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Repositioning of anti-viral drugs as therapy for cervical cancer
Pharmacological Reports 68 (2016) 983–989
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Repositioning of anti-viral drugs as therapy for cervical cancer Sapna Sharmaa , Ruma Baksib , Milee Agarwala,b,* a Department of Pharmacology and Toxicology, NIPER-Ahmedabad, C/o B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Thaltej-Gandhinagar Highway, Ahmedabad 380054, Gujarat, India b Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Thaltej-Gandhinagar Highway, Ahmedabad 380054, Gujarat, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 September 2015 Received in revised form 16 May 2016 Accepted 16 May 2016 Available online xxx
Background: Increase in expression of eIF4E (Eukaryotic translation initiation factor 4E) protein is mediated by oncogenic proteins of Human Papilloma Virus (HPV). Increased expression of eIF4E plays an important role in HPV induced carcinogenesis. Ribavirin and Indinavir are known inhibitors of eIF4E activity. Methods: The effect of the drugs on HeLa cells was assessed by in vitro assays including cell viability using MTT and Neutral red assay, apoptotic potential using Caspase-3, Caspase-8 and Caspase-9 activity assays and MMP-2 and MMP-9 secretion by determination of Gelatinase activity. The in vivo effect of Ribavirin treatment on tumor volume was assessed in human xenograft in immunocompromised C57BL/6 mice. Results: In vitro analyses indicate that Ribavirin and Indinavir reduce viability of HeLa cells, induce apoptosis and decrease secretion of MMPs. Treatment with Ribavirin at a dose of 50 mg/kg and 100 mg/kg daily led to significant decrease in tumor volume in vivo. Conclusion: The study thus provides evidence that Ribavirin and Indinavir can be explored as therapy against HPV-18 induced cervical cancer. ã 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o.. All rights reserved.
Keywords: HeLa HPV Ribavirin Indinavir
Introduction Cervical cancer is most common cause of cancer related deaths among women worldwide [1,2]. About 86% of cervical cancer related deaths occur in the low and middle income group population of developing countries due to paucity of affordable medical facilities for prevention, diagnosis and treatment [2]. In India and many Asian countries, socio-cultural factors also hamper early diagnosis of the infection. Infection with Human Papilloma Virus (HPV) particularly HPV16 and HPV18 have been found in 99% of cervical cancers, thus making it the main causative agent for cervical cancer [3]. With the advent of low cost diagnostic tools and increased awareness with regard to HPV infection has decreased the number of cases and deaths related to cervical cancer [3,4]. Prophylactic vaccines against HPV have been developed that will
* Corresponding author at: B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Thaltej-Gandhinagar Highway, Ahmedabad 380054, Gujarat, India. E-mail addresses: [email protected], [email protected] (M. Agarwal).
potentially decrease the future incidences of cervical cancer [5]. However, these vaccines are effective for the HPV naïve population as these vaccines do not enhance the clearance of pre-existing HPV infection [5]. Current therapies for advanced cervical cancer include platinum based chemotherapy along with radiotherapy [2,6]. The response rate for cervical cancer is approximately 25% and even these responses are short-lived and thus have little or no effect on the cervical cancer survival [7]. Resistance of advanced and invasive cervical cancer to radiotherapy is responsible for decreased survival of cervical cancer patients [8]. Thus, there is a need for new strategies and therapeutic targets for better treatment of cervical cancer. Eukaryotic translation initiation factor 4E (eIF4E) is required for cap dependent translation and has oncogenic potential [9]. In normal cells, a minimal amount of eIF4E is required for cellular maintenance [9,10]. However, in cancer cells, increased expression of eIF4E has been reported to increase the translation of proteins related to malignancy [9,10]. Over expression of eIF4E has been reported in HPV induced cancerous lesions of cervix [11,12]. Recent research has provided the evidence that HPV E6 protein induces the expression of eIF4E and thus abrogating
http://dx.doi.org/10.1016/j.pharep.2016.05.007 1734-1140X/ ã 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o.. All rights reserved.
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eIF4E may reduce the malignant potential of HPV infection [11]. Ribavirin and Indinavir are anti-virals which are known to inhibit eIF4E activity [13]. The presnt study is based on the hypothesis that treatment with Ribavirin and Indinavir will inhibit the proliferation, migration and induce apoptosis in HPV infected cervical cancer cells. HeLa cell line which is derived from an adenocaricoma of cervix epithelia containing HPV-18 sequences was used to analyse the efficacy of Ribavirin and Indinavir against HPV induced cervical cancer. The purpose of this study was to assess the possible repositioning of the drugs already in clinical use and having the ability of inhibiting eIF4E activity for therapy against HPV induced cervical malignancy.
water/acetic acid (50:49:1) solution was used to dissolve the Neutral Red stained cells and color intensity was measured at 540 nm in a micro-plate reader. For MTT analysis, 104 cells/well were plated in 96 well plate and after 24 h of incubation, cells were treated with different concentrations of Indinavir, Ribavirin and Cisplatin. After continuous exposure of 48 h, the plates were analyzed for cell viability using MTT assay. Each assay was done at least three times and each time, every concentration was analyzed in triplicates. From the absorbance of each well, the absorbance of blank well was subtracted. Relative percent cell viability was calculated by taking the absorbance of the medium control well or vehicle control well to be 100%.
Materials and methods
MMPs (Gelatinase) activity determination
Chemicals and reagents
Activity of the gelatinases was estimated in the cell culture medium of the cells treated with different concentration of Indinavir, Ribavirin and Cisplatin as described previously [16]. Briefly, 105 cells/well were plated in 12 well plates. After 24 h, media was replaced with serum free media and cells were treated with different drug concentrations. The spent media was collected after 48 h of drug exposure and centrifuged at 1000g, 4 C for 5 min. The protein in the collected supernatant was then estimated by Bradford assay [17]. 50 mg of protein was used for each analysis and corresponding volume of supernatant was incubated with gelatin for 2 h. The quantity of gelatin remaining after incubation with media was determined using the heated Coomassie Brilliant Blue R-250, resulting in visible blue precipitate. The precipitates were collected by centrifugation and dimethyl sulfoxide was added to dissolve the dye for the precipitate and measurement of the absorbance at 630 nm was done. Appropriate controls were included and the entire assay was repeated thrice with each sample being assayed in triplicates. Relative percent enzyme inhibition was calculated with respect to control without any addition of supernatant.
Marketed preparations of Indinavir (Capsules, Mepro pharmaceuticals India), Ribavirin (Capsules, Cipla Ltd., India), Ketoconazole (Injections, Albatross pharmaceuticals India), cyclosporine (Capsules, Biocon Ltd., India) and ampoxin (Injections, Unichem laboratories India) were used in the study. Solvents such as chloroform, glacial acetic acid, isopropyl alcohol, methanol, dimethylsulfoxide and phenol were procured from Merck Limited, India and Qualigens Fine Chemicals, India. Chemicals like Calcium Chloride, Ethylene Diamine Tetraacetic acid (EDTA), Glycerol, Glucose, Potassium Acetate, Sodium Dodecyl Sulphate (SDS), Potassium Dihydrogen Phosphate, Dipotassium Phosphate, Sodium Acetate, Sodium Hydroxide, Tris Base, Cyclophosphamide were obtained from Sigma–Aldrich, India. Components of nutrient medium including Dulbecco’s Modified Eagle Media (DMEM), Fetal Bovine Serum (FBS), antibiotic and antimycotic preparations (for animal cell culture), Caspase activity assay kits were obtained from Life Technologies, India. Gelatin, 0.2% Trypsin EDTA solution, MTT and Neutral Red, Coomassie Brilliant Blue G-250 and Coomassie Brilliant Blue R-250 were obtained from HiMedia laboratories Pvt. Ltd., India. Cell culture HeLa cell line was procured from cell repository at National Centre for Cell Sciences (NCCS), Pune, India. The HeLa cells were placed into 25 cm2 tissue culture flasks and grown at 37 C under a humidified 5% CO2 atmosphere in 90% DMEM with 2 mM Lglutamine adjusted to contain 2.0 g/l sodium bicarbonate and supplemented with 10% fetal bovine serum, and penicillinstreptomycin (100 U/ml penicillin and 0.1 mg/ml streptomycin) and 250 ng/ml Amphotericin B. The cells were routinely sub cultured by treatment of Trypsin-EDTA solution and all the assays have been performed with the cells between the range 45– 60 passage numbers. Cell viability determinations The cytotoxicity of Indinavir and Ribavirin was tested against HeLa cells by the Neutral Red Uptake (NRU) assay and MTT assay as previously described [14,15]. For neutral red assay, 2000 cells/ well were plated in 96 well plates. After 24 h of incubation, cells were exposed to different concentrations of Indinavir and Ribavirin for 48 h in appropriate condition (5% (v/v) CO2 atmosphere at 37 C). Cisplatin was used as positive control and cell free control and medium control and vehicle controls were also included in each assay. After several washings with Phosphate Buffered Saline (PBS), cells were exposed to 0.0075% NR solution for 2 h in humidified atmosphere of 5% (v/v) CO2 at 37 C and then subsequently again washed with PBS. Ethanol/
Determination of Caspase activity Caspase-3, Caspase-8 and Caspase-9 activity was assessed using colorimetric Caspase activity kits (Life Technologies, India). The assays are based on the cleavage of Caspase specific substrate (DEVD-pNA), composed of the chromophore p-nitroanilide (pNA), and a synthetic tetrapeptide, DEVD (Asp-Glue-Val-Asp) for Caspase-3; IETD-pNA, composed of the chromophore, p-nitroanilide (pNA), and a synthetic tetrapeptide, IETD ((Ile-Glu-Thr-Asp) for Caspase-8; LEHD-pNA, composed of the chromophore, pnitroanilide (pNA), and a synthetic tetrapeptide, LEHD (Leu-GluHis-Asp) for Caspase-9). The cleavage by the Caspases releases a colorimetric product (pNA) that is estimated spectrophotometrically at 405 nm. Cell lysates of the treated and non-treated cells corresponding to 100 mg protein was used for the Caspase activity assays. The assays were performed as per the supplier’s instructions. Determination of anti-cancer potential in vivo Female C57BL/6 female mice weighing 20–25 g were obtained from Mahaveera Enterprises, Hyderabad (Registration number: 1661/PO/a/12/CPCSEA, Date: 26th September, 2013) and were housed in the animal house of Institute. Animal housing and handling were performed in accordance with Good Laboratory Practice (GLP) mentioned in CPCSEA guidelines. The protocol for the in vivo experimentation was approved by the Institutional Animal Ethics Committee (IAEC) (Protocol No. IAEC/2014/010). The human tumor xenograft model for HeLa cells was developed in immunocompromised C57BL/6 female mice as described
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previously [18]. Once the xenograft tumors reached the volume of 90–100 mm3, the mice were randomized into three groups (n = 6/group). The animals of first group I were administered 0.1 ml of PBS daily and the animals of group II and group III were administered 50 mg/kg/day and 100 mg/kg/day Ribavirin intraperitoneally (ip). The tumor volume was measured on alternate days using Vernier calipers. The tumors upon cessation of the treatment were excised, weighed, measured and then examined histopathologically. Statistical analysis All the data were analyzed by statistical software GraphPad Prism 5.01. Data are expressed as mean SEM (Standard error of the mean) or mean SD (Standard Deviation). Multi-group comparisons were performed through One-way ANOVA test, while pairwise was performed through t-test,p value <0.05 was considered to be significant. Results Ribavirin and Indinavir are cytotoxic to HeLa cells The effect of Ribavirin and Indinavir on the viability of HeLa cells was examined by MTT assay and NRU assay. The viability of HeLa cells was expressed as percent of untreated control cells. The 50% growth inhibition concentration (IC50) upon 48 h incubation with Cisplatin, Indinavir and Ribavirin as estimated by MTT assay and NRU assay was as described in Table 1. MTT and neutral red assay are based on different principles. MTT assay is based on reduction of the MTT reagent by the mitochondrial dehydrogenases and if the treatment itself can modulate cellular ROS (Reactive oxygen species), the observed results may vary [15]. Thus, NRU assay which is based on entrapment of the supravital dye neutral red in the lysosomes of the viable cells, can be used to reconfirm the results obtained by MTT assay [14]. Cisplatin is known to increase mitochondrial ROS [19]. However, there are no such reports for Indinavir and Ribavirin. There is significant difference in the IC50 obtained for Cisplatin, Ribavirin and Indinavir by both MTT and NRU assay (Fig. 1 and Table 1). The treatment of Ribavirin and Indinavir significantly decreased cell viability of HeLa cells. Ribavirin and Indinavir induces apoptosis in HeLa cells The aim of every anti-cancer therapy is to induce apoptosis in cancer cells [20]. Thus, induction of apoptosis in cancer cells is a sign of effective anti-cancer therapy. The pro-apoptotic potential of Indinavir and Ribavirin was assessed by estimation of Caspase3, Caspase-8 and Caspase-9 enzymes. Caspase-9 and Caspase8 are involved in the initiation of Caspase dependent apoptosis process and Caspase-3 is involved in formation of apoptotic bodies and is a central point for any apoptotic signaling pathway [21]. In normal physiological condition, caspases are present as zymogens in cells and do not have any proteolytic activity until they are activated. The presence of the activated Caspase-3,
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Caspase-8 and Caspase-9 in HeLa cells upon exposure to Cisplatin, Ribavirin and Indinavir was analyzed. After 8 h of treatment, Caspase-9 activity was significantly more in cells treated with Cisplatin (10 mg/ml), Indinavir (100 mg/ml) and Ribavirin (500 mg/ml) as compared to that of untreated HeLa cells (Fig. 2). Upon treatment for 16 h, Caspase-9 activity in HeLa cells treated with different drugs was not different from that observed in untreated HeLa cells (Fig. 2). However, the Caspase-8 activity was found to be significantly increased in the cells treated with different drugs at both timepoints of analysis (8 h and 16 h) except upon treatment with Cisplatin for 16 h (Fig. 2). Upon 8 h treatment, Caspase-3 activity was significantly increased in cells treated with Ribavirin (Fig. 2). Although, there was an increase in the levels of Caspase-3 activity in cells treated with Cisplatin and Indinavir, it was not significant. The increase in Caspase-3 activity was significant in cells treated with Indinavir and Ribavirin after 16 h treatment (Fig. 2). Indinvair, Cisplatin and Ribavirin induce the apoptosis by activation of both extrinsic (Caspase-8) and intrinsic pathway (Caspase-9) of apoptosis, although the increase in the activity of these caspases by different drugs is distinct. The activation of the Caspase-8 by the different drugs was more prominent and prolonged (Fig. 2). Activated Caspase-8 initiated the apoptotic pathway by activating the Caspase-3 leading to the cell death. Ribavirin and Indinavir treatment attenuates the MMP-2 and MMP9 activity Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9) have been linked to metastasis of cervical cancer cells [22,23]. The effect of the treatment of Indinavir and Ribavirin on the MMP-2 and MMP-9 activity was assayed using gelatin as substrate. Indinavir and Ribavirin treatment significantly reduces the MMP-2 and MMP-9 activity in the spent medium indicating their efficacy in attenuating migrating and invasion of HeLa cells (Fig. 3). In vivo Ribavirin treatment leads to tumor regression Immunocompromised mice (n = 18) having subcutaneous human tumor xenograft of HeLa cells of about 90–100 mm3 volume were randomly divided into three equal groups. Ribavirin was administered at a dose of 50 mg/kg/day (ip) and 100 mg/kg/ day (ip) for seven days to the animals of two treatment groups. The animals of the control group were administered 0.1 ml PBS ip daily. A significant decrease in tumor volume was observed in the animals of the treated groups with a significant more decrease in the animals being treated with 100 mg/kg/day Ribavirin (Fig. 4A). The tumor continued to increase in size in the animals of control group. At the end of the study, the remnant tumors were excised and weighed. The weight of the tumors excised from the animals of the treated groups was significantly less as compared to that of tumors removed from the animals of control group (Fig. 4B). Comparison of microphotographs of the skin tissue of mice and tumor revealed that the tumor was formed due to growth of HeLa cells (Fig. 4C).
Table 1 50% growth inhibition concentration (IC50) of different drugs for HeLa cells. Drug Name
Cisplatin (mM)
Indinavir (mM)
Ribavirin (mM)
IC50 (NRU assay) (Mean SEM)
161.33 0.132
2660 2.34
IC50 (MTT assay) (Mean SEM)
262.69 0.257
378.71 0.07 (by extrapolating) 365.4 2.01 (by extrapolating)
2254 4.35
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Fig. 1. The effect of the Ribavirin and Indinavir on the viability of HeLa cells was determined by MTT and Neutral Red Assay. The exposure time was 48 h. The results were calculated by taking the cell viability for the untreated sample as 100%. Each concentration was assayed in triplicates and each assay was repeated three times. Cisplatin was taken as positive control for the assays. The error bars indicate Standard error of the mean. (A) MTT assay (B) Neutral Red assay.
Discussion Cancer is a devastating disease that continues to be an immense disease burden to patients, human society as well as health care machinery. 15–20% of all human cancers are due to viral infections [19]. Viruses have been reported to reprogram host signaling pathways pertaining to cellular maintenance, genomic integrity and immune surveillance leading to sustenance of aberrant cells that initiates the development of malignancy [24]. Viruses also control the translational machinery of host cells in order to replicate themselves. It has been proved evidently that E6 protein increases the expression of eIF4E (a major player in cap-dependent translation). EIF4E has been implicated in increased translation of proteins related to malignancy [9]. Thus,
targeting eIF4E may lead to efficacious treatment for cervical cancer. Rather than synthesizing and evaluating newer molecules, it is more prudent to use approved drugs for different indications as it decreases the time required for the therapy to reach clinics [25,26]. Ribavirin and Indinavir have been reported to inhibit the activity of eIF4E and are being clinically used to treat viral infections. Ribavirin has been explored as treatment for breast cancer and acute lymphoid leukemia [27,28]. Indinavir has been reported to have anti-metastatic potential in mouse model of hepato carcinoma with no effect on cell proliferation [29]. In addition, previous screening report has found Indinavir to be a non-effective therapy for HPV induced cervical cancer [30].
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Fig. 2. Determination of Caspase-3, Caspase-8 and Caspase-9 activity in HeLa Cells upon treatment with Indinavir and Ribavirin. 10 mg/ml of Cisplatin was used as a positive control. The cells were assayed for the Caspase activity after 8 h and 16 h of treatment. Each concentration was assayed in triplicates. The error bars indicate Standard Deviation (SD). **Indicates p value <0.0001 upon comparison with Untreated Control.
However, in vitro analyses of our study revealed that Ribavirin and Indinavir effectively induce cell death through apoptosis induction in HeLa cells. In addition, they also reduce the metastatic potential by decreasing the activity of MMP-2 and/MMP-9. In tumor xenograft model, seven day treatment with Ribavirin at a dose of 50 mg/kg/day and 100 mg/kg/day led to regression of the tumor size by 65% and 90%, thus indicating that this strategy
may be useful as treatment for cervical cancer. The doses used in the study will convert to human dose of 250 mg/day and 500 mg/day for a healthy individual of 60 kg [31]. The bioavailability of Ribavirin by oral dose is 64% and recommended human dose by this route is 600 mg/day for a 60 kg adult, thus effective drug amounts to 384 mg/day [32,33]. Keeping that the bioavailability of Ribavirin by intraperitoneal route is almost equal
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Fig. 3. Effect of Ribavirin and Indinavir on MMP-2 and MMP-9 activity. The gelatinase activity in the spent medium was estimated after 48 h of exposure. The inhibition of the gelatinase activity was depicted by taking the absorbance of the sample without media as 100%. Each concentration was assayed in triplicates and each assay was repeated three times. Cisplatin was taken as positive control for the assays. The error bars indicate Standard error of the mean (SEM). **Indicates p value <0.0001 upon comparison with Untreated Control.
Fig. 4. In vivo study to assess the anti-tumoral effect of Ribavirin in human xenograft model. Mice was divided into six groups with n = 6 per group. (A) Tumor volume measured by Vernier calipers. The data is represented as mean tumor volume SD (mm3) for different group of animals at different days during treatment regimen. (B) The mean weight (mg) of the tumors excised from the animals of different groups after the completion of the treat regimen. The error bars indicate SD. (C) Microphotographs of Hematoxylin and eosin stained sections of subcutaneous skin and tumor at 100 magnification. **Indicates p value <0.0001 upon comparison with Untreated Control group. # Indicates p value <0.005 and ## indicates p value <0.0001 upon comparison with 50 mg/kg treated group.
to that of intravenous route, the lower dose used in the in vivo study i.e. 250 mg/day can be achieved by the prescribed clinical administration of Ribavirin orally. In vivo efficacy of Indinavir remains to be tested. Topical formulations of Ribavirin or Indinavir that can be directly applied to the affected cervical
area or HPV infected premalignant lesions can be explored as therapy. The same therapeutic strategies can be also tested for their efficacy for squamous cancer of oral cavity caused due to HPV infection.
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Conflict of interest None. Funding body The manuscript is partially based on M.S. Pharm. Dissertation project of Ms. Sapna Sharma, a student of NIPER-Ahmedabad. The authors acknowledge NIPER-Ahmedabad and B. V. Patel PERD Centre, Ahmedabad for institutional support. Acknowledgements We acknowledge the support of the staff and students of the Pharmacology and Toxicology department of NIPER-Ahmedabad and B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Ahmedabad for their assistance during our study. The Institutional Communication Number for the manuscript is NIPER-A/59/07/2015. References [1] Aswathy S, Reshma J, Avani D. Epidemiology of cervical cancer with special focus on India. Int J Women’s Health 2015;40:5 10.2147/IJWH.S50001. [2] Denny L. Cervical cancer prevention and treatment. Discov Med 2012;14:125– 31. [3] Schiffman M, Wentzensen N, Wacholder S, Kinney W, Gage JC, Castle PE. Human papillomavirus testing in the prevention of cervical cancer. J Natl Cancer Inst 2011;103:368–83, doi:http://dx.doi.org/10.1093/jnci/djq562. [4] Gaffikin L, McGrath JA, Arbyn M, Blumenthal PD. Visual inspection with acetic acid as a cervical cancer test: accuracy validated using latent class analysis. BMC Med Res Methodol 2007;7:36, doi:http://dx.doi.org/10.1186/1471-22887-36. [5] Castle PE, Zhao F-H. Population effectiveness, not efficacy should decide who gets vaccinated against human papillomavirus via publicly funded programs. J Infect Dis 2011;204:335–7, doi:http://dx.doi.org/10.1093/infdis/jir287. [6] Rosa DD, Medeiros LR, Edelweiss MI, Pohlmann PR, Stein AT. Adjuvant platinum-based chemotherapy for early stage cervical cancer. Cochrane Database Syst Rev 2012, doi:http://dx.doi.org/10.1002/14651858.cd005342. pub3 ;6:CD005342. [7] Alberts DS, Garcia D, Mason-Liddil N. Cisplatin in advanced cancer of the cervix: an update. Semin Oncol 1991;18:11–24. [8] Li X, Meng Q, Fan S. Adenovirus-mediated expression of UHRF1 reduces the radiosensitivity of cervical cancer HeLa cells to g-irradiation. Acta Pharmacol Sin 2009;30:458–66, doi:http://dx.doi.org/10.1038/aps.2009.18. [9] Mamane Y, Petroulakis E, Rong L, Yoshida K, Ler LW, Sonenberg N. eIF4E—from translation to transformation. Oncogene 2004;23:3172–9, doi:http://dx.doi. org/10.1038/sj.onc.1207549. [10] Carroll M, Borden KLB. The oncogene eIF4E: using biochemical insights to target cancer. J Interferon Cytokine Res 2013;33:227–38, doi:http://dx.doi.org/ 10.1089/jir.2012.0142. [11] Wang S, Pang T, Gao M, Kang H, Ding W, Sun X, et al. HPV E6 induces eIF4E transcription to promote the proliferation and migration of cervical cancer. FEBS Lett 2013;587:690–7, doi:http://dx.doi.org/10.1016/j.febslet.2013.01.042. [12] Oh K-J, Kalinina A, Park N-H, Bagchi S. Deregulation of eIF4E: 4E-BP1 in differentiated human papillomavirus-containing cells leads to high levels of expression of the E7 oncoprotein. J Virol 2006;80:7079–88, doi:http://dx.doi. org/10.1128/JVI.02380-05.
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Repositioning of anti-viral drugs as therapy for cervical cancer Sapna Sharmaa , Ruma Baksib , Milee Agarwala,b,* a Department of Pharmacology and Toxicology, NIPER-Ahmedabad, C/o B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Thaltej-Gandhinagar Highway, Ahmedabad 380054, Gujarat, India b Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Thaltej-Gandhinagar Highway, Ahmedabad 380054, Gujarat, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 September 2015 Received in revised form 16 May 2016 Accepted 16 May 2016 Available online xxx
Background: Increase in expression of eIF4E (Eukaryotic translation initiation factor 4E) protein is mediated by oncogenic proteins of Human Papilloma Virus (HPV). Increased expression of eIF4E plays an important role in HPV induced carcinogenesis. Ribavirin and Indinavir are known inhibitors of eIF4E activity. Methods: The effect of the drugs on HeLa cells was assessed by in vitro assays including cell viability using MTT and Neutral red assay, apoptotic potential using Caspase-3, Caspase-8 and Caspase-9 activity assays and MMP-2 and MMP-9 secretion by determination of Gelatinase activity. The in vivo effect of Ribavirin treatment on tumor volume was assessed in human xenograft in immunocompromised C57BL/6 mice. Results: In vitro analyses indicate that Ribavirin and Indinavir reduce viability of HeLa cells, induce apoptosis and decrease secretion of MMPs. Treatment with Ribavirin at a dose of 50 mg/kg and 100 mg/kg daily led to significant decrease in tumor volume in vivo. Conclusion: The study thus provides evidence that Ribavirin and Indinavir can be explored as therapy against HPV-18 induced cervical cancer. ã 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o.. All rights reserved.
Keywords: HeLa HPV Ribavirin Indinavir
Introduction Cervical cancer is most common cause of cancer related deaths among women worldwide [1,2]. About 86% of cervical cancer related deaths occur in the low and middle income group population of developing countries due to paucity of affordable medical facilities for prevention, diagnosis and treatment [2]. In India and many Asian countries, socio-cultural factors also hamper early diagnosis of the infection. Infection with Human Papilloma Virus (HPV) particularly HPV16 and HPV18 have been found in 99% of cervical cancers, thus making it the main causative agent for cervical cancer [3]. With the advent of low cost diagnostic tools and increased awareness with regard to HPV infection has decreased the number of cases and deaths related to cervical cancer [3,4]. Prophylactic vaccines against HPV have been developed that will
* Corresponding author at: B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Thaltej, Thaltej-Gandhinagar Highway, Ahmedabad 380054, Gujarat, India. E-mail addresses: [email protected], [email protected] (M. Agarwal).
potentially decrease the future incidences of cervical cancer [5]. However, these vaccines are effective for the HPV naïve population as these vaccines do not enhance the clearance of pre-existing HPV infection [5]. Current therapies for advanced cervical cancer include platinum based chemotherapy along with radiotherapy [2,6]. The response rate for cervical cancer is approximately 25% and even these responses are short-lived and thus have little or no effect on the cervical cancer survival [7]. Resistance of advanced and invasive cervical cancer to radiotherapy is responsible for decreased survival of cervical cancer patients [8]. Thus, there is a need for new strategies and therapeutic targets for better treatment of cervical cancer. Eukaryotic translation initiation factor 4E (eIF4E) is required for cap dependent translation and has oncogenic potential [9]. In normal cells, a minimal amount of eIF4E is required for cellular maintenance [9,10]. However, in cancer cells, increased expression of eIF4E has been reported to increase the translation of proteins related to malignancy [9,10]. Over expression of eIF4E has been reported in HPV induced cancerous lesions of cervix [11,12]. Recent research has provided the evidence that HPV E6 protein induces the expression of eIF4E and thus abrogating
http://dx.doi.org/10.1016/j.pharep.2016.05.007 1734-1140X/ ã 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o.. All rights reserved.
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eIF4E may reduce the malignant potential of HPV infection [11]. Ribavirin and Indinavir are anti-virals which are known to inhibit eIF4E activity [13]. The presnt study is based on the hypothesis that treatment with Ribavirin and Indinavir will inhibit the proliferation, migration and induce apoptosis in HPV infected cervical cancer cells. HeLa cell line which is derived from an adenocaricoma of cervix epithelia containing HPV-18 sequences was used to analyse the efficacy of Ribavirin and Indinavir against HPV induced cervical cancer. The purpose of this study was to assess the possible repositioning of the drugs already in clinical use and having the ability of inhibiting eIF4E activity for therapy against HPV induced cervical malignancy.
water/acetic acid (50:49:1) solution was used to dissolve the Neutral Red stained cells and color intensity was measured at 540 nm in a micro-plate reader. For MTT analysis, 104 cells/well were plated in 96 well plate and after 24 h of incubation, cells were treated with different concentrations of Indinavir, Ribavirin and Cisplatin. After continuous exposure of 48 h, the plates were analyzed for cell viability using MTT assay. Each assay was done at least three times and each time, every concentration was analyzed in triplicates. From the absorbance of each well, the absorbance of blank well was subtracted. Relative percent cell viability was calculated by taking the absorbance of the medium control well or vehicle control well to be 100%.
Materials and methods
MMPs (Gelatinase) activity determination
Chemicals and reagents
Activity of the gelatinases was estimated in the cell culture medium of the cells treated with different concentration of Indinavir, Ribavirin and Cisplatin as described previously [16]. Briefly, 105 cells/well were plated in 12 well plates. After 24 h, media was replaced with serum free media and cells were treated with different drug concentrations. The spent media was collected after 48 h of drug exposure and centrifuged at 1000g, 4 C for 5 min. The protein in the collected supernatant was then estimated by Bradford assay [17]. 50 mg of protein was used for each analysis and corresponding volume of supernatant was incubated with gelatin for 2 h. The quantity of gelatin remaining after incubation with media was determined using the heated Coomassie Brilliant Blue R-250, resulting in visible blue precipitate. The precipitates were collected by centrifugation and dimethyl sulfoxide was added to dissolve the dye for the precipitate and measurement of the absorbance at 630 nm was done. Appropriate controls were included and the entire assay was repeated thrice with each sample being assayed in triplicates. Relative percent enzyme inhibition was calculated with respect to control without any addition of supernatant.
Marketed preparations of Indinavir (Capsules, Mepro pharmaceuticals India), Ribavirin (Capsules, Cipla Ltd., India), Ketoconazole (Injections, Albatross pharmaceuticals India), cyclosporine (Capsules, Biocon Ltd., India) and ampoxin (Injections, Unichem laboratories India) were used in the study. Solvents such as chloroform, glacial acetic acid, isopropyl alcohol, methanol, dimethylsulfoxide and phenol were procured from Merck Limited, India and Qualigens Fine Chemicals, India. Chemicals like Calcium Chloride, Ethylene Diamine Tetraacetic acid (EDTA), Glycerol, Glucose, Potassium Acetate, Sodium Dodecyl Sulphate (SDS), Potassium Dihydrogen Phosphate, Dipotassium Phosphate, Sodium Acetate, Sodium Hydroxide, Tris Base, Cyclophosphamide were obtained from Sigma–Aldrich, India. Components of nutrient medium including Dulbecco’s Modified Eagle Media (DMEM), Fetal Bovine Serum (FBS), antibiotic and antimycotic preparations (for animal cell culture), Caspase activity assay kits were obtained from Life Technologies, India. Gelatin, 0.2% Trypsin EDTA solution, MTT and Neutral Red, Coomassie Brilliant Blue G-250 and Coomassie Brilliant Blue R-250 were obtained from HiMedia laboratories Pvt. Ltd., India. Cell culture HeLa cell line was procured from cell repository at National Centre for Cell Sciences (NCCS), Pune, India. The HeLa cells were placed into 25 cm2 tissue culture flasks and grown at 37 C under a humidified 5% CO2 atmosphere in 90% DMEM with 2 mM Lglutamine adjusted to contain 2.0 g/l sodium bicarbonate and supplemented with 10% fetal bovine serum, and penicillinstreptomycin (100 U/ml penicillin and 0.1 mg/ml streptomycin) and 250 ng/ml Amphotericin B. The cells were routinely sub cultured by treatment of Trypsin-EDTA solution and all the assays have been performed with the cells between the range 45– 60 passage numbers. Cell viability determinations The cytotoxicity of Indinavir and Ribavirin was tested against HeLa cells by the Neutral Red Uptake (NRU) assay and MTT assay as previously described [14,15]. For neutral red assay, 2000 cells/ well were plated in 96 well plates. After 24 h of incubation, cells were exposed to different concentrations of Indinavir and Ribavirin for 48 h in appropriate condition (5% (v/v) CO2 atmosphere at 37 C). Cisplatin was used as positive control and cell free control and medium control and vehicle controls were also included in each assay. After several washings with Phosphate Buffered Saline (PBS), cells were exposed to 0.0075% NR solution for 2 h in humidified atmosphere of 5% (v/v) CO2 at 37 C and then subsequently again washed with PBS. Ethanol/
Determination of Caspase activity Caspase-3, Caspase-8 and Caspase-9 activity was assessed using colorimetric Caspase activity kits (Life Technologies, India). The assays are based on the cleavage of Caspase specific substrate (DEVD-pNA), composed of the chromophore p-nitroanilide (pNA), and a synthetic tetrapeptide, DEVD (Asp-Glue-Val-Asp) for Caspase-3; IETD-pNA, composed of the chromophore, p-nitroanilide (pNA), and a synthetic tetrapeptide, IETD ((Ile-Glu-Thr-Asp) for Caspase-8; LEHD-pNA, composed of the chromophore, pnitroanilide (pNA), and a synthetic tetrapeptide, LEHD (Leu-GluHis-Asp) for Caspase-9). The cleavage by the Caspases releases a colorimetric product (pNA) that is estimated spectrophotometrically at 405 nm. Cell lysates of the treated and non-treated cells corresponding to 100 mg protein was used for the Caspase activity assays. The assays were performed as per the supplier’s instructions. Determination of anti-cancer potential in vivo Female C57BL/6 female mice weighing 20–25 g were obtained from Mahaveera Enterprises, Hyderabad (Registration number: 1661/PO/a/12/CPCSEA, Date: 26th September, 2013) and were housed in the animal house of Institute. Animal housing and handling were performed in accordance with Good Laboratory Practice (GLP) mentioned in CPCSEA guidelines. The protocol for the in vivo experimentation was approved by the Institutional Animal Ethics Committee (IAEC) (Protocol No. IAEC/2014/010). The human tumor xenograft model for HeLa cells was developed in immunocompromised C57BL/6 female mice as described
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previously [18]. Once the xenograft tumors reached the volume of 90–100 mm3, the mice were randomized into three groups (n = 6/group). The animals of first group I were administered 0.1 ml of PBS daily and the animals of group II and group III were administered 50 mg/kg/day and 100 mg/kg/day Ribavirin intraperitoneally (ip). The tumor volume was measured on alternate days using Vernier calipers. The tumors upon cessation of the treatment were excised, weighed, measured and then examined histopathologically. Statistical analysis All the data were analyzed by statistical software GraphPad Prism 5.01. Data are expressed as mean SEM (Standard error of the mean) or mean SD (Standard Deviation). Multi-group comparisons were performed through One-way ANOVA test, while pairwise was performed through t-test,p value <0.05 was considered to be significant. Results Ribavirin and Indinavir are cytotoxic to HeLa cells The effect of Ribavirin and Indinavir on the viability of HeLa cells was examined by MTT assay and NRU assay. The viability of HeLa cells was expressed as percent of untreated control cells. The 50% growth inhibition concentration (IC50) upon 48 h incubation with Cisplatin, Indinavir and Ribavirin as estimated by MTT assay and NRU assay was as described in Table 1. MTT and neutral red assay are based on different principles. MTT assay is based on reduction of the MTT reagent by the mitochondrial dehydrogenases and if the treatment itself can modulate cellular ROS (Reactive oxygen species), the observed results may vary [15]. Thus, NRU assay which is based on entrapment of the supravital dye neutral red in the lysosomes of the viable cells, can be used to reconfirm the results obtained by MTT assay [14]. Cisplatin is known to increase mitochondrial ROS [19]. However, there are no such reports for Indinavir and Ribavirin. There is significant difference in the IC50 obtained for Cisplatin, Ribavirin and Indinavir by both MTT and NRU assay (Fig. 1 and Table 1). The treatment of Ribavirin and Indinavir significantly decreased cell viability of HeLa cells. Ribavirin and Indinavir induces apoptosis in HeLa cells The aim of every anti-cancer therapy is to induce apoptosis in cancer cells [20]. Thus, induction of apoptosis in cancer cells is a sign of effective anti-cancer therapy. The pro-apoptotic potential of Indinavir and Ribavirin was assessed by estimation of Caspase3, Caspase-8 and Caspase-9 enzymes. Caspase-9 and Caspase8 are involved in the initiation of Caspase dependent apoptosis process and Caspase-3 is involved in formation of apoptotic bodies and is a central point for any apoptotic signaling pathway [21]. In normal physiological condition, caspases are present as zymogens in cells and do not have any proteolytic activity until they are activated. The presence of the activated Caspase-3,
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Caspase-8 and Caspase-9 in HeLa cells upon exposure to Cisplatin, Ribavirin and Indinavir was analyzed. After 8 h of treatment, Caspase-9 activity was significantly more in cells treated with Cisplatin (10 mg/ml), Indinavir (100 mg/ml) and Ribavirin (500 mg/ml) as compared to that of untreated HeLa cells (Fig. 2). Upon treatment for 16 h, Caspase-9 activity in HeLa cells treated with different drugs was not different from that observed in untreated HeLa cells (Fig. 2). However, the Caspase-8 activity was found to be significantly increased in the cells treated with different drugs at both timepoints of analysis (8 h and 16 h) except upon treatment with Cisplatin for 16 h (Fig. 2). Upon 8 h treatment, Caspase-3 activity was significantly increased in cells treated with Ribavirin (Fig. 2). Although, there was an increase in the levels of Caspase-3 activity in cells treated with Cisplatin and Indinavir, it was not significant. The increase in Caspase-3 activity was significant in cells treated with Indinavir and Ribavirin after 16 h treatment (Fig. 2). Indinvair, Cisplatin and Ribavirin induce the apoptosis by activation of both extrinsic (Caspase-8) and intrinsic pathway (Caspase-9) of apoptosis, although the increase in the activity of these caspases by different drugs is distinct. The activation of the Caspase-8 by the different drugs was more prominent and prolonged (Fig. 2). Activated Caspase-8 initiated the apoptotic pathway by activating the Caspase-3 leading to the cell death. Ribavirin and Indinavir treatment attenuates the MMP-2 and MMP9 activity Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9) have been linked to metastasis of cervical cancer cells [22,23]. The effect of the treatment of Indinavir and Ribavirin on the MMP-2 and MMP-9 activity was assayed using gelatin as substrate. Indinavir and Ribavirin treatment significantly reduces the MMP-2 and MMP-9 activity in the spent medium indicating their efficacy in attenuating migrating and invasion of HeLa cells (Fig. 3). In vivo Ribavirin treatment leads to tumor regression Immunocompromised mice (n = 18) having subcutaneous human tumor xenograft of HeLa cells of about 90–100 mm3 volume were randomly divided into three equal groups. Ribavirin was administered at a dose of 50 mg/kg/day (ip) and 100 mg/kg/ day (ip) for seven days to the animals of two treatment groups. The animals of the control group were administered 0.1 ml PBS ip daily. A significant decrease in tumor volume was observed in the animals of the treated groups with a significant more decrease in the animals being treated with 100 mg/kg/day Ribavirin (Fig. 4A). The tumor continued to increase in size in the animals of control group. At the end of the study, the remnant tumors were excised and weighed. The weight of the tumors excised from the animals of the treated groups was significantly less as compared to that of tumors removed from the animals of control group (Fig. 4B). Comparison of microphotographs of the skin tissue of mice and tumor revealed that the tumor was formed due to growth of HeLa cells (Fig. 4C).
Table 1 50% growth inhibition concentration (IC50) of different drugs for HeLa cells. Drug Name
Cisplatin (mM)
Indinavir (mM)
Ribavirin (mM)
IC50 (NRU assay) (Mean SEM)
161.33 0.132
2660 2.34
IC50 (MTT assay) (Mean SEM)
262.69 0.257
378.71 0.07 (by extrapolating) 365.4 2.01 (by extrapolating)
2254 4.35
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Fig. 1. The effect of the Ribavirin and Indinavir on the viability of HeLa cells was determined by MTT and Neutral Red Assay. The exposure time was 48 h. The results were calculated by taking the cell viability for the untreated sample as 100%. Each concentration was assayed in triplicates and each assay was repeated three times. Cisplatin was taken as positive control for the assays. The error bars indicate Standard error of the mean. (A) MTT assay (B) Neutral Red assay.
Discussion Cancer is a devastating disease that continues to be an immense disease burden to patients, human society as well as health care machinery. 15–20% of all human cancers are due to viral infections [19]. Viruses have been reported to reprogram host signaling pathways pertaining to cellular maintenance, genomic integrity and immune surveillance leading to sustenance of aberrant cells that initiates the development of malignancy [24]. Viruses also control the translational machinery of host cells in order to replicate themselves. It has been proved evidently that E6 protein increases the expression of eIF4E (a major player in cap-dependent translation). EIF4E has been implicated in increased translation of proteins related to malignancy [9]. Thus,
targeting eIF4E may lead to efficacious treatment for cervical cancer. Rather than synthesizing and evaluating newer molecules, it is more prudent to use approved drugs for different indications as it decreases the time required for the therapy to reach clinics [25,26]. Ribavirin and Indinavir have been reported to inhibit the activity of eIF4E and are being clinically used to treat viral infections. Ribavirin has been explored as treatment for breast cancer and acute lymphoid leukemia [27,28]. Indinavir has been reported to have anti-metastatic potential in mouse model of hepato carcinoma with no effect on cell proliferation [29]. In addition, previous screening report has found Indinavir to be a non-effective therapy for HPV induced cervical cancer [30].
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Fig. 2. Determination of Caspase-3, Caspase-8 and Caspase-9 activity in HeLa Cells upon treatment with Indinavir and Ribavirin. 10 mg/ml of Cisplatin was used as a positive control. The cells were assayed for the Caspase activity after 8 h and 16 h of treatment. Each concentration was assayed in triplicates. The error bars indicate Standard Deviation (SD). **Indicates p value <0.0001 upon comparison with Untreated Control.
However, in vitro analyses of our study revealed that Ribavirin and Indinavir effectively induce cell death through apoptosis induction in HeLa cells. In addition, they also reduce the metastatic potential by decreasing the activity of MMP-2 and/MMP-9. In tumor xenograft model, seven day treatment with Ribavirin at a dose of 50 mg/kg/day and 100 mg/kg/day led to regression of the tumor size by 65% and 90%, thus indicating that this strategy
may be useful as treatment for cervical cancer. The doses used in the study will convert to human dose of 250 mg/day and 500 mg/day for a healthy individual of 60 kg [31]. The bioavailability of Ribavirin by oral dose is 64% and recommended human dose by this route is 600 mg/day for a 60 kg adult, thus effective drug amounts to 384 mg/day [32,33]. Keeping that the bioavailability of Ribavirin by intraperitoneal route is almost equal
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Fig. 3. Effect of Ribavirin and Indinavir on MMP-2 and MMP-9 activity. The gelatinase activity in the spent medium was estimated after 48 h of exposure. The inhibition of the gelatinase activity was depicted by taking the absorbance of the sample without media as 100%. Each concentration was assayed in triplicates and each assay was repeated three times. Cisplatin was taken as positive control for the assays. The error bars indicate Standard error of the mean (SEM). **Indicates p value <0.0001 upon comparison with Untreated Control.
Fig. 4. In vivo study to assess the anti-tumoral effect of Ribavirin in human xenograft model. Mice was divided into six groups with n = 6 per group. (A) Tumor volume measured by Vernier calipers. The data is represented as mean tumor volume SD (mm3) for different group of animals at different days during treatment regimen. (B) The mean weight (mg) of the tumors excised from the animals of different groups after the completion of the treat regimen. The error bars indicate SD. (C) Microphotographs of Hematoxylin and eosin stained sections of subcutaneous skin and tumor at 100 magnification. **Indicates p value <0.0001 upon comparison with Untreated Control group. # Indicates p value <0.005 and ## indicates p value <0.0001 upon comparison with 50 mg/kg treated group.
to that of intravenous route, the lower dose used in the in vivo study i.e. 250 mg/day can be achieved by the prescribed clinical administration of Ribavirin orally. In vivo efficacy of Indinavir remains to be tested. Topical formulations of Ribavirin or Indinavir that can be directly applied to the affected cervical
area or HPV infected premalignant lesions can be explored as therapy. The same therapeutic strategies can be also tested for their efficacy for squamous cancer of oral cavity caused due to HPV infection.
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Conflict of interest None. Funding body The manuscript is partially based on M.S. Pharm. Dissertation project of Ms. Sapna Sharma, a student of NIPER-Ahmedabad. The authors acknowledge NIPER-Ahmedabad and B. V. Patel PERD Centre, Ahmedabad for institutional support. Acknowledgements We acknowledge the support of the staff and students of the Pharmacology and Toxicology department of NIPER-Ahmedabad and B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Ahmedabad for their assistance during our study. The Institutional Communication Number for the manuscript is NIPER-A/59/07/2015. References [1] Aswathy S, Reshma J, Avani D. Epidemiology of cervical cancer with special focus on India. Int J Women’s Health 2015;40:5 10.2147/IJWH.S50001. [2] Denny L. Cervical cancer prevention and treatment. Discov Med 2012;14:125– 31. [3] Schiffman M, Wentzensen N, Wacholder S, Kinney W, Gage JC, Castle PE. Human papillomavirus testing in the prevention of cervical cancer. J Natl Cancer Inst 2011;103:368–83, doi:http://dx.doi.org/10.1093/jnci/djq562. [4] Gaffikin L, McGrath JA, Arbyn M, Blumenthal PD. Visual inspection with acetic acid as a cervical cancer test: accuracy validated using latent class analysis. BMC Med Res Methodol 2007;7:36, doi:http://dx.doi.org/10.1186/1471-22887-36. [5] Castle PE, Zhao F-H. Population effectiveness, not efficacy should decide who gets vaccinated against human papillomavirus via publicly funded programs. J Infect Dis 2011;204:335–7, doi:http://dx.doi.org/10.1093/infdis/jir287. [6] Rosa DD, Medeiros LR, Edelweiss MI, Pohlmann PR, Stein AT. Adjuvant platinum-based chemotherapy for early stage cervical cancer. Cochrane Database Syst Rev 2012, doi:http://dx.doi.org/10.1002/14651858.cd005342. pub3 ;6:CD005342. [7] Alberts DS, Garcia D, Mason-Liddil N. Cisplatin in advanced cancer of the cervix: an update. Semin Oncol 1991;18:11–24. [8] Li X, Meng Q, Fan S. Adenovirus-mediated expression of UHRF1 reduces the radiosensitivity of cervical cancer HeLa cells to g-irradiation. Acta Pharmacol Sin 2009;30:458–66, doi:http://dx.doi.org/10.1038/aps.2009.18. [9] Mamane Y, Petroulakis E, Rong L, Yoshida K, Ler LW, Sonenberg N. eIF4E—from translation to transformation. Oncogene 2004;23:3172–9, doi:http://dx.doi. org/10.1038/sj.onc.1207549. [10] Carroll M, Borden KLB. The oncogene eIF4E: using biochemical insights to target cancer. J Interferon Cytokine Res 2013;33:227–38, doi:http://dx.doi.org/ 10.1089/jir.2012.0142. [11] Wang S, Pang T, Gao M, Kang H, Ding W, Sun X, et al. HPV E6 induces eIF4E transcription to promote the proliferation and migration of cervical cancer. FEBS Lett 2013;587:690–7, doi:http://dx.doi.org/10.1016/j.febslet.2013.01.042. [12] Oh K-J, Kalinina A, Park N-H, Bagchi S. Deregulation of eIF4E: 4E-BP1 in differentiated human papillomavirus-containing cells leads to high levels of expression of the E7 oncoprotein. J Virol 2006;80:7079–88, doi:http://dx.doi. org/10.1128/JVI.02380-05.
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