Different Stages Of Malignancy Research Articles

Modeling Physical Forces Experienced by Cancer and Stromal Cells Within Different Organ-Specific Tumor Tissue.

Mechanical force exerted on cancer cells by their microenvironment have been reported to drive cells toward invasive phenotypes by altering cells' motility, proliferation, and apoptosis. These mechanical forces include compressive, tensile, hydrostatic, and shear forces. The importance of forces is then hypothesized to be an alteration of cancer cells' and their microenvironment's biophysical properties as the indicator of a tumor's malignancy state. Our objective is to investigate and quantify the correlation between a tumor's malignancy state and forces experienced by the cancer cells and components of the microenvironment. In this study, we have developed a multicomponent, three-dimensional model of tumor tissue consisting of a cancer cell surrounded by fibroblasts and extracellular matrix (ECM). Our results on three different organs including breast, kidney, and pancreas show that: A) the stresses within tumor tissue are impacted by the organ specific ECM's biophysical properties, B) more invasive cancer cells experience higher stresses, C) in pancreas which has a softer ECM (Young modulus of 1.0 kPa) and stiffer cancer cells (Young modulus of 2.4 kPa and 1.7 kPa) than breast and kidney, cancer cells experienced significantly higher stresses, D) cancer cells in contact with ECM experienced higher stresses compared to cells surrounded by fibroblasts but the area of tumor stroma experiencing high stresses has a maximum length of 40 μm when the cancer cell is surrounded by fibroblasts and 12 μm for when the cancer cell is in vicinity of ECM. This study serves as an important first step in understanding of how the stresses experienced by cancer cells, fibroblasts, and ECM are associated with malignancy states of cancer cells in different organs. The quantification of forces exerted on cancer cells by different organ-specific ECM and at different stages of malignancy will help, first to develop theranostic strategies, second to predict accurately which tumors will become highly malignant, and third to establish accurate criteria controlling the progression of cancer cells malignancy. Furthermore, our in silico model of tumor tissue can yield critical, useful information for guiding ex vivo or in vitro experiments, narrowing down variables to be investigated, understanding what factors could be impacting cancer treatments or even biomarkers to be looking for.

Aptamers for the diagnosis and treatment of human glial tumors

Purpose of the study: to evaluate the feasibility of using functional analogues of protein antibodies – dNa/ RNa aptamers in diagnostics, treatment and prognosis of human brain glial tumors.Material and Methods. The relevant literature sources were searched in scopus, Web of science, pubmed, elibrary with inclusion of publications from 2000 to 2023. sixty articles are presented in the review.Results. The analysis of the literature devoted to classification, diagnostics and therapy of brain glioblastomas was carried out and the feasibility of using for in vivo diagnostics and therapy of this disease aptamers, which are molecular recognition elements based on DNA/RNA oligonucleotides, capable of binding to the given molecular targets and distinguishing even separate functional groups in them, was studied. A list of aptamers to human glial brain tumors and their molecular targets that can be used for diagnostics and therapy of glioblastoma, including tumor imaging by pet/ct, mRi, plasmon resonance, fluorescence and confocal microscopy, etc., is presented. literature data suggest that DNA/RNA aptamers can be used to search for circulating tumor cells in the blood of glioblastoma patients, to target therapeutic drugs to the tumor and to inhibit tumor growth.Conclusion. Brain glioblastoma is a heterogeneous tumor consisting of cells at different stages of malignancy and, accordingly, with a different set of oncogenes. For this reason, a multitarget strategy that includes combined suppression of angiogenesis, invasion, metastasis, proliferation and survival of tumor cells should be proposed for the therapy of this disease. DNA/RNA aptamers tailored to key proteins involved in oncogenic transformation may be suitable candidates for the implementation of multitarget therapy for brain glioblastoma.

Abstract 3680: The expression profiles of activins and follistatin at the different stages of colorectal cancer

Abstract Background: Activins belong to the family of transforming growth factor-β and the mature proteins (activin-A, -B and AB) are formed by the dimerization of two β-subunits (βA & βB). The biological activities of activins are tightly regulated by their binding protein, follistatin. Activin-A is the most studied isoform in the biology of colorectal cancer (CRC) and none of the earlier studies explored the expression profiles of the other activins as well as follistatin. Objectives: This study measured the gene and protein expressions of activin βA- and βB-subunits together with follistatin in paired malignant and remote non-cancerous specimens that were obtained at the different stages of malignancy from patients diagnosed with primary sporadic CRC. Materials and methods: Archived paired cancerous and non-cancerous colonic specimens were obtained from a total of 30 patients diagnosed with primary CRC. Following the identification of cancer stages by expert histopathologists, the genes and proteins of the targeted molecules were measured by quantitative RT-PCR and immunohistochemistry, respectively. Results: The genes and proteins of both activin βA- and βB-subunits alongside follistatin were comparable between the non-cancerous specimens obtained at the different stages of cancer. Nevertheless, significant up-regulations in the genes and proteins of βA-subunit and follistatin simultaneously with a marked inhibition of the βB-subunit were detected in the malignant specimens compared with their counterpart non-malignant tissues. Additionally, the βA-subunit and follistatin showed substantial increases in the malignant sites as cancer progressed and the highest levels were detected in stage IV compared with the earlier stages. Opposingly, the βB-subunit decreased gradually and significantly with cancer advancement and the protein expression was completely lost at stage IV. Conclusions: This study suggests that the overexpression of activin βA-subunit and follistatin may contribute to colon carcinogenesis and tumor progression, whereas the βB-subunit could be associated with anti-tumorigenic activities. However, more studies are still needed to measure the levels of the activin mature proteins (e.g. activin-A, -B and -AB) together with follistatin to precisely characterize their roles in CRC tumorigenesis. Acknowledgment: This project was funded by the DEANSHIP OF SCIENTIFIC RESEARCH AT UMM AL-QURA UNIVERSITY; Project (17-MED-1-01-0068). Citation Format: Mohammed Abbas Baghdadi, Jawwad Ahmad, Shakir Idris, Jamal Zekri, Abdelrazak Meliti, Hosam Alardati, Aiman Alsaegh, Mohammed Aslam, Bassem Refaat. The expression profiles of activins and follistatin at the different stages of colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3680.

Atomic force microscopy for cell mechanics and diseases

Abstract Atomic Force Microscopy (AFM) is a powerful technique widely employed in biophysics, for instance to study topography of living cells and cell mechanics. Cell mechanics is a very interesting, biophysical parameter of cells, because it is strongly changed by various cellular processes, for example during cell division, cell movement, differentiation, aging, and also various diseases. Since cancer is a prominent example of changes in mechanical properties of diseases, the concept of the mechanical fingerprint has developed, which makes it possible to distinguish between healthy and diseased cells. In this article we report on various studies of cell mechanics with the AFM. We will first give a brief introduction on AFM principles and operational modes and then we will report on some applications of AFM in the field of cellular biophysics, like discriminating between healthy and cancer cells, as well as distinguishing cancer cells at different stages of malignancy. Overall, we will show that AFM has made a significant contribution in studying the biophysics of cancer and the concept of mechanical fingerprints could find a wide variety of applications in biomedicine and medical diagnostics.

Effect of Absorption and Scattering on Fluorescence of Buried Tumours

Fluorescence spectroscopy is widely used for biomedical optical diagnosis and surgical resection of tumours. This work investigates laser-induced fluorescence spectroscopy of fluorescence inclusions that are embedded in turbid media. 405 nm laser diode is used for exciting buried protoporphyrin- (PpIX) based inclusions in brain-like optical phantoms. Effects of scattering and absorption of the turbid medium on the recorded fluorescence signal and depth-resolved fluorescence were studied. Results show that optical properties of the surrounding turbid medium influence the intensity of the fluorescence signal. Absorption coefficient of the surrounding medium is the major contributor to the fluorescent signal. Analysis of the recorded fluorescence spectra shows that the effect of absorption coefficient is larger than the effect of scattering coefficient on the fluorescence intensity by nearly fivefold. The findings indicate that the fluorescence signal could be used as a biomarker of optical property variations through different stages of malignancy. This can enhance the detectability of malignant tissue for diagnostic and surgical purposes as well.

Epithelial Ovarian Cancer Diagnosis of Second-Harmonic Generation Images: A Semiautomatic Collagen Fibers Quantification Protocol.

A vast number of human pathologic conditions are directly or indirectly related to tissular collagen structure remodeling. The nonlinear optical microscopy second-harmonic generation has become a powerful tool for imaging biological tissues with anisotropic hyperpolarized structures, such as collagen. During the past years, several quantification methods to analyze and evaluate these images have been developed. However, automated or semiautomated solutions are necessary to ensure objectivity and reproducibility of such analysis. This work describes automation and improvement methods for calculating the anisotropy (using fast Fourier transform analysis and the gray-level co-occurrence matrix). These were applied to analyze biopsy samples of human ovarian epithelial cancer at different stages of malignancy (mucinous, serous, mixed, and endometrial subtypes). The semiautomation procedure enabled us to design a diagnostic protocol that recognizes between healthy and pathologic tissues, as well as between different tumor types.

Decellularized matrices as in vitro models of extracellular matrix in tumor tissues at different malignant levels: Mechanism of 5-fluorouracil resistance in colorectal tumor cells

Chemoresistance is a major barrier for tumor chemotherapy. It is well-known that chemoresistance increases with tumor progression. Chemoresistance is altered by both genetic mutations and the alteration of extracellular microenvironment. Particularly, the extracellular matrix (ECM) is remodeled during tumor progression. Therefore, ECM remodeling is expected to cause the acquisition of chemoresistance in highly malignant tumor tissue. Here, we prepared cultured cell-derived decellularized matrices that mimic native ECM in tumor tissues at different stages of malignancy, and 5-fluorouracil (5-FU) resistance was compared among these matrices. 5-FU resistance of colorectal tumor cells increased on the matrices derived from highly malignant tumor HT-29 cells, although the resistance did not increase on the matrices derived from low malignant tumor SW480 cells and normal CCD-841-CoN cells. The resistance on HT-29 cell-derived matrices increased through the activation of Akt and the upregulation of ABCB1 and ABCC1 without cell growth promotion, suggesting that ECM remodeling plays important roles in the acquisition of chemoresistance during tumor progression. It is expected that our decellularized matrices, or “staged tumorigenesis-mimicking matrices”, will become preferred cell culture substrates for in vitro analysis of comprehensive ECM roles in chemoresistance and the screening and pharmacokinetic analysis of anti-cancer drugs.

Monitoring Tyrosinase Expression in Non-metastatic and Metastatic Melanoma Tissues by Scanning Electrochemical Microscopy.

Although tremendous progress has been made in the diagnosis of melanoma, the identification of different stages of malignancy in a reliable way remains challenging. Current strategies rely on optical monitoring of the concentration and spatial distribution of specific biomarkers. State-of-the-art optical methods can be affected by background-color interference and autofluorescence. We overcame these shortcomings by employing scanning electrochemical microscopy (SECM) to map the prognostic indicator tyrosinase (TyR) in non-metastatic and metastatic melanoma tissues by using soft-stylus microelectrodes. Electrochemical readout of the TyR distribution was enabled by adapting an immunochemical method. SECM can overcome the limitations of optical methods and opens unprecedented possibilities for improved diagnosis and understanding of the spatial distribution of TyR in different melanoma stages.

Three-Dimensional In Vitro Skin and Skin Cancer Models Based on Human Fibroblast-Derived Matrix.

Three-dimensional in vitro skin and skin cancer models help to dissect epidermal-dermal and tumor-stroma interactions. In the model presented here, normal human dermal fibroblasts isolated from adult skin self-assembled into dermal equivalents with their specific fibroblast-derived matrix (fdmDE) over 4 weeks. The fdmDE represented a complex human extracellular matrix that was stabilized by its own heterogeneous collagen fiber meshwork, largely resembling a human dermal in vivo architecture. Complemented with normal human epidermal keratinocytes, the skin equivalent (fdmSE) thereof favored the establishment of a well-stratified and differentiated epidermis and importantly allowed epidermal regeneration in vitro for at least 24 weeks. Moreover, the fdmDE could be used to study the features of cutaneous skin cancer. Complementing fdmDE with HaCaT cells in different stages of malignancy or tumor-derived cutaneous squamous cell carcinoma cell lines, the resulting skin cancer equivalents (fdmSCEs) recapitulated the respective degree of tumorigenicity. In addition, the fdmSCE invasion phenotypes correlated with their individual degree of tissue organization, disturbance in basement membrane organization, and presence of matrix metalloproteinases. Together, fdmDE-based models are well suited for long-term regeneration of normal human epidermis and, as they recapitulate tumor-specific growth, differentiation, and invasion profiles of cutaneous skin cancer cells, also provide an excellent human in vitro skin cancer model.

Expression and subcellular localization of RAGE in melanoma cells

The receptor for advanced glycation end products (RAGE) is involved in multiple stages of tumor development and malignization. To gain further knowledge on the RAGE role in tumor progression, we investigated the receptor expression profile and its subcellular localization in melanoma cells at different stages of malignancy. We found that RAGE clustered at membrane ruffles and leading edges, and at sites of cell-to-cell contact in primary melanoma cells (e.g., MelJuSo), in contrast with a more dispersed localization in metastatic cells (e.g., SK-Mel28). RAGE silencing by RNAi selectively inhibited migration of MelJuSo cells, whilst having no influence on SK-Mel28 cell migration, in a "wound healing" assay. Western blot detection of RAGE showed a more complex RAGE oligomerization in MelJuSo cells compared to melanocytes and SK-Mel28 cells. By competing the binding of antibodies with recombinant soluble RAGE, an oligomeric form running at approximately 200 kDa was detected, as it was the monomeric RAGE of 55-60 kDa. SDS-PAGE electrophoresis under reducing versus nonreducing conditions indicated that the oligomer of about 200 kDa is formed by disulfide bonds, but other interactions are likely to be important for RAGE multimerization in melanoma cells. Immunofluorescence microscopy revealed that treatment with two cholesterol-chelating drugs, nystatin and filipin, significantly affected RAGE localization in MelJuSo cells. SK-Mel28 cells showed a reduced RAGE glycosylation and association with cholesterol-rich membranes and also a considerable downregulation of the soluble forms. Our results indicate that RAGE isoform expression and subcellular localization could be important determinants for the regulation of its function in tumor progression.

A Solid Mass in the Head of the Pancreas with Intense 18FDG Uptake: Intraductal Tubulopapillary Neoplasm

Context 18 FDG-PET/CT has emerged as a useful diagnostic modality for the staging of different malignancies. Its role in the characterization of both cystic and solid pancreatic tumors is debated, especially in the differential diagnosis between malignant and benign/borderline tumors. Case report A 66-year-old man complained of abdominal discomfort with no other symptoms. An abdominal US showed a solid mass in the head of the pancreas. He underwent contrast medium CT scan and MRI that showed a pseudonodular mass of 35x40 mm in the pancreatic head without dilatation of the common bile duct/main pancreatic duct. There was no infiltration of the surrounding pancreatic parenchyma, duodenum and peripancreatic vessels. Serum CA 19.9 was 795 U/mL. A 18 FDG PET/CT scan showed an intense uptake by the mass. He underwent pylorus-preserving pancreaticoduodenectomy. Macroscopic examination of the specimen revealed a 4 cm tumor with endocystic growth without infiltration of surrounding structures, including common bile duct and main pancreatic duct. Histologically the tumor was composed of cubic to cylindrical well-differentiated cells, with low/intermediate grade atypia. Tumor cells formed tubulopapillae and contained little cytoplasmic mucin. Immunohistochemical profile showed positivity only for cytokeratin 7 and cytokeratin 19. Ki67 index was 25-30%. 37 lymph nodes were removed without metastases. A diagnosis of intraductal tubulopapillary neoplasm (ITPN) with low-grade/intermediate dysplasia was made. Conclusion ITPN is a rare primary pancreatic neoplasm accounting for less than 1% of all pancreatic exocrine neoplasms. ITPN can show a different biological behavior, from low-grade to high-grade dysplasia and invasive carcinoma. Presence of high proliferative index, high-grade atypia, and, obviously, infiltration of surrounding structures/lymph node metastases, indicate malignancy. Differential diagnosis include mainly solid-pseudopapillary tumor and acinar cell carcinoma. In this case intense 18 FDG uptake and high CA 19.9 level prompted for resection, although only low-grade/intermediate dysplasia was present.

Global microRNA profiling of well-differentiated small intestinal neuroendocrine tumors

Well-differentiated small intestinal neuroendocrine tumors are rare malignancies. They arise from enterochromaffin cells and very little is known about differential microRNA (miRNA) expression. The aim of this study was to identify the miRNA profile of well-differentiated small intestinal neuroendocrine tumors, which may have a critical role in tumor development, progression and potentially develop miRNAs as novel clinical biomarkers. Specimens from two test groups, 24 small intestinal neuroendocrine tumor specimens at different stages of malignancy, are included in this study. Total RNA from the first test group, five primary tumors, five mesentery metastases and five liver metastases was hybridized onto the Affymetrix Genechip miRNA arrays to perform a genome-wide profile. The results were validated by using quantitative real-time PCR (QRT-PCR) and northern blot analyses. We then expanded the investigation to laser capture microdissected small intestinal neuroendocrine tumor cells and immuno-laser capture microdissected normal enterochromaffin cells of the first test group. Furthermore, a second test group, three primary tumors, three mesentery metastases and three liver metastases, was included in the study. Thus, two independent test groups validated the data by QRT-PCR. Moreover, we characterized nine miRNAs, five (miR-96, -182, -183, -196a and -200a), which are upregulated during tumor progression, whereas four (miR-31, -129-5p, -133a and -215) are downregulated. Several online software programs were used to predict potential miRNA target genes to map a number of putative target genes for the aberrantly regulated miRNAs, through an advanced and novel bioinformatics analysis. Our findings provide information about pivotal miRNAs, which may lead to further insights into tumorigenesis, progression mechanisms and novel therapeutic targets recognition.

A hybrid FLIM-elastic net platform for label free profiling of breast cancer

We report a label-free fluorescence lifetime profiling strategy to classify breast cancer cells, MCF10CA1h (malignant), MCF10A (nonmalignant), and MCF10AneoT (premalignant) in different stages of malignancy. Fluorescence Lifetime Imaging Microscopy (FLIM) was used to record the lifetime of autofluorescence of endogenous flavin in MCF10 cells in different stages of malignancy. Predominant differences in lifetimes ascertained by multi-exponential fitting curves can be attributed to the different forms of flavin protein; flavin mononucleotide (FMN), free flavin adenine dinucleotide (FAD), semiquinone, and bound FAD. A lifetime map of the metabolite was derived from the contribution of the lifetime of each metabolite by iterative reconvolution fitting of the Time Correlated Single Photon Counting (TCSPC) decay curves. Lifetime maps were constructed by mapping the average lifetime values pixel by pixel using MATLAB. The FLIM image (150 × 150 pixels) of each cell was extracted, resized and centered into 100 × 100 pixels using the nearest neighbor algorithm. Principal Component Analysis (PCA) in conjunction with Elastic net Analysis (EnA) was then used to classify the different stages of MCF10 cell lines based on average lifetime values. The EnA model provided an excellent classification of the cells at different stages of tumorigenesis yielding 100% accuracy.

374. Photoacoustic Nodal Metastases Detection in a Rat Model Using a Clinical Superparamagnetic Iron Oxide Nanoparticle Dispersion

Background: Detection of tumor metastases in the lymphatic system is essential for accurate staging of different malignancies, however fast and accurate pre- or intra-operative evaluation of the nodal status remains difficult to perform with common available medical imaging techniques. In recent years, numerous studies have confirmed the additional value of superparamagnetic iron oxide dispersions (SPIOs) for nodal staging purposes, prompting the clearance of different SPIO dispersions for clinical practice. Here we evaluate whether a combination of photoacoustic (PA) imaging and a clinically approved SPIO dispersion, could have the potential for sensitive assessment of the nodal status both pre- and intra-operatively. Materials and methods: Prostate adenocarcinoma cells (MAT-lylu) were inoculated in the footpad of Copenhagen rats. For lymph node imaging animals received a subcutaneous injection of a SPIO dispersion (Endorem®, Guerbet, France) either 5 (n=4) or 8 (n=4) days after tumor inoculation. The resected nodes were photoacoustically imaged at 720 nm using 20 mJ/cm2, 24 hours later, and imaging results were compared to 14 Tesla MR imaging and histology. Control animals were imaged without SPIO injection in the involved leg or received SPIO injections on the contra lateral site harboring no tumor inoculation and hence normal lymph nodes. Results: In the tumor groups clear irregularities, as small as 1 mm, were observed in the PA contrast pattern of the nodes. These irregularities could be correlated to the absence of contrast in the MR images and could directly be linked to metastatic deposits seen on the histological slides. The PA and MR images of the control animals did not show these irregularities. Conclusions: We conclude that the combination of photoacoustic imaging with a clinically approved iron oxide nanoparticle dispersion is able to detect lymph node metastases as small as 1 mm. This approach opens up new possibilities for pre- or intra-operative nodal staging.

PMO-088 A large proportion of colorectal tumour-infiltrating CD4+ T cells are suppressive irrespective of FOXP3 expression

IntroductionThe presence of increased numbers of CD3+ T cells in colorectal cancer (CRC) correlates with improved prognosis. However, it is difficult to measure anti-tumour responses in tumour-infiltrating lymphocytes (TILs) suggesting...

Spheroid assay to measure TGF-β-induced invasion.

TGF-β has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage(1,2). Moreover, TGF-β is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis (3,4). The mechanisms by which TGF-β induces invasion are not well understood. TGF-β elicits its cellular responses via TGF-β type II (TβRII) and type I (TβRI) receptors. Upon TGF-β-induced heteromeric complex formation, TβRII phosphorylates the TβRI. The activated TβRI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes(5). In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways(6). To study the role of TGF-β in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells(7), the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice(8), and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung(9). This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background. For the analysis of TGF-β-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid(10). Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures(11). This MCF10 3D model system allowed us to investigate the impact of TGF-β signaling on the invasive properties of breast cells in different stages of malignancy.

Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD.

Human papillomavirus (HPV) E6 oncoproteins target many cellular proteins for ubiquitin-mediated proteasomal degradation. In the case of p53, this is mediated principally by the E6AP ubiquitin ligase. Several studies have reported that E6 can target certain of its substrates in an apparently E6AP-independent fashion and that several of these substrates vary in the degree to which they are degraded by E6 at different stages of malignancy. To more fully understand the regulation of the E6AP/E6 proteolytic targeting complex, we performed a mass spectroscopic analysis of HPV type 18 (HPV-18) E6 protein complexes and identified the HECT domain-containing ubiquitin ligase EDD as a new HPV-18 E6 binding partner. We show that EDD can interact independently with both E6 and E6AP. Furthermore, EDD appears to regulate E6AP expression levels independently of E6, and loss of EDD stimulates the proteolytic activity of the E6/E6AP complex. Conversely, higher levels of EDD expression protect a number of substrates from E6-induced degradation, partly as a consequence of lower levels of E6 and E6AP expression. Intriguingly, reduction in EDD expression levels in HPV-18-positive HeLa cells enhances cell resistance to apoptotic and growth arrest stimuli. These studies suggest that changes in the levels of EDD expression during different stages of the viral life cycle or during malignancy could have a profound effect upon the ability of E6 to target various substrates for proteolytic degradation and thereby directly influence the development of HPV-induced malignancy.

Toll-like Receptor 3 Regulates Angiogenesis and Apoptosis in Prostate Cancer Cell Lines through Hypoxia-Inducible Factor 1α

Toll-like receptors (TLRs) recognize microbial/viral-derived components that trigger innate immune response and conflicting data implicate TLR agonists in cancer, either as protumor or antitumor agents. We previously demonstrated that TLR3 activation mediated by its agonist poly(I:C) induces antitumor signaling, leading to apoptosis of prostate cancer cells LNCaP and PC3 with much more efficiency in the former than in the second more aggressive line. The transcription factor hypoxia-induciblefactor 1 (HIF-1)regulates several cellular processes, includingapoptosis, in response to hypoxia and to other stimuli also in normoxic conditions. Here we describe a novel protumor machinery triggered by TLR3 activation in PC3 cells consisting of increased expression of the specific 1.3 isoform of HIF-1α and nuclear accumulation of HIF-1 complex in normoxia, resulting in reduced apoptosis and in secretion of functional vascular endothelial growth factor (VEGF). Moreover, we report that, in the less aggressive LNCaP cells, TLR3 activation fails to induce nuclear accumulation of HIF-1α. However, the transfection of 1.3 isoform of hif-1α in LNCaP cells allows poly(I:CI-induced HIF-1 activation, resulting in apoptosis protection and VEGF secretion. Altogether, our findings demonstrate that differences in the basal level of HIF-1α expression in different prostate cancer cell lines underlie their differential response to TLR3 activation, suggesting a correlation between different stages of malignancy, hypoxic gene expression, and beneficial responsiveness to TLR agonists.

Effect of phytic acid and inositol on the proliferation and apoptosis of cells derived from colorectal carcinoma.

We characterized the effect of phytic acid (inositol hexaphosphate, IP6) as a potential adjuvant in treatment of colorectal carcinoma and evaluated the optimal concentration and treatment time to produe the maximal therapeutic effect. There is some evidence that myoinositol (Ins) can potentiate anti-cancer effects of IP6. Therefore, we tested both IP6 and Ins individually and in combination on human cell lines HT-29, SW-480 and SW-620 derived from colorectal carcinoma in different stages of malignancy. The effect of tested chemicals on the cells was measured using metabolic activity assay (WST-1), DNA synthesis assay (BrdU), protein synthesis assay (Brilliant Blue) and apoptosis (caspase-3 activity). We tested IP6 and Ins at three concentrations: 0.2, 1 and 5 mM for 24, 48 and 72 h. The concentrations and incubation periods were chosen according to low toxicity of the tested substance that was observed in a long-term clinical study. We found that all employed concentrations of IP6 or IP6/Ins decreased proliferation of the cell lines, with the maximum decrease being observed in HT-29 cells. Metabolic activity of treated cells differed in response to IP6 and IP6/Ins treatment; in HT-29 and SW-620 significant decrease was observed only at the highest concentration, whereas in SW-480 cells metabolic activity was lower at each concentration except 0.2 and 1 mM IP6 or IP6/Ins in 24-h incubation. The results from protein content assay corresponded to the results obtained from WST assay. In addition, we found maximum increase in caspase-3 activity at concentration 5 mM IP6 or IP6/Ins in HT-29 cells and with IP6 at concentration of 0.2 mM or IP6/Ins in SW-480 cells with clear indication of Ins enhancing the proapoptotic effect of IP6 in all the cell lines studied.

Chromosomes in preinvasive, microinvasive and invasive cervical carcinoma.

The chromosomes in 32 preinvasive, 5 microinvasive, and 10 invasive cervical carcinomas were studied in direct fixations. The chromosome pattern was surveyed in relation to earlier results obtained in this field. Of the preinvasive lesions 66% had a diploid, 13% a triploid and 22% a tetraploid modal population. In 3 of the microinvasive tumours the modal population was diploid and in 2, triploid. In the diploid subgroup of the preinvasive tumours without and with microinvasion the chromosome number 46 was predominant. In the polyploid subgroup all were hypertriploid to hypotetraploid. Of the invasive carcinomas 7 exhibited a diploid modal population and 3 a triploid. The modes were spread throughout the ploidy regions. The normal diploid karyotype was common in carcinoma in situ, but absent in the microinvasive and invasive carcinomas. Gains and losses in the modal population were associated with the same chromosome types in the different stages of malignancy. Chromosomes were usually in excess in group C, and deficits were found in group B and, more often, in groups D and G. Structural aberrations appeared occasionally in preinvasive and microinvasive lesions and more often in invasive tumours, usually as an integrated part of the modal karyotype. A statistical analysis of the mean karyotype deviations in the entire material revealed a non-random pattern, and a significant increase in the severity of the chromosome changes with degree of malignancy.