Significance of CD133 positive cells in four novel HPV-16 positive cervical cancer-derived cell lines and biopsies of invasive cervical cancer.

BACKGROUND: Cervical cancer is a major cause of cancer-related mortality in women in the developing world. Cancer Stem cells (CSC) have been implicated in treatment resistance and metastases development; hence understanding their significance is important. METHODS: Primary culture from tissue biopsies of invasive cervical cancer and serial passaging was performed for establishing cell lines. Variable Number Tandem Repeat (VNTR) assay was performed for comparison of cell lines with their parental tissue. Tumorsphere and Aldefluor assays enabled isolation of cancer stem cells (CSC); immunofluorescence and flow cytometry were performed for their surface phenotypic expression in cell lines and in 28 tissue samples. Quantitative real-time PCR for stemness and epithelial-mesenchymal transition (EMT) markers, MTT cytotoxicity assay, cell cycle analysis and cell kinetic studies were performed. RESULTS: Four low-passage novel cell lines designated RSBS-9, - 14 and - 23 from squamous cell carcinoma and RSBS-43 from adenocarcinoma of the uterine cervix were established. All were HPV16+. VNTR assay confirmed their uniqueness and derivation from respective parental tissue. CSC isolated from these cell lines showed CD133+ phenotype. In tissue samples of untreated invasive cervical cancer, CD133+ CSCs ranged from 1.3-23% of the total population which increased 2.8-fold in radiation-resistant cases. Comparison of CD133+ with CD133- bulk population cells revealed increased tumorsphere formation and upregulation of stemness and epithelial-mesenchymal transition (EMT) markers with no significant difference in cisplatin sensitivity. CONCLUSION: Low-passage cell lines developed would serve as models for studying tumor biology. Cancer Stem Cells in cervical cancer display CD133+ phenotype and are increased in relapsed cases and hence should be targeted for achieving remission.

Minimal-deviation Endometrioid Adenocarcinoma of the Cervix: A Case Report With Ultrastructural Analysis Demonstrating Abnormal Ciliation of the Tumor Cells.

Minimal-deviation endometrioid adenocarcinoma (MDEA) of the uterine cervix is a rare tumor that may be confused histologically with a number of benign lesions as well as other types of endocervical neoplasia. The histologic and immunohistochemical features of MDEA have been described in case reports and in small series, but correlation of these findings with ultrastructural examination has not been documented. Herein we report a 51-yr-old patient who underwent hysterectomy for menorrhagia and was found to have a clinically unsuspected, stage IB cervical MDEA. The light microscopic, immunohistochemical, and electron microscopic features of the tumor are described, with the most significant ultrastructural abnormality being the presence of abnormal cilia and ciliogenesis.

Light scattering from normal and cervical cancer cells.

The light scattering characteristic plays a very important role in optic imaging and diagnostic applications. For optical detection of the cell, cell scattering characteristics have an extremely vital role. In this paper, we use the finite-difference time-domain (FDTD) algorithm to simulate the propagation and scattering of light in biological cells. The two-dimensional scattering cell models were set up based on the FDTD algorithm. The cell models of normal cells and cancerous cells were established, and the shapes of organelles, such as mitochondria, were elliptical. Based on these models, three aspects of the scattering characteristics were studied. First, the radar cross section (RCS) distribution curves of the corresponding cell models were calculated, then corresponding relationships between the size and the refractive index of the nucleus and light scattering information were analyzed in the three periods of cell canceration. The values of RCS increase positively with the increase of the nucleo-cytoplasmic ratio in the cancerous process when the scattering angle ranges from 0° to 20°. Second, the effect of organelles in the scattering was analyzed. The peak value of the RCS of cells with mitochondria is higher than the cells without mitochondria when the scattering angle ranges from 20° to 180°. Third, we demonstrated that the influence of cell shape is important, and the impact was revealed by the two typical ideal cells: round cells and oval cells. When the scattering angle ranges from 0° to 80°, the peak values and the frequencies of the appearance of the peaks from the two models are roughly similar. It can be concluded that: (1) the size of the nuclei and the change of the refractive index of cells have a certain impact on light scattering information of the whole cell; (2) mitochondria and other small organelles contribute to the cell light scattering characteristics in the larger scattering angle area; and (3) the change of the cell shape significantly influences the value of scattering peak and the deviation of scattering peak position. The results of the numerical simulation will guide subsequent experiments and early diagnosis of cervical cancer.

Effects of Propofol on Several Membrane Characteristics of Cervical Cancer Cell Lines.

BACKGROUND: Although significant advances have been made toward understanding the molecular mechanisms underlying the effect of propofol on tumor cell metastasis, less is known regarding how cell membrane and cytoskeletal ultrastructure are affected in this process. Here, we investigated the relationship between cell morphology and cell size, which are features mainly defined by the cytoskeleton. METHODS: To confirm the effects of propofol on the migratory ability of human cervical carcinoma cells, cell migration and invasion were examined through scratch wound healing and transwell membrane assays. Furthermore, HeLa cells cultivated with different concentrations of propofol were examined by confocal microscopy and atomic force microscopy (AFM), and the mean optical density and migration ability of these cells were also assessed. In addition, cell membrane morphology was inspected using AFM. RESULTS: The results of the wound healing and transwell membrane assays indicated that propofol decreases the migratory ability of cervical carcinoma cells compared to control cells. A comparative analysis of the test results revealed that short-term (3 h) exposure to propofol induced marked changes in cell membrane microstructure and in the cytoskeleton in a dose-dependent manner. These morphological changes in the cell membrane were accompanied by cytoskeleton (F-actin) derangement. The present findings demonstrate a close relationship between changes in cell membrane ultrastructure and cytoskeletal alterations (F-actin) in propofol-treated HeLa cells. AFM scanning analysis showed that cell membrane ultrastructure was significantly changed, including a clear reduction in membrane roughness. CONCLUSION: The influence of propofol on the HeLa cell cytoskeleton can be directly reflected by changes in cellular morphology, as assessed by AFM. Moreover, the use of AFM is a good method for investigating propofol-mediated changes within cytoskeletal ultrastructure.

Towards early detection of cervical cancer: Fractal dimension of AFM images of human cervical epithelial cells at different stages of progression to cancer.

We used AFM HarmoniX modality to analyse the surface of individual human cervical epithelial cells at three stages of progression to cancer, normal, immortal (pre-malignant) and carcinoma cells. Primary cells from 6 normal strains, 6 cancer, and 6 immortalized lines (derived by plasmid DNA-HPV-16 transfection of cells from 6 healthy individuals) were tested. This cell model allowed for good control of the cell phenotype down to the single cell level, which is impractical to attain in clinical screening tests (ex-vivo). AFM maps of physical (nonspecific) adhesion are collected on fixed dried cells. We show that a surface parameter called fractal dimension can be used to segregate normal from both immortal pre-malignant and malignant cells with sensitivity and specificity of more than 99%. The reported method of analysis can be directly applied to cells collected in liquid cytology screening tests and identified as abnormal with regular optical methods to increase sensitivity. FROM THE CLINICAL EDITOR: Despite cervical smear screening, sometimes it is very difficult to differentiate cancers cells from pre-malignant cells. By using AFM to analyze the surface properties of human cervical epithelial cells, the authors were able to accurately identify normal from abnormal cells. This method could augment existing protocols to increase diagnostic accuracy.

Blockage of cisplatin-induced autophagy sensitizes cervical cancer cells to cisplatin.

Development of chemoresistance is a major obstacle that leads to the recurrence and progression of cervical cancer (CC). Autophagy, meaning, "eating of self", has shown paradoxical functions in tumors. In this study, we first investigated the process of autophagy induction by cisplatin in CC cells. Next, we investigated the role of autophagy in cisplatin-sensitivity of CC cells via blockage of cisplatin-induced autophagy. The results demonstrated that cisplatin induces autophagy in CC HeLa cells via upregulating the formation of autophagic vesicle, promoting the conversion of LC-I to LC-II, and increasing the expression of autophagy-related gene 7 (Atg-7). On the other hand, the autophagy inhibitor, 3MA, downregulated cisplatin-induced formation of autophagic vesicles, reduced the conversion of LC-I to LC-II, and decreased Atg-7 expression. Moreover, 3MA reversed the reduction in cellular viability and induction of apoptosis by cisplatin in HeLa cells. Our results imply that autophagy blockage may play a key role in the chemosensitivity of cervical cancers.

Functional properties of a novel hybrid antimicrobial peptide NS: potent antitumor activity and efficient plasmid delivery.

Antimicrobial peptides have been widely recognized as potential candidates for treating tumor, especially for defending against multidrug-resistant cells. Previously, based on the structure of substance P, we have designed a novel class of hybrid antimicrobial peptide NS, which possesses potent antimicrobial activity against a broad spectrum of bacterial pathogens. In this study, we evaluated its cytotoxicity to tumor cells and studied the possible mechanism of action. We showed that NS could efficiently kill tumor cells by rapidly disrupting the tumor cell membrane and inhibiting the DNA synthesis. In addition, we also found that NS could efficiently deliver plasmids into cells and exhibit high transfection efficiency after the introduction of a stearyl moiety to its N-terminus, like many reported cell-penetrating peptides. Taken together, this study revealed the potential multiple functions of NS, providing fundamental support for further therapeutic application as potential antitumor agent.

Investigation of CPD and HMDS sample preparation techniques for cervical cells in developing computer-aided screening system based on FE-SEM/EDX.

This paper investigated the effects of critical-point drying (CPD) and hexamethyldisilazane (HMDS) sample preparation techniques for cervical cells on field emission scanning electron microscopy and energy dispersive X-ray (FE-SEM/EDX). We investigated the visualization of cervical cell image and elemental distribution on the cervical cell for two techniques of sample preparation. Using FE-SEM/EDX, the cervical cell images are captured and the cell element compositions are extracted for both sample preparation techniques. Cervical cell image quality, elemental composition, and processing time are considered for comparison of performances. Qualitatively, FE-SEM image based on HMDS preparation technique has better image quality than CPD technique in terms of degree of spread cell on the specimen and morphologic signs of cell deteriorations (i.e., existence of plate and pellet drying artifacts and membrane blebs). Quantitatively, with mapping and line scanning EDX analysis, carbon and oxygen element compositions in HMDS technique were higher than the CPD technique in terms of weight percentages. The HMDS technique has shorter processing time than the CPD technique. The results indicate that FE-SEM imaging, elemental composition, and processing time for sample preparation with the HMDS technique were better than CPD technique for cervical cell preparation technique for developing computer-aided screening system.

Anticancer activity and mechanism of juglone on human cervical carcinoma HeLa cells.

Induction of apoptosis in tumor cells has become the major focus of anti-tumor therapeutics development. Juglone, a major chemical constituent of Juglans mandshurica Maxim, possesses several bioactivities, including anti-tumor. In the present study, HeLa cells were incubated with juglone at various concentrations. The proliferation inhibition of juglone on HeLa cells was tested by the MTT assay. Occurrence of apoptosis was detected by Hoechst 33258 staining, flow cytometry, and transmission electron microscopy. The expression of apoptotic-related proteins was examined by Western blot. The results showed that juglone inhibits the growth of HeLa cells in dose-dependent manner. Topical morphological changes of apoptotic body formation after juglone treatment were observed. The percentages of early apoptosis of Annexin V-FITC were 5.23%, 7.95%, 10.69%, and 20.92% with the concentrations of juglone (12.5, 25, 50, and 100 µmol/L), respectively. After cells were treated with juglone at the different dose for 24 h, the expression of Bcl-2 was significantly down-regulated and the expression of Bax was significantly up-regulated compared with the control. These events paralleled with activation of caspase-9, -8, -3, and PARP cleavage. The results suggest that juglone may be effective for the treatment of HeLa cells.

[The cytological diagnostics of dysplasia and epidermoid carcinoma of the cervix in studying the nucleolar-organizing regions of chromosomes].

The study of the nucleolar-organizing regions of chromosomes in the cells of dys-plastically modified squamous epithelium and epidermoid cancer of cervix was carried out. The successive increase of quantita-tive content of main morphofunctional types of nucleoli is established including active compact and transient nucleolonemic-compact ones in accordance with in-crease of dysplastic modifications and appearance of signs of epidermoid cancer of cervix. The dominance of percentage content of large argyrophilic granules in nu-cleoli of nucleolonemic type under epidermoid cancer of cervix as compared with dysplasia is established. The algorithm of differentiating cytological diagnostics of the degree of dysplasia and epidermoid cancer of cervix is developed. This algo-rithm reflects the degree of structural functional modifications of nucleolar-organizing regions of chromosomes.

Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods.

Assessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

Selective suppression of cervical cancer Hela cells by 2-O-ß-D-glucopyranosyl-L-ascorbic acid isolated from the fruit of Lycium barbarum L.

Lycium barbarum fruit has been used as a Chinese traditional medicine and dietary supplement for centuries. 2-O-ß-D-Glucopyranosyl-L-ascorbic acid (AA-2ßG), a novel stable vitamin C analog, is one of the main biologically active components of the fruit. In this report, we investigated the cytotoxic and antiproliferative effect of AA-2ßG against cancer cells in vitro and identified the proteins with significantly differential expression in the cervical cancer cells (Hela) cultured in the presence of AA-2ßG proteomic analysis. Our results demonstrated that the cytotoxic and antiproliferative activity of AA-2ßG on cancer cell lines were in a cell type-, time-, and dose-dependent manner. Similar to vitamin C, the AA-2ßG selectively induced cell death repressed the proliferation of Hela cells by the mechanism of cell apoptosis and cell cycle arrest induced by AA-2ßG through a mechanism of stabilizing p53 protein. However, the biological activity of inhibition of cell proliferation in other malignant cancer cell lines or primary cells were varied, as demonstrated by either moderate inhibition or slight promotion following treatment with AA-2ßG. Comparative analysis of the proteomic profiles and immunoblot analysis identified 15 proteins associated with repressing cell apoptosis and/or stimulating cell proliferation in Hela cells that were downregulated in the presence of AA-2ßG or vitamin C. These data indicate that a mechanism of the AA-2ßG and vitamin C mediated antitumor activity by downregulating the expression of proteins involved in cell apoptosis and proliferation and consequently inducing Hela cell apoptosis and cell cycle arrest, suggesting that AA-2ßG and vitamin C may share a similar mechanism of inducing Hela cell apoptosis. These results also suggest that the L. barbarum fruit may be a potential dietary supplement and anticancer agent aimed at the prevention and treatment of cervical cancer.

Histone deacetylase inhibitor vorinostat suppresses the growth of uterine sarcomas in vitro and in vivo.

BACKGROUND: Uterine sarcomas are very rare malignancies with no approved chemotherapy protocols. Histone deacetylase (HDAC) inhibitors belong to the most promising groups of compounds for molecular targeting therapy. Here, we described the antitumor effects of suberoylanilide hydroxamic acid (SAHA; vorinostat) on MES-SA uterine sarcoma cells in vitro and in vivo. We investigated effects of vorinostat on growth and colony forming ability by using uterine sarcoma MES-SA cells. We analyzed the influence of vorinostat on expression of different HDACs, p21(WAF1) and activation of apoptosis. Finally, we examined the antitumor effects of vorinostat on uterine sarcoma in vivo. RESULTS: Vorinostat efficiently suppressed MES-SA cell growth at a low dosage (3 microM) already after 24 hours treatment. Decrease of cell survival was even more pronounced after prolonged treatment and reached 9% and 2% after 48 and 72 hours of treatment, respectively. Colony forming capability of MES-SA cells treated with 3 microM vorinostat for 24 and 48 hours was significantly diminished and blocked after 72 hours. HDACs class I (HDAC2 and 3) as well as class II (HDAC7) were preferentially affected by this treatment. Vorinostat significantly increased p21(WAF1) expression and apoptosis. Nude mice injected with 5 x 106 MES-SA cells were treated for 21 days with vorinostat (50 mg/kg/day) and, in comparison to placebo group, a tumor growth reduction of more than 50% was observed. Results obtained by light- and electron-microscopy suggested pronounced activation of apoptosis in tumors isolated from vorinostat-treated mice. CONCLUSIONS: Our data strongly indicate the high therapeutic potential of vorinostat in uterine sarcomas.

Ratio-contrast imaging of dual-energy absorption for element mapping with a scanning transmission X-ray microscope.

The detection of chemical mapping with a spatial resolution of 30 nm has been achieved with a scanning transmission X-ray microscope at the Shanghai Synchrotron Radiation Facility. For each specimen, two absorption images were scanned separately with energies E(1) and E(2): E(1) was focused on the absorption edge of the chosen element and E(2) was focused below the edge. A K-edge division method is proposed and applied to obtain the element mapping. Compared with the frequently used K-edge subtraction method, this ratio-contrast method is shown to be more accurate and sensitive in identifying the elements of interest, where the definition of the contrast threshold is simple and clear in physics. Several examples are presented to illustrate the effectiveness of the method.

[Human papilloma virus and cervical cancer. An historical review on the development of research on cancer of the cervix uteri in Venezuela]. / El virus del papiloma humano y el cáncer cervical. Una revisión de la historia actualizada sobre la investigación del cáncer del cuello uterino en Venezuela.

The history on the relationship of VPH infection and cervical cancer was examined. Findings were initially reported in Maracaibo(1971), later in Mexico(1973) and thereafter several studies on the ultrastructure and immunohistochemistry of VPH infection and its role on cervical cancer were described. The ultrastructural findings of viral particles of HPV and their proteins, as well as their role in the incorporation of the viral genome to the human cervical cells were also described. Glycoproteins on the surface of cervical cells were reviewed and their importance on HPV infection was related to p16, blood group antigens and early genetic changes in the cell cycle with loss of heterozigocity, all of which, stimulated by the high risk HPV infection lead to cervical cancer.

Spatial allelic imbalance of BCL2 genes and chromosome 18 territories in nonneoplastic and neoplastic cervical squamous epithelium.

Several studies suggest a correlation between genome architecture and gene function. To elucidate mechanisms of gene positioning during cell differentiation and malignant transformation we investigated the nuclear positions of the BCL2 alleles and chromosome 18 territories in different layers of nonneoplastic cervical squamous epithelium and cervical squamous carcinomas in relation to gene expression. Fluorescence in situ hybridization and three-dimensional (3D) image analysis using tissue sections revealed that one BCL2 allele was located more peripherally than the other one in nuclei of the basal layer of nonneoplastic epithelium. During terminal cell differentiation the outer BCL2 allele showed a shift towards the nuclear center. In BCL2-expressing carcinomas the inner BCL2 allele was located more peripherally compared with the basal layer of nonneoplastic epithelium. Our results suggest a functional relevance of unequal allelic BCL2 gene positioning and support the hypothesis that transcriptional BCL2 activation is associated with BCL2 relocation towards the nuclear periphery.

AgNOR polymorphism association with squamous intraepithelial lesions and invasive carcinoma with HPV infection.

OBJECTIVE: Evaluate the relationships between AgNORs polymorphisms and squamous intraepithelial lesions (SIL) and squamous cell carcinoma (SCC) with HPV infection. MATERIALS AND METHODS: A study was carried out on sixty women from the state of Guerrero, Mexico. HPV detection was performed by PCR. AgNORs were identified by argentic impregnation. One hundred cells per slide were counted and classified according to the polymorphism of AgNORs dots; typical (spherical) and atypical (large, kidney-shaped and clustered). RESULTS: A total of 100% of the cases were positive for HPV infection. Nine different high-risk HPV genotypes were found, type 16 was the most common (48.6%). The AgNORs showed a significant decrease in spherical shape according to neoplastic development. The three atypical shapes showed a significant increase in SIL and SCC (p-trend<0.001). CONCLUSIONS: AgNORs polymorphism rises progressively according to the grade of histological lesions that can be useful as a prognosis for progression of SCC.

Lesions of the subepithelial stromal zone of the lower female genital tract. An immunopathological study.

The stroma often fibroepithelial and ten adenomatous cervical polyps, 12 fibro-epithelial vaginal polyps, 20 non neoplastic cervices, 15 cone specimens with CIN changes and five specimens with infiltrative squamous cell carcinoma was investigated. Vimentin, desmin, actin, collagen III, CD34, ER and PgR were studied using the Ventana Automatic Immunostaining System. A subepithelial stromal layer was detected in 17/20 normal specimens, 16/20 cervical polyps, 10/12 vaginal polyps and in 6/15 cone biopsies but not in neoplastic specimens. Vimentin staining was positive in all fibroepithelial polyps, normal specimens and in 6/10 adenomatous polyps. Actin was negative in the loose subepithelial zone but positive in the underlying stroma. Desmin, alpha-1-antitrypsin and CD34 were detected in isolated cells in normal specimens. ER and PgR were observed in isolated subepithelial cells, with greater density in the cells of the fibromuscular wall. In conclusion, loose mesenchymal stroma in the lower female genital tract of a fibrohistiocytic nature was observed with minimal smooth muscle participation--which is the matrix of vaginal and cervical polyps.

Mesonephric adenocarcinoma of the uterine cervix: a case report with immunohistochemical and ultrastructural studies.

A very rare case of mesonephric adenocarcinoma with lobular mesonephric hyperplasia in the uterine cervix of a 46-year-old female is presented. The lesion was a 4 cm, exophytic, almost circumferential, whitish yellow, friable mass in the uterine cervix. Microscopically, the tumor was composed predominantly of atypical round to polygonal cells arranged in a ductal, tubular, or papillary pattern. The tumor involved the entire cervix with varying depths of penetration. Lobular mesonephric hyperplasia was also observed in the lateral cervical wall and adjacent to the tumor. Immunohistochemically, the tumor was positive for CAM5.2, CK7, epithelial membrane antigen, calretinin, and chromogranin A, and was negative for vimentin, carcinoembryonic antigen, estrogen and progesterone receptors, and CD10. An ultrastructural analysis showed telolysomes, which were characteristic features of mesonephric epithelium. The patient was alive without disease at 4 months after surgery.

Increasing the uptake of cervical screening programmes.

Successful cervical screening programmes depend on the degree of coverage and the rate of attendance. There are many demographic reasons why some women fail to attend for cervical screening, including lack of knowledge and education and socioeconomic status. Moreover, a woman's ethnicity and her age also play a role in screening uptake. Community and practice nurses are ideally positioned to identify women's information needs and provide appropriate information to overcome barriers to screening attendance. This article discusses the main predictors of participation in cervical screening programmes and interventions that can be used to increase cervical screening uptake.