Lovastatin

sensitized

human

glioblastoma

cells

to

TRAIL-induced

apoptosis

Synergy

study

with

chemotherapeutic

agents

is

a

common

in

vitro

strategy

in

the

search

for

effective

cancer

therapy

For

non-chemotherapeutic

agents

efficacious

synergistic

effects

are

uncommon

Here

we

have

examined

two

non-chemotherapeutic

agents

for

synergistic

effects

lovastatin

and

Tumor

Necrosis

Factor

-LRB-

TNF

-RRB-

related

apoptosis-inducing

ligand

-LRB-

TRAIL

-RRB-

for

synergistic

effects

on

three

human

malignant

glioblastoma

cell

lines

and

Cells

treated

with

lovastatin

plus

TRAIL

for

h

showed

apoptotic

cell

death

whereas

TRAIL

alone

-LRB-

ng/ml

-RRB-

did

not

suggesting

that

lovastatin

sensitized

the

glioblastoma

cells

to

TRAIL

attack

Cell

cycle

analysis

indicated

that

lovastatin

increased

arrest

in

these

cells

Annexin

V

study

demonstrated

that

apoptosis

was

the

predominant

mode

of

cell

death

We

conclude

that

the

combination

of

lovastatin

and

TRAIL

enhances

apoptosis

synergistically

Moreover

lovastatin

sensitized

glioblastoma

cells

to

TRAIL

suggesting

a

new

strategy

to

treat

glioblastoma

Introduction

Glioblastomas

are

the

most

common

intracranial

brain

tumors

Its

prognosis

is

usually

poor

with

survival

times

of

less

than

months

from

first

diagnosis

-LSB-

-RSB-

Surgical

resection

and

chemotherapy

are

common

treatments

-LSB-

-RSB-

Despite

recent

advances

in

the

understanding

of

the

molecular

mechanism

of

tumourogenesis

the

outcome

of

malignant

glioma

remains

poor

-LSB-

-RSB-

Thus

new

effective

forms

of

therapy

are

needed

The

Tumor

Necrosis

Factor-Related

Apoptosis-Inducing

Ligand

-LRB-

TRAIL

-RRB-

-LSB-

-RSB-

a

member

of

the

TNF

superfamily

can

bind

with

death

receptors

and

-LSB-

-RSB-

and

induces

apoptosis

in

a

wide

range

of

cancer

cells

without

harming

normal

cells

The

specific

property

of

TRAIL

has

attracted

many

researchers

to

look

for

new

treatments

by

combining

it

with

chemotherapeutic

agents

such

as

phenoxazine

derivatives

-LSB-

-RSB-

doxorubicin

and

cisplatin

-LSB-

-RSB-

Such

combinations

have

shown

synergistic

effects

on

different

types

of

cancer

cells

in

vitro

Lovastatin

a

CoA

-LRB-

HMG

CoA

-RRB-

reductase

inhibitor

is

a

commonly

used

cholesterol-lowering

agent

for

prevention

of

atherosclerotic

cardiovascular

diseases

-LSB-

-RSB-

Lovastatin

blocks

the

mevalonate

pathway

and

reduces

the

formation

of

the

downstream

products

cholesterol

geranylgeranyl

proteins

farnesylated

-LSB-

-RSB-

Recently

both

in

vitro

and

in

vitro

studies

have

found

that

lovastatin

has

antiproliferative

proapoptotic

and

anti-invasive

properties

in

a

wide

range

of

cancer

cell

types

-LSB-

-RSB-

Lovastatin

is

known

to

have

an

apoptotic

effect

on

tumor

cells

and

its

combination

with

chemotherapeutics

and

cytokines

often

exert

a

synergistic

effect

against

tumor

growth

-LSB-

-RSB-

The

mechanism

that

leads

to

lovastatin-induced

apoptosis

is

not

yet

clear

but

the

main

event

is

thought

to

be

associated

with

the

alteration

of

mitochondrial

stress

which

releases

cytochrome

C

activates

pro-caspase

cascade

and

finally

leads

to

apoptotic

cell

death

Escape

from

apoptotic

regulation

is

one

of

the

major

characteristics

of

cancer

-LSB-

-RSB-

and

many

successful

anti-cancer

agents

induce

apoptosis

by

damaging

DNA

Unfortunately

such

agents

may

also

severely

affect

normal

cells

Given

the

fact

that

both

lovastatin

and

TRAIL

are

non-chemotherapeutic

agents

and

capable

of

inducing

apoptosis

in

different

types

of

cancer

cells

it

is

important

to

determine

whether

the

combination

of

these

two

agents

would

produce

synergistic

effects

that

may

be

lighten

for

a

novel

therapeutic

application

in

gliomas

We

therefore

hypothesized

that

the

combination

of

TRAIL

and

lovastatin

neither

of

which

alone

has

noxious

effects

on

healthy

cells

could

generate

a

regime

that

was

effective

in

killing

cancer

cells

but

caused

minimal

insult

to

normal

healthy

cells

In

this

study

we

report

the

effects

of

TRAIL

in

combination

with

a

non-chemotherapeutic

drug

lovastatin

on

glioblastoma

cells

Materials

and

methods

Reagents

-LSB-

-LRB-

tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl

-RRB-

ethyl

-RSB-

ester

butanoic

acid

-LRB-

Lovastatin

-RRB-

DL-Mevalonic

acid

lactone

and

-LRB-

-RRB-

bromide

-LRB-

MTT

-RRB-

were

purchased

from

Sigma

-LRB-

St

Louis

MO

-RRB-

Lovastatin

was

dissolved

in

DMSO

for

stock

and

adjusted

to

final

concentrations

using

complete

medium

or

serum

free

medium

Soluble

Human

TRAIL

-LRB-

-RRB-

was

affinity

purified

from

lysates

of

bacteria

transformed

with

pET

plasmid

containing

TRAIL

-LSB-

-RSB-

Cellular

DNA

fragmentation

ELISA

kit

-LRB-

Roche

Mannheim

Germany

-RRB-

RNeasy

kit

DNA

extraction

kit

-LRB-

Qiagen

Germany

-RRB-

and

RT-PCR

kit

-LRB-

Promega

Madison

WI

-RRB-

were

used

Three

primary

antibodies

used

were

as

follows

rabbit

polyclonal

antibody

to

-LRB-

Chemicon

International

dilution

-RRB-

rabbit

polyclonal

antibody

to

-LRB-

Cell

Signaling

Technology

dilution

-RRB-

and

rabbit

polyclonal

antibody

to

β-tubulin

-LRB-

Santa

Cruz

Biotechnology

dilution

-RRB-

Goat

anti-rabbit

secondary

antibody

was

obtained

from

Santa

Cruz

Biotechnology

Cell

culture

Three

human

glioblastoma

cell

lines

and

were

purchased

from

American

Type

Culture

Collection

-LRB-

Rockville

MD

-RRB-

The

glioblastoma

cells

were

kept

in

Dulbecco

's

modified

Eagle

's

medium

-LRB-

-RRB-

-LRB-

GIBCO

BRL

Grand

Island

NY

-RRB-

with

or

without

fetal

bovine

serum

penicillin

and

streptomycin

at

°C

under

Media

were

changed

every

days

Measurement

of

cell

viability

We

measured

cell

viability

using

the

MTT

assay

MTT

is

a

water-soluble

tetrazolium

salt

that

is

metabolized

by

viable

cells

to

a

colored

water-insoluble

formazan

salt

Thus

the

salt

allows

cell

viability

measurements

In

short

cells

were

cultured

in

the

serum

free

medium

in

the

presence

of

lovastatin

with

or

without

TRAIL

in

a

well

microtiter

plate

for

designated

time

periods

The

medium

was

aspirated

and

μl

MTT

-LRB-

mg/ml

in

PBS

-RRB-

were

added

to

each

well

and

the

cells

incubated

for

h

After

MTT

medium

was

aspirated

the

cells

were

solubilized

in

μl

DMSO

The

optical

density

of

each

sample

at

nm

-LRB-

reference

nm

-RRB-

was

measured

using

a

microplate

reader

The

optical

density

of

the

media

was

proportional

to

the

degree

of

viable

cells

Cell

cycle

analysis

Propidium

iodide

-LRB-

PI

-RRB-

staining

and

flow

cytometry

were

used

to

determine

the

stage

of

the

cell

cycle

Cells

were

treated

with

μM

lovastatin

for

h

at

°C

and

the

control

cells

were

treated

with

normal

medium

-LRB-

DMSO

-RRB-

or

serum

free

medium

-LRB-

DMSO

-RRB-

treated

cells

were

washed

with

ml

PBS

and

then

were

trypsinized

at

°C

for

min

Cells

were

spun

down

and

washed

with

ml

PBS

Finally

cells

were

resuspended

in

μl

PBS

and

fixed

with

ml

Ethanol

with

gentle

vortexing

Cells

were

allowed

to

sit

in

°C

for

overnight

Fixed

cells

were

spun

down

and

washed

with

ml

PBS

and

then

cells

were

resuspended

in

μl

PI

-LRB-

μg/ml

-RRB-

Triton

-LRB-

v/v

-RRB-

staining

solution

with

RNase

A

-LRB-

μg/ml

-RRB-

in

dark

and

analyzed

by

a

flow

cytometer

The

staining

solution

was

purchased

from

Chemicon

-LRB-

Temecula

CA

-RRB-

Apoptosis

assay

Apoptotic

cells

were

determined

by

two

methods

Annexin-V

and

PI

stained

cells

by

flow

cytometry

and

DNA

fragmentation

assay

During

apoptosis

translocation

of

phosphatidylserine

from

inner

membrane

to

outer

membrane

is

a

common

phenomenon

Cells

were

stained

with

Annexin

V

for

analysis

of

phosphoserine

inversion

which

was

considered

to

be

a

sensitive

marker

of

apoptosis

Using

an

Annexin

V-FITC

apoptosis

detection

kit

-LRB-

Molecular

Probe

Inc

Eugene

OR

-RRB-

the

levels

of

binding

of

Annexin

V

and

staining

with

PI

were

measured

for

the

detection

of

early

and

late

apoptosis

respectively

All

of

the

procedures

were

preformed

under

manufacturer

's

guidelines

Cells

were

treated

with

lovastatin

or/and

TRAIL

for

h

and

then

stained

with

Annexin-V

and

PI

Viable

cells

were

recognized

as

negative

for

both

Annexin-V

and

PI

early

apoptotic

events

were

positive

for

Annexin-V

but

negative

for

PI

staining

Late

apoptotic

events

were

positive

to

both

Annexin

V

and

PI

Necrotic

cells

were

positive

for

PI

staining

only

DNA

fragmentation

determination

was

carried

out

with

the

ELISA

assay

kit

After

cells

-LRB-

-RRB-

were

treated

with

lovastatin

and/or

TRAIL

for

h

DNA

fragmentation

was

detected

using

wells

microplate

reader

RT-PCR

Total

RNA

was

isolated

using

Qiagen

RNeasy

extraction

kit

and

performed

according

to

the

manufacturer

's

protocol

Total

RNA

-LRB-

μg

-RRB-

was

reversely

transcribed

using

Promega

RT-PCR

kit

and

thermal

program

was

set

at

°C

for

min

and

°C

for

min

PCR

reaction

was

performed

using

the

following

primers

which

have

previously

been

tested

successfully

CTG

AGC

AAC

GCA

GAC

TCG

CTG

TCC

and

TCC

AAG

GAC

ACG

GCA

GAG

CCT

GTG

CCA

GCC

TCA

TGG

ACA

ATG

AGA

TAA

AGG

TGG

and

CCA

AAT

CTC

AAA

GTA

CGC

ACA

AAC

GAA

GAA

TTT

GGT

GCC

AAT

GCC

ACT

and

CTC

TTG

GAC

TTG

GCT

GGG

AGA

TGT

CTT

TTC

CGG

CGG

CGT

TCA

TGT

CCT

and

GTT

TCT

TCC

AGG

CTG

CTT

CCC

TTT

GTA

The

thermal

program

was

set

up

as

one

cycle

at

°C

for

min

cycles

at

°C

for

min

°C

for

min

°C

for

min

and

one

cycle

at

°C

for

min

PCR

products

were

resolved

and

visualized

on

a

agarose

gel

stained

with

ethidium

bromide

Western

blot

analysis

Western

blot

was

performed

according

to

previous

description

-LSB-

-RSB-

Briefly

after

cells

were

treated

for

h

total

protein

was

isolated

and

reacted

with

the

relevant

antibodies

The

probed

proteins

were

visualized

using

the

enhanced

chemiluminescence

Western

blotting

detection

system

-LRB-

ECL

Western

Blotting

Detection

Amersham

Biosciences

-RRB-

Statistics

The

statistical

significance

was

analyzed

using

one-way

ANOVA

analysis

and

Student

's

t-test

All

statistical

work

was

carried

out

using

the

SPSS

software

for

Windows

-LRB-

Release

Chicago

IL

-RRB-

Differences

were

considered

to

be

significant

when

P

Results

Cell

viability

measured

by

MTT

assay

We

first

examined

the

anti-proliferation

effect

of

TRAIL

on

three

glioblastoma

cell

lines

and

glioblastoma

cells

were

resistant

to

TRAIL-induced

cell

death

and

remained

cell

viable

following

treatment

in

both

normal

and

serum

free

conditions

-LRB-

Fig.

b

-RRB-

Only

showed

minor

cell

death

after

TRAIL

treatment

-LRB-

Fig.

-RRB-

Fig.

effects

on

glioblastoma

cell

lines

and

MTT

assays

were

preformed

after

h

incubation

time

treated

with

ng/ml

TRAIL

in

normal

medium

-LRB-

a

-RRB-

or

serum

free

medium

-LRB-

b

-RRB-

subjected

to

minor

TRAIL-induced

cell

death

while

and

cells

were

resistant

to

TRAIL

Same

trend

of

data

were

obtained

in

both

normal

medium

and

serum

free

condition

Experiment

set

were

repeated

at

least

three

times

with

triplicate

wells

for

each

condition

-LRB-

mean

SD

-RRB-

Synergistic

effects

by

TRAIL

and

lovastatin

were

then

examined

and

glioblastoma

cells

were

incubated

in

and

μM

lovastatin

alone

or

together

with

ng/ml

TRAIL

for

h

In

glioblastoma

cells

and

μM

lovastatin

with

ng/ml

TRAIL

promoted

significant

cell

death

when

compared

with

lovastatin

control

-LRB-

Fig.

-RRB-

and

μM

lovastatin

alone

induced

and

of

cell

death

respectively

however

in

the

presence

of

ng/ml

TRAIL

lovastatin

induced

and

of

cell

death

respectively

and

μM

lovastatin

with

ng/ml

TRAIL

also

synergistically

promoted

cell

death

in

and

In

cells

and

μM

lovastatin

alone

caused

and

cell

death

whereas

in

the

presence

of

ng/ml

TRAIL

lovastatin

induced

and

cell

death

respectively

-LRB-

Fig.

-RRB-

There

were

and

cell

death

induced

by

and

μM

lovastatin

alone

in

in

the

presence

of

ng/ml

TRAIL

lovastatin

at

all

concentration

tested

caused

about

of

cell

death

-LRB-

Fig.

-RRB-

Collectively

the

combination

of

TRAIL

and

lovastatin

was

much

more

effective

than

lovastatin

alone

in

the

induction

of

cell

death

in

all

three

glioblastoma

cells

tested

and

the

result

indicated

that

there

was

a

synergistic

effect

when

TRAIL

and

lovastatin

were

used

together

Fig.

anti-proliferation

effects

on

glioblastoma

cell

lines

and

Glioblastoma

cells

were

treated

with

and

μM

lovastatin

alone

or

μM

lovastatin

plus

ng/ml

TRAIL

μM

lovastatin

plus

ng/ml

TRAIL

μM

lovastatin

plus

ng/ml

TRAIL

and

μM

lovastatin

plus

ng/ml

TRAIL

for

h.

Percentage

of

viable

glioblastoma

cells

showed

synergistic

anti-proliferation

effects

by

combined

the

two

agents

In

glioblastoma

cells

-LRB-

a

-RRB-

ng/ml

TRAIL

with

and

μM

lovastatin

induced

cell

death

significantly

when

compared

with

lovastatin

only

groups

In

-LRB-

b

-RRB-

and

-LRB-

c

-RRB-

ng/ml

TRAIL

with

and

μM

lovastatin

induced

cell

death

significantly

when

compared

with

lovastatin

only

groups

ANOVA

were

used

for

statistics

analysis

and

P

P

Experimental

set

were

repeated

for

at

least

three

times

with

triplicate

wells

for

each

condition

-LRB-

mean

SD

-RRB-

Cell

cycle

determination

by

PI

staining

Propidium

iodide

staining

and

flow

cytometry

were

used

to

determine

the

degree

of

cell

synchronization

by

μM

lovastatin

Cells

were

incubated

with

normal

serum

medium

serum

free

medium

and

μM

lovastatin

with

normal

serum

medium

for

h.

PI-stained

cells

were

analyzed

using

flow

cytometry

to

quantify

cells

in

certain

cell

cycle

stages

A

significant

increase

in

cell

population

at

phase

was

observed

when

the

cells

were

treated

with

μM

lovastatin

indicating

that

lovastatin

was

able

to

arrest

the

cells

at

stage

cell

population

was

also

increased

by

serum

free

medium

conditions

-LRB-

positive

control

-RRB-

and

the

increase

reached

a

significant

level

except

in

cells

-LRB-

Fig.

-RRB-

Fig.

glioblastoma

cells

arrested

in

Phase

Propidium

Iodide

staining

for

cell

cycle

analysis

were

performed

after

glioblastoma

cells

were

treated

with

normal

medium

without

lovastatin

serum

free

medium

without

lovastatin

and

μM

lovastatin

for

h.

Serum

free

condition

was

used

as

a

positive

control

which

is

commonly

known

to

induce

cell

arrest

in

phase

Serum

free

condition

and

lovastatin

increased

cell

arrest

in

all

glioblastoma

cells

and

reached

significant

level

-LRB-

except

serum

free

condition

in

-RRB-

ANOVA

were

used

for

statistics

analysis

and

P

P

Experimental

set

were

repeated

for

at

least

three

times

with

triplicate

wells

for

each

condition

-LRB-

mean

SD

-RRB-

Apoptosis

is

the

major

mode

of

cell

death

In

order

to

determine

the

mode

of

cell

death

induced

by

lovastatin

and

TRAIL

Annexin

V

and

PI

staining

was

employed

for

this

purpose

Cells

were

incubated

with

or

μM

of

lovastatin

in

the

presence

or

absence

of

ng/ml

TRAIL

for

h

The

percentage

of

cell

death

of

glioblastoma

cells

treated

with

both

lovastatin

and

TRAIL

was

significantly

higher

than

that

with

either

agent

alone

-LRB-

Fig.

b

and

c

-RRB-

glioblastoma

cells

were

vulnerable

to

TRAIL

induced

apoptosis

-LRB-

Fig.

-RRB-

but

significantly

more

apoptotic

cells

were

observed

following

treatment

with

both

TRAIL

and

lovastatin

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

TRAIL

was

able

to

induce

apoptosis

in

of

glioblastoma

cells

However

TRAIL

in

combination

with

and

μM

could

induce

apoptosis

in

approximately

and

cells

respectively

indicating

a

synergistic

effect

occurred

Similarly

a

significant

synergistic

effect

was

also

observed

in

and

cells

-LRB-

P

-RRB-

-LRB-

Fig.

c

-RRB-

with

up

to

nearly

a

increase

in

apoptotic

cells

μM

lovastatin

only

induced

apoptosis

in

and

and

respectively

which

were

not

different

from

the

control

-LRB-

without

any

treatment

-RRB-

μM

lovastatin

plus

ng/ml

TRAIL

dramatically

increased

apoptotic

cells

to

and

in

and

respectively

cells

which

lack

of

DNA-dependent

protein

kinase

expression

-LSB-

-RSB-

were

less

vulnerable

to

the

two

agents

implying

that

DNA-dependent

protein

kinase

may

play

a

role

in

apoptosis

induced

by

TRAIL

and

lovastatin

Fig.

synergistic

apoptotic

effects

were

quantified

by

flow

cytometry

using

Annexin

V

and

PI

staining

Glioblastoma

cells

were

treated

with

DMSO

-LRB-

Control

-RRB-

μM

lovastatin

μM

lovastatin

ng/ml

TRAIL

μM

lovastatin

plus

ng/ml

TRAIL

and

μM

lovastatin

plus

ng/ml

TRAIL

for

h

Then

cells

were

stained

with

Annexin

V

and

PI

to

determine

percentage

of

apoptotic

cell

death

using

flow

cytometry

Synergistic

apoptotic

effects

were

observed

in

three

glioblastoma

cell

lines

-LRB-

a

-RRB-

-LRB-

b

-RRB-

and

-LRB-

c

-RRB-

reached

significant

level

The

portion

of

apoptotic

cell

death

was

indicated

in

low-right

quarter

of

the

flow-cytometry

scatter

plot

and

the

trend

of

apoptotic

cells

between

groups

ANOVA

were

used

for

statistics

analysis

and

P

P

-LRB-

compared

to

control

group

-RRB-

P

P

-LRB-

compared

to

TRAIL

only

group

-RRB-

Experimental

set

were

repeated

for

at

least

three

times

-LRB-

mean

SD

-RRB-

DNA

fragmentation

in

glioblastoma

cells

DNA

fragmentation

is

one

of

the

hallmarks

when

cells

undergo

apoptosis

We

performed

ELISA

assay

to

confirm

DNA

fragmentation

was

induced

in

the

cells

treated

with

both

lovastatin

and

TRAIL

A

synergistic

effect

on

DNA

fragmentation

by

both

agents

was

demonstrated

in

all

three

cell

lines

tested

-LRB-

Fig.

b

and

c

-RRB-

The

level

of

DNA

fragmentation

was

increased

in

treated

glioblastoma

cells

but

it

was

only

statistically

significant

in

the

cells

treated

with

TRAIL

plus

μM

lovastatin

In

and

cells

however

there

was

a

dramatic

elevation

of

DNA

fragmentation

in

a

dose-dependent

manner

when

both

of

the

agents

were

applied

This

observation

supports

the

argument

that

the

cell

death

induced

by

the

two

agents

was

apoptotic

and

that

both

agents

were

able

to

function

in

a

synergistic

manner

Fig.

fragmentation

was

detected

in

combination

of

lovastatin

and

TRAIL

Same

treatment

in

apoptotic

cell

staining

was

preformed

for

DNA

fragmentation

ELISA

detection

Significant

level

of

DNA

fragmentation

was

detected

in

all

glioblastoma

cells

when

combined

with

lovastatin

and

TRAIL

ANOVA

were

used

for

statistics

analysis

and

P

P

-LRB-

compared

to

control

group

-RRB-

P

P

-LRB-

compared

to

TRAIL

only

group

-RRB-

Experimental

set

were

repeated

for

at

least

three

times

-LRB-

mean

SD

-RRB-

The

expression

of

TRAIL

receptors

in

glioblastoma

cells

Resistance

to

cell

death

induced

by

TRAIL

may

be

adapted

by

an

altered

level

of

TRAIL

receptors

-LRB-

-RRB-

and

-LRB-

-RRB-

are

known

to

attenuate

TRAIL-induced

apoptosis

whereas

-LRB-

-RRB-

and

-LRB-

-RRB-

promote

TRAIL-induced

apoptosis

-LSB-

-RSB-

The

effect

of

lovastatin

treatment

on

the

TRAIL

receptors

was

not

determined

RT-PCR

was

performed

to

investigate

the

expression

profiles

on

three

glioblastoma

cell

lines

tested

Cells

were

treated

with

normal

serum

medium

serum

free

medium

serum

medium

with

or

μM

lovastatin

for

h.

and

were

not

detected

in

all

conditions

and

cell

types

tested

was

only

detected

on

cells

with

serum

free

medium

or

lovastatin

-LRB-

Fig.

-RRB-

was

expressed

on

all

cell

types

of

cells

with

or

without

lovastatin

and

its

level

was

not

significantly

different

between

lovastatin-treated

cells

and

the

controls

Therefore

the

expression

of

did

not

contribute

to

the

lovastatin-induced

cell

death

in

glioblastoma

cells

tested

Fig.

expression

of

TRAIL

receptor

mRNA

in

glioblastoma

cells

The

cells

were

treated

with

normal

serum

medium

serum

free

medium

and

μM

lovastatin

with

normal

serum

medium

At

the

end

of

the

treatment

RNA

was

isolated

for

the

detection

of

-LRB-

-RRB-

-LRB-

a

-RRB-

and

-LRB-

-RRB-

-LRB-

b

-RRB-

by

RT-PCR

After

we

tested

the

mRNA

expression

of

TRAIL

receptors

on

glioblastoma

cells

the

further

investigation

was

performed

to

determine

the

active

protein

expression

of

and

matured

by

Western

blot

The

result

showed

that

the

protein

expression

profiles

of

and

were

found

in

three

glioblastoma

cells

with

or

without

lovastatin

suggesting

the

protein

of

these

two

receptors

was

underwent

post-translational

modifications

The

significantly

differences

were

only

found

on

the

expression

of

cells

with

μM

lovastatin

-LRB-

P

-RRB-

and

the

expression

of

in

cells

with

lovastatin

-LRB-

P

-RRB-

-LRB-

Fig.

-RRB-

Fig.

expression

of

-LRB-

-RRB-

and

-LRB-

-RRB-

proteins

in

glioblastoma

cells

The

cells

were

treated

with

normal

serum

medium

serum

free

medium

and

μM

lovastatin

with

normal

serum

medium

for

h

At

the

end

of

the

treatment

proteins

were

isolated

for

the

detection

of

-LRB-

a

b

-RRB-

and

-LRB-

c

d

-RRB-

by

Western

blot

Representative

Western

blots

were

shown

-LRB-

a

c

-RRB-

The

target

bands

were

scanned

and

normalized

to

β-tubulin

The

index

of

densities

was

calculated

-LRB-

b

d

-RRB-

P

P

Discussion

Malignant

glioblastoma

is

one

of

the

major

causes

of

brain

tumors

morbidity

Aggressive

infiltration

in

the

CNS

ultimately

leads

to

death

in

nearly

all

cases

-LSB-

-RSB-

Malignant

glioblastoma

carries

with

aberrant

biological

and

biochemical

properties

including

several

activating

mutations

that

can

lead

to

chemotherapeutics

resistance

-LSB-

-RSB-

Targeting

apoptotic

signaling

machinery

is

thought

to

be

a

promising

alternative

for

glioblastoma

treatment

-LSB-

-RSB-

The

preliminary

data

presented

in

this

report

indicate

that

lovastatin

a

blood

cholesterol

lowering

medicine

sensitizes

glioblastoma

cells

to

TRAIL-mediated

apoptosis

Earlier

reports

show

statin-induced

cell

death

through

a

mitochondrial-mediate

pathway

-LRB-

intrinsic

pathway

-RRB-

that

is

closely

related

to

the

family

protein

Bid

and

activation

of

caspase

and

-LSB-

-RSB-

However

the

mechanism

for

statin

activation

of

caspase

remains

unknown

because

caspase

is

normally

activated

by

receptor-mediated

signals

such

as

Fas

ligand

and

TRAIL

-LSB-

-RSB-

Lovastatin

has

been

found

to

enhance

TRAIL-induced

cytotoxicity

in

a

synergistic

manner

in

glioblastoma

cells

We

demonstrated

a

synergistic

effect

produced

by

the

combination

of

lovastatin

and

TRAIL

on

glioblastoma

cells

Lovastatin

was

found

to

sensitize

the

cells

to

cell

death

induced

by

TRAIL

The

mode

of

cell

death

induced

by

both

agents

in

combination

was

apoptosis

as

demonstrated

by

two

different

methods

Annexin

V

and

PI

staining

and

DNA

fragmentation

assay

We

also

demonstrated

that

two

of

the

glioblastoma

cell

lines

tested

were

resistant

to

TRAIL

induced

apoptosis

From

this

we

inferred

that

lovastatin

not

only

sensitized

these

glioblastoma

cells

through

its

effects

on

the

TRAIL

receptor

pathway

but

also

triggered

an

unknown

mechanism

Lovastatin

served

as

a

cytostatic

agent

and

turned

on

an

unknown

mechanism

to

support

TRAIL-induced

apoptosis

in

these

glioblastoma

cells

It

has

been

shown

that

colon

and

lung

tumor

cells

arrested

in

stages

are

vulnerable

to

TRAIL-induced

cell

death

-LSB-

-RSB-

However

our

arrested

glioblastoma

cell

lines

remained

resistant

to

TRAIL-induced

cell

death

in

the

serum

free

control

Our

finding

suggests

that

a

combination

of

TRAIL

and

lovastatin

together

may

form

a

new

treatment

for

glioblastoma

multiforme

The

mechanism

by

which

lovastatin

sensitizes

glioblastoma

cells

to

TRAIL

induced-apoptosis

remains

unknown

In

human

glioblastoma

lovastatin

has

been

shown

to

induce

or

to

enhance

apoptosis

by

altering

a

number

of

apoptotic

molecules

For

example

it

can

induce

apoptosis

or

downregulate

cell

proliferation

by

targeting

Ras

in

primary

cultured

human

glioblastoma

cells

-LSB-

-RSB-

and

increase

pro-apoptotic

Bim

in

and

glioblastoma

cells

-LSB-

-RSB-

Lovastatin

has

also

been

shown

to

downregulate

RhoA

and

increase

iNOS

in

and

glioblastoma

cells

-LSB-

-RSB-

Additionally

Lovastatin

may

induce

apoptosis

by

increasing

and

the

apoptosis

induced

can

be

prevented

by

the

overexpression

of

-LSB-

-RSB-

suggesting

a

mitochondrial-related

apoptosis

It

is

noted

that

this

study

by

Schmidt

et

al.

fails

to

document

that

lovastatin

can

enhance

death

receptor

-LRB-

-RRB-

mediated

apoptosis

in

glioblastoma

cells

and

-LSB-

-RSB-

The

result

from

our

study

indicate

that

lovastatin

in

combination

with

TRAIL

can

synergistically

induce

apoptosis

in

and

glioblastoma

cells

The

apoptosis

induced

is

associated

with

arrest

but

not

with

pro-apoptotic

Bid

-LRB-

data

not

shown

-RRB-

It

is

well

known

that

TRAIL-induced

apoptosis

takes

place

via

a

death

receptor-mediated

pathway

-LSB-

-RSB-

Our

study

showed

that

mRNA

expressed

in

but

was

hardly

detected

in

the

other

two

cell

lines

However

protein

was

detectable

in

all

three

cell

lines

suggesting

that

the

was

modified

by

a

post-translational

mechanism

in

the

cells

tested

The

level

of

protein

was

higher

in

cells

treated

with

μM

of

lovastatin

However

such

an

elevation

of

is

unlikely

to

be

responsible

for

apoptosis

induced

by

lovastatin

because

the

apoptotic

rate

between

was

not

different

from

the

other

cell

lines

can

be

detected

at

both

protein

and

RNA

levels

The

level

of

mRNA

was

not

different

in

the

cells

treated

with

lovastatin

but

its

protein

level

was

much

higher

in

cells

treated

with

either

or

mM

lovastatin

the

result

of

which

indicated

that

was

modified

by

a

post-translational

mechanism

in

cells

It

is

noted

that

the

base

level

of

in

cells

does

not

differ

from

other

two

cell

lines

tested

Therefore

it

is

unlikely

that

could

count

for

the

relatively

higher

sensitivity

of

cells

to

TRAIL

stimulation

than

the

other

two

cell

lines

All

three

cell

lines

underwent

a

similar

level

of

cell

death

when

they

were

treated

by

lovastatin

Therefore

the

contribution

of

lovastatin-induced

protein

to

the

cell

death

in

cells

seems

to

be

minimal

Such

results

appear

to

be

in

line

with

a

study

using

mevastatin

a

similar

HMG-CoA

reductase

inhibitor

Mevastatin

can

significantly

induce

apoptosis

of

myeloma

cells

in

a

pathway

independent

of

death

receptors

including

-LSB-

-RSB-

Nevertheless

further

quantitative

tests

are

needed

in

glioblastoma

cells

to

verify

the

result

obtained

Apoptosis

induced

by

lovastatin

is

generally

considered

to

be

via

the

mitochondrial-mediated

pathway

-LSB-

-RSB-

It

is

possible

for

both

pathways

to

talk

to

each

other

to

amplify

apoptotic

signals

and

this

is

indeed

a

case

for

TRAIL

since

TRAIL

is

capable

of

inducing

either

mitochondrial-independent

or

dependent

apoptotic

pathways

in

some

types

of

cells

-LSB-

-RSB-

The

Bid

is

a

molecule

functions

as

a

bridge

that

links

death

receptor

and

mitochondrial-mediated

pathways

Therefore

without

involvement

of

Bid

apoptosis

induced

by

TRAIL

and

lovastatin

in

combination

in

the

present

study

seem

to

be

separately

mediated

by

these

two

pathways

However

considering

the

fact

that

two

out

of

three

glioblastoma

cells

tested

are

insensitive

to

TRAIL

treatment

but

they

become

responsive

in

the

presence

of

lovastatin

it

can

be

hypothesized

that

lovastatin

treatment

may

remove

an

unknown

inhibitory

factor

-LRB-

s

-RRB-

that

overcomes

the

TRAIL-mediated

pathway

or

that

lovastatin

may

wake

up

an

activator

-LRB-

s

-RRB-

that

normally

remains

in

a

resting

condition

One

of

inhibitory

factors

known

to

involve

TRAIL-mediated

pathways

is

its

decoys

and

-LSB-

-RSB-

However

it

can

be

seem

from

our

study

that

this

inhibitory

factor

-LRB-

s

-RRB-

if

any

should

not

be

the

decoys

and

since

both

are

not

detectable

in

the

cells

tested

One

possible

explanation

for

this

synergistic

effect

is

the

activation

of

caspase

an

initiator

caspase

in

death

receptor-mediated

pathways

Lovastatin

has

been

shown

to

enhance

caspase

activity

-LSB-

-RSB-

It

is

possible

that

is

the

activator

factors

waken

up

by

lovastatin

Whatever

it

is

the

mechanism

accounting

for

the

synergistic

effect

of

TRAIL

and

lovastatin

against

glioblastoma

cells

in

the

present

study

is

complicated

and

remains

to

be

uncovered

Mevalonate

is

a

critical

component

of

a

complex

biochemical

pathway

and

its

products

are

vital

for

a

variety

of

important

cellular

functions

including

cell

signaling

protein

synthesis

and

cell

cycle

regulation

-LSB-

-RSB-

Little

is

known

about

the

molecular

events

leading

to

apoptosis

of

cancer

cells

due

to

lovastatin

exposure

It

is

likely

that

apoptosis

is

abrogated

by

mevalonate

and

GGPP

and

is

partially

reversed

by

FPP

-LSB-

-RSB-

Our

results

also

reinforced

the

conclusion

of

our

previous

study

that

DNA-dependent

protein

kinase

-LRB-

DNA-PK

-RRB-

plays

an

important

role

in

cell

apoptosis

cells

that

lack

of

DNA-PK

activity

are

resistant

not

only

to

total

cell

death

but

also

apoptosis

-LSB-

-RSB-

Experts

disagree

over

the

functions

of

DNA-PK

in

cell

regulation

DNA-PK

has

been

reported

to

promote

cell

death

by

interacting

with

telomeres

whereas

other

reports

suggest

that

DNA-PK

protects

cells

from

cell

death

via

caspase-independent

or

independent

pathways

The

reason

for

this

paradoxical

finding

remains

unknown

This

study

demonstrated

a

synergistic

interaction

between

lovastatin

and

TRAIL

but

the

mechanisms

of

action

by

which

lovastatin

sensitized

glioblastoma

cells

remains

unknown

Our

results

are

in

agreement

with

the

concept

of

combined

cancer

therapeutic

action

via

both

intrinsic

and

extrinsic

apoptotic

cell

death

pathways

This

combination

of

non-chemotherapeutic

agents

TRAIL

and

lovastatin

may

offer

a

potential

regime

for

glioblastoma

treatment

B O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O B O O O O B O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O B O O O O B O O O O O O O B O O O O O O O B O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O B O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O B O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O B O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O B O B O O O O O O O O O B O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O B O O B O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O

tumor necrosis factor (tnf)-related apoptosis-inducing ligand (trail)


Preconception Care for Improving Perinatal Outcomes : The Time to Act Introduction A healthy baby and a healthy mother are valued hopes and dreams of families of all cultural heritages . National health goals in many countries around the world prioritize infant and maternal mortality and morbidity . In the United States , as in many other industrialized countries , pregnancy outcomes and maternal and infant health indicators have improved dramatically . At the turn of the 20th century , 7.28 women and 96 infants died for every 1,000 babies born alive -LSB- 1 -RSB- . By the end of the year 2000 , the maternal mortality rate had decreased to less than 0.08 and infant mortality had decreased to less than 7 deaths per 1,000 live births -LSB- 2 -RSB- . These changes were characterized as one of the ten `` great public health achievements '' of the 20th century -LSB- 3 -RSB- . Much of this progress can be attributed to changes in social and living conditions as well as the development and implementation of effective medical interventions . Despite this progress , during the last two decades of the 20th century , even in the presence of significant breakthroughs in medical technology and its application , improvements in maternal and infant pregnancy ouctomes slowed down significantly , and in some cases , outcomes deteriorated . For example , from 1960 to 1980 , the maternal mortality rate decreased from 32.1 to 9.4 deaths per 100,000 live births , a decrease of 70.7 % . From 1980 to 2000 , the maternal mortality rate decreased only 12.8 % , from 9.4 to 8.2 -LRB- Figure 1 -RRB- -LSB- 2 -RSB- . At the same time , from 1980 to 2000 , the proportion of babies born preterm , very preterm , low birth weight , and very low birth weight increased by 26 % , 8.2 % , 14.7 % and 25.9 % , respectively -LRB- Figure 2 -RRB- -LSB- 4 -RSB- . However , unlike other maternal and infant health indicators , from 1980 to 2000 the infant mortality rate continued to decrease almost at a similar pace as that from 1960 to 1980 , dropping by 45.2 % from 12.6 to 6.9 infant deaths per 1,000 live births , compared with a drop of 51.5 % from 26.0 to 12.6 per 1,000 live births for 1960 to 1980 -LRB- Figure 1 -RRB- -LSB- 2 -RSB- . Finally , it is estimated that the proportion of babies born in the United States who have a serious or major structural defect that can have adverse effects on their health or development continues to be about 3 % -LSB- 5 -RSB- . Associated with this slowing rate of improvement -LRB- and , in some cases , deterioration -RRB- in pregnancy outcomes and maternal and infant health indicators is a shift in the leading causes of infant mortality . In 1960 , maternal complications of pregnancy -LRB- including complications of placenta , cord , and the membranes -RRB- did not appear on the list of the 10 leading causes of infant mortality -LSB- 1 -RSB- . By 1980 , maternal complications of pregnancy became the fifth leading cause of infant mortality , and the third leading cause of infant death in 2002 , after congenital anomalies and low birth weight/preterm delivery -LSB- 6 , 7 -RSB- . In 2002 , congenital anomalies , low birth weight , preterm delivery , and maternal complications of pregnancy accounted for 46.4 % of all infant deaths in the United States -LRB- 12,996 infant deaths -RRB- -LRB- Figure 3 -RRB- -LSB- 7 -RSB- . Although some of these infant deaths might have been prevented through interventions targeted at improving the health of mothers and modifying behaviors contributing to adverse pregnancy outcomes , poor maternal health , behaviors contributing to adverse pregnancy outcomes , and maternal complications of pregnancy continue to be prevalent . For example , 28.6 % of women who gave birth between 1993 and 1997 were reported to have an obstetric complication , 4.1 % had a preexisting medical condition , and 43.0 % experienced some form of maternal morbidity -LRB- i.e. , an obstetric complication , a preexisting medical condition , a cesarean section , or any combination of the three types of morbidity -RRB- -LSB- 8 -RSB- . In 2002 , 26.1 % of all deliveries were performed using cesarean section , presumably because of maternal or infant complications -LSB- 8 -RSB- . Fig. 1Maternal and infant mortality rates , United States , 1960 -- 2002Fig . 2Percent of all live births that are preterm delivery , very preterm delivery , low birth weight , and very low birth weight , United States , 1980 -- 2002 Early prenatal care is too late One of the reasons that progress in improving pregnancy outcomes has slowed down , and in some cases reversed direction , is that we have failed to intervene before pregnancy to detect , manage , modify , and control maternal behaviors , health conditions , and risk factors that contribute to adverse maternal and infant outcomes . Although we know many interventions that , if delivered before pregnancy , could improve pregnancy outcomes , we have failed to make those services and interventions available to couples and women in need . Women of childbearing age suffer from a variety of chronic conditions that could potentially contribute to adverse pregnancy outcomes . For example , in 2002 , the U.S. Department of Health and Human Services reported that 6.1 % of women of reproductive age have asthma , 5 % are obese , 3.4 % have cardiac disease , 3.0 % are hypertensive , 9.3 % are diabetic , and 1.4 % have thyroid disorder -LSB- 9 -RSB- . Moreover , a substantial proportion of women continue to enter pregnancy with risks proven to contribute to adverse pregnancy outcomes : in 2002 , 11.4 % of pregnant women smoked during pregnancy , a risk factor for low birth weight -LSB- 4 -RSB- ; at the same time , 10.1 % of pregnant women and 54.9 % of women at risk of getting pregnant consumed alcohol , a risk factor for fetal alcohol syndrome -LSB- 10 -RSB- . Finally , using the Perinatal Periods of Risk approach , researchers in three cities -LRB- New York City ; Tulsa , Oklahoma ; and Kansas City , Missouri -RRB- concluded that racial and ethnic disparities in feto-infant mortality were largely related to maternal health , and , interventions to reduce feto-infant mortality should include preconception care and improvements in women 's health -LSB- 11 -- 13 -RSB- . Fig. 3Leading causes of infant mortality , United States , 1960 , 1980 , and 2002 . * IMR = Infant Mortality Rate The prenatal care `` revolution '' of the 1980s resulted in an increase in the proportion of women receiving early prenatal care -LRB- defined as prenatal care begun in the first trimester -RRB- -LSB- 2 -RSB- . After decreasing from 76.3 % in 1980 to 75.8 % in 1990 , the proportion of women receiving early prenatal care increased to 83.2 % in 2000 -LSB- 2 -RSB- . However , for many women , `` early prenatal care is too late '' -LSB- 14 -RSB- . By the time a pregnant woman makes it to her first early prenatal visit , most fetal organs are already been formed , and many interventions to prevent birth defects or adverse maternal and infant outcomes come too late to have any effect . As a result , many national organizations now recommend routine preconception care . For example , the March of Dimes recommends that `` as the key physician/primary care provider , the obstetrician/gynecologists must take advantage of every health encounter to provide preconception care and risk reduction before and between conceptions -- the time when it really can make a difference '' -LSB- 14 -RSB- . The American Academy of Pediatrics -LRB- AAP -RRB- and the American College of Obstetricians and Gynecologists -LRB- ACOG -RRB- recommend that `` all health encounters during a woman 's reproductive years , particularly those that are a part of preconception care , should include counseling on appropriate medical care and behavior to optimize pregnancy outcomes '' -LSB- 15 -RSB- . Preconception interventions work Interventions designed to lower preconception risks are known collectively as preconception care . These interventions are characterized by the need to start -- and sometimes complete -- a designated intervention before conception occurs . To simplify matters , ACOG and AAP have grouped the main components of preconception care under four categories of interventions : maternal assessment -LRB- e.g. , family history , behaviors , obstetric history , general physical exam -RRB- ; vaccinations -LRB- e.g. , rubella , varicella and hepatitis B -RRB- ; screening -LRB- e.g. , HIV , STD , genetic disorders -RRB- ; and counseling -LRB- e.g. , folic acid consumption , smoking and alcohol cessation , weight management -RRB- -LSB- 15 -RSB- . Many of these interventions are currently available to women and , when indicated , to couples ; however , they are not systematically delivered . A comprehensive review of the literature identified a long list of risk factors suggested to be included in comprehensive preconception care -LRB- Table 1 -RRB- . The strength of evidence supporting the efficacy of the various components of preconception care varies greatly -LSB- 16 -RSB- . Clinical practice guidelines -LRB- CPGs -RRB- have been developed for reducing the risk related to some of these factors . We identified 14 conditions for which CPGs exist and for which there is scientific evidence demonstrating effectiveness in improving pregnancy outcomes -LRB- Table 2 -RRB- -LSB- 17 -- 74 -RSB- . Table 1Risk factors -LRB- by Category -RRB- identified in the literature as needing attention during the preconception period1Chronic diseases : Diabetes ; heart disease ; high blood pressure ; thyroid disease ; asthma ; anemia ; kidney disease ; metabolic and hematological disorders ; depression and other mental disorders ; autoimmune disease ; and physical disability -LRB- access -RRB- 2Infectious diseases : Vaccine-preventable diseases -LRB- rubella , hepatitis B , varicella , influenza , and tetanus -RRB- ; HIV/AIDS ; syphilis , chlamydia , and other sexually transmitted diseases ; periodontal disease ; toxoplasmosis , and cytomegalic inclusion virus3Reproductive concerns : Unplanned pregnancies ; contraception ; infertility ; adverse past pregnancy outcomes -LRB- preterm delivery , birth defects , fetal/infant death , maternal complications -RRB- 4Genetic/inherited conditions : Sickle cell anemia ; thalassemia ; Tay-Sachs disease ; fragile X syndrome ; Down syndrome ; cystic fibrosis ; muscular dystrophy ; hearing and vision loss associated with genetic predisposition5Medications and medical treatment : Prescription medications contraindicated in pregnant women -LRB- FDA 's Category X Drugs , 117 products in 2001 PDR , antiepileptic drugs , oral anticoagulants for maternal clotting disorders , and Accutane -RRB- ; diagnostic radiation exposures6Personal behaviors and exposures : Smoking ; alcohol consumption ; illicit drug use ; overweight/underweight ; folic acid supplement use ; domestic violence ; eating disorders ; exposure to infections ; exposures to chemicals and other environmental toxins ; consumption of over-the-counter medications ; hyperthermia -LRB- e.g. , from sauna use -RRB- Note . This list is not exhaustiveTable 2Selected preconception risk factors for adverse pregnancy outcomes for which clinical practice guidelines have been developed • Folic acid . Daily use of vitamin supplements containing folic acid has been demonstrated to reduce the occurrence of neural tube defects by two thirds -LSB- 17 -- 24 -RSB- • Rubella seronegativity . Rubella vaccination provides protective seropositivity and prevents the occurrence of congenital rubella syndrome -LSB- 25 -- 27 -RSB- • Diabetes -LRB- preconception -RRB- . The three-fold increase in the prevalence of birth defects among infants of women with type 1 and type 2 diabetes is substantially reduced through proper management of diabetes -LSB- 28 -- 31 -RSB- • Hypothyroidism . The dosages of Levothyroxine ™ required for treatment of hypothyroidism increases in early pregnancy . Levothyroxine ™ dosage needs to be adjusted for proper neurologic development -LSB- 32 -- 34 -RSB- • HIV/AIDS . If HIV infection is identified before conception , timely treatment can be administered and women -LRB- or couples -RRB- can be given additional information that can influence the timing of the onset of pregnancy -LSB- 35 -- 41 -RSB- • Maternal phenylketonurea -LRB- PKU -RRB- . Women diagnosed with PKU as infants have infants with mental retardation . However , this adverse outcome can be prevented when mothers adhere to a low phenylalanine diet before conception and continue it throughout their pregnancy -LSB- 42 , 43 -RSB- • Oral anticoagulant . Warfarin , which is used for the control of blood clotting , has been demonstrated to be a teratogen . To avoid exposure to warfarin during early pregnancy , medications can be changed to a nonteratogenic anticoagulant before the onset of pregnancy -LSB- 15 , 44 -- 46 -RSB- • Anti-epileptic drugs . Certain anti-epileptic drugs are known as teratogens . Before conception , women who are on a regimen of these drugs and who are contemplating pregnancy should be prescribed a lower dosage of these drugs -LSB- 15 , 47 -- 51 -RSB- • Isotretinoins -LRB- Accutane ® -RRB- : Use of isotretinoins in pregnancy to treat acne results in miscarriage and birth defects . Effective pregnancy prevention should be implemented to avoid unintended pregnancies among women with childbearing potential who use this medication -LSB- 15 , 52 -- 54 -RSB- • Smoking . Preterm birth , low birthweight , and other adverse perinatal outcomes associated with maternal smoking in pregnancy can be prevented if women stop smoking during early pregnancy . Because only 20 % of women successfully control tobacco dependence during pregnancy , cessation of smoking is recommended before pregnancy -LSB- 55 -- 58 -RSB- • Alcohol misuse . No time during pregnancy is safe to drink alcohol , and harm can occur early , before a woman has realized that she is or might be pregnant . Fetal alcohol syndrome and other alcohol-related birth defects can be prevented if women cease intake of alcohol before conception -LSB- 59 -- 64 -RSB- • Obesity . Adverse perinatal outcomes associated with maternal obesity include neural tube defects , preterm delivery , diabetes , cesarean section , and hypertensive and thromboembolic disease . Weight loss before pregnancy reduces these risks -LSB- 15 , 65 -- 69 -RSB- • STD . Chlamydia trachomatis and Neisseria gonorrhea have been strongly associated with ectopic pregnancy , infertility , and chronic pelvic pain . STDs during pregnancy might result in fetal death or substantial physical and developmental disabilities , including mental retardation and blindness -LSB- 70 , 71 -RSB- • Hepatitis B. Vaccination is recommended for men and women who are at risk for acquiring hepatitis B virus -LRB- HBV -RRB- infection . Preventing HBV infection in women of childbearing age prevents vertical transmission of infection to infants and eliminates risk for infection and sequelae , including hepatic failure , liver carcinoma , cirrhosis , and death -LSB- 72 -- 74 -RSB- However , the best evidence for the effectiveness of specific components of preconception care is seen when the focus was on a single intervention and often not in the context of improving pregnancy outcomes . For example , effectiveness documented by the U.S. Preventive Services Task Force for interventions related to smoking , alcohol misuse , and obesity are based on studies of interventions delivered in primary care settings that was not complicated by the additional delivery of other components of preconception care -LSB- 56 , 61 , 66 -RSB- . One study reported the effectiveness of comprehensive preconception care but inferences for the United States are limited because the setting of intervention delivery was in Hungary -LSB- 75 -RSB- . Priorities for preconception care and preconception health The immediate priority is to ensure that evidence-based preconception interventions are implemented to further improve infant and maternal pregnancy outcomes . Many national professional organizations have developed and published CPGs for specific components of preconception care . For example , the American Diabetes Association developed CPGs to be started before pregnancy for women with preconception diabetes -LSB- 28 -RSB- . The American Association of Clinical Endocrinologists has developed preconception CPGs for women with hypothyroidism -LSB- 34 -RSB- . CPGs have also been developed for women being treated with teratogenic medications to guide the transition to safer medications . CPGs for women using antiepileptic drugs or oral anticoagulants have been developed by the American Academy of Neurology -LSB- 50 -RSB- , the American Heart Association , and the American College of Cardiologists , respectively -LSB- 44 , 46 -RSB- . Guidelines , recommendations , and strategies for reducing alcohol misuse and prevention of fetal alcohol syndrome have been developed and published by the U.S. Public Health Service , U.S. Preventive Service Taskforce , ACOG , and the National Taskforce on Fetal Alcohol Syndrome and Fetal Alcohol Effects -LSB- 55 -- 59 -RSB- . However , moving forward towards more universal preconception care is not without its challenges . There is no national policy , nor do standard tools exist , for the delivery of these services . Some have suggested that challenges could be overcome by promoting preconception health to all women of reproductive age at each and every encounter with the health care system . The generally recommended mechanisms would include risk assessment -LRB- screening -RRB- , health promotion -LRB- education and counseling -RRB- , and intervention or referral . However , the existing recommendations to provide these three essential components have not been translated into practical tools for action and decision-making in the clinical setting . Stated another way , primary care providers do not have the tools they need to implement what is known to work for improving preconception health . Challenges and opportunities Whereas clinical practice and the promotion of preconception care services could potentially be shaped by guidelines , widespread adoption of the guidelines requires a more active approach -LSB- 76 , 77 -RSB- . The literature on diffusion of innovation and translation of research into practice clearly indicates that changing primary care providers ' knowledge , attitudes , and practices requires multiple steps including consolidation and distillation of guidelines , active organizational support , clinically relevant decision tools , use of quality improvement techniques , and performance monitoring -LSB- 78 , 79 -RSB- . A growing body of research indicates that providers are more likely to engage in evidence-based practices following participation in quality improvement projects -LRB- e.g. , rapid improvement cycles or collaborative groups -RRB- . The federally funded Health Disparities Collaborative , the Perinatal Care and Patient Safety Collaboratives , and similar quality improvement efforts for community health centers demonstrate how such approaches are being used to better serve low-income and uninsured patients . The need to develop innovative approaches to deliver and finance bundles of preconception interventions must be viewed as a high priority . However , although strong evidence exists for many preconception care interventions and shows they are effective , discussion continues regarding the benefits of delivering comprehensive preconception care -LSB- 16 , 80 , 81 -RSB- , and the effectiveness or added value of `` packaging '' or `` bundling '' these interventions remains to be determined . For example , preconception care interventions could potentially be bundled into behavioral modification services -LRB- e.g. , smoking and alcohol cessation , weight control , exercise -RRB- , screening and assessment -LRB- e.g. , vaccines , general physical examination -RRB- , and specialized care -LRB- e.g. , management of diabetes and hypertension -RRB- . Alternatively , interventions could be bundled into visits -LRB- e.g. , do x at each well-woman visit , do y every 2 years , do z only for women at risk -RRB- . Some evidence already suggests that bundling could be of added value . For example , an evaluation of the National Centers of Excellence in Women 's Health found that , compared with other settings , women received better-quality primary health care at those centers having better integrated and coordinated services -LSB- 82 -RSB- . However , more such research , focused specifically on integration of preconception care components into primary care , is clearly warranted . Efforts to promote healthy behavior and improve service delivery should be accompanied by improvements in health care coverage and financing for women of childbearing age . Affordability of care is a major concern for many women -LSB- 83 -RSB- , and more can be done to improve access to preconception care . Many women under age 65 are uninsured , including 40 % of poor women , one third of near-poor women -LRB- with income between 100 % and 200 % of the federal poverty level -RRB- , 50 % of women with disabilities , and 29 % of young women ages 19 -- 24 years -LSB- 84 -RSB- . Expanding health care coverage for low-income women , through public programs such as Medicaid , Medicaid waivers , the State Children 's Health Insurance Program -LRB- SCHIP -RRB- , is particularly important . Medicaid offers particularly important opportunities . In 2003 , 12 % of all women of childbearing age -LRB- 15 -- 44 years -RRB- and 37 % of poor women in that age group relied on Medicaid for health care coverage -LSB- 83 -RSB- . Many low-income women do not , however , qualify for Medicaid because they do not have children under age 18 , are not over age 65 , or are undocumented . As states seek to expand Medicaid coverage to low-income , uninsured adults , women of childbearing age should receive high priority . Although two thirds of women in Medicaid are of childbearing age -LSB- 19 -- 44 , 85 -RSB- , many additional low-income women do not qualify for Medicaid benefits under current state eligibility rules unless they are pregnant . Without coverage before and between pregnancies , low-income women typically miss preventive visits -LSB- 83 -RSB- . Recognizing these gaps and missed opportunities for prevention , 21 states have developed special programs -LRB- under federal Medicaid waivers -RRB- that cover some women who do not otherwise qualify for Medicaid , particularly to offer family planning and interconception care . Specifically , these states extend eligibility to women who lose coverage after the birth of a baby or starting a job , while other states offer family planning coverage based on income status to men and/or women -LSB- 86 -RSB- . An evaluation of `` family planning waiver '' projects prepared for the federal Center for Medicare and Medicaid Services -LRB- CMS -RRB- found that they resulted in significant savings to both the federal and state governments -LSB- 87 -RSB- . Greater potential savings and prevention , however , could undoubtedly result if states offered more preconception risk screening , health promotion , and interventions ; to do so , however , states need permission from the federal government to include such services in their waivers or need for Congress to approve interconception care as an optional benefit . Finally , special attention should be given to the continuing , and sometimes increasing , racial gap in poor outcomes , especially in terms of access to services , and quality of care . These priorities can potentially complicate decision-making for public health policy . The best evidence of intervention effectiveness is often based on studies of health care systems . However , in developing programs and policies for the delivery of preconception services , one must consider the fact that poor access to health care can be a major determinant of health disparities . Preconception care : the next maternal and child health frontier The significant improvements in infant and maternal pregnancy outcomes during the past 40 years have resulted largely from the intensified focus on family planning in the 1960s and 1970s , on child health in the 1970s and 1980s , and on prenatal care in the 1980s and 1990s . Over the past 40 years , many public health and clinical care providers and their organizations have also recognized that , although it is important to offer family planning services to prevent unplanned pregnancies , it is not enough to worry about a pregnant woman 's health only during her pregnancy or a child 's health only after its birth , because many of the factors that contribute to the mother 's and child 's health can be identified and often successfully managed before pregnancy . Today , the greatest opportunities for further improvement in pregnancy outcomes -- in improving the health of women and their children -- lie in prevention strategies that must be implemented prior to conception to be effective . In 1989 , the U.S. Public Health Service Expert Panel identified preconception care as an essential part of prenatal care -LSB- 88 -RSB- . National organizations of health professionals , such as the AAP , ACOG , and the American Academy of Family Physicians -LRB- AAFP -RRB- , all recommend specific components of preconception care that should be delivered within the context of health care systems -LSB- 15 , 89 -RSB- . The time has come to move forward to the next maternal and child health frontier of `` prevention '' by acting on the recommendations of professional organizations and implementing scientifically proven interventions to further improve pregnancy outcomes . Action is urgently required in an array of clinical settings , in health promotion campaigns , and in academic research settings . Because of the wide range of interventions included under the umbrella of preconception care , such interventions may be delivered in both primary care and specialty care practices . However , opportunities also exist to improve preconception health through wellness care , through maintenance care for women with chronic health conditions associated with increased preconception risk -LRB- e.g. , maternal diabetes -RRB- , and in settings where women seek medical support for one specific risk such as smoking or obesity . In addition , more research is needed to understand how a women 's motivation for healthy behavior can be used to initiate the reduction of a number of preconception risks . For example , comparison of concurrent versus sequential interventions for smoking and weight control requires additional clarification -LSB- 90 -RSB- . Above all , health care providers and health agencies must understand that early prenatal care is too late for many women and babies . In particular , some evidence-based interventions recommended for implementation during pregnancy could be more effective and beneficial if implemented before conception . For example , although smoking cessation is recommended for all pregnant women , more than one attempt to stop may be necessary to achieve success . Only about 20 % of pregnant women who smoke are able to stop smoking during pregnancy -LSB- 90 -RSB- . Starting smoking cessation treatment -- and successfully completing it -- should be recommended before pregnancy is begun . Similarly , the only definitive way of preventing fetal alcohol syndrome is to stop drinking even before pregnancy . In the same manner , HIV testing and genetic counseling are recommended during pregnancy , but additional options are available to women who use these interventions prior to conception , including the decision not to become pregnant . Conclusion There is ample evidence that individual elements of preconception care work . Equally important , however , is the evidence that some interventions work best and others only work if provided before pregnancy . Given these realities , to further improve perinatal outcomes , the United States must commit to improving preconception health and to providing preconception care to all women of reproductive age . Even though sufficient knowledge and evidence is present to take action now , further efforts are needed to identify best practices and the most effective means of delivering integrated preconception services .

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