Heparin (GAG-hed) inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding
© Villanueva et al; licensee BioMed Central Ltd. 2006
Received: 12 May 2006
Accepted: 31 August 2006
Published: 31 August 2006
High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line.
Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA.
We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR.
Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data suggest that GAG-hed could have antitumoral and antiviral activity mainly by inhibiting AP1 binding to the HPV18-LCR.
Cervical cancer represents the second most frequent malignant tumour found in women worldwide, with an estimated frequency of approximately 440,000 new cases per year, corresponding to about 5.8% of global cancer incidence . In countries like Mexico, cervical carcinoma stands as the leading cause of death among the female population, with 14 deaths per 100,000 women (15 years old or more), representing 34% of all new female cancer cases reported [2, 3]. Human papillomaviruses (HPVs), especially the high risk types 16 and 18, have been identified as causative agents of at least 90% of cervical cancer cases and are also linked to more than 50% of other anogenital cancers . The HPV genome consists of around 8000 base pairs (bp) of closed-circular double-stranded DNA containing up to nine genes, functionally divided into three regions: a long control region (LCR) covering about 10% of the genome, and early (E) and late (L) regions . The regulation of viral gene expression is complex and is controlled by multiple cellular and viral transcription factors. Most of the regulation occurs within the LCR, which varies substantially in nucleotide composition between individual HPV types. Within the LCR, cis-active elements regulate transcription of the E6/E7 genes, which represent the transforming genes for immortalization and for maintenance of the malignant phenotype in HPV-positive cervical cancer cells [5–7].
A number of cellular transcription factors, such as NF1, AP1, KRF1, Oct1, SP1, YY1, and the glucocorticoid receptor, have been shown to bind and regulate HPV18-LCR activity [8–15]. AP1 represents a key regulatory protein in the maintenance of E6/E7 gene expression in almost all HPV types hitherto investigated [14, 16]. HPV18-LCR contains two identical AP1 binding sites (TGACTAA) in opposite orientations, one located in the promoter (nucleotides 7792–7798) and the other one in the enhancer (nucleotides 7607–7613) . Both sites are essential for HPV18 transcription from the early P105 promoter [8, 13, 16, 18] and AP1 transactivation is required for tumour promotion in vivo . AP1 also appears to be involved in negative regulation of HPV transcription, since treatment of HPV16 immortalized human keratinocytes with the anti-oxidant pyrrolidine-dithio-carbamate (PDTC) selectively reduced the amount of viral mRNA by blocking transcription initiation, an effect that is profoundly associated with alterations of the AP1 heterodimerization pattern . Therefore, there is considerable interest in identifying compounds able to down-regulate AP1 activity for the treatment of HPV related malignant lesions.
Glycosaminoglycans (GAGs) are unbranched polysaccharide chains composed of repeated disaccharide sequences that consist of sulphate groups in various positions ; these groups give the GAG chains a net negative charge. In 1989, Regelson reported that polyanionic substances such as heparin, a member of the GAG group, are tumour inhibitors . This effect may result from the binding of anionic heparins to a wide range of proteins and molecules, thus affecting their biological activities. As a consequence, heparins have a wide variety of biological properties other than their anticoagulant effects, and those properties may interfere with the malignant processes . Heparin can affect proliferation, migration, and invasiveness of cancer cells in various cell types, including those derived from epithelial cells [24–27]. It has been shown that heparins selectively inhibit the phosphorylation of mitogen activated protein kinases [28, 29], and there is direct evidence that heparin penetrates into the cell nucleus and causes inhibition of Fos-Jun/AP1 activity as a direct result of its nuclear localization in HeLa cells . Additionally, heparin and the heparinoids dextran sulfate and pentosan polysulfate, potently and selectively inhibit the in vitro replication of herpes simplex virus 2 (HSV-2), cytomegalovirus (CMV), AIDS virus (HIV), vesicular stomatitis viruses, respiratory syncytial, influenza type A, Sendai, Junin, and Tacaribe viruses [31–35]. The growth of rat vascular smooth muscle cells transformed by SV40 was also inhibited by heparin .
In this work, we use the heparin analogue GAG-hed to determine its effect on HPV18 early expression in two murine models and in cultured HeLa cells. Our results demonstrate that GAG-hed inhibits tumoral growth in a model generated using HeLa cells in nu/nu mice. A direct inhibitory effect of GAG-hed on the activity of HPV18-LCR was also noticed, as shown by the inhibition of β-galactosidase expression in HPV18-LCR-LacZ transgenic mice. Additionally, in HeLa cells, Northern and RT-PCR analysis showed that GAG-hed treatment resulted in a suppressive effect on E6/E7 viral expression and also GAG-hed has a significant negative effect on cell viability in HeLa cultures. Finally, this product also inhibited the sequence specific binding of the nuclear factor AP1 to HPV18-LCR. When tested by an in vitro approach, we found a blockade in protein/DNA binding activity due to GAG-hed treatment. All of these data suggest a potential antitumoral and antiviral application for GAG-hed.
GAG-hed was isolated from porcine intestine mucosa and obtained from PROBIOMED laboratories (México). In comparison with standard heparins, this molecule has a higher average in the amount of sulfate groups with a molecular weight ranging from 3–14 kDa.
Cell culture and cell proliferation assay
HeLa cells (HPV18 positive cervical carcinoma derived cell line), C33-A (HPV negative cervical carcinoma derived cell line) and SW480 (colorectal adenocarcinoma) were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen Gaithersburg, MD) supplemented with 10% fetal calf serum, with the appropriate antibiotic mix at 37°C in a 5% CO2 atmosphere. Culture media was replaced every two days.
Cell viability was measured by the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) reduction assay, performed as first described by Mosmann . Cells were seeded on a 24-well plate in 500 μl culture media with or without GAG-hed, incubated for indicated times and analyzed (quadruplicates). After incubation of the cells with the MTT reagent (5 mg/ml) for approximately 4 h, an isopropanol: HCl solution was added to lyse the cells and solubilize the coloured crystals. The samples were read using an ELISA plate reader (wavelength of 630 nm) Opsys MR (Dynex Technologies).
Athymic nude mice were obtained from Instituto Nacional de la Nutrición animal facility, bred and maintained in the animal facilities at the Instituto Nacional de Cancerología of México. Athymic female mice, 5–6 week old were housed (3 animals/cage, n = 12) in holding rooms that were kept at 21–25°C, and 40–60% relative humidity.
HeLa cell cultures at 80% of confluence were trypsinized, washed twice and resuspended in 200–500 μl phosphate buffered saline (PBS). HeLa cells were subcutaneously (S.C.) inoculated in the dorsal position in only one inoculation place in each female nu/nu mice. After a period of 10 to 16 days, when small nodules were already palpable, GAG-hed was administrated at three different concentrations of GAG-hed, two doses per week, all applied intra-tumoral and tumour growth was monitored. Tumours were measured twice a week and tumour volume in cubic millimetres was calculated as vol = (d1d2d3π)/6, where the width of the tumour is used twice as d1 and d2 and the length as d3 . The two dimensions were measured at least twice using Venire callipers.
Transgenic mice and GAG-hed treatment
Female mice expressing the LacZ gene under the control of the HPV18-LCR were reported previously [39, 40]. Transgenic mice (line 406) were crossed back to C57Bl/6J X C3HeB/FeJ F1 no transgenic strain and hemizygous 3 to 6 months old F1 females were obtained for our experiments. A volume of 50 μl of GAG-hed (10 mg/ml) or physiological solution as placebo, were introduced into the vagina of the female mice with a small probe attached to an insulin syringe. Doses were applied at noon and at night along 6 days. The seventh day vaginal smears were spread in slides for fixation and staining with hematoxylin-eosin and the phase of the oestrous cycle was determined by microscopic examination of the cells. Immediately after, the animals were sacrificed, dissected, and organs were frozen for transgene activity quantification. Two groups of three females each in estrogenic phase (proestrous-oestrous) were selected for these experiments.
Determination of β-galactosidase activity was described by Cid-Arregui et al. . Briefly, crude extracts from organs were prepared homogenizing with polytron in PM-2 buffer containing 33 mM NaH2PO4, 66 mM Na2HPO4, 0.1 mM MnCl2, 2 mM MgSO4, 40 mM β-mercaptoethanol, pH 7.3 and centrifuging twice for 10 min in a microfuge, discarding the pellet and lipid layer. Reactions were carried out with 200 μg of protein and 800 μg of ONPG (o-nitrophenyl-β-D-galactopyranoside) as substrate in a final volume of 1 ml of PM-2, and incubated at 37°C for 1 h. Color development was measured at A420 against a blank without protein. β-galactosidase-specific activities were calculated, after subtracting the initial absorbance of the reaction, using the formula: units = 380 X A420/time (min), such that 1 unit is equivalent to the conversion of 1 nM of ONPG per min at 37°C . For whole-organ staining vaginas were dissected and immediately fixed in 1% formaldehyde, 0.2% glutaraldehyde in PBS (pH 7.3) for 1 h at room temperature and then washed 4–5 times in PBS. Staining was performed with 1 mg/ml of X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; Sigma) in 0.01% sodium deoxycolate, 0.02% Nonidet P-40 in PBS at 30°C for 6 h in the dark. Finally, a second fixation was performed in 1% formaldehyde/2% glutaraldehyde in PBS.
Total RNA was extracted from HeLa cells employing the Chomczynski method , with minimal modifications. RNA was run in de-naturalizing gels, checked for equally loading RNA amounts, transferred to Hybond-N nylon membranes and fixed by baking membranes to 80°C for 2 h. As probe we used the fragment BamH1/EcoR1 containing the viral early region, obtained from plasmid pBR2.4 reported by Lazo . Labelling was performed by nick translation (Amersham Biosciences, Buckinghamshire, UK) using 32P-dCTP. Filters were hybridized for 16–24 h at 65°C, washed and analyzed by autoradiography.
Total RNA was isolated from confluent C33-A cells, and from treated and non treated HeLa cells. Treatment was performed with increasing GAG-hed concentrations for 48 h. Total RNA was extracted from cells using the Trizol method (Invitrogen Gaithersburg, MD). All samples were treated with RNAse-free DNAse (Invitrogen Gaithersburg, MD) to prevent genomic DNA contamination during PCR amplification. First strand cDNA was prepared from 1 μg of total cellular RNA using First Strand cDNA Synthesis Kits with oligo dT as a primer (Invitrogen Gaithersburg, MD). All PCR reactions were carried out in a 25 μl total volume containing 1 X PCR buffer, 4 mM MgCl2, 200 μM dNTP, 100 ng each of forward and reverse primers (Sigma Aldrich St. Louis, MO), and 1 unit of Taq polymerase in a Peltier Thermaln Cycle (Perkin-Elmer). Amplification conditions for both E6/E7 and β2-microglobulin were as follow: after an initial 94°C incubation for 3 min, reactions were amplified for 35 cycles at 94°C for 30 s, 58°C for 60 s and 72°C for 90 s. The reactions were then incubated at 72°C for 10 min. PCR amplification products were separated on a 1% agarose gel and visualized by ethidium bromide staining. Primers used to amplify the E6/E7 gene were as follows: forward, 5'-TGTCAAAAACCGTTGTGTCC-3', and reverse, 5'-GAGCTGTCGCTTAATTGCTC-3' . Primers used to amplify the β2-microglobulin were: forward 5'-ACCCCCACTGAAAAAGATGAGTAT-3', and reverse, 5'- ATGATGCTGCTTACATGTCTCGAT-3'.
Cell cycle analysis
For determination of cell percentage in each phase of the cell cycle a standard procedure based on the established method of whole-cell staining with propidium iodide (PI) was followed . Briefly, cells were trypsinized, fixed and permeabilized in 70% ice cold ethanol to make them accessible PI. Once fixed, cells were rinsed with PBS and stained with a PBS solution containing 0.1% Triton X-100, 0.2 mg/ml DNase free RNase A and 0.02 mg/ml of PI. Triton X-100 was included to decrease the cell loss resulting from electrostatic cell attachment to tubes and the RNase to digest the double stranded sections of RNA that might stain with PI. Measurements were done on a FACSCalibur Instrument (Becton-Dickinson) with an excitation of 488 nm (argon-ion laser line). Data were analyzed using the ModFit LT V2.0 (PMac) DNA content histogram de-convolution software.
Translocation of phosphatidylserine (PS) to the external surface of the membrane was determined using the Annexin V-FLUOS staining kit (Roche Applied Science) according to the manufacturer's instructions. HeLa cells were treated for 12 hours with staurosporine (1 μM, Sigma) as a positive control of apoptosis induction. The percentage of early apoptotic and apoptotic lysed cells was determined on a Becton-Dickinson FACS Vantage SE flow cytometer.
Chromatin condensation and/or nucleus fragmentation were investigated morphologically by DAPI (4',6-Diamidine-2'-phenylindole dihydrochloride) staining. Apoptotic cells were estimated by counting cells on UV microscopy after staining.
Electrophoretic Mobility Shift Assay
We performed gel mobility shift experiments (EMSA) utilizing nuclear extracts of HeLa cells prepared as previously described . All buffers contained a protease inhibitory cocktail to prevent nuclear factor proteolysis. Protein concentration was measured by the Bradford method using the Bio-Rad protein assay reagent . Double stranded oligonucleotides were end-labelled with [α-32P] dATP or [α-32P] dCTP (3000 Ci/mMol) and Klenow enzyme. Labelled oligonucleotides were incubated with up to 7–8 μg nuclear protein in a reaction mixture with 2x BDG buffer containing 24 mM HEPES, pH 7.8, 20% glycerol, 0.1 mM EDTA, 8 mM MgCl2, 20 mM KCl, 2 mM dithiothreitol, 4 mM spermidine for 10 min on ice; 0.5 μg poly [dI-dC] was added as unspecific competitor (Pharmacia Biotech). After probe addition, the reaction mixtures were incubated for 10 min on ice, electrophoresed in 6% polyacrylamide gels using a low ionic strength 0.5X TBE buffer. The gels were dried and exposed to an autoradiography film. The double stranded oligonucleotides used were:
AP1 from HPV18  5'- CTAGAATATGACTAAGCT-3' CTTATACTGATTCGAGATC;
SP1 (bona fide SV40) 5'-CTAGATTCGATCGGGGCGGGGCGA-3' TAAGCTAGCCCCGCCCCGCTGATC;
Egr-1 (bona fide)  5'-CTAGGATCCAGCGGGGGCGAGCGGGGGCGA-3' CCTAGGTCGCCCCCGCTCGCCCCCGCTGATC
GAG-hed inhibits tumour growth in nu/nu mice
In vivo effect of GAG-hed on HPV18-LCR activity
Effect of GAG-hed on E6/E7 expression in HeLa cells
GAG-hed affects cell proliferation rates but did not induce apoptosis
Furthermore, flow-cytometric analysis showed that cells treated for 48 h with GAG-hed underwent a change in cell cycle progression. Cells accumulated in the G2/M phase after GAG-hed treatment depending on applied dose (Figure 5C and 5D). The percentage of cells in G2/M increased from 4 % in the control to 18.43% in those treated with 10 mg/ml of GAG-hed.
Sequence specific binding of nuclear factor AP1 is inhibited by GAG-hed
The present study provides a closer look into the effects of heparinoids over the expression of HPV type 18, employing different methods and experimental systems. All assays were performed with GAG-hed, a highly sulphated heparinoid formulation which is heterogeneous in molecular weight (3–14 kDa), that has been used here to provide four lines of evidence suggesting that this compound blocks AP1 binding to HPV18 LCR. First, HeLa derived tumours developed in athymic mice grew significantly less under treatment with GAG-hed. Second, in transgenic female mice containing the HPV18-LCR-LacZ transgene, GAG-hed negatively affects the LCR activity based on the reduction of β-galactosidase reporter expression after treatment. Third, GAG-hed abolished the expression of HPV18 E6/E7 genes in HeLa cells and cell proliferation was profoundly affected. Finally, GAG-hed also reduced the binding of AP1 to its DNA sequence in HPV18 LCR, separately shown both by direct in vitro application and when cells in culture were previously treated for two days.
In HPV containing carcinoma cells, the GAG-hed antiproliferative effect may also be the result of an antiviral effect. The transcription of the HPV18 P105 promoter, which controls expression of the E6 and E7 transforming genes, is regulated by a combination of viral and cellular factors. The transcription factor AP1 is essential for the activity of this promoter, a finding mainly based on the observation that mutation of the corresponding binding sites within the HPV18-LCR completely abolishes P105 promoter activity in human keratinocytes [14, 16]. Indeed, it has been shown that AP1 also plays a central role in positive transcriptional regulation of several other human pathogenic HPVs [52, 53]. JunB is an important factor in HPV18 transcription in keratinocytes. In nuclear extracts prepared from human keratinocytes, JunB was the predominant Jun component bound to the DNA probe containing the same cis element we tested here (Figure 6) . It is therefore reasonable to assume that all selection mechanisms during HPV-linked carcinogenesis which enhance AP1 activity are required for HPV to exert its function as a DNA tumour virus. In HPV-positive carcinoma cells, GAG-hed causes a strong inhibition of AP1 binding to specific sites located on the viral LCR (Figure 6), which probably explains the decrease in E6/E7 mRNA synthesis. Diminution in β-galactosidase activity observed here (Figure 2), in addition to Northern blot and RT-PCR experiments in HeLa cells (Figure 3), are both highly suggestive of a direct affectation in AP1 regulatory activity within HPV18 LCR. Diminished expression in E6/E7 transcripts has been shown to be associated with inhibition of proliferation of cervical tumour cells [7, 51, 54]. Consistently, we noticed a significant loss in HeLa proliferation rates (Figure 4), which may be associated with the importance of AP1's role in E6/E7 expression. AP1 also plays a crucial role in cell proliferation , regulating gene expression in the pre neoplastic-to-neoplastic progression in cell culture models [56–63]. It is also possible that AP1 binding is blocked at the level of several cellular promoter regions of genes involved in cell proliferation, enhancing the antitumoral capabilities of GAG-hed. Heparin may have multiple targets for its antiproliferative activity; it can bind to Fos and Jun peptides, rendering the AP1 factor unable to bind DNA. Heparin selectively blocks the induction of immediate-early genes like c-fos and c-jun and other genes which are involved in cell cycle progression, including c-myc, c-myb, tissue plasminogen activator and ornithine decarboxylase . Heparin suppresses PMA induced expression of c-jun and Jun B mRNA and protein in baboon VSMC . Blocking c-fos and c-jun transcription by heparin is particularly important for transit through the cell cycle. However, the most significant effect of heparin on Jun family members occurs post-translationally. As Jun B is synthesized, it is converted to a higher molecular weight form by phosphorylation(s). Heparin prevents the transition to the higher molecular weight species, which is presumably the active form of Jun B, suggesting that heparin- mediated inhibition of Jun B may account for the reduced AP1-binding to the phorbol ester response element found on promoter regions upstream of the collagenase and tissue-type plasminogen activator (TPA) genes . Highly specific effects of heparin on signal transduction pathways involving MAPK, PKC and CaMK II have also been reported and may be reflected in the effects of heparin on gene regulation.
GAG-hed treatment affects cell proliferation but did not induce apoptosis (Figure 5). In HPV positive cells, p53 levels are regulated by the continuous expression of E6. The E6 oncoprotein has been shown to recruit the cellular ubiquitin-protein ligase E6-AP to target the tumor-suppressor protein p53 for ubiquitin-proteasome-mediated degradation [65, 66]. Treatment with antiE6 resulted in down-regulation of E6/E7 mRNA and an increase in p53 levels accompanied with a significant decrease in the growth rate . Part of the mechanism by which p53 blocks cells at the G2 checkpoint involves inhibition of Cdc2, the cyclin-dependent kinase required to enter mitosis. Cdc2 is inhibited simultaneously by three transcriptional targets of p53, Gadd45, p21, and 14-3-3σ . We observed an increase in the percentage of HeLa cells in G2/M phase together with a slight decrease in the number of cells in G1 and S phases with GAG-hed treatment, which may be associated with a recovery in cell cycle control by the increase in p53 after lowering E6/E7 expression. Current work in our laboratory is exploring this point.
GAGs are acidic and highly negatively charged molecules, which interact with a large number of proteins and basic molecules through ionic and hydrogen bonding interactions . There is direct evidence for heparin incorporation into cell cytoplasm and its presence in the nucleus [30, 69]. There is also evidence that heparan sulfate and glycosaminoglycans in the Extracellular Matrix and on the cell surface can be internalized by cells while bound to receptors. The uptake of heparin involves complexation and internalization with fibroblast growth factor and fibroblast growth factor receptor [70, 71]. Once in nuclei, heparin binds AP1; in 1992, Busch showed that 125I-labeled-heparin also binds directly to Fos and Jun peptides. In line with these observations, important changes in the levels of "free" transcription factors can be observed as a result of heparin internalization. It has been recently shown that heparin complexation with transcription factors may result in their inability to bind DNA regulatory elements, increasing factor levels in the cytoplasm and nuclei and culminates in apoptosis and cell death [69, 72]. Heparin also inhibits other important transcription factors in VSMC, such as c-myb and Oct-1, which presumably play a role in cell proliferation [73–75].
Model depicted in Figure 7 summarizes our current understanding of how GAG-hed may block HPV18. Based on previous studies [30, 69, 72], plus our present data, we suggest a possible molecular mechanism by which GAG-hed displays the effects described here, supporting the idea of heparinoids as plausible anti-viral and anti-tumoral drugs.
This work was supported by grants from CONACyT to E.L.B. (41273-A and 50414) and P.G. (38463-M). A.G.C was recipient of a Grant from the Scientific Collaborative Joint Program of the German BMBF (MXI6GDA5A). The authors would like to thank Ing. Jaime Uribe de la Mora (PROBIOMED Inc.) for generously providing the GAG-hed formulation. R. V. and R. T. are recipients of a CONACyT Doctoral Fellowship. Technical assistance of Matilde Corona, Georgina Díaz-Herrera, Blanca Estela Reyes, Victor Hugo Rosales García, Gerardo Marmolejo and Ing. Guillermo Benitez is acknowledged.
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