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Artikel Penelitian

Maj Kedokt Indon, Volum: 59, Nomor: 6, Juni 2009

Prevention of Malignancy FollowingHydatidiform Mole With Vitamin A

Andri Andrijono,* Muhammad Muhilal,** Emil Taufik,*** Meny Hartati,***

Ria Kodariah,*** Wan Leli Heffen****

*Department of Obstetrics and Gynecology, Faculty of Medicine University of Indonesia/

Dr. Cipto Mangunkusumo GeneralHospital,

**Center for Research and Development of Nutrition, Ministry of Health,

***Department of Anatomic Pathology, Faculty of Medicine University of Indonesia,

****Center for Research and Development, Dharmais Cancer Hospital

Abstract: The aim of this study was to demonstrate the role of vitamin A as chemoprevention for

malignancy following hydatidiform mole. This study was a series of trials. Two supporting stud-

ies, i.e. the study on the expression of RPB (retinol binding protein) receptor, the study on

apoptosis activity in trophoblastic cells with the administration of retinoic has been conducted.

The main study is clinical trial on the prevention of malignancy following hydatidiform mole

with vitamin A. The study was a randomized double-blind clinical trial. Subjects of the study

were patients with complete hydatidiform mole. Placebo and vitamin A were administered at

200,000 IU per day until patients were declared cured or MTD. Variables of outcome were the

incidence of regression and MTD which were established based on WHO criteria. From as many

as 21 specimens of with immunohistochemistry study, we found RBP receptor expression in

trophoblastic cells. In the study on apoptosis activity in the culture of trophoblastic cells receiv-

ing retinoic, we found apoptosis activity. Apoptosis activity in the control was 60%, in ATRA of

50 g/ml was 89.54%, of 100 g/ml was 87.23%, of 150 g/ml was 94.63%, and of 200 g/ml was

94,83%. The clinical trial found 67 cases admitted to the study. As many as 2 cases were lost

from observation, and 3 cases experienced pregnancy during observation. The incidence rate of

MTD in the control group was 28.57%, and in the therapy group 6.25%.

Keywords: vitamin A, Malignant trophoblastic Disease, trophoblastic cells, hydatidiform mole

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Pencegahan Keganasan Pascamola Hidatidosa dengan Vitamin A

Andri Andrijono,* Muhammad Muhilal,** Emil Taufik,*** Meny Hartati,***Ria Kodariah,*** Wan Leli Heffen****

*Departemen Obstetri dan Ginekologi Fakultas Kedokteran Universitas Indonesia/

Rumah Sakit Umum Dr. Cipto Mangunkusumo

**Pusat Penelitian dan Pengembangan Gizi Departemen Kesehatan

*** Departemen Patologi Anatomi Fakultas Kedokteran Universitas Indonesia

**** Pusat Penelitian dan Pengembangan Rumah Sakit Kanker Dharmais

Abstrak: Penelitian ini bertujuan membuktikan vitamin A sebagai kemoprevensi keganasan

pascamola hidatidosa. Penelitian ini merupakan rangkaian penelitian. Dua penelitian yang

mendukung penelitian yaitu penelitian ekspresi reseptor retinol binding protein dan penelitian

aktivitas apoptosis pada pemberian asam retinoat pada sel trofoblas. Penelitian utama adalah

penelitian pencegahan keganasan pascamola dengan vitamin A. Penelitian adalah uji klinik acak

double-blind. Plasebo atau vitamin A dengan dosis 200 000 IU perhari sampai dinyatakanregresi atau PTG (penyakit trofoblas ganas). Variabel keluaran adalah regresi atau keganasan

yang ditetapkan berdasarkan criteria WHO. Dengan imunohistokimia terdapat ekspresi reseptor

RBP pada 21 sampel sel trofoblas. Aktivitas apoptosis dijumpai pada penelitian kultur sel trofoblas

yang diberikan retinoat. Aktivitas apoptosis pada kontrol 60%, 89,54% pada dosis ATRA 50 g/

ml , 87,23% pada 100 g/ml, 94,63% pada dosis 150 g/ml, dan 94,83% pada dosis 200 g/ml.

Sejumlah 67 kasus masuk dalam uji klinik. Dua kasus hilang pada pengamatan, 3 kasus hamil.

Kejadian PTG pada kelompok control 28,57%, dan pada kelompok terapi 6,25%.

Kata kunci: vitamin A, penyakit tropoblas ganas, se; trofoblas, mola hudatidosa.

Introduction

Hydatidiform mole showed complaints and signs of

pregnancy, with several more dominant symptoms, such as

symptoms of vomiting, more rapid uterus enlargement. Fre-

quently, it is accompanied by symptoms of bleeding, or

symptoms of thyrotoxicosis. With the adjunct of ultra-

sonographic examination, hydatidiform mole could be diag-

nosed earlier.1-3 Histopathologically, datidiform mole is an

abnormal pregnancy characterized by the proliferation of

trophoblastic cells and hydrophic chorionic villi and with or

without fetus.1,2Diagnosis of hydatidiform mole could be

made on the basis of clinical findings.

The incidence of MTD following hydatidiform mole was

approximately 15-28%. Several factors are implicated in the

occurrence of MTD, i.e clinical factor and molecular factor.

The clinical factors which are suspected as the risk factors

for the occurrence of MTD include, among others, pre-evacu-

ation HCG level and uterus size. Uterus size which is larger

than the uterus of gestational age of 20 weeks is a risk factor.

Uterus size is a factor that is easy to examine.4

Trophoblastic cells have several activities; however,

two primary activities of trophoblastic cells are proliferation

activity and apoptosis. If the proliferation following curet-

tage continues, a malignancy degeneration will occur which

is known as persistent hydatidiform mole or malignant tro-

phoblastic disease (MTD). If the activity of apoptosis is

dominant, a spontaneous regression will occur.

Etiology of hydatidiform mole is still unknown. Molecu-

lar factor, which induces cell cycle15suspected as risk factor.

Gene c-erbB2 is a receptor gene of epithelial growth factor

(EGF). Disruption of c-erbB2expression is suspected to have

strong correlation with malignancy degeneration following

hydatidiform mole.5PCNA(proliferating cell nuclear anti-

gen) is one of the genes that plays a role in metastasis. Ma-

lignancy following hydatidiform mole has potential to me-

tastasize, particularly into the lungs. Manifestation of the

PCNAexpression is a marker of malignancy risk of tropho-

blastic cells.6

Expression of human telomerase reverse transcriptase

(hTERT), ribonucleoprotein telomeraseplays a role in the

survival or carcinogenesis. Telomerase expression is discov-

ered in hydatidiform mole and choriocarcinoma, and is not

found in the partial hydatidiform mole or normal pregnancy.6,7

Activation of this enzyme is frequently found in malignancy.

The role of telomerase in hydatidiform mole is still unclear

yet. It was suspected that this enzyme plays a role in the

possible occurrence of malignancy following hydatidiform

mole.

Prevention of Malignancy Following Hydatidiform Mole With Vitamin A

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Apoptosis is controlled or stimulated by several genes,

such asBcl-2and other genes that work to inhibit apoptosis.

Expression of apoptosis gene proved to be higher in hyda-

tidiform mole trophoblastic cells than in the trophoblasticcells of normal placenta.8

Vitamin A works to control cell proliferation and stimu-

late apoptosis. Vitamin A intake from food would be metabo-

lized intro retinol. Inside the liver, retinol takes the form of

retinyl ester. Retinol in the plasma is bound by receptor on

the cell surface. Retinol would enter cytoplasm with the aid

of receptor. In the cytoplasm, retinol is metabolized into

retinoic acid. Retinoic acid in the cytoplasm would enter cell

nucleus and form a complex of retinoic receptor.11

Retinoic acid plays a role in controlling cell cycle by

arresting cell cycle at G1phase and S phase. The cell arrest

by retinoic is achieved through the activation of p53, p21,

p27, and it inhibits cyclin.12, Retinoic acid also plays a role in

inducing apoptosis. Apoptosis induction by retinoic occurs

through the induction of caspase, dab and p53.13,14

Proliferation and apoptosis are the activities of tropho-

blastic cells and constitute the main activities of vitamin A.

There might be a relationship between vitamin A intake and

hydatidiform mole. This relationship was identified in the

epidemiological study of vitamin A level in hydatidiform mole

patients, which showed that level of vitamin A was lower in

hydatidiform mole patients than in pregnant women. The

RETINOIC + RAR

APOPTOSISCELL CYCLEG1

S

G2

M

CASPASE-7 CASPASE-9

(+)

(+)

CRABP

Apaff-1

(+)

(+)(+)

Bcl-2

(-)

(+)

(-)

CYCLIN-D1,

A, E

P53

(+)

(-)(+)

(-)

P21

(+)

(-)

P27

E2F

pRb-E2F

+

(-)

(-)

(+)

(+)

CYCLIN D3

(-)

Cmyc,Cjun

(+)

(-)

GATA6 dab2(+)

(+)

(+)

Figure 1. Activity of Cell Cycle Arrest and Apoptosis by Retinoic

risk for developing hydatidiform mole in women less than 24

years of age and with vitamin A deficiency was 6.29 times as

high. This risk increased by 7 times if the pregnancy experi-

enced was the first pregnancy.15

Based on this study, there were two questions raised:

could vitamin A be one of the factors responsible for the

occurrence of hydatidiform mole, and could therapy of vita-

min A reduce the risk for developing MTD.

Methods

In order to demonstrate the benefits of vitamin A ad-

ministration in reducing the incidence of MTD, it is neces-

sary to conduct a series of studies. This series of studies

needs to be performed since such studies have not yet been

reported by previous investigators.

Study on the expression of retinol receptor in tropho-

blastic cells. The presence of retinol receptor in trophoblas-

tic cells is extremely important. This is because retinol could

enter trophoblastic cell by active mechanism with the aid of

receptor. Diffusion mechanism is difficult to demonstrate.

Active mechanism could be demonstrated by the presence

of receptor in the cell.16 The demonstration of retinol recep-

tor in trophoblastic cells could be carried out by immunohis-

tochemistry examination.17With the absence of retinol re-

ceptor, the role of vitamin A in trophoblastic cells was rela-

tively small. The presence of retinol receptor in trophoblas-

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tic cells should be demonstrated because such a study has

not yet been reported by previous investigators.

Materials of the study were fixed and paraffinized speci-

mens. The immunohistochemistry examination was indirect.

We used secondary antibody of Retinol Binding Protein,

and it was evaluated by anatomic pathologist at the Depart-

ment of Anatomic Pathology, Faculty of Medicine Univer-

sity of Indonesia. The variables evaluated were the pres-

ence of RBP expression, the strength and position of RBP

receptor expression in trophoblastic cells.Study on apoptosis signals by retinoic acid in tropho-

blastic cells.Apoptosis signals were better in identifying

the activity of medication used as chemoprevention.

Apoptosis was considered to be better because it would

occur if the arrest of cell cycle took place. The presence of

retinol receptor in trophoblastic cells showed that retinol

could enter the cell. The study on various cells demonstrated

that the activity of retinoic could cause apoptosis. In tro-

phoblastic cells, the activity of retinoic in trophoblastic cells

has not yet been reported by previous investigators. Samples

of the study were trophoblastic cells. We performed the cul-

ture of trophoblastic cells, the presence of trophoblastic cells

in the cell culture was demonstrated by hCG examination of

PROBLEM SOLVING

------------------------------------

VITAMIN A HYDATIDIFORM MOLE

EPIDEMIOLOGICAL STUDY

----------------------------------------

LEVEL OF RETINOL IN HYDATIDIFORM MOLE

LABORATORY STUDY

------------------------------------------

EXPRESSION OF RBP RECEPTOR

AT HYDATIDIFORM MOLE

TROFOBLASTIC CELL

CLINICAL TRIAL

----------------------------------------

CHEMOPREVENTION BY VITAMIN A

LABORATORY STUDY

---------------------------------------------------------

APOPTOSIS INDUCTION OF

TROFOBLASTIC CELL BY

RETINOIC ACID

culture media. The cell culture was administered ATRA at

doses of 50 g/ml, 100 g/ml, 150 g/ml, and 200 g/ml. The

outcome variables evaluated were percentages of cells un-

dergoing apoptosis. Evaluation of apoptosis was made withflowcytometry examination in 24 hours following the admin-

istration. The percentage of cells undergoing apoptosis was

recorded in cytogram by flow-cytometry at the lower right

quadrant.18

Study on the prevention of malignancy following hy-

datidiform mole with vitamin A. Vitamin A could be catego-

rized as chemoprevention. As a medication, vitamin A was

also a metabolite of natural substance, easily afforded, inex-

pensive with mild side effect, and worked at precancerous

stage. The work mechanism of vitamin A in trophoblastic

cells was demonstrated by laboratory studies. If vitamin A

plays a role in trophoblastic cells, it is necessary to demon-

strate that vitamin A is capable to work as chemoprevention

in hydatidiform mole. Design of this study was randomized

clinical trial, double blind study. Samples of the study were

patients with complete hydatidiform mole who met the inclu-

sion criteria. We performed treatment by administering pla-

cebo and vitamin A 200.000 IU per day until regression or

degeneration of MTD was observed. Diagnosis of MTD and

regression was established on the basis of WHO criteria.3

The interfering variables were age, education, gestational

age, uterine size, and retinol deposit in the liver. The vari-

ables were dependent on the incidence of regression and

MTD.

Results

Expression of retinol receptor in trophoblastic cells.

We performed the examinations of receptors by indirect

immunohistochemistry.

25 4

SERUM LEVELOF RETINOL

< N

HYDATIDIFORM MOLE

MALIGNANT OF TROPHOBLASTICDISEASE

PROLIFERATION

THERAPY OF VITAMIN A

SERUM LEVEL OF VITAMIN A : N

PROLIFERATION (-)

APOPTOSIS

SERUM LEVEL OF VITAMIN A

APOPTOSIS

60,64% VS 94,83%

RETINOIC

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Trophoblastic cells had a relatively high level of apop-

tosis activity. This spontaneous regression process may be

due to the fact that trophoblastic cells had apoptosis acti-

vity. In laboratory studies the apoptosis activity that wasobserved in trophoblastic cells was relatively high, i.e.

60.64%.

Hydatidiform mole has two main activities, i.e. prolifera-

tion and apoptosis. The increase of cell proliferation and

decrease of apoptosis constituted the risk for continued pro-

liferation of trophoblastic cells which clinically known as

MTD.

Vitamin A has two main activities: i.e. controlling and

arresting cell proliferation and inducing apoptosis. These

two main activities of vitamin A constituted the rationale for

administering preventive therapy for malignancy following

hydatidiform mole with vitamin A.

The administration of vitamin A would increase retinol

level in the serum. The increase of retinol level in the serum

would increase the amount of retinol entering trophoblastic

cells. The increase of retinol in cytoplasm of trophoblastic

cells would enhance the metabolism of retinoic acid.

The increase of retinoic acid would enhance the sig-

nals controlling cell proliferation and increase apoptosis

activity. Clinically, the arrest of cell cycle and increase of

apoptosis was considered as the increased incidence of re-

gression following hydatidiform mole.

The increased incidence of regression by vitamin A

was demonstrated by identifying the decreased incidence

of malignancy following hydatidiform mole during the ad-ministration of vitamin A.

The incidence rates of malignancy following hydatidi-

form mole in the control group was 28.57%, and in the group

receiving the therapy of vitamin A was 6.25%. These find-

ings were nearly the same as those obtained at the study on

chemoprevention following hydatidiform mole with actino-

mycin (the control group was 29% and the therapy group

6.9%).19

The risk for developing malignancy following hyda-

tidiform mole when vitamin A was not administered was 8.4

times as high as when hydatidiform mole patients received

therapy of vitamin A. In addition, the administration of vita-min A therapy did not result in different side effects as when

vitamin A was not administered. However, the administra-

tion of vitamin A improved SGOT level of hydatidiform mole

patients.

Randomized clinical trial, double blind study (clinical

trial) showed that the incidence rates of malignancy follow-

ing hydatidiform mole receiving vitamin A therapy were lower

than hydatidiform mole that did not receive vitamin A.

Conclusions

The study showed existed a receptor of retinol binding

in trophoblastic cells. The laboratory study showed that

25 8

trophoblastic cells of hydatidiform mole had apoptosis ac-

tivity of 60.64% and retinoic acid increased apoptosis activ-

ity of trophoblastic cells. The clinical trial showed that the

incidence rates of malignancy following hydatidiform mole(MTD) receiving vitamin A was 6.25%, and in the control

group was 28.57%.

Recommendations

Further studies should be performed on the doses of

vitamin A, correlation of vitamin A and ovulation disorder,

correlation of vitamin A and ovum abnormalities, correlation

of vitamin A and invasive mole, and correlation of vitamin A

and choriocarcinoma.

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