Investigation of the influence of high-risk human papillomavirus on the biochemical composition of cervical cancer cells using vibrational spectroscopy

The main aetiology of cervical cancer is infection with high-risk human papillomavirus (HPV). Cervical cancer is almost 100% curable if detected in the early stages. Thus, information about the presence and levels of HPV in patient samples has high clinical value. As current screening methods, such as the Pap smear test, are highly subjective and in many cases show low sensitivity and specificity, new supportive techniques are desirable to improve the quality of cervical cancer screening. In this study, vibrational spectroscopic techniques (Raman and Fourier Transform Infra Red absorption) have been applied to the investigation of four cervical cancer cell lines: HPV negative C33A, HPV-18 positive HeLa with 20-50 integrated HPV copies per cell, HPV-16 positive SiHa with 1-2 integrated HPV strands per cell and HPV-16 positive CaSki containing 60-600 integrated HPV copies per cell. Results show that vibrational spectroscopic techniques can discriminate between the cell lines and elucidate cellular differences originating from proteins, nucleic acids and lipids. Similarities between C33A and SiHa cells were exhibited in the Raman and infrared spectra and were confirmed by Principal Component Analysis (PCA). Analysis of the biochemical composition of the investigated cells, with the aid of PCA, showed a clear discrimination between the C33A-SiHa group and HeLa and CaSki cell lines indicating the potential of vibrational spectroscopic techniques as a support to current methods for cervical cancer screening.     

Improved immunodetection of human papillomavirus E7

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell lines which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.     

HPV E6 antisense induces apoptosis in CaSki cells via suppression of E6 splicing

Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.     

DNA vaccines for cervical cancer: from bench to bedside

More than 99% of cervical cancers have been associated with human papillomaviruses (HPVs), particularly HPV type 16. The clear association between HPV infection and cervical cancer indicates that HPV serves as an ideal target for development of preventive and therapeutic vaccines. Although the recently licensed preventive HPV vaccine, Gardasil, has been shown to be safe and capable of generating significant protection against specific HPV types, it does not have therapeutic effect against established HPV infections and HPV-associated lesions. Two HPV oncogenic proteins, E6 and E7, are consistently co-expressed in HPV-expressing cervical cancers and are important in the induction and maintenance of cellular transformation. Therefore, immunotherapy targeting E6 and/or E7 proteins may provide an opportunity to prevent and treat HPV-associated cervical malignancies. It has been established that T cell-mediated immunity is one of the most crucial components to defend against HPV infections and HPV-associated lesions. Therefore, effective therapeutic HPV vaccines should generate strong E6/E7-specific T cell-mediated immune responses. DNA vaccines have emerged as an attractive approach for antigen-specific T cell-mediated immunotherapy to combat cancers. Intradermal administration of DNA vaccines via a gene gun represents an efficient way to deliver DNA vaccines into professional antigen-presenting cells in vivo. Professional antigen-presenting cells, such as dendritic cells, are the most effective cells for priming antigen-specific T cells. Using the gene gun delivery system, we tested several DNA vaccines that employ intracellular targeting strategies for enhancing MHC class I and class II presentation of encoded model antigen HPV-16 E7. Furthermore, we have developed a strategy to prolong the life of DCs to enhance DNA vaccine potency. More recently, we have developed a strategy to generate antigen-specific CD4(+) T cell immune responses to further enhance DNA vaccine potency. The impressive pre- clinical data generated from our studies have led to several HPV DNA vaccine clinical trials.     

The differences in heparin binding for the C-terminal basic-sequence-rich peptides of HPV-16 and HPV-18 capsid protein L1

The high-risk types of human papillomaviruses (HPV) HPV-16 and -18 are the predominant types associated with cervical cancer. HPV-16 and -18 account for about 50% and 20%, respectively, of cervical cancers worldwide. While the reason and molecular mechanism of the distinct prevalence and distributions between them remain poorly understood, the binding affinity of cell surface receptor with capsid proteins, especially L1, may be involved. We examined heparin binding with two synthetic peptides corresponding to the 14 amino acid C-terminal peptides of HPV-16 and -18 L1 with the goal of comparing the equivalent residues in different HPV types. Using isothermal titration calorimetry (ITC) and static right-angle light scattering (SLS), we determined the binding constant K, reaction enthalpy ΔH, and other thermodynamic parameters in the interaction. Especially, we assessed the role of specific residues in binding with heparin by comparing the NMR spectra of free and heparin-bound peptides.     

八种黄酮类天然产物对高危型HPV16/18 E6蛋白的分子对接研究

宫颈癌以其高发病率和高死亡率严重危害女性健康,传统治疗手段有效率低且治疗过程给患者带来极大痛苦。近年来,许多天然植物来源化合物已被确定为治疗和预防宫颈癌的有希望的药物来源,但天然产物治疗癌症的具体作用机制尚未明确。因此,本文选取了8种黄酮类天然小分子抑制剂,分别与高危型HPV16/18 E6蛋白重要位点LxxLL疏水口袋进行分子对接研究,以期探索天然产物抗宫颈癌的作用机制。对接分析显示,这些天然产物均与HPV18E6蛋白LxxLL疏水口袋产生较强相互作用,与HPV16 E6蛋白对接时,木犀草素则比其他7种黄酮类化合物结合得更为深入,这些相互作用可能有助于p53功能的恢复。对接分析有助于理解蛋白质-配体相互作用的分子机制,为设计治疗HPV感染的新药提供依据。     

HPV在宫颈炎、宫颈癌前病变、宫颈癌中的检测及其临床意义研究

目的研究并探讨人乳头状瘤病毒(human papillomavirus,简称HPV)在宫颈炎、宫颈癌前病变、宫颈癌中的检测价值及临床意义。方法选取菏泽市妇幼保健院2014年1月—2016年12月期间收治的120例慢性宫颈炎患者、90例宫颈癌前病变患者、60例宫颈癌患者作为研究对象,分别设置为宫颈炎组、癌前病变组、宫颈癌组,并将癌前病变组分为CIN Ⅰ级、CIN Ⅱ级、CIN Ⅲ级,所有患者均接受HPV检测,比较其HPV阳性率、HPV DNA负荷载量、HPV感染持续时间,计算HPV感染持续时间、HPV DNA负荷载量与宫颈癌发生的相关性。结果宫颈炎组、癌前病变组、宫颈癌组的HPV阳性率、HPV DNA负荷载量、HPV感染持续时间比较,差异均具有统计学意义(P0.05),从高至低依次为宫颈癌组、癌前病变组、宫颈炎组,且不同分级宫颈癌前病变患者的HPV DNA负荷载量、HPV感染持续时间均存在显著差异(P0.05);经相关性分析,HPV感染持续时间、HPV DNA负荷载量均与宫颈癌的发生密切相关,呈正相关。结论HPV感染可能参与到宫颈癌前病变、宫颈癌的发生、发展中,开展HPV检测可对宫颈癌前病变、宫颈癌进行筛查,有利于宫颈癌的防治。     

四川地区宫颈癌组织HPV18和HPV45 E6基因突变分析

采用聚合酶链反应技术对四川地区2003~2004年收集的60例宫颈癌患者的癌组织DNA进行人乳头瘤病毒(HPV) E6基因扩增, 获得HPV18和45型E6基因. 序列分析发现, 18型三例E6基因有同样的两处同义突变; 45型两例E6基因发生突变, 一例有两处碱基突变, 另一例发生六处碱基突变, 其中两处涉及氨基酸变化, 均位于E6抗原决定簇区. HPV45型E6基因中134位c→t, 157位c→t, 259位g→t和341位t→c的碱基点突变未见报道. 另外， 该地区HPV18和45型突变株之间存在碱基互变,  它们之间的最小差异比野生型HPV18和HPV45之间的差异小4.05%, 该数值比在非洲发现的突变株的要小很多, 该结果支持HPV18和HPV45可能起源于非洲的观点.     

A simple and efficient method for potential point-of-care diagnosis of human papillomavirus genotypes: combination of isothermal recombinase polymerase amplification with lateral flow dipstick and reverse dot blot

Cervical cancer is the second most common cancer in the world’s woman population with a high incidence in developing countries where diagnostic conditions for the cancer are poor. The main culprit causing the cancer is the human papillomavirus (HPV). HPV is divided into three major groups, i.e., high-risk (HR) group, probable high-risk (pHR) group, and low-risk (LR) group according to their potential of causing cervical cancer. Therefore, developing a sensitive, reliable, and cost-effective point-of-care diagnostic method for the virus genotypes in developing countries even worldwide is of high importance for the cancer prevention and control strategies. Here we present a combined method of isothermal recombinase polymerase amplification (RPA), lateral flow dipstick (LFD), and reverse dot blot (RDB), in quick point-of-care identification of HPV genotypes. The combined method is highly specific to HPV when the conserved L1 genes are used as targeted genes for amplification. The method can be used in identification of HPV genotypes at point-of-care within 1 h with a sensitivity of low to 100 fg of the virus genomic DNA. We have demonstrated that it is an excellent diagnostic point-of-care assay in monitoring the disease without time-consuming and expensive procedures and devices.

     

Treatment of HPV infection-associated cervical condylomata acuminata with 5-aminolevulinic acid-mediated photodynamic therapy

The aim of this study was to investigate the efficacy of 5-aminolaevulinic acid (ALA)-mediated photodynamic therapy (PDT) in treatment of human papillomavirus (HPV)-associated cervical condylomata. A total of 56 patients with cervical and external condylomata lesions were recruited for this open-label study. HPV genotyping of exfoliated cells collected from the cervix and external lesions was performed. Cervical lesions were treated with PDT by applying ALA gel (10%) to the surface of the cervix for 4 h followed by irradiating with a 635 nm laser at 100 J cm(-2). PDT was repeated at 2-week intervals if lesion and HPV infection remained. Patients were followed up for 6-24 months. Genotyping analysis revealed four HPV subtypes (HPV6, 11, 16 and 18). The overall complete remission rate of 1-4 sessions of treatments was 98.2% and the corresponding HPV clearance rate was 83.9%. Ten cases showed complete removal of cervical lesions and HPV infection after a single treatment. Recurrence rate was 3.6%. Adverse effects were minimal and no structural complications were reported. In conclusion, topical ALA PDT is safe and effective for eradicating cervical HPV infection and eliminating condylomata lesion. Its definitive role in treating cervical condylomata deserves further investigation.     

人乳头瘤病毒HPV-16衣壳蛋白的结构特征

人乳头瘤病毒HPV-16是引发子宫颈癌的主要元凶.研究HPV-16病毒的结构特点,对于二十面体病毒几何结构的认识、描述和疫苗设计具有重要意义.综述了HPV-16病毒的衣壳结构特征、衣壳蛋白,分析了它们之间的相互作用,并试探性地指出了HPV-16衣壳几何结构的数学问题.     

In Silico Comparison of Low- and High-Risk Human Papillomavirus Proteins

Human papillomavirus (HPV) is an important pathogen which is classified into two, high- and low-risk groups. The proteins of high-risk and low-risk HPV types have different functions. Therefore, there is a need to develop a computational method for predicting these two groups. In the present study, the physiochemical properties of all early (E1, E2, E4, E5, E6, and E7) and late (L1 and L2) proteins in high- and low-risk HPV types have been studied. The concept of receiver operating characteristic analysis and support vector machines methods has been used for comparison of high- and low-risk HPV types. The results demonstrate that amino acid composition, physiochemical, and secondary structure of E2 protein are significantly different between these two groups. The results demonstrate that in silico properties can create useful information to predict high-risk and low-risk HPV types.     

Electrochemical genosensor array for the simultaneous detection of multiple high-risk human papillomavirus sequences in clinical samples

An electrochemical genosensor array for the simultaneous detection of three high-risk human papillomavirus (HPV) DNA sequences, HPV16, 18 and 45, exhibiting high sensitivity and selectivity is presented. The electrodes of a 4 × 4 array were modified via co-immobilization of a 1:100 (mol/mol) mixture of a thiolated probe and an oligoethyleneglycol-terminated bipodal thiol. Detection of synthetic and PCR products was carried out in a sandwich type format, with the target hybridized between a surface immobilized probe and a horseradish peroxidase-labelled secondary reporter probe. The detection limits obtained in the detection of each individual target were in the pM range, allowing the application of this sensor for the detection of samples obtained from PCR amplification of cervical scrape samples. The results obtained exhibited an excellent correlation with the HPV genotyping carried out within a hospital laboratory. Multiplexing and cross-reactivity studies demonstrated high selectivity over potential interfering sequences, facilitating application of the developed platform for the high-throughput screening of multiple high-risk DNA sequences.     

Effectiveness of Photodynamic Therapy in Elimination of HPV‐16 and HPV‐18 Associated with CIN I in Mexican Women

This study aimed to determine the effectiveness of photodynamic therapy (PDT), using δ‐aminolevulinic acid (5‐ALA), in the elimination of premalignant cervical lesions in Mexican patients with human papillomavirus (HPV) infection and/or cervical intraepithelial neoplasia (CIN). Thirty women diagnosed with CIN I and/or positive for HPV participated in the study. Topical 6% 5‐ALA in gel form was applied to the uterine cervix; after 4 h, the lesion area was irradiated with a light dose of 200 J cm^?2 at 635 nm. This procedure was performed three times at 48‐h intervals. Clinical follow‐up was performed at 3, 6, and 12 months after the initial PDT administration, by colposcopy, cervical cytology, histopathological analysis, polymerase chain reaction, and hybrid capture. Of HPV‐infected patients without evidence of CIN I, 80% cleared the infection, while HPV associated with CIN I was eliminated in 83% of patients (P < 0.05). At 12 months, CIN I had regressed in 57% of patients, although this response was not statistically significant. PDT using 6% 5‐ALA is concluded to be effective in eliminating HPV infection associated or not with CIN I.     

Correlation of p16(INK4A) expression and HPV copy number with cellular FTIR spectroscopic signatures of cervical cancer cells

Cervical cancer, a potentially preventable disease, has its main aetiology in infection by high risk human papillomavirus (HR-HPV). Approaches to improving cervical cancer screening and diagnostic methodologies include molecular biological analysis, targeting of biomarker proteins, but also exploration and implementation of new techniques such as vibrational spectroscopy. This study correlates the biomarker protein p16(INK4A) expression levels dependent on HPV copy number with the infrared absorption spectral signatures of the cervical cancer cell lines, HPV negative C33A, HPV-16 positive SiHa and CaSki and HPV-18 positive HeLa. Confocal fluorescence microscopy demonstrated that p16(INK4A) is expressed in all investigated cell lines in both nuclear and cytoplasmic regions, although predominantly in the cytoplasm. Flow cytometry was used to quantify the p16(INK4A) expression levels and demonstrated a correlation, albeit nonlinear, between the reported number of integrated HPV copies and p16(INK4A) expression levels. CaSki cells were found to have the highest level of expression, HeLa intermediate levels, and SiHa and C33A the lowest levels. FTIR spectra revealed differences in nucleic acid, lipid and protein signatures between the cell lines with varying HPV copy number. Peak intensities exhibited increasing tendency in nucleic acid levels and decreasing tendency in lipid levels with increasing HPV copy number, and although they were found to be nonlinearly correlated with the HPV copy number, their dependence on p16(INK4A) levels was found to be close to linear. Principal Component Analysis (PCA) of the infrared absorption spectra revealed differences between nuclear and cytoplasmic spectroscopic signatures for all cell lines, and furthermore clearly differentiated the groups of spectra representing each cell line. Finally, Partial Least Squares (PLS) analysis was employed to construct a model which can predict the p16(INK4A) expression level based on a spectral fingerprint of a cell line, demonstrating the diagnostic potential of spectroscopic techniques.     

Raman spectroscopic study on classification of cervical cell specimens

Cervix-cancer is the third most common female cancer worldwide. Papanicolaou (Pap) test, a well-recognized screening tool, is labor intensive, time consuming and prone to subjective interpretations. Optical spectroscopic methods, sensitive to molecular changes are being pursued as potential diagnostics tool. In this study we have explored Raman spectroscopic approach to differentiate exfoliated cell pellets using 94 cervical cell specimens (45-normal and 49-abnormal specimens). Study was carried out by two approaches. In the first approach, spectral data from 37 cell specimens were acquired and analyzed by Principal Component-Linear Discriminant Analysis (PC-LDA), which yielded classification efficiencies of 86% and 84% for normal and abnormal specimens, respectively. Mean and difference spectra suggest presence of blood in abnormal specimen as a major cause of discrimination. However, as tumor is vascular, bleeding was observed during abnormal sample collection. Hence, spectra of abnormal specimens show heme and fibrin features, and this can lead to false interpretations, as bleeding also occur in several non-cancerous conditions. Therefore, remaining 57 specimens were treated with Red Blood Corpuscles (RBC) lysis buffer in order to remove the RBC influence. PC-LDA resulted classification efficiency of about 79% and 78% for normal and abnormal smear, respectively – comparable to Pap test. Thus finding of the study suggests feasibility of Raman spectroscopic classification of normal and cancerous exfoliated cervical cell specimens.     

Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 μM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.     

HPV E6, E6AP and cervical cancer

Abstract

Every year, approximately 470,000 new cases of cervical cancer are diagnosed and approximately 230,000 women worldwide die of the disease, with the majority (~80%) of these cases and deaths occurring in developing countries. Human papillomaviruses (HPVs) are the etiological agents in nearly all cases (99.7%) of cervical cancer, and the HPV E6 protein is one of two viral oncoproteins that is expressed in virtually all HPV-positive cancers. E6 hijacks a cellular ubiquitin ligase, E6AP, resulting in the ubiquitylation and degradation of the p53 tumor suppressor, as well as several other cellular proteins. While the recent introduction of prophylactic vaccines against specific HPV types offers great promise for prevention of cervical cancer, there remains a need for therapeutics. Biochemical characterization of E6 and E6AP has suggested approaches for interfering with the activities of these proteins that could be useful for this purpose.

Publication history

Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

     

Effects of Hydroxyapatite Nanoparticles on Apoptosis and Invasion of Human Renal Cell Carcinoma 786-0 Cells

Renal cell carcinoma is the most common cancer of the kidney, and resistant to traditional therapies. The aim of this study is to investigate the effects of hydroxyapatite nanoparticles on human renal cell carcinoma 786-0 cells. Cell proliferation was assessed with an 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide(MTT) staining kit. The apoptosis assay was assessed with an FITC Annexin V Apoptosis Detection Kit. Caspase-3 and caspase-12 were detected by immunocytochemical staining and semi-qua...