Adenoviral p53 effects and cell-specific E7 protein-protein interactions of human cervical cancer cells

We investigated the time-course tumor growth suppression effects of recombinant adenovirus expressing p53 on human cervical cancer cells and cell-specific E7 protein-protein interactions in cell lysates using surface plasmon resonance (SPR) biosensor. Six HPV-infected human cervical cancer cell lines (HPV 16-positive cells, CaSki and SiHa cells; HPV 18-positive cells, HeLa and HeLaS3 cells; and HPV negative C33A and HT3 cells) were used. After infection with AdCMVp53, the cell-specific growth inhibition was studied in vitro and in vivo. Also, we produced the recombinant E7 oncoprotein of HPV 16 type and tested chip-based protein-protein interactions with each cell lysate. For each cervical cancer cell, differential cell growth inhibitions were shown via cell count assay and MTT assay. Note that the same trend in suppression levels was shown in CaSki, HeLa and in SiHa, HeLaS3, respectively. In contrast, infection with AdCMVLacZ showed increased cell growth in a manner similar to the negative control group. The levels of p53 protein were notably expressed in CaSki and HeLa more than in SiHa and HeLaS3 for 4 days. In contrast, p53 expression was continually maintained in C33A and HT3 for 6 days. After transfection AdCMVp53 into CaSki- and SiHa-xenografted nude mice, the size of tumor was remarkably decreased in SiHa cells as compared to AdCMVLacZ transfection. The SPR sensor surface was successfully modified with the recombinant E7 oncoprotein and showed cell-specific interactions between E7 and its target proteins from cell lysates. The anti-tumor effects were accomplished via differential role of p53-specific apoptotic cell death, which is dependent upon the cervical cancer cell line. Also, a molecular level understanding of cell-dependent protein interaction effects of recombinant E7 was shown.     

Degradation of p53 by Human Alphapapillomavirus E6 Proteins Shows a Stronger Correlation with Phylogeny than Oncogenicity

Background

Human Papillomavirus (HPV) E6 induced p53 degradation is thought to be an essential activity by which high-risk human Alphapapillomaviruses (alpha-HPVs) contribute to cervical cancer development. However, most of our understanding is derived from the comparison of HPV16 and HPV11. These two viruses are relatively distinct viruses, making the extrapolation of these results difficult. In the present study, we expand the tested strains (types) to include members of all known HPV species groups within the Alphapapillomavirus genus.

Principal Findings

We report the biochemical activity of E6 proteins from 27 HPV types representing all alpha-HPV species groups to degrade p53 in human cells. Expression of E6 from all HPV types epidemiologically classified as group 1 carcinogens significantly reduced p53 levels. However, several types not associated with cancer (e.g., HPV53, HPV70 and HPV71) were equally active in degrading p53. HPV types within species groups alpha 5, 6, 7, 9 and 11 share a most recent common ancestor (MRCA) and all contain E6 ORFs that degrade p53. A unique exception, HPV71 E6 ORF that degraded p53 was outside this clade and is one of the most prevalent HPV types infecting the cervix in a population-based study of 10,000 women. Alignment of E6 ORFs identified an amino acid site that was highly correlated with the biochemical ability to degrade p53. Alteration of this amino acid in HPV71 E6 abrogated its ability to degrade p53, while alteration of this site in HPV71-related HPV90 and HPV106 E6s enhanced their capacity to degrade p53.

Conclusions

These data suggest that the alpha-HPV E6 proteins'' ability to degrade p53 is an evolved phenotype inherited from a most recent common ancestor of the high-risk species that does not always segregate with carcinogenicity. In addition, we identified an amino-acid residue strongly correlated with viral p53 degrading potential.     

Suppression of HPV E6 and E7 expression by BAF53 depletion in cervical cancer cells

Deregulation of the expression of human papillomavirus (HPV) oncogenes E6 and E7 plays a pivotal role in cervical carcinogenesis because the E6 and E7 proteins neutralize p53 and Rb tumor suppressor pathways, respectively. In approximately 90% of all cervical carcinomas, HPVs are found to be integrated into the host genome. Following integration, the core-enhancer element and P105 promoter that control expression of E6 and E7 adopt a chromatin structure that is different from that of episomal HPV, and this has been proposed to contribute to activation of E6 and E7 expression. However, the molecular basis underlying this chromatin structural change remains unknown. Previously, BAF53 has been shown to be essential for the integrity of higher-order chromatin structure and interchromosomal interactions. Here, we examined whether BAF53 is required for activated expression of E6 and E7 genes. We found that BAF53 knockdown led to suppression of expression of E6 and E7 genes from HPV integrants in cervical carcinoma cell lines HeLa and SiHa. Conversely, expression of transiently transfected HPV18-LCR-Luciferase was not suppressed by BAF53 knockdown. The level of the active histone marks H3K9Ac and H4K12Ac on the P105 promoter of integrated HPV 18 was decreased in BAF53 knockdown cells. BAF53 knockdown restored the p53-dependent signaling pathway in HeLa and SiHa cells. These results suggest that activated expression of the E6 and E7 genes of integrated HPV is dependent on BAF53-dependent higher-order chromatin structure or nuclear motor activity.     

Cisplatin restores p53 function and enhances the radiosensitivity in HPV16 E6 containing SiHa cells

Most HPV-positive cervical cancer cells possess wild type p53 gene, but its normal p53 functions are disrupted by expression of HPVs E6. Treatment with 0-20 microM cisplatin for 24 h in HPV16 E6 containing SiHa cells suppressed E6 mRNA, reduced E6 protein, and restored p53 expression in dose-dependent manners. Dual-parameter flow cytometric analysis indicated that sub-G(1) apoptotic cells, but not necrotic cells were the major species for cisplatin-induced cytotoxicity in SiHa cells. After 0-10 microM cisplatin treatment, slightly more apoptotic cells appeared from SiHa cells than those from dominant negative p53-transfected SiHa cells. There was no different ionizing radiation (IR)-induced apoptosis in these two different cells. On the other hand, cisplatin enhanced more IR-induced sub-G(1) apoptosis in SiHa than mp53-SiHa cells. These accompanied with prolonged p53 restoration in irradiated-SiHa cells after 24 h cisplatin treatment and thereafter. In contrast, it was not found in cells after irradiation alone. Similar results were also shown in Mdm2 expression in SiHa cells after combined treatment. Therefore, cisplatin restored p53 expression and prolonged IR-induced p53 restoration would be possible candidates to response more sub-G(1) apoptosis in irradiated SiHa cells. These results provided another new explanation on cisplatin sensitizing radiotherapy for HPV16 E6 containing cancer cells.     

p53 and PPP1R13L (alias iASPP or RAI) form a feedback loop to regulate genotoxic stress responses

Background

PPP1R13L gene has been found to be over-expressed in variety of cancers and its expression in p53 wild-type background is sufficient to promote tumor growth in vivo. However, in the non-transformed cells it acts as a tumor suppressor which suggests that the role of PPP1R13L is multifaceted.

Methods

We have used siRNA optimized for inhibition of p53, PPP1R13L, BAX and GADD45 alpha expression and investigated the role of those gene products for PPP1R13L expression and induction in a variety of mouse and human cells with different p53 status. In addition we have applied Western Blot, Q-PCR and proteasome inhibition analysis to further ascertain the link between PPP1R13L induction and p53 status.

Results

We show that the pattern and extent of the PPP1R13L expression depend on the presence of active p53. Downregulation of p53 target genes BAX and/or GADD45 alpha led to decreased in PPP1R13L activation after adriamycin and/or etoposide treatments. Treatment of the cells with the proteasome inhibitor MG-132 resulted in the accumulation of both p53 and PPP1R13L proteins.

Conclusions

We have provided evidence that endogenous PPP1R13L acts as a negative regulator of p53 function, presumably by direct binding. p53 accumulation and activity after DNA damage is compromised by PPP1R13L expression. We suggest that PPP1R13L and p53 form a negative feedback loop which regulates their amount and activity.

General significance

The profound modulatory effect of the PPP1R13L protein on the ability of p53 to cause cellular apoptosis has important implications in cancer and presents new therapeutic possibilities.     

Effects of celastrol on human cervical cancer cells as revealed by ion-trap gas chromatography–mass spectrometry based metabolic profiling

Background

Celastrol, a quinine methide triterpene extracted from a Chinese medicine (Trypterygium wilfordii Hook F.), has the potential to become an anticancer drug with promising prospects. Cell culture metabolomics has been a powerful method to study metabolic profiles in cell line after drug treatment, which can be used for discovery of drug targets and investigation of drug effects.

Methods

We analyzed the metabolic modifications induced by celastrol treatment in human cervical cancer cells, using an ion-trap gas chromatography–mass spectrometry based metabolomics combined with multivariate statistical analysis, which allows simultaneous screening of multiple characteristic metabolic pathways related to celastrol treatment. Three representative apoptosis-inducing cytotoxic agents, namely cisplatin, doxorubicin hydrochloride and paclitaxel, were selected as positive control drugs to validate reasonableness and accuracy of our metabolomic investigation on celastrol.

Results

Anti-proliferation and apoptotic effects of celastrol were demonstrated by CCK-8 assay, Annexin-V/PI staining method, mitochondrial membrane potential (ΔΨm) assay and caspase-3 assay. Several significant metabolites involved in energy, amino acid and nucleic acid metabolism in HeLa cells induced by celastrol and positive drugs were reported. Our method is proved to be effective and robust to provide new evidence of pharmacological mechanism of celastrol.

Conclusions

The metabolic alterations induced by drug treatment showed the impaired physiological activity of HeLa cells, which also indicated anti-proliferative and apoptotic effects of celastrol and these positive drugs.

General significance

GC/MS-based metabolomic approach applied to cell culture could give valuable information on the systemic effects of celastrol in vitro and help us to further study its anticancer mechanism.     

The Ubiquitin-Specific Peptidase USP15 Regulates Human Papillomavirus Type 16 E6 Protein Stability

Proteomic identification of human papillomavirus type 16 (HPV16) E6-interacting proteins revealed several proteins involved in ubiquitin-mediated proteolysis. In addition to the well-characterized E6AP ubiquitin-protein ligase, a second HECT domain protein (HERC2) and a deubiquitylating enzyme (USP15) were identified by tandem affinity purification of HPV16 E6-associated proteins. This study focuses on the functional consequences of the interaction of E6 with USP15. Overexpression of USP15 resulted in increased levels of the E6 protein, and the small interfering RNA-mediated knockdown of USP15 decreased E6 protein levels. These results implicate USP15 directly in the regulation of E6 protein stability and suggest that ubiquitylated E6 could be a substrate for USP15 ubiquitin peptidase activity. It remains possible that E6 could affect the activity of USP15 on specific cellular substrates, a hypothesis that can be tested as more is learned about the substrates and pathways controlled by USP15.Human papillomaviruses (HPVs) are associated with several human cancers, most notably human cervical cancer, the second most common cancer among women worldwide (43). Papillomaviruses cause proliferative squamous epithelial lesions, and more than 100 HPV types have been described (14). The HPV types associated with mucosal squamous epithelial lesions have been further classified into high- or low-risk types based on the propensity for the lesions with which they are associated to progress to cancer. Among the high-risk HPV types, HPV type 16 (HPV16) and HPV18 account for approximately 70% of cervical cancers (43). The high-risk HPV types carry two genes, the E6 and E7 genes, which have oncogenic properties and are always expressed in HPV-positive cancers. E6 and E7 interfere with the p53 and retinoblastoma (pRB) tumor suppressor pathways, respectively, and contribute directly to cell cycle alterations, protection from apoptosis, and transformation (14). The dysregulated expression of the E6 and E7 oncoproteins is an important step in the progression from a preneoplastic stage to cancer in HPV-infected cells and is often a consequence of the integration of the viral genome into the host chromosome.The interaction between E6 and p53 is mediated by the E3 ubiquitin ligase E6AP (15). E6, p53, and E6AP form a complex in which E6 directs the ligase activity of E6AP to p53, thereby targeting p53 for ubiquitin-mediated degradation (36). E6, however, has a number of other cellular partners and other functions. For instance, the C terminus of the high-risk E6 protein contains a PDZ binding motif (20, 25) that mediates the interaction with several PDZ domain-containing proteins, including discs large (Dlg), Scribble (Scrib), the MAGI family of proteins, MUPP1, and PATJ (9, 10, 29). Some of these proteins are also targeted for degradation in an E6AP-dependent manner (22, 29). While the major mechanism of oncogenesis revolves around E6''s ability to inhibit the proapoptotic effects of p53, recent work involving the PDZ domain proteins indicates that these interactions are also important to the oncogenic potential of E6 (38, 41). Furthermore, E6 has been reported to bind a number of other cellular proteins, including but not limited to Bak, CBP/p300, c-Myc, E6TP1, hADA3, IRF3, MCM7, PTPH1, and TNF-R1 (7, 8, 17, 23, 24, 32, 35, 39, 40). The importance of the binding of several of these proteins with regard to the transformation or other functions of E6 remains to be established. E6 itself is thought to be targeted for degradation by an ubiquitin-proteasome pathway (18), although how E6 protein stability is regulated has not been well studied.Many of the E6 binding partners have been identified using purified bacterially expressed E6 fusion proteins and cell lysates from various cell types or using yeast two-hybrid screenings. While some of these interactions with E6 have been validated, the physiologic relevance of a number of proposed E6 targets remains undetermined. In an effort to identify E6-interacting proteins, perhaps under more physiologic conditions, we employed tandem affinity purification (TAP) using tagged HPV16 E6 stably expressed in the HPV16-positive cervical cancer cell line SiHa. We have discovered several new interacting proteins, including an interaction between E6 and the cellular deubiquitylating enzyme (DUB) USP15. USP15 is not targeted for degradation by E6, but we found that USP15 stabilizes E6 protein levels, suggesting that E6 may itself be a target for USP15 DUB activity.     

The <Emphasis Type="Italic">PIK3CA</Emphasis> E542K and E545K mutations promote glycolysis and proliferation via induction of the β-catenin/SIRT3 signaling pathway in cervical cancer

Background

The study aims to present the effect of PIK3CA E542K and E545K mutations on glucose metabolism and proliferation and identify their underlying mechanisms in cervical cancer.

Methods

The maximum standard uptake value (SUV_max) of tumors was detected by¹⁸F-FDG PET/CT scan. In vitro, glycolysis analysis, extracellular acidification rate analysis, and ATP production were used to evaluate the impact of PIK3CA E542K and E545K mutations on glucose metabolism. The expression level of key glycolytic enzymes was evaluated by western blotting and immunohistochemical staining in cervical cancer cells and tumor tissues, respectively. Immunofluorescence analysis was used to observe the nuclear translocation of β-catenin. The target gene of β-catenin was analyzed by using luciferase reporter system. The glucose metabolic ability of the xenograft models was assessed by SUV_max from microPET/CT scanning.

Results

Cervical cancer patients with mutant PIK3CA (E542K and E545K) exhibited a higher SUV_max value than those with wild-type PIK3CA (P =?0.037), which was confirmed in xenograft models. In vitro, enhanced glucose metabolism and proliferation was observed in SiHa and MS751 cells with mutant PIK3CA. The mRNA and protein expression of key glycolytic enzymes was increased. AKT/GSK3β/β-catenin signaling was highly activated in SiHa and MS751 cells with mutant PIK3CA. Knocking down β-catenin expression decreased glucose uptake and lactate production. In addition, the nuclear accumulation of β-catenin was found in SiHa cells and tumors with mutant PIK3CA. Furthermore, β-catenin downregulated the expression of SIRT3 via suppressing the activity of the SIRT3 promotor, and the reduced glucose uptake and lactate production due to the downregulation of β-catenin can be reversed by the transfection of SIRT3 siRNA in SiHa cells with mutant PIK3CA. The negative correlation between β-catenin and SIRT3 was further confirmed in cervical cancer tissues.

Conclusions

These findings provide evidence that the PI3K E542K and E545K/β-catenin/SIRT3 signaling axis regulates glucose metabolism and proliferation in cervical cancers with PIK3CA mutations, suggesting therapeutic targets in the treatment of cervical cancers.

Trial registration

FUSCC 050432–4-1212B. Registered 24 December 2012 (retrospectively registered).

     

Expression and Functional Analysis of Toll-like Receptor 4 in Human Cervical Carcinoma

Toll-like receptors are expressed in human immune cells and many tumors, but the role of toll-like receptor 4 (TLR4) in the development of tumors is controversial. We demonstrated the expression, distribution, and functional activity of TLR4 in tissues of normal cervix, cervical intraepithelial neoplasia (CIN), invasion cervical cancers (ICC), and different human papillomavirus (HPV)-infected cervical cancer cells. The results showed that TLR4 expression was in accordance with the histopathological grade: higher in ICC than in CIN, and low in normal cervical tissues and malignant cervical stroma. Expression was higher in SiHa (HPV16+) than in HeLa (HPV18+) cells, but was not observed in C33A (HPV?) cells. After treatment with its agonist, lipopolysaccharide (LPS), the expression levels of TLR4 was increased and apoptosis resistance was induced in SiHa cells, but not in HeLa or C33A cells. Meanwhile, LPS treatment did not alter the cell cycle distribution in SiHa cells. The mechanism of apoptosis resistance may be related to HPV16 infection and not correlated with the cell cycle distribution. Targeting TLR4 in combination with traditional drug treatment may serve as a novel strategy for more effectively killing cancer cells.     

Simvastatin induced HCT116 colorectal cancer cell apoptosis through p38MAPK-p53-survivin signaling cascade

Background

Statins, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors with cholesterol-lowering properties, were recently shown to exhibit anti-cancer effects. However, the molecular mechanism underlying statin-induced cancer cell death remains to be elucidated. Elevated level of survivin is often found over-expressed in human cancers and has been implicated in the progression of tumorigenesis. Given its central role in cell division and action as an apoptosis suppressor, survivin represents a potential molecular target in cancer management.

Methods

In this study, we explored the underlying mechanisms in simvastatin-induced HCT116 colorectal cancer cell apoptosis.

Results

Simvastatin decreased cell viability and induced cell apoptosis in HCT116 cells. These results are associated with the modulation of p21^cip/Waf1 and survivin. Survivin knockdown using survivin siRNAs also decreased cell viability and induced cell apoptosis. Simvastatin's actions on p21^cip/Waf1, survivin and apoptosis were reduced in p53 null HCT116 cells. Simvastatin caused an increase in p53 phosphorylation and acetylation. In addition, simvastatin activated p38 mitogen-activated protein kinase (p38MAPK), whereas an inhibitor of p38MAPK signaling abrogated simvastatin's effects of increasing p53 and p21^cip/Waf1 promoter luciferase activity. Cell viability and survivin promoter luciferase activity in the presence of simvastatin were also restored by p38MAPK inhibitor. Furthermore, Sp1 binding to the survivin promoter region decreased while p53 and p63 binding to the promoter region increased after simvastatin exposure.

Conclusions

Simvastatin activates the p38MAPK-p53-survivin cascade to cause HCT116 colorectal cancer cell apoptosis.

General significance

This study delineates, in part, the underlying mechanisms of simvastatin in decreasing survivin and subsequent colorectal cancer cell apoptosis.     

<Emphasis Type="Italic">Human papillomavirus</Emphasis> type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response

Background

Despite vaccination and screening measures, anogenital cancer, mainly promoted by HPV16 oncoproteins, still represents the fourth tumor and the second cause of death among women.Cell replication fidelity is the result of the host DNA damage response (DDR). Unlike many DNA viruses that promote their life cycle through the DDR inactivation, HR-HPVs encourage cells proliferation despite the DDR turned on. Why and how it occurs has been only partially elucidated.During HPV16 infection, E6 links and degrades p53 via the binding to the E6AP LXXLL sequence; unfortunately, E6 direct role in the DDR response has not clearly identified yet.Similarly, E7 increases DDR by competing with E2F1-pRb interaction, thus leading to the inactivation of pRb, and promotion, E2F1 mediated, of DDR genes translation, by binding to the pRb-like proteins CBP/p300 and p107, that also harbour LXXLL sequence, and via the interaction and activation of several DDR proteins.

Methods

To gain information regarding E6 and E7 contribution in DDR activation, we produced an in vitro 3D HPV16-E6E7 infected epithelium, already consolidated study model for HPVs, and validated it by assessing H&E staining and BrdU, HPV16 DNA, E6E7 proteins and γH2A.X/53BP1 double-strand break (DSBs) sensors expression; then we made an immuno-colocalization of E6 and E7 with cyclin E2 and B1.Since 53BP1, like E6 and E7, also binds p53 and pRb, we supposed their possible direct binding. To explore this hypothesis, we performed a double immunofluorescence of E6 and E7 with 53BP1, a sequence analysis of 53BP1 within its BRCT2 domain and then an in situ PLA within CaSki, E6E7HPV16 NHEKs and the 3D model.

Results

The in vitro epithelium resembled the histology and the events typical of in vivo infected tissues. E6E7HPV16 were both expressed in basal and differentiated strata and induced H2A.X phosphorylation and 53BP1 increment into nuclear foci.After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the bindings via the PLA technique.

Conclusions

Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.

     

GRIM-19 Disrupts E6/E6AP Complex to Rescue p53 and Induce Apoptosis in Cervical Cancers

Background

Our previous studies showed a down-regulation of GRIM-19 in primary human cervical cancers, and restoration of GRIM-19 induced tumor regression. The induction of tumor suppressor protein p53 ubiquitination and degradation by E6 oncoportein of high risk-HPV through forming a stable complex with E6AP is considered as a critical mechanism for cervical tumor development. The aims of this study were to determine the potential role of GRIM-19 in rescuing p53 protein and inducing cervical cancer cell apoptosis.

Methodology/Principal Findings

The protein levels of GRIM-19 and p53 were detected in normal cervical tissues from 45 patients who underwent hysterectomy for reasons other than neoplasias of either the cervix or endometrium, and cervical cancer tissues from 60 patients with non-metastatic squamous epithelial carcinomas. Coimmunoprecipitation and GST pull-down assay were performed to examine the interaction of GRIM-19 with 18E6 and E6AP in vivo and in vitro respectively. The competition of 18E6 with E6AP in binding GRIM-19 by performing competition pull-down assays was designed to examine the disruption of E6/E6AP complex by GRIM-19. The augment of E6AP ubiquitination by GRIM-19 was detected in vivo and in vitro ubiquitination assay. The effects of GRIM-19-dependent p53 accumulation on cell proliferation, cell cycle, apoptosis were explored by MTT, flow cytometry and transmission electron microscopy respectively. The tumor suppression was detected by xenograft mouse model.

Conclusion/Significance

The levels of GRIM-19 and p53 were concurrently down regulated in cervical cancers. The restoration of GRIM-19 can induce ubiquitination and degradation of E6AP, and disrupt the E6/E6AP complex through the interaction of N-terminus of GRIM-19 with both E6 and E6AP, which protected p53 from degradation and promoted cell apoptosis. Tumor xenograft studies also revealed the suppression of p53 degradation in presence of GRIM-19. These data suggest that GRIM-19 can block E6/E6AP complex; and synergistically suppress cervical tumor growth with p53.     

Specific apoptosis induction in human papillomavirus-positive cervical carcinoma cells by photodynamic antisense regulation

Human papillomavirus type 18 (HPV18) is frequently detected in cervical cancer cells. The viral proteins E6 and E7 are expressed consistently and have oncogenic activities. The E7 protein binds to a tumor suppressor, the retinoblastoma gene product (pRB), however, leading to the stabilization of tumor suppressor, p53 protein. On the other hand, another viral product, E6, forms complexes with p53 and abrogates its function, resulting in tumor progression. These facts imply that the E6 oncogene is one of the ideal targets for directed gene therapy in HPV-positive cervical cancer. In this study, we tried photodynamic antisense regulation of the antiapoptotic E6 expression using a photocross-linking reagent, 4,5',8-trimethylpsoralen, conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo). This photodynamic antisense strategy effectively elicited the apoptotic death of HPV18-positive cervical cancer cells through the selective repression of E6 mRNA and consequent stabilization of p53 protein. E7-mediated signals potentially activated the p53 function and mobilized the p53 pathway to deliver pro-apoptotic signals to the cancer cells, leading to the suppression of in vivo tumorigenesis. An extremely low concentration of cisplatin in addition to Ps-S-Oligos further up-regulated p53 activity, provoking massive apoptotic induction. These results suggest that the photodynamic antisense strategy has the great therapeutic potential in HPV-positive cervical cancers.     

Premature senescence in human breast cancer and colon cancer cells by tamoxifen-mediated reactive oxygen species generation

Aims

Cellular senescence is an important tumor suppression process in vivo. Tamoxifen is a well-known anti-breast cancer drug; however, its molecular function is poorly understood. Here, we examined whether tamoxifen promotes senescence in breast cancer and colon cancer cells for the first time.

Main methods

Human breast cancer MCF-7, T47D, and MDA-MB-435 and colorectal cancer HCT116 cells were treated with tamoxifen. Cellular senescence was measured by SA-β-gal staining and based on the protein expression of p53 and p21^Cip1/WAF1. The production of reactive oxygen species (ROS) was determined by staining with CM-H₂DCFDA and dihydroethidium (DHE). CK2 activity was assessed with a specific peptide substrate.

Key findings

Tamoxifen promoted senescence phenotype and ROS generation in MCF-7 and HCT116 cells. The ROS scavenger, N-acetyl-l-cysteine (NAC), and the NADPH oxidase inhibitor, apocynin, almost completely abolished this event. Tamoxifen inhibited the catalytic activity of CK2. Overexpression of CK2α antagonized senescence mediated by tamoxifen, indicating that tamoxifen induced senescence via a CK2-dependent pathway. A well-known CK2 inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), also stimulated ROS production and senescence in MCF-7 cells. Finally, experiments using T47D (wild-type p53) and MDA-MB-435 (mutant p53) cell lines suggested that tamoxifen induces p53-independent ROS production as well as p53-dependent senescence in breast cancer cells.

Significance

These results demonstrate that tamoxifen promotes senescence through a ROS–p53–p21^Cip1/WAF1 dependent pathway by inhibiting CK2 activity in breast cancer and colon cancer cells.     

Transducing properties of a pre-structured α-helical DPT-peptide containing a short canine adenovirus type 2 E4orf4 PP2A1-binding sequence

Background

Induction of the death pathway resulting from the specific interaction of the PP2A₁ phosphatase with adenoviral E4orf4 protein is a promising approach for cancer therapy. With the aim of deregulating tumor pathways, and mimicking E4orf4 anti-cancer signal, we have previously proposed the DPT technology concept, based on design of specific PP1/PP2A interacting penetrating peptides.

Methods

Using biochemical, structural and cell survival experiments, we have characterized new DPT-peptides containing short PP2A binding sequences.

Results

We identified overlapping sequences, located within the N-terminal domain E4orf4_23-46 of canine adenoviral E4orf4 protein, that interact with the PP2A-Bα subunit of PP2A₁ holoenzyme. We characterized DPT-E4orf4₄ and TAT-E4orf4₄, two bi-partite cell penetrating peptides containing the 12 PP2A₁ binding residues of the canine type 2 E4orf4_27-38 sequence, respectively fused to the DPT-sh1 and TAT shuttle sequences. Surprisingly DPT-E4orf4₄, in contrast to inactive TAT-E4orf4₄, adopted a well defined α-helical structure and co-precipitated PP2A₁ from HeLa cell extracts. DPT-E4orf4₄ also internalized streptavidin-HRP and inhibited survival of HeLa cells more efficiently than TAT, TAT-E4orf4₄ or the previously published anti-tumor TAT-derived peptide shepherdin. DPT-E4orf4₄ also efficiently inhibited the survival of human adherent transformed cells, including wild type and p53 mutated colonic HCT116 cells, without affecting survival of human non-transformed fibroblasts.

Conclusions

We characterized the transducing properties of a new α-helical DPT-E4orf4₄ peptide containing a short PP2A-interacting sequence from canine Adenoviral E4orf4 protein.

General significance

Our results suggest that α-helical structured DPT peptides specifically interacting with PP2A could be a valuable anti-cancer drug design scaffold.     

Distribution of Carcinogenic Human Papillomavirus Genotypes and Association to Cervical Lesions among Women in Fez (Morocco)

Objectives

To determine the distribution of cervical high-risk human papillomavirus genotypes and their association to cellular abnormalities in women from Fez and its neighborhood.

Methods

Women attending the Hassan II University Hospital for cervical pap smears were recruited after an informed consent. Interviews and two cervical samples were performed for each woman. Cervical samples were used for cytological analysis and HPV DNA detection. HPV was typed using a method based on multiplex PCR with fluorescently labeled specific primers followed by capillary electrophoresis. The study was approved by the ethics committee of the Faculty of Medicine and Pharmacy of Fez.

Results

The HPV prevalence in the studied population was 43.1% and the most prevalent types were HPV 53 (23 cases); HPV 16 (20 cases); HPV 35 (18 cases); HPV 51 (10 cases) and HPV 56 (7 cases). From the 619 confirmed pap smears, 20% were abnormal. The cytological abnormalities were significantly associated to HPV infection, women age, number of pregnancies and parity (p < 0.05).

Conclusion

More attention should be given to HPV in Morocco because it represents an important public health concern. The distribution of carcinogenic HPV types in the studied population is different from the data in other regions but epidemiological studies in other Moroccan regions are required.