Short half-life of HPV16 E6 and E7 mRNAs sensitizes HPV16-positive tonsillar cancer cell line HN26 to DNA-damaging drugs.

Here we show that treatment of the HPV16-positive tonsillar cancer cell line HN26 with DNA alkylating cancer drug melphalan-induced p53 and activated apoptosis. Melphalan reduced the levels of RNA polymerase II and cellular transcription factor Sp1 that were associated with HPV16 DNA. The resulting inhibition of transcription caused a rapid loss of the HPV16 early mRNAs encoding E6 and E7 as a result of their inherent instability. As a consequence of HPV16 E6 and E7 down-regulation, the DNA damage inflicted on the cells by melphalan caused induction of p53 and activation of apoptosis in the HN26 cells. The BARD1-negative phenotype of the HN26 cells may have contributed to the failure to repair DNA damage caused by melphalan, as well as to the efficient apoptosis induction. Finally, nude mice carrying the HPV16 positive tonsillar cancer cells responded better to melphalan than to cisplatin, the chemotherapeutic drug of choice for tonsillar cancer. We concluded that the short half-life of the HPV16 E6 and E7 mRNAs renders HPV16-driven tonsillar cancer cells particularly sensitive to DNA damaging agents such as melphalan since melphalan both inhibits transcription and causes DNA damage.

Smokeless tobacco increases aneuploidy in oral HPV16 E6/E7-transformed keratinocytes in vitro.

BACKGROUND: The scope of this work was to study synergism between human papillomavirus (HPV) infection and tobacco in vitro, both known to be independent risk factors for oral cancer. METHODS: HPV-positive and HPV-negative oral keratinocytes and oral HPV-negative fibroblasts were exposed to smokeless tobacco extract (STE) prepared from the Scandinavian (STE1) and US-type (STE2) snuff. Cell cycle profiles were determined with flow cytometry, and HPV E6/E7 mRNA expression in HPV-positive cells was assayed using RT-qPCR. RESULTS: The exposure of HPV-positive keratinocytes with STE2 increased the number of aneuploid cells from 27% to 80% of which 44% were in S-phase, while none of the diploid cells were in S-phase. The changes after STE1 exposure were less than seen after STE2: from 27% to 31% of which 34% were in S-phase. STE had no effect on HPV16 E6/E7 expression in HPV-positive keratinocytes. In oral spontaneously transformed, HPV-negative keratinocytes, the number of aneuploid cells at G2-M stage increased after STE1 and STE2 exposure from 3% to 9% and 7%, respectively. In HPV-negative oral fibroblasts, the number of cells at G2-M phase increased from 11% to 21% after STE1 and 29% after STE2 exposure. CONCLUSIONS: The effect of STE varied in the cell lines studied. STE2 increased significantly the proportion of aneuploid cells in HPV-positive oral keratinocytes, but not HPV16 E6/E7 expression. This indicates that tobacco products may enhance the effects of HPV 16 and the risk of DNA aneuploidy increasing risk to malignant transformation.

Sensitivity to the non-COX inhibiting celecoxib derivative, OSU03012, is p21(WAF1/CIP1) dependent.

OSU03012 is a non-COX inhibiting celecoxib derivative with growth inhibiting and apoptotic activity in many cancer cell lines. To investigate mechanisms related to cell cycle proteins in growth inhibition and apoptosis induced by OSU03012, the primary human oral epithelial cell line, TE1177, was transformed with HPV16 E6 (TE/E6), HPV16 E7 (TE/E7) or empty vector (TE/V). TE/E6 cell lines exhibiting low levels of p53 and undetectable levels of p21(WAF1/CIP1) were sensitized to the growth inhibiting and apoptotic effects of OSU03012. The TE/E7 cell lines expressing low levels of Rb and elevated levels of p53 and p21(WAF1/CIP1) were resistant. OSU03012 reduced the number of cells in the S phase of the TE/E7 and TE/V cell lines with intact p53-p21(WAF1/CIP1) checkpoint, but not in the checkpoint defective TE/E6 cell lines. Treatment with OSU03012 also markedly reduced the levels of cyclin A and Cdk2 in TE/E7 and TE/V, but not in TE/E6 cell lines, which had significantly enhanced basal levels of cyclin A and Cdk2. Consistent with the TE/E6 cell line, p21(WAF1/CIP1)-/- mouse embryo fibroblasts were more sensitive to OSU03012-induced apoptosis as evidenced by PARP and caspase 3 cleavages. These data suggest that p21(WAF1/CIP1) is an important factor in the sensitivity of cells to the growth inhibiting and apoptotic effects of OSU03012.

Podophyllotoxin directly binds a hinge domain in E2 of HPV and inhibits an E2/E7 interaction in vitro.

Podophyllotoxin (PT), a strong cytotoxic agent from berberidaceae, has been known to inhibit tubulin polymerization. Although PT has been used for developing anticancer drugs as one of seed compounds, clinical treatment by itself has been unsuccessful because of the side effects, except one example in the treatments of warts. In this study, we screened peptides binding to PT with T7 phage display clonings in order to obtain more information about molecular mechanism of the action. A selected phage clone has a specific amino acid sequence to be SVPSRRRPDGRTHRSSRG. A homology search by protein database BLAST showed that this sequence had a similarity to a hinge domain (HD) of E2 protein in human papillomavirus (HPV) type 1a which is known to cause plantar warts. Surface plasmon resonance (SPR) analysis showed that PT bound to a recombinant HPV 1a E2 protein giving a K(D)=24.1microM which has compared with those of other domains of E2 protein. Also we demonstrated whether PT inhibited HD interaction or not. E7 protein of HPV has been known to be an oncoprotein and was reported to interact with HD of E2 protein. We demonstrated that an E2/E7 interaction was inhibited by the addition of PT in this report. And we showed the bindings of PT to other types of HPV. Our results suggest that PT is potential as a tool for clarifying the molecular mechanism of HPV.

CuZn-SOD suppresses the bovine papillomavirus-induced proliferation of fibroblasts.

Eukaryotic cells continuously produce reactive oxygen species (ROS) and have mechanisms to control ROS levels. ROS have been shown to mediate cell proliferation and transformation. We studied the effect of CuZn-superoxide dismutase (CuZnSOD) on the focus-forming ability of bovine papillomavirus (BPV-1) wtDNA and hypertransforming mutant of its major oncoprotein E5, E5-17S. We found that CuZnSOD suppresses the focus-forming ability of BPV-1 wtDNA and E5 oncoprotein. Significantly fewer foci were detected in pCGCuZnSOD- and BPV-1 DNA-cotransfected cell culture compare to BPV-1 DNA-transfected cell culture (p<0.001). CuZnSOD decreases the rate of cell proliferation in both non-transformed C127 and BPV-1- and E5-transformed cell lines. CuZnSOD decelerates cell entry into the S phase of the cell cycle and has a suppressing effect on the actively dividing cells. As the transformed cells proliferate faster than normal cells when confluent, CuZnSOD inhibits the growth of foci. These results indicate that superoxide radicals may be involved in signaling for cell proliferation and that SOD suppresses cell proliferation.

Specific HIV protease inhibitors inhibit the ability of HPV16 E6 to degrade p53 and selectively kill E6-dependent cervical carcinoma cells in vitro.

Although HIV protease inhibitor (PI) drugs predominantly target HIV proteases 1 and 2, it is also known that part of their efficacy is due to selective inhibition of the proteasome. The pathogenicity of high-risk human papilloma virus (HPV) is dependent on expression of viral E6 proteins which inappropriately activate the 26S proteasome to degrade p53 and other cellular proteins that are detrimental to viral replication. Comparison of the ability of the PIs indinavir, ritonavir, amprenavir, lopinavir, atazanavir, nelfinavir and saquinavir to inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells showed that 15 microM lopinavir, 1 mM indinavir or 125 microM ritonavir treatment for 24 h produced a stable increase in the level of nuclear p53 in these cells with minimal cell death. After 4 h exposure of HPV16+ve SiHa cells to 15 microM lopinavir, a transient increase in wild-type p53 expression was observed associated with a 7% reduction in the chymotryptic activity of the 205 proteasome and apoptosis after 24h. Comparison of growth rates of PI treated SiHa, CaSki, C33A, C33A-E6 and non-transformed NIH/3T3 cells showed that SiHa were the most sensitive, whereas NIH/3T3 were least affected. In conclusion, these data show that specific HIV PIs such as lopinavir and possibly indinavir, can induce selective toxicity of HPV-transformed cervical carcinoma cells expressing wild-type p53 and may form the basis of a topically applied alternative to surgery for the treatment of HPV-related premalignant lesions of the cervix.

Downregulation of Epstein-Barr virus-encoded latent membrane protein-1 by arsenic trioxide in nasopharyngeal carcinoma cells.

AIMS AND BACKGROUND: It was documented that nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV) and that EBV-encoded latent membrane protein-1 expression (LMP1) plays an important role in the pathogenesis of NPC. In preclinical studies, arsenic trioxide (As2O3) has been identified as a promising anticancer agent for treatment of NPC. The purpose of this study is to investigate if this agent can inhibit the expression of LMP1 and therefore lead to growth inhibition of NPC cells in vitro. METHODS: LMP1-positive NPC cells, HNE1-LMP1, were treated with 3 micromol/L of As2O3 for 96 hours. The LMP1 protein expression and mRNA level in HNE1-LMP1 cells were determined by western blot, confocal immunofluorescence staining and semiquantitative reverse transcriptase reaction (RT-PCR). Apoptosis was determined by light microscopy and the TUNEL method. Alterations in the cell cycle distribution were also investigated by flow cytometry. MTT assay and colony formation assay were used to detect the proliferation of the cells. The LMP1-negative parental cell lines HNE1 and HNE2 were used as control in an attempt to elucidate the role of LMP1 in the anticancer effect of As2O3 on NPC cells. RESULTS: The expression of LMP1 at the protein and mRNA level was reduced after exposure to 3 micromol/L As2O3. This dose of As2O3 significantly induced apoptosis and growth retardation of HNE1-LMP1 cells. In addition, more HNE1-LMP1 cells were induced to G0/G1 and G2/M arrest. The same dose of As2O3 had a moderate effect on HNE1 and HNE2 cells. CONCLUSION: Arsenic trioxide can inhibit LMP1 expression and dictate apoptosis and alterations of cell cycle distribution as well as growth retardation. LMP1-positive NPC cells are more sensitive to As2O3 treatment than LMP1-negative NPC cells.

Potential drugs against cervical cancer: zinc-ejecting inhibitors of the human papillomavirus type 16 E6 oncoprotein.

BACKGROUND: The principal agent in the etiology of cervical cancer, i.e., human papillomavirus (HPV) type 16, encodes three oncoproteins, E5, E6, and E7. Structural and mutational studies have identified two potential zinc-finger domains as critical for E6 protein function. We investigated several assays to identify and characterize compounds that interfere with the binding of zinc to E6. METHODS: Thirty-six compounds were selected on the basis of their structure, which would facilitate their participation in sulfhydryl residue-specific redox reactions, and were tested for their ability to release zinc from E6 protein. The zinc-ejecting compounds were then tested for their ability to inhibit E6 binding to E6-associated protein (E6AP) and E6-binding protein (E6BP), two coactivators of E6-mediated cellular transformation. The binding of E6 to E6BP and E6AP was measured by use of surface plasmon resonance (a technique that monitors molecular interactions by measuring changes in refractive index) and by use of in vitro translation assays. The compounds were also tested for their effects on the viability of HPV-containing cell lines. RESULTS: Nine of the 36 tested compounds ejected zinc from E6. Two of the nine compounds inhibited the interaction of E6 with E6AP and E6BP, and one of these two, 4, 4'-dithiodimorpholine, selectively inhibited cell viability and induced higher levels of p53 protein (associated with the induction of apoptosis [programmed cell death]) in tumorigenic HPV-containing cells. CONCLUSION: We have described assay systems to identify compounds, such as 4,4'-dithiodimorpholine, that can potentially interfere with the biology and pathology of HPV. These assay systems may be useful in the development of drugs against cervical cancer, genital warts, and asymptomatic infections by genital HPVs.

Retinoic acid inhibition of human papillomavirus type 16-mediated transformation of human keratinocytes.

We previously reported that human keratinocytes (HKc) immortalized by transfection with human papillomavirus type 16 DNA (HKc/HPV16) are more sensitive than normal HKc to growth inhibition by retinoic acid (RA), and that RA treatment of HKc/HPV16 inhibits HPV16 E6/E7 mRNA expression (L. Pirisi et al., Cancer Res., 52: 187-193, 1992). We now demonstrate that HPV16 E2 and E5 mRNAs are also decreased by RA treatment of HKc/HPV16, indicating a general inhibition by RA on the expression of HPV16 early genes. In addition, protein levels of E6 and E7, as measured by immunofluorescence, are also decreased in a dose-dependent manner following RA treatment of HKc/HPV16. Since E6 and E7 are considered the oncogenes of HPV16, we explored the possibility that RA may interfere with HPV16-mediated immortalization of HKc. RA treatment (1 nM) of normal HKc, immediately following transfection with HPV16 DNA, inhibited immortalization by about 95%. Overall, these results provide a direct biochemical basis for a role of RA in the chemo-prevention of human papillomavirus-induced cancers.

UVB irradiation reduces the half-life and transactivation potential of the human papillomavirus 16 E2 protein.

Human papillomaviruses (HPV) are causative agents of human cancers including those of the cervix and also of the head and neck; HPV16 is the most commonly found type in these diseases. The viral E2 protein regulates transcription from the viral genome by interacting with DNA-binding sequences in the HPV transcriptional control region; it also regulates replication by interacting with and recruiting the HPV replication factor E1 to the viral origin. Therefore, E2 is essential for the viral life cycle. The E2 protein interacts with several proteins involved in the cellular response to DNA damage including p53, TopBP1, and PARP. We therefore set out to establish whether DNA-damaging agents can regulate E2 activity. Here we show that UVB irradiation downregulates transcriptional activity of both HPV16 and HPV8 E2, while hydroxyurea and etoposide do not. This downregulation of E2 activity is independent of p53 function as it occurs in p53 wild type and null cell types as well as in the presence of functional HPV16 E6 that degrades p53. Using stable cell lines expressing E2 we show that this downregulation of E2 function by UVB is due to a reduction of the E2 protein half-life. The identification of the pathway(s) through which UVB downregulates E2 transcriptional activity and protein levels will present a novel target for the treatment of HPV-related diseases.

Quercetin elevates p27(Kip1) and arrests both primary and HPV16 E6/E7 transformed human keratinocytes in G1.

Our previous work with primary bovine fibroblasts demonstrated that quercetin, a potent mutagen found in high levels in bracken fern (Pteridium aquilinum), arrested cells in G1 and G2/M, in correlation with p53 activation. The expression of bovine papillomavirus type 4 (BPV-4) E7 overcame this arrest and lead to the development of tumorigenic cells lines (Beniston et al., 2001). Given the possible link between papillomavirus infection, bracken fern in the diet and cancer of the upper gastrointestinal (GI) tract in humans, we investigated whether a similar situation would occur in human cells transformed by human papillomavirus type 16 (HPV-16) oncoproteins. Quercetin arrested primary human foreskin keratinocytes in G1. Arrest was linked to an elevation of the cyclin-dependent kinase inhibitor (cdki) p27(Kip1). Expression of the HPV16 E6 and E7 oncoproteins in transformed cells failed to abrogate cell cycle arrest. G1 arrest in the transformed cells was also linked to an increase of p27(Kip1) with a concomitant reduction of cyclin E-associated kinase activity. This elevation of p27(Kip1) was due not only to increased protein half-life, but also to increased mRNA transcription.

siRNA targeting of the viral E6 oncogene efficiently kills human papillomavirus-positive cancer cells.

The targeted inhibition of antiapoptotic factors in tumour cells may provide a rational approach towards the development of novel anticancer therapies. Using human papillomavirus (HPV)-transformed cells as a model system, we investigated if RNA interference (RNAi)-mediated gene silencing can be employed in order to overcome the apoptosis resistance of cancer cells. We found that both vector-borne and synthetic small interfering (si)RNAs, specifically directed against the antiapoptotic HPV E6 oncogene, restored dormant tumour suppressor pathways in HPV-positive cancer cells that are otherwise inactive in the presence of E6. This ultimately resulted in massive apoptotic cell death, selectively in HPV-positive tumour cells. These findings show that RNAi provides a powerful molecular strategy to inactivate intracellular E6 function efficiently. Moreover, they define E6 as a most promising therapeutic target to eliminate HPV-positive tumour cells specifically by RNAi. Thus, by sequence-specific targeting of antiapoptotic genes, siRNAs may be developed into novel therapeutics that can efficiently correct the apoptosis deficiency of cancer cells.

Strategies for human papillomavirus therapeutic vaccines and other therapies based on the E6 and E7 oncogenes.

High-risk human papillomaviruses (HR-HPVs) are one of the most devastating oncogenic viruses worldwide and have been causally linked with the development of human cervical cancer. Several prophylactic and therapeutic clinical HPV vaccine trials are in progress. Although prophylactic vaccines are useful in preventing the incidence of cervical cancer, the elimination of existing HPV infections needs to be addressed, because cervical cancer is the leading female cancer in developing countries. Several different and encouraging strategies have been investigated in a preclinical and clinical setting for the treatment and elimination of existing HPV-induced infection. This review summarizes the therapeutic clinical trials and the different preclinical research strategies that are under investigation whereby HR-HPV E6 and E7 oncogenes are delivered in a nucleic acid form, in viral and bacterial vectors, or as peptide- and protein-based vaccines.

Inhibitory effect of jaceosidin isolated from Artemisiaargyi on the function of E6 and E7 oncoproteins of HPV 16.

Jaceosidin (4',5,7-trihydroxy-3',6-dimethoxyflavone) was isolated from Artemisia argyi as a putative oncogene inhibitor. Jaceosidin inhibited binding between oncoprotein E6 of the human papillomavirus and the p53 tumor suppressor protein. In addition, jaceosidin inhibited binding between the E7 oncoprotein and the Rb tumor suppressor protein, and also inhibited the function of HPV-16 harboring cervical cancer cells, including SiHa and CaSki. Collectively, jaceosidin inhibited the functions of the E6 and E7 oncoproteins of the human papillomavirus, suggesting that this compound might be used as a potential drug for the treatment of cervical cancers associated with the human papillomavirus.

Activation of src tyrosine kinases by peroxynitrite.

In this study, we demonstrate that the phosphorylation activity of five tyrosine kinases of the src family from both human erythrocytes (lyn, hck and c-fgr) and bovine synaptosomes (lyn and fyn) was stimulated by treatment with 30-250 microM peroxynitrite. This effect was not observed with syk, a non-src family tyrosine kinase. Treatment of kinase immunoprecipitates with 0.01-10 microM peroxynitrite showed that the interaction of these enzymes with the oxidant also activated the src kinases. Higher concentrations of peroxynitrite inhibited the activity of all kinases, indicating enzyme inactivation. The addition of bicarbonate (1.3 mM CO2) did not modify the upregulation of src kinases but significantly protected the kinases against peroxynitrite-mediated inhibition. Upregulation of src kinase activity by 1 microM peroxynitrite was 3.5-5-fold in erythrocytes and 1.2-2-fold in synaptosomes, but this could be the result, at least in part, of the higher basal level of src kinase activity in synaptosomes. Our results indicate that peroxynitrite can upregulate the tyrosine phosphorylation signal through the activation of src kinases.

Interleukin-10 induces transcription of the early promoter of human papillomavirus type 16 (HPV16) through the 5'-segment of the upstream regulatory region (URR).

The effects of various proinflammatory cytokines on the transcription of human papillomaviruses (HPVs) have been demonstrated. On the other hand, the role of anti-inflammatory cytokines has not been elaborated, despite the fact that levels of interleukin-10 (IL-10) have been found significantly elevated in cervical dysplasias or carcinomas as well as in the cervix of HIV-positive individuals. These conditions are also associated with elevated viral transcription. Thus, the impact of IL-10 on HPV transcription might be important in pathogenesis of cervical lesions in both immunocompetent or immunosuppressed individuals. In this paper we describe the effects of IL-10 on the transcription of HPV type 16. We found that treatment of HPV 16-positive cervical carcinoma cells with IL-10 increased mRNA levels of the E7 early gene at the level of transcription. Similarly, IL-10 significantly and dose-dependently induced the transcription from the HPV early promoter in a reporter system. Employing deletion mutants we determined that this induction is mapped to the 5' segment of the URR. Transient transfection of an antisense-STAT3-expression vector abolished IL-10-induced reporter activity as well as HPV 16 E7 expression. This suggests that STAT3 either directly binds to the URR and stimulates transcription or affects expression and/or binding of transcription factors that bind to the 5'-region. Our findings suggest a mechanism by which--in addition to its immunosuppressive effects--IL-10 might enhance persistence and progression of HPV-related lesions under conditions (e.g. dysplastic progression, HIV infection) when the cytokine expression in the cervical microenvironment changes.

Effects of liposomal antisense oligonucleotides on mRNA and protein levels of the HPV 16 E7 oncogene.

Despite the known association of human papillomavirus (HPV) infection with cervical cancer there is no specific antiviral treatment for HPV infection. Antisense oligode-oxynucleotides (AS-ODNs) may offer an effective way to treat HPV infections as the stability and delivery have been improved using modified ODNs or carrier systems. In this study we investigated the effects of liposomal AS-ODNs (0.1, 1 and 5 microM) on HPV 16 E7 mRNA and protein levels in CaSki cells. We used cationic liposomes (10 microM) containing dimethyldioctadecylammonium bromide (DDAB) or 2,3-dioleyloxy-N-[2(sperminecar-boxamido)ethyl]-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA). Both these liposomes had dioleoylphosphatidyl-ethanolamine (DOPE) as a helper lipid. The target of the AS-ODNs was E7 protein because it is the one of the two oncoproteins of HPV 16. Only liposomal AS-ODNs which were targeted to the initiation codon of E7, had an effect on E7 mRNA expression; two shorter transcripts were detected, suggesting that RNase H degradation was activated. Liposomal random ODN or liposomal ODN targeted downstream from the initiation site of E7 did not affect the mRNA pattern. However, no change was found in the E7 protein levels detected by immunoprecipitation. Further studies showed that AS-ODNs inhibited the translation of E7 mRNA in a rabbit reticulocyte lysate assay. This data, together with the changes in mRNA levels, proved that the AS-ODNs reached the target mRNA. One possible explanation for the unchanged protein level of E7 in CaSki cells might be that immunoprecipitation is not sensitive enough to detect minor changes in protein levels. However, further progress is still needed in the field of carrier systems and modifications of AS-ODNs before non-sequence specific effects can be avoided.

HPV E6 oncoprotein as a potential therapeutic target in HPV related cancers.

INTRODUCTION: Human Papillomaviruses (HPVs) are the main etiological agents for the development of most ano-genital cancers and for a subset of head and neck neoplasias. The oncogenic capacity of HPV is due to the combined activity of the viral oncoproteins E6 and E7. A defining feature of all HPV associated cancers is the continued retention and expression of these two viral oncoproteins throughout the development of the disease, and this highlights their value as potential targets for therapeutic intervention, in HPV-induced malignancies. AREAS COVERED: In this review, the authors focus on the HPV E6 oncoprotein functions and its interactions with cellular targets containing either LxxLL motifs or PDZ domains. New approaches leading to the prevention such interactions are described, showing the advantage of E6 as a target for therapeutic intervention against malignant transformation and cancer. EXPERT OPINION: The high degree of conservation in E6-LxxLL interactions across multiple HPV types makes this a compelling therapeutic target for pathologies caused by diverse HPV types. Combining this with therapeutics directed against E6-PDZ interactions offers great promise for the treatment of malignancies caused by high-risk HPV types.

Anticancer drugs aimed at E6 and E7 activity in HPV-positive cervical cancer.

Standard treatment of locally advanced cervical cancer currently consists of concurrent chemoradiation, leading to a 5-year disease-free survival of 66-79%, indicating that there is still ample room for improvement. Characteristic of cervical cancer is the presence of high risk (HR) human papillomavirus (HPV) DNA in more than 99% of these tumors. When the HR HPV genome integrates into the host genome, oncogenic E6 and E7 proteins become constitutively expressed. These oncogenes are also active earlier in the infection cycle and hence are available as therapeutic targets at the preneoplastic stages as well. E7 plays an important role in the early stage of carcinogenesis by stimulating proliferation. HR HPV E6-induced proteasomal degradation of p53 hampers p53 functionality in cell cycle arrest and apoptosis. As p53 plays a key role in the intrinsic apoptotic pathway, current chemoradiation cannot optimally activate this pathway. In this review, we focus on targeted anticancer drugs to eliminate the consequences of HR HPV E6 and E7 activity. Strategies for direct and indirect targeting of HR HPV E6 and E7, including RNA interference, small molecules, proteasome inhibitors, and histone deacetylase inhibitors, are described. In addition, the extrinsic apoptotic pathway as possible alternative therapeutic target for apoptosis induction is reviewed. The rational for implementing recombinant human TRAIL and death receptor agonists and the latest developments on combining these drugs with standard treatment in preclinical settings as well as clinical trials are discussed.

Methyl jasmonate reduces the survival of cervical cancer cells and downregulates HPV E6 and E7, and survivin.

The present study further investigated the mode of action of methyl jasmonate (MJ) in different cervical cancer cell lines. We show that in addition to the short term cytotoxicity, MJ effectively reduced the survival of cervical cancer cells (clonogenicity assays). MJ induced apoptosis in all cervical cancer cells. In some cell lines, MJ caused elevation of the mitochondrial superoxide anion, notably, in HeLa and CaSki. Changes in the expression of p53 and bax were variable, yet, downregulation of survivin was common to all cervical cancer cells. MJ significantly reduced the levels of the human papillomavirus (HPV) E6 and E7 proteins without alteration of the mRNA levels. Moreover, ectopic expression of E6, E7 or both in cervical cancer cells that lack HPV (C33A), did not alter significantly their response to MJ. Our studies point to MJ as an effective anticancer agent against a variety of cervical cancer cells acting through shared and different pathways to induce cell death regardless of the presence of HPV.