Cancer Letters 158 (2000) 6164

www.elsevier.com/locate/canlet

Serum a-N-acetylgalactosaminidase is associated with

diagnosis/prognosis of patients with squamous cell carcinoma
of the uterine cervix
Alagarsamy Lakku Reddi a,*, Kannan Sankaranarayanan a, Harold Stephan Arulraj b,
Niranjali Devaraj c, Halagowder Devaraj a
a
Unit of Biochemistry, Department of Zoology, University of Madras, Guindy Campus, Chennai 600 025, India
Barnard Institute of Radiology and Oncology, General Hospital, Madras Medical College, Chennai 600 001, India
c
Department of Biochemistry and Molecular Biology, University of Madras, Guindy Campus, Chennai 600 025, India
b

Received 18 September 1999; received in revised form 27 May 2000; accepted 31 May 2000

Abstract
Serum a-N-acetylgalactosaminidase (NaGalase) is responsible for the deglycosylation of vitamin D3-binding protein
(Gc protein). The deglycosylated Gc protein cannot be converted into major macrophage-activating factor (MAF), leading
to immunosuppression. NaGalase is universally detected in a variety of cancer patients, but not in healthy individuals (Cancer
Res. 56 (1997) 28272831). However, the diagnostic/prognostic utility of NaGalase in squamous cell carcinoma (SCC) of the
uterine cervix is not known. To address this issue, the serum NaGalase was quantitatively determined in 210 patients with
different stages of SCC of the uterine cervix. NaGalase levels were increased with the progression of the cancer. After
radiotherapy, the increased levels returned toward or to normal levels in early stages (FIGO stage IIIB) but not in advanced
stages (FIGO stage IIIIV). The present study revealed that the amount of NaGalase in the patient's bloodstream reects the
tumor burden and aggressiveness of the disease. We conclude that NaGalase is an independent predictor of diagnosis/prognosis
in SCC of the uterine cervix, and therefore suggest that quantitative NaGalase alteration may reect important differences in the
immunological functions of these neoplasms. q 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords: a-N-Acetylgalactosaminidase; Immunosuppression; Uterine cervix; Squamous cell carcinoma; Macrophage activating factor

1. Introduction
Cervical carcinoma is a major public health
problem in India, as not only the incidence is high
but also because most cases (70%) present themselves
in the advanced stages of the disease at the time of
diagnosis [1]. Cancer patients in the advanced stages,
* Corresponding author. Division of Immunology, Department of
Biotechnology, All India Institute of Medical Sciences, New Delhi
110 029, India. Tel.: 191-11-653-2891; fax: 191-11-685-2286.
E-mail address: vkmalr@hotmail.com (A.L. Reddi).

often suffer from a deciency of immunity and

increased susceptibility to infection with concomitant
depression of antibody production that becomes more
evident as the disease progresses [24]. Recently, a
defective macrophage activation cascade has been
demonstrated in all types of cancer patients [5]. Particularly advanced cancer patients have a severe defect
in the macrophage activation process. As macrophage
activation for phagocytosis and antigen presentation is
the rst step in the immune developmental cascade, a
defect in the macrophage activation cascade leads to

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A.L. Reddi et al. / Cancer Letters 158 (2000) 6164

immunosuppression [6]. Korbelik et. al. [7],

suggested that the secretion of a-N-acetylgalactosaminidase (NaGalase) from cancer cells to the bloodstream was responsible for deglycosylation of patient
serum Gc protein (vitamin D3-binding protein is
known as Gc protein), a serum protein that is the
precursor for the major macrophage-activating factor
(MAF) [7]. The deglycosylated Gc protein cannot be
converted to MAF. The progress of malignancy was
found to be associated with an increase in the serum
NaGalase activity and concomitant decrease in the
precursor activity of serum Gc protein. Some reports
have revealed that loss of the precursor activity was
due to deglycosylation of Gc protein by NaGalase
activity detected in a variety of cancer patients [4,5].
But the reports are lacking information regarding the
level of serum NaGalase in patients having SCC of the
uterine cervix and whether effective treatment could
be assessed by such monitoring. The objective of the
present study was to determine the diagnostic/prognostic value of NaGalase in sera of patients with squamous cell carcinoma (SCC) of the uterine cervix.
2. Materials and methods
This study involved in 210 patients with uterine
cervical cancer undergoing treatment at the Barnard
Institute of Radiology and Oncology, General Hospital, Madras Medical College, Chennai, India, on the
recommendation by the physician after approval by
the ethical board. The clinical staging and histological
classication were determined according to the
system of International Federation of Gynaecology
and Obstetrics (FIGO) [8] and World Health Organization (WHO) [9]. The 210 patients were histologically diagnosed with SCC. The patient's
characteristics are given in Table 1. All patients
were treated by external whole-pelvis irradiation
followed by two insertions of intracavitory bracytherapy. The total dose of irradiation to point A of 6000
cGy in stage IB, 7600 cGy in stages IIA, IIB and IIIB
and 45005500 cGy in stage IVA. The patients blood
sample were collected 1 day after the radiotherapy
was nished. Normal individuals consisted of
women who were in the age group of 2963 years,
with no evidence of any disease. Normal and patients
serum were separated from 10 ml of blood collected

Table 1
Patient's characteristics
Sl. No.

Variable

1.
2.

Mean age in years (range)

No. of evaluable patients

7.

Menopause status
Pre
Post

160
50

4.

Group I (normal)

85

Group II
FIGO stage
FIGO stage
FIGO stage
FIGO stage

I
II
III
IV

30
73
89
18

Group III
FIGO stage
FIGO stage
FIGO stage
FIGO stage

I
II
III
IV

30
73
89
18

47 (2963)
210

Histological type
Squamous cell carcinoma (SCC)

210

Cervical inltration
.5 mm
5 mm

25
185

Tumor size
.5 cm
5 cm

25
185

by veno-puncture. Normal individuals and patients

were placed in three groups as follows:
Group I: normal (healthy volunteers);
Group II: patients with different stages of SCC of
the uterine cervix before radiotherapy;
Group III: patients with different stages of SCC of
the uterine cervix after radiotherapy.

2.1. Assay for a -N-acetylgalactosaminidase in sera

One milliliter of sera was precipitated by 70%
ammonium sulfate saturation. The precipitate was
dissolved in 50 mM citrate-phosphate buffer (pH

A.L. Reddi et al. / Cancer Letters 158 (2000) 6164

6.0) and dialyzed against the same buffer at 48C, overnight. The dialysates were made up to 1 ml with buffer
and assayed for the enzyme activity [6,10]. The
substrate solution (1 ml) contained 5 mmol of p-nitrophenyl N-acetyl-a-d-galactosaminidase in 50 mM
citrate buffer (pH 6.0). The reaction was initiated by
addition of 100 ml of the dialyzed samples kept at
378C for 60 min, and terminated by adding 200 ml
of 10% trichloroacetic acid. After centrifugation of
the reaction mixture, 500 ml of 0.5 M Na2CO3 solution
was added to the supernatant. The amount of p-nitrophenol released was determined spectrophotometrically at 420 nm and expressed as nmol/mg per min
of serum protein. Protein concentrations were determined by the Bradford method [11].
2.2. Statistical analysis
Student's t-test was performed to examine the relationship of the expression of NaGalase in patients
with SCC of the uterine cervix against healthy
controls.
3. Results and discussion
The present study showed increased expression of
NaGalase activity with increasing stages of SCC of the
uterine cervix. Sera of age-matched controls contained
an extremely low level (0.26 ^ 0.1 nmol/mg per min)
of NaGalase (Table 2). NaGalase expression was

63

detected in all the stages of different types of cancer

patient's bloodstream, but not in healthy humans [5].
The patient's serum NaGalase deglycosylates Gc
protein. Deglycosylated Gc protein cannot be
converted to MAF. Thus, this serum enzyme inactivates the MAF precursor activity of Gc protein.
Advanced cancer patient serum contains high NaGalase activity, leading to no macrophage activation [5].
Because, macrophage activation is the rst step in the
immune development cascade, such cancer patients
become severely immunosuppressed. This may
explain, at least in part, why cancer patients die due
to an overwhelming infection [10].
AIDS patients also carry NaGalase in their bloodstream [12]. This enzyme seems to play a role in
immunosuppression in HIV-infected patients. The
enzyme in cancer patients appears to be coded by an
oncogene, whereas the enzyme in HIV-infected
patients is coded by the viral genome [5]. An impairment of immune function of the host will enhance
opportunities for survival and proliferation of
neoplastic cells [13]. Thus, AIDS patients are at an
extremely high risk for the development of various
forms of malignant tumors [14].
Circulating levels of NaGalase was signicantly
reduced after radiotherapy in all stages of SCC of
the uterine cervix (Table 2). NaGalase levels reached
near normal level in early stages (stage I and II) of the
SCC of the uterine cervix after radiotherapy but not in
advanced stages (stage III and IV). The present study

Table 2
Levels of serum a-N-acetylgalactosaminidase in the SCC of the uterine cervix, before and after radiotherapy a
No. of patients

Group I (normal)
Group II
Stage I
Stage II
Stage III
Stage IV
Group III
Stage I
Stage II
Stage III
Stage IV
a

a-N-Acetyl galactosaminidase (nmol/mg per min)

Mean ^ SD

Range

85

0.26 ^ 0.1

0.180.41

30
73
89
18

0.72 ^ 0.12
1.54 ^ 0.44*
3.78 ^ 1.03**
5.05 ^ 0.17***

0.310.89
0.622.91
1.215.16
3.565.91

30
73
89
18

0.39 ^ 0.08#
0.64 ^ 0.23#
1.38 ^ 0.33##
3.14 ^ 0.86###

0.230.45
0.371.53
0.931.92
2.623.58

*P , 0:05, **P , 0:01, ***P , 0:001 group II vs. group I. #P , 0:05, ##P , 0:01, ###P , 0:001 group III vs. group II.

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A.L. Reddi et al. / Cancer Letters 158 (2000) 6164

revealed that the levels of NaGalase in the bloodstream reects the tumor burden and aggressiveness
of the disease. A recent experimental study proved
that the amount of NaGalase in the bloodstream is
directly proportional to the number of cancerous
cells or the size of tumor in the hosts [6]. Surgical
removal of malignant lesions resulted in a subtle
decrease in serum NaGalase activity and concomitant
increase in precursor activity. However, even after
surgical removal of tumor lesions, the majority of
postoperative patients carried signicant amounts of
NaGalase activity in their bloodstream, suggesting
that the enzyme is derived from the remnant cancerous lesions and/or metastasized tumor lesions [6]. We
support the recommendation made by Korbelik et al.
[7] that the NaGalase measurement could serve as a
diagnostic and prognostic index that might allow
oncologist to design the dose and the nature of treatment.
Acknowledgements
We thank the Director and Assistant Professors of
Barnard Institute of Radiology and Oncology, General
Hospital, Madras Medical College, Chennai, for
providing blood samples from the cancer patients.
One of the authors Dr A.L. Reddi, thanks the University Grants Commission (UGC), India for nancial
assistance.
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