HPV-positive Cervical Carcinoma Cells Research Articles

Plumbagin as a preferential lead molecule to combat EGFR-driven matrix abundance and migration of cervical carcinoma cells.

The handshake between the complex networks of matrix components in the tumor micro-environment (TME) is considered as a crucial event in the progression of several cancers including cervical carcinoma (CC). A number of studies report a connection between epidermal growth factor (EGF) and matrix component production. Studies demonstrate that the mechano-transduction trigger by collagen, influences the tumor cells to undergo epithelial-mesenchymal transition (EMT) and block the entryof drugs. We hypothesize that the intervention to prevent EGF triggered deposition of matrix components could sensitize several therapies for CC cells. We utilized morphological assessment, MTT assay, mitored tracking, acridine orange (AO)/ ethidium bromide (EtBr) staining and bromodeoxyuridine (BrdU) assay to measure the cell viability, mitochondrial activity, cellular apoptosis, and DNA synthesis. Clonogenic assay and scratch healing assay were executed to address the stemness and migratory potential. Detection of glycosaminoglycan's (GAGs), collagen, matrix metalloproteinase (MMP)-2/9 secretion and calcium (Ca2+) ions were performed to assess the production of matrix components. Finally, the interaction between EGFR and plumbagin was evaluated by employing molecular dynamics (MD) simulation. Pre-treating the cells with plumbagin inhibited the EGF-induced EMT along with reduction in cell proliferation, migration, clonogenesis and depletion of matrix components. The actions of EGF and plumbagin were more pronounced in HPV-positive CC cells than HPV-negative CC cells. This study identified that increased matrix production triggered by EGF-rich milieu is inhibited by plumbagin in human papilloma viral (HPV) 68 positive ME180, HPV 16 positive SiHa and HPV-negative C33A cell lines. Delivery of plumbagin directly to TME would effectively accelerate the clearance of CC cells, reduce metastasis and matrix abundance by employing targeted delivery to minimize the undesired effects of plumbagin.

A One-Pot CRISPR/Cas9-Typing PCR for DNA Detection and Genotyping

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9 (Cas9) has high specificity to its target DNA as a gene editing tool. This characteristic makes it useful for DNA detection. Combining the advantages of CRISPR/Cas9 and PCR, this study establishes a novel CRISPR/Cas9-based DNA detection method, named CRISPR/Cas9-typing PCR version 4.0 (ctPCR4.0). This method can detect target DNA in one pot with high specificity and sensitivity. In a homogenous reaction, the target DNA is first cleaved by a pair of Cas9- single-guide RNA complexes and thus releases two single strands with free 3' ends, allowing a pair of oligonucleotides to anneal with the strands. The annealed oligonucleotides provide templates for DNA polymerization from the free 3' ends. A universal primer annealing site is thus produced at the end of two single strands. The target DNA is then amplified by PCR using a universal primer. This method was first verified by accurately detecting the cloned L1 fragments of 10 genotypes of high-risk human papilloma viruses (HPVs). This method was then validated by detecting the L1 fragments of two highest-risk HPVs, HPV 16 and HPV 18, in the genomic DNA of two HPV-positive cervical carcinoma cells, HeLa and SiHa. Finally, this method was further validated by accurately detecting 10 high-risk HPVs in 30 clinical samples.

LncRNA SNHG8 accelerates proliferation and inhibits apoptosis in HPV-induced cervical cancer through recruiting EZH2 to epigenetically silence RECK expression.

Infection of human papillomaviruses (HPVs), such as subtypes HPV16 and HPV18 is carcinogenic to human and is prominent cause of HPV-positive cervical carcinoma (CC). A closer investigation into the mechanism of HPV-induced CC may stimulate the generation of an improved therapy treating cervical cancer. Our study herein interrogated the function of a small nucleolar RNA host gene 8 (SNHG8) in HPV-induced CC. As a result, a notable increase of SNHG8 in HPV-induced CC cells was found compared with HPV-negative CC cells. Functionally, it identified that SNHG8 aggravated the cell proliferation and migration in Cell Counting Kit-8 and transwell assays. Besides, flow cytometry apoptosis assay displayed that blockade of SNHG8 exacerbated apoptosis of HPV-positive CC cells. As detected by fluorescence in situ hybridization analysis and subcellular fractionation assay, SNHG8 was primarily expressed in the nucleus and exerted suppressive role on reversion inducing cysteine-rich protein with kazal motifs (RECK) expression, which implied a potential transcriptional regulation of SNHG8 on RECK level. Mechanically, SNHG8 was disclosed to interact with enhancer of zeste homolog 2 (EZH2) based on RNA immunoprecipitation assay. ChIP assay further unveiled the occupancy of EZH2 in the promoter region of RECK. An additional chromatin immunoprecipitation assay highlighted that SNHG8 intensified the enrichment of EZH2 and H3K27me3 in RECK promoter region. Altogether, it reflected that SNHG8 recruited EZH2 to downregulate RECK expression, leading to HPV-induced CC aggravation.

Long non-coding RNA-OIS1 inhibits HPV-positive, but not HPV-negative cervical squamous cell carcinoma by upregulating MTK-1.

Long non-coding RNA-oncogene-induced senescence 1 (lncRNA-OIS1) is a novel lncRNA that is involved in oncogene-induced senescence, while its functionality in cervical squamous cell carcinoma is unknown. In the present study, 68 human papillomavirus (HPV)-positive and 22 HPV-negative patients with cervical squamous cell carcinoma were recruited. Additionally, 40 healthy females were employed as healthy controls. Tumor tissues and adjacent healthy tissues were collected from all patients with cervical squamous cell carcinoma, and blood samples were obtained. Expression of OIS1 was detected by reverse transcription-quantitative polymerase chain reaction. Receiver operating characteristic curve analysis was used to evaluate the diagnostic value of OIS1 for cervical squamous cell carcinoma. HPV-positive and HPV-negative cervical squamous cell carcinoma and normal cervical cell lines were used, and the effects of OIS1 or mitogen-activated protein kinase kinase kinase 4, (MTK-1) expression vector transfection on the proliferation of cell lines and MTK-1 expression were detected by CCK-8 assay and western blotting, respectively. It was established that a reduction in OIS1 expression level in tumor tissues was apparent only in HPV-positive patients. Serum levels of OIS1 were lower in HPV-positive patients compared with that in HPV-negative patients and healthy controls, and no significant differences were observed between HPV-negative patients and healthy controls. Serum levels of OIS1 were significantly associated with tumor size, but not distant tumor metastasis. OIS1 expression level was lower in HPV-positive cancer cell lines compared with that in HPV-negative cancer cell lines, while no significant differences were observed between HPV-positive and HPV-negative normal cell lines. OIS1 overexpression inhibited and MTK-1 overexpression promoted the proliferation of HPV-positive, but not HPV-negative cancer or normal cell lines. OIS1 transfection also decreased the expression of MTK-1 in HPV-positive cancer cell lines, but not in any of the other cell lines. Therefore, it was concluded that OIS1 inhibited HPV-positive, but not HPV-negative cervical squamous cell carcinoma by upregulating MTK-1.

Detecting and typing target DNA with a novel CRISPR-typing PCR (ctPCR) technique

This study develops a new method for detecting and typing the interested DNAs based on CRISPR, which was named as ctPCR3.0, representing CRISPR- or Cas9/sgRNA-typing PCR, version 3.0. This technique detects target DNA in just one homogeneous step: quantitative PCR (qPCR) amplifying the Cas9/sgRNA-cleaved DNA samples. By directly adding Cas9 and sgRNA into the qPCR reaction and giving an additional isothermal incubation before qPCR program, the target DNA can be homogeneously detected in as few as 2 h. Without opening the detecting tube in the whole detection process, ctPCR3.0 can be used to detect target DNA as the traditional qPCR detection. The technique was fully verified by detecting the cloned HPV L1 genes of 10 high-risk human papillomavirus (HPV) subtypes. The technique also successfully detected the L1 and E6-E7 genes of two highest-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells, HeLa and SiHa. Finally, the ctPCR3.0 method was validated by successfully detecting HPVs in many clinical samples. By performing these detections, this study thus provides a new CRISPR-based DNA detection and typing platform and a ready-to-use HPV clinical detection technique. The platform has wide application in clinical diagnosis.

Detection of target DNA with a novel Cas9/sgRNAs-associated reverse PCR (CARP) technique.

This study develops a new method for detecting target DNA based on Cas9 nuclease, which was named as CARP, representing CRISPR- or Cas9/sgRNAs-associated reverse PCR. This technique detects target DNA in three steps: (1) cleaving the detected DNA sample with Cas9 in complex with a pair of sgRNAs specific to target DNA; (2) ligating the cleaved DNA with DNA ligase; (3) amplifying target DNA with PCR. In the ligation step, the Cas9-cut target DNA was ligated into intramolecular circular or intermolecular concatenated linear DNA. In the PCR step, the ligated DNA was amplified with a pair of reverse primers. The technique was verified by detecting HPV16 and HPV18 L1 genes in nine different human papillomavirus (HPV) subtypes. The technique also detected the L1 and E6-E7 genes of two high-risk HPVs, HPV16 and HPV18, in the genomic DNA of two HPV-positive cervical carcinoma cells (HeLa and SiHa), in which no L1 and E6-E7 genes were detected in the HPV-negative cervical carcinoma cell, C-33a. By performing these proof-of-concept experiments, this study provides a new CRISPR-based DNA detection and typing method. Especially, the CARP method developed by this study is ready for the clinical HPV detection, which was supported by the final clinical sample detection. Graphical abstract CRISPR-associated reverse PCR (CARP) can be used to detect and type target DNA in a simple three-step procedure, cutting, ligation, and amplification.

Alterations of MicroRNA Expression Patterns in Human Cervical Carcinoma Cells (Ca Ski) toward 1′S-1′-Acetoxychavicol Acetate and Cisplatin

The aims of this study were to investigate the combined effects of a natural compound 1'S-1'-acetoxychavicol acetate (ACA) with cisplatin (CDDP) on HPV-positive human cervical carcinoma cell lines (Ca Ski-low cisplatin sensitivity and HeLa-high cisplatin sensitivity), and to identify microRNAs (miRNAs) modulated in response toward ACA and/or CDDP. It was revealed that both ACA and CDDP induced dose- and time-dependent cytotoxicity when used as a stand-alone agent, while synergistic effects were observed when used in combination with a combination index (CI) value of 0.74 ± 0.01 and 0.85 ± 0.01 in Ca Ski and HeLa cells, respectively. A total of 25 miRNAs were found to be significantly differentially expressed in response to ACA and/or CDDP. These include hsa-miR-138, hsa-miR-210, and hsa-miR-744 with predicted gene targets involved in signaling pathways regulating apoptosis and cell cycle progression. In conclusion, ACA acts as a chemosensitizer which synergistically potentiates the cytotoxic effect of CDDP in cervical cancer cells. The altered miRNA expression upon administration of ACA and/or CDDP suggests that miRNAs play an important role in anticancer drug responses, which can be manipulated for therapeutic purposes.

Lopinavir Up-Regulates Expression of the Antiviral Protein Ribonuclease L in Human Papillomavirus-Positive Cervical Carcinoma Cells

We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism. SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used. Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression. These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.

Protein tyrosine phosphatase H1 is a target of the E6 oncoprotein of high-risk genital human papillomaviruses

The E6 proteins of high-risk genital human papillomaviruses (HPV), such as HPV types 16 and 18, possess a conserved C-terminal PDZ-binding motif, which mediates interaction with some cellular PDZ domain proteins. The binding of E6 usually results in their ubiquitin-mediated degradation. The ability of E6 to bind to PDZ domain proteins correlates with the oncogenic potential. Using a yeast two-hybrid system, GST pull-down experiments and coimmunoprecipitations, we identified the protein tyrosine phosphatase H1 (PTPH1/PTPN3) as a novel target of the PDZ-binding motif of E6 of HPV16 and 18. PTPH1 has been suggested to function as tumour suppressor protein, since mutational analysis revealed somatic mutations in PTPH1 in a minor fraction of various human tumours. We show here that HPV16 E6 accelerated the proteasome-mediated degradation of PTPH1, which required the binding of E6 to the cellular ubiquitin ligase E6-AP and to PTPH1. The endogenous levels of PTPH1 were particularly low in HPV-positive cervical carcinoma cell lines. The reintroduction of the E2 protein into the HPV16-positive cervical carcinoma cell line SiHa, known to lead to a sharp repression of E6 expression and to induce growth suppression, resulted in an increase of the amount of PTPH1. Our data suggest that reducing the level of PTPH1 may contribute to the oncogenic activity of high-risk genital E6 proteins.

Role of p53 in antioxidant defense of HPV-positive cervical carcinoma cells following H2O2 exposure

In HPV-positive cervical carcinoma cells, p53 protein is functionally antagonized by the E6 oncoprotein. We investigated a possible role of p53 in antioxidant defense of HPV-positive cervical cancer cell lines. We found that SiHa cells containing integrated HPV 16 had higher expression of p53 and exhibited the greatest resistant to H2O2-induced oxidative damage, compared with HeLa, CaSki and ME180 cell lines. Downregulation of p53 resulted in the inhibition of p53-regulated antioxidant enzymes and elevated intracellular ROS in SiHa cells. By contrast, the ROS level was not affected in HeLa, CaSki and ME180 cell lines after inhibition of the p53 protein. Under mild or severe H2O2-induced stress, p53-deficient SiHa cells exhibited much higher ROS levels than control SiHa cells. Furthermore, we analyzed cell viability and apoptosis after H2O2 treatment and found that p53 deficiency sensitized SiHa cells to H2O2 damage. Inhibition of p53 resulted in excessive oxidation of DNA; control SiHa cells exhibited a more rapid removal of 8-oxo-7,8-dihydro-2'-deoxyguanosine from DNA compared with p53-deficient SiHa cells exposed to the same level of H2O2 challenge. These data collectively show that endogenous p53 in SiHa cells has an antioxidant function and involves in the reinforcement of the antioxidant defense.

Genetic redundancy in human cervical carcinoma cells: Identification of cells with “normal” properties

Although it is generally assumed that cancer arises from a singular cell, a tumor must be considered as a dynamic and emergent biological structure, whose organizing principle is determined by genetic and epigenetic modifications, occurring variably in response to microenvironmental selection conditions. As previously shown, HPV-positive cervical carcinoma cells have lost their ability to induce IFN-beta upon TNF-alpha treatment. However, regarding cancer as a non-linear system, which may, even in the absence of an apparent selection pressure, fluctuate between different "metastable" phenotypes, we demonstrate that TNF-alpha mediated IFN-beta induction is not irreversibly disturbed in all cells. Using the IFN-beta sensitive Encephalomyocarditis virus (EMCV) as a tool to monitor antiviral activity in long-term established malignant HeLa cells, rare IFN-beta expressing clones were rescued from a population of non-responsive and EMCV-sensitive cells. Antiviral activity was mediated by the re-expression of IRF-1 and p48 (IRF-9), both key regulatory molecules normally found to be suppressed in cervical carcinoma cells. Upon inoculating of selected clones into immunocompromised animals, a reduced or even an absence of tumorigenicity of initially highly malignant cells could be discerned. These data indicate that both the absence of interferon signaling and the ability to form tumors were reversed in a minority of cells. We provide a paradigm for the existence of innate genetic redundancy mechanisms, where a particular phenotype persists and can be isolated without application of drugs generally changing the epigenetic context.

Antiproliferative and antiviral mechanisms of ursolic acid and dexamethasone in cervical carcinoma cell lines

The chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid. Herein, we investigated the antiproliferative and antiviral effects of ursolic acid and dexamethasone in human papillomavirus (HPV)-associated cervical cancer cells. We performed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide assay to measure antiproliferative activity, and also characterized apoptosis by DNA fragmentation, 4'-6-diamidino-2-phenylindole (DAPI) staining, and flow cytometry (FACS) analysis. We investigated apoptosis-related proteins using western blots. After in vitro treatment, we used reverse transcription-polymerase chain reaction for the expression of the HPV E6/E7 gene to observe the antiviral effects. Ursolic acid suppressed the growth of HPV-positive cervical carcinoma cells (HeLa, CaSki, and SiHa) in a dose- and time-dependent manner, but not the HPV-negative cervical cancer cell line (C33A). Ursolic acid-treated HeLa cells showed typical apoptosis characteristics in DNA fragmentation, DAPI staining, and FACS analysis. The expression of Fas protein was induced, and caspase-8, caspase-3, and poly ADP-ribose polymerase (PARP) proteins were cleaved after ursolic acid treatment. HPV-18 E6/E7 gene expression decreased after ursolic acid treatment in HeLa cells, but the levels of p53 and Rb proteins did not change. In contrast, dexamethasone, which has a similar structure, did not inhibit proliferation. Our findings may offer new insight into the mechanism of antiproliferative and antiviral effect of ursolic acid. Also, these results suggest that ursolic acid might be a useful anticancer drug in treatment of HPV-associated cervical neoplasia.

Oncolytic Adenovirus Expressing a p53 Variant Resistant to Degradation by HPV E6 Protein Exhibits Potent and Selective Replication in Cervical Cancer

Rationale in the development of novel treatment strategies for HPV-associated cancers is targeting on the basis of the presence of HPV in (pre)malignant cells. Here, we designed a new conditionally replicating adenovirus (CRAd) for selective and effective oncolytic replication in HPV-containing cells. As the backbone, we used the CRAd AdCB016, which replicates selectively in cells expressing HPV E6 and E7 proteins. To enhance its oncolytic potency, we armed AdCB016 with p53 variant mp53(268N), which is resistant to HPV E6-mediated degradation. The new CRAd AdCB016-mp53(268N) was analyzed for its lytic replication properties in cervical carcinoma cell lines, HPV-immortalized keratinocyte cell lines representing dysplastic cells, and primary human keratinocytes. AdCB016-mp53(268N) exhibited 10- to 1000-fold greater efficacy than AdCB016 on high-risk HPV-positive cervical carcinoma cells and HPV-immortalized keratinocytes. Importantly, infection with AdCB016-mp53(268N) did not affect primary nonmalignant human keratinocytes. This favorable efficacy and safety profile was confirmed in organotypic raft cultures. Our findings suggest that AdCB016-mp53(268N) is a promising new agent for treatment of HPV-associated human cancers.

Expression of membrane type 1 matrix metalloproteinase in papillomavirus-positive cells: role of the human papillomavirus (HPV) 16 and HPV8 E7 gene products

Matrix metalloproteinases (MMPs) degrade extracellular matrix. They are involved in cellular proliferation, migration, angiogenesis, invasion and metastasis. MT-1 MMP, a membrane-bound MMP, is expressed in carcinomas of the uterine cervix in vivo. This type of cancer is associated with human papillomavirus (HPV) infection. Here it was shown that keratinocytes transformed with HPV16 or HPV18 in vitro, and HPV-positive cervical carcinoma cell lines, constitutively expressed MT-1 MMP. Expression of the E7 protein from the mucosal and cutaneous high-risk types HPV16 and HPV8, but not from the cutaneous low-risk type HPV1, was sufficient to induce MT-1 MMP expression in primary human keratinocytes and HaCaT cells. As a consequence, MMP-2 was activated. MT-1 MMP expression might play a role in the HPV life cycle by promoting proliferation of host cells and might contribute to their invasive phenotype during malignant progression.

Transition to an irreversible state of senescence in HeLa cells arrested by repression of HPV E6 and E7 genes

Inhibition of human papillomavirus (HPV) E6 and E7 transcription by means of the E2 protein of bovine papillomavirus 1 (BPV1) has been shown to induce acute growth arrest in HPV-positive cervical carcinoma cells. This state of arrest is marked by the expression of senescence phenotypes including SA β-Gal activity and lipofuscin accumulation. In this study, we examined the reversibility of these phenotypes by exogenously expressing the E6 and E7 genes into HeLa cells growth-arrested by the depletion of E6/E7. Re-expression of E7 (but not E6) in 2 days following E2 transduction induced the cells to resume growth. The proliferating cells manifested the phenotype of untreated HeLa cells, suggesting that E7 is the major factor responsible for the continued proliferation and the suppression of the senescence phenotype in cervical carcinoma cells. However, E7 in 5 days following E2 transduction did not prevent HeLa cells from entering the senescent state, indicating that the arrested state becomes irreversible. Our results suggest that, upon depletion of the viral oncoproteins, a senescent state is irreversibly induced in HeLa cells after a period of commitment. The status and cellular location of certain factors involved in signal transduction and cell cycle control was altered as well along with this irreversibility transition.

Selective suppression of monocyte chemoattractant protein-1 expression by human papillomavirus E6 and E7 oncoproteins in human cervical epithelial and epidermal cells.

Infection of cervical keratinocytes by high-risk HPV is involved in the etiology of cervical carcinoma. Since viral products are immunogenic, development of cancer may require suppression of immune responses directed against infected epithelial cells. Many markers of host immune effector responses decrease as cervical intraepithelial neoplasia progresses. Among these is epithelial cell expression of the chemokine MCP-1, though the mechanism for its suppression is unclear. Here, we show that the E6 and E7 viral oncogenes from high-risk HPV, individually and together, suppress MCP-1 expression in primary epithelial cells derived from the female genital tract. This is not a consequence of global suppression of chemokine expression since other chemokines, including IP-10, IL-8 and RANTES, were less affected. Furthermore, 4 of 6 HPV-positive cervical carcinoma cell lines did not express MCP-1. Our data indicate that suppression of MCP-1 expression is part of the program of high-risk HPV E6/E7-induced transformation of primary epithelial cells. These observations are consistent with a model in which MCP-1 expression by infected keratinocytes, which would stimulate an immune attack on HPV-transformed cells, is suppressed for invasive cervical cancer to appear.

Iron withdrawal strategies fail to prevent the growth of SiHa-induced tumors in mice

Objective Cervical carcinoma is a human papillomavirus (HPV)-associated cancer for which treatment options still mainly rely on surgical procedures, with or without adjuvant radiotherapy and chemotherapy. We have previously shown that the chemically unrelated iron chelators desferrioxamine and deferiprone inhibit the growth and induce the apoptosis of HPV-positive cervical carcinoma cell lines, suggesting that iron chelators may represent a potential therapeutic approach for the treatment of cervical carcinoma. The present study was designed to investigate the effect of iron deprivation on the growth of human cervical carcinoma xenografts in athymic nude mice. Methods Nude mice (nu/nu) of BALB/c background were treated with iron chelators [desferrioxamine (DFO), deferiprone (L1), or starch-DFO conjugate] or were fed with an iron-poor diet 6 weeks prior to subcutaneous injection of Si-Ha cells. These treatments were continued for 5 weeks after injection of the tumor cells. Results Treatment with the maximum tolerated doses of DFO, L1, or starch-DFO conjugate induced no significant iron deprivation in non-iron-overloaded mice, while an iron-poor diet led to a dramatic decrease in serum iron, transferrin iron saturation, and ferritin levels. However, neither iron chelators nor an iron-poor diet could significantly inhibit tumor growth. Conclusion Despite a potent antitumor effect in vitro, iron chelators fail to prevent the growth of cervical carcinoma xenografts in mice. On the basis of these results, clinical trials with iron chelators in patients with cervical carcinoma appear inappropriate.

Antiproliferative and Antiviral Mechanism of Ursolic Acid in Cervical Carcinoma Cell Lines

Objective : Ursolic acid which has been used as a herb medication, was reported for anti-cancer and anti-inflammatory effects. We tried to analyze the anti-proliferative and anti-viral effects of ursolic acid in Human papillomavirus (HPV) associated cervical carcinoma cell lines. Methods : We treated ursolic acid or dexamethasone to cervical carcinoma cell lines. We carried out MTT assay to observe the anti-proliferative effects and tried to find out the characteristics of apoptosis by DNA fragmentation, DAPI staining and FACS analysis. By means of western blotting, we investigated proteins related with apoptosis. After treating of ursolic acid, we used RT-PCR for the expression of HPV E6 and E7 gene to observe the anti-viral effects. Through proteomics analysis including two-dimensional electrophorosis (2DE) and matrix-associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we found the interaction between cell cycle regulatory factor and apoptosis related protein, and also demonstrated anti-cancer and anti-viral mechanism. Results : While the growth of HPV-positive cervical carcinoma cell lines (HeLa, CaSki, SiHa) is suppressed by ursolic acid in a dose- and time-dependent manner, that of HPV-negative cervical carcinoma cell line (C33A) did not have any inhibitory effects on proliferation. As we increased the concentrations of ursolic acid in HeLa cell, the typical characteristics of apoptosis were noted in DNA fragmentation, DAPI staining and FACS analysis, The expression of Fas protein was induced and caspase-8, caspase-3 and PARP proteins changed into cleavaged forms after treating ursolic acid. HPV-18 E6/E7 gene expression decreased after treating ursolic acid in HeLa cells, but the levels of p53 and Rb proteins did not changed. Total 30 spots were detected by proteomics analysis, along with 20 up-regulated, 6 down-regulated and 4 unknown protein were founded. Conclusion : These results suggest that ursolic acid inhibit the cell proliferation through apoptosis in HPV-associated cervical carcinoma cell lines. The apoptotic mechanism of ursolic acid in HeLa cells might be through the signal pathway “Fas→caspase-8→PARP”. In addition, the antiviral effect of ursolic acid through suppression of HPV E6/E7 gene expression would contribute to growth inhibition in HeLa cells. These results suggest that ursolic acid could be useful for an effective anticancer drug in treatment of HPV-associated cervical neoplasia.

Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference.

Selective silencing of mammalian gene expression has recently been achieved using short interfering RNA (siRNA). Synthetic siRNA targets homologous mRNA for degradation and the process is highly efficient. Here we demonstrate siRNA silencing of pathogenic viral gene expression. As a well characterized model we chose cervical carcinoma cells positive for human papillomavirus type 16. Over 90% of human cervical cancers are positive for papillomavirus and abnormal cell proliferation is driven by co-operative effects of viral E6 and E7 genes. We sought to silence HPV E6 and E7 gene expression using siRNAs to target the respective viral mRNAs. Our results indicate selective degradation of E6 and E7 mRNAs. Silencing was sustained for at least 4 days following a single dose of siRNA. E6 silencing induced accumulation of cellular p53 protein, transactivation of the cell cycle control p21 gene and reduced cell growth. In contrast, E7 silencing induced apoptotic cell death. HPV-negative cells appeared unaffected by the anti-viral siRNAs. Thus we demonstrate for the first time (i) that siRNA can induce selective silencing of exogenous viral genes in mammalian cells, and (ii) that the process of siRNA interference does not interfere with the recovery of cellular regulatory systems previously inhibited by viral gene expression.

Antiviral agent Cidofovir restores p53 function and enhances the radiosensitivity in HPV-associated cancers.

High-risk human papillomaviruses (HPVs) have been associated to the development of cervical and some other human cancers. Most of them express E6 and E7 oncoproteins, able to bind to p53 and retinoblastoma (pRb) tumor suppressor proteins respectively and neutralize their function. Restoration of these pathways by blocking E6 and E7 expression would provide a selective therapeutic effect. Here, we show that a clinically approved antiviral agent Cidofovir reduced E6 and E7 expression in cervical carcinoma Me180 and head and neck squamous cell carcinoma HEP2 cells at the transcriptional level. Cidofovir induced the accumulation of active p53 and pRb associated to induction of cyclin dependent kinase inhibitor p21(WAF1/CIP1) in Me180 and HEP2 cells. p53 induction was also shown in Hela HPV-positive cervical carcinoma cell line. In addition, S phase cell cycle accumulation with concomitant decrease of cyclin A expression were associated to the antiproliferative activity of Cidofovir in HPV-treated cells. Combining Cidofovir to irradiation both in vivo and in nude mice xenografts resulted in a marked radiosensitization in HPV-positive cells, which was not observed in virus negative cells. This study provides the basis for a new anticancer strategy to enhance the antitumor effect of ionizing radiation in HPV-related cancers, without increase deleterious effects.