Vitamin C Augments Chemotherapeutic Response of
Cervical Carcinoma HeLa Cells by Stabilizing P53
Vijay G. Reddy, Neeru Khanna, and Neeta Singh1
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India
Received February 20, 2001
Human Papilloma Virus (HPV) is associated in most
instances with cervical cancer. The HPV oncoproteins
target P53 protein for degradation, leading to deregu-
lation of cell cycle. We investigated whether stabiliza-
tion of P53 in cervical cancer cells, by downregulating
HPV transcription would restore the apoptotic ability
of these cells. Our findings show that vitamin C down-
regulates the redox sensitive transcription factor AP-1
and decreases one of its transcription targets HPV E6,
and stabilizes P53. This was associated with an in-
crease in Bax and decrease in Bcl-2 and telomerase
activity. Accumulation of P53 and its target gene bax
then sensitized HeLa cells to cell-cycle arrest, cell
death/apoptosis induced by cisplatin, and etoposide.
Increasing drug sensitivity of cervical carcinoma cells
by stabilizing P53 using vitamin C is a novel approach
and has potential clinical relevance. © 2001 Academic Press
Key Words: vitamin C; cervical cancer; P53, HPV; E6;
chemotherapy.
Cervical cancer has a high prevalence especially
amongst women in Asia and is a leading cause of can-
cer death. It primarily has a viral etiology and HPVs
have been shown to be involved in the pathogenesis of
cervical, vulval, penile, and perianal cancer (1). The
oncogenic potential of high risk HPV types 16, 18, and
to some extent types 33, 45, 52, 58 is attributed to E6
and E7 oncogene products, which participate in trans-
forming the infected cell. The E6 oncoprotein targets
P53 protein, induces its ubiquitin-mediated destruc-
tion (2) and affects its cell-cycle regulation. E6 and E7
oncoproteins are also involved in the activation of te-
lomerase, which contributes to the immortalization of
transformed cells (3). It is shown that absence of P53
leads to abolition of G1 arrest or apoptosis in response
to ionizing radiation and DNA damaging agents (4).
Some reports have associated loss of P53, with in-
creased sensitivity to chemotherapy, whereas others to
increased chemoresistance (4–6). Cervical cancer che-
motherapy in vivo improved in cases with high P53
expression in the tumor tissue (7, 8). It has been shown
that transactivation and DNA-binding affinity of acti-
vator protein (AP-1) as well as P53 can be modulated
by both posttranslation modifications as well as by
alteration of intracellular redox state (9, 10). Cervical
cancer is associated with low antioxidant status (11).
Vitamin C has been suggested to play a protective role
in development of cervical intraepithelial neoplasia
(CIN) (12), however, its role in the treatment of cervical
cancer has not been studied.
To investigate the effect of vitamin C on the tran-
scriptional regulation of HPV E6/E7 oncogene expres-
sion, HPV-18 positive Hela cells were treated with
noncytotoxic amounts of vitamin C. The findings sug-
gest that vitamin C caused downregulation of the viral
oncoprotein E6, which was paralleled by a decrease of
AP-1 members c-jun and c-fos in a dose- and time-
dependent manner. The downregulation of E6 resulted
in the up regulation of proapoptotic, P53 and Bax pro-
teins but downregulation of apoptotic inhibitor Bcl-2.
There was also significant decrease in telomerase ac-
tivity. Vitamin C also increased the susceptibility/
apoptosis induced by cisplatin and etoposide.
MATERIALS AND METHODS
Cell culture. The human cervical carcinoma cell line (Hela) was
obtained from National Centre for Cell Science, Pune, India and
maintained in DMEM medium containing 10% fetal calf serum and
antibiotics in a humidified atmosphere of 5% CO2in air at 37°C.
Logarithmically growing cells were used for all experiments.
Drug treatment and cell viability assay. 1⫻104cells seeded in
96-well microtiter plates were treated with in vivo achievable con-
centrations of vitamin C ranging from 0.1
M to 10 mM and/or
chemotherapeutic drugs i.e., cisplatin (2–100
M), etoposide (2–100
M), adriamycin (0.01–10
M), bleomycin (0.0004– 0.4 U/ml). The
growth inhibitory effect of vitamin C and chemotherapeutic drugs
was assessed by the MTT assay. After 48 h of incubation, 100
lof5
mg/ml of MTT was added followed by incubation for4hat37°C. The
1To whom correspondence should be addressed at Department of
Biochemistry, Room No 3027-A, All India Institute of Medical Sci-
ences, Ansari Nagar, New Delhi-110029, India. Fax: 91-11-6862663.
E-mail: singh_neeta@hotmail.com.
Biochemical and Biophysical Research Communications 282, 409– 415 (2001)
doi:10.1006/bbrc.2001.4593, available online at http://www.idealibrary.com on
409 0006-291X/01 $35.00
Copyright © 2001 by Academic Press
All rights of reproduction in any form reserved.
