Vitamin C Augments Chemotherapeutic Response of Cervical Carcinoma HeLa Cells by Stabilizing P53 Vijay G. Reddy, Neeru Khanna, and Neeta Singh 1 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% CO 2 in air at 37°C. Logarithmically growing cells were used for all experiments. Drug treatment and cell viability assay. 1  10 4 cells 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 l of 5 mg/ml of MTT was added followed by incubation for 4 h at 37°C. The 1 To 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.