Antiangiogenic Potential of Three Triterpenic Acids in Human Liver Cancer Cells

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Anti-angiogenic potential of three triterpenic acids in human liver cancer cells

1

2 3

CHUN-CHE LINø,,#, CHUN-YIN HUANG‡,#, MEI-CHIN MONG†, CHIEN-YI CHAN‡, 4

MEI-CHIN YIN†,‡,* 5

ø

Department of Internal Medicine, Chung Shan Medical University Hospital, Taichung City, 6

Taiwan 7



Institute of Medicine, Chung Shan Medical University, Taichung City, Taiwan 8

‡

Department of Nutrition, China Medical University, Taichung City, Taiwan 9

†

Department of Health and Nutrition Biotechnology, Asia University, Taichung County, 10

Taiwan 11

12 13

running title: anti-angiogenic potential of triterpenic acids 14

#

These two authors contributed equally to this study. 15

*To whom correspondence should be addressed: Dr. Mei-chin Yin, Professor, Department 16

of Nutrition, China Medical University, 16th Floor, 91, Hsueh-shih Rd, Taichung City, 17

Taiwan, ROC 18

TEL: 886-4-22053366 ext. 7510, FAX: 886-4-22062891 19

Email: [email protected] 20

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ABSTRACT 1

Three triterpenic acids, oleanolic acid, ursolic acid and maslinic acid, at 2 or 4 mol/L 2

were used to study their anti-angiogenic potential in human liver cancer Hep3B, Huh7 and 3

HA22T cell lines. The effects of these compounds upon the level and/or expression of 4

hypoxia-inducible factor (HIF)-1, basic fibroblast growth factor (bFGF), vascular 5

endothelial growth factor (VEGF), interleukin (IL)-8, urokinase plasminogen activator 6

(uPA), reactive oxygen species (ROS), nitric oxide (NO) and cell invasion and migration 7

were examined. Results showed that these triterpenic acids at 4 mol/L significantly 8

suppressed HIF-1expression in three cell lines (P<0.05); and these compounds at test 9

doses failed to affect bFGF expression (P>0.05). Three triterpenic acids 10

dose-dependently decreased production and expression of VEGF and IL-8, retained 11

glutathione level, lowered ROS and NO levels, and declined cell invasion and migration in 12

test cell lines (P<0.05). These compounds also dose-dependently reduced uPA production 13

and expression in Hep3B and Huh7 cell lines (P<0.05); but these agents only at 4mol/L 14

significantly suppressed uPA production and expression in HA22T cells (P<0.05). These 15

findings suggest that these triterpenic acids are potent anti-angiogenic agents to retard 16

invasion and migration in liver cancer cells. 17

18

KEYWORDS: angiogenesis; triterpenic acid; liver cancer cell; hypoxia-inducible factor; 19

vascular endothelial growth factor 20

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INTRODUCTION 1

Angiogenesis, also called as neovascularization, is an important process for tumor 2

growth and metastasis. Liver cancer, also called as hepatocellular carcinoma (HCC), is a 3

hypervascular tumor characterized by massive angiogenesis (1). The increased 4

circulating levels and/or expression of several angiogenetic factors such as vascular 5

endothelial growth factor (VEGF) and interleukin (IL)-8 in patients with HCC have been 6

reported (2, 3). Hypoxia-inducible factor (HIF)-1and basic fibroblast growth factor 7

(bFGF) are two crucial activators responsible for cancer angiogenesis, in which HIF-1 8

regulates the essential adaptive responses of cancer cells to hypoxia, the major 9

pathophysiological condition promoting angiogenesis; and bFGF mediates extracellular 10

matrix degradation (4, 5). In addition, VEGF through increasing vascular permeability 11

could enhance endothelial cell migration and proliferation, which are required for vascular 12

remodeling and sprouting development of new blood vessels (6). Overexpression of 13

urokinase plasminogen activator (uPA), a gene related to invasion, also contributed to HCC 14

angiogenesis (7). On the other hand, reactive oxygen species (ROS) and nitric oxide (NO) 15

play important roles in cancer angiogenesis because these free radicals enhance oxidative 16

stress and stimulate VGEF production, which in turn favors cancer progression (8, 9). 17

Obviously, these factors provide vascular support for the growth of already established 18

malignant tumor. Thus, any agents with suppressive effects on the production or 19

expression of these angiogenetic factors may be beneficial in retarding tumor progression. 20

Oleanolic acid, ursolic acid and maslinic acid are pentacyclic triterpenic acids 21

naturally occurring in many herbs and plant foods such as glossy privet fruit (Ligustrum 22

lucidum Ait.), hawthorn fruit (Crataegi Pinnatifidae Fructus) and olive (10, 11). The 23

inhibitory effects of oleanolic acid and ursolic acid against the growth of human bladder, 24

prostate and colorectal cancer cells have been observed (12, 13). Our previous study (14) 25

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also found that these two triterpenic acids at 8mol/L caused markedly apoptotic effects in 1

four human liver cancer HepG2, Hep3B, Huh7 and HA22T cell lines via increasing DNA 2

fragmentation, decreasing mitochondrial membrane potential and lowering 3

Na+-K+-ATPase activity. Juan et al. (15) reported that maslinic acid, another triterpenic 4

acid, could inhibit cell proliferation in human colon cancer cell, HT-29. These studies 5

suggest that these triterpenic acids are potent anti-cancer agents to cause apoptosis or 6

retard metastasis for liver cancer. Therefore, a further study was conducted to examine 7

the anti-angiogenic potential of these triterpenic acids. The major purpose of our present 8

study was to investigate the effects of triterpenic acids on the production and/or expression 9

of HIF-1, bFGF, VEGF, IL-8 and uPA in liver cancer cell lines. The influence of these 10

compounds upon the production of ROS and NO in these cell lines was also evaluated. 11

On the other hand, the presence of ursolic acid and maslinic acid in apple peel has 12

been considered as major compounds responsible for the anti-cancer activity of apple (16). 13

So far, less information is available regarding the presence of these triterpenic acids in 14

fresh vegetables. In order to enrich the food component database, eleven locally available 15

fresh vegetables in Taiwan were used to analyze the content of oleanolic acid, ursolic acid 16

and maslinic acid. 17

18

MATERIALS AND METHODS 19

Chemicals. Oleanolic acid (OA, 99%), ursolic acid (UA, 98%) and maslinic acid 20

(MA, 98.5%) were purchased from Aldrich Chemical Co. (Milwaukee, WI, USA). 21

Medium, plates, antibiotics and chemicals used for cell culture were purchased from Difco 22

Laboratory (Detroit, MI, USA). All chemicals used in these measurements were of the 23

highest purity commercially available. 24

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Content of Triterpenic Acids. Eleven locally available fresh vegetables, carrot 1

(Daucus carota L.), celery (Apium graveolens L.), okra (Hibiscus esculentus), eggplant 2

(Solanum melongena L.), spinach (Spinacia oleracea L.), leaf mustard (Brassica juncea), 3

gynura (Gynura bicolor DC.), Chinese chive (Allium tuberosum Rottler), fennel 4

(Foeniculum vulgare), basil (Ocimum basilicum L.) and daylily (Hemerocallis fulva L.), 5

were used to analyze the content of OA, UA and MA. These vegetables, harvested in 6

summer of 2010, were purchased from farms in central area of Taiwan. The content of 7

three triterpenic acids in these vegetables was analyzed by HPLC methods described in Liu 8

et al. (17). 9

Cell Culture. Human normal liver cell line (L-02 cell) and liver cancer Hep3B, Huh7 10

and HA22T cell lines were obtained from American Type Culture Collection (ATCC, 11

Rockville, MD, USA). All cells were cultured in RPMI 1640 medium, containing 10% 12

fetal bovine serum (BSA), 100 units/mL of penicillin and 100 units/mL of streptomycin 13

(pH 7.4). Hypoxia (1% O2) was created using an anaerobic jar (AnaeroPack Series, 14

Mitsubishi Gas Chemical, Tokyo, Japan) with an AnaeroPack disposable O2-absorbing and 15

CO2-generating agent. The culture medium was changed every three days, and cells were 16

subcultured once a week. A phosphate buffer saline (PBS, pH 7.2) was added to adjust 17

the cell number to 105/mL for various experiments and analyses. 18

Experimental Design. Stock solution of OA, UA or MA was prepared in dimethyl 19

sulfoxide (DMSO) and diluted with medium. An equal volume of DMSO (final 20

concentration <0.5%) was added to the controls. Our previous study (14) found that OA 21

and UA at 2 and 4mol/L caused 55-70% cell viability in Hep3B, Huh7 and HA22T cells 22

(8 mol/L caused 20-30% cell viability). Thus, in order to observe the effects of these 23

triterpenic acids upon angiogenic factors in living cells, these compounds at these two 24

doses were used for present study. The cells at 105/mL were treated with OA, UA or MA 25

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at 2 or 4mol/L under hypoxia at 37 °C for 72 h. Control group contained no OA, UA 1

or MA. 2

Measurement of Glutathione (GSH), Reactive Oxygen Species (ROS) and Nitrite. 3

After 72 h hypoxic treatment, cells were washed and suspended in RPMI 1640 medium. 4

GSH concentration (ng/mg protein) was determined by a commercial colorimetric GSH 5

assay kit (OxisResearch, Portland, OR, USA). The dye DCFH2-DA was used to measure 6

level of intracellular cellular ROS according to the method of Fu et al. (18). In briefly, 7

cells were incubated with 50 mol/L dye for 30 min and washed with PBS. After 8

centrifugation at 412 xg for 10 min, the medium was removed and cells were dissolved 9

with 1% Triton X-100. Fluorescence changes were measured at an excitation wavelength 10

of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader. 11

The production of NO was determined by measuring the formation of nitrite. Briefly, 12

100 L of supernatant was mixed with 100 L Griess reagent after centrifuged at 10,000 13

xg for 15 min at 4 ºC. The optical absorbance at 540 nm was measured and compared 14

with a sodium nitrite standard curve. 15

Determination of Angiogenic Factors. VEGF and IL-8 levels in cell homogenates 16

were quantified by commercial ELISA kits (R&D Systems, Minneapolis, MN, USA). 17

uPA level was assayed by commercial ELISA kit purchased from American Diagnostica 18

(Greenwich, CT, USA). Each measurement was made in duplicate, and the level of each 19

angiogenic factor was determined from a standard curve. 20

Real Time Polymerase Chain Reaction (RT-PCR) for mRNA Expression. Total 21

RNA was isolated from cells using Trizol reagent (Invitrogen, Life Technologies, Carlsbad, 22

CA, USA). Two g of total RNA was used to generate cDNA, which was amplified 23

using Taq DNA polymerase. PCR was carried out in 50 L of reaction mixture 24

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dNTP, 2.5 mM MgCl2, 0.5 mM of each primer) and 2.5 U Taq DNA polymerase. The 1

specific oligonucleotide primers for HIF-1, bFGF, VEGF, IL-8, uPA and 2

glyceraldehyde-3-phosphate dehydrogenase (GAPDH, the housekeeping gene) are as 3

follows. HIF-1: forward, 5'-CTG CTG TCT TAC TGG TCC TT-3', reverse, 5'-GTC 4

GCT TCT CCA ATT CTT AC-3'; bFGF: forward, 5’-GTG TGC TAA CCG TAC CTG 5

GC-3’, reverse, 5’-CTG GTG ATT TCC TTG ACC GG-3’; VEGF, forward, 5'-ATG AAC 6

TTT CTG CTC TCT GG-3', reverse, 5'-TCA TCT CTC CTA TGT GCT GGC-3'; IL-8, 7

forward,5’-CAC TCT CAA TCA CTC TCA GTT CTT TGA T-3’;reverse,5’-CTT CCT 8

GAT TTC TGC AGC TCT GT-3’; uPA, forward, 5′-TCA CAC CAA GGA AGA GAA 9

TGG CCT-3′, reverse 5′-AAT GAC AAC CAG CAA GAA AGC GGG-3′;GAPDH: 10

forward,5’-TGA TGA CAT CAA GAA GGT GGT GAA G-3’,reverse,5’-CCT TGG 11

AGG CCA TGT AGG CCA T-3’. The cDNA was amplified under the following 12

reaction conditions: 95 ºC for 1 min, 55 ºC for 1 min, and 72 ºC for 1 min. 28 cycles 13

were performed for GAPDH and 35 cycles were performed for HIF-1, bFGF, VEGF, 14

IL-8 and uPA. Generated fluorescence from each cycle was quantitatively analyzed by 15

using the Taqman system based on real-time sequence detection system (ABI Prism 7700; 16

Perkin-Elmer Inc., Foster City, CA, USA). In this study, mRNA level was calculated as 17

percentage of the control group. 18

Invasion Assay. Invasion was measured using 8 m pore BD BioCoat Matrigel

19

Invasion Chambers (BD Biosciences, San Jose, CA, USA), in which membrane was 20

coated with 500 ng/μL Matrigel. After 72 h hypoxic treatment, cells were resuspended in 21

RPMI 1640 medium containing 1% BSA, and at 105/100 L were seeded into the upper 22

chamber. The chambers were incubated for 24 h at 37 ºC in a humidified, 5% CO2 23

atmosphere for cell invasion. Non-invasive cells were removed by Q-tip. Invasive cells 24

were stained with 0.2% crystal violet in 10% ethanol. Four independent fields of 25

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invasive cells per well were photographed under the microscope to determine the number 1

of invasive cells. Data were calculated as percentage of the control group. 2

Migration assay. Migration was measured using a transwell chamber (Corning, NY, 3

USA). The underside of transwell membranes was precoated with fibronectin (10 4

g/mL). After 72 h hypoxic treatment, cells were resuspended in RPMI 1640 medium 5

containing 1% BSA, and at 105/100 L were seeded into the upper chamber of transwell 6

chambers separated by inserts with 6.5 mm polycarbonate filters of 8m pore size. After 7

24 h incubation, non-migratory cells on the upper surface of membranes were removed 8

and migratory cells were stained with 0.1% crystal violet. Four independent fields of 9

migratory cells per well were photographed under the microscope to determine the number 10

of migratory cells. Data were calculated as percentage of the control group. 11

Statistical Analysis. The effect of each treatment was analyzed from nine different 12

preparations (n=9). Data were reported as means  standard deviation (SD), and 13

subjected to analysis of variance (ANOVA). Differences among means were determined 14

by the Least Significance Difference Test with significance defined at P<0.05. 15

16

RESULTS 17

The content of three triterpenic acids in eleven fresh vegetables is shown in Table 1. 18

Among test vegetables, spinach had the highest content of OA and UA, and leaf mustard 19

had the highest MA content. As shown in Figure 1, HIF-1mRNA expression of three 20

liver cancer cells under hypoxic condition was greater than normoxic condition (P<0.05), 21

which indicated that hypoxia had been created. 22

OA, UA and MA at 4 mol/L significantly suppressed HIF-1mRNA expression in 23

three cell lines (Figure 2, P<0.05); and these compounds at test doses failed to affect bFGF 24

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mRNA expression (Figure 3, P>0.05). Three triterpenic acids dose-dependently 1

decreased production and mRNA expression of VEGF and IL-8 in test cell lines (Figures 4 2

and 5, P<0.05). As shown in Table 2, three cancer cells had significantly lower GSH 3

level and higher production of ROS and NO than normal liver cells (P<0.05); however, test 4

compounds dose-dependently retained GSH level, and lowered production of ROS and NO 5

in test liver cancer cells (P<0.05). These compounds also dose-dependently diminished 6

invasion and migration in three test cell lines (Figure 6, P<0.05). As shown in Figure 7, 7

three test triterpenic acids dose-dependently reduced uPA production and mRNA 8

expression in Hep3B and Huh7 cell lines; but only at 4 mol/L significantly 9

down-regulated uPA production and mRNA expression in HA22T cells (P<0.05). 10

11

DISCUSSION 12

Our previous study already indicated that OA and UA exhibited marked apoptotic 13

effects in liver cancer cell lines (14). The results of our present study further found that 14

these two triterpenic acids and MA, another triterpenic acid, effectively down-regulated 15

mRNA expression of HIF-1, VEGF, IL-8, uPA, retained GSH level, decreased ROS and 16

NO production, as well as declined invasion and migration in three liver cancer cell lines 17

under hypoxic condition. These findings support that these triterpenic acids could 18

mitigate angiogenesis, which might attenuate liver cancer metastasis and deterioration. 19

Furthermore, we found that spinach, leaf mustard and daylily are rich in these triterpenic 20

acids. It has been suggested that vegetable intake could reduce cancer risk (19, 20). The 21

results of our present study further suggest that the vegetables rich in these triterpenic acids 22

may offer more protection against liver cancer. 23

Malignant tumor growth causes hypoxic condition within the tumor, which in turn 24

enhances the expression of HIF-1in order to mediate an adaptive response through 25

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increasing angiogenesis. Subsequently, HIF-1, as an transcriptional activator, 1

up-regulates VEGF expression and leads to tumor neovascularization (21, 22). Thus, 2

regulation of HIF-1and/or VEGF has been considered as promising target for cancer 3

therapy. In our present study, three test triterpenic acids down-regulated HIF-1 4

expression at high dose, but decreased VEGF expression and production in dose-dependent 5

manner. Thus, the anti-angiogenic activity of these triterpenic acids could be partially 6

ascribed to these agents suppress HIF-1 and VEGF expression in test cell lines. 7

However, it is interesting to find that these agents at lower dose (2mol/L) failed to affect 8

HIF-1 expression, but they lowered VEGF expression. Obviously, other upstream 9

factor(s) was involved for VEGF transcription in these triterpene-treated cancer cells. 10

bFGF, another angiogenic activator, is more potent than VEGF for stimulating the vascular 11

endothelial mitogenesis because bFGF enhances the release and activity of collagenases, 12

proteases and integrins on the extracellular membrane to form nascent microvascular 13

networks (23). In our present study, three triterpenic acids at test doses failed to lower 14

bFGF expression. These results suggested that the anti-angiogenic activity of test 15

triterpenic acids did not involve bFGF associated pathway. 16

IL-8 from tumor cells is an autocrine growth factor and acts on endothelial cells 17

through their receptors to promote migration, invasion and angiogenesis, which 18

consequently elevated metastatic potential (24). Thus, decreasing IL-8 expression and 19

production might also alleviate cancer progression. In our present study, test triterpenic 20

acids reduced IL-8 expression and production in dose-dependent manner, which 21

subsequently contributed to diminish angiogenesis. IL-8 expression could be evoked by 22

HIF-1and NO (25, 26). Since these triterpenic acids could suppress HIF-1expression 23

and reduce NO production, it seems reasonable to observe the declined expression of 24

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downstream target gene, IL-8. On the other hand, enhanced IL-8 expression could 1

increase the activity of some matrix metalloproteins and facilitate tumor cell invasion (27). 2

Thus, the lowered cell invasion as we observed in triterpene-treated liver cancer cells could 3

be partially ascribed to these agents already down-regulated IL-8. 4

It has been indicated that oxidative stress enhanced the expression of both HIF-1and 5

VEGF in cancer cells (28). Intracellular ROS derived from NADPH oxidase or 6

mitochondria, acting a signaling messenger, is able to activate critical target molecules 7

such as intercellular adhesion molecule-1 and trigger cell adhesion, 8

epithelial-mesenchymal transition and migration in tumor cells (29). Nitrite is capable of 9

inducing VEGF expression and favors nitrite-driven formation of endothelial tubes in 10

hypoxic tumor microenvironment (30). Obviously, liver cancer cells released ROS and 11

NO to promote their migration and angiogenesis. In our present study, triterpenic acids 12

treatments effectively retained GSH level, and reduced ROS and NO release in test cell 13

lines, which in turn alleviated oxidative stress and diminished the expression of angiogenic 14

factors such as VEGF. Several studies have reported that OA, UA and MA could 15

attenuate oxidative stress via sparing GSH, inhibiting NO production and/or scavenging 16

ROS (31-33). Thus, the lower ROS and NO levels in triterpenic acid-treated groups as 17

we observed could be explained. These findings revealed that these triterpenic acids 18

could mitigate angiogenesis in liver cancer cells through their anti-oxidative actions. 19

uPA converts plasminogen to plasmin, which is responsible for the degradation of 20

fibrin and extracellular matrix components, and finally promotes cell migration and 21

invasion (34). In our present study, the production and expression of uPA in the liver 22

cancer cell lines could be down-regulated by triterpenic acid treatments, which 23

subsequently decreased plasminogen conversion and degradation of fibrin and matrix 24

proteins. In addition, ROS could stimulate uPA expression in tumor cells (35). Thus, 25

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the reduced uPA expression from triterpenic acids could be partially due to these 1

compounds already decrease ROS production. Since uPA expression was suppressed, the 2

observed lower cell invasion and migration in these triterpenic acid-treated groups could be 3

explained. Furthermore, test triterpenic acids at 4 mol/L inhibited 50-70% cell invasion 4

and migration in these three cell lines. Thus, these agents might be able to retard HCC 5

progression via blocking metastatic process. 6

It is interesting to find that three test compounds at equal dose exhibited greater 7

suppressive effects upon VEGF and uPA expression in Hep3B and Huh7 cells than in 8

HA22T cells, but these agents were greater in declining IL-8 expression in HA22T cells 9

than in other two cell lines. It has been reported that Hep3B expressed more 10

mesenchymal proteins for mesenchymal transition, Huh7 was highly associated with 11

hepatitis B virus- or C virus-related HCC, and HA22T was a poorly-differentiated cell line 12

(36-38). Thus, these cells, based on different pathological features and biochemical 13

behavior, might exhibit different response to certain chemical(s). These findings implied 14

that selecting any agent for anti-hepatoma therapy should consider the responsible cancer 15

cell line, and/or target molecules. In addition, the results of our present study suggested 16

that any agent, like these triterpenic acids, with multiple anti-cancer actions toward 17

multiple HCC cell lines might be appropriate choice for anti-HCC therapy. 18

In summary, the content of oleanolic acid, ursolic acid and maslinic acid in daylily, 19

spinach and leaf mustard was in the range of 76-174 mg/100 g dry weight. These three 20

triterpenic acids at 4mol/L markedly suppressed mRNA expression of angiogenic factors, 21

HIF-1, VEGF, IL-8 and uPA. These triterpenic acids also effectively alleviated 22

oxidative stress via retaining GSH level, lowering ROS and NO production, as well as 23

decreased cell invasion and migration in Hep3B, Huh7 and HA22T cell lines. These 24

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findings suggest that these triterpenic acids are potent anti-angiogenic agents to retard 1

invasion and migration in liver cancer cells. 2

3

Acknowledgement 4

This study was supported by a grant from China Medical University, Taichung City, 5

Taiwan, ROC (Grant CMU99-asia-01). 6

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Table 1. Content (mg/100 g dry weight) of oleanolic acid (OA), ursolic acid (UA) and 1

maslinic acid (MA) in eleven vegetables, carrot, celery, okra, eggplant, spinach, leaf 2

mustard, gynura, Chinese chive, fennel, basil and daylily. Data are meanSD, n=6. 3 OA UA MA carrot 254 316 -celery 172 - -okra 616 233 404 eggplant 534 323 847 spinach 16713 1338 12611 leaf mustard 13511 926 17412 gynura -a 456 375 Chinese chive - - 384 fennel 548 175 -basil 967 8312 354 daylily 11810 765 1059 a

means too low to be detected. 4

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Figure 1. HIF-1mRNA expression in human Hep3B, Huh7 and HA22T cell lines under 1

normoxia and hypoxia condition. Data are meanSD (n = 9). a,bMeans among bars 2

without a common letter differ, P<0.05. 3

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1 b a b a b a 0 50 100 150 200 250 300 350 400 normoxia hypoxia re la ti v e m R N A ex p re ss io n

Hep3B Huh7 HA22T 2

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Figure 2. Effect of oleanolic acid (OA), ursolic acid (UA) and maslinic acid (MA) at 0 1

(control), 2 or 4 mol/L upon HIF-1mRNA expression in human Hep3B, Huh7 and 2

HA22T cell lines. Data are meanSD (n = 9). a-cMeans among bars without a common 3

letter differ, P<0.05. 4

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c _c b b b b b a a c b b a a a c b b b a a 0 20 40 60 80 100 120

Hep3B Huh7 HA22T

re la ti v e m R N A ex p re ss io n (% o f co n tr o l)

control OA, 2 OA, 4 UA, 2 UA, 4 MA, 2 MA, 4 1

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(control), 2 or 4mol/L upon bFGF mRNA expression in human Hep3B, Huh7 and HA22T 2

cell lines. Data are meanSD (n = 9). aMeans among bars without a common letter differ, 3

P<0.05.

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a a a a a a a a a a a _a a a a a a a _a a a 0 20 40 60 80 100

Hep3B Huh7 HA22T

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(control), 2 or 4mol/L upon VEGF level (upper part) and mRNA expression (lower part) in 2

human Hep3B, Huh7 and HA22T cell lines. Data are meanSD (n = 9). a-dMeans among 3

bars without a common letter differ, P<0.05. 4

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d c c c b b b a a b b b a a a b b b a a a 0 50 100 150 200 250

Hep3B Huh7 HA22T

p g /m g p ro te in

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c c c b b b a a a b b b a a a b b b a a a 0 20 40 60 80 100 120

Hep3B Huh7 HA22T

control OA, 2 OA, 4 UA, 2 UA, 4 MA, 2 MA, 4

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(control), 2 or 4 mol/L upon IL-8 level (upper part) and mRNA expression (lower part) in 2

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d d d b c b a b a b c b a a a c c c b b b 0 50 100 150 200 250 300

Hep3B Huh7 HA22T

p g /m g p ro te in

1 2 3

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d c c b b b a a a b b b a a a c b b a a a 0 20 40 60 80 100 120

Hep3B Huh7 HA22T

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Table 2. Effect of oleanolic acid (OA), ursolic acid (UA) and maslinic acid (MA) at 0 1

(control), 2 or 4 mol/L upon level of GSH, ROS and NO in human normal liver cell line 2

(L-02 cells), Hep3B, Huh7 and HA22T cell lines. Data are meanSD (n = 9). 3

GSH ng/mg protein

L-02 Hep3B Huh7 HA22T

Control 804a 312a 233a 384a OA, 2 845a 423b 364b 475b OA, 4 883a 575c 505c 605c UA, 2 826a 404b 403b 454b UA, 4 853a 564c 604d 582c MA, 2 795a _475b _334b _503b MA, 4 874a _693d _482c _635c

ROS nmol/mg protein

L-02 Hep3B Huh7 HA22T

Control 0.350.04a 2.060.26d 2.140.13c 1.900.18c OA, 2 0.330.06a 1.670.20c 1.750.17b 1.580.10b OA, 4 0.310.02a 1.030.14a 1.390.15a 1.270.12a UA, 2 0.290.05a 1.720.21c 1.650.09b 1.650.15b UA, 4 0.320.03a 1.350.16b 1.230.10a 1.190.07a MA, 2 0.300.06a 1.700.18c 1.720.15b 1.520.14b MA, 4 0.330.05a _1.380.13b _1.400.11a _1.230.09a NO M/mg protein

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Control 1.30.3a _15.71.1d _14.61.0c _15.31.4d OA, 2 1.60.5a _10.50.7b _12.10.6b _10.81.0b OA, 4 1.40.2a 8.30.9a 8.51.1a 7.90.8a UA, 2 1.20.4a 12.60.8c 11.80.9b 12.91.2c UA, 4 1.50.2a 8.01.1a 8.30.7a 7.60.5a MA, 2 1.40.5a 13.11.0c 12.21.2b 13.00.9c MA, 4 1.30.6a 8.20.6a 9.00.8a 7.40.6a a-d

Means in a column without a common letter differ, P<0.05. 1

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(control), 2 or 4 mol/L upon invasion and migration in human Hep3B, Huh7 and HA22T 2

cell lines. Data are meanSD (n = 9). a-dMeans among bars without a common letter 3

differ, P<0.05. 4

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invasion c c c b b b a a a b b b a a a b b b a a a 0 20 40 60 80 100 120

Hep3B Huh7 HA22T

% o f co n tr o l

1 2

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migration c c d b b c a _a b b b b a a a b b c a a b 0 20 40 60 80 100 120

Hep3B Huh7 HA22T

% o f co n tr o l

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(control), 2 or 4mol/L upon uPA level (upper part) and mRNA expression (lower part) in 2

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1 d d b c c b b b a c c b a b a c c b a a a 0 2 4 6 8 10 12 14 16

Hep3B Huh7 HA22T

n g /m g p ro te in

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b d d b c c a b b b c c a b a b b c a a a 0 20 40 60 80 100 120

Hep3B Huh7 HA22T