Conformational Preferences and Antiproliferative Activity of Peptidomimetics Containing Methyl 1′-Aminoferrocene-1-carboxylate and Turn-Forming Homo- and Heterochiral Pro-Ala Motifs

The concept of peptidomimetics is based on structural modifications of natural peptides that aim not only to mimic their 3D shape and biological function, but also to reduce their limitations. The peptidomimetic approach is used in medicinal chemistry to develop drug-like compounds that are more active and selective than natural peptides and have fewer side effects. One of the synthetic strategies for obtaining peptidomimetics involves mimicking peptide α-helices, β-sheets or turns. Turns are usually located on the protein surface where they interact with various receptors and are therefore involved in numerous biological events. Among the various synthetic tools for turn mimetic design reported so far, our group uses an approach based on the insertion of different ferrocene templates into the peptide backbone that both induce turn formation and reduce conformational flexibility. Here, we conjugated methyl 1′-aminoferrocene-carboxylate with homo- and heterochiral Pro-Ala dipeptides to investigate the turn formation potential and antiproliferative properties of the resulting peptidomimetics 2–5. Detailed spectroscopic (IR, NMR, CD), X-ray and DFT studies showed that the heterochiral conjugates 2 and 3 were more suitable for the formation of β-turns. Cell viability study, clonogenic assay and cell death analysis showed the highest biological potential of homochiral peptide 4.

In vitro modified microdosimetric kinetic model-based predictions for B14-150 cells survival in 450 MeV/u carbon ion beam with aluminum ridge filter for biologically optimized spread-out Bragg peak

The relative biological efficiency of particle irradiation could be predicted with a wide variety of radiobiological models for various end-points. We validate the forecast of modified Microdosimetric Kinetic Model in vitro using combined data of reference Co-60 radiation and carbon ion plateau data for specific cell line to optimize the survival function in spread-out Bragg Peak obtained with an especially designed ridge filter. We used Geant4 Monte-Carlo software to simulate the fragment contribution along Bragg curve inside water phantom, open-source toolkit Survival to predict the expected linear-quadratic model parameters for each fragment, and in-house software to form the total survival curve in spread-out Bragg Peak. The irradiation was performed at U-70 synchrotron with an especially designed Aluminum ridge filter under the control of PTW and in-house ionization chambers. The cell clonogenic assay was conducted with the B14-150 cell line. The data analysis was accomplished using scipy and CERN ROOT. The clonogenic assay represents the survival in spread-out Bragg Peak at different points and qualitatively follows the modeled survival curve very well. The quantitative difference is within 3σ, and the deviation might be explained by the uncertainties of physical modeling using Monte-Carlo methods. Overall, the obtained results are promising for further usage in radiobiological studies or carbon ion radiotherapy. Shaping the survival curve in the region of interest (i.e., spread-out Bragg Peak) is a comprehensive task that requires high-performance computing approaches. Nevertheless, the method's potential application is related to the development of next-generation treatment planning systems for ion beams. This can open a wide range of improvements in patient treatment outcome, provide new optimized fractionation regimes or optimized dose delivery schemes, and serve as an entrance point to the translational science approach.

CBIO-02. IN VITRO TUMOR TREATING FIELDS (TTFields) REDUCE PROLIFERATION AND ALTER MLH1 EXPRESSION IN TEMOZOLOMIDE RESISTANT (TMZR) PATIENT-DERIVED GLIOBLASTOMA (GBM) CELLS

BACKGROUND TTFields therapy is approved as a monotherapy for the treatment of recurrent GBM, tumors that often have acquired resistance to TMZ. To examine TTFields efficacy in the context of TMZR in vitro, TTFields were applied to TMZR GBM cells to assess proliferation, clonogenicity, and changes in expression of O6-methylguanine-DNA methyltransferase (MGMT) that confers TMZ resistance and DNA Mismatch Repair Protein Mlh1 (MLH1) that may confer TMZ sensitivity. METHODS 15-037 GBM cells were isolated from a newly-diagnosed patient tumor (IDH-WT, unmethylated MGMT promoter. Cells were grown in DMEM/F12 media with 10% FBS and gentamicin. U-251 MG cells and 15-037 cells were incubated with 100 µM TMZ for three 5-day cycles to establish TMZR. TMZR cells were plated on coverslips (1×104cells/coverslip) and incubated overnight prior to TTFields application. TTFields were applied at 200 kHz (field intensity of ~1.6 V/cm) for 14 days. Then coverslips (n=3/group) were harvested, counted, and replated for clonogenic assay at 1000 cells/35mm2 well. Clonogenic assay plates were stained with crystal violet, imaged, and colonies counted. Additional coverslips (n=3/group) were harvested and MGMT and MLH1 expression were assessed by qRT-PCR with the delta-delta Ct method using GAPDH as housekeeping control. Cell counts and colony counts were compared with two-tailed t-test with significance at p< 0.05. RESULTS In U-251 MG and 15-037 TMZR cells, TTFields application significantly reduced both cell counts and clonogenic assay colony counts compared to untreated controls (U-251 MG: 45.4% and 26.5%; 15-037: 96.2% and 60.9%, respectively, p< 0.05). In both TMZR cell lines, MGMT expression was unchanged by TTFields, but MLH1 expression increased 2.2 fold in U-251 MG cells and 7.1 fold in 15-037 cells after TTFields application. CONCLUSIONS Not only are TMZR GBM cells sensitive to TTFields in vitro, but TTFields increased expression of MLH1 which may be able to reduce resistance to TMZ.

Quercetin inhibits intrahepatic cholangiocarcinoma by inducing ferroptosis and inhibiting epithelial-mesenchymal transformation via the NF-κB pathway

Intrahepatic cholangiocarcinoma (ICC) is a rare high-fatal hepatobiliary malignancy, the treatment option of ICC is very limited, and the prognosis is also poor. Recently, emerging evidence has shown the potential of quercetin (QE) for cancer therapy. We explored the effect and mechanism of QE on ICC in vitro and in vivo. CCK-8 assay and Clonogenic assay showed that QE could inhibit ICC cells proliferation and survival. PI staining suggested QE could induce ICC cells arrest in G1 phase. AV/PI staining suggested QE could promote ICC cells apoptosis. Wound Healing Assay and Transwell chamber experiment suggested QE could inhibit ICC cells EMT. RNA-seq, the changes in the structure of mitochondria by electron microscopy and the key markers of ferroptosis (free iron ions, MDA, SOD, GPX4) were supported QE could promote ferroptosis in ICC cells. Molecular docking showed that QE had direct interaction with NF-κB and GPX4. In vivo, treatment with QE inhibited tumor growth and prolonged survival time of tumor-bearing nude mice. Our data for the first time suggest that QE is a new ferroptosis inducer and combinative treatment of inhibiting NF-κB in ICC cells by inducing ferroptosis and inhibiting EMT, which will hopefully provide a prospective strategy for ICC patients.

Ανάπτυξη κυτταρογενετικών μεθοδολογιών για την εκτίμηση της ατομικής ακτινοευαισθησίας και αξιολόγηση σύγχρονων εξατομικευμένων τεχνικών ακτινοθεραπείας

Παρόλο που οι σύγχρονες τεχνικές ακτινοθεραπείας επιτρέπουν την αύξηση της δόσης στον όγκο-στόχο για μεγιστοποίηση του ποσοστού ίασης της νόσου και ελαχιστοποίηση της δόσης στους υγιείς ιστούς, υπάρχει σημαντική ανάγκη να κατανοήσουμε καλύτερα τους μηχανισμούς που εμπλέκονται στο πολύπλοκο δίκτυο της κυτταρικής απόκρισης στις ακτινοεπαγόμενες βλάβες του DNA γνωστό ως “DNA Damage response” (DDR), καθώς και να υπερνικήσουμε την ακτινοαντοχή του όγκου. Οι ATM, ATR και Chk1 είναι οι βασικές κινάσες που εμπλέκονται άμμεσα στην ενεργοποίηση του DDR και την αντοχή του όγκου στην ακτινοβολία. Είναι σημαντικό επομένως, να καθιερωθεί μία κυτταρογενετική δοκιμασία (assay) έναντι της συμβατικής κλωνογόνου δοκιμασίας (clonogenic assay) που να προσφέρει αξιόπιστα και γρήγορα αποτελέσματα για την εκτίμηση τόσο της ακτινοευαισθησίας όσο της ακτινοευαισθητοποίησης των κυττάρων με τη χρήση αναστολέων DDR. Η παρούσα διατριβή στοχεύει: (1) Στην ανάπτυξη μιας κυτταρογενετικής μεθόδου (G2-assay) για την εκτίμηση της χρωμοσωματικής ακτινοευαισθησίας χρησιμοποιώντας αναστολείς του DDR και του σημείου ελέγχου G2/M του κυτταρικού κύκλου. (2) Στην αξιολόγηση του G2-assay, ως εναλλακτική έναντι της συμβατικής κλωνογόνου δοκιμασίας (clonogenic assay) ως προς το χρόνο που απαιτείται για την ανάλυση των αποτελεσμάτων. (3) Στην ανάδειξη των πλεονεκτημάτων της μεθοδολογίας που αναπτύσεται στα πλαίσια της παρούσας διατριβής ως εργαλείο για τη διερεύνηση της αποτελεσματικότητας διαφορετικών αναστολέων του DNA Damage response – DDR για την ακτινοευαισθητοποίηση των κυττάρων. Σε αυτό το μέρος της πειραματικής διαδικασίας, ανθρώπινες κυτταρικές σειρές (ινοβλάστες 82-6 hTERT και επιθηλιακά κύτταρα RPE) ακτινοβολήθηκαν κατά τη διάρκεια της μετάβασής τους από τη φάση G2 του κυτταρικού τους κύκλου στη Μίτωση (Μ), και αξιολογήθηκε η συμβολή της αναστολής των οδών ATR, ATM και Chk1 αναλύοντας τις επαγόμενες χρωματιδικές αλλοιώσεις. Τα πειράματα διεξήχθησαν με κύτταρα που επωάστηκαν με καφεΐνη (αναστολέας ATM/ATR), με VE-821 (αναστολέας της ATR) και με UCN-01 (αναστολέας Chk1) ώστε να επιτευχθεί η κατάργηση της ενεργοποίησης του G2/Μ σημείου ελέγχου. Τα πειραματικά αποτελέσματα οδήγησαν στην ανάπτυξη και βελτιστοποίηση ενός τροποποιημένου G2-assay που υιοθετεί τον ATR αναστολέα VE-821, επιτρέποντας τη σύντομη χρονικά εκτίμηση της ακτινοευαισθησίας των κυττάρων. Η συνδυασμένη αυτή μεθοδολογία θα μπορούσε επίσης να χρησιμοποιηθεί ως ένα αξιόπιστο εργαλείο για τον έλεγχο της ακτινοευαισθητοποίησης που θα μπορούσαν να επιφέρουν μελλοντικοί αναστολείς DDR. Επίσης, τα αποτελέσματά του μέρους αυτού της διατριβής ενισχύουν την ιδέα ότι τα ATM, ATR και Chk1 αποτελούν ελκυστικούς στόχους φαρμάκων στην ογκολογία.Επιπλέον, υπάρχει σημαντική ανάγκη για περαιτέρω διερεύνηση της αποτελεσματικότητας των νέων τεχνικών ακτινοθεραπείας εκτιμώντας την απορροφούμενη δόση με κυτταρογενετικές μεθόδους βιοδοσιμετρίας, καθώς και για τη διερεύνηση της πιθανής επίδρασης του ρυθμού δόσης των νέων τεχνικών στο βιολογικό αποτέλεσμα. Στο πλαίσιο της παρούσας διατριβής εξήχθησαν αποτελέσματα σχετικά με την απορροφούμενη δόση σε ανθρώπινα λεμφοκύτταρα περιφερικού αίματος και σε κύτταρα καρκίνου του προστάτη PC3, μετά από ακτινοβόληση με τρεις διαφορετικές τεχνικές ακτινοθεραπείας (3D-CRT, IMRT και VMAT) με συνταγογραφούμενη δόση 2Gy. Για κάθε δείγμα αίματος ασθενούς και για τα κύτταρα PC3, πραγματοποιήθηκαν τρεις διαφορετικές ακτινοβολήσεις χρησιμοποιώντας τις τεχνικές 3D-CRT, IMRT και VMAT με δόση 2 Gy. Η απορροφούμενη δόση υπολογίστηκε με τη μέθοδο βιοδοσιμετρίας η οποία βασίζεται στην ανίχνευση δικεντρικών χρωµοσωµάτων σε κύτταρα. Τα αποτελέσματα της διατριβής έδειξαν μία υποεκτίμηση της απορροφούμενης δόσης της τάξης του ~4% για τις τεχνικές IMRT και VMAT συγκριτικά με τη συμβατική 3D-CRT ενώ δεν παρατηρήθηκε στατιστικά σημαντική διαφορά για τις τεχνικές VMAT και IMRT. Η παρατηρούμενη υποεκτίμηση της απορροφούμενης δόσης στα κύτταρα με τις τεχνικές VMAT και IMRT, πιθανόν να οφείλεται στο μικρότερο ρυθμό δόσης που επιτυγχάνεται με τις τεχνικές αυτές.

Design, Synthesis and Biological Activities of New Phthalimide and Thiazolidine Derivatives

The combination of pharmacophoric nuclei with different targets has been a strategy for the development of new drugs aimed at improving cancer treatment. A series of ten novel phthalimido-thiazolidine-2-4-dione derivatives were synthesized by two different synthetic routes. The compounds were tested and evaluated in vitro, through antineoplastic activities against cancer cells. Cell cycle analyzes and clonogenic assay were also performed. In addition to these tests, in silico predictions were performed. The synthesized FT-12 compound (9j) exhibited antiproliferative activity against Panc-1, Sk-mel-28 and PC-3 cells. FT-12 reduced the ability to form new clones, also caused irreversibility in cell cycle, inducing arrest in phase S. Besides, the compound (FT-12) caused necrosis and apoptosis. The results suggest that phthalimido-thiazolidine derivatives may be useful in cancer therapy, highlighting compound FT-12 (9j) as a promising candidate. More studies must be carried out to confirm the viability.

Design, Synthesis and Biological Activities of New Phthalimide and Thiazolidine Derivatives

The combination of pharmacophoric nuclei with different targets has been a strategy for the development of new drugs aimed at improving cancer treatment. A series of ten novel phthalimido-thiazolidine-2-4-dione derivatives were synthesized by two different synthetic routes. The compounds were tested and evaluated in vitro, through antineoplastic activities against cancer cells. Cell cycle analyzes and clonogenic assay were also performed. In addition to these tests, in silico predictions were performed. The synthesized FT-12 compound (9j) exhibited antiproliferative activity against Panc-1, Sk-mel-28 and PC-3 cells. FT-12 reduced the ability to form new clones, also caused irreversibility in cell cycle, inducing arrest in phase S. Besides, the compound (FT-12) caused necrosis and apoptosis. The results suggest that phthalimido-thiazolidine derivatives may be useful in cancer therapy, highlighting compound FT-12 (9j) as a promising candidate. More studies must be carried out to confirm the viability.

Anti-Tumor Activity of Metformin in Human Epidermal Growth Factor Receptor 2 Positive Breast Cancer Cells

Breast Cancer (BC) is the leading cause of cancer death in women. One BC subtype is very aggressive with amplification of human epidermal growth factor receptor 2 (HER2) protein. Although specific HER2+ targeting agents are available, most of HER2+ BC patients develop resistant to these agents. Recent studies show that metformin, is able to become anti-tumor in various cancer cells. This research aims to evaluate anti-tumor activities of metformin to HER2+ BC cells in both sensitive and resistant to trastuzumab. A series of assays were performed to evaluate metformin anti-tumor activities in HCC-1954 and SKBR-3 HER2+ BC cells. MTT assay was performed to evaluate cell death, and inhibitory concentration (IC50), while scratch assay was performed to assess inhibition of cell migration and clonogenic assay to assess cell proliferation. p<0.05 was considered to be significant. Metformin could suppress the number of HER2+ BC cells. Viability assay showed suppression of viable cells after metformin incubation of 60 and 600 µM compared to control, 30 and 90%, respectively. Surprisingly, IC50 of metformin was smaller in HER2+ BC HCC-1954 cells that resistant to trastuzumab compare to the sensitive one (SKBR-3). Both were below 1 µM, with R2 more than 0.95. Additionally, clonogenic assay showed less colony number and colony area with at least p < 0.05 in colony number and p < 0.01 in the area. In addition, metformin inhibited cell migration of HER2+ BC cells. Metformin shows a potency as anti-tumor by inducing cell death, inhibiting cell proliferation and cell migration of HER2+ BC cells.

Anticancer effects of 7,8-dihydromethysticin in human leukemia cells are mediated via cell-cycle dysregulation, inhibition of cell migration and invasion and targeting JAK/STAT pathway

The main focus of this research work was to study the anti-cancer properties of 7,8-dihydromethysticin against HL-60 leukemia cells. Investigations were also performed to check its impact on the phases of the cell cycle, cell migration and invasion, JAK/STAT signalling pathway and intracellular mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). Cell proliferation was assessed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and effects on colony formation were examined via clonogenic assay. Flow cytometry and Western blott analysis were performed to investigate the distribution of cell cycle phases. Flow cytometric analysis was performed for the examination of MMP and ROS production. The effect on JAK/STAT signalling pathway was examined through Western blot analysis. Results depicted that 7,8-dihydromethysticin induced concentration- as well as time-dependent inhibition of cell proliferation in leukemia HL-60 cells. Clonogenic assay indicated potential suppression in leukemia HL-60 cell colonies. The 7,8-dihydromethysticin molecule also caused cell cycle arrest at G2/M-phase along with concentration-dependent inhibition of cyclin B1, D1 and E. ROS and MMP measurements indicated significant ROS enhancement and MMP suppression with increasing 7,8-dihydromethysticin concentrations. Additionally, 7,8-dihydromethysticin led to remarkable dose-reliant inhibition of cell invasion as well as cell migration. Therefore, 7,8-dihydromethysticin should be considered a valuable candidate for leukemia research and chemoprevention.

Ursodeoxycholic Acid Suppresses The Malignant Progression of Colorectal Cancer Through TGR5-YAP Axis

Background: The Hippo/YAP pathway plays an important role in the development of cancers. Previous studies have reported that bile acids can activate YAP (Yes Associated Protein) to promote tumorigenesis and tumor progression. Ursodeoxycholic acid (UDCA) is a long-established old drug used for cholestasis treatment. So far, the effect of UDCA on YAP signaling in colorectal cancer (CRC) is not well defined. The study means to explore relationship of UDCA and YAP in CRC.Methods: The effect of UDCA on CRC cells proliferation was examined by MTT and clonogenic assay, western blotting (WB), real-time PCR, seperation of nucleoprotein and cytoplasmic protein and immunofluorescence (IF) assays were used to detect expression of YAP, WB, real-time PCR, GST-pull down assay and rhodamine-labeled phalloidin staining assays were used to detect activity of RhoA. After transfection of YAP5SA plasmids and RhoAV14 plasmids respectively, MTT, clonogenic assay, EdU incorporation assays and WB were performed. WB and ELISA were used to detect activity of cAMP/PKA axis, after blocking cAMP/PKA axis with siRNA and inhibitors, MTT, WB, IF and clonogenic assay were performed to test effect of UDCA on CRC cells. WB, IF and real-time PCR assays were used to detect expression of TGR5 in CRC cells after UDCA treatment, and then TGR5 agonist, TGR5 antagonist and siRNA were used to detect effect of UDCA on CRC cells. AOM/DSS-induced primary model was used to detect effect of UDCA, WB and Immunohistochemistry (IHC) were used to detect expression of related proteins.Results: UDCA suppressed YAP signaling by activating the membrane G‐protein‐coupled bile acid receptor (TGR5). TGR5 mainly regulated cAMP/PKA signaling pathway to inhibit RhoA activity, thereby suppressing YAP signaling. Moreover, the restoration of YAP expression alleviated the inhibitory effect of UDCA on CRC cell proliferation. In AOM/DSS-induced CRC model, UDCA inhibited tumor growth in a concentration-dependent manner and decreased expression of YAP and Ki67.Conclusion: UDCA plays a distinguished role in regulating YAP signaling and CRC growth from the primary bile acids and partial secondary bile acids, demonstrating the importance of maintaining normal intestinal bile acid metabolism in cancer patients. It also presents a potential therapeutic intervention for CRC.