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Page 1: Aspergillus fumigatus and Cylindrocarpon candidum …egyptianjournal.xyz/27_4.pdf · 1- Trypan blue exclusion test. 0.05 % trypan blue solution was added to cell suspension (v/v)

The Egyptian Journal of Hospital Medicine (2007) Vol., 27: 163– 175

Aspergillus fumigatus and Cylindrocarpon candidum fungi induced apoptosis

in HepG 2 cell line through activation of caspases enzymes

Osman, M.E. *, L,A,R, Sallam**, Wafaa Abdallah Ahmed***, Khttab, O.H.*,

Abo EL-Nasser, A.A* * Department of Botany and Microbiology, Faculty of science, Helwan University.

**National Research Center, Department of Microbiology and Natural Products Chemistry

Egypt.***Department of Cancer Biology, Cancer Institute, Cairo University.

Abstract

Introduction: Many investigations are now interested to discover naturally occurring

compounds, which can be used for the prevention and treatment of cancer. Most natural products which may be used as adjuvant therapy or to reduce the side effect of chemotherapy

and radiotherapy. More than 300 products obtained from microorganisms have antitumor

activities.

Results: In the study we isolated N-(3-4-Dichlorophenyl) 2-Methyl, 2,3Dihydroxypropio amide from Aspergillus fumigatus and 2.4.6. Triphenyl pyridine from Cylindrocarpon

candidum and investigate the cytotoxic effect and apoptotic effect on HepG2 cell line. The

results revealed high cytotoxic effect at the concentration of 400µg/ml for both N-(3-4-Dichlorophenyl) 2-Methyl, 2,3Dihydroxypropio amide and 2.4.6. Triphenyl pyridine and effect

is increase with time of incubation. The apoptotic effect of both products were investigated by

measurement the caspase enzymes, the results showed highest activity of caspase 3 and caspase 9. Also at concentration 400µg/ml in both products.

Conclusion: From this data we observe that two isolated product have antitumor effect

and this effect is related to the concentration of the products and incubation period. Also, the

two products induce apoptosis through increase activation of caspase 3 and caspase 9 which lead to programmed of cell death. This study need to furthermore study on experimental animal

to confirm our results.

Introduction The first line of cancer treatment is

surgery which has significantly reduced the

cancer mortality. The use of additional

treatment such as radiotherapy and chemo-therapy has resulted in no more than 5%

reduction of death (Samantha et al., 2003).

The development of tumor drug resistance during treatment is the major factor

limiting the success of chemotherapeutic

management of tumors. Such resistance may occur during primary therapy or be

acquired during subsequent treatment (Curt

et al., 1984 and Cairo et al., 1989).

Many investigations are now interested to discover naturally occurring

compounds, which can be used for the

prevention and treatment of cancer despite little understanding about their molecular

and cellular basis of action Hala et al .

(2004). Wainwright (1992) reported that

300 antitumor agents have been isolated

from microorganisms, 13% have been

obtained from fungi. Of 43 antitumor

agents obtained from fungi, 23 come from the imperfect fungi, 15 from the Basid-

iomycetes and 5 from the Ascomycetes.

Zhang et al. (1994) isolated sixteen polys-accharides from the mycelium of G. tsngac

three of them showed antitumor activity

against the solid cancer sarcoma 180/mice. Polysaccharides extracted from

Sargassum thunbergii have strong antitu-

mor effects against transplanted tumors

such as sarcoma 180 and Ehrlich solid carcinoma (Zhuang et al., 1995).

Acebal et al. (1999) isolated agroc-

helin as a new alkaloid cytotoxic substance by the fermentation of Agrob-acterium sp.

The compound was obtained from the bac-

terial cell by solvent extraction and purified

163

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Aspergillus fumigatus and Cylindrocarpon candidum………

164

by silica gell chromatography. Rimpler et

al. (1996) described the ability of fungi in

the families Tricholomataceae and Polyporacea to produce antitumor activity.

The effects of a polysaccharide-

protein complex (PSPC) isolated from the

culture filtrate of T. lobayense were inves-tigated in mice injected with S-180 cells.

PSPC restored the phagocytic function of

peritoneal exudate cells (PEC) and T-cell mitogenic activity in tumor-bearing mice.

(Liu 1996).

Gary et al. (1997) purified a series of

peptide antifungal – anticancer agents kno-wn as leucinostatin A from Acremo-nium

sp. Leucinostatin A possess activity against

certain human cancer cell lines. Raha et al. (1990) found that L-Aspara-ginase from

Cylindrocarpon obtusisporum MB-10 inhi-

bits the growth of ascites fibro sarcoma and Dalton's Lymphoma tumor cells in vivo

and significantly increases the survi-val

rate of tumor bearing mice. The enzymetr-

eated normal mice become healthier and survive longer than their usual life span.

In this work we continue the

previous study on the Aspergillus fumig-atus and Cylindrocarpon candidum to emp-

hasis has been imposed for isolation, puri-

fication and identification active principles in the both extracts of the two mentioned

organisms. Furthermore we study the

cytotoxic effect of the two organisms on

HepG2 cells and trying to identify their mechanism of action.

Material and methods

Microorganisms.

Experimental fungal species

Two fungal species were used in the study:

1- Aspergillus fumigatus was obtained

during the studies curried by Mahmod

(2004)

2- Cylindrocarpon candidum was obtained

during the studies curried by Abo EL-

Nasser (2000).

Culture media. -Liquid Malt extract for Production of

antitumor agents.

This medium was described by Fang, et al. (1997). It has the following compo-

sition (g/L): malt extract, 20; peptone, l;

glucose, 20 and distilled water, 1000 ml.

Production and Extraction of antitumor

agents. Aliquots of 1L liquid malt extract

medium placed in conical flask (2L) and

autoclaved at 121 0C for 20 min. Each flask

was inoculated by 5ml spore suspension of

the experimental organism and incubated at

25 0C for 5 days. The broth media was

separated into a supernatant and mycelial

cake. The mycelial cake was extracted with

acetone and concentrated under vacuum

and the supernatant was extracted with ethyl acetate and also concentrated under

vacuum. The resulting residues were

combined and extracted with ethyl acetate to obtain a crude material. This residue was

tested for its toxicity against tumor cells.

Purification and analysis of the extracts

obtained from Aspergillus fumigates

and Cylindrocarpon candidum. 100 liters of the broth media was

separated into a supernatant and mycelial

cake. The mycelial cake was extracted with acetone and concentrated under vacuum

condition and the supernatant was extra-

cted with ethyl acetate and concentrated under vacuum condition. The resulting

residues were combined and extracted with

ethyl acetate to obtain a crude material.

The residue was fractionated on column chromatography packed with silical gell 60

G F254 for column chromatography and

eluted with ethyl acetate flowed by an increasing proportion of methanol.

Fractions were collected at 15 0C.

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Osman, M.E et al

165

Fig (1) Purification of antitumor agent of Aspergillus fumigates crud.

F1 F2 F3 F4 F5 F6 F7

Extraction with acetone

Resinous residue (3.5g)

100L Culture broth

Aqueous filtrate (97L) Mycelium (225g)

separation

Extraction with EtOAc

Resinous residue (5g)

Extraction with EtOAc

Residue (7g)

Fractionation on silical gell column

by EtOAc: methanol (1:1)

All fractions were tested using HepG2 cell line

Active fraction

F5 (0.83g)

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Aspergillus fumigatus and Cylindrocarpon candidum………

166

Fig (2) Purification of antitumor agent of Cylindrocarpon candidum crud.

F1 F2 F3 F4 F5

Extraction with acetone

100L Culture broth

Aqueous filtrate (98.4L) Mycelium (150g)

separation

Extraction with EtOAc

Resinous residue (3.7g) Resinous residue (1.5g)

Extraction with EtOAc

Residue (4.2g)

Fractionation on silical gell column

by EtOAc: methanol (1:1)

All fractions were tested using HepG2 cell line

Active fraction

F3 (0.5g)

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Osman, M.E et al

167

Standard Protein Assay

The protein concentration in the

investigated samples was determined according to the methods of (Bradford

1976).

Cell line

Human liver carcinoma cell line

HepG2 obtained from American type culture collection.

Cell culture

HepG2 cell line was maintained and sub culture in 75 cm

2 cell culture flask

(Fisher Scientific Pittsburgh, PA). Using

10 ml of RPMI – 1640 ]Roswell park Memorial Institute medium[.

(Supplemented with 1 % (2 mM) glutamic

acid, 10 % unheated fetal bovine serum

(FBS), 100 µ/ ml penicillin and 100 µg/ ml streptomycin). All cells were grown at 37 0C in atmosphere 5% CO2 – 95 % air in a

high humidity atmosphere in water – Jacteted incubator.

Cell viability assays. 1- Trypan blue exclusion test.

0.05 % trypan blue solution was

added to cell suspension (v/v) from each

control and treated cells. Cells were examined under the light microscope. Total

cell counts and viable cell number

(survival) were determined by a standard Heamocytometer procedure. Live viable-

cells were seen colorless (impermeable to

the dye due to intact cell membrane) and dead cells were seen as blue (permeable to

dye due to disruption of cell membrane):

% live (Recovery rate) =

Count of living cells X 100

Count of total cells

2- MTT assay Cytotoxicity was measured using the

MTT ]3-(4,5-dimethyl thiazol)-2,5-

diphenyltetrazolum bromide[ cell viability assay which depend on the ability of active

mitochondria dehydrogenase enzyme of

living cells to clave the tetrazolium rings of

the yellow MTT and form a dark blue in

solution formazan crystals which is largely impermeable to cell membranes, resulting

in it accumulation within healthy cells.

Solublization of crystals, which are then

solublized. The number of viable cells is directly proportional to the level of soluble

formazan dark blue color. The extent of the

reduction of MTT was quantified by measuring the optical absorbance (OD) at

570nm (Mosmann, 1998).

Procedure HepG2 cells (0.5 X 10

5 cells/ well) in

serum. Free media were plated in fetal

bottom 96.well microtiter plate, and treated with compound for 24h at 37

0C in humi-

dified 5% CO2 atmosphere. After incuba-

tion media were removed and 40 µl MTT solution /well was added and incubated for

an 4hrs. MTT crystals were solublized by

adding 180 µl of acidified isopropanal

/well and plate was shacked at room temperature. The absorbance was read at

570 nm using ELISA reader (meter tech

960, USA). Triplicate wells were prepared for control and each individual dose and

the average was calculated. Data were

expressed as the percentage of relative viability comparison, the untreated cells

compared with control. The cytotoxicity

indicated by < 100 % relative viability.

Percentage of relative viability was calculated using the following equation:

Absorbance of treated cells /

Absorbance of control X 100. Then the half maximum inhibitory

concentration IC 50 was calculated from

the equation of the dose response curve.

3- Colorimetric Protease assays.

Caspase 3 and Caspase 9 (the

ApoTarget) The ApoTarget Colorimetric Protease

assays Catalog # KHZ0101 and KHZ0102.

Caspase 3 and caspase 9 protease assays are to be used for in vitro determination of

proteolytic activity in lysates of mamm-

alian cells (biosource International, Inc.542

Flynn Road Camarillo, California 93012, USA). Assay procedure briefly. Induce

apoptosis in cells by different

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Aspergillus fumigatus and Cylindrocarpon candidum………

168

concentration of each compound. Cells

were counted and pellet 3 – 5 million cells

per sample. Resuspend cells in 50 µl of chilled cell lysis buffer and cells were

incubated on ice for 10 min. then

centrifuged and the protein concentration

was assayed by the Brad ford methods (FISI) in cytosol extract. Each cytosol was

a diluted to a concentration of 50–200µg

protein per 50 µl. cell lysis buffer. Reaction buffer ]containing DDT (dithiothreitol)[

was added to each sample. 5µl of the 4 mµ

DEVD – pNA ]amino acid sequence

composed of the chromophore, p-nitroanalinide (pNA)[ substrate (200µm

final concentration) was added and

incubated at 37 0C for 2 hrs in dark. Then

the absorbance was recorded at 405 nm

using spectrophotometer.

Results

Fractionation, purification and

Identification of the anticancer agents

from Aspergillus fumigates and

Cylindrocarpon candidum.

The culture broth of both organisms

were extracted to obtain crude antitumor active agents (figs1,2). These crude

fractions were fractionated by silica gell

column chromatography using ethyl acetate: methanol 1:1 as eluting agents.

Seven fractions and five fractions were

obtained from Aspergillus fumigates and

Cylindrocarpon candidum respectively (table 1). These different fractions were

examined for their anticancer activity

against HepG2 cell line. It was found that F5 from Aspergillus fumigates and F3 from

Cylindrocarpon candidum showed the

highest activity (fig 3). Each product was tested by Infrared

absorption spectra (IR) were measured with

FI.IR spectrophotometer. NEXUS- 460

(USA) using KB6 as standard material, Ultra violet-visible spectra (UV spectra)

were measured with Microlet Evolution 300.

UV and IR were determined in Desert Research Center (DRC). Micro analytical

unit and G.C Mass spectra were measured

with Fimmigan mat. SSQ 7000 (Thermo. Inst. Sys. Ine. U.S.A. mass spectrometer at

an-ionization Voltage 870 ev and El mode.

These measured in Ain Shams University,

Faculty of Science, Central Laboratory, to

know the structure of each product. The first products was the (N-(3-4-

Dichlorophenyl)2-Methyl 2, 3 Dihydroxyp-

ropio amide) produced from Aspergillus

fumigates it shows ג max in the UV regin at 266nm indication of substituted was being

(NH.AC) (Fig 4). In IR spectrum showed

absorption bands at 3621cm-1

OH, 3421 cm-1

NH and 1693 CO cm-1

. Mass spectra

showed M/Z 229, 149, 73 and 41 the spectra

data confirm assigned structure I.

The second product (Fig 5) was the (2.4.6. Triphenyl pyridine) produced from

Cylindrocarpon candidum. In IR spectrum

showed absorption band at 3405 cm-1 NH.

Mass spectra showed M/Z 307, 242,206,154

and 125 the spectra data confirm assigned

structure II.

Measurement of anticancer activity of

the two active fractions (DMD and TPP)

on HepG2 cell line (liver cancer). HepG2 cells were treated with graded

concentrations (25- 400 µg/ml) of DMD and

TPP for 12-24 h. and then the cell proliferation and cell viability were

monitored. The results showed that

treatment of HepG2 cells with two compounds of DMD and TPP resulted in

significant decrease viability of the cells in a

dose and time- dependent manner (12, 24 h)

as compared with control (Figs 6,7). The effect of DMD and TPP on cell proliferation

was found to remarkably inhibit the

proliferation of HepG2. The result showed that Cylindrocarpon candidu had cytotoxic

effect more than Aspergillus fumigates in

all concentration as shown in (Fig 8). Cell

proliferation was determined by the cell titer 96 TM non-radioactive cell proliferation

assay MTT. Each value is the mean of

triplicates + SD. Induction of apoptosis in HepG2 cells by DMD and TPP. The apopt-

otic effect was measured through activation

of caspase enzymes. The activation of caspase 3 and caspase 9 proteases were

measured at different concentrations of

protein contents (25-250µg) of cell treated

with graded dose of each DMD and TPP products. The results showed that highly

significant increase in activity of caspase 3

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Osman, M.E et al

169

enzymes in the cell treated with DMD at

concentration (400 µM) whereas the activity

of enzyme was decreased at less concent-ration (Fig 9). The same result was obtained

when monitored the activity of caspase 9 in

HepG2 cells treated with different

concentration of DMD (Fig 10). On the

other hand, when measured the activation of

caspase 3 and caspase 9 in HepG2 cells treated with TPP was lesser than that of

DMD. The result showed dose dependent

activity of both enzymes (Figs 11,12).

Table (1): Fractionation of Aspergillus fumigatus and Cylindrocarpon candidum crude

extracts by column chromatography.

Aspergillus fumigatus

Fractions ml EtoAc: methanol

F1 100 100%EtoAc

F2 75 5% methanol

F3 150 10% methanol

F4 100 15% methanol

F5 50 20% methanol

F6 175 25% methanol

F7 100 30% methanol

Cylindrocarpon candidum

Fractions ml EtoAc : methanol

F1 75 100% EtoAc

F2 50 5% methanol

F3 100 10% methanol

F4 150 15% methanol

F5 100 20% methanol

Fig (4): Structure of active fraction of Aspergillus fumigatus.

(N-(3-4-Dichlorophenyl)2-Methyl ,2,3Dihydroxypropio amide)

OH O Cl

CH C C NH O Cl

OH CH3

Fig (4): Structure of active fraction of Aspergillus fumigatus.

F2 F3 F4 F5 F6 F7

55

60

65

70

75

80

85

90

95

100

Aspergillus fumigatus

Cylindrocarpon candidum

Sur

viva

l cel

ls (

% c

ontr

ol).

Fig(3): Effect of different fraction of Aspergillus fumigatus and

Cylindrocarpon candidum extract on cancer cells.

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Aspergillus fumigatus and Cylindrocarpon candidum………

170

(N-(3-4-Dichlorophenyl)2-Methyl ,2,3Dihydroxypropio amide)

OH O

Cl

CH C C NH O Cl

OH CH3

Fig (5): Structure of active fraction of Cylindrocarpon candidum. (2.4.6. Triphenyl pyridine )

N

25 50 100 200 400

60

65

70

75

80

85

90

Fig (7): Dose response curve of TPP effect on Hep G2 cells after 12 and 24 h. of exposure

determined by trypan blue exculsion test.

after12h.

after24h.

Via

bili

ty (

contr

ol%

)

concentration of the drug (l/ml)

25 50 100 200 400

60

65

70

75

80

85

90

Fig (6): Dose response curve of DMD effect on HepG2 cells after 12 and 24 h. of exposure

determined by trypan blue exculsion test.

After12h

After24h

Via

bili

ty (

con

tro

l%)

concentration of the drug (l/ml)

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Osman, M.E et al

171

100 150 200 250 300 350 400

0

10

20

30

40

50

60

DMD

TPP

Cel

l pro

lifer

atio

n (%

of c

ontro

l)

Concentration (g/ml).

Fig (8): MTTassay for determination of proliferation of HepG2 cell line

under different concentration of DMD and TPP.

0 50 100 150 200 250

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

Fig (9): The activity of Caspase 3 at different concentration

of cell protein treated with DMD

control

treate100

treate200

treate400

optical denasity o

f caspase 3

.

protin concentration (g)

0 50 100 150 200 250

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Fig (10): The activity of Caspase 9 at different concentration

of cell protein treated with DMD

control

tr100

tr200

tr400

Obtical denasity o

f caspase 9

.

protin concentration (g)

0 50 100 150 200 250

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

0.26

0.28

0.30

0.32

0.34

Fig (11):The activity of Caspase 3 at different concentration

of cell protein treated with TPP

control

tr100

tr200

tr400

OP

tical denasity o

f caspase 3

protin concentration (g)

0 50 100 150 200 250

0.00

0.05

0.10

0.15

0.20

0.25

Fig (12): The activity of Caspase 9 at different concentration

of cell protein treated with TPP

Control

Treat100

Treat200

Treat400

Obtical denasity o

f caspase 9

.

protin concentration (g)

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Aspergillus fumigatus and Cylindrocarpon candidum………

172

Discussion Although, the most common fungi

causes an important source of morbidity

and mortality in immunocompromized hosts but its toxin may be useful to inhibit

the growth of other pathogenic organisms

inside the host. Some mycotoxin which

produced from fungi have cytotoxic effect on cancer cells when tested in vitro on

different cancer cell lines (Lewis et al.,

2006). Gliotoxin is a mycotoxtin produced by several moluds such as Aspergillus

fumigatus produce cytotoxic effect in liver

cancer (Stanzani et al., 2006). Many

investigations are now being carried out to discover naturally occurring compounds

and biological response modifiers (BRMs)

which can suppress and prevent many diseases including cancer (Shi et al 1992,

Samaranayake et al., 2000, Thapliyal et al.,

2002 and Samantha et al., 2003). In fact, many effective chemotherapeutic are

agents that induced programmed of cell

death (apoptosis) via activation of different

pathways (Goli et al., 2002, Kaufmann and Earnshaw 2000 and Reed, 2002) and the

caspase enzymes are the key executors of

apoptosis. In this study, we determined the role of caspases in signaling of Aspergillus

fumigates and Cylindrocarpon candidu

apoptosis in liver cancer cells ( HepG2 ) and the data revealed significant inhibition

of cell growth and proliferation in both

culture treated with N-(3-4-Dichloro-

phenyl) 2- Methyl ,2,3 Dihydroxypropio amide and 2.4.6. Triphenyl pyridine

products. This inhibition was associated

with the incubation time and the conc-entration of products. In vitro cytotoxicity

and cell proliferation were measured by

trypan blue exclusion and MMT assays.

Similar results were reported by (Leah et al., 2006), he study in vitro cytotoxic

activities of some fungal products against

different cell lines including heptoma cell line HepG2 (Saint et al., 2006).

Wright et al. (2004) demonstrated

that liver cells are able to react specifically to a fungal pathogen through increase

expression of specific receptors. Stationary

phase cultures of Aspergillus fumigates

were associated with the appearance of

typical markers of apoptosis including

elevated proteolytic enzyme activity. In these study the activities of caspase 3 and

caspase 9 were measured in HepG2 cells

after treatment with different concentr-

ations of N-(3- 4-Dichlorophenyl) 2-Methyl, 2,3 Dihydroxypropio amide and

2.4.6. Triphenyl pyridine products. The

result obtained was revealed an elevation of both enzyme activities in culture treated

with Aspergillus fumigates and Cylindro-

carpon candidu. The elevation of caspase

activity was associated with increased concentration. This result was not in

agreement with Berkova (2006), who

reported that , Aspergillus fumigates inhibit tumor necrosis factor (TNF) alpha, a

known inducer of apoptosis he also

observed that, the anti apoptotic effect of Aspergillus fumigates was associated with

a significant reduction of caspase 3. In

conclusion, invitro of anticancer activities

of the tested components; N-(3- 4-Dichlorophenyl) 2-Methyl, 2,3 Dihydroxy-

propio amide and 2.4.6. Triphenyl pyridine

which produced from Aspergillus fumig-ates and Cylindrocarpon candidum against

liver cells. Also these two products were

strong induced apoptotic effects through activation of caspase 3and caspase 9

enzymes. This study needs furthermore

confirmation in vivo study on experimental

animal module to provide accuracy of the phase one trial on the two products of

Aspergillus fumigates and Cylindrocarpon

candidu.

References

1. Abo EL-Nasser A. (2000): Eco

physiological studies of aquatic fungi

isolated from River Nile. M.Sc. Thesis.

Botany and Microbiology Department,

Faculty of Science, Helwan University.

2. Acebal C, Librada M, Jose L, Puentes

F Baz J and Romero F. (1999): Agrochelin, a new cytotoxic antibiotic

from a marine Agrobacterium. Taxonomy,

Fermentation, Isolation, Physico-chemical

Page 11: Aspergillus fumigatus and Cylindrocarpon candidum …egyptianjournal.xyz/27_4.pdf · 1- Trypan blue exclusion test. 0.05 % trypan blue solution was added to cell suspension (v/v)

Osman, M.E et al

173

Properties and Biological Activity. The

Journal of Antibiotics , 52 (11): 983-987.

3. Berkova1 N, Lair S, Féménia F, Huet1,

H, Marie C, Gorna K, Tournier F,

Ibrahim O, Guillot J, Chermette R,

Boireau, P. and Paul J. (2006): Aspergillus fumigatus conidia inhibit

tumour necrosis factor- or staurosporine-

induced apoptosis in epithelial cells.

International Immunology Advance,

18(1):139-150. 4. Bradford M. (1976): A Rapid and

Sensitive Method for the Quantities of

Microgram Quantities of Protein Utilizing

the Principle of Protein-Dye Binding"

Anal. Biochem., 72:248-254.

5. Cairo M, Siegel S, Hnas N. and Nder S.

(1989): Clinical trial of continuous

infusion verapamil, bolus vinblastine and

continuous infusion VP-16 in drug

resistant pediatric tumors. Cancer Res.

49:1036- 1066. 6. Charles R. (2006): Apoptosis of breast

cancer MCF-7 cells in vitro is induced

specifically by yeast and not by fungal

mycelia. Anticancer Res. 2006 May-Jun.,

26(3A):2013-2022.

7. Curt G A, Clendeninn N J. and Chabner

B A. (1984): Drug resistance in cancer.

Cancer Treat. Rep., 28:87.

8. Fang F, Hideaki U, Shiomi K, Masuma,

R, Yamaguchi Y, Zhang C, Wu X,

Tanaka Y. and Omura, S. (1997): Two New Components of the Aspochalasins

Produced by Aspergillus sp. The Journal

of Antibiotics ,vol. 50 No. (11) 919 - 925.

9. Goli M, Mahatsib H. and Bakkar A.

(2002): Modulating cell cycle. Current

application and prospect for future drug

development. Carr cancer drug Target

2:309-336.

10. Gary A, Torczynski R. and Bollon A.

(1997): Acremonium sp.-a Leucinostatin

A producing endophyte of European yew.

Plant Science, 128: 97-108.

11. Hala G, Mona D, Carsten B, Josianne,

A, Roland H, Albert R. and Reging S.

(2004): thymoquinon extracted from black

seed triggers apoptosis cell death in human

colorectal cancer.Inter.J. of Onco., 25: 857.

12. Kaufmann S H. and Earnshaw W.C

(2000): Induction of apoptosis by cancer

chemotherapy. EXP cell Ros 256: 42.49.

13. Leah B, Strickland M, Ramon L, Sandin

M, John N, Greene M, Ahmad M. and

Lee M. (2006): Anew diphenyl ether from marine derived fungus Aspergillus sp.

Infect Med., 59 (6): 362-365.

14. Lewis R, Wiederhold N, Lionakis M,

and Kontoyiannis R. (2006): Frequency

and species distribution of gliotoxin,

Aspergillus isolates recovered from

patients cancer center. Clin. Microbiol.,

43(12):612-622.

15. Liu F, Ooi V, Liu W. and Chang S.

(1996): Immunomodulating and antitumor

activity of polysaccharide protein complex

from the culture filtrates of a local edible

mushroom, Trichoioma lobayense. General- Pharmacology., 27: (4): 621-624

16. Mahmod R. (2004): fungal application in

the removal of certain elements from iron

ore. M.Sc. Thesis. Botany and microb-

iology Department, Faculty of Science,

Helwan University.

17. Mosmnn T. (1983): Rapid colorimetric

assay for cellular growth and survival

application to proliferation and cytotoxicity

assay. J. Immunol. Methods 65,p 55

Abstract and Reference PDF (436K).

18. Raha S K, Dey S K, Roy S K, Chaudhuri

S. and Chakrabarty, S.L. (1990): Antitumor activity of L-asparagi-nase from

Cylindrocarpon obtusisporum MB-10 and

its effect on the immune system. Biochem-

Int.1990 Sep; 21(6): 1001-11, 0158-5231.

19. Reed J. (2002): Apoptosis based therapies.

Nat Rev Drug Discov 1:111-121.

20. Rimpler M, Polzl M, LiChun H, Bong,

H. and Ho L. (1996): Antitumor activity

of substances in fungi. Biologische-Med-izin., (25): 5, 217-224;

21. Saint A. Bauvy C, Codogno P. and

Stuart E. (2006): Cytotoxic biotransfo-

rmed products from ciobufagin by Mucor

spinosus and Aspergillus niger. The

National Library of Medeseine and the

National Institute of Health., 71 (5): 392-

402.

22. Samantha S, Nalinie W, Ira T,

Neelakanthi R. and Mayuri, G. (2003): Protection against diethylnitrosoamine-

induced hepatocarcinogenesis by an indig-enous medicine comprised of Nigella

sativa, Hemidesmus indicus and smilax

glabra: a preliminary study.J. of carcinoge-

nesis., 2:6

23. Samaranayake M, Wickramasinghe S,

Angunawela P. and Jayasekara S.

(2000): Antimicrobial activity of O-carbor-

anylalanine. Ammino- Acids. 17: (4): 357-

368.

24. Shi Q, Chen K, Toshihiro F. and Yoshiki

K. (1992): Antitumour agent 135 Structure and stereochemistry of polacan-drin, A

new cytotoxic triterpene from Polacandrin,

Page 12: Aspergillus fumigatus and Cylindrocarpon candidum …egyptianjournal.xyz/27_4.pdf · 1- Trypan blue exclusion test. 0.05 % trypan blue solution was added to cell suspension (v/v)

Aspergillus fumigatus and Cylindrocarpon candidum………

174

A nem cytotoxin triterpene from Polamsia

Dodecandria. Journal of Natural products,

55: 1488-1497.

25. Thapliyal R, Deshpande S, Maru G.

(2002): Mechanism(s) of turmeric-medi-

ated protective effects against benzopyr-

ene-derived adducts. Cancer Letters 2002,

175: 79-88.

26. Whainwaright N, Miura N, Sugawara N,

Tokunaka K, Kirigaya N. and Yadmae

T. (1992): Immunom-odulation by hot water and ethanol extracts of Ganoderma

lucidum. Pharm. Pharmacol. Lett., 8 (4):

174-177

27. Wright M, Clusen H. and Abrahamsen

T. (2004): Liver cells respond to

Aspergillus fumigatus increase in C3

secretion and gene expression as well as

an expression increase in TLR2. Immunol

Lett., 95(1): 25-30.

28. Zhang C, Mizuno T, Ito H, Shimura K

Sumiya T. and Kawade M. (1994):

Antitumor activity and immunological pro-

perty of polysaccharides from the myce-

lium of liquidcultured Grifola frondosa.

Journal o-the-Japanese-Society-for-Food-

Science-and-Technology, 41 (10): 724-

732.

29. Zhuang H, Mizuno T. and Hiloshi I.

(1995): Antitumor active fucoidan from

jlie brown seaweed, umitoranoo

{Sargassum thunbergii}. Biosci. Biotic.

Biochem., 59 (4). 563—567.

Page 13: Aspergillus fumigatus and Cylindrocarpon candidum …egyptianjournal.xyz/27_4.pdf · 1- Trypan blue exclusion test. 0.05 % trypan blue solution was added to cell suspension (v/v)

Osman, M.E et al

175

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ينوسيليدروكربوكاديد

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