Carboplatin-induced upregulation of pan ß-tubulin and class III ß-tubulin is implicated in acquired resistance and cross-resistance of ovarian cancer.

Resistance to platinum- and taxane-based chemotherapy represents a major obstacle to long-term survival in ovarian cancer (OC) patients. Here, we studied the interplay between acquired carboplatin (CBP) resistance using two OC cell models, MES-OV CBP and SK-OV-3 CBP, and non-P-glycoprotein-mediated cross-resistance to paclitaxel (TAX) observed only in MES-OV CBP cells. Decreased platination, mesenchymal-like phenotype, and increased expression of α- and γ-tubulin were observed in both drug-resistant variants compared with parental cells. Both variants revealed increased protein expression of class III ß-tubulin (TUBB3) but differences in TUBB3 branching and nuclear morphology. Transient silencing of TUBB3 sensitized MES-OV CBP cells to TAX, and surprisingly also to CBP. This phenomenon was not observed in the SK-OV-3 CBP variant, probably due to the compensation by other ß-tubulin isotypes. Reduced TUBB3 levels in MES-OV CBP cells affected DNA repair protein trafficking and increased whole-cell platination level. Furthermore, TUBB3 depletion augmented therapeutic efficiency in additional OC cells, showing vice versa drug-resistant pattern, lacking ß-tubulin isotype compensation visible at the level of total ß-tubulin (TUBB) in vitro and ex vivo. In summary, the level of TUBB in OC should be considered together with TUBB3 in therapy response prediction.

MiRNA-mRNA integrative analysis reveals epigenetically regulated and prognostic miR-103a with a role in migration and invasion of carboplatin-resistant ovarian cancer cells that acquired mesenchymal-like phenotype.

BACKGROUND: DNA methylation, histone modifications, and miRNAs affect ovarian cancer (OC) progression and therapy response. PURPOSE: Identification of epigenetically downregulated miRNAs in drug-resistant OC cell lines with a possible role in drug resistance and/or drug-induced mesenchymal-like phenotype. METHODS: MiRNA profiling was performed on parental and carboplatin-resistant OC cells, MES-OV and MES-OV CBP. RT-qPCR validation, epigenetic modulation and other CBP-resistant OC cell lines were used to select miRNAs of interest. The integration of miRNA-predicted target genes and differentially expressed genes (DEGs), pathway and functional analysis were used for forecasting their biological role. Data mining was performed to determine their possible prognostic and predictive values. RESULTS: MiRNA profiling revealed 48 downregulated miRNAs in OC cells whose drug sensitivity and metastatic potential were impacted by epigenetic modulators. Of the fourteen selected, nine were validated as changed, and seven of these restored their expression upon treatment with epigenetic inhibitors. Only three had similar expression patterns in other OC cell lines. MiRNA-mRNA integrative analysis resulted in 56 target DEGs. Pathway analysis revealed that these genes are involved in cell adhesion, migration, and invasion. The functional analysis confirmed the role of miR-103a-3p, miR-17-5p and miR-107 in cell invasion, while data mining showed their prognostic and predictive values. Only miR-103a-3p was epigenetically regulated at the constitutive level. CONCLUSION: High throughput miRNA and cDNA profiling coupled with pathway analysis and data mining delivered evidence for miRNAs which can be epigenetically regulated in drug-resistant, mesenchymal-like OC cells as possible markers to combat therapy-induced short overall survival and tumor metastatic potential.

Transcriptome analysis of newly established carboplatin-resistant ovarian cancer cell model reveals genes shared by drug resistance and drug-induced EMT.

BACKGROUND: In ovarian cancer (OC) therapy, even initially responsive patients develop drug resistance. METHODS: Here, we present an OC cell model composed of variants with differing degrees of acquired resistance to carboplatin (CBP), cross-resistance to paclitaxel, and CBP-induced metastatic properties (migration and invasion). Transcriptome data were analysed by two approaches identifying differentially expressed genes and CBP sensitivity-correlating genes. The impact of selected genes and signalling pathways on drug resistance and metastatic potential, along with their clinical relevance, was examined by in vitro and in silico approaches. RESULTS: TMEM200A and PRKAR1B were recognised as potentially involved in both phenomena, also having high predictive and prognostic values for OC patients. CBP-resistant MES-OV CBP8 cells were more sensitive to PI3K/Akt/mTOR pathway inhibitors Rapamycin, Wortmannin, SB216763, and transcription inhibitor Triptolide compared with parental MES-OV cells. When combined with CBP, Rapamycin decreased the sensitivity of parental cells while Triptolide sensitised drug-resistant cells to CBP. Four PI3K/Akt/mTOR inhibitors reduced migration in both cell lines. CONCLUSIONS: A newly established research model and two distinct transcriptome analysis approaches identified novel candidate genes enrolled in CBP resistance development and/or CBP-induced EMT and implied that one-gene targeting could be a better approach than signalling pathway inhibition for influencing both phenomena.

Styryl dyes with N-Methylpiperazine and N-Phenylpiperazine Functionality: AT-DNA and G-quadruplex binding ligands and theranostic agents.

New monomethine, unsymmetrical styryl dyes consisting of benzothiazole and N-methylpiperazine or N-phenylpiperazine scaffolds were synthesized, and their binding affinities for different ds-polynucleotides and G-quadruplex were studied. Substitution of piperazine unit with methyl or phenyl group strongly influenced their binding modes, binding affinities, spectroscopic responses and antiproliferative activities. Compounds with N-methylpiperazine substituents showed a significant preference for AT-DNA polynucleotides and demonstrated AT-minor groove binding, which manifested in strong fluorescence increase, significant double helix stabilization, and positive induced circular dichroism spectra. These compounds formed complexes with G-quadruplex by π-π stacking interactions of dye with the top or bottom G-tetrad. Bulkier compounds with N-phenylpiperazine function are probably bound to ds-polynucleotide by partial intercalation between base pairs. On the other hand, they showed stronger stabilization of G-quadruplex compared to methyl-substituted compounds. Fluorimetric titrations pointed to possible mixed stoichiometry's: 1:1 complex with π-π stacking interactions of dye on the top or bottom G-tetrad and 1:2 complex with dye positioned between two G-quadruplex molecules. Bulkier dyes with N-phenylpiperazine fragments demonstrated micromolar and submicromolar antiproliferative activity that was especially pronounced for leukaemia and lymphoma. Flow cytometric assay shows dose- and time-dependent increase in SubG0/G1 phase. Furthermore, the compounds enter the cells readily and accumulate in the mitochondrial space, co-localize with the standard mitochondrial markers.

Organometallic ruthenium(II)-arene complexes with triphenylphosphine amino acid bioconjugates: Synthesis, characterization and biological properties.

(p-Cymene)-ruthenium bioconjugates ML (1) and ML2 (2), bearing phosphane ligands substituted with chiral or non-chiral amino acid esters, L, were synthetized and characterized by instrumental methods (NMR, CD, MS) and DFT calculations (using the wB97xD functional). Cytotoxic activity of complexes 1 and 2 was investigated by using human cervical carcinoma cell line (HeLa) and MTT assay. Four (2pG, 2pA, 2mG and 2mA) out of ten synthesized ruthenium complexes showed significant toxicity, with IC50 values of 5-30 µM. Evaluation of the potential biomolecular targets of bioconjugates 2 by UV-Vis, fluorescence and CD spectroscopy revealed no measurable interaction with DNA, but micromolar affinity for proteins. The cytotoxicity of bioconjugates 2 is in correlation with their BSA binding constants, i. e. bioconjugates with lower IC50 values show higher binding affinities towards BSA. Compound 2mG with value of IC50 16 µM was selected for further biological characterization. The higher level of toxicity towards tumor compared to normal cell lines indicates its selective activity, important characteristic for potential medical use. It was detected 2mG caused increase of cells in the S phase of cell cycle and consequential decrease of cells in G0/G1 phase. Additionally, 2mG caused dose- and time-dependent increase of SubG0/G1 cell population, suggesting its ability to induce programmed cell death. Further investigation determined autophagy as the mode of cell death. The role of GSH in HeLa cells response to investigated organometallic ruthenium complexes was confirmed using specific regulators of GSH synthesis, buthionine sulfoximine and N-acetyl-cysteine. Pre-treatment of cells with ethacrynic acid and probenecid emphasized the role of GSH in detoxification of 2mG compound. The amount of total ruthenium accumulation in the cell did not correlate with toxicity of 2pG, 2pA, 2mG and 2mA, suggesting structure dependent differences in either cell uptake or kinetics of ruthenium complexes detoxification. We speculate that ruthenium complexes bind protein-based biomolecules further triggering cell death. Based on the gained knowledge, the synthesis and development of more tumor-specific ruthenium-based complexes as potential anticancer drugs can be expected.

Biological Activity of Flavonoids and Rare Sesquiterpene Lactones Isolated From Centaurea ragusina L.

The endemic Croatian species Centaurea ragusina L., like other species from the genus Centaurea, has been traditionally used in Croatia as an antibacterial agent and for the treatment of gastrointestinal and urogenital disorders. In several chromatographic steps, three flavonoids and three sesquiterpene lactones (STLs) were isolated and identified from the most active fractions of the ethanol extract. Two STLs, one for which we created the trivial name ragusinin, and hemistepsin A are here reported for the first time as constituents of the genus Centaurea. All six compounds were screened for their effect on several tumor and one normal cell lines. Among them, ragusinin showed the best bioactivity and high specificity to affect tumor murine SCCVII, human HeLa and Caco-2 cell lines, but not the viability of normal V79 fibroblasts. Due to these characteristics the action of ragusinin was investigated in more detail. Since DNA is the primary target for many drugs with antibacterial and anticancer activity, we studied its interaction with ragusinin. Rather moderate binding affinity to DNA excluded it as the primary target of ragusinin. Due to the possibility of STL interaction with glutathione (GSH), the ubiquitous peptide that traps reactive compounds and other xenobiotics to prevent damage to vital proteins and nucleic acids, its role in deactivation of ragusinin was evaluated. Addition of the GSH precursor N-acetyl-cysteine potentiated the viability of HeLa cells, while the addition of GSH inhibitor L-buthionine sulfoximine decreased it. Moreover, pre-treatment of HeLa cells with the inhibitor of glutathione-S-transferase decreased their viability indicating the detoxifying role of GSH in ragusinin treated cells. Cell death, derived by an accumulation of cells in a G2 phase of the cell cylce, was shown to be independent of poly (ADP-ribose) polymerase and caspase-3 cleavage pointing toward an alternative cell death pathway.