Cathepsins H and L in colorectal cancer

Cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biologi­cal processes while they also play an essential role in cancer progression, invasion and metastasis. The purpose of our study was: first to compare the expression levels of cathepsins H and L in the supernatants of colon cancer tissues from 74 patients versus the same enzymic expressions of the supernatants of the adjacent normal colorectal tissues and second to correlate our findings to the grade of the malignancy by using an enzyme-linked immunosorbent assay (ELISA). The results indicated that the cathepsins H and L of all malignant tissues presented significant higher expression’s values than the corresponding control. Specifically the concentration of cathepsin H that has been found increased significantly as malignancy proceeded, was higher than the corresponding control as following: 155% in B1 stage and 204,44% in D stage. Between the two inves­tigated proteases cathepsin L has showed the greatest increase, which in D stage was 261,03% higher than the corresponding control. According to these results, the expression of cysteine proteases H and L could be of critical value in the diagnosis and progression of colon cance

Role of Vanadium in Cellular and Molecular Immunology: Association with Immune-Related Inflammation and Pharmacotoxicology Mechanisms

Over the last decade, a diverse spectrum of vanadium compounds has arisen as anti-inflammatory therapeutic metallodrugs targeting various diseases. Recent studies have demonstrated that select well-defined vanadium species are involved in many immune-driven molecular mechanisms that regulate and influence immune responses. In addition, advances in cell immunotherapy have relied on the use of metallodrugs to create a “safe,” highly regulated, environment for optimal control of immune response. Emerging findings include optimal regulation of B/T cell signaling and expression of immune suppressive or anti-inflammatory cytokines, critical for immune cell effector functions. Furthermore, in-depth perusals have explored NF-κB and Toll-like receptor signaling mechanisms in order to enhance adaptive immune responses and promote recruitment or conversion of inflammatory cells to immunodeficient tissues. Consequently, well-defined vanadium metallodrugs, poised to access and resensitize the immune microenvironment, interact with various biomolecular targets, such as B cells, T cells, interleukin markers, and transcription factors, thereby influencing and affecting immune signaling. A synthetically formulated and structure-based (bio)chemical reactivity account of vanadoforms emerges as a plausible strategy for designing drugs characterized by selectivity and specificity, with respect to the cellular molecular targets intimately linked to immune responses, thereby giving rise to a challenging field linked to the development of immune system vanadodrugs

Interaction studies of metal ions with H-ras and K-ras oncogenes: focus on carcinogenic cadmium and development of anticancer vanadium metallodrugs

In this thesis we studied the interaction of metal ions with H-ras and K-ras oncogenes, focusing on the carcinogenic cadmium and the development of vanadium-based anticancer drugs. The results shed light on the functional link between oncogenes and interleukins, inflammation, the ras-signaling and carcinogenesis in the colon, thereby contributing to the future development of cancer detection methods and oligonucleotide therapies for K-ras. For this purpose, specially designed vanadodrugs targeting mutated K-ras in various types of cancer have emerged as a promising therapeutic approach.The work presented here suggests that cadmium acts as an inducer of caspase-3 apoptosis, while promoting variable H-ras protein expression. The observed variable behavior in the presence and absence of H-ras(wt) and H-ras(G12V) attests to that and points to an interplay of the two factors in a form-specific (H-ras(wt) vs. H-ras(G12V)) and concentration-dependent fashion. Studies directed toward understanding a) the modular interplay and molecular interactions involved in the case of cadmium with H-ras and its mutant form(s), b) the concentration-dependent effects of cadmium at low concentrations reaching pM levels, and c) the linkage of the chemical reactivity of the inorganic metallotoxin to genetically induced instability effects linked to carcinogenic phenotypes are currently under investigation.The present study also demonstrates that the employed ternary vanadium-peroxido-betaine [VO(O2)2{(CH3)3NCH2CO2}]– species exhibits effectively antitumor properties. The observed antitumor activity projects association with the ras-MAPK-MMP2 inhibition pathway in cancer cells through oxidative stress related apoptosis, thereby necessitating investigation into interactions of vanadium with more molecular targets in signaling pathways. It, therefore, stands plausible that this ternary vanadium-peroxido compound represents a class of important anticancer molecules, which can be appropriately modulated so as to enhance their activity and specificity toward a variety of cancer tissues. In addition, the results highlight the critical role that specific interleukins (IL-17, IL-22 and IL-23) play in inflammation-driven tumorigenesis and provide new insights into the aberrant cytokine signaling in oncogenic K-ras associated colorectal carcinogenesis. Given the role of ras signaling in cancer progression and metastasis, this study can serve as a platform for the identification and validation of synergistic biomarkers with oncogenic K-ras toward development of therapeutically efficacious molecular agents targeting these mutations. The findings a) project important implications for further design of novel therapies for patients with colorectal adenocarcinoma, and b) shed light on molecular mechanisms involving crosstalk between tumor cells and the immune system by targeting exclusive members of the cytokine superfamily that regulate tumor growth and surveillance.Στην παρούσα έρευνα μελετήθηκε η αλληλεπίδραση μεταλλοϊόντων με τα Η-ras και Κ-ras ογκογονίδια, εστιάζοντας στο καρκινογόνο κάδμιο και στην ανάπτυξη αντικαρκινικών φαρμάκων βαναδίου. Τα αποτελέσματα αναδεικνύουν τη σχέση μεταξύ των ογκογονιδίων και ιντερλευκινών, στη φλεγμονή, στη ras-σηματοδότηση και στην καρκινογένεση του παχέος εντέρου, συμβάλλοντας στην ανάπτυξη μεθόδων ανίχνευσης του καρκίνου και της ολιγονουκλεοτιδικής θεραπείας κατά του K-ras. Για το σκοπό αυτό, ειδικά σχεδιασμένα βαναδοϋλικά έχουν ως στόχο το μεταλλαγμένο K-ras σε διάφορους τύπους καρκίνου και έχουν αναδυθεί ως μια πολλά υποσχόμενη θεραπευτική προσέγγιση. Αφού εξετάσθηκε ο μοριακός μηχανισμός ενεργοποίησης της καρκινογένεσης από το κάδμιο, αποφασίσθηκε να μελετηθεί η αντικαρκινική δράση του βαναδίου και κατά πόσον αυτή συνδέεται με τους κυτταρικούς μηχανισμούς ενεργοποίησης του καδμίου. Αποδείχτηκε τελικά ότι και οι δύο μηχανισμοί (καρκινογένεση και αναστολή της) χρησιμοποιούν τα ίδια κυτταρικά μονοπάτια έκφρασης με κοινούς κυτταρικούς στόχους. Τα αποτελέσματα έδειξαν ότι η χρησιμοποιούμενη τριαδική ένωση βαναδιο-υπεροξο-βεταΐνη [VO(O2)2{(CH3)3NCH2CO2}]– εμφανίζει πολύ αξιόλογες ιδιότητες. Συγκεκριμένα, παρατηρήθηκε αναστολή του κυτταρικού μονοπατιού RAS-ΜΑΡΚ-MMP2, οδηγώντας τα καρκινικά κύτταρα σε απόπτωση μέσω οξειδωτικού stress και παράλληλα μείωσε αισθητά την έκφραση της μεταστατικής πρωτεΐνης MMP-2, που αντικατοπτρίζει και τον κύριο δείκτη μετάστασης των καρκινικών κυττάρων. Οι μετρήσεις έδειξαν ότι το βανάδιο αναστέλλει τον κυτταρικό πολλαπλασιασμό και τη βιωσιμότητα των καρκινικών κυττάρων και στις δύο καρκινικές σειρές, χωρίς ωστόσο να βλάπτει τα υγιή φυσιολογικά κύτταρα. Ως εκ τούτου, αποδεικνύεται ότι το βανάδιο αντιπροσωπεύει μια τάξη σημαντικών αντικαρκινικών μορίων, τα οποία μπορεί να είναι κατάλληλα διαμορφωμένα έτσι ώστε να ενισχυθεί η δραστικότητά τους και η εξειδίκευσή τους προς μια ποικιλία μορφών καρκίνου. Στο τρίτο μέρος της διδακτορικής έρευνας αποφασίσθηκε να μελετηθεί η έκφραση των ογκογονιδίων ras σε ζωντανά δείγματα ιστών από ασθενείς με καρκίνο του παχέος εντέρου και να εξετασθεί η έκφραση του ογκογονιδίου K-ras σε ασθενείς που εμφανίζουν τη μετάλλαξη G12V στο ογκογονίδιο K-ras σε σχέση με φυσιολογικά δείγματα. Μελετήθηκε, επίσης, η έκφραση των κυτταροκινών ιντερλευκινών IL-17, IL-22 και IL-23, της ιντερφερόνης γ (IFNγ) και του αυξητικού παράγοντα GM-CSF, που αποτελούν δείκτες φλεγμονώδους αντίδρασης, μιας φυσιολογικής λειτουργίας του ανοσολογικού συστήματος που αναπτύσσεται κατά τη διάρκεια των μολύνσεων από ιούς ή βακτήρια, με την υπερέκφραση ή αναστολή τους να συνδέεται άμεσα με την καρκινογένεση

Differential expression of IL-17, 22 and 23 in the progression of colorectal cancer in patients with K-ras mutation: Ras signal inhibition and crosstalk with GM-CSF and IFN-γ.

Recent studies have suggested that aberrant K-ras signaling is responsible for triggering immunological responses and inflammation-driven tumorigenesis. Interleukins IL-17, IL-22, and IL-23 have been reported in various types of malignancies, but the exact mechanistic role of these molecules remains to be elucidated. Given the role of K-ras and the involvement of interleukins in colorectal tumorigenesis, research efforts are reported for the first time, showing that differentially expressed interleukin IL-17, IL-22, and IL-23 levels are associated with K-ras in a stage-specific fashion along colorectal cancer progression. Specifically, a) the effect of K-ras signaling was investigated in the overall expression of interleukins in patients with colorectal cancer and healthy controls, and b) an association was established between mutant K-ras and cytokines GM-CSF and IFN-γ. The results indicate that specific interleukins are differentially expressed in K-ras positive patients and the use of K-ras inhibitor Manumycin A decreases both interleukin levels and apoptosis in Caco-2 cells by inhibiting cell viability. Finally, inflammation-driven GM-CSF and IFN-γ levels are modulated through interleukin expression in tumor patients, with interleukin expression in the intestinal lumen and cancerous tissue mediated by aberrant K-ras signaling. Collectively, the findings a) indicate that interleukin expression is influenced by ras signaling and specific interleukins play an oncogenic promoter role in colorectal cancer, highlighting the molecular link between inflammation and tumorigenesis, and b) accentuate the interwoven molecular correlations as leads to new therapeutic approaches in the future

Mechanisms and Αpplications of Ιnterleukins in Cancer Immunotherapy

Over the past years, advances in cancer immunotherapy have resulted in innovative and novel approaches in molecular cancer diagnostics and cancer therapeutic procedures. However, due to tumor heterogeneity and inter-tumoral discrepancy in tumor immunity, the clinical benefits are quite restricted. The goal of this review is to evaluate the major cytokines-interleukins involved in cancer immunotherapy and project their basic biochemical and clinical applications. Emphasis will be given to new cytokines in pre-clinical development, and potential directions for future investigation using cytokines. Furthermore, current interleukin-based approaches and clinical trial data from combination cancer immunotherapies will also be discussed. It appears that continuously increasing comprehension of cytokine-induced effects, cancer stemness, immunoediting, immune-surveillance as well as understanding of molecular interactions emerging in the tumor microenvironment and involving microRNAs, autophagy, epithelial-mesenchymal transition (EMT), inflammation, and DNA methylation processes may hold much promise in improving anti-tumor immunity. To this end, the emerging in-depth knowledge supports further studies on optimal synergistic combinations and additional adjuvant therapies to realize the full potential of cytokines as immunotherapeutic agents

GM-CSF and IFN-γ levels of PBMCs from patients before surgery.

<p>In (<b>A</b>) GM-CSF ELISPOT of PBMCs was obtained from patients before surgery. Mononuclear cells were stimulated and were investigated for GM-CSF secretion. All experiments were carried out in triplicate. Results are presented as mean ± SD of the different groups. (<b>B</b>) IFN-γ ELISPOT of PBMCs was obtained from patients before surgery. Mononuclear cells were stimulated and were investigated for IFN-γ secretion. All experiments were carried out in triplicates. Results are presented as mean ± SD of the different groups. Large bar indicates comparison between patient stage D and stage D+Manumycin treatment samples.</p

Protein and mRNA expression levels of IL-17, IL-22, IL-23, GM-CSF, and IFN-γ.

<p>(<b>A</b>) Protein expression levels of IL-17, IL-22, IL-23, GM-CSF, and IFN-γ. GAPDH was used as a loading control for Western blot analysis. Data are mean ± SD of three independent experiments. Densitometric analysis of each protein level was calculated from the average of three experiments. Each value was expressed as the ratio of the measured protein to GAPDH level (P<0.001). (<b>B</b>) mRNA expression levels of IL-17, IL-22, IL-23, GM-CSF, and IFN-γ from colon cancer tissues performed by RT-PCR analysis. 5 µg of total RNA was isolated from cancer tissues. Values represent the mean ± SD of three independent experiments.</p

Immunohistochemical analysis of IL-17, IL-22, and IL-23 in human colon biopsies.

<p>Immunohistochemical staining of (<b>A</b>) IL-17, (<b>B</b>) IL-22, and (<b>C</b>) IL-23 in colon biopsies from patients with colorectal cancer. Paraffined sections of human colon biopsies were obtained from patients with colorectal cancer (<b>b</b>), and non-cancerous tissue (<b>a</b>). Human IL-17, IL-22, and IL-23, were detected using anti-human monoclonal antibodies from Santa Cruz. Staining with mouse IgG1 isotype was used as the negative control. Stained tumor cells are shown at a final magnification of ×400.</p

Effect of Manumycin A on Caco-2 cell viability and apoptosis.

<p>(<b>A</b>) MTT assay showing the effect of Manumycin A on cancer cell viability in Caco-2 cells. Cells were treated with different concentrations of Manumycin A for 24 hr. Cell viability was measured with an ELISA plate reader at 570 nm. Image magnification ×200. (<b>B</b>) The results indicate that Manumycin A inhibits the viability of human colon adenocarcinoma Caco-2 cells in a dose-dependent fashion. Values represent the mean ± SD of three independent experiments. Manumycin A induces apoptosis in (<b>C</b>) Caco-2 human colon adenocarcinoma cancer cells. Caco-2 cell apoptosis was detected using the TUNEL detection assay from Roche. Manumycin A concentrations: 10, 50, 100, 200, and 300 μΜ. The cancerous cell line was exposed to Manumycin A at different concentrations for 24 hr and stained. Red apoptotic cells were viewed using a Carl Zeiss (Zeiss Europe) fluorescent microscope. Image magnification ×200. (<b>D</b>) The graphs represent the quantitative results of apoptosis. Data are mean ± SD of three independent experiments.</p