Human Cervical Tissue Samples Research Articles

Cervical Squamous Cell Carcinoma Diagnosis by FTIR Microspectroscopy.

Cervical cancer was considered the fourth most common cancer worldwide in 2020. In order to reduce mortality, an early diagnosis of the tumor is required. Currently, this type of cancer occurs mostly in developing countries due to the lack of vaccination and screening against the Human Papillomavirus. Thus, there is an urgent clinical need for new methods aiming at a reliable screening and an early diagnosis of precancerous and cancerous cervical lesions. Vibrational spectroscopy has provided very good results regarding the diagnosis of various tumors, particularly using Fourier transform infrared microspectroscopy, which has proved to be a promising complement to the currently used histopathological methods of cancer diagnosis. This spectroscopic technique was applied to the analysis of cryopreserved human cervical tissue samples, both squamous cell carcinoma (SCC) and non-cancer samples. A dedicated Support Vector Machine classification model was constructed in order to categorize the samples into either normal or malignant and was subsequently validated by cross-validation, with an accuracy higher than 90%.

Cisplatin treatment modulates Annexin A1 and inhibitor of differentiation to DNA 1 expression in cervical cancer cells

Cisplatin (Cis) is a choice chemotherapy approach to cervical cancer by inducing DNA adducts and subsequent apoptosis. We have investigated the effects of Cis on Annexin A1 (ANXA1) and inhibitor of DNA binding 1 (ID1) proteins expression to elucidate further mechanisms of Cis actions. Human cervical tissue samples from twenty-four patients, with Cervical Intraepithelial Neoplasia (CIN, stage I, II and III), were evaluated to quantified ANXA1 and ID1 expressions. In vitro, human epidermoid carcinoma of the cervix (SiHa cell line) were treated with Annexin A1 peptide (ANXA12−26), Cis or Cis + ANXA12−26 to evaluate cell proliferation and migration, cytotoxicity of treatments as well as ANXA1 and ID1 modulations by mRNA and protein expression. Our findings showed expression of ID1 and ANXA1 proteins in tissue samples from Cervical Intraepithelial Neoplasia (CIN) patients, with intense immunological identification of ID1 in the CIN III stage. In SiHa cells, treatments with Cis alone or Cis + ANXA12−26, increase mRNA expressions of the ANXA1 and reduced the ID1. In agreement, Cis + ANXA12−26 enhanced ANXA1 protein expression and Cis or Cis + ANXA12−26 abolished ID1 protein expression. Cell proliferation was reduced after treatment with ANXA12−26 peptide and more significant after Cis or Cis + ANXA12−26 treatments. These two last treatments reduced cell viability, by inducing late apoptosis, and impaired cell migration. Together, our data highlight endogenous ANXA1 is involved in Cis therapy for cervical cancer.

Molecular Effects and Tissue Penetration Depth of Physical Plasma in Human Mucosa Analyzed by Contact- and Marker-Independent Raman Microspectroscopy.

Noninvasive epithelial tissue treatment with cold atmospheric plasma (CAP) is a promising option for local treatment of chronic inflammatory and precancerous lesions as well as various mucosal cancer diseases. Atmospheric pressure plasma jets (APPJ) are well-characterized and medically approved plasma sources. There are numbers of medically approved plasma sources for the treatment of epithelial diseases; however, little is known about the biochemical effects of CAP at the plasma-tissue interface. Furthermore, the actual penetration depth of CAP into tissue is currently unclear. Noninvasive and marker-independent Raman microspectroscopy was employed to assess the molecular effects of CAP on single cells and primary human cervical tissue samples. CAP treatment showed immediate and persisting changes of specific molecular tissue components determined by multivariate analysis. Raman imaging identified CAP-dependent changes in the morphology of the tissue, as well as molecular tissue components. The expression of the different components was not significantly altered within 24 h of incubation. DNA and lipids showed the strongest changes upon CAP treatment, which were traced to the basal cell layer of cervical epithelium, corresponding to an average functional plasma penetration depth of roughly 270 μm. In this study, Raman microspectroscopy is shown to be a promising method for molecular single-cell and solid tissue characterization. Regarding CAP treatment of tissues, Raman microspectroscopy could be suitable for the screening of biological mechanisms as well as for future contact- and marker-independent monitoring of plasma tissue effects.

High-speed collagen fiber modeling and orientation quantification for optical coherence tomography imaging.

Quantifying collagen fiber architecture has clinical and scientific relevance across a variety of tissue types and adds functionality to otherwise largely qualitative imaging modalities. Optical coherence tomography (OCT) is uniquely suited for this task due to its ability to capture the collagen microstructure over larger fields of view than traditional microscopy. Existing image processing techniques for quantifying fiber architecture, while accurate and effective, are very slow for processing large datasets and tend to lack structural specificity. We describe here a computationally efficient method for quantifying and visualizing collagen fiber organization. The algorithm is demonstrated on swine atria, bovine anterior cruciate ligament, and human cervical tissue samples. Additionally, we show an improved performance for images with crimped fiber textures and low signal to noise when compared to similar methods.

Changes in Activities of Caspase-8 and Caspase-9 in Human Cervical Malignancy

The apoptotic process of programmed cell death and its dysfunctions in a variety of human diseases, including cervical cancer, has become the focus of extensive scientific research. Caspases are considered key factors in the execution of apoptosis, although there are many aspects of their role to be elucidated. It has been found that disturbance of initiator caspase-8 and caspase-9 expression or function may contribute to cancer formation/progression, and inactivation of them could promote resistance to current treatment approaches. In our research, the activities of caspase-8 and caspase-9 have been estimated during the progression of human cervical malignancy. The experimental material includes human cervical tissue samples (normal and pathological), in which enzyme activities have been measured colorimetrically. Activities of caspase-8 and caspase-9 presented the highest increase, compared to the controls, in the low-grade squamous intraepithelial lesion samples (statistically significant, P < 0.01 by t test). The activities diminished in the high-grade squamous intraepithelial lesion and even more in the cancer samples but remained higher than the controls. The observed changes in the activities of caspase-8 and caspase-9 could be attributed to their involvement in the cervical tissue's effort to resist malignancy progression.

Indication of participation of caspase-2 and caspase-5 in mechanisms of human cervical malignancy.

When apoptosis is disrupted, the transformed cells can survive, proliferate, and evolve into a malignancy. The strictly conserved caspase genes and the reliable experimental data clearly show that some caspases play a crucial role in apoptosis even if some of them have no apoptotic activity and others exhibit both apoptotic and nonapoptotic properties. Although caspase-2 belongs to initiator caspases, its normal role remains unclear. Experimental studies have shown that it is primarily necessary for the execution of apoptosis in mutagenic cells. Human caspase-5 is classified as an inflammatory caspase, although its substrate has not been identified yet. In this research, the activities of caspase-2 and caspase-5 have been estimated during the progression of human cervical malignancy. The experimental material includes human cervical tissue samples (normal and pathological) and blood serum samples of the corresponding tissue donors, where enzyme activities have been measured colorimetrically. Both caspases' activities showed the highest increase, statistically significant (P < 0.01, by t test) compared with the controls, in the low-grade squamous intraepithelial lesion tissues. Caspase-2 of all pathological tissues was proved more active than the controls. Serum caspases' activities were significantly lower than those of the tissues. Serum caspase-2's activity in patients with low-grade squamous intraepithelial lesion stage showed no statistically significant increase compared with the controls. Serum caspase-5's activity of all patients with malignancy stages was presented elevated, whereas that of the serum of patients with cervical cancer had the highest activity (P < 0.01, by t test). The changes of caspase-2 and caspase-5 activities could be indicative of their involvement in the cervical malignancy mechanisms.

Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker analysis in a collection of cervical tumours and matched controls

BackgroundUltra-high throughput sequencing technologies provide opportunities both for discovery of novel molecular species and for detailed comparisons of gene expression patterns. Small RNA populations are particularly well suited to this analysis, as many different small RNAs can be completely sequenced in a single instrument run.ResultsWe prepared small RNA libraries from 29 tumour/normal pairs of human cervical tissue samples. Analysis of the resulting sequences (42 million in total) defined 64 new human microRNA (miRNA) genes. Both arms of the hairpin precursor were observed in twenty-three of the newly identified miRNA candidates. We tested several computational approaches for the analysis of class differences between high throughput sequencing datasets and describe a novel application of a log linear model that has provided the most effective analysis for this data. This method resulted in the identification of 67 miRNAs that were differentially-expressed between the tumour and normal samples at a false discovery rate less than 0.001.ConclusionsThis approach can potentially be applied to any kind of RNA sequencing data for analysing differential sequence representation between biological sample sets.

Alternative splicing variants of IRF-1 lacking exons 7, 8, and 9 in cervical cancer

The two previously identified major splice variants of interferon regulatory factor 1 (IRF-1) do not appear to affect IRF-1-mediated gene activation. We searched for additional splice variants and examined their effect on wild-type IRF-1. RT-PCR experiments using normal and malignant human cervical tissue samples revealed five variants lacking some combination of exons 7, 8, and 9; their expression levels were higher in the malignant samples. These variants had predicted deletions of the functional domain or truncated protein isoforms, had different transcriptional activities, and attenuated transcriptional activity of IRF-1. Unlike the cell cycle-dependent IRF-1 transcript, the splice variant mRNA levels remained consistent throughout the cell cycle. The variant proteins were more stable than the IRF-1 protein, which may explain the strong inhibition of IRF-1 transcription in the presence of relatively small quantities of the alternative transcripts. In conclusion, alternative splicing in exons 7, 8, and 9 is an important mechanism for negatively regulating IRF-1 in cervical cancer.

Loss of expression, and mutations of Smad 2 and Smad 4 in human cervical cancer.

Mutations in Smads, intermediates of transforming growth factor-beta signaling, are known to contribute to the loss of sensitivity to transforming growth factor-beta, a common feature of many neoplastic cells. However, not much information is available on Smad alterations in cervical cancer and so we probed, for the first time, for alterations in Smad 2 and Smad 4 genes using human cervical cancer cell lines and human cervical tissue samples. Using PCR/reverse transcription-PCR, single-stranded conformation polymorphism analysis and DNA sequencing, we observed a deletion of 'G' in the L3 loop (crucial in Smad-receptor interaction) in C-33A cells, and an insertion of 'A' in codon 122 (loss of MH2 domain) from a cervical tumor sample, both of which caused frame shift and pretermination in Smad 2. In addition, a G/A transition at 31 bp upstream-nontranslated regions of exon 8 of Smad 4 was found in Bu 25TK cells. Smad 2 expression was less in some of the cervical tumor samples than that of nonmalignant samples and six cancer samples showed C-terminal deletions that abolish Smad 2 phosphorylation sites. The loss of expression of Smad 4 found in some cervical tumor samples was due to transcription loss rather than deletion of the gene. Our results highlight an important role for Smad 2 and Smad 4 in human cervical tumors.

Histopathologic correlation of high-resolution magnetic resonance imaging of human cervical tissue samples at 3 tesla: validation of a technique.

High-resolution magnetic resonance (HR-MR) images of human cervical tissue samples were correlated with low magnification histology to investigate the MR presentation of normal and pathologic anatomic structures in cervical tissue samples at 3T. Tissue samples were obtained from five large loop excision of the transformation zone (LLETZ) cone biopsies and one surgical specimen. HR-MR images (3D-Spin Echo sequence: TR/TE: 1500/60 milliseconds, voxel size of 117 x 208 x 234 microm) and low magnification (5x) histology sections were acquired in the same anatomical planes and correlated. In vitro HR-MR imaging of cervical tissue samples correlated well with low magnification microscopic histology, demonstrating normal anatomy (epithelium and its mucus layer, connective tissue of the stroma, glands, blood vessels). In vitro HR-MR imaging adds information about tumor content and margins. High resolution MR imaging of cervical tissue samples assists in the identification of both normal structures and pathologic changes.

A bioadhesive patch cervical drug delivery system for the administration of 5-fluorouracil to cervical tissue

A novel bioadhesive cervical patch drug delivery is described, containing 5-fluorouracil for the treatment of cervical intraepithelial neoplasia (CIN). The patch was of bilaminar design, with a drug-loaded bioadhesive film cast from a gel containing 2% (w/w) Carbopol® 981 plasticised with 1% (w/w) glycerin. The casting solvent was ethanol/water 30:70, chosen to give a non-fissuring film with an even particle size distribution. The film, which was mechanically stable on storage under ambient conditions, was bonded directly to a backing layer formed from thermally-cured polyvinyl chloride emulsion. Bioadhesive strength was independent of drug loading in the bioadhesive matrix over the range investigated but was influenced by both the plasticiser concentration in the casting gel and the thickness of the final film. Release of 5-fluorouracil from the bioadhesive layer into an aqueous sink was rapid but was controlled down to an undetectable level through the backing layer. The latter characteristic was desirable to prevent drug spill from the device onto vaginal epithelium in vivo. Despite the relatively hydrophilic nature of 5-fluorouracil, substantial drug release through human cervical tissue samples was observed over approximately 20 h. Drug release, which was clearly tissue rather than device dependent, may have been aided by a shunt diffusion route through aqueous pores in the tissue. The bioadhesive and drug release characteristics of the 5-fluorouracil cervical patch indicated that it would be suitable for further clinical investigation as a drug treatment for CIN.