Integrin expression and usage by prostate cancer cell lines on laminin substrata.

During prostate cancer progression, invasive glandular epithelial cells move out of the ductal-acinar architecture and through the surrounding basement membrane. Extracellular matrix proteins and associated soluble factors in the basal lamina and underlying stroma are known to be important regulators of prostate cell behaviors in both normal and malignant tissues. In this study, we assessed cell interactions with extracellular matrix and stromal factors during disease progression by characterizing integrin usage and expression in a series of parental and lineage-derived LNCaP human prostate cancer cell lines. Although few shifts in integrin expression were found to accompany disease progression, integrin heterodimer usage did change significantly. The more metastatic sublines were distinct in their use of alphavbeta3 and, when compared with parental LNCaP cells, showed a shift in alpha6 heterodimerization, a subunit critical not only for interaction with prostate basal lamina but also for interaction with the bone matrix, a favored site of prostate cancer metastases.     

Incorporation of an (125)I-labeled hexa-iodinated diglyceride analog into low-density lipoprotein and high specific uptake by cells of cervical carcinoma cell lines.

The feasibility of using low-density lipoprotein (LDL) to deliver cytotoxic drugs to tumor cells has been explored since the 1980s, when cells of a number of cancer cell lines were found to have higher LDL receptor activity than normal cells. Such differential uptake between tumor and normal cells may provide a unique opportunity to use LDL as a tumor-specific carrier of radiopharmaceuticals for the clinical management of cancer. In this study, an (125)I-labeled hexa-iodinated diglyceride analog, 1, 3-dihydroxypropan-2-one 1,3-diiopanoate (DPIP), was synthesized and incorporated into LDL using a fusion technique. It was found that approximately 500 [(125)I] DPIP molecules were incorporated into each LDL particle. Cells of three human cervical tumor cell lines, HeLa, SiHa and C-33A, were used to examine the cellular uptake of the [(125)I]DPIP-LDL conjugate. It was shown that the [(125)I]DPIP-LDL conjugate was specifically bound to and taken up by cervical tumor cells through an LDL receptor-mediated endocytosis pathway. The results suggest that LDL may be a selective carrier for delivering hydrophobic radiopharmaceuticals to cancer cells and particularly for the diagnosis of cervical tumors.     

Modification of gap junctions in cells transformed by a temperature-sensitive mutant of Rous sarcoma virus

Summary Prompted by our observation that a reduction in junctional permeance is one of the earlier events in the process of neoplastic transformation of a cell line by Rous sarcoma virus, we analyzed the gap junctions, from these cells to determine if the basis of the reduction is a loss of junctional channels. The cells (normal rat kidney, or NRK) are infected with a temperature-sensitive mutant of Rous sarcoma virus, allowing one easily to manipulate the cells into and out of the transformed state, and hence also to manipulate the junctional permeance. Using freeze-fracture electron microscopy, we found that the number and size of the junctions did not change in parallel with the permeance changes we had previously characterized. There is, however, a significant rearrangement of the junctional particles to a more random configuration when the cells are transformed and a reversal to the more ordered pattern when the cells are shifted back to the normal phenotype. These changes do parallel the changes in junctional permeance. We conclude that the permeance of existing junctional channels is modified and that the change in permeance may involve a change in the interaction of the junctional channels with each other and/or the surrounding lipid domain.     

CircRNA hsa_circRNA_101996 increases cervical cancer proliferation and invasion through activating TPX2 expression by restraining miR-8075

In recent years, circular RNAs have been shown to serve as essential regulators in several human cancers. Nevertheless, the function and mechanism of CircRNA in cervical cancer remain elusive. In the present study, we showed that hsa_circRNA_101996 was highly expressed in cervical cancer tissues compared with matched normal tissues by bioinformatics analysis. We showed that the expression level of hsa_circRNA_101996 in cervical cancer tissues was positively correlated with TNM stage, tumor size, and lymph node metastasis. Moreover, higher levels of hsa_circRNA_101996 were related to poor outcomes of cervical cancer patients. We found that knockdown of hsa_circRNA_101996 significantly inhibited the proliferation, cell cycle, migration, and invasion of cervical cancer cells. Mechanistically, we demonstrated that hsa_circRNA_101996 served as a sponge of miR-8075, which targeted TPX2 in cervical cancer cells. We showed that miR-8075 that was downregulated in cervical cancer tissues repressed cervical cancer cell proliferation, migration, and invasion. Furthermore, we validated that upregulation of TPX2 by hsa_circRNA_101996-mediated inhibition of miR-8075 contributed to cervical cancer proliferation, migration, and invasion. Taken together, our findings revealed a novel mechanism that hsa_circRNA_101996-miR-8075-TPX2 network promoted cervical cancer progression.     

Temporal variation in morphine-mast cell interaction

The possible rhythmic interaction between morphine and mast cell was investigated in mice. It has been found that both at normal and drug-induced state, mast cells varied as a function of time and the number of mast cells was significantly affected at the time in which the number of mast cells was highest over a 24-h period. The exact underlying mechanism of the phenomenon still remains to be investigated.     

Cervical cancer vaccines: emerging concepts and developments

Certain human cancers are linked to infection by oncogenic viruses that are able to cause transformation of the normal host cell into a cancerous cell. Human papillomavirus (HPV) DNA and expression of viral transforming proteins are found in virtually all cervical cancer cells, indicating an important role of this virus in the pathogenesis of the disease. Evidence exists that the immune response to cancer cells can play a major role in determining the outcome of disease. The fact that HPV is a necessary cause for cervical cancer provides a clear opportunity to develop a therapeutic vaccine against the virus to treat patients with cervical cancer at its early and late stages. Development of a prophylactic vaccine for HPV would also reduce the incidence of cervical neoplasias by preventing virus infection. Various candidate HPV vaccines are being developed and tested in animal models and/or in human clinical trials. These HPV vaccines, both preventive and therapeutic, are the subjects of this review.     

Probing the extent of randomness in protein interaction networks

Protein-protein interaction (PPI) networks are commonly explored for the identification of distinctive biological traits, such as pathways, modules, and functional motifs. In this respect, understanding the underlying network structure is vital to assess the significance of any discovered features. We recently demonstrated that PPI networks show degree-weighted behavior, whereby the probability of interaction between two proteins is generally proportional to the product of their numbers of interacting partners or degrees. It was surmised that degree-weighted behavior is a characteristic of randomness. We expand upon these findings by developing a random, degree-weighted, network model and show that eight PPI networks determined from single high-throughput (HT) experiments have global and local properties that are consistent with this model. The apparent random connectivity in HT PPI networks is counter-intuitive with respect to their observed degree distributions; however, we resolve this discrepancy by introducing a non-network-based model for the evolution of protein degrees or "binding affinities." This mechanism is based on duplication and random mutation, for which the degree distribution converges to a steady state that is identical to one obtained by averaging over the eight HT PPI networks. The results imply that the degrees and connectivities incorporated in HT PPI networks are characteristic of unbiased interactions between proteins that have varying individual binding affinities. These findings corroborate the observation that curated and high-confidence PPI networks are distinct from HT PPI networks and not consistent with a random connectivity. These results provide an avenue to discern indiscriminate organizations in biological networks and suggest caution in the analysis of curated and high-confidence networks.     

Plasma proteome analysis of cervical intraepithelial neoplasia and cervical squamous cell carcinoma

Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.     

Assessment of the antiproliferative and apoptotic roles of sulfonamide carbonic anhydrase IX inhibitors in HeLa cancer cell line

Carbonic anhydrase IX (CA IX) has recently been validated as an antitumor/antimetastatic drug target. In this study, we examined the underlying molecular mechanisms and the anticancer activity of sulfonamide CA IX inhibitors against cervical cancer cell lines. The effects of several sulfonamides on HeLa, MDA-MB-231, HT-29 cancer cell lines, and normal cell lines (HEK-293, PNT-1A) viability were determined. The compounds showed high cytotoxic and apoptotic activities, mainly against HeLa cells overexpressing CA IX. We were also examined for intracellular reactive oxygen species (ROS) production; intra-/extracellular pH changes, for inhibition of cell proliferation, cellular mitochondrial membrane potential change and for the detection of caspase 3, 8, 9, and CA IX protein levels. Of the investigated sulfonamides, one compound was found to possess high cytotoxic and anti-proliferative effects in HeLa cells. The cytotoxic effect occurred via apoptosis, being accompanied by a return of pHe/pHi towards normal values as for other CA IX inhibitors investigated earlier.     

Seminal plasma induces the expression of IL-1α in normal and neoplastic cervical cells via EP2/EGFR/PI3K/AKT pathway

Background

Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation.

Methods and results

Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE₂ and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling.

Conclusion

SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.

     

Two L1-peptides are excellent tools for serological detection of HPV-associated cervical carcinoma lesions

A persistent high risk human papillomavirus (HR-HPV) infection causes cervical intraepithelial lesions and cervical carcinoma. There is evidence that detecting anti-L1 antibodies could be successfully used for discriminating between cervical lesion patients and women having normal cytology. It was found that peptides 18283 (55PNNNKILVPKVSGLQYRVFR74) and 18294 (284LYIKGSGSTANLASSNYFPT300) from the L1-surface exposed regions were specifically recognised by antibodies from the cervical lesion patient sera. These peptides were tested against 165 womens' normal cytology sera and 148 cervical lesion or cervical cancer patients' sera. Less than 3.6% of women's normal cytology sera recognised peptides 18283 or 18294; on the contrary, 91% to 96% of the cervical lesion (CIN I to CIN III) or cervical cancer patient sera recognised peptides 18283 and 18294. These data show that anti-peptide 18283 and 18294 antibodies in the patients' sera are strongly associated with the presence of HR-HPV associated cervical lesions, showing 92-97% sensitivity and 89-95% specificity in recognising precancerous and cervical cancer patients. These two peptides could be excellent tools for use in large-scale serological screening of women populations at risk of developing cervical carcinoma.     

Cathepsin B may be a potential biomarker in cervical cancer

Cathepsin B is a protease which is able to digest extracellular matrix. It is currently unknown whether cathepsin B plays a role in cervical cancer development and progression. With Q-PCR and Western blotting, we observed cathepsin B expression in cervical cancer cell line Hela cells. After the gene was silenced in HeLa cells with SiRNA, we confirmed that cathepsin B expressions at both mRNA and protein levels were significantly reduced. At the same time, cell proliferation, migration and invasion of the HeLa cells were significantly decreased compared to control cells. In addition, a significant regression of tumor growth in nude mice which received the siRNA targeted cathepsin B HeLa cells was observed. We further studied the expression of cathepsin B in a series of 169 clinical samples, including 56 invasive cervical squamous carcinoma, 85 CINs and 28 normal cervical tissues. It was found that cathepsin B expression in invasive carcinomas was significantly higher than that in the CINs and normal tissues (P<0.01). In addition, cathepsin B expression in the invasive carcinomas was positively correlated to tumor invasion depth and lymphatic metastasis. Our results indicate that cathepsin B may be a potential biomarker for further strategical clinical studies in cervical cancer.     

The Cytological Prediction of Cervical Intraepithelial Neoplasia In Colposcopically Directed Biopsies

The tissue sections and preceding cervical smears of 1262 women who had colposcopic cervical biopsies were reviewed and the reports correlated. Close correlation between the cytological and histological findings, to within one histological grade of cervical intraepithelial neoplasia (CIN), was noted in 86% of cases. However, the biopsy was negative, or contained evidence of wart virus infection only, in 24% of cases where dyskaryotic cells had been observed in the cervical smear. Of particular concern was the fact that negative histological findings were recorded in 13% of cases where the smear contained cells showing a moderate dyskaryosis and in 1.26% of cases where the smear showed severe dyskaryosis. This suggests that colposcopically directed biopsies do not always reflect the underlying pathological changes in the cervix. Management of these cases is discussed. In 45 women with a normal cervical smear prior to biopsy, histology revealed seven cases of CIN 3 and one case of invasive squamous carcinoma. This indicates that referral for colposcopy is advisable whenever there is clinical suspicion of cancer, even if the cervical smear report is normal.     

microRNA-802 inhibits cell proliferation and induces apoptosis in human cervical cancer by targeting serine/arginine-rich splicing factor 9

microRNAs (miRNAs) play crucial roles in cancer development and progression by targeting mRNAs for degradation and/or translational repression. microRNA-802 (miR-802) has been reported as a tumor suppressor and its deregulation is observed in various human cancers. However, the prognostic value of miR-802 and its underlying mechanisms involved in human cervical cancer are poorly investigated. The purposes of this study were to explore the role of miR-802 in cervical cancer and to clarify the regulation of serine/arginine-rich splicing factor 9 (SRSF9) by miR-802. Here, we found that miR-802 was downregulated in both cervical cancer tissues and cell lines. Transfection of a miR-802 mimic into cervical cancer cells inhibited their proliferation and colony formation, and promoted cell cycle arrest at the G0/G1 phase and cell apoptosis. In addition, we found that miR-802 could directly target the 3′-untranslated region of SRSF9 and suppress SRSF9 expression. Rescue experiments revealed that overexpression of SRSF9 partially reversed the inhibition effect of miR-802 in cervical cancer cells. Overall, these findings demonstrate that miR-802 functions as a tumor suppressor in cervical cancer by targeting SRSF9, suggesting that miR-802 might serve as a potential therapeutic target in cervical cancer.     

Analysis of normal-tumour tissue interaction in tumours: prediction of prostate cancer features from the molecular profile of adjacent normal cells

Statistical modelling, in combination with genome-wide expression profiling techniques, has demonstrated that the molecular state of the tumour is sufficient to infer its pathological state. These studies have been extremely important in diagnostics and have contributed to improving our understanding of tumour biology. However, their importance in in-depth understanding of cancer patho-physiology may be limited since they do not explicitly take into consideration the fundamental role of the tissue microenvironment in specifying tumour physiology. Because of the importance of normal cells in shaping the tissue microenvironment we formulate the hypothesis that molecular components of the profile of normal epithelial cells adjacent the tumour are predictive of tumour physiology. We addressed this hypothesis by developing statistical models that link gene expression profiles representing the molecular state of adjacent normal epithelial cells to tumour features in prostate cancer. Furthermore, network analysis showed that predictive genes are linked to the activity of important secreted factors, which have the potential to influence tumor biology, such as IL1, IGF1, PDGF BB, AGT, and TGFβ.     

Driving Protein Conformational Cycles in Physiology and Disease: “Frustrated” Amino Acid Interaction Networks Define Dynamic Energy Landscapes

A general framework by which dynamic interactions within a protein will promote the necessary series of structural changes, or “conformational cycle,” required for function is proposed. It is suggested that the free-energy landscape of a protein is biased toward this conformational cycle. Fluctuations into higher energy, although thermally accessible, conformations drive the conformational cycle forward. The amino acid interaction network is defined as those intraprotein interactions that contribute most to the free-energy landscape. Some network connections are consistent in every structural state, while others periodically change their interaction strength according to the conformational cycle. It is reviewed here that structural transitions change these periodic network connections, which then predisposes the protein toward the next set of network changes, and hence the next structural change. These concepts are illustrated by recent work on tryptophan synthase. Disruption of these dynamic connections may lead to aberrant protein function and disease states.     

Therapeutic efficacy of natural dipeptide carnosine against human cervical carcinoma cells

Natural substances have been attracted several researchers in the recent years, because of its potential antioxidant, anti‐inflammatory and anti‐cancer properties. We have investigated the effect of carnosine on cell viability, apoptosis, DNA damage, reactive oxygen species (ROS) and caspase 3 enzyme expression in human cervical carcinoma and Madin‐Darby Kidney Cells (MDCK) cells . Carnosine inhibited cancer cell growth up to 23%. ROS level was increased up to 30 and 31% in MDCK and HeLa cells respectively. Tunnel assay showed 42 and 14% of positive apoptotic cells in cancer and normal cells respectively. The alteration in mitochondrial and nuclear morphology was determined. The extended lace‐like network of normal mitochondria found in control cells. Carnosine treatment significantly altered the mitochondrial morphology of normal cervical carcinoma cell. Mitochondria were condensed clump structures in carnosine treated cancer cells. Carnosine reduced the number of colonies of cervical carcinoma cells. Caspase 3 expression was corresponded to the appearance of immunofluorescence in the cytoplasm. Caspase 3 expression was gradually increased in cervical carcinoma cells. In Silico, docking study was performed to recognize the binding activity of carnosine against a subunit of the caspase 3 , and carnosine was able to bind to the drug binding pocket of caspase 3. The glide energy is ?5.2 kcal/mol, suggesting the high binding affinity of carnosine to caspase 3. Taking all these data together, the natural dipeptide L‐carnosine could be a suitable antiproliferative agent in cervical carcinoma cells. Copyright © 2016 John Wiley & Sons, Ltd.     

A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer.

DNA of a new papillomavirus type was cloned from a cervical carcinoma biopsy. Two EcoRI clones of 7.8 and 6.9 kb in length were obtained, the latter contained a 900-bp deletion. The BamHI fragments of both clones were used to characterize the DNA. It represents a distinct type of papillomavirus as determined by its size, its cross-hybridization with DNA of other papillomavirus types under conditions of low stringency only, the co-linear alignment of its genome with HPV 6 and HPV 16 prototypes and its occasional occurrence as oligomeric episomes. We tentatively propose to designate it as HPV 18. DNA hybridizing with HPV 18 under stringent conditions was detected in 9/36 cervical carcinomas from Africa and Brazil, in 2/13 cervical tumors from Germany and 1/10 penile carcinomas. Benign tumors (17 cervical dysplasias, 29 genital warts), eight carcinomata in situ and 15 biopsies of normal cervical tissue were devoid of detectable HPV 18 DNA. HPV 18-related DNA was found, however, in cells of the HeLa, KB and C4-1 lines all derived from cervical cancer. The state of the viral DNA was investigated in four cervical cancer biopsies. The data reveal that the DNA might be integrated into the host cell genome. One tumor provided evidence for head to tail tandem repeats some of which persisted as circular episomes.     

Proteomics characterization of the cytotoxicity mechanism of ganoderic acid D and computer-automated estimation of the possible drug target network

Triterpenes isolated from Ganoderma lucidum could inhibit the growth of numerous cancer cell lines and were thought to be the basis of the anticancer effects of G. lucidum. Ganoderic acid D (GAD) is one of the major components in Ganoderma triterpenes. GAD treatment for 48 h inhibited the proliferation of HeLa human cervical carcinoma cells with an IC(50) value of 17.3 +/- 0.3 microM. Flow cytometric analysis and DNA fragmentation analysis indicated that GAD induced G(2)/M cell cycle arrest and apoptosis. To identify the cellular targets of GAD, two-dimensional gel electrophoresis was performed, and proteins altered in expressional level after GAD exposure of cells were identified by MALDI-TOF MS/MS. The regulation of proteins was also confirmed by Western blotting. The cytotoxic effect of GAD was associated with regulated expression of 21 proteins. Furthermore these possible GAD target-related proteins were evaluated by an in silico drug target searching program, INVDOCK. The INVDOCK analysis results suggested that GAD could bind six isoforms of 14-3-3 protein family, annexin A5, and aminopeptidase B. The direct binding affinity of GAD toward 14-3-3 zeta was confirmed in vitro using surface plasmon resonance biosensor analysis. In addition, the intensive study of functional association among these 21 proteins revealed that 14 of them were closely related in the protein-protein interaction network. They had been found to either interact with each other directly or associate with each other via only one intermediate protein from previous protein-protein interaction experimental results. When the network was expanded to a further interaction outward, all 21 proteins could be included into one network. In this way, the possible network associated with GAD target-related proteins was constructed, and the possible contribution of these proteins to the cytotoxicity of GAD is discussed in this report.