects of AA and Its Derivatives Be-
electrophoresis (SDS-PAGE) and transferred to polyvinylidene cause cell culture medium supplemented with 10% FBS could
difluoride membranes. After blocking for 1h in 5% skimmed provide the cells with an excess of growth factors overriding
milk in Tris-buffered saline, the membrane was incubated possible effects of drugs.23) We treated A549 and PC9/G cell
with the desired primary antibody (1:2000 dilution) overnight lines under normal serum conditions (10% FBS) and reduced
at 4°C, followed by treatment with horseradish peroxidase- serum conditions (1% FBS). Exposure of NSCLC to AA and
coupled secondary antibody (1:5000 dilution) for 1h at room its derivatives for 48h revealed considerably higher inhibi-
temperature. Immunoreactive proteins were detected using an tion of cell proliferation by the AA derivatives, relative to the
enhanced chemiluminescence kit (Millipore, Bedford, MA, parent compound, AA (Table 1). Under low serum conditions
U.S.A.) and visualized using the Versa Doc Imaging System (1% FBS) the IC50 values of A-3 (9.51 4.38, 9.73 0.11 µM)
(BioRad Model 4000, CA, U.S.A.). β-Actin was used as an and A-5 (11.52 3.58, 13.11 2.69µM) on A549 and PC9/G cell
internal control. Antibodies used for Western blotting, spe- lines were significantly lower than those of AA (44.95 5.14,
cifically, EGFR, p-EGFR, Akt, p-Akt, ERK1/2, p-ERK1/2, 58.04 2.90µM). Under normal serum conditions (10% FBS)
mammalian target of rapamycin (mTOR), p-mTOR, Cyclin B, both A549 and PC9/G cells were significantly affected by
Cyclin E, CDK1, CDK2, and β-actin, were obtained from Cell A-3 compared with AA, the IC50 values of A-3 were 26.03
Signaling Technology Inc. (Beverly, MA, U.S.A.).
2.47 and 25.57 0.51, respectively. A-3 exerted a dose- and
Statistical Analysis All experiments were repeated three time-dependent antitumor effect on A549 and PC9/G, and the
times, and data expressed as means S.D. Statistical compari- optimal duration of action was 48h (Figs. 2A,B). Compounds
sons of the results were made using Student’s t-test. The level A-1 and A-2 also exhibited good inhibitory activity on A549
of significance was taken at p<0.05.
and PC9/G cell lines in 1% FBS, the IC50 of A-1 and A-2 were
10.13 0.51 µM and 11.57 0.37 µM, respectively.
A-3 Induced Gene Expression Changes in A549 and
Results
Synthesis of AA Derivatives AA was prepared from PC9/G Cells The NSCLC cell lines, A549 and PC9/G, were
hydrolysis of asiaticoside with 5M NaOH in methanol, with treated with 10µM A-3 for 48h, and total RNA extracted.
a 90–97% yield. Using AA as the lead compound, struc- Microarray experiments were conducted using Affymetrix
tures were modified at C-2, C-3, C-23, and C-28. The syn- GeneChip® Human Genome U133 Plus 2.0 Array to deter-

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Table 1. IC50 Values of AA and Derivatives on A549 and PC9/G Cells Proliferation
IC50 (µM)
Compounds
A549
PC9/G
1% FBS
10% FBS
1% FBS
10% FBS
AA
A-1
A-2
A-3
A-4
A-5
44.95 5.14
10.13 0.51
11.57 0.37
9.51 4.38
>60
>60
58.04 2.90
10.58 0.63
11.02 0.66
9.73 0.11
>60
>60
34.73 1.74
48.86 2.93
26.03 2.47
>60
52.45 1.31
47.61 1.67
25.57 0.51
>60
11.52 3.58
40.21 2.01
13.11 2.69
21.66 0.97
Fig. 2. Inhibitory Activities of AA and Its Derivatives on A549 and PC9/G Cell Proliferation
(A) Inhibitory activity of A-3 on A549 cells. (B) Inhibitory activity of A-3 on PC9/G cells. For (A) and (B), cells treated for the indicated times at different concentra-
tions of A-3 were analyzed for viability with the CCK-8 assay.
mine changes in the specific genes encoding proteins related cules would be found together by chance. Accordingly, a high-
to pathological alterations in intracellular signaling pathways er extent correlation indicates greater statistical significance
in response to the AA derivative, A-3. As shown in Fig. 3A, that molecules depicted in the network are interconnected.
after A-3 treatment, 1121 genes in A549 cells and 1873 genes Network contains significant deregulated genes and identified
in PC9/G cells were differentially expressed, compared with around ERK are shown in Fig. 3D. Genes are colored accord-
controls, by at least two-fold. Venn diagram analysis facili- ing to gene expression value; red gene symbols indicate up-
tated the identification of a subset of 357 genes with similar regulation and green gene symbols indicate down-regulation.
functions between A549 and PC9/G cells treated with A-3 Nodes are displayed using various shapes that represent the
(complete list of the 357 genes is presented in Table S1).
functional class of the gene product. Dashed lines show indi-
Hierarchical clustering of the 357 genes led to segregation rect interaction, while a continuous line represents direct in-
of A549 and PC9/G cells treated with A-3 as a separate group teractions. This result suggested that A-3 exerted its inhibition
from control cells, according to gene expression profiles (Fig. on proliferation of A549 and PC9/G through inactivation of
3B). A549 and PC9/G cells treated with A-3 clustered togeth- ERK associated network, which is an important central factor
er, suggesting that A-3 has a transcriptomic effect on both cell in NSCLC cell growth and survival pathway.
types. Significant gene alterations were observed in A549 and
PC9/G cells following treatment with A-3, indicating that the cell cycle regulation, signaling and other functions as shown
compound induces global changes in gene expression.
in Fig. 3C (the complete ingenuity pathway analysis is present-
The broad categories included genes with specific roles in
Ingenuity Pathway Analysis (IPA) of Differentially Ex- ed in Fig. S1). Network analysis was additionally performed
pressed Variant Genes A gene network of 10 central nodes to generate a graphical representation of genes with a known
was constructed by using the differentially expressed genes in biological relationship. Expression levels of a number of genes
A549, PC9/G and controls, as described in Experimental. A in the EGFR and ERK networks were downregulated, depict-
dataset containing the differentially expressed genes, called ed as green shading of gene icons in the network (Fig. 3D).
the focus molecules, between NSCLC cell lines and controls Additional network analysis disclosed that A-3 interferes with
was overlaid onto a global molecular network developed from the same pathways as the MAPK/ERK kinase-specific inhibi-
information contained in the Ingenuity Pathways Knowledge tor, PD98059 (Fig. S2).
Base. Networks of these focus molecules were then algorith-
Effects of A-3 on Signaling Pathways and Cell Cycle
mically generated based on their connectivity. Network mole- Regulation Cells were treated as for microarray analysis,

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Fig. 3. Whole-Transcriptome Analysis of Cancer Cell Lines Treated with A-3
(A) Venn diagram of overlapping genes with differential expression in response to A-3. (B) Heat map of the 357 overlapping genes. Red/green indicates an increase/de-
crease in gene expression, relative to the universal mean for each gene. (C) Canonical pathway-based analysis of gene expression data from A549 and PC9/G cells treated
with A-3. Ingenuity Pathway Analysis (IPA; Ingenuity® Systems, www.ingenuity.com) was used to identify the genes significantly associated with canonical pathways in
the Ingenuity Pathways Knowledge Base. The significance of association between the significantly altered genes (fold change ≥2) and the canonical pathway was deter-
mined based on p values calculated with IPA statistic analysis methods. (D) EGFR and ERK signaling pathways in response to A-3 in A549 and PC9/G cells. Differential
gene expression was based on treatment versus control expression changes (2-fold and p<0.05, t-test). Red icons indicate higher expression, while green icons indicate de-
creased expression. Blue lines are activation functions and orange lines are inactivation. While yellow line indicates opposite result of A-3 comparing with IPA knowledge
base. (Color images were converted into gray scale.)
collected, and the expression levels of phosphorylated signal- kinase components of Cyclin E/CDK2 and Cyclin B/CDK1
ing molecules analyzed via western blotting to examine the are predominantly involved in cellular G1/S and G2/M arrest
effects of A-3 on the EGFR downstream signaling pathway after A-3 treatment.
(Fig. 4A). Following treatment of cells with 10µM A-3 for
48h, EGFR phosphorylation levels of A549 and PC9/G cells
Discussion
were decreased, along with c-Raf and p-ERK1/2 levels, com-
Non-small cell lung cancer (NSCLC) is the leading cause of
pared with their control counterparts. However, no significant cancer-related deaths worldwide. Although a number of drugs
variations in the phosphorylation levels of Akt and mTOR targeting EGFR mutations have been developed to date, most
were evident in either A549 or PC9/G cell lines. Expression advanced cases are still incurable.6,24,25) More than 50% of pa-
levels of cell cycle regulatory proteins were additionally ex- tients with NSCLC are not suitable for erlotinib and gefitinib
amined via western blotting. Since transcriptional profiling treatment,3,4) and almost all patients with initial sensitivity to
using microarray and functional analysis revealed changes gefitinib develop resistance to these drugs. Novel synthetic
in expression levels of genes involved in cell cycle (Figs. 3C, triterpenoids mimicking physical and chemical properties of
S1), we examined the effects on cell cycle checkpoints (G1/S natural triterpenoids are effective in inducing apoptosis in
and G2/M) in the two cell lines after exposure to A-3. Varia- a wide variety of tumor cells via diverse mechanisms and
tions in the expression levels of different kinase components sensitizing tumor cells that do not respond to conventional
were observed following A-3 treatment, including Cyclin E/ chemotherapy.26,27)
CDK2 and Cyclin B/CDK1, which mainly activate cell cycle
In this study, five AA derivatives with the modification of
transition from G1 to S and G2 to M. As shown in Fig. 4B, the functional groups of C-2, C-3, C-23, and C-28 were pre-
A-3 induced dramatic downregulation of Cyclin E/CDK2 and pared, and their antitumor activities on a gefitinib-resistant
Cyclin B/CDK1 in both cell lines. Our results suggest that the variant (PC9/G) and the gefitinib-intermediate-sensitive cell

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Fig. 4. Western Blot Analysis
(A) Effects of A-3 on expression levels of EGFR downstream signaling pathways in A549 and PC9/G cells. (B) Effects of A-3 on expression levels of cell cycle regula-
tory proteins. NSCLC cells, A549 and PC9/G, were treated with 10µM A-3 for 48h. β-Actin was used as the loading control.
Fig. 5. Potential Mechanisms of A-3 as an Anti-tumor Agent against NSCLC Cell, A549 and PC9/G
A-3 induces inhibition of cell proliferation via downregulation of the Ras/Raf/MEK/ERK pathway and cell cycle arrest at G1/S and G2/M.
line (A549) were evaluated. Among them, compound A-3 methyl ester at C-28 for biological activity. Our data suggested
showed the most significant antiproliferative effects on tumor that AA derivatives with cross-link at C-3 and C-23 resulted
cells in 10% and 1% FBS. The nearly identical cytotoxicity in stronger cytotoxic activities than AA against the two cell
of compounds A-1 and A-2 suggested the unimportance of lines, and modification of AA into D-phenylglycine at C2 po-

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Vol. 61, No. 10
sition increased the antitumor activity such as in compound
A-5. However, compound A-4 showed no activity, which
might be effected by Fmoc.
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Acknowledgment This work was jointly supported by the
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