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The role of vitamin c in enhancing the response of cervical carcinoma hela cells to chemotherapeutic drugs by stabilizing the p53 protein. The study suggests that vitamin c downregulates the viral oncoprotein e6, decreases ap-1 members c-jun and c-fos, and upregulates p53, bax, and downregulates bcl2. The findings have potential clinical relevance as they indicate that vitamin c can increase the susceptibility of cervical carcinoma cells to chemotherapeutic drugs.
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Received February 20, 2001
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 E 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 E 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 P
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 P 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 3 10 4 cells seeded in 96-well microtiter plates were treated with in vivo achievable con- centrations of vitamin C ranging from 0.1 mM to 10 mM and/or chemotherapeutic drugs i.e., cisplatin (2–100 mM), etoposide (2– mM), adriamycin (0.01–10 mM), 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 ml 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
Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
formazan crystals thus formed were dissolved in DMSO and the absorbance measured at 570 nm with an ELISA reader. Immunocytochemistry. HeLa cells were grown on glass cover slips and treated with vitamin C (0.1 mM–10 mM). The cells were washed in PBS, fixed in ice-cold acetone, and methanol (1:1) for 10 min, and incubated for 1 h with primary antibodies c-Jun, c-Fos, P53, HPV-18-E6, Bax, and Bcl2 (Santa Cruz Biotechnology, USA). Sub- sequently the respective secondary antibody conjugated with alka- line phosphatase (AP) was added, the colour developed using BCIP and NBT (Promega, USA), and visualized under a light microscope (200 3 magnification). Western blot analysis. Equal amounts of protein extract was elec- trophoresed on 12–15% SDS–PAGE gels and electrotransferred to nitrocellulose membrane. Western blotting was done using the kit from Promega, USA, as described by the manufacturer. The blots were scanned by UMAX Gemini D-16 scanner and comparative den- sitometric analysis was done using NIH 1.52 image analysis soft- ware. Detection of apoptosis. Apoptotic cells were visualized by TUNEL assay using the DeadEnd colorimetric detection system (Promega, USA) and by flow cytometry as described earlier (13). Flow cytometry was performed using an EPICS XL-MCL flow cytometer (Coulter Electronics, Miami, FL) and the data was analyzed using WinMDI 2.8 software. Assay of telomerase activity. This was measured using PCR- based ELISA kit (Roche Molecular Biochemicals, Germany). The samples were lysed, and an aliquot containing 2 mg of protein was used for the assay. Telomerase positive embryonic kidney cell line (293) and untreated Hela cells were used as positive controls while heat inactivated Hela cell extract was used as negative control. The activity detected was expressed in relation to the positive control Hela cells.
RESULTS
Vitamin C Downregulates E6 Oncoprotein
To study the effect of vitamin C on transcriptional activity of HPV, HeLa cells were treated with vitamin
C doses ranging from 0.1 mM to 10 mM for 3 to 48 h. A dose of 1 mM vitamin C downregulated E6 without being toxic, as observed by immunocytochemistry and Western blot analysis (Figs. 1 and 2), and hence this dose was used for all further experiments. Immunocy- tochemistry revealed nuclear and cytoplasmic expres- sion of P53 (Fig. 1). b-Actin levels in all the treatments were found to be normal.
Vitamin C Downregulates c-Jun/c-Fos
Since HPV is under the transcriptional control of AP-1, we looked at the protein expression of c-jun/c-fos AP-1 heterodimer, a predominant form of AP-1 com- plex in Hela cells. Vitamin C treatment decreased the expression of both c-fos and c-jun, but more signifi- cantly that of c-fos (Fig. 2), starting at 3– 6 h and reaching the lowest level of 8.96% for c-fos and 47.3% for c-jun at 36 h, but recovered at 48 h.
Vitamin C Upregulates P53, Bax, and Downregulates Bcl
Since bax and bcl2 are the down stream targets of P53, we looked at their protein expression. The ob- served upregulation of P53 was followed by increase in Bax protein expression which reached a maximum of 328% at 24 h as compared to the control and this was sustained till 36 h. Concurrently, the expression of Bcl2 showed a 50% decrease after an initial increase at 3– 6 h (Fig. 2).
Vitamin C Produces Partial G1/S Block
The dose of vitamin C (1 mM) which was able to upregulate P53, induced a partial G1/S block, a 14%
FIG. 1. E6 and P53 protein expression as detected by immuno- cytochemistry (a) E6 expression in untreated control Hela cells shows predominance of perinuclear and cytoplasmic expression (b) E6 expression 24 h after treatment with 1 mM vitamin C (c) P expression in untreated control Hela cells (d) P53 expression 24 h after 1 mM vitamin C treatment shows both nuclear and cytoplasmic expression, nuclear expression is more in cells undergoing apoptosis.
FIG. 2. Western blot analysis of E6, c-Fos, c-Jun, P53, Bax, and Bcl2 protein expression in Hela cells after treatment with 1 mM vitamin C for various time periods, and their percentage expression compared to the control taken as 100%.
5B). There was no increase in G1/S arrest, however, partial S/G2 block was seen on vitamin C priming for 12 h. Interestingly, vitamin C priming for 36 h po- tentiated the G2/M arrest produced by cisplatin and this was accompanied by maximum apoptosis. This indicates that the upregulated P53 is not sufficient to produce a G1/S arrest, but is sufficient to trigger increased apoptosis. Priming with high dose vitamin C (1 mM) produced increased cell death when combined with low dose of cisplatin (2–10 mM), but cell death was decreased on increasing the dose of cisplatin (25–100 mM) (Fig. 4B). The percentage apoptosis decreased from 74.4 to 56% after simultaneous administration of 50 mM cisplatin with 1 mM vitamin C, and decreased from 97 to 73.6% when cisplatin (50 mM) was administered after prim- ing with vitamin C (1 mM) for 24 h (Fig. 4B). This indicates that the dose of vitamin C and cisplatin as well as the duration of priming was critical for the additive effect. The percentage apoptosis increased from 43.1% with 2 mM etoposide to 66% after priming with 1 mM vitamin C for 36 h (Fig. 4c). Similarly priming with 1 mM vitamin C for 36 h increased the percentage apoptosis by 16% with 0.1 mM adriamycin and by 18% with .0004 U/ml of bleomycin (data not shown). Thus the nature of cell-cycle arrest and the induction of apoptosis appears to be dependent on the type of chemotherapeutic drug.
Vitamin C Treatment Decreases Telomerase Activity
Reactivation of telomerase is a significant step in tumorigenesis and downregulation of telomerase activ- ity is a new approach of cancer control. Perhaps this is the first demonstration showing a significant decrease
in telomerase activity following vitamin C (1 mM) treat- ment, which was detectable as early as 6 h and reached 50% of control by 36 h (Fig. 6). The decrease in telo- merase activity coincided with the decrease in E6 and increase in P53 protein expression. This indicates that restoration of normal cell-cycle control mechanism by decreasing HPV transcription can decrease the telo- merase activity.
DISCUSSION
The cause for poor responsiveness to chemotherapy lies in the etiopathogenesis of cervical cancer i.e., HPV infection and loss of tumor suppressor gene function due to inactivation of P53 and Rb by HPV encoded viral oncoproteins. The restoration of P53 levels could be a potential strategy to increase chemoresponsiveness. However, there are conflicting reports regarding the role of P53 and chemosensitivity (4 – 6). Different au- thors have adopted different strategies to decrease E (14, 15) and stabilize P53 levels. We selected vitamin C based on the fact that it prevents the development of CIN to cervical cancer and decreases methylcholan- threne (MCA) induced cervical cancer in mice (12, 16). Vitamin C at low doses was seen to significantly de- crease c-Fos/c-Jun AP-1 dimer followed by decrease in E6 and increase in p53 protein. The level of Fra-1 was not estimated since it expression is low in Hela cells. We found that the increase in P53 resulted in upregu- lation of pro-apoptotic Bax and downregulation of anti apoptotic Bcl2, but was not sufficient to induce apop- tosis. Cisplatin, the single most active drug against cervi- cal cancer, was found to produce maximum additive affect in vitro on vitamin C pretreatment, followed by etoposide, whereas adriamycin and bleomycin showed only moderate effects. The probable reason for this could be that adriamycin and bleomycin themselves produce reactive oxygen species and are known to show chemoresistance in the presence of increased antioxi- dant status of the cell (17). Another important finding of our study was that the combination of high doses of vitamin C and cisplatin could decrease the effect of chemotherapy. The reason for this is not known al- though it is known that vitamin C at low doses acts as antioxidant and at high doses as a pro-oxidant (18). However, this finding is in congruence with a report, which states that hydrogen peroxide lowers the degree of cell killing by cisplatin (19). Our findings suggest that priming with low dose of vitamin C can have a significant additive effect particularly with low dose of in vivo achievable chemotherapeutic drugs, as shown by increased apoptosis. Although vitamin C showed increase in the levels of P53, it was unable to produce a very significant G1/S arrest. There was also no G1/S arrest in re- sponse to chemotherapeutic drugs indicating that
Vitamin C dose/ priming duration
0 mM
2 mM
10 mM
25 mM
100 mM
0 mM 100 88.5 58.3 38.3 29. 1 mM/0 h 116 83.16 50.48 36.68 30. 1 mM/6 h 156.7 90.2 57.2 31.8 30. 1 mM/12 h 97.7 54.4 46.48 26.4 21. 1 mM/24 h 93.7 *39.6 *36.5 31.7 21. 1 mM/36 h 90.7 *37.4 *35.3 30.1 19.
Note. Viability of Hela cells after priming with 1 mM vitamin C for 6 –36 h followed by cisplatin treatment with various doses for 48 h. The percentage viability is expressed in comparison to untreated controls. The data is the mean of 3 separate experiments.
FIG. 4. (A) Effect of 1 mM vitamin C priming on cisplatin induced apoptosis (a) Control: 0 – 48 h (b) Vitamin C 1 mM: 0 – 48 h (c) Cisplatin 2 mM: 0 – 48 h (d) Vitamin C 1 mM: 0 –12 h 1 Cisplatin 2 mM: 12– 60 h (e) Vitamin C 1 mM: 0 –24 h 1 Cisplatin 2 mM: 24 – h (f) Vitamin C 1 mM: 0 –36 h 1 Cisplatin 2 mM: 36 – 84 h. (B) Effect of high dose of vitamin C (1 mM) on cisplatin induced apoptosis (a) Cisplatin 50 mM: 0 – 48 h (b) Vitamin C 1 mM 1 Cisplatin 50 mM: 0 – 48 h (c) Cisplatin 50 mM: 24 –72 h (d) Vitamin C 1 mM: 0 – h 1 Cisplatin 50 mM: 24 –72 h. (C) Effect of 1 mM vitamin C priming on etoposide induced apoptosis (a) etoposide 2 mM: 0 – 48 h (b) vitamin C 1 mM: 0 –12 h 1 etoposide 2 mM: 12– 60 h (c) vitamin C 1 mM: 0 –24 h 1 etoposide 2 mM: 24 –72 h (d) vitamin C 1 mM: 0 – h 1 etoposide 2 mM: 36 – 84 h.
num complexes in a human ovarian cancer cell line. Mol. Phar- macol. 57, 503–511.