apoptotic effects of a high performance liquid chromatography (hplc) fraction of antrodia camphorata...

1
Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata are mediated via down-regulation of the expressions of four tumor-related genes in human non-small cell lun g carcinoma A549 cell Lan-Hsiang Chien ( 簡簡簡 ), Yu-Yi Chan ( 簡簡簡 ), Chun-Sheng Chang ( 簡簡簡 ), Ting-Feng Wu* ( 簡簡簡 ) Department of Biotechnology and Biotechnology Research Center, Southern-Taiwan University, Tainan, Taiwan Abstract Antrodia camphorata (niu-chang-chih) is a fu ngus native to Taiwan which is believed to be effective in preventing diseases. Recen t reports demonstrate that A. camphorata pro ducts induce the apoptosis of various kind s of tumor cells. In this study we determi ned the inhibitory effects of alcohol extr act and individual fractions of alcohol ex tract on the proliferation of human non-sm all cell lung carcinoma A549 cell and clar ified the mechanism underlying the anti-ca ncer activities. Three HPLC fractions, fractions 5 to 7, had robust inhibition of human A549 cells and among them fraction 6 (Fr-6) possessed the most potent effectiveness. Apoptotic a ssay showed that Fr-6 induced human A549 c ell apoptosis by triggering the mitochondr ial pathway and endothelium reticulum (ER) stress. Immunoblotting results demonstrate d that Fr-6 possibly activated the mitocho ndrial pathway and ER stress by lowering t he expression level of calpain 1/2 small s ubunit and Fr-6-mediated decrease in cell proliferation might attribute to the suppr essive effect on the Erk 1/2 pathway, whic h arose from Fr-6-derived low galectin-1 e xpression. Furthermore Fr-6 could diminish Rho GDP dissociation inhibitor (RhoGDI- ) expression and subsequently activated c-Jun NH2-terminal kinase (JNK) pathway, w hich is linked to cell apoptosis. Fr-6 als o could decrease the production level of e ukaryotic translation initiation factor 5A, which is a potential cancer intervention target. Introduction Antrodia camphorata is the traditional T aiwan crude drug for treating diarrhea, hypertension , inflammation and cancer. Previous studies have shown that the Antrod ia camphorata crude extract has anti-prolifer ation and anti-migration on the transitiona l cell carcinomas (Chiung, et al., 2007) and ethanol extract from wild fruiting bodie s could induce leukemia HL-60 cells apoptos is ( Mei et al., 2008 ). Recent studies has identified that lanostane and ergostane - type triterpenes isolated from the fru iting bodies of A. camphorata could inhib it proliferation of human colon, live, br east, lung cancer cells ( Chi et al., 200 9). However, the wild fruiting bodies o f A. camphorata are rarity in nature, ver y expensive and the failure of artificial cultivation . Therefore , using a subm erged cultured method to obtain useful cell ular materials or to produce effective s ubstances from cultured mycelia , might b e a possible way to overcome the disadvanta ge of the fruiting bodies ( Sone et al., Agricultural and Biological Chemist ry , 1985 ). The mycelia of Antrodia camphor ata in submerged culture induced apoptos is in human hepatoma cells ( Tuzz e t al., Journal of Ethnopharmacology , 200 5 ). A. camphorata inhibited proliferation and induced apoptosis of human breast c ancer cells in vitro and in vivo ( Hseu et al., Food and Chemical Toxicology , 2008 ) . Our previous study demonstrates that ethanol extracts of A. camphorata cu ltivated by solid -state fermentation coul d effectively impede the proliferation of human lung cancer A549 cells and inhibi t the expression of galectin-1, eIF-5a, R hoGDI-α and calpain-1/2 ( Hung et al., Pro teomics, 2006). However, no information abo ut their mechanism of action and signific ant compounds. To explore their significa nt compounds , the ethanol extracts of A. camphorata (AC-EtOH) were separated by H PLC and collect to one fraction every fifte en minutes. This study evaluates the whethe r the eight fractions also have the inhibit ory effect on A549 cell line and expl ore their mechanism of action. Results and discussion Figure 1. Preparation for Antrodia camphorata ex tracts ( A ) The extraction flow chart ( B ) The ethanol extracts of A. camphorata were separated using HPLC column and the elut e was collected every fifteen minutes. Total eight fractions were obtained. Methods Preparation for Antrodia camphorata extract s A. camphorata mycelia were provided by Bio technology Center, Southern Taiwan Univer sity. A. camphorata mycelia (about 555 g) w ere sequentially extracted using the sol vent with increasing polarity, including n-hexane, ethylacetate, ethanol and wtaer (Figure 1A). 20.1 g, 16 g and 127.6 g of the products were obtained respectively i n the EtOAc, EtOH and water extraction ex periments. Separation of the ethanol extract of A. camphorata by high performance liquid chr omatography (HPLC) The ethanol extracts were separated us ing HPLC column (250 x 4.6 簡 , Hypersil, 5μm ) with a linear solvent gradient elution system composed of solvents A an d B ( A H2O B 100 % acetonitrite ) The elute was collected every 15 minutes and totally eight fractions were obtain ed. Each fraction was dissolved in dim ethyl sulfoxide ( DMSO ) and control cultures received the carrier solvent ( 0.1 % DMSO ). Cell viability Cell viability was measured by MT T assay. Apoptosis analysis SubG 1 analysis and Annexin V/propid ium iodide (PI) assay were used to observe human A549 cell apoptosis Western blotting After treatment as indicated above , cells were collected and l ysed in the lysis buffer. Total cell lysates were separated by 12.5 % SDS- PAGE and transferred onto PVDF membranes. The m embrane was blotted with primary antibody overnight , followed by incubation with HRP-conjugated secondary antib ody and visualized using chemilumine Hexane A. camphorata mycelia Sup. ppt. EtoAc Sup. ppt. EtoH Sup. ppt. H 2 O Sup. ppt. Assay Assay Assa y Assay A M inutes 0 25 50 75 100 125 m AU 0 1000 2000 m AU 0 1000 2000 1 2 3 4 5 6 7 8 B Figure 2. The inhibitory effects of HPLC fract ions on human A549 cells. (A) Treatment w ith 200μg/ml for 72 h, the maximum inhibit ory effect on proliferation was fractions 5-7. ( B ) Treatment with various conce ntration of fraction 5-7 for 72 h, 25 μg / m l fraction 6 inhibited cell proliferation at 72 % , indicating that human A549 cell w as more sensitive to fraction 6 than to fract ion 5 and 7. ( C ) Treatment with 50μg / ml of fraction 6 for 0~72 h, which inhibite d cell proliferation at 80% and the result wa s similar with Fig. 2B. (D) Treatment with v arious concentration of fraction 6 on MRC-5 c ell, the inhibitory effect was observed with 100μg/ml. These results suggested that norma l ( MRC-5 ) and cancer (A549) cell lines exhi bit differential sensitivity to fraction 6. 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 Fraction Survival ratio (% ofcontrol) 0 20 40 60 80 100 120 140 5μg/m l 10μg/m l 25μg/m l 50μg/m l 100μg/m l 150μg/m l 200μg/m l Concentration Survival ratio (% ofcontrol) Fraction-5 Fraction-6 Fraction-7 0 20 40 60 80 100 120 0hr 4hr 8hr 12hr 24h 48h 72hr Treatm entTimes Survival ratio (% ofcontrol) 0 50 100 150 200 250 5μg/m l 10μg/m l 25μg/m l 50μg/m l 100μg/m l 150μg/m l 200μg/ml Concentration Survival ratio (% ofcontrol) A B D C Figure 3. Apoptosis assay ( A ) , ( B ) The remarkable accumulation o f sub-G1 was considered apoptosis. Fr action- 6 (50μg/ml) elevated the percen tage of human A549 cells in the sub-G1 phase from 24% to 34%, 64% (24h~72h) wh en compared with the control group. (C),(D) Compared with vehicle-treated cells, 50μg / ml fraction 6 induced 1 7%, 29%, 13% of early apoptosis cell s and 6%, 11%, 75% of late apoptosis c ells at 24h, 48h and 72 h. These resul ts suggest edthat fraction 6 could indu ce apoptosis on A549 cell. However, cyt ometric analysis could not determine wh ether Fr-6 leaded to the apoptosis by d eath receptor signaling, mitochondrial damage or ER stress. To explore the mec hanism by which Fr-6 invoked the apopto sis, western blotting with anti-cappas e-3, -12 and bax/bcl-2 were carried out in Fig. 4. 24h 48h 72h control 50μg/ml 0 10 20 30 40 50 60 70 80 90 24hr 48hr 72hr Treatm entTime sub-G 1(% oftotalcel Control Fraction-6 control 50μg/ml 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 C ontrol- 24hr Fraction 6-24hr C ontrol- 48hr Fraction 6-48hr C ontrol- 72hr Fraction 6-72hr Apoptotic cells (% oftatal cells Late apoptosis Early apoptosis A B C D 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0hr 24hr 48hr 72hr caspase3 /ac Bcl-2 Actin Bax 0hr 4hr 8hr 12hr 24hr 0.00 0.50 1.00 1.50 2.00 2.50 3.00 0hr 4hr 8hr 12hr 24hr B a x / B c 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 0hr 4hr 8hr 12hr 24hr c a s p a s e 1 2 / a 0hr 24hr 48hr 72hr Pro-caspase3 Actin Pro- caspase12 Acti n 0hr 4hr 8hr 12hr 24hr A B C Figure 4. Effects of fraction 6 on caspas e-3, caspase-12 activation and Bax, Bc l-2 expression ( A ) A549 cells treat ed with fraction 6 caused a time-dependen t decrease in pro-caspase3. (B) Bax coul d induce apoptosis, but Bcl-2 could inhib it apoptosis on the mitochondria pat hway. The up-regulation of Bax / Bc l-2 expression was detected at time-dep endent after treatment. (C) ER-induc ed apoptosis is associated with the act ivation of caspase12. Fraction 6 also cau sed a time-dependent decrease in pro-caspase12. These results suggested t hat fraction 6 could induce apoptosis on A549 cells through mitochondria pat hway and ER-induced pathway. Our previ ous examinations suggested that the ethan ol extracts of A. camphorata (SACE) can ev oke the apoptosis of human A549 cells pos sibly by the down-regulation of human gal ectin-1, human eIF5A, human Rho GDI-, hu man calpain small (regulatory) subunit ce lls (Wu et al., 2006). 1.50 1.60 1.70 1.80 1.90 2.00 2.10 0hr 15min 30min 1hr 2hr 4hr 8hr R hoG D I/ac Actin RhoGDI-α 0hr 15min 30min 1hr 2hr 4hr 8hr Acti n eIF- 5a 0hr 15min 30min 1hr 2hr 4hr 8hr 0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 0hr 15min 30min 1hr 2hr 4hr 8hr eIF -5 a / a c 0.00 0.20 0.40 0.60 0.80 1.00 1.20 0hr 15min 30min 1hr 2hr 4hr 8hr galectin-1 /actin 0hr 15min 30min 1hr 2hr 4hr 8hr Galectin -1 Actin Calpain-1/2 (small subunit) Actin 0hr 15min 30min 1hr 2hr 4hr 8hr 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0hr 15min 30min 1hr 2hr 4hr 8hr calpain-1/2 / actin A B C D Figure 5. Effect of fraction 6 on galect in-1, calpain-1/2, RhoGDI-α and eIF-5a expression To investigate if Fr-6 had t he same effect on the expression of thes e four tumor-related genes, western blot ting with the antibody against each of t hese four proteins was carried out. (A), (B), (C), (D) Fraction 6 (50μg / ml) cau sed a time-dependent decrease in galec tine-1, calpain-1 / 2, RhoGDI-α an d eIF-5a expression after treatme nt. These results suggest ed that fra ction 6 could reduce the expression of the proteins as indicated above on A5 49 cell. 0hr 15min 30min 1hr 2hr 4hr 8hr ERK-1 ERK-2 Acti n 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 0hr 15min 30min 1hr 2hr 4hr 8hr ER K /actin ERK-1 ERK-2 JNK Acti n 0hr 4hr 8hr 12hr 24hr 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0hr 4hr 8hr 12hr 24hr JN K / ac 0.0 0.3 0.6 0.9 0hr 15m in 30m in 1hr 2hr 4hr 8hr p-E R K / ac t p-ERK-1 p-ERK-2 p-ERK- 1 p-ERK- 2 Acti n 0hr 15min 30min 1hr 2hr 4hr 8hr p-JNK1 p- JNK2/3 Acti n 0hr 4hr 8hr 12hr 24hr 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0hr 4hr 8hr 12hr 24hr p-JN K /actin p-JNK-1 p-JNK-2/3 A B C D Figure 6. Effect of fraction 6 on ERK and JN K activation. Recent findings showed that th e recruiting of H-Ras to the cell membrane i s dependent on galectin-1 and galectin-1 bin ds H-Ras to stimulate the transformation (Bel anis et al., 2008; Paz et al., 2001). Since Ras take part in Erk pathway, western blotti ng with the antibodies against Erk and phosp ho-Erk (p-Erk) to examine if Fr-6 could inte rfere with Erk pathway. (A), (B) The dow n-regulation of ERK and phospho-ERK expr ession were detected at time - de pendent after treatment. Recent investigat ions showed that he inhibition of RhoGDI- p roduction invokes the apoptosis of insulin-s ecreting cells by the activation of c-Jun NH 2-terminal kinase (JNK) pathway. (C) The dow n-regulation of JNK expression was detected at time-dependent after treatment. (D) The up-regulation of phospho-JNK expressi on was detected at time-dependent after t reatment. These results suggest that fractio n 6 could prevent the activation of ERK, bu t promote the activation of JNK. 0 20 40 60 80 100 120 140 0hr 24hr 48hr 72hr Treatm entTim e Survivalratio (% ofcontrol) 50μg/mlfraction-6 fraction-6+15uM SP600125 0hr 4hr 8hr 12hr Acti n JNK P-JNK1 P-JNK2/3 Fraction- 6+SP600125 RhoGDI A F-6 Fraction- 6+SP600125 0hr 2hr 4hr 8hr 8hr Actin B C Figure 7. Effect of fraction 6 on activated JNK expression in relation to RhoGDI-α Cells were treated with 50μg / ml fraction 6 alone or 15μM JNK inhi bitor ( SP600125 ) for the time periods in dicated. (A), (B) The SP600125 was admi nistered to cells 24 h prior to fraction- 6 treatment. Pre-treatment with JNK inhibitor could prevent the activation of JNK and the reduction in RhoGDI-α expression caused by fraction 6. ( C ) Viability of cells treated with both fraction 6 and the JNK inhibitor was 42 % greater than tha

Upload: annabelle-norman

Post on 29-Dec-2015

219 views

Category:

Documents


3 download

TRANSCRIPT

Page 1: Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata are mediated via down- regulation of the expressions

Apoptotic effects of a high performance liquid chromatography (HPLC) fraction of Antrodia camphorata are mediated via down-regulation of the expressions of four tumor-related genes in human non-small cell lung carcinoma A549 cell

Lan-Hsiang Chien (簡嵐翔 ), Yu-Yi Chan (詹于宜 ), Chun-Sheng Chang (張春生 ), Ting-Feng Wu* (吳定峰 )

Department of Biotechnology and Biotechnology Research Center, Southern-Taiwan University, Tainan, TaiwanAbstract Antrodia camphorata (niu-chang-chih) is a fungus native to Taiwan which is believed to be effective in preventing diseases. Recent reports demonstrate that A. camphorata products induce the apoptosis of various kinds of tumor cells. In this study we determined the inhibitory effects of alcohol extract and individual fractions of alcohol extract on the proliferation of human non-small cell lung carcinoma A549 cell and clarified the mechanism underlying the anti-cancer activities. Three HPLC fractions, fractions 5 to 7, had robust inhibition of human A549 cells and among them fraction 6 (Fr-6) possessed the most potent effectiveness. Apoptotic assay showed that Fr-6 induced human A549 cell apoptosis by triggering the mitochondrial pathway and endothelium reticulum (ER) stress. Immunoblotting results demonstrated that Fr-6 possibly activated the mitochondrial pathway and ER stress by lowering the expression level of calpain 1/2 small subunit and Fr-6-mediated decrease in cell proliferation might attribute to the suppressive effect on the Erk 1/2 pathway, which arose from Fr-6-derived low galectin-1 expression. Furthermore Fr-6 could diminish Rho GDP dissociation inhibitor (RhoGDI-) expression and subsequently activated c-Jun NH2-terminal kinase (JNK) pathway, which is linked to cell apoptosis. Fr-6 also could decrease the production level of eukaryotic translation initiation factor 5A, which is a potential cancer intervention target.

Introduction

Antrodia camphorata is the traditional Taiwan crude drug for treating diarrhea, hypertension , inflammation and cancer. Previous studies have shown that the Antrodia camphorata crude extract has anti-proliferation and anti-migration on the transitional cell carcinomas (Chiung, et al., 2007) and ethanol extract from wild fruiting bodies could induce leukemia HL-60 cells apoptosis ( Mei et al., 2008 ). Recent studies has identified that lanostane and ergostane - type triterpenes isolated from the fruiting bodies of A. camphorata could inhibit proliferation of human colon, live, breast, lung cancer cells ( Chi et al., 2009).

However, the wild fruiting bodies of A. camphorata are rarity in nature, very expensive and the failure of artificial cultivation . Therefore , using a submerged cultured method to obtain useful cellular materials or to produce effective substances from cultured mycelia , might be a possible way to overcome the disadvantage of the fruiting bodies ( Sone et al., Agricultural and Biological Chemistry , 1985 ). The mycelia of Antrodia camphorata in submerged culture induced apoptosis in human hepatoma cells ( Tuzz et al., Journal of Ethnopharmacology , 2005 ). A. camphorata inhibited proliferation and induced apoptosis of human breast cancer cells in vitro and in vivo ( Hseu et al., Food and Chemical Toxicology , 2008 ) . Our previous study demonstrates that ethanol extracts of A. camphorata cultivated by solid -state fermentation could effectively impede the proliferation of human lung cancer A549 cells and inhibit the expression of galectin-1, eIF-5a, RhoGDI-α and calpain-1/2 ( Hung et al., Proteomics, 2006). However, no information about their mechanism of action and significant compounds. To explore their significant compounds , the ethanol extracts of A. camphorata (AC-EtOH) were separated by HPLC and collect to one fraction every fifteen minutes. This study evaluates the whether the eight fractions also have the inhibitory effect on A549 cell line and explore their mechanism of action.

Results and discussion

Figure 1. Preparation for Antrodia camphorata extracts ( A ) The extraction flow chart ( B ) The ethanol extracts of A. camphorata were separated using HPLC column and the elute was collected every fifteen minutes. Totally eight fractions were obtained.

Methods

Preparation for Antrodia camphorata extracts

A. camphorata mycelia were provided by Biotechnology Center, Southern Taiwan University. A. camphorata mycelia (about 555 g) were sequentially extracted using the solvent with increasing polarity, including n-hexane, ethylacetate, ethanol and wtaer (Figure 1A). 20.1 g, 16 g and 127.6 g of the products were obtained respectively in the EtOAc, EtOH and water extraction experiments.

Separation of the ethanol extract of A. camphorata by high performance liquid chromatography (HPLC) The ethanol extracts were separated using HPLC column (250 x 4.6 ㎜ , Hypersil, 5μm ) with a linear solvent gradient elution system composed of solvents A and B ( A: H2O; B: 100 % acetonitrite ) The elute was collected every 15 minutes and totally eight fractions were obtained. Each fraction was dissolved in dimethyl sulfoxide ( DMSO ) and control cultures received the carrier solvent ( 0.1 % DMSO ).Cell viability Cell viability was measured by MTT assay.

Apoptosis analysis

SubG1 analysis and Annexin V/propidium iodide (PI) assay were used to observe human A549 cell apoptosis

Western blotting

After treatment as indicated above , cells were collected and lysed in the lysis buffer. Total cell lysates were separated by 12.5 % SDS- PAGE and transferred onto PVDF membranes. The membrane was blotted with primary antibody overnight , followed by incubation with HRP-conjugated secondary antibody and visualized using chemiluminescence.

HexaneA. camphorata mycelia

Sup.ppt.EtoAc

Sup.ppt.EtoH

Sup.ppt.H2O

Sup.ppt.

Assay

Assay

Assay

Assay

A

Minutes

0 25 50 75 100 125

mA

U

0

1000

2000

mA

U

0

1000

2000

1 2 3 4 5 6 7 8

B

Figure 2. The inhibitory effects of HPLC fractions on human A549 cells. (A) Treatment with 200μg/ml for 72 h, the maximum inhibitory effect on proliferation was fractions 5-7. ( B ) Treatment with various concentration of fraction 5-7 for 72 h, 25 μg / ml fraction 6 inhibited cell proliferation at 72 % , indicating that human A549 cell was more sensitive to fraction 6 than to fraction 5 and 7. ( C ) Treatment with 50μg / ml of fraction 6 for 0~72 h, which inhibited cell proliferation at 80% and the result was similar with Fig. 2B. (D) Treatment with various concentration of fraction 6 on MRC-5 cell, the inhibitory effect was observed with 100μg/ml. These results suggested that normal ( MRC-5 ) and cancer (A549) cell lines exhibit differential sensitivity to fraction 6.

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8

Fraction

Surv

ival r

atio

(%

of contr

ol)

0

20

40

60

80

100

120

140

5μg/ml 10μg/ml 25μg/ml 50μg/ml 100μg/ml 150μg/ml 200μg/ml

Concentration

Sur

viva

l rat

io (

% o

f con

trol

)

Fraction-5

Fraction-6

Fraction-7

0

20

40

60

80

100

120

0hr 4hr 8hr 12hr 24h 48h 72hr

Treatment Times

Surv

ival r

atio

(%

of contr

ol)

0

50

100

150

200

250

5μg/ml 10μg/ml 25μg/ml 50μg/ml 100μg/ml 150μg/ml 200μg/ml

Concentration

Surv

ival r

atio

(%

of contr

ol)

A B

DC

Figure 3. Apoptosis assay ( A ) , ( B ) The remarkable accumulation of sub-G1 was considered apoptosis. Fraction- 6 (50μg/ml) elevated the percentage of human A549 cells in the sub-G1 phase from 24% to 34%, 64% (24h~72h) when compared with the control group. (C),(D) Compared with vehicle-treated cells, 50μg / ml fraction 6 induced 17%, 29%, 13% of early apoptosis cells and 6%, 11%, 75% of late apoptosis cells at 24h, 48h and 72 h. These results suggest edthat fraction 6 could induce apoptosis on A549 cell. However, cytometric analysis could not determine whether Fr-6 leaded to the apoptosis by death receptor signaling, mitochondrial damage or ER stress. To explore the mechanism by which Fr-6 invoked the apoptosis, western blotting with anti-cappase-3, -12 and bax/bcl-2 were carried out in Fig. 4.

24h

48h

72h

control 50μg/ml

0

10

20

30

40

50

60

70

80

90

24hr 48hr 72hr

Treatment Time

sub-G

1(%

of to

tal c

ells

) Control

Fraction-6

control 50μg/ml

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

100.00

Control-24hr

Fraction6-24hr

Control-48hr

Fraction6-48hr

Control-72hr

Fraction6-72hr

Apopto

tic c

ells

(%

of ta

tal c

ells

)

Late apoptosis

Early apoptosis

A

B

C

D

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0hr 24hr 48hr 72hr

caspase3 / a

ctin

Bcl-2

Actin

Bax

0hr 4hr 8hr 12hr 24hr

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0hr 4hr 8hr 12hr 24hr

Bax / B

cl-2

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0hr 4hr 8hr 12hr 24hr

caspase12 / a

ctin

0hr 24hr 48hr 72hr

Pro-caspase3

Actin

Pro-caspase12

Actin

0hr 4hr 8hr 12hr 24hr

A

B

C

Figure 4. Effects of fraction 6 on caspase-3, caspase-12 activation and Bax, Bcl-2 expression ( A ) A549 cells treated with fraction 6 caused a time-dependent decrease in pro-caspase3. (B) Bax could induce apoptosis, but Bcl-2 could inhibit apoptosis on the mitochondria pathway. The up-regulation of Bax / Bcl-2 expression was detected at time-dependent after treatment. (C) ER-induced apoptosis is associated with the activation of caspase12. Fraction 6 also caused a time-dependent decrease in pro-caspase12. These results suggested that fraction 6 could induce apoptosis on A549 cells through mitochondria pathway and ER-induced pathway. Our previous examinations suggested that the ethanol extracts of A. camphorata (SACE) can evoke the apoptosis of human A549 cells possibly by the down-regulation of human galectin-1, human eIF5A, human Rho GDI-, human calpain small (regulatory) subunit cells (Wu et al., 2006).

1.50

1.60

1.70

1.80

1.90

2.00

2.10

0hr 15min 30min 1hr 2hr 4hr 8hr

RhoG

DI / actin

Actin

RhoGDI-α

0hr 15min 30min 1hr 2hr 4hr 8hr

Actin

eIF-5a

0hr 15min 30min 1hr 2hr 4hr 8hr

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

0hr 15min 30min 1hr 2hr 4hr 8hr

eIF

-5a / a

ctin

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0hr 15min 30min 1hr 2hr 4hr 8hr

gale

ctin

-1 / a

ctin

0hr 15min 30min 1hr 2hr 4hr 8hr

Galectin-1

Actin

Calpain-1/2

(small subunit)

Actin

0hr 15min 30min 1hr 2hr 4hr 8hr

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0hr 15min 30min 1hr 2hr 4hr 8hr

calp

ain

-1/2

/ a

ctin

A B

C D

Figure 5. Effect of fraction 6 on galectin-1, calpain-1/2, RhoGDI-α and eIF-5a expression To investigate if Fr-6 had the same effect on the expression of these four tumor-related genes, western blotting with the antibody against each of these four proteins was carried out. (A), (B), (C), (D) Fraction 6 (50μg / ml) caused a time-dependent decrease in galectine-1, calpain-1 / 2, RhoGDI-α and eIF-5a expression after treatment. These results suggest ed that fraction 6 could reduce the expression of the proteins as indicated above on A549 cell.

0hr 15min 30min 1hr 2hr 4hr 8hr ERK-1

ERK-2

Actin

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

0hr 15min 30min 1hr 2hr 4hr 8hr

ER

K / a

ctin

ERK-1

ERK-2

JNK

Actin

0hr 4hr 8hr 12hr 24hr

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0hr 4hr 8hr 12hr 24hr

JN

K / a

ctin

0.0

0.3

0.6

0.9

0hr 15min 30min 1hr 2hr 4hr 8hr

p-E

RK

/ a

cti

np-ERK-1

p-ERK-2

p-ERK-1

p-ERK-2

Actin

0hr 15min 30min 1hr 2hr 4hr 8hr

p-JNK1

p-JNK2/3

Actin

0hr 4hr 8hr 12hr 24hr

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0hr 4hr 8hr 12hr 24hr

p-JN

K /

actin

p-JNK-1

p-JNK-2/3

A B

C D

Figure 6. Effect of fraction 6 on ERK and JNK activation. Recent findings showed that the recruiting of H-Ras to the cell membrane is dependent on galectin-1 and galectin-1 binds H-Ras to stimulate the transformation (Belanis et al., 2008; Paz et al., 2001). Since Ras take part in Erk pathway, western blotting with the antibodies against Erk and phospho-Erk (p-Erk) to examine if Fr-6 could interfere with Erk pathway. (A), (B) The down-regulation of ERK and phospho-ERK expression were detected at time - dependent after treatment. Recent investigations showed that he inhibition of RhoGDI- production invokes the apoptosis of insulin-secreting cells by the activation of c-Jun NH2-terminal kinase (JNK) pathway. (C) The down-regulation of JNK expression was detected at time-dependent after treatment. (D) The up-regulation of phospho-JNK expression was detected at time-dependent after treatment. These results suggest that fraction 6 could prevent the activation of ERK, but promote the activation of JNK.

0

20

40

60

80

100

120

140

0hr 24hr 48hr 72hr

Treatment Time

Surv

ival

ratio

(% o

f con

trol)

50μ g/ml fraction-6

fraction-6+15uM SP600125

0hr 4hr 8hr 12hr

Actin

JNK

P-JNK1

P-JNK2/3

Fraction-6+SP600125

RhoGDI

A

F-6Fraction-6+SP600125

0hr 2hr 4hr 8hr 8hr

Actin

B

C

Figure 7. Effect of fraction 6 on activated JNK expression in relation to RhoGDI-α Cells were treated with 50μg / ml fraction 6 alone or 15μM JNK inhibitor ( SP600125 ) for the time periods indicated. (A), (B) The SP600125 was administered to cells 24 h prior to fraction- 6 treatment. Pre-treatment with JNK inhibitor could prevent the activation of JNK and the reduction in RhoGDI-α expression caused by fraction 6. ( C ) Viability of cells treated with both fraction 6 and the JNK inhibitor was 42 % greater than that of cells treated with fraction 6 alone at 72 h. These results suggested that fraction 6-induced cell death might occur through an increase in the levels of activated JNK.